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植物BBX转录因子基因家族的研究进展

  生物工程学报  ${metaVo.year}, Vol. 36 Issue (4): 666-677

http://dx.doi.org/10.13345/j.cjb.190302

中国科学院微生物研究所、中国微生物学会主办

0

文章信息

杨宁, 从青, 程龙军

Ning Yang, Cong Qing, Cheng Longjun

植物BBX转录因子基因家族的研究进展

BBX transcriptional factors family in plants–a review

生物工程学报, ${metaVo.year}, 36(4): 666-677

Chinese Journal of Biotechnology, ${metaVo.year}, 36(4): 666-677

10.13345/j.cjb.190302

文章历史

Received: July 8, 2019

Accepted: September 29, 2019

Published: October 25, 2019

 Abstract

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引用本文

杨宁, 从青, 程龙军, 等. 植物BBX转录因子基因家族的研究进展. 生物工程学报, ${metaVo.year}, 36(4): 666-677

Yang N, Cong Q, Cheng LJ, et al. BBX transcriptional factors family in plants–a review. Chinese Journal of Biotechnology, ${metaVo.year}, 36(4): 666-677.

植物BBX转录因子基因家族的研究进展

杨宁

,

从青

,

程龙军

浙江农林大学 亚热带森林培育国家重点实验室，浙江 杭州 311300

收稿日期：2019-07-08；接收日期：2019-09-29；网络出版时间：2019-10-25

基金项目：浙江省科技厅林木新品种选育重大科技专项(No. 2016C02056-9)；国家自然科学基金(No. 31270657)资助

摘要：转录因子在调控植物生长、发育及环境适应性等方面发挥重要作用。具有B-box结构域的一类锌指结构转录因子称为BBX，它们通过调控基因转录，与同类或其他转录因子的互作参与植物光形态建成、花发育、避荫效应、植物信号转导以及非生物和生物逆境响应等。文中从BBX蛋白结构、分类以及其功能方面对该类转录因子在植物中的作用进行了综述。

关键词：B-box    光形态建成    成花    避荫效应    逆境    

BBX transcriptional factors family in plants–a review

Yang Ning

,

Qing Cong

,

Longjun Cheng

State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Hangzhou 311300, Zhejiang, China

Received: July 8, 2019; Accepted: September 29, 2019; Published: October 25, 2019

Supported by: Zhejiang Province Key Project for Science and Technology Grant (No. 2016C02056-9); National Natural Science Foundation of China (No. 31270657)

Corresponding author:

Longjun Cheng. Tel: +86-571-63743855; E-mail: ljcheng@zju.edu.cn.

Abstract: Transcriptional factors play important roles in plant growth, development and responses to stresses. BBX transcriptional factors are characterized with one or two B-box domains in the protein sequence. They are comprehensively involved in photomorphogenesis, flowering, shade avoidance, signal transduction of phytohormones, biotic and abiotic stress responses in plants by regulating gene transcription and interacting with other transcription factors. The classification, structure and functions of BBX of plants are reviewed in this paper.

Keywords:

B-box    photomorphogenesis    flowering    shade avoidance    stress    

转录因子是生物中重要的调控因子，广泛参与生物生长、发育、代谢及环境响应的调控过程。锌指蛋白(Zinc finger protein, ZFP)类转录因子是生物中的一大类转录因子，其含有由组氨酸(His)、半胱氨酸(Cys)和锌离子构成的锌指结构域，能够与DNA、RNA及蛋白质互作，发挥对转录、RNA包装、细胞凋亡、蛋白折叠组装等的调控作用。根据锌指结构转录因子蛋白结构的不同，该蛋白家族又分为若干亚家族[1-2]。

BBX (B-box)是锌指结构蛋白家族的一个亚家族，其氨基酸序列中包含1个或2个参与蛋白质-蛋白质之间互作的B-box基序。含B-box结构域的蛋白广泛存在于真核生物中，在动物中B-box结构域经常与RING结构域和卷曲螺旋结构域形成三价体结构蛋白TRIM (Tripartite motif)/RBCC (RING-B-box-coiled-coil)。在细胞中泛肽化过程、蛋白运输以及转录调控等方面发挥作用[3-4]。在植物中，拟南芥Arabidopsis thaliana有32个BBX蛋白，其中21个含2个B-box基序，另外11个仅含一个B-box基序[5]。水稻(Oryza sativa) 30个BBX蛋白中则有17个在N末端含有2个B-box基序[6]。B-box结构域往往单独存在于N末端，或者和存在于C末端的CCT (Conserved carboxy- terminal)结构域一起出现，含B-box结构域的蛋白与其他具有卷曲螺旋结构域的蛋白互作能产生等同于TRIM/RBCC的功能[5-6]。拟南芥中发现的第一个BBX蛋白CONSTANS (CO/AtBBX1)就能够与含有卷曲螺旋结构域的蛋白SPA1 (Suppressor of phyA-105 1)互作，参与光周期影响的成花控制过程[7]。

与动物中BBX蛋白研究相比[3, 8]，植物中BBX蛋白功能的研究比较晚。但近年来研究发现，植物B-box结构域在介导蛋白互作和基因表达调控方面发挥着极为重要的作用。在植物中，B-box结构域能够在BBX蛋白家族内部或和其他蛋白形成异源二聚体的形式，在基因转录调控中发挥重要作用。拟南芥中AtBBX24和AtBBX25通过形成一个非活性异源二聚体的形式，干扰HY5 (ELONGATED HYPOCOTYL 5)功能的发挥，进而影响AtBBX22/LZF1 (LIGHT-REGULATED ZINC FINGER PROTEIN 1)表达，抑制幼苗的光形态建成过程[9]。而AtBBX24还可以通过和HY5形成异源二聚体的作用，影响HY5与花青素合成酶基因启动子的结合抑制它们的表达[10]。甚至不同植物之间的BBX蛋白也可以产生互作效应。拟南芥中的AtBBX32蛋白就能够和大豆Glycine max中的GmBBX62互作[11]。另一方面，作为转录因子，BBX蛋白本身也调控着很多基因的表达，参与植物的生长、发育、光形态建成、激素信号转导和逆境响应等生命过程。随着研究的深入，BBX基因在植物中发挥的重要功能和分子调控机制不断被揭示出来，在植物分子遗传学领域其被重视的程度越来越高。

1 BBX蛋白的结构、分类及进化 B-box结构域包含1个或2个长约40个氨基酸残基的B-box基序。根据B-box基序氨基酸序列的一致性差异以及锌离子结合氨基酸残基的特异性，分为B-box1 (B1)和B-box2 (B2)两种类型。但组成B-box结构域的氨基酸残基序列仍具有相当的保守性。CCT结构域所含氨基酸残基为42–43个，其序列也具有高度保守性，参与BBX蛋白转录调控和核蛋白转运等功能的发挥[12-14]。

根据B-box结构域的数目和含有CCT结构域的情况，BBX蛋白可以分为5种结构类型。类型Ⅰ和类型Ⅱ都含有2个B-box和1个CCT结构域(B1+B2+CCT)，但类型Ⅰ、Ⅱ的B-box2在氨基酸序列上有差异；类型Ⅲ仅有B-box1和1个CCT结构域(B1+CCT)；类型Ⅳ只含有2个B-box结构域(B1+B2)；类型Ⅴ只有1个B-box结构域B1 (图 1)。除了B-box和CCT结构域外，Ⅰ–Ⅳ类中的不同BBX蛋白在C末端还含有CO、CO-like、TOC (Translocons the outer membrane of the Choroplast)以及M1–M7这7个功能尚不明确的结构域；有些BBX蛋白在C末端还存在1个由6个氨基酸组成的缬氨酸-脯氨酸(VP)基序，其一致性序列为：G-I/V-V-P-S/T-F。VP基序离CCT结构域一般约16–20个氨基酸残基，该基序在BBX蛋白与卷曲螺旋蛋白互作中发挥重要作用[12, 15-16]。COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1)作为泛肽连接酶组分参与HY5的降解，抑制植物光形态建成[17]。研究发现，具有差异的VP基序可以使不同的BBX以不同的亲和力结合COP1的WD40 (Trp-Asp-40)结构域，进而调控光形态建成[18]。

图 1 BBX蛋白的结构类型

Fig. 1 Structure class of BBX protein.

图选项

在BBX蛋白进化中，2个B-box结构域的氨基酸序列属于严谨性保守序列，而VP基序、核定位序列(Nuclear localization sequence, NLS)则具有辐射性变异特征[19]。例如BBX蛋白类型Ⅰ、Ⅱ和Ⅲ的核定位序列均为双组分型核定位序列，位于CCT结构域内，而没有CCT结构域的类型Ⅳ和Ⅴ也存在双组分或单组分核定位信号序列[12]。从BBX蛋白进化角度上看，动物中B-box1和B-box2结构域氨基酸序列的一致性具有较大差异。而植物中B-box1和B-box2结构域氨基酸序列尽管也有一定的差异，但它们的拓扑结构是相同的。另外，大多数绿藻的BBX蛋白只有1个B-box结构域，但较为原始的莱茵衣藻Chlamydomonas reinhrdtti BBX蛋白CrBBX1有2个B-box结构域，这说明最初的BBX蛋白只有1个B-box结构域，只是在后来的进化中B-box结构域所在的基因组序列发生了复制事件，而该事件很可能发生在绿色植物登陆之前，然后才是CCT结构域的形成。而2个B-box和CCT结构域在随后进化过程中的删除，以及B-box结构域的进一步复制事件，帮助BBX蛋白完成了其不同结构类型的演化[12]。

2 BBX蛋白的功能 BBX基因家族编码的蛋白作为重要的转录因子，一方面直接作用于相关基因的启动子区域，调控基因的表达；另外还以蛋白互作的形式调控其他蛋白的功能，参与植物的光形态建成、成花生理、避荫效应、激素信号转导及对逆境胁迫的响应等重要生长、发育过程。

2.1 BBX蛋白与光形态建成 BBX蛋白在光形态建成中发挥重要作用。拟南芥bbx4突变体幼苗在红光下表现为胚轴伸长；bbx20在红光和蓝光下胚轴伸长；bbx21和bbx2无论在红光、远红光还是蓝光中都表现为胚轴伸长[14, 16, 20-21]。抑制光形态建成的BBX蛋白则与光受体类型关系不大，bbx24、bbx25和bbx32突变体在红光、远红光和蓝光下，胚轴都受到明显抑制[9, 22-23]。这说明促进光形态建成的BBX蛋白往往在不同光敏色素和细胞色素途径的下游起作用。

不同的BBX蛋白在光形态建成中发挥的作用也不同，既有协同作用，又有拮抗作用。尤其是类型Ⅳ，该类型拟南芥中的8个BBX蛋白有6个(BBX20–BBX25)参与HY5依赖性的光形态建成过程，HY5是对光形态建成过程起促进作用的核心调控因子[24]。其中，AtBBX20–23是光形态建成的正调控因子；而AtBBX24和AtBBX25则是光形态建成负调控因子。AtBBX21和AtBBX22互作又可以直接作用于HY5基因表达的启动子区域，增强其表达活性[16, 25]，AtBBX24和AtBBX25则通过形成异源二聚体的形式抑制HY5的转录活性[9]。不同的BBX蛋白对COP1也有不同的调控。AtBBX20、AtBBX21和AtBBX22抑制COP1的功能发挥；AtBBX24和AtBBX25则能提高COP1的活性，而COP1在有光的条件下又能够结合到BBX24和BBX25上减弱它们的功能[9, 14, 16, 21, 26]。在发挥作用的方式方面，BBX20、BBX24和BBX25能直接与COP1进行互作，而BBX21和BBX22则需要被COP1招募进入核散斑才能发挥作用。另外，紫外受体UVR8 (UV-B resistance 8)在紫外辐射下，能够在核内积累并激活COP1，进而调控紫外响应基因，抑制拟南芥幼苗胚轴的生长，但BBX24能延缓由COP1活化引起的HY5积累造成的光形态建成效应[27]。有意思的是，BBX24还可以调节另外一个紫外信号响应调节蛋白RCD1 (Radical-induced cell death 1)，抑制BBX24的表达，表明BBX24在紫外辐射引起的光形态建成效应中，可以通过负反馈调节途径精准地调控这种效应[28]。对于结构相似的BBX蛋白为何产生完全不同的功能，近来的研究认为这可能与BBX蛋白C末端序列的多样性有密切的关系，该区域松散的保守型导致了不同BBX蛋白功能的多样性，例如BBX21和BBX24都可以在转录后调控HY5的活性，但方式是相反的，这种相反的调控方式就是由它们C末端序列的不同导致的[10, 29]。

其他BBX蛋白也参与光形态建成过程，如AtBBX4可以在COP1下游作为光形态建成的正调控因子而发挥作用[14]；AtBBX32能够与AtBBX21结合后，进而与HY5互作并降低其转录活性[23]；低剂量紫外辐射条件下，COP1还可以促进BBX5和BBX18的表达而抑制BBX7和BBX8的表达[30]。BBX30和BBX31在正常光下都抑制植物光形态建成，但在紫外光下BBX31却作为光形态建成的正调控因子而存在[31-32]。

另外，光诱导的花色素苷合成作为光形态建成的一部分，也受到BBX蛋白调控。其中，AtBBX21、AtBBX22和AtBBX23是花色素苷合成的正调控因子[16, 25, 33-34]，AtBBX24、AtBBX25和AtBBX32则抑制花色素苷的生物合成和积累[9-10, 23]。梨Pyrus pyrifolia中PpBBX16也是果实中花青素苷的正调控因子[35]。BBX蛋白往往通过直接或者与其他蛋白互作间接调控花青素苷生物合成的相关基因，AtBBX21可以通过直接结合在HY5启动子区域，激活其表达，影响花青素苷的合成；番茄Solanum lycopersicum中SlBBX20也可以直接结合在类胡萝卜素合成关键酶基因PHYTOENE SYNTHASE1启动子上，诱导其表达，参与类胡卜素的合成[36]。而AtBBX24则可能通过与HY5形成异源二聚体的方式干扰HY5与花青素苷合成基因启动子的结合[10]。

由此可见，BBX蛋白参与光形态建成既有表达层面上的基因调控起作用，也有蛋白层面的互作效应的参与，甚至结构相似的蛋白也会发挥完全不同的功能，它们之间的互作机制更是极其复杂。

2.2 BBX蛋白在植物成花过程中的作用 植物成花过程受多种条件控制。其中，光周期对植物成花的影响与BBX蛋白关系密切。长日照下，拟南芥的CO/AtBBX1能够直接结合在FT (FLOWERING LOCUS T)基因启动子上，激活FT基因的表达促进植物的开花[13, 37]。同时，针对CO的抑制因子COP1，光诱导的FKF1能够阻止COP1二聚体的形成，使COP1发挥作用的四聚体形式[(COP1)2(SPA1)2]不能正常形成，从而使CO能顺利地作用于成花途径，短日照条件下，FKF1 (FLAVIN-BINDING, KELCH REPEAT，F-BOX 1)主要在夜间表达，不能被光激活，因此也就不能发挥抑制CO的抑制子COP1的作用[38-40]。另外，HOS1 (HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1)也能与CO互作，介导长日照条件下CO蛋白的降解，通过调控CO的表达量精确植物的成花时间[41-42]。

除了CO外，其他BBX蛋白也参与植物成花的调控过程。拟南芥bbx4/col3突变体长日照、短日照条件下都能提前开花[14]。进一步研究表明，AtBBX4 (COL3)可能是通过与AtBBX32互作进一步调控FT的表达实现对植物成花调控的[43]。AtBBX32还能够与EMF1 (EMBRYONIC FLOWER1)互作调控拟南芥中成花时间[44]。短日照条件下，AtBBX6也能通过促进FT、SOC基因的表达导致植株提前开花，co突变体中超表达AtBBX6也能一定程度上抑制晚花效应。但有意思的是，BBX6缺失的突变体中，成花时间并不发生改变。暗示其在调控植物成花方面的功能上可能具有冗余性[45]。bbx7/col8突变体开花早于野生型，而BBX7超表达株系长日照下则延长拟南芥成花时间，说明BBX7可能通过抑制CO和FT基因表达影响植物的成花[46]。AtBBX19、AtBBX13也都影响成花。超表达AtBBX19抑制拟南芥转基因株系的成花，它能够通过与CO竞争性结合FT的启动子，抑制FT及其下游基因的表达，从而抑制成花[47]；COL12 (AtBBX13)则通过与CO的互作，改变CO的活性，影响植物的成花发育[48-49]。

在成花途径的影响方面，BBX蛋白在不同植物之间具有很强的保守性。水稻中CO同源基因Hd1在短日照条件下促进开花而长日照条件下抑制开花，其功能发挥不依赖于其转录水平的高低，而与其蛋白功能的调控密切相关[50]。Hd1 (Heading date 1)可能通过与OsHAL3 (Halotolerance protein 3)或者GHD7 (Grain number, plant height, and heading date 7)形成蛋白复合物的形式，抑制长日照下水稻的成花，另外OsPPR37 (Pentatricopeptide repeat- containing protein 37)可能也存在与Hd1的互作效应[51-53]。水稻中另外两个BBX蛋白也参与了光周期途径，OsBBX5、OsBBX27和OsCOL15短日照条件下均抑制成花，OsBBX5在光敏色素-B受体的下游发挥作用；OsBBX27通过负调控Hd3a和FTL (FLOWERING LOCUS T-like)基因的表达而发挥作用[54-55]；OsCOL15则可以上调GHD7或者下调成花激活基因RID1 (Rice Indeterminate 1)的作用来实现其功能[56]。大麦Hordeum vulgare L.中的HvCO1基因也是BBX基因家族的同源基因，它能够通过上调HvFT基因的表达而促进植株的成花[57]。中国大白菜Brassica rapa中的BrBBX32也能通过其B-box结构域与BrAGL24 (AGAMOUS-LIKE 24)互作促进植株的开花过程[58]。菊花Chrysanthemum morifolium中的CmBBX24则对开花有抑制作用[59]。

但影响植物花发育的BBX蛋白的保守性特点也有一定的局限。甜菜Beta vulgaris中的BvCOL1与拟南芥中的CO亲缘关系最近，但它并不调控植株的成花效应。而BvBBX19则可能与BTC1 (Bolting time control 1)互相作用调控BvFT1和BvFT2参与花发育，这条途径很可能与拟南芥中CO介导的成花发育途径有所不同[60]。

2.3 BBX蛋白在避荫响应中的作用 植物生长密度过高时，其生长环境的红光/远红光(R/FR: red/far red)比值会下降，这种下降成为植物产生避荫响应的一种重要信号，从而导致植物的竞争性生长，如胚轴和茎的伸长、分枝减少、叶片相对与水平方向的生长角度加大和加速开花等[61-62]。BBX蛋白能够介导遮荫环境中的细胞伸长。cop1突变体严重抑制避荫响应，而在突变体中双突变bbx1和bbx2则可以恢复cop1的避荫响应，这说明BBX21/LHUS和BBX22作用在COP1的下游，在遮荫环境条件下通过负反馈机制调节植株的避荫响应[63]。bbx24突变体遮荫条件下胚轴缩短，bbx24和bbx25双突变体中，胚轴缩短的表型被加强。研究表明BBX24和BBX25蛋白对遮荫的响应也是依赖于COP1蛋白的[9]。另外，PIF4转录因子在遮荫环境中能结合在细胞伸长相关基因的启动子上，促进细胞伸长，而这种效应需要BBX24与DELLA蛋白的结合，以阻止DELLA介导的PIF4活性抑制[64]。BBX16/COL7能够通过上调PIL1的表达在高R/FR比值下促进拟南芥的分枝，在低R/FR比值下增强植株的避荫效应[65]。

研究表明，不同的BBX蛋白在避荫响应中发挥的作用是不同的，甚至是相反的，如AtBBX19、AtBBX21、AtBBX22能抑制避荫响应，而AtBBX18、AtBBX24对避荫响应则有促进作用，参与避荫响应的植物激素相关基因的表达也受到BBX蛋白的调控[63]。不同BBX基因在避荫响应发挥的具体功能和作用机制都需要进一步的研究进行揭示。

2.4 生物和非生物逆境响应中的BBX蛋白 逆境因子一直是影响植物生长发育的重要因素。大部分转录因子家族的基因都参与逆境响应调控。BBX蛋白在植物生长的生物逆境和非生物逆境响应方面都发挥一定的作用。

在光形态建成发挥负调控作用的AtBBX18还参与了拟南芥耐热响应。AtBBX18基因表达被热胁迫诱导。AtBBX18 RNA干涉转基因株系其耐热性得到提高，而超表达AtBBX18的转基因株系则热耐受性下降。AtBBX18对热胁迫响应基因DGD1 (Digalactosyldiacyglycerol synthase 1)、HsfA2 (Heat stress transcription factor A2)和Hsp101 (Heat shock protein 101)都有下调作用，暗示该基因在热胁迫响应中发挥负调控作用[66]。AtBBX24则参与盐逆境信号的传递作用。在酵母Saccharomyces cerevisiae中，AtBBX24 cDNA转化细胞能够提高其耐盐能力，拟南芥中超表达AtBBX24的株系耐盐能力也得到提高，但BBX24基因表达并不受盐的诱导。另外，AtBBX24蛋白能结合到一个H-蛋白基因HPPBF-1的启动子区域，促进其表达，该基因同样受盐胁迫诱导，说明AtBBX24间接参与了植物耐盐性提高相关的分子途径[67]。苹果Malus domestica中相当一部分BBX基因在渗透压、高盐、低温和脱落酸(Abscisic acid, ABA)处理下表达上调[68]。其中，MdBBX10在大肠杆菌Escherichia coli中表达增强了细胞对盐和渗透胁迫的耐受性，研究表明拟南芥中超表达该基因也能增强转基因植株对干旱、盐等非生物逆境的抗性，这种抗性的增加与超表达MdBBX10导致的活性氧清除能力增强密切相关[69-70]。水稻中BBX基因Ghd2超表达后，植株对干旱变得敏感，进一步研究发现，超表达Ghd2转基因株系衰老相关基因表达上调，表明Ghd2在加速干旱诱导的水稻叶片衰老中起重要作用[71]。菊花的CmBBX24、CmBBX22也都参与了植株对低温、干旱等的响应过程[59, 72-73]。另外，高盐和PEG处理能够诱导马铃薯Solanum sogarandinum中SsBBX24基因的表达和蛋白积累，且日照时间长短还能调控SsBBX24对盐胁迫的响应[74]。由此可见，BBX在广泛参与了植物的干旱、低温、高盐和氧化胁迫等非生物逆境响应。对它们进行具体分子调控机制的研究，将有利于提高作物的非生物逆境抗性。

机械损伤响应和生物侵害的保卫反应中也有BBX蛋白的参与。茉莉酸(Jasmonic acid, JA)是机械损伤响应中的重要信号分子，用JA的前体12-氧-植物二烯酸(12-oxo-phytodienoic acid, OPDA)处理拟南芥时，AtBBX32表达被强烈上调。几丁质也可以上调AtBBX32的表达，响应几丁质的转录因子是参与植物保卫反应的关键因子[75]。油菜Brassica napus在遭受跳甲危害时，BBX基因的表达也发生改变[76]。水稻中敲除OsCOL9会增加转基因株系对稻瘟病的易感性，而超表达该基因，则可以增强植株对稻瘟病的抗性[77]。这表明BBX在机械损伤和生物胁迫中也发挥了一定作用。

2.5 BBX与激素信号转导 除了直接在转录和蛋白水平上的调控外，BBX蛋白已经被证实在生长素(Indole-3-acetic acid, IAA)、赤霉素(Gibberellic acid, GA)、脱落酸和油菜素内酯(Brassinosteroid, BR)等激素信号转导中发挥了重要的作用[78]。

AtBBX21能够分别与HY5和ABI5 (ABA insensitive 5)形成异源二聚体，调控光介导的ABA信号转导作用，影响光形态建成[79]；AtBBX18则可以通过促进赤霉素的活性促进胚轴的生长，这种促进作用是通过调控GA代谢基因的活性来实现的[80]。AtBBX20可以通过抑制参与信号途径的基因BZR1 (BRASSINAZOLE-RESISTANT 1)，来抑制胚轴的伸长[81]。水稻中的OsBBX8、OsBBX27和OsBBX30对光信号和IAA、GA等激素信号都有响应，表明这些基因可能在光形态建成和激素信号的交叉互作中发挥了重要的作用[6]。

在避荫响应中，AtBBX21作为负调控因子能够下调生长素、乙烯和油菜素内酯相关的基因，影响长期遮荫条件下的植物生长[63]。AtBBX16能够上调生长素合成抑制因子SUR2 (Superroot 2)的表达，调控植物的分枝特性以应对遮荫条件[65, 82]。与BBX24结合调控PIF4 (Phytochrome interacting factors 4)的DELLA因子，是GA信号转导途径的重要负调控因子，说明GA信号也参与了避荫响应的调控过程[64]。

干旱环境下，ABA可以通过调控CO转录后的功能或者活性，进而促进FT基因的表达而实现“干旱逃逸” (干旱条件下加速开花的现象)[83]。菊花中Cm-BBX24 RNA干涉株系开花提前，但很多GA生物合成途径的相关基因被上调，外施GA4/7也会影响Cm-BBX24表达，暗示GA可能参与了Cm-BBX24对菊花成花的调控过程[59]。拟南芥中CO还可以通过与介导水杨酸(Salicylic acid, SA)信号转导的TGA4 (TGACG MOTIF-BINDING FACTOR 4)蛋白互作调控植物的成花发育[84]。这说明在植物成花发育过程中，植物激素同样在BBX扮演的角色中发挥了一定作用。

激素信号同样参与了BBX在植物逆境响应中作用。OsCOL9能够与OsRACK1 (Receptor for activated C-Kinase 1)互作，通过水杨酸和乙烯(Ethylene, ET)信号通路增强水稻对稻瘟病的抗性[77]。用ABA处理拟南芥植株时，叶片中BBX11、BBX13、BBX22的表达在大幅上调，而BBX2、BBX3、BBX16、BBX18、BBX19则能被ABA以及参与ABA早期信号转导的环腺苷二磷酸核糖cADPR (Cyclic adenosine diphosphate ribose)下调[85]。拟南芥中超表达CmBBX22，转基因植株对ABA敏感程度下降，且对干旱耐受能力增强。暗示ABA可能介导了CmBBX22响应干旱的能力。中国白梨Pyrus bretschneideri和苹果中BBX基因在非生物逆境和激素处理下表达变化，也说明了BBX在非生物逆境响应下功能的发挥需要植物激素协助[72, 86]。

3 总结与展望 BBX基因家族作为锌指结构转录因子中一类重要成员，广泛参与了植物生长和发育以及对环境的响应过程。在发挥作用的方式上，不仅存在转录活性的调控，BBX还往往通过复杂的蛋白互作效应实现其功能。同一个基因家族不同成员在调控同一个生理过程时，正效应和负效应共存，这对植物生长、发育的精准调控非常有利。研究这类基因的功能，有利于了解植物对复杂生理过程和环境响应的分子机制。目前，尽管对BBX基因家族成员功能开始有了一定了解，但囿于其功能的复杂性，这种认知仍非常有限。特别是在木本植物中，相应BBX基因功能的研究更少，而BBX参与的成花效应、避荫响应和逆境胁迫响应等对林木植物的生产和育种也具有非常重要的意义。很有必要对重要林木植物的BBX基因功能进行深入的研究。

笔者所在的研究团队从巨桉Eucalyptus grandis全基因组范围内鉴定得到了21个BBX基因，21个EgrBBX蛋白序列的B1、B2和CCT结构域均非常保守，并且和拟南芥B1、B2和CCT结构域保守氨基酸序列的相似性非常强。说明BBX的结构域在进化过程中具有很强的保守性。在这些启动子序列上，分布着大量的：光响应元件：ACE、Sp1、G-box等；激素响应元件：脱落酸响应元件ABRE (ABA-responsive element)、茉莉酸响应元件CGTCA-motif、水杨酸响应元件TCA；以及非生物逆境响应元件：干旱诱导的MYB结合序列(MYB binding sequence，MBS)、低温响应元件(Low temperature response，LTR)和热激响应元件(Heat shock elements，HSE)等。暗示这些基因在光信号、激素和逆境响应中都可能发挥了重要功能。进一步的表达分析也表明，EgrBBX基因对光照强度和光周期有明显的响应；低温(4 ℃)和高盐(200 mmol/L NaCl)处理对大部分EgrBBX基因能产生瞬时性的诱导表达。尤其在高盐条件下，除了EgrBBX7、EgrBBX9和EgrBBX13外，其他EgrBBX基因均在24 h内有一个明显的诱导表达峰出现，茉莉酸甲酯处理的植株中EgrBBX基因的表达也有类似的特点。这些结果表明，桉树中的BBX蛋白可能也是整合光形态建成、激素信号转导和逆境响应等的关键因子，进一步研究这些基因的功能具有重要意义。

BBX功能的复杂性主要在于不同BBX蛋白之间的功能差异，及由它们之间的不同互作引起的不同生物学效应方面。因此，下一步研究中，一方面可以根据基因在不同环境和处理下的表达情况筛选相应生物学相关的重要基因，通过遗传转化手段，进一步明确这些基因的功能。同时结合蛋白互作技术平台，分析不同BBX之间的互作对它们功能发挥的影响。而转录组等组学分析手段的利用，将为解析BBX参与的重要分子调控网络提供有力的支持。

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The Plant Cell | 邓兴旺研究组揭示植物光形态建成的重要分子机理

日期： 2018-08-13

2018年8月10日，北京大学现代农学院邓兴旺教授研究组在国际知名学术期刊《The Plant Cell》在线发表题为“B-BOX

DOMAIN PROTEIN28 Negatively Regulates Photomorphogenesis by Repressing the

Activity of Transcription Factor HY5 and Undergoes COP1-mediated Degradation”的研究成果。

太阳光不仅被植物通过光合作用转化为有机能，同时作为一个重要的外源环境信号调控植物的多个生长发育过程，包括种子的萌发，幼苗的形态建成，植株的开花以及生物周期节律等。在自然界中，埋在土壤中的种子萌发后，将进行暗形态建成，破土而出见光后，则能够进行光形态建成，长成健康的幼苗。CONSTITUTIVELY

PHOTOMORPHOGENIC 1（COP1）是光形态建成的重要核心抑制子，它是一个含有RING结构域的E3泛素连接酶，可以直接泛素化下游光形态建成促进因子如HY5等，并促使它们通过26S蛋白酶体所降解，以此来抑制植物的光形态建成。BBX（B-box）蛋白是一类含有B-box结构域的转录调节子，已有文献报道，多个BBX蛋白在COP1-HY5核心调控组分所介导的光形态建成的过程中行使了重要功能。

结合遗传学和生物化学的方法，邓兴旺教授团队鉴定了参与光形态建成的一个新的抑制因子，BBX28。BBX28是含有一个B-box结构域的转录调节子，属于B-box蛋白第五亚家族的成员。在黑暗下，BBX28与COP1相互作用，并且COP1 会促进BBX28通过26S蛋白酶体所降解。在光照下，由于COP1蛋白的活性受到多种分子机制的抑制，使得COP1蛋白的底物如HY5和BBX28等得以积累。而此时积累的BBX28和HY5在细胞核内形成异源二聚体，通过抑制HY5对下游目的基因启动子上特异DNA基序的结合能力，来抑制HY5的转录活性，最终抑制植物的光形态建成。该项原创性的研究揭示了植物光形态建成中一项新的分子调控机制，为进一步深入理解光调控植物生长发育信号通路具有重要意义。

COP1-BBX28-HY5调控植物光形态建成的工作模型

本项研究工作以北京大学现代农学院、生命科学学院为第一单位。北京大学现代农学院、生命科学学院邓兴旺教授和美国得克萨斯大学奥斯汀分校分子生物科学系许冬清博士是本文共同通讯作者；北京大学现代农学院、生命科学学院林芳博士、南方科技大学生物系江燕博士为本文共同第一作者；北京大学现代农学院、生命科学学院范六民教授和李健博士；南方科技大学生物系梁建生教授和严婷婷以及美国得克萨斯大学奥斯汀分校分子生物科学系 Jeffey Chen教授参与了本项目的部分研究工作。

这项研究得到了北大-清华生命科学联合中心、蛋白质与植物基因研究国家重点实验室、南方科技大学、国家自然科学基金委员会以及中国博士后科学基金的资助。

文章链接: http://www.plantcell.org/content/early/2018/08/10/tpc.18.00226

上一篇：PLOS Biology | 唐世明研究组报道清醒猴长时期全光学光遗传技术 下一篇：EUR J HUM GENET | 饶毅研究组通过多层次基因组分析揭示参与人类记忆的遗传基因

友情链接：

北京大学国家级生物学实验教学示范中心

蛋白质与植物基因研究国家重点实验室

膜生物学国家重点实验室

细胞增殖与分化教育部重点实验室

北京大学生命科学学院校友尊师基金

北京大学生物医学前沿创新中心

北京大学高通测序中心

生命科学联合中心

北京大学生态中心

北京生命科学研究所

北大工会

清华大学生命科学学院

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Plant Cell | 兰州大学林芳课题组揭示BBX28和BBX29整合光信号和油菜素内酯信号调控植物生长发育的新机制 - 知乎

Plant Cell | 兰州大学林芳课题组揭示BBX28和BBX29整合光信号和油菜素内酯信号调控植物生长发育的新机制 - 知乎切换模式写文章登录/注册Plant Cell | 兰州大学林芳课题组揭示BBX28和BBX29整合光信号和油菜素内酯信号调控植物生长发育的新机制BioArt植物​已认证账号责编 | 王一光信号作为重要的环境信号影响着幼苗的形态建成，油菜素内酯是众多植物激素中调控幼苗形态建成最典型的植物激素，光信号与油菜素内酯信号在幼苗的形态建成中存在着紧密的交互作用，然而其具体的分子机制还不是很清楚。 2022年3月16日，兰州大学生命科学学院/细胞活动与逆境适应教育部重点实验室林芳教授课题组在The Plant Cell在线发表了题为The photomorphogenic repressors BBX28 and BBX29 integrate light and brassinosteroid signaling to inhibit seedling development in Arabidopsis的研究论文。该研究发现光形态建成的抑制因子BBX28和BBX29能够正向调控油菜素内酯信号抑制拟南芥幼苗的生长发育过程。BBX (B-BOX DOMAIN PROTEIN) 蛋白是一类含有B-box结构域的锌指蛋白，在拟南芥中共有32个成员，主要含有B-box结构域、CCT结构域和VP元件。拟南芥BBX蛋白作为重要的转录调节因子参与植物幼苗的光形态建成、植株开花、避荫反应和各种生物或非生物胁迫等。先前的文献报道，BBX28和BBX29能够与HY5相互作用，通过抑制HY5对下游基因启动子的结合能力进而抑制HY5的转录活性，以此来抑制拟南芥幼苗光形态建成的过程（Lin et al., 2018 The Plant Cell; Song et al., 2020 Plant Journal）。然而BBX28和BBX29是否参与植物生长发育的其他生物学过程还不清楚。为了进一步探究BBX28和BBX29参与植物幼苗形态建成的分子机制。作者利用各种植物激素对BBX28和BBX29的突变体和过量表达转基因植株进行处理，发现油菜素内酯合成的抑制剂Brz (Brassinazole) 处理后，bbx28 bbx29子叶张开的角度与野生型相比明显变大，而BBX28和BBX29过量表达转基因植株子叶张开的角度与野生型相比明显变小，预示着BBX28和BBX29是油菜素内酯信号通路的正调节因子。通过表型分析发现，BBX28和BBX29部分位于BRI1 (BRASSINOSTEROID INSENSITIVE 1)，BIN2 (BR-INSENSITIVE 2)和BZR1 (BRASSINAZOLE RESISTANT 1) 的遗传学下游。油菜素内酯处理后BBX28和BBX29蛋白量明显增加，而BRI1和BIN2部分参与油菜素内酯促进BBX28和BBX29蛋白积累的过程。进一步通过酵母双杂交文库的筛选发现BBX28和BBX29能够与油菜素内酯信号通路的正调节因子BEE1/2/3 (BR ENHANCED EXPRESSION 1) 相互作用，并且BBX28/29通过促进BEE1/2/3的转录活性来调节下游目的基因的表达，以此来参与油菜素内酯信号通路所介导的植物生长发育的过程。因此，BBX28和BBX29是新发现的整合光信号和油菜素内酯信号调控植物形态建成的重要调节因子。兰州大学生命科学学院林芳教授为该论文的通讯作者，兰州大学生命科学学院博士研究生曹婧、硕士研究生梁宇霞和南方科技大学博士研究生严婷婷为论文的共同第一作者。北京大学邓兴旺教授、兰州大学黎家教授、南京农业大学许冬清教授和厦门大学黄烯教授等参与了该工作。该研究得到了国家自然科学基金、中国科协“青年人才托举工程”项目和兰州大学中央高校基本科研业务费等的资助。参考文献：Cao, J., Liang, Y.X., Yan, T.T., Wang, X.C., Zhou, H., Chen, C., Zhang, Y.L., Zhang, B.H., Zhang, S.H., Liao, J.C., Cheng, S.J., Chu, J.F., Huang, X., Xu, D.Q., Li, J., Deng, X.W., and Lin, F. (2022). The photomorphogenic repressors BBX28 and BBX29 integrate light and brassinosteroid signaling to inhibit seedling development in Arabidopsis. Plant Cell https://doi.org/10.1093/plcell/koac092. Lin, F.#, Jiang, Y.#, Li, J., Yan, T., Fan, L., Liang, J.S, Chen, Z.J., Xu, D., and Deng, X.W. (2018) B-BOX DOMAIN PROTEIN28 negatively regulates photomorphogenesis by repressing the activity of transcription factor HY5 and undergoes COP1-mediated degradation. The Plant Cell 30, 2006-2019. (#co-first author).Song, Z. #, Yan, T. #, Liu, J., Bian, Y., Heng, Y., Lin, F., Jiang, Y., Deng, X.W., and Xu, D. (2020). BBX28/BBX29-HY5-BBX30/31 form a feedback loop to fine-tune photomorphogenic development. Plant Journal. (#co-first author)BioArt植物原文链接：https://doi.org/10.1093/plcell/koac092编辑于 2022-03-19 17:50植物​赞同 3​​添加评论​分享​喜欢​收藏​申请

【BBX盘点】世锦赛冠军们！！丨2005-2018的Beatbox世界冠军合集丨顶尖Beatboxer_哔哩哔哩_bilibili

【BBX盘点】世锦赛冠军们！！丨2005-2018的Beatbox世界冠军合集丨顶尖Beatboxer_哔哩哔哩_bilibili 首页番剧直播游戏中心会员购漫画赛事投稿【BBX盘点】世锦赛冠军们！！丨2005-2018的Beatbox世界冠军合集丨顶尖Beatboxer

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787624☞Human Beatbox丨上世纪80年代的新兴hiphop元素ᝰ通俗讲作节奏口技 但区别于中国传统口技

【订阅】此频道℡，持续输出最新最dope的BBX生态ᝰ

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原作者：DR Production

网址：https://youtu.be/aNWPXUlcOag

注：搬运视频如涉及版权纠纷，本账号会立即处理音乐演奏BBOXBEATBOXBATTLEHIPHOP比赛现场

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赛事库 课堂 2021

Plant Cell | 河南大学/中科院植物所合作团队揭示植物生物钟的精细运行机制 - 知乎

Plant Cell | 河南大学/中科院植物所合作团队揭示植物生物钟的精细运行机制 - 知乎首发于BioArt植物 · 前沿切换模式写文章登录/注册Plant Cell | 河南大学/中科院植物所合作团队揭示植物生物钟的精细运行机制BioArt植物​已认证账号责编 | 王一生物钟 (Circadian clock) 作为有机体内在的时间调控机制，产生在分子、生化、生理及动物行为上的近24小时节律 (Circadian rhythms)，与环境中授时因子信号的周期性变化维持同步 (Synchronization)，保证有机体的环境适应能力。构成植物生物钟的诸多核心振荡子 (Core oscillators) 具有精细的时空表达特性，但分子模型有待深入解析【1】。此前的研究表明，生物钟组分CCA1, LHY, RVE8, RVE4与LNK1, 2在清晨时段相互作用，RVE4, 8通过LNK1, 2激活生物钟的重要抑制组分——PSEUDO-RESPONSE REGULATORs (PRR) 家族中PRR1/TOC1与PRR5在夜间的转录【2】；PRR9,7,5,3,1基因从清晨到夜间时序性达到转录高峰【3】，持续发挥转录抑制功能，目前的研究表明，PRRs可以与PIFs和TOPLESS家族互作共同抑制CCA1等在清晨的表达【4,5】，但有关PRRs抑制靶基因转录的作用机理仍不清楚。5月14日，河南大学省部共建作物逆境适应与改良国家重点实验室徐小冬教授、谢启光教授与中科院植物所王雷研究员合作在The Plant Cell在线发表了题为 BBX19 fine-tunes the circadian rhythm by interacting with PSEUDO-RESPONSE REGULATOR proteins to facilitate their repressive effect on morning-phased clock genes的研究论文。该研究发现，锌指结构转录因子BBX18、BBX19从清晨到傍晚次序招募PRR9, PRR7, PRR5蛋白，在白天形成BBX19-PRRs转录复合体，直接抑制CCA1和RVE8等生物钟核心基因的表达进而维持近24小时节律，揭示了植物生物钟转录-翻译反馈环路中新的运行机制。该研究发现，BBX (B-box) 转录因子第四亚家族的BBX18, BBX19与CCA1, LHY的表达具有较高的关联性，且转录本富集呈现近日节律性，峰值出现在上午；BBX19基因缺失突变体呈现出短于24小时的节律表型，而BBX18和BBX19表达量增多则引起长于24小时的节律表型。蛋白-蛋白间动态互作分析发现，BBX18与BBX19蛋白间可形成同源和异源二聚体，互作峰值在清晨；BBX18, BBX19分别与PRR9,7,5从清晨至傍晚依时间次序动态互作；BBX19,18-PRRs蛋白复合体主要定位于细胞核中。利用RNA-seq转录组数据分析，发现受BBX19抑制的靶基因多集中在黎明前后表达，而受BBX19诱导的靶基因的转录本多在傍晚时段富集。利用雌二醇诱导型启动子驱动BBX19表达的转基因材料，对BBX19蛋白的靶基因的转录时间特异性进行分析，发现在夜间诱导BBX19高表达可显著抑制CCA1, LHY和RVE8等清晨基因的转录；而在prr7-3 prr9-1和prr5-1 prr7-3突变体中，BBX19对CCA1, LHY和RVE8转录抑制作用减弱。ChIP-qPCR的实验结果证实，PRR9,7,5介导了BBX19蛋白与CCA1, LHY和RVE8基因启动子区的结合。锌指蛋白BBX 家族诸多成员参与到植物的光形态建成、庇荫反应及开花时间调控【6】。该研究绘制了生物钟的光信号响应曲线，发现BBX19基因缺失突变体中近日节律相位对红光及蓝光授时因子的响应能力与野生型中的一致。生物钟BBX19-PRRs复合体抑制清晨基因转录的分子模型综上所述，该研究发现锌指结构转录因子BBX18, 19参与植物近日节律的维持；BBX19按时间次序动态招募PRR9,7,5蛋白，形成的日间蛋白复合体直接抑制CCA1、LHY和RVE8在清晨时段的转录。该项成果揭示出植物生物钟精细的分子调控模型。该项研究为河南大学省部共建作物逆境适应与改良国家重点实验室与中科院植物所共同协作完成，河南大学博士后袁力与植物所在读博士生于英俊为共同第一作者，河南大学徐小冬教授、中科院植物所王雷教授和河南大学谢启光教授为共同通讯作者，河南大学“黄河学者”特聘青年学者刘明明博士、河南大学在读研究生宋扬、李红民、孙军秋，河北师范大学毕业研究生王俏参与了该研究。该课题得到国家自然科学基金委、河北省自然科学基金重点项目及中国科学院战略先导研究计划项目的资助。参考文献：[1] C.R. McClung, The Plant Circadian Oscillator, Biology (Basel) 8(1) (2019) 14.[2] Q. Xie, P. Wang, X. Liu, L. Yuan, L. Wang, C. Zhang, Y. Li, H. Xing, L. Zhi, Z. Yue, C. Zhao, C.R. McClung, X. Xu, LNK1 and LNK2 are transcriptional coactivators in the Arabidopsis circadian oscillator, Plant Cell 26(7) (2014) 2843-57.[3] T. Mizuno, N. Nakamichi, Pseudo-Response Regulators (PRRs) or True Oscillator Components (TOCs), Plant Cell Physiol. 46 (2005) 677-685.[4] L. Wang, J. Kim, D.E. Somers, Transcriptional corepressor TOPLESS complexes with pseudoresponse regulator proteins and histone deacetylases to regulate circadian transcription, Proc. Natl. Acad. Sci. USA 110 (2013) 761-766.[5] Y. Zhang, A. Pfeiffer, J.M. Tepperman, J. Dalton-Roesler, P. Leivar, E. Gonzalez Grandio, P.H. Quail, Central clock components modulate plant shade avoidance by directly repressing transcriptional activation activity of PIF proteins, Proc Natl Acad Sci U S A 117(6) (2020) 3261-3269.[6] Z. Song, Y. Bian, J. Liu, Y. Sun, D. Xu, B-box proteins: Pivotal players in light-mediated development in plants, J Integr Plant Biol 62(9) (2020) 1293-1309.原文链接：https://doi.org/10.1093/plcell/koab133发布于 2021-05-15 13:56生物钟生物化学生物钟颠倒​赞同 4​​添加评论​分享​喜欢​收藏​申请转载​文章被以下专栏收录BioArt植物 · 前沿专注植物科学领域前

【BBX反应视频】十大让人爽到炸的Beatbox合集_哔哩哔哩_bilibili

【BBX反应视频】十大让人爽到炸的Beatbox合集_哔哩哔哩_bilibili 首页番剧直播游戏中心会员购漫画赛事投稿【BBX反应视频】十大让人爽到炸的Beatbox合集

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554392☞Human Beatbox丨上世纪80年代的新兴hiphop元素ᝰ通俗讲作节奏口技 但区别于中国传统口技

【订阅】此频道℡，持续输出最新最dope的BBX生态ᝰ

【欢迎】在评论区交流你的想法ᝰ

【期待】你对频道的意见或建议♬

原作者：Young Recluse

网址：https://youtu.be/Y4oSwJgwPsc音乐演奏HIPHOP音乐BEATBOX合集REACTION街头文化

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IJMS | Free Full-Text | Genome-Wide Identification and Analysis of the BBX Gene Family and Its Role in Carotenoid Biosynthesis in Wolfberry (Lycium barbarum L.)

IJMS | Free Full-Text | Genome-Wide Identification and Analysis of the BBX Gene Family and Its Role in Carotenoid Biosynthesis in Wolfberry (Lycium barbarum L.)

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Open AccessArticle

Genome-Wide Identification and Analysis of the BBX Gene Family and Its Role in Carotenoid Biosynthesis in Wolfberry (Lycium barbarum L.)

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Yue YinYue Yin

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1,2,†, Hongyan ShiHongyan Shi

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State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling, Xianyang 712100, China

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National Wolfberry Engineering Research Center, Ningxia Academy of Agriculture and Forestry Sciences, Yinchuan 751002, China

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Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742, USA

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These authors contributed equally to this work.

Int. J. Mol. Sci. 2022, 23(15), 8440; https://doi.org/10.3390/ijms23158440

Submission received: 9 July 2022

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Revised: 27 July 2022

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Accepted: 27 July 2022

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Published: 29 July 2022

(This article belongs to the Section Molecular Plant Sciences)

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Abstract:

The B-box proteins (BBXs) are a family of zinc-finger transcription factors with one/two B-Box domain(s) and play important roles in plant growth and development as well as stress responses. Wolfberry (Lycium barbarum L.) is an important traditional medicinal and food supplement in China, and its genome has recently been released. However, comprehensive studies of BBX genes in Lycium species are lacking. In this study, 28 LbaBBX genes were identified and classified into five clades by a phylogeny analysis with BBX proteins from Arabidopsis thaliana and the LbaBBXs have similar protein motifs and gene structures. Promoter cis-regulatory element prediction revealed that LbaBBXs might be highly responsive to light, phytohormone, and stress conditions. A synteny analysis indicated that 23, 20, 8, and 5 LbaBBX genes were orthologous to Solanum lycopersicum, Solanum melongena, Capsicum annuum, and Arabidopsis thaliana, respectively. The gene pairs encoding LbaBBX proteins evolved under strong purifying selection. In addition, the carotenoid content and expression patterns of selected LbaBBX genes were analyzed. LbaBBX2 and LbaBBX4 might play key roles in the regulation of zeaxanthin and antheraxanthin biosynthesis. Overall, this study improves our understanding of LbaBBX gene family characteristics and identifies genes involved in the regulation of carotenoid biosynthesis in wolfberry.

Keywords: Lycium barbarum; LbaBBX gene family; LbaBBX gene expression; carotenoid biosynthesis; protein subcellular localization

1. IntroductionZinc finger transcription factors (TFs) are some of the most abundant TFs in plants and play a vital regulatory role in the regulation of transcription and various biological functions [1,2]. B-Box (BBX) proteins are a class of zinc-finger TFs possessing one or two B-box domains (CX2CX8CX7CX2CX4HX8H) in the N-terminus; some have an additional CCT (CONSTANS, CO-like, and TOC1) conserved domain or VP (valine–proline) motifs in the C-terminus. The B-box domains can be classified into two types: B-box1(B1) and B-box2 (B2). Two B-box conserved domains are recognized based on their consensus sequence and the distance between the zinc-binding residues [3]. Potential segmented duplication and deletion events result in differences in the consensus sequences and space between the zinc-binding residues in the two B-box domains [3,4]. In addition, the highly conserved CCT domain is comprised of 42–43 amino acids and is important for the regulation of transcription and nuclear protein transport [5,6]. According to the existence of BBX and CCT domains, 32 BBX proteins have been identified and classified into five subgroups in Arabidopsis [7]. Therefore, members of BBX proteins are divided into five categories depending on the presence of B-Box domains along with the CCT domain and have been reported in multiple species [4].Subsequently, many studies have shown that plant BBX proteins play important roles in diverse physiological and biochemical processes, such as flowering time regulation, photomorphogenesis, shade avoidance, secondary metabolism, and biotic and abiotic stress responses [8,9,10,11,12]. The first BBX gene (CONSTANS (CO), known as AtBBX1) was identified and characterized in Arabidopsis; it can activate FLOWERING LOCUS T (FT) by binding to its promoter under a long day length [13]. Other BBX genes were subsequently discovered and functionally characterized, including BBX4, BBX6, BBX7, and BBX32, with roles in the regulation of flowering time [14,15,16]. Recently, BBX proteins have been reported to regulate secondary metabolism in fruits, especially anthocyanin and carotenoid biosynthesis. In Arabidopsis, BBX21/22/23 are positive regulators of anthocyanin accumulation [10], while BBX24/25/31 negatively regulate anthocyanin biosynthesis in response to several environmental factors. The overexpression of VvBBX44 decreased the expression of VvHY5 and VvUFGT and reduced the anthocyanin content in grape calli [17]. In pear, PpBBX16, a homolog of AtBBX22, is a positive regulator of light-induced anthocyanin accumulation [18]. In apple, MdBBX1/20/21/22 and MdBBX33 promote anthocyanin biosynthesis by light-induced anthocyanin accumulation, whereas MdBBX37 is a negative regulator of anthocyanin accumulation via light signaling. In tomatoes, SlBBX20 regulates the synthesis of carotenoids by directly binding to the promoter of the gene encoding the carotenoid biosynthesis enzyme PHYTOENESYNTHASE 1 [11]. However, studies of BBX genes in wolfberry are rare. Wolfberry (Lycium barbarum L.; 2n = 2x = 24), a fruit tree in the family of Solanaceae, is an important medicinal and edible plant in China. L. barbarum is a rich source of carotenoid esters, which are mainly composed of zeaxanthin dipalmitate, lutein palmitate, antheraxanthin, and β-cryptoxanthin. Therefore, carotenoids are responsible for the orange, yellow, and red colors of L. barbarum fruits [19,20]. During the past few decades, many studies have identified TFs families with important roles in the regulation of carotenoid biosyntheses, such as SlMYB72, SlWRKY35, MdAP2, and SlBBX20 [11,21,22,23]. Two R2R3-MYB family members, LbaMYB26(Lba02g01219) and LbaMYB123 (Lba11g01830), are candidate genes involved in the regulation of carotenoid biosynthesis in L. barbarum fruits [24]. The BBX gene family has been identified and evaluated in many plant species, such as Solanum lycopersicum [25], Capsicum annuum [26], Iris germanica [27], Prunus avium [28], Vitis Vinifera [29], and Arabidopsis thaliana [30], and has diverse functions. Our understanding of the functions of the BBX gene family, such as roles in responses to biotic and abiotic stresses and secondary metabolite biosynthesis, has advanced. However, the mechanism by which BBX genes contribute to the regulation of carotenoid biosynthesis is still unclear.Comprehensive studies of BBX genes in wolfberry have not been reported to date. The recent completion of the L. barbarum genome provides a basis for investigating the BBX gene family in the species at the genome level [31]. To further characterize the BBX gene family in Lycium, we performed systematic genome-wide identification and analyses of the BBX gene family, bridging the research gap in BBX gene family studies. Analyses of physical and chemical characteristics, collinearity analysis, phylogenetic and evolutionary relationships, conserved domains, gene structures, cis-regulatory networks, subcellular localizations, and expression patterns of LbaBBX genes were performed. This study lays a foundation for further analyses of the roles of LbaBBX genes in carotenoid biosynthesis and fruit development in wolfberry. 2. Results 2.1. Identification and Characteristics of LbaBBX Genes To identify BBX genes in the wolfberry genome, hidden Markov model (HMM) searches with the B-box domain HMM profile (PF00643) and BLSATP using 32 BBX protein sequences from Arabidopsis thaliana as queries were performed. The candidate BBX protein sequences were used to detect the presence of B-box conserved domains by the Simple Modular Architecture Research Tool (SMART) and the National Center for Biotechnology Information (NCBI) batch CD-Search. A total of 28 putative LbaBBX genes were identified (Table 1). These BBX genes were named LbaBBX1 to LbaBBX28 according to their location on the L. barbarum chromosomes. The coding sequences (CDS) of BBX genes ranged from 330 bp to 1374 bp. They encoded proteins that were 109 to 457 amino acids (AA) in length, with predicted putative molecular weights ranging from 12.49 kDa to 51.73 KDa. The grand average of hydropathicity (GRAVY) values for all BBXs were negative, indicating that the BBX proteins were hydrophilic. The subcellular localization results showed that most of the LbaBBX proteins were found in the nucleus. 2.2. Protein Domains and Phylogenetic Analysis of LbaBBX ProteinsThe conserved sequences of B-Box domains (B-Box1 and B-Box2), CCT domain, and VP motif in wolfberry BBX proteins were identified, and sequence logos are shown in Figure S1. Out of 28 LbaBBXs, ten contained two B-box domains and a conserved CCT domain, whereas three members had a valine–proline (VP) motif. Three and six members contained one B-box domain plus a CCT domain and only one B-Box domain, respectively, and the remaining nine members contained two B-Box domains. Conserved structures of LbaBBX members were found with B-box1 sequence (C-X2-C-X8-C-X2-D-X4-C-X2-C-D-X3-H-X8-H-X-R-X, X represents any amino acid) and B-box2 (C-X2-X8-C-X8-C-C-X3-X9-H-X-R-X4). Additionally, the CCT domain was highly conserved. Multiple sequence alignments of B-box1, B-box2, CCT domain, and VP motif for all LbaBBX proteins were also generated (Figure S2). Based on the alignments, some absolutely conserved amino acid residues were found, such as the Cysteine (C) and Histidine (H) residues in the B-box domain, Arginine (R) and Lysine (K) residues in the CCT domain, and Valine (V) and Proline (P) residues in the VP motif. The full-length amino acid sequences were used to construct a phylogenetic tree by the maximum likelihood (ML) method using IQ-TREE. As shown in Figure 1a, the LbaBBX family was divided into five subgroups, consistent with previous studies of the gene family in tomato, pepper, and Arabidopsis [25,26,30]. We found that LbaBBX proteins assigned to the same group possess similar domain organizations. For example, in subgroup I, three LbaBBXs contained two B-Box domains, an additional CCT domain, and a VP domain (Figure 1a). In order to confirm the subfamily clustering of BBX members in wolfberry, a phylogenetic tree of LbaBBX together with SlBBX, CaBBX, StBBX, SmBBX, IcBBX, and AtBBX was also constructed by using the ML method (Figure S3 and Table S1). All BBX proteins were also divided into five subfamilies. Furthermore, the sequences of B-box 1 (Figure 1b), B-box 2 (Figure 1c), and CCT (Figure 1d) domains were also evaluated. The members of subgroups I and II contained both B-Box and CCT domains, except for LbaBBX23, which harbored only two B-Box domains. Members of subgroup III had one B-box domain and one CCT domain. Members of subgroups IV and V had no CCT domain and only two or one B-Box domain(s), respectively. 2.3. Gene Structure and Motif Composition of the LbaBBX Gene FamilyThe exon–intron structures and conserved motifs were examined to gain insight into the structural diversity of LbaBBX genes. As shown in Figure 2b, the number of exons ranged from one to five, with an average of 2.9. Additionally, wolfberry BBX genes in clades I, II, III, and Ⅳ exhibited highly similar gene structures; however, LbaBBX genes in clade V showed highly variable structures. For example, most of the LbaBBX genes in clades I, II, III, and Ⅳ possessed two, four (except LbaBBX23), two, and four (except LbaBBX16) genes, respectively (Figure 2a). These results suggested that exon losses or gains occurred during the evolution of the gene family and resulted in functional divergence among closely related LbaBBXs. To further examine the structural features of LbaBBX proteins, the conserved motif compositions were analyzed using MEME. Fifteen conserved motifs were predicted and named motifs 1–15 (Figure 2c). Motifs one and four were found in all LbaBBX proteins except LbaBBX3. Most of the LbaBBX genes assigned to the same group shared similar motif compositions and arrangements, which further validated the classification results. For example, motifs 6 and 13 were detected only in groups II and I, respectively. Three LbaBBX members (LbaBBX9, LbaBBX10, and LbaBBX12) from group II possessed maximum motifs, containing motifs 1, 2, 3, 4, 6, 8, and 14. Except for LbaBBX7, LbaBBX26 and LbaBBX28 harbored only two motifs (motif one and motif four) in group V. The detailed sequence information for these 15 motifs is shown in Table S2. 2.4. Chromosomal Location and Duplication of LbaBBX GenesWe plotted the LbaBBX genes on the chromosomes of the wolfberry genome (Figure 3a). A total of 28 LbaBBX genes were evenly distributed on 11 of 12 wolfberry chromosomes, and the number of LbaBBX genes on each chromosome was not related to the chromosome size (Figure S4). Each LbaBBX gene name corresponds to its physical position from the top to the bottom of L. barbarum chromosome 1 to chromosome 12. Chromosome 4 contained the largest number of LbaBBX genes (6 genes, ~21.4%), followed by chromosome 5 (5 genes, ~17.9%) and chromosome 11 (5 genes, ~17.9%). Only one LbaBBX gene was located on each of chromosomes 1, 2, 3, and 12, and only two were detected on chromosomes 6, 9, and 10. No LbaBBX genes were located on chromosome 8.Different patterns of gene duplication contributed to the evolution of the BBX gene family, including whole-genome duplication (WGD) as well as segmental duplication, tandem duplication (TD), proximal duplication (PD), transposed duplication (TRD), and dispersed duplication (DSD). We used DupGen_finder [32] to detect duplicated BBX family gene pairs in wolfberry. The numbers of DSD, WGD, TRD, PD, and TD duplication events in wolfberry were 23, 12, 4, 1, and 1, respectively (Figure 3b). These results indicated that DSDs and WGDs explained the majority of gene duplication events in the LbaBBX gene family.In addition, collinearity was analyzed among homologous regions in other species, including Solanum lycopersicum, Capsicum annuum, Solanum melongena, and Arabidopsis thaliana. The interspecific collinearity results revealed 56 orthologous pairs (Figure 3c). Orthologous relationships were detected between LbaBBX genes and genes in four species belonging to Solanaceae and A. thaliana, including L. barbarum–S. lycopersicum (23 pairs), L. barbarum–S. melongena (20 pairs), L. barbarum–C. annuum (8 pairs), and L. barbarum–A. thaliana (5 pairs) (Table S3). The numbers of orthologous events of LbaBBX-SlBBX, LbaBBX-SmeBBX, and LbaBBX-CaBBX were greater than that of LbaBBX-AtBBX. These results indicated that wolfberry was closely related to the other three species in Solanaceae. The high numbers of orthologous events of LbaBBX-SlBBX identified in our study suggest that LbaBBX genes in wolfberry share a similar structure and function to those of SlBBX genes in tomato. 2.5. Nonsynonymous (Ka) and Synonymous (Ks) Substitutions per Site, and Ka/Ks Analysis of BBX Family GenesWe estimated rates of synonymous (Ks) and nonsynonymous (Ka) substitutions for 56 duplicated gene pairs. As illustrated in Figure 4, the Ka/Ks values for WGD-derived gene pairs in wolfberry ranged from 0.172 to 0.403, and the Ka/Ks values for gene pairs derived from DSD, TRD, PD, and TD were 0.098–0.527, 0.199–0.227, 0.357–0.357, and 0.444–0.444, respectively (Figure 4 and Table S6). In general, Ka/Ks value greater than 1.0 provide evidence for positive selection, values less than 1.0 suggest purifying selection, and values equal to 1.0 suggest neutral evolution. In our study, all LbaBBX gene pairs had Ka/Ks values less than 1, indicating that these genes primarily underwent strong purifying selection. 2.6. Cis-Regulatory Elements in the Promoters of LbaBBX GenesThe 1500 bp upstream sequences of the 28 LbaBBX genes were extracted for analyses of the cis-regulatory elements in the promoter regions. In total, 482 cis-acting elements were identified and classified into three basic categories, including plant growth and development, phytohormone, and stress responses (abiotic/biotic) (Figure 5 and Table S4). In the first subgroup (i.e., plant growth and development), the majority (84.5%) of elements were light-responsive elements, such as GT1-motif, Box 4, G-box, and I-box, which are widespread in plants (Figure 5c). The second subgroup included elements involved in phytohormone responses; the ABREs for abscisic acid (ABA) responsiveness were the most common elements, appearing 55 times in 28 LbaBBXs, accounting for 27.9% of the hormone-responsive cis-regulatory elements (Figure 5c). The others were the CGTCA-motif and TGACG-motif for MeJA-responsiveness elements, TATC-box and P-box for gibberellin-responsive elements, and TGA-element for auxin-responsive elements, suggesting that LbaBBXs are regulated by various hormones (Figure 5c). The last subgroup included elements related to different stress responses. A cis-acting regulatory element essential for anaerobic induction (ARE) was identified in 19 LbaBBX gene promoters (Table S4), suggesting that these genes might be induced by low oxygen levels. W box (wounding and pathogen responsiveness), LTR (low-temperature responsiveness), and MBS (MYB binding site involved in drought-inducibility) were also found. Furthermore, MYC was found in 24 LbaBBX genes, suggesting that LbaBBXs contribute to the response to abiotic stress (Table S4). 2.7. Expression Patterns of LbaBBX Genes in Different TissuesTo further understand the dynamic gene expression patterns of BBX gene family members in L. barbarum, we evaluated expression profiles in four tissues (leaf, stem, flower, and fruit) with RNA-seq analysis. The LbaBBX genes exhibited tissue-specific expression and were further divided into three groups (Figure 6 and Table S5). In Group I, six genes (LbaBBX16, LbaBBX25, LbaBBX1, LbaBBX19, LbaBBX20, and LbaBBX21) presented high overall expression levels in all four organs, suggesting that these LbaBBX genes play important roles in the formation of these tissues, except two genes (LbaBBX15 and LbaBBX25) in leaves with relatively low expression. Of 28 genes, seven BBXs were assigned to Group II. Remarkably, not all homologous gene pairs exhibited similar patterns of expression; for example, LbaBBX9 had the highest transcript abundance in the leaf, and LbaBBX26 expression was highest in the stem (Figure 6b). In Group III, the remaining 14 genes shared similar low expression levels in these tissues, except LbaBBX4 (which was highly expressed in fruits). Additionally, several genes that were highly expressed in the fruit were identified, including LbaBBX1, LbaBBX4, LbaBBX16, and LbaBBX25 (Figure 6b). 2.8. Identification LbaBBX Genes Related to Carotenoid ContentsIn order to ascertain how LbaBBX gene expression (FPKM values) may be predictive of carotenoid accumulation in mature wolfberry fruit, a Pearson correlation analysis was performed based on estimates at the mature (S5) stages. First, the carotenoid content was analyzed by HPLC at five developmental stages in L. barbarum var. auranticarpum (Figure 7a). During fruit development, carotenoid contents (zeaxanthin, antheraxanthin, β-cryptoxanthin, and lutein palmitate) increased sharply as maturation progressed (Figure 7b). Among the four types of carotenoid metabolites, zeaxanthin was the most abundant in all stages of fruit development. Correlation tests were performed to evaluate relationships between abundances of various carotenoids (zeaxanthin, antheraxanthin, β-cryptoxanthin, and lutein palmitate) and transcript abundances of LbaBBX genes. As shown in Figure 7c, we observed a positive correlation between LbaBBX25 with zeaxanthin (r = 0.967, p < 0.05) (Table S7). Strong positive correlations were observed between transcript levels of LbaBBX1 and LbaBBX2 with antheraxanthin contents (r = 0.993, p < 0.001; r = 0.989, p < 0.001, respectively). Significant correlations were also observed between LbaBBX11 (r = 0.985), LbaBBX16 (r = 0.981), and LbaBBX25 (r = 0.985) expression levels and antheraxanthin accumulation (all p < 0.05). Significant correlations were also observed between LbaBBX11, LbaBBX16, and LbaBBX25 expression levels and antheraxanthin accumulation (r = 0.985, p < 0.05; r = 0.981, p < 0.05; r = 0.985, p < 0.05, respectively). However, weak negative correlations of LaBBX9, LbaBBX12, and LbaBBX13 expression levels with antheraxanthin contents were observed. These correlations indicate that carotenoid accumulation in wolfberry fruits is correlated with the expression patterns of LbaBBXs.To further investigate the regulation of LbaBBX genes in wolfberry, a correlation network was constructed combining four metabolites, 14 structural genes, and 13 LbaBBX TFs related to carotenoid biosynthesis. Only the pairs with a Pearson correlation coefficient >0.8 were included in this analysis (Figure 8). The network (visualized using Cytoscape) included 31 nodes connected by 123 edges. The pairwise correlations between genes (FPKM values) and between gene and metabolite levels revealed that 74 and 49 pairs of nodes, respectively, showed positive and negative correlations. As shown in Figure 8, all nine carotenoid biosynthesis genes exhibited positive correlations with carotenoid contents, with LbaCYP97A29 showing the highest positive correlation (Table S8). For the 13 LbaBBX TFs, the transcript changes in LbaBBX1, LbaBBX2, LbaBBX4, LbaBBX11, LbaBBX16, LbaBBX18, and LbaBBX25 showed positive correlations, while LbaBBX9, LbaBBX12, and LbaBBX13 showed negative correlations (Table S7). For relationships between levels of carotenoid biosynthesis genes and BBX TFs, the highest positive correlation was observed between LbaBBX2 and LbaPDS, followed by LbaBBX1 and LbaCRTISO, while the highest negative correlation was found between LbaBBX11 and LbaLCYE (Table S9). It is worth noting that LbaBBX1, LbaBBX2, LbaBBX11, and LbaBBX16 levels showed strong positive correlations with levels of nine carotenoid biosynthesis genes each (Table S9). These results indicated that these five LbaBBXs (LbaBBX1, LbaBBX2, LbaBBX4, LbaBBX11, and LbaBBX16) might be involved in the regulation of carotenoid biosynthesis. 2.9. Gene Expression Analyses by qRT-PCRNine potential LbaBBXs that showed strong correlations with the carotenoid content during fruit development we further evaluated by qRT-PCR. The expression patterns of several individual genes were highly correlated with the carotenoid content during wolfberry fruit development. Our results indicated that the expression levels of LbaBBX2 and LbaBBX4 increased sharply from S1 (12 DAF) to S3 (25 DAF) and reached peak values (Figure 9). The trends in the expression levels of these genes were consistent with trends in zeaxanthin content. Taken together, LbaBBX2 and LbaBBX4 were identified as important candidate genes involved in carotenoid biosynthesis and should be the focus of further functional research. 2.10. Subcellular Localization of LbaBBX2 and LbaBBX4The candidate carotenoid-related genes LbaBBX2 and LbaBBX4 were selected for further analyses of subcellular localization. Their proteins were predicted to be located in the nucleus. To observe the subcellular localization of LbaBBX2 and LbaBBX4, 35S-LbaBBX2::GFP and 35S-LbaBBX4:GFP were constructed and transiently expressed in tobacco leaves, and 35S-GFP was used as a negative control. As determined by fluorescence microscopy, the 35S-LbaBBX2::GFP and 35S-LbaBBX4::GFP fusion proteins were located exclusively in the nucleus, whereas the 35S-GFP control was distributed in the tobacco leaf protoplasts (Figure 10). These results indicate that both LbaBBX2 and LbaBBX4 encode nuclear-localized proteins. 3. DiscussionThe BBX gene family has recently been identified in many higher plants, such as Arabidopsis, tomato, and pepper [7,25,26]. The quantity of BBX genes varies among species. For example, 32 BBX genes were identified in Arabidopsis [7], 31 in tomato [25], 24 in pepper [26], and 30 in potato [33]. In this study, 28 LbaBBX genes were identified in the wolfberry genome. The number of BBX genes in the four species in the family Solanaceae (tomato, pepper, potato, and wolfberry) was relatively conserved. However, there are 64 BBX members in apple [34]. Of note, the wolfberry genome (1.67 Gb) [31] is larger than those of Arabidopsis (134 Mb) [35] and tomato (900 Mb) [36], although it was smaller than the pepper genome (3.48 Gb) [37]. These results indicated that the number of BBX gene family members might not be directly related to the plant genome size. Furthermore, the composition of the BBX genes in different subclades also differed among species (Figure S3). In wolfberry, the numbers of BBX members with two tandem B-boxes plus the CCT domain, two tandem B-boxes, box 1 plus CCT, and B-box 1 were only 7, 9, 6, and 6, respectively. The corresponding counts were 13, 8, 4, and 7 in Arabidopsis [30] and 8, 11, 5, and 7 in tomatoes [25]. These results suggested that BBX genes shared a common ancestor and underwent an independent expansion after the divergence between monocots and dicots [38].Previous phylogenetic analyses have verified that most plant BBX genes can be divided into five subgroups (Ⅰ–IV) [4,38]. In this present study, a phylogenetic tree based on BBX protein sequences from Arabidopsis, tomato, pepper, potato, eggplant, Iochroma cyaneum, and wolfberry also supported their clustering into five subfamilies (Figure S3), consistent with previous results [38]. On the other hand, BBX proteins were grouped into five groups based on structure, depending on the presence of at least one B-box domain and a CCT domain. For example, 32 Arabidopsis BBXs were divided into five subclades according to a combination of conserved domains. The conserved domain-based classification of BBX proteins in L. barbarum was rather complex. As shown in Table 1, LbaBBX17, LbaBBX19, and LbaBBX21 were classified into group I, which had two B-boxes and a CCT plus a VP domain. Eight BBX members were classified into group II, including three LbaBBXs (LbaBBX9, LbaBBX10, and LbaBBX12) with one B-box plus a CCT domain, four LbaBBXs (LbaBBX8, LbaBBX13, LbaBBX20, and LbaBBX27) with two B-boxes and a CCT domain, and one LbaBBX (LbaBBX23) with two B-boxes. Group five contained only one B-box. A sequence alignment of LbaBBXs revealed a high degree of conservation of the B-Box1 domain among LbaBBX1 to LbaBBX28 (Figure S2). Thus, the clustering results were similar to those based on B-box 1. These results revealed that some LbaBBX proteins lost the B-box2 domain during evolution.Gene duplication is one of the key factors responsible for the generation of novel genes, including WGD, TD, PD, TRD, and DSD, contributing to the expansion of gene family members in many species [32]. WGD, TD, and DSD are the main events in eukaryotic genome evolution and drove the development of new functions and genetic evolutionary systems [39]. Gene families, such as R2R3-MYB and BAHD acyltransferase families, expanded primarily through WGD and DSD [24,40]. WGD and TD are the main duplication events in the PMEI gene family [41]. In this study, DSD and WGD were the main factors driving BBX expansion in wolfberry, with relatively minor contributions from other replication modes.The diversity of the biochemical functions of BBX genes has been identified in different species, including roles in plant photomorphogenesis, growth, development, metabolism, and responses to biotic or abiotic stresses [30]. For example, a number of BBXs, such as AtBBX21, AtBBX22, and AtBBX25, are involved in photomorphogenesis in Arabidopsis [42]. In apple, MdBBX37 is a negative regulator of anthocyanin accumulation via light signaling [43]. In tomatoes, SlBBX17 is a positive regulator of heat stress tolerance [12]. Despite the diverse functions of BBX gene family members, we focus on their roles in carotenoid biosynthesis. Few studies have reported that BBX genes are involved in the regulation of carotenoid metabolism. In tomatoes, SlBBX20 enhances carotenoid accumulation by activating SlPSY1 promoter activity [11]. The 28 LbaBBX genes identified in this study showed variation in expression levels across the five stages of wolfberry fruit development. Based on transcriptome expression profiles combined with a correlation network analysis, expression levels of two candidate genes (LbaBBX2 and LbaBBX4) belonging to clade Ⅳ were strongly correlated with carotenoid contents during fruit ripening. qRT-PCR analyses of LbaBBX2 and LbaBBX4 expression yielded consistent results, supporting the validity of the RNA-seq data. In addition, phylogenetic analyses indicated that LbaBBX2 and LbaBBX4 share high protein sequence homology with SlBBX20 (Figure S3). Together, we speculate that LbaBBX2 and LbaBBX4 are involved in the regulation of carotenoid biosynthesis in wolfberry. A limitation of this study is that a genetic transformation system for wolfberry plants has not been established. Therefore, the mechanism underlying LbaBBX gene expression dynamics in wolfberry plants still needs to be fully elucidated. 4. Materials and Methods 4.1. Plant MaterialsFruits of Lycium barbarum var. auranticarpum (with yellow fruit) were picked from the wolfberry germplasm of the National Wolfberry Engineering Research Center located at Yinchuan in Ningxia, China (38°38′49″ N, 106°9′10″ E). Fruit samples were harvested at five developmental stages (12, 19, 25, 30, and 37 days after full bloom, DAF). These fruits were immediately ground in liquid nitrogen and stored at −80 °C until further analysis. 4.2. Identification and Characterization of LbaBBX Genes in the L. barbarum GenomeIn order to identify all possible BBX TFs in wolfberry, two strategies were used. In the first strategy, 31 BBX genes in the Arabidopsis thaliana genome were downloaded from the Arabidopsis Information Resource (TAIR, https://www.arabidopsis.org/, accessed on 4 January 2022) and were used as queries to search for potential BBXs in the L. barbarum genome database [31] by BLSATP with e value cutoff set at 1 × 10−5. In the second strategy, the HMMs of the B-box domain (PF00643) were downloaded from the Pfam database (https://pfam.xfam.org/, accessed on 8 January 2022), and HMMER 3.2 was used to identify BBX genes from the BLASTP alignments with default parameters. Subsequently, the presence of the BBX domain in each of the putative gene family members was further verified using the Pfam database (https://pfam.xfam.org/, accessed on 8 January 2022) [44], SMART database [45] (http://smart.embl-heidelberg.de/, accessed on 8 January 2022), and Conserved Domain Database (https://www.ncbi.nlm.nih.gov/cdd, accessed on 8 January 2022) [46]. Genes encoding proteins containing B-box domains were identified as BBX genes. The chemical properties, including the number of amino acids (aa), isoelectric point (pI), molecular weight (MW), and grand average of hydropathicity (GRAVY), were obtained from the ExPasy website (https://web.expasy.org/protparam/, accessed on 15 January 2022) [47]. The subcellular localizations of LbaBBX genes were predicted using WoLF PSORT (https://www.genscript.com/wolf-psort.html?src=leftbar, accessed on 15 January 2022) [48]. 4.3. Multiple Sequence Alignment and Phylogenetic Analysis of LbaBBX ProteinsThe BBX sequences for five species in Solanaceae, including tomato, pepper, eggplant, potato, and Iochroma cyaneum, were obtained from the Solanaceae Genomics Network (https://solgenomics.net/, accessed on 20 January 2022). First, full-length BBX protein sequences for the six species in Solanaceae and Arabidopsis were aligned by using Muscle v3.8 [49]. The deduced amino acid sequences in the B-box1, B-box2, and CCT domains were then adjusted manually using GeneDoc [50]. IQ-TREE [51] was used to construct a maximum likelihood (ML) phylogenetic tree based on all 194 full-length protein sequences. The best-fit substitution model, JTT+G, was determined using MEGA 6.06 [52]. The number of bootstrap replicates was 1000. The phylogenetic trees were visualized using iTOL v5 (https://itol.embl.de/, accessed on 20 January 2022) [53]. 4.4. Gene Structure and Motif Composition of the LbaBBX Gene FamilyThe BBX genomic sequence and corresponding coding regions retrieved from the wolfberry genome were sent to the Gene Structure Display Server (http://gsds.gao-lab.org/, accessed on 25 January 2022) [54] to investigate exon–intron structures. MEME (https://meme-suite.org/meme/tools/meme, accessed on 25 January 2022) [55] was used to predict conserved motifs with a maximum number of motifs of 15 and optimum width of 3 to 50 bp. 4.5. Chromosomal Location and Gene Duplication Analysis of LbaBBX GenesThe chromosomal distribution of LbaBBX genes was visualized using TBtools [56]. To examine duplication events for LbaBBX genes in wolfberry and other plants, including A. thaliana, S. lycopersicum, and C. annuum, TBtools were used. The gene duplication pairs were visualized in Tbtools [56]. The whole-genome sequences of three species in Solanaceae, including L. barbarum, S. lycopersicum, C. annuum, and A. thaliana, were used to analyze collinearity. The detected syntenic blocks were visualized using Tbtools [56]. Furthermore, Ka and Ks substitution rates were calculated for each syntenic pair using KaKs_Calculator 3.0 [57]. 4.6. Cis-Regulatory Elements in the Promoters of LbaBBX GenesThe 1500 bp genomic DNA sequences upstream of the start codon (ATG) of LbaBBX genes were extracted from the wolfberry genome database using Tbtools [56]. The cis-regulatory elements in these LbaBBX gene promoters were predicted by using PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 3 March 2022). 4.7. RNA Isolation, cDNA Library Construction, and RNA-Seq AnalysisTotal RNA was extracted independently from different wolfberry tissues using an RNA Kit (Tiangen, Beijing, China), according to the manufacturer’s instructions. RNA purity, concentration, and integrity were measured using a Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). High-quality RNAs were used to construct a cDNA library. First- and second-strand cDNAs were synthesized using Superscript II reverse transcriptase and random hexamer primers. Double-strand cDNA was fragmented by nebulization and used to generate RNA-seq libraries, as described previously [58]. Three biological replicates of cDNA libraries were sequenced using the Illumina HiSeq 4000 platform (Illumina Inc., San Diego, CA, USA) with a paired-end read length of 150 bp. 4.8. Expression Profiles of LbaBBXsIn order to study the expression profiles of LbaBBX genes, RNA-Seq data were downloaded from the NCBI database (PRJNA845109), including data for various tissues (stems, leaves, flowers, and fruits). The estimated expression levels of the BBX genes were represented and normalized in the form of fragments per kilobase of transcript per million mapped (FPKM). The heatmap for LbaBBX genes was visualized using Tbtools [56]. 4.9. Quantitative Real-Time PCR (qRT-PCR) AnalysisRNA extraction and qRT-PCR were performed as previously described [24]. The primers for LbaBBX genes were designed using Primer Premier 5 and are listed in Table S10. The wolfberry Actin gene was used as an internal control [20]. Three independent biological replications were conducted. 4.10. Carotenoid Extraction and HPLC AnalysisThe extraction steps were as follows. Freeze-dried fruits were homogenized (30 Hz, 1.5 min) to a powder with a grinder (MM 400; Retsch, Haan, Germany). A mixture of n-hexane: acetone: ethanol (1:1:1, v/v/v) was prepared as an extraction solution, and then 0.01% BHT (g/mL) and 50 mg of power were mixed with an appropriate amount of extraction solution and internal standard. The extract was vortexed for 20 min at room temperature. The mixture was then centrifuged at 12,000 rpm/min for 5 min at 4 °C, and the supernatant was removed. The residue was re-extracted by repeating Steps under the same conditions. The supernatants were combined and evaporated to dryness. A mixture of methanol and methyl tert butyl ether was prepared; the sample was resuspended with an appropriate amount of the solution, vortexed thoroughly until it was fully dissolved, and centrifuged. The solution was filtered through a 0.22 μm membrane filter for further LC-MS/MS analysis [59]. Carotenoid contents were detected using the AB Sciex WTRAP 6500 LC-MS/MS platform by MetWare (Wuhan, China). 4.11. Correlation Network ConstructionExpression patterns were explored based on RNA-seq data for five stages. The correlation coefficients for relationships between gene pairs and the carotenoid content were measured based on Pearson’s correlation coefficients (PCC). These values were screened using Excel (threshold > 0.8). A network including carotenoid contents, LbaBBX TFs, and structural genes was constructed and visualized using Cytoscape [60]. 4.12. Subcellular LocalizationFor subcellular location assays, the 35S-LbaBBX2::GFP, 35S-LbaBBX4::GFP, and 35S::NLS-RFP (control) constructs were introduced into tobacco (Nicotiana benthamiana) epidermal cells via Agrobacterium infiltrated tobacco leaves. Samples transformed with 35-GFP were used as controls. After 2 days, GFP and RFP signals from the tobacco leaves inoculated with A. tumefaciens were detected by fluorescence microscopy (Olympus, BX63; Tokyo, Japan). Three independent experiments were performed for each gene. 4.13. Statistical AnalysesThe data are presented as means ± SD of at least three independent experiments. Differences were evaluated by the Student’s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001). 5. ConclusionsOur study provided the first genome-wide analysis of the BBX gene family in L. barbarum. A total of 28 LbaBBXs were identified and were unevenly distributed across the whole genome. A systematic and comprehensive analysis of the LbaBBX gene family was performed, including analyses of phylogenetic relationships, conserved domains, gene structure, motif composition, chromosome location, gene duplication, cis-acting elements, and expression patterns. Many cis-acting elements were found in the LbaBBX promoter sequences, indicating that LbaBBX genes are involved in complex regulatory networks controlling development. Correlation and qRT-PCR analyses revealed that LbaBBX genes might be involved in the regulation of carotenoid synthesis. Therefore, our genome-wide analysis of the BBX family provides a foundation for further studies of the molecular mechanisms underlying carotenoid synthesis in wolfberry.

Supplementary MaterialsThe following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms23158440/s1.Author ContributionsConceptualization, Y.Y. and H.S.; methodology, J.M.; software, Y.Y.; validation, D.Z. and C.G.; investigation, X.H. and X.Q.; resources, J.Z. (Jianhua Zhao) and W.A.; data curation, Y.Y. and J.M.; Writing—original draft preparation, Y.Y. and Y.C.; visualization, Y.C.; supervision and manuscript revisions, Y.C., J.Z. (Jianhua Zhu) and X.Z. All authors have read and agreed to the published version of the manuscript.FundingThis research was funded by the Key Research and Development projects of Ningxia Hui Autonomous Region (No.2022BBF02008), the Natural Foundation of Ningxia (No.2020AAC03284), and the Employee Innovation Project of All-China Federation of Trade Unions (No.2018300002).Institutional Review Board StatementNot applicable.Informed Consent StatementNot applicable.Data Availability StatementThe wolfberry genome datasets used during the current study are available in NCBI database (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA640228, accessed on 20 December 2021). The tomato, pepper, eggplant, and potato genome sequences were downloaded from the Genome Database for the Solanaceae (https://solgenomics.net/, accessed on 20 December 2021). The sequence of Arabidopsis was downloaded from the Arabidopsis Information Resource (https://www.arabidopsis.org/, accessed on 20 December 2021). The raw data of the transcriptome analysis used in this study were submitted to the Sequence Read Archive (SRA) at a NCBI database (PRJNA845109).Conflicts of InterestThe authors declare no conflict of interest.AbbreviationsTFsTranscription factorsHMMHidden Markov modelCDSCoding sequence GRAVYGrand average of hydropathicityMWMolecular weightFPKMFragments per Kilobase MillionKsSynonymous substitutionsKaNonsynonymous substitutionsGFPGreen Fluorescent ProteinRFPRed Fluorescent ProteinNCBINational Center for Biotechnology InformationqRT-PCRQuantitative Real-Time PCRWGDWhole genome duplicationTDTandem duplicationPDProximal duplicationTRDTransposed duplicationDSDDispersed duplicationReferencesTakatsuji, H. Zinc-finger transcription factors in plants. Cell. Mol. Life Sci. CMLS 1998, 54, 582–596. [Google Scholar] [CrossRef]Kielbowicz-Matuk, A. Involvement of plant C2H2-type zinc finger transcription factors in stress responses. Plant Sci. 2012, 185–186, 78–85. 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Figure 1.

Phylogenetic tree analysis of BBX proteins in wolfberry. (a) The trees shown were based on the alignments of the protein sequences of the full length, and the phylogenetic tree was constructed using maximum likelihood method with 1000 bootstrap replicates by IQ-TREE. (b–d) The tree shown were based on the alignments of the protein’s sequences of the B-box 1 domain, B-box 2 domain and CCT domain, respectively. The members marked in black triangle contain two B-Box and one CCT domains.

Figure 1.

Phylogenetic tree analysis of BBX proteins in wolfberry. (a) The trees shown were based on the alignments of the protein sequences of the full length, and the phylogenetic tree was constructed using maximum likelihood method with 1000 bootstrap replicates by IQ-TREE. (b–d) The tree shown were based on the alignments of the protein’s sequences of the B-box 1 domain, B-box 2 domain and CCT domain, respectively. The members marked in black triangle contain two B-Box and one CCT domains.

Figure 2.

Phylogenetic relationships and motif composition of the LbaBBX proteins, and gene structure of the LbaBBX genes. (a) The phylogenetic tree was constructed based on the full-length sequences using IQ-TREE software by maximum likelihood (ML) method and 1000 bootstrap replicates. (b) Exon/intron structures of BBX genes from wolfberry. The exons and introns are represented by yellow boxes and black lines, respectively. The sizes of exons and introns can be estimated using the scale below. (c) The conserved motifs of wolfberry proteins were elucidated by MEME. The 15 motifs were displayed by the different colored rectangles. The sequence information for each motif is provided in Table S2. The length of protein can be estimated using the scale at the bottom.

Figure 2.

Phylogenetic relationships and motif composition of the LbaBBX proteins, and gene structure of the LbaBBX genes. (a) The phylogenetic tree was constructed based on the full-length sequences using IQ-TREE software by maximum likelihood (ML) method and 1000 bootstrap replicates. (b) Exon/intron structures of BBX genes from wolfberry. The exons and introns are represented by yellow boxes and black lines, respectively. The sizes of exons and introns can be estimated using the scale below. (c) The conserved motifs of wolfberry proteins were elucidated by MEME. The 15 motifs were displayed by the different colored rectangles. The sequence information for each motif is provided in Table S2. The length of protein can be estimated using the scale at the bottom.

Figure 3.

Chromosomal location and duplicated genes among LbaBBX genes. (a) Intraspecific collinearity analysis. A total of 28 LbaBBX genes were mapped onto the chromosomes based on their physical location. The red lines indicate duplicated LbaBBX gene pairs. (b) Different models of gene duplication in LbaBBX family. The x-axis represents the duplication type. The y-axis represents the number of duplicated gene pairs. Whole genome duplication (WGD), tandem duplication (TD), proximal duplication (PD), transposed duplication (TRD), dispersed duplication (DSD). (c) Analysis of collinearity between two different species. The gray lines indicated duplicated blocks, while the red lines indicated the syntenic BBX gene pairs. Chromosome numbers are indicated above or below each chromosome.

Figure 3.

Chromosomal location and duplicated genes among LbaBBX genes. (a) Intraspecific collinearity analysis. A total of 28 LbaBBX genes were mapped onto the chromosomes based on their physical location. The red lines indicate duplicated LbaBBX gene pairs. (b) Different models of gene duplication in LbaBBX family. The x-axis represents the duplication type. The y-axis represents the number of duplicated gene pairs. Whole genome duplication (WGD), tandem duplication (TD), proximal duplication (PD), transposed duplication (TRD), dispersed duplication (DSD). (c) Analysis of collinearity between two different species. The gray lines indicated duplicated blocks, while the red lines indicated the syntenic BBX gene pairs. Chromosome numbers are indicated above or below each chromosome.

Figure 4.

Different models of gene duplication in the LbaBBX family. The x-axis represents the duplication type. The colorful dots represent duplicated gene pairs. Whole genome duplication (WGD), tandem duplication (TD), proximal duplication (PD), transposed duplication (TRD), and dispersed duplication (DSD).

Figure 4.

Different models of gene duplication in the LbaBBX family. The x-axis represents the duplication type. The colorful dots represent duplicated gene pairs. Whole genome duplication (WGD), tandem duplication (TD), proximal duplication (PD), transposed duplication (TRD), and dispersed duplication (DSD).

Figure 5.

Identification of cis-elements in promoter regions of LbaBBXs. (a) Three categories of cis-acting elements in the LbaBBXs. Different numbers and color of the gird representing the number of different elements in these LbaBBXs. (b) Histogram of the cis-acting elements in each LbaBBX gene. The blue rectangle represents plant growth and development responsive cis-elements, the orange rectangle represents phytohormone responsive cis-elements, and the gray rectangle represents abiotic and biotic stress responsive cis-elements. (c) Pie charts of different sizes indicated the ration of each promoter element in each category, respectively.

Figure 5.

Identification of cis-elements in promoter regions of LbaBBXs. (a) Three categories of cis-acting elements in the LbaBBXs. Different numbers and color of the gird representing the number of different elements in these LbaBBXs. (b) Histogram of the cis-acting elements in each LbaBBX gene. The blue rectangle represents plant growth and development responsive cis-elements, the orange rectangle represents phytohormone responsive cis-elements, and the gray rectangle represents abiotic and biotic stress responsive cis-elements. (c) Pie charts of different sizes indicated the ration of each promoter element in each category, respectively.

Figure 6.

Expression pattern of LbaBBX genes. (a) Tissue-specific expression pattern of LbaBBX genes in four tissues: leaf, stem, flower, and fruits, including five development stages. Blue and red color indicated lower and higher transcript abundance, respectively. (b) Identification of highly expressed BBX genes in L. barbarum. Blue, green, orange, red indicated low (1–7.3 FPKM), mid-low (7.3–48 FPKM), mid-high (48–114 FPKM), and high (114–317 FPKM) expression, respectively.

Figure 6.

Expression pattern of LbaBBX genes. (a) Tissue-specific expression pattern of LbaBBX genes in four tissues: leaf, stem, flower, and fruits, including five development stages. Blue and red color indicated lower and higher transcript abundance, respectively. (b) Identification of highly expressed BBX genes in L. barbarum. Blue, green, orange, red indicated low (1–7.3 FPKM), mid-low (7.3–48 FPKM), mid-high (48–114 FPKM), and high (114–317 FPKM) expression, respectively.

Figure 7.

Identification LbaBBX genes related to carotenoid biosynthesis. (a) Fruits of Lycium barbarum var. auranticarpum at different stages of development. S1, S2, S3, S4, and S5 period represent 12, 19, 25, 30, and 37 days after full bloom (DAF), respectively. Scale bars represent 1 cm. (b) Trends in carotenoids (zeaxanthin, antheraxanthin, β–cryptoxanthin and lutein palmitate) at five developmental stages. The data contain the averages and standard deviations of three individual replicates. Asterisks indicate a significant difference (* p < 0.05, ** p < 0.01, *** p < 0.001) compared with S1 at the different time points during development. (c) Correlation analysis was constructed using the expression levels of LbaBBX genes and carotenoids content in five different developmental stages. The blue color means negative correlation, the red color means positive correlation. Asterisks indicate a significant difference (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 7.

Identification LbaBBX genes related to carotenoid biosynthesis. (a) Fruits of Lycium barbarum var. auranticarpum at different stages of development. S1, S2, S3, S4, and S5 period represent 12, 19, 25, 30, and 37 days after full bloom (DAF), respectively. Scale bars represent 1 cm. (b) Trends in carotenoids (zeaxanthin, antheraxanthin, β–cryptoxanthin and lutein palmitate) at five developmental stages. The data contain the averages and standard deviations of three individual replicates. Asterisks indicate a significant difference (* p < 0.05, ** p < 0.01, *** p < 0.001) compared with S1 at the different time points during development. (c) Correlation analysis was constructed using the expression levels of LbaBBX genes and carotenoids content in five different developmental stages. The blue color means negative correlation, the red color means positive correlation. Asterisks indicate a significant difference (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 8.

Correlation network analysis for structural genes, LbaBBX transcription factors and carotenoid content. The red ellipse boxes indicated that carotenoid metabolism, the green ellipse boxes indicated that LbaBBX transcription factors, and the purple diamond boxes indicated carotenoid biosynthetic genes, respectively. The black solid lines indicated positive regulation, while the dot lines indicated negative regulation, respectively. The edges are drawn when the linear correlation coefficient is >0.8 with p-value < 0.05. The related correlation coefficients were shown in Table S8.

Figure 8.

Correlation network analysis for structural genes, LbaBBX transcription factors and carotenoid content. The red ellipse boxes indicated that carotenoid metabolism, the green ellipse boxes indicated that LbaBBX transcription factors, and the purple diamond boxes indicated carotenoid biosynthetic genes, respectively. The black solid lines indicated positive regulation, while the dot lines indicated negative regulation, respectively. The edges are drawn when the linear correlation coefficient is >0.8 with p-value < 0.05. The related correlation coefficients were shown in Table S8.

Figure 9.

The relative expression levels of nine LbaBBX genes at different fruit developmental stages. Actin gene was used as reference gene to measure expression levels in each period. The x-axis indicates the five distinct fruit developmental stages (12 DAF, 19 DAF, 25 DAF, 30 DAF and 37 DAF). The y-axis indicates the relative expression. Data represent the means ± SDs (n = 3).

Figure 9.

The relative expression levels of nine LbaBBX genes at different fruit developmental stages. Actin gene was used as reference gene to measure expression levels in each period. The x-axis indicates the five distinct fruit developmental stages (12 DAF, 19 DAF, 25 DAF, 30 DAF and 37 DAF). The y-axis indicates the relative expression. Data represent the means ± SDs (n = 3).

Figure 10.

Subcellular localization of LbaBBX4 protein. (a) Schematic diagram of the 35S-GFP, 35S-LbaBBX2::GFP, and 35S-LbaBBX4::GFP fusion protein constructs used for transient expression. (b) The LbaBBX2-GFP and LbaBBX4-GFP fusion proteins were transiently expressed in N. benthamiana leaves and observed by fluorescence microscopy 48 h later. The 35S-GFP was used as positive control. From left to right, bright field, red fluorescent protein (RFP) (nuclear localization signal (NLS)-RFP), green fluorescent protein (GFP), and merge image of RFP and GFP image. Scale bars =20 μm.

Figure 10.

Subcellular localization of LbaBBX4 protein. (a) Schematic diagram of the 35S-GFP, 35S-LbaBBX2::GFP, and 35S-LbaBBX4::GFP fusion protein constructs used for transient expression. (b) The LbaBBX2-GFP and LbaBBX4-GFP fusion proteins were transiently expressed in N. benthamiana leaves and observed by fluorescence microscopy 48 h later. The 35S-GFP was used as positive control. From left to right, bright field, red fluorescent protein (RFP) (nuclear localization signal (NLS)-RFP), green fluorescent protein (GFP), and merge image of RFP and GFP image. Scale bars =20 μm.

Table 1.

Information on the BBX gene family in wolfberry.

Table 1.

Information on the BBX gene family in wolfberry.

Gene IDGene NameCDSAApIMW (kDa)GRAVYSubcellular LocalizationDomainsStructureLba01g02500LbaBBX17052344.9125.99−0.393nucleus2BBXⅣLba02g02688LbaBBX29033006.2033.42−0.522nucleus2BBXⅣLba03g02797LbaBBX312244075.2346.60−0.86nucleus1BBX + CCTⅢLba04g02191LbaBBX49002995.1332.15−0.272nucleus2BBXⅣLba04g02506LbaBBX56662215.0324.38−0.582nucleus1BBXⅤLba04g02507LbaBBX65911965.1021.65−0.683chloroplast1BBXⅤLba04g02527LbaBBX73691227.5213.83−0.241nucleus1BBXⅤLba04g02528LbaBBX812694226.0846.01−0.564nucleus2BBX + CCTⅡLba04g02630LbaBBX912394125.1345.55−0.656nucleus1BBX + CCTⅡLba05g00735LbaBBX1012724235.2446.57−0.508nucleus1BBX + CCTⅡLba05g00905LbaBBX116932305.6325.67−0.343nucleus2BBXⅣLba05g01291LbaBBX1212274085.6444.95−0.601nucleus1BBX + CCTⅡLba05g01679LbaBBX1313384457.0548.87−0.603nucleus2BBX + CCTⅡLba05g02193LbaBBX1413744575.2451.73−0.741nucleus1BBX + CCTⅢLba06g03364LbaBBX156001995.8821.86−0.518nucleus2BBXⅣLba06g03380LbaBBX165851945.8821.54−0.573chloroplast2BBXⅣLba07g00041LbaBBX1711403796.7041.94−0.35chloroplast2BBX + CCTⅠLba07g01710LbaBBX189603198.4035.23−0.479nucleus2BBXⅣLba07g01848LbaBBX1910683555.9639.31−0.561chloroplast2BBX + CCTⅠLba09g00845LbaBBX2011403795.2742.66−0.728nucleus2BBX + CCTⅡLba09g01983LbaBBX219663215.6935.91−0.592cytoplasmic2BBX + CCTⅠLba10g01709LbaBBX2212634205.3748.09−0.824nucleus1BBX + CCTⅢLba10g01753LbaBBX2312574184.9446.75−0.506nucleus2BBXⅡLba11g00500LbaBBX248822934.9831.56−0.404nucleus2BBXⅣLba11g00948LbaBBX256242074.7123.43−0.916nucleus1BBXⅤLba11g00982LbaBBX263451148.5612.96−0.421cytoplasmic1BBXⅤLba11g01258LbaBBX279813264.6535.69−0.423nucleus2BBX + CCTⅡLba12g01725LbaBBX283301099.2112.49−0.446cytoplasmic1BBXⅤ

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Yin, Y.; Shi, H.; Mi, J.; Qin, X.; Zhao, J.; Zhang, D.; Guo, C.; He, X.; An, W.; Cao, Y.;

et al. Genome-Wide Identification and Analysis of the BBX Gene Family and Its Role in Carotenoid Biosynthesis in Wolfberry (Lycium barbarum L.). Int. J. Mol. Sci. 2022, 23, 8440.

https://doi.org/10.3390/ijms23158440

AMA Style

Yin Y, Shi H, Mi J, Qin X, Zhao J, Zhang D, Guo C, He X, An W, Cao Y,

et al. Genome-Wide Identification and Analysis of the BBX Gene Family and Its Role in Carotenoid Biosynthesis in Wolfberry (Lycium barbarum L.). International Journal of Molecular Sciences. 2022; 23(15):8440.

https://doi.org/10.3390/ijms23158440

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Yin, Yue, Hongyan Shi, Jia Mi, Xiaoya Qin, Jianhua Zhao, Dekai Zhang, Cong Guo, Xinru He, Wei An, Youlong Cao,

and et al. 2022. "Genome-Wide Identification and Analysis of the BBX Gene Family and Its Role in Carotenoid Biosynthesis in Wolfberry (Lycium barbarum L.)" International Journal of Molecular Sciences 23, no. 15: 8440.

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Yin, Y.; Shi, H.; Mi, J.; Qin, X.; Zhao, J.; Zhang, D.; Guo, C.; He, X.; An, W.; Cao, Y.;

et al. Genome-Wide Identification and Analysis of the BBX Gene Family and Its Role in Carotenoid Biosynthesis in Wolfberry (Lycium barbarum L.). Int. J. Mol. Sci. 2022, 23, 8440.

https://doi.org/10.3390/ijms23158440

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Yin Y, Shi H, Mi J, Qin X, Zhao J, Zhang D, Guo C, He X, An W, Cao Y,

et al. Genome-Wide Identification and Analysis of the BBX Gene Family and Its Role in Carotenoid Biosynthesis in Wolfberry (Lycium barbarum L.). International Journal of Molecular Sciences. 2022; 23(15):8440.

https://doi.org/10.3390/ijms23158440

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Yin, Yue, Hongyan Shi, Jia Mi, Xiaoya Qin, Jianhua Zhao, Dekai Zhang, Cong Guo, Xinru He, Wei An, Youlong Cao,

and et al. 2022. "Genome-Wide Identification and Analysis of the BBX Gene Family and Its Role in Carotenoid Biosynthesis in Wolfberry (Lycium barbarum L.)" International Journal of Molecular Sciences 23, no. 15: 8440.

https://doi.org/10.3390/ijms23158440

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Light and ripening-regulated BBX protein-encoding genes in Solanum lycopersicum | Scientific Reports

Light and ripening-regulated BBX protein-encoding genes in Solanum lycopersicum | Scientific Reports

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Light and ripening-regulated BBX protein-encoding genes in Solanum lycopersicum

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Published: 06 November 2020

Light and ripening-regulated BBX protein-encoding genes in Solanum lycopersicum

Bruno Silvestre Lira1, Maria José Oliveira1 na1, Lumi Shiose1 na1, Raquel Tsu Ay Wu1, Daniele Rosado1 nAff2, Alessandra Cavalcanti Duarte Lupi1, Luciano Freschi1 & …Magdalena Rossi 

ORCID: orcid.org/0000-0003-3650-772X1 Show authors

Scientific Reports

volume 10, Article number: 19235 (2020)

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Light responsesPlant physiology

AbstractLight controls several aspects of plant development through a complex signalling cascade. Several B-box domain containing proteins (BBX) were identified as regulators of Arabidopsis thaliana seedling photomorphogenesis. However, the knowledge about the role of this protein family in other physiological processes and species remains scarce. To fill this gap, here BBX protein encoding genes in tomato genome were characterised. The robust phylogeny obtained revealed how the domain diversity in this protein family evolved in Viridiplantae and allowed the precise identification of 31 tomato SlBBX proteins. The mRNA profiling in different organs revealed that SlBBX genes are regulated by light and their transcripts accumulation is directly affected by the chloroplast maturation status in both vegetative and fruit tissues. As tomato fruits develops, three SlBBXs were found to be upregulated in the early stages, controlled by the proper chloroplast differentiation and by the PHYTOCHROME (PHY)-dependent light perception. Upon ripening, other three SlBBXs were transcriptionally induced by RIPENING INHIBITOR master transcriptional factor, as well as by PHY-mediated signalling and proper plastid biogenesis. Altogether, the results obtained revealed a conserved role of SlBBX gene family in the light signalling cascade and identified putative members affecting tomato fruit development and ripening.

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IntroductionZinc finger transcription factors (TFs) comprise one of the most important families of transcriptional regulators in plants and play a central role in plant growth and development regulation, as well as in biotic and abiotic stress responses1,2. Among these TFs, B-box domain containing proteins (BBX) belong to a subclass characterised by the presence of one or two zinc finger B-box domains, which are predicted to be involved in protein–protein interactions3. BBX proteins were classified into five structure groups, according to the number of B-box and CCT (CONSTANS, CONSTANS-like and TIMING OF CAB1) domains and VP (valine-proline) motifs. Members of group I are characterised by the presence of two B-box domains in tandem, one CCT domain and one VP motif. Group II is similar to group I, also presenting two B-box domains and one CCT domain, but no VP motif. Group III contains a single B-box domain and a CCT. Group IV is characterised by the presence of two B-box domains but without CCT domain. Finally, group V is composed by proteins with just one B-box domain3,4. Although the VP is mentioned as a group I exclusive motif, it has already been identified in several proteins belonging to group III, IV and V; thus, the presence of the VP motif differs members from structure group I from II, but evidences show that it is not exclusive to the first5.Out of the 32 BBX proteins identified in Arabidopsis thaliana, 21 have already been functionally characterised, being described as regulators of various processes such as seedling photomorphogenesis6,7, photoperiodic flowering regulation8, shade avoidance9, and responses to biotic and abiotic stresses10. Interestingly, 14 BBX proteins were also found to be components of the light signalling transduction pathway4,6,11,12, with 12 of them belonging to groups IV (8 proteins) and V (4 proteins). Four of the light-signalling group IV proteins act as positive regulators—AtBBX2013, AtBBX2114, AtBBX2215 and AtBBX2316—and the other four play a negative role—AtBBX1817, AtBBX1918, AtBBX2419 and AtBBX2520,21. In the case of group V, only repressors of light signal transduction were reported, AtBBX286, AtBBX307, AtBBX317 and AtBBX3222.BBX proteins act by the direct or indirect interaction with central components of the light signal transduction network, including the transcription factors ELONGATED HYPOCOTYL 5 (HY5), HOMOLOG OF HY5 (HYH) and PHYTOCHROME INTERACTING FACTORs (PIFs), and the protein-ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1)4,23. For instance, AtBBX21 and AtBBX22 promote HY5 transcript accumulation and can be tagged for proteasomal degradation via COP1-mediated ubiquitination14,24,25. In contrast, AtBBX24 and AtBBX25 downregulate light signalling by the physical interaction with HYH and HY520,26. Interestingly, AtBBX28 was characterised as a light-induced light repressor, as it physically represses HY5 transcriptional regulatory activity and is marked for degradation in darkness by COP16. Yet, it was demonstrated that PIF3 and PIF1 transcription factors signalling cascade regulates AtBBX23 transcription, whose product physically interacts with HY5 inducing photomorphogenesis in A. thaliana seedlings12.The above-described links between BBXs and light signalling have been almost exclusively explored in seedling photomorphogenesis, and their role in other light-controlled physiological processes, such as plastid development and maintenance, plant architecture and fruit development, which are important determinants of crop yield and nutritional quality27, remains elusive. In this context, although the effect of light perception and signalling in tomato (Solanum lycopersicum L.) fruit productivity and nutraceutical composition has been increasingly demonstrated28,29,30,31,32,33,34,35, the association of the BBX protein family with light in this species is still elusive. In tomato, 29 BBX domain encoding genes were identified and reported to be modulated by abiotic stress and phytohormones36. Additionally, the Solyc01g110180 locus encodes the only deeply characterised tomato BBX, which is a positive regulator of fruit carotenogenesis37.Here, a comprehensive genome survey allowed the identification of 31 BBX protein-encoding loci in tomato genome. A robust phylogenetic reconstruction corroborated the monophyletic nature of the five previously identified structure groups and allowed the proposition of a new interpretation of the evolutionary history of this protein family. Further, we focused on the transcriptional profile of the 15 genes belonging to groups IV and V, revealing their association with organ greening and light signalling. Additionally, six genes were either up- or downregulated from immature fruit stages towards ripening. Finally, it was addressed whether the mRNA accumulation of these six genes is regulated by PHYTOCHROME (PHY)-mediated light perception and/or plastid development and differentiation.Materials and methodsPlant material, growth conditions and samplingDifferent tomato (Solanum lycopersicum L.) cv. Micro-Tom genotypes were used for SlBBXs transcriptional analysis: control genotype harbouring the wild-type GOLDEN-2 LIKE 2 (SlGLK2) allele (WT)38; uniform ripening Slglk2 mutant, which is deficient in SlGLK2, the master transcription factor controlling fruit chloroplast differentiation and maintenance33 and; fruit-specific transgenic lines silenced for SlPHYA (SlphyA) and SlPHYB2 (SlphyB2)30. Although Micro-Tom cultivar is deficient in brassinosteroid biosynthesis due to the weak mutation dwarf (d), it has been extensively demonstrated that represents a convenient and adequate model system to study fruit biology39. In this work we used Micro-Tom variety because we have all the germplasm collection in this background, including Slglk2 mutant and the fruit-specific SlPHY-silenced transgenic lines.For the experiments with seedlings, seeds were in vitro germinated in the darkness as described in40. After 2 days, seedlings were either kept in the darkness or transferred to the light (12 h photoperiod) for another 7 days, when hypocotyls and cotyledons were sampled.Leaves and fruits were harvested from plants cultivated in 2L rectangular plastic pots containing a 1:1 mixture of substrate and vermiculite supplemented with NPK 10:10:10, dolomite limestone (MgCO3 + CaCO3) and magnesium thermophosphate (Yoorin), under controlled temperature (between 23 °C and 27 °C), daily automatically irrigation by capillarity, and under natural light conditions (13 h photoperiod and 250–350 μmolm−2 s−1 of incident photo-irradiance) in a biosafety level 1 greenhouse.Source and sink leaves were harvested from 4 and 8th phytomer closest to the base of the plant, respectively, of plants with 40-day-old plants34. Fruit pericarp, without placenta and locule walls, was collected from fruits at different stages: (i) immature green 3 (IG3, approximately 8 days post-anthesis); (ii) immature green 5 (IG5, approximately 15 days post-anthesis); (iii) mature green (MG, when the placenta displays a gelatinous aspect, approximately 26 days post-anthesis); (iv) breaker (Br, beginning of ripening process when the fruit begins to present a yellowish coloration, approximately 32 days post-anthesis); (v) Br3 (three days after breaker stage, the fruits presents orange coloration); (vi) Br5 (5 days after breaker stage). Fruits were sectioned in three parts: (i) pedicellar, also known as the green shoulder, where developed chloroplast are predominately located, (ii) stylar region, which lacks developed chloroplasts), and (iii) the middle region that was discarded. For all the experiments, at least four pools of fruits (biological replicates) were harvested from at least five plants. Samples were frozen in liquid nitrogen and stored at − 80 °C freezer until processing. Mature green fruits were used for chromatin immunoprecipitation assay.Phylogenetic analysisFor phylogenetic analysis BBX proteins from plant species representing angiosperms and Chlorophyta, as well as from Homo sapiens (as outgroup) were used. The loci encoding BBX proteins were retrieved from: Phytozome 12.1 (https://phytozome.jgi.doe.gov) database for Arabidopsis thaliana, Chlamydomonas reinhardtii, Solanum lycopersicum and Volvox carteri and, from NCBI ref-seq database (https://www.ncbi.nlm.nih.gov/refseq/) for Chlorella variabilis, Coccomyxa subellipsoidea C-169, Homo sapiens, Micromonas commode, Micromonas pusilla CCMP1545, Ostreococcus lucimarinus CCE9901, Ostreococcus tauri and Volvox carteri f. nagariensis (Supplementary Table S1).Sequences from A. thaliana3 and tomato36 were named as previously reported. Amino acid sequences were aligned with Expresso T-COFFEE41 and the phylogeny was reconstructed as described in42. Briefly, the protein alignment was subjected to maximum likelihood phylogenetic reconstruction (PHYML 3.0) by JTT model with the proportion of invariable sites and gamma shape parameter estimated from the data sample. The obtained tree was optimized by tree topology and branch length, improved by subtree pruning and regrafting, and the branch support was calculated by the approximate likelihood-ratio test Shimodaira-Hasegawa-like (aLTR SH-like).Reverse transcriptase quantitative PCR analysis (RT-qPCR)RNA extraction, complementary DNA (cDNA) synthesis, primer design and RT-qPCR assays were performed as described by43. Primer sequences used are detailed in Supplementary Table S2. qPCR reactions were carried out in a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) using 2X Power SYBR Green Master Mix reagent (Life Technologies) in a 10 µL final volume. Absolute fluorescence data were analysed using the LinRegPCR software package44 in order to obtain quantitation cycle (Cq) values and calculate PCR efficiency. Expression values were normalised against the geometric mean of two reference genes, TIP41 and EXPRESSED, according to43. A permutation test lacking sample distribution assumptions45 was applied to detect statistical differences (P < 0.05) in expression ratios using the algorithms in the fgStatistics software package version 17/05/201246.Chromatin immunoprecipitation assay (ChIP)Full‐length cDNA encoding RIPENING INHIBITOR transcription factor (SlRIN, Solyc05g012020) without the stop codon was amplified with the primers listed in Supplementary Table S2. The fragment was cloned into pENTR/DTOPO using Gateway technology (Invitrogen). The entry plasmids were recombined into pK7FWG247 using LR Clonase (Invitrogen) to produce 35S::SlRIN-GFP fusion protein. The construct obtained was introduced into Agrobacterium tumefaciens (GV3101) for further infiltration. ChIP assay followed by qPCR was performed as described in34. Briefly, MG fruits were agroinfiltrated with 35S::SlRIN-GFP construct, kept for 3 days under 16 h/8 h photoperiod, and fixed with formaldehyde to promote the cross-linking between DNA and proteins. Following nuclei enrichment with a Percoll (GE Healthcare) gradient, the chromatin was fragmented by sonication (10 s on/20 s off, amplitude 70, during 10 min using QSonica700 device) and then incubated with Dynabeads Protein‐A (Invitrogen) with either anti-GFP or anti‐HA antibodies (Invitrogen). Next, the immunoprecipitated DNA was purified by phenol:chloroform:isoamyl alcohol extraction and used as template for qPCR analysis. Specific primer pairs flanking the predicted TF binding motif for each promoter region and the coding region of SlACTIN4 gene48 as control non-binding region (Supplementary Table S2) were used.Data analysesDifferences in parameters were analysed using Infostat software version 17/06/201549. When the data set showed homoscedasticity, Student’s t-test (P < 0.05) was performed to compare transgenic lines against the control genotype. In the absence of homoscedasticity, a non-parametric comparison was performed by applying the Mann–Whitney test (P < 0.05). All values represent the mean of at least three biological replicates.Transcription factor binding motifs were identified on the 3000 bp upstream of the transcription initiation site using PlantPAN 2.050.ResultsSolanum lycopersicum harbours similar diversity of BBX protein-encoding genes than A. thaliana

The BBX TF family has been extensively studied in A. thaliana, whose proteins were classified into five groups accordingly to the domain structure3,5. Similar classification was reported for other species such as tomato36, potato51, rice52 and grapevine53. However, not all provided a phylogeny with high branch support for the groupings and the lack of outgroup led the evolutionary history of the protein family ambiguous.To provide robust phylogenetic information, BBX domain-containing protein sequences from tomato and A. thaliana were retrieved from Phytozome database (https://phytozome.jgi.doe.gov) (Supplementary Table S1). This survey led to the identification of two additional loci encoding BBX proteins in the tomato genome, that were named SlBBX30 and SlBBX31, following the previously nomenclature published for this species36. A. thaliana sequences were named according to the nomenclature adopted by3 (Supplementary Table S1).The phylogenetic reconstruction (Fig. 1a) grouped the sequences according to their domain structure as previously reported in A thaliana4, confirming the monophyletic nature of the five structure groups. Regarding the tree topology, structure group IV appeared isolated from the other four groups, while groups II and V clustered together. Interestingly, AtBBX26 and AtBBX27 were previously classified in the structure group V4, while SlBBX27 was found clustered with group III proteins36; the three were described as a single B-box domain containing protein. Here, it was found that these three proteins contain indeed two BBX domains and grouped together as a subclade of structure group II without CCT domain, being referred as structure group VI (Fig. 1a). When the structure group VI sequences (i.e. AtBBX26, AtBBX27 and SlBBX27) and three representative sequences of structure group II (i.e. AtBBX10, AtBBX11 and AtBBX12) were aligned, the CCT motif could be clearly identified in the latter and some conserved residues could also be found in structure group VI sequences (Fig. 1b). Thus, this result indicates that the structure group VI diverged from structure group II sequences that lost the CCT motif.Figure 1Phylogenetic presentation of A. thaliana and tomato BBX proteins. (a) Phylogenetic reconstruction obtained from the alignment of A. thaliana and tomato BBX proteins. The clusters were named accordingly to the structure groups described for A. thaliana and the domain architecture of each clade was determined using the consensus sequence. (b) A highlight of CCT motif alignment of structure group II representatives and the corresponding region of structure group VI sequences. Shading threshold = 60%.Full size imageThe above described topology is in agreement with the one obtained for grapevine53, but is not with two other well supported phylogenies5,54. The approach applied here differed from the previously reported in two methodological aspects: human (H. sapiens) B-box domain-containing proteins were obtained from NCBI ref-seq database (https://www.ncbi.nlm.nih.gov/refseq/) (Supplementary Table S1) and used as an outgroup in the analysis; and the structure-based multiple sequence alignment whose accuracy surpass sequence-based only packages was applied41.Thus, to further confirm the obtained topology and bring information about the evolutionary history of this protein family, another phylogenetic analysis was performed including sequences from Chlorophyta species (Supplementary Table S1). The same above described topology for only tomato and A. thaliana was obtained. As the structure group VI was identified as a subclade of group II, group VI was collapsed with group II sequences to simplify the visualization (Fig. 2a). Moreover, two Chlorophyta clusters were observed, one grouping with the structure group IV and other with the clade composed of structure groups I/II/III/V. This indicated that the Viridiplantae ancestral, as means before the divergence of Chlorophyta and land plants, had two BBX-coding genes, one of which was subjected to three duplication events along land plants evolution.Figure 2Evolution of BBX proteins. (a) Phylogenetic reconstruction obtained from the alignment of A. thaliana, tomato, chlorophyta and human B-box domain containing proteins. The clusters were named accordingly to the structure groups described for A. thaliana. The sequences information is available in Supplementary Table S1. (b) Consensus sequence for B-box and CCT domains (identity ≥ 60%). (c) Proposed hypothesis for domain evolution in the BBX protein family. While the B-box1 and CCT domains appear to have single origins along the evolution of these proteins, the B-box2 domain evolved independently three times.Full size imageThe consensus sequence for the B-box and CCT domains was identified for each group (Fig. 2b). The CCT domain appear to have one single origin in the ancestral sequence of the structure groups I/II/III/V, before the divergence of Chlorophyta and land plants. It is not clear whether the ancestral proteins had one or two BBX domains. Based on the domain consensus, B-box1 seems to have a single origin, while B-box2 may have arisen several times independently, i.e. in the ancestral of the structure group IV clade, in structure group I group and in the ancestral of the structure groups II/V. Regarding the latter, the alignment of the sequences of both groups revealed that some B-box2 domain conserved residues could be still identified in structure group V members, however none could be identified in structure group III (Supplementary Fig. S1). Thus, this indicates that B-box2 appeared in the ancestral of structure group II and V after the divergence from group III. The occurrence of only B-box1 domain in structure group V is the consequence of the divergence of B-box2 and a deletion in the ancestral sequence that resulted in the loss of the CCT domain.Concluding, these results bring evidences that the ancestral Viridiplanteae harboured two B-box containing proteins; the ancestral of group IV with two B-box domains and the ancestral of group I/II/III/V-like clade with a single B-box domain. This later, after the divergence of land plants and Chlorophyta, diverged into four structure groups in which B-box2 domain arose two times independently (Fig. 2c).The expression pattern of groups IV and V SlBBX genes is influenced by the stage of plastid development in both vegetative and fruit tissuesTo gain insight into the link between BBX proteins and light signalling in tomato, we explored the transcription pattern of SlBBX genes that belong to the structure groups IV and V in organs bearing chloroplast at distinct light-regulated developmental stages, such as source and sink leaves, etiolated and de-etiolated seedlings and, fruits from immature to ripe stages29,34,40.As shown in Fig. 3a, SlBBX genes were significantly more expressed in source leaves than in sink counterparts, excepting SlBBX25 and SlBBX30 whose mRNA remained invariable. SlBBX20 was the gene that showed the most expressive induction, approximately six times (Supplementary Table S3).Figure 3Transcript profile of structure group IV and V SlBBX genes. (a) Heatmap representation of the relative transcript ratio of SlBBXs in sink and source leaves from the 8th and the 4th phytomers of 40-day-old plants, respectively. Values are means of at least three biological replicates. Colored squares represent statistically significant differences in relation to the sink leaf sample (P < 0.05). Relative transcript values are detailed in Supplementary Table S3. (b) Heatmap representation of the relative transcript ratio of SlBBXs in etiolated and de-etiolated hypocotyls and cotyledons. Values are means of at least three biological replicates. Different letters represent statistically significant differences among the samples within each gene (P < 0.05). Relative transcript values are detailed in Supplementary Table S3. (c) Relative transcript ratio of SlBBXs in the pedicellar (top) portion throughout fruit development and ripening. Data were normalised against the IG3 sample. Values are means ± SE of at least three biological replicates. Different letters indicate statistically significant differences between fruit stages (P < 0.05). IG3: immature green 3; IG5: immature green 5; MG: mature green; Br: breaker; Br3: 3 days after Br; Br5: 5 days after Br.Full size imageTranscript abundance of these SlBBX genes was also analysed under etiolation (skotomorphogenesis) and de-etiolation (photomorphogenesis) conditions in hypocotyls and cotyledons (Fig. 3b, Supplementary Table S3). Interestingly, most of the SlBBX genes showed higher levels of mRNA in cotyledons compared to hypocotyls, both in dark-grown (SlBBX18, SlBBX19, SlBBX20, SlBBX22, SlBBX23, SlBBX24, SlBBX25, SlBBX26, SlBBX28 and SlBBX30) and light-grown (SlBBX18, SlBBX21, SlBBX23, SlBBX24, SlBBX25, SlBBX26, SlBBX28 and SlBBX29) seedlings. Light exposure upregulated five (SlBBX18, SlBBX24, SlBBX17, SlBBX28 and SlBBX30) and eight (SlBBX18, SlBBX21, SlBBX16, SlBBX17, SlBBX26, SlBBX28, SlBBX29 and SlBBX31) genes in hypocotyls and cotyledons, respectively.Finally, the transcript pattern of SlBBXs belonging to structure groups IV and V was profiled throughout fruit development and ripening. Since there is a chloroplast development gradient along the longitudinal axis in wild type (WT) tomato fruits55, they were sectioned in pedicellar (with more and more developed chloroplasts) and stylar (with less and poorly developed chloroplasts) portions. As the profiles from both sections were mostly similar (Supplementary Fig. S2), we focused the analysis on the pedicellar portion (Fig. 3c, Supplementary Table S4). Most SlBBX genes exhibited substantial variations in the mRNA accumulation within the analysed stages. Interestingly, six genes showed clear association with either early development or ripening of fruits: SlBBX19 (Solyc01g110370), SlBBX20 (Solyc12g089240) and SlBBX26 (Solyc10g006750) were strongly upregulated upon ripening triggering, as means from MG to Br stage; while, the amount of SlBBX16 (Solyc12g005750), SlBBX28 (Solyc12g005660) and SlBBX29 (Solyc02g079430) mRNA was higher at green stages of fruit development gradually declining afterwards. The most expressive fold changes were observed for SlBBX20 and SlBBX16, which were eight times more and ten times less expressed from IG3 towards fully ripe Br5 fruits, respectively.The comparison of the relative mRNA accumulation levels of groups IV and V SlBBX genes among all the four organs analysed displayed no evident organ or structural specificity; however, except for SlBBX20 and SlBBX22, they showed the highest expression either in source leaves or cotyledons (Supplementary Fig. S3). To sum up, the results showed that the plastid type and developmental stage (i.e. proplastid, chloroplast or chromoplast) seem to affect the transcript accumulation pattern of these 15 SlBBX genes in leaves, hypocotyls, cotyledons and fruits.SlBBX genes associated with fruit early development or ripening are regulated by SlPHY and/or SlGLK2The identification of SlBBXs whose transcript profile is associate with fruit development and the importance of plastidial metabolism for determining nutraceutical content of tomato fruit, led to the investigation whether SlGLK2, a transcription factor essential for fruit chloroplast differentiation and activity maintenance33,55, and PHY-mediated light perception29 participate in the transcriptional regulation of the six above highlighted SlBBX genes (i.e. SlBBX16, SlBBX19, SlBBX20, SlBBX26, SlBBX28 and SlBBX29). The hypothesis that SlGLK2- and/or PHYs regulate these genes was reinforced by the finding, in their promoter regions, of at least one HY5 (key inductor of PHY-mediated photomorphogenesis56,57), PHYTOCHROME INTERACTING FACTORs (PIF; key repressor of PHY-mediated photomorphogenesis58), or GLK binding motifs59 (Supplementary Fig. S4). Slglk2 mutant, which encodes a truncated and inactive version of the protein55, and two fruit-specific SlPHY-silenced transgenic genotypes were used for the mRNA profiling. Out of the five tomato PHYs60, fruit-specific functional characterization highlighted two as major contributors to fruit physiology: SlPHYA, a positive regulator of tomato plastid division machinery; SlPHYB2, a negative regulator of chlorophyll accumulation30 and; both, inductors of fruit carotenogenesis.Among the SlBBX genes downregulated during fruit development, SlBBX28, regardless punctual fluctuations, did not show clear pattern of SlPHY- and SlGLK2-dependent regulation (Fig. 4). In the case of SlBBX29, while the lack of SlGLK2 led to a reduced transcript amount at IG3; SlPHYs have opposite effects at MG stage. Yet, SlBBX16 regulation appears to be more complex, at the peak of expression (i.e. IG3 stage) SlPHYA- and SlPHYB2-deficiency enhanced mRNA accumulation level. On the contrary, SlGLK2 seemed to have an inductive effect at green stages of fruit development (Fig. 4, Supplementary Table S5). The biological significance of the transcript level differences in the tested genotypes from Br to Br5 is questionable due to the extremely low amount of mRNA detected in ripening stages of WT genotype (i.e. the mRNA level of SlBBX16 at Br stage is only 3% of the IG3 value, Supplementary Table S4).Figure 4Transcriptional profile of SlBBXs in developing fruits of tomato lines impaired in light perception or chloroplast differentiation. The relative mRNA abundance of the six SlBBXs modulated by ripening was addressed in fruits of wild type plants (WT), SlGLK2-deficient mutant (Slglk2, Lupi et al. 2019), and fruit-specific SlPHYA- and SlPHYB2-silenced (SlphyA and SlphyB2) lines30. Values were normalised against the respective WT sample and are means of at least three biological replicates. The relative transcript values are detailed in Supplemental Table S5. Statistically significant differences relative to WT samples are colored (P < 0.05). IG: immature green 3; MG: mature green; Br: breaker; Br3: 3 days after Br; Br5: 5 days after Br.Full size imageThe ripening induction observed in SlBBX19, SlBBX20 and SlBBX26 was attenuated in SlPHYA- and SlPHYB2-silenced fruits as well as in the SlGLK2-deficient genotype. This is clearly shown by the downregulation of their expression from Br towards Br5, suggesting that SlGLK2- and SlPHY-mediated signalling cascade stimulate the expression of these genes.RIPENING INHIBITOR (SlRIN) regulates ripening-dependent expression of SlBBXsSlRIN is a master TF controlling tomato fruit ripening61 whose binding motif C(CT)(AT)6(AG)G was identified after a genome wide ChIP-Seq experiment62,63. On the promoter region (3000 bp upstream the transcription initiation site) of the three ripening-induced SlBBX genes (i.e. SlBBX19, SlBBX20 and SlBBX26), putative RIN binding motifs were identified (Fig. 5a). To address whether SlRIN directly interacts with the promoter of the aforementioned genes, a 35S::SlRIN‐GFP construct was transiently expressed in WT mature green tomato fruits followed by a ChIP-qPCR assay with anti-GFP or negative control anti-HA antibodies. The anti-GFP immunoprecipitated chromatin showed to be enriched for all SlBBX promoters tested (Fig. 5b), demonstrating that SlRIN physically binds the regulatory region of SlBBX19, SlBBX20 and SlBBX26, explaining the above-mentioned ripening-associated upregulation.Figure 5SlRIN binds to the ripening-induced SlBBXs promoter. (A) SlRIN binding motifs (C(CT)(AT)6(AG)G) blue triangles) in the promoter region (3000 bp upstream of the + 1 base) of the three ripening-induced SlBBX genes. Arrows indicate the positions of the primers used for ChIP‐qPCR assay. (B) ChIP‐qPCR experiment performed in tomato fruits transiently expressing 35S::SlRIN‐GFP using anti‐GFP and anti‐HA (as negative control) antibodies. Asterisks denote statistically significant differences (P < 0.05) to the respective anti-HA sample.Full size imageDiscussionOver the past years, BBX protein family was surveyed in several species such as apple64, A. thaliana4, grapevine53, orchids65, pear54, rice52, potato51, Arachis duranensis66 and tomato36, being classified in five groups accordingly to the domain composition of the proteins. The comprehensive phylogenetic analysis performed in this work (Fig. 2a) provided evolutionary validation of this classification by revealing that the structure groups corresponded to well sustained monophyletic clusters. A foundational work3 performed a phylogenetic analysis of A. thaliana BBX protein family that was further revised by5, which proposed a model for BBXs evolutionary trajectory in green plants. Although the phylogeny topology obtained here does not reflect the evolutionary model proposed by5, two pieces of evidences showed by the phylogenetic analysis of B-box domains reported by these authors support the clustering observed here: (i) B-box2 domain from groups IV and I are more closely related than group II B-box2 and; (ii) B-box1 domain from groups II and V are the most closely related. Moreover, some methodological differences might have increased the accuracy of the topology obtained here: i) the incorporation of an outgroup; (ii) the multiple sequence alignment carried out with structure-based information41 and; (iii) the algorithm used for the multiple sequence alignment is consistency-based, whose accuracy is increased in comparison to matrix-based ones such as ClustalW67.Our analysis showed that some A. thaliana and tomato proteins, previously reported as members of the structure group V3,4 and II36, respectively, are actually members of a new structure group, VI, which is diverging from group II after the loss of the CCT domain. As also observed for punctual examples belonging to groups II and V4, these results suggest that some BBX proteins lost a domain in a recent evolutionary event, but conserve other common characteristics of their structure group.Concluding, based on phylogenetic and domain structure analyses, we propose that the ancestral Viridiplanteae harboured two B-box domain containing proteins that originated structure group IV-like and structure group I/II/III/V-like clades, respectively. Moreover, while B-box1 and CCT domains seem to single origins in the evolutionary history of this protein family, B-box2 arose three time, independently (Fig. 2c).Functional studies regarding B-box domain encoding genes were performed almost exclusively in A. thaliana seedlings and, interestingly, especially members of structure group IV and V, were characterised as components of the light signalling cascade13,14,16,18,19,20,21,24. By employing different photoreceptors, plants can track light intensity, quality, periodicity and direction. Among photoreceptors, PHYs are codified by a small gene family, with members playing different roles gathering information for adjusting plant development and metabolism to the changing environment68. Once activated by light, PHYs phosphorylate several nuclear proteins controlling their function69. Among them, E3 ubiquitin ligase COP1 activity and stability is negatively modulated by PHYs70. Free of COP1 repression, the transcription factor HY5 is able to induce and repress the expression of photomorphogenesis- and skotomorphogenesis-related genes, respectively57. Several reports have pinpointed the major contribution of the above described light signal transduction pathway for determining tomato fruit yield and nutritional quality30,31,32,33,34,35,71,72. However, regarding SlBBX genes, only the locus Solyc01g110180, here named as SlBBX25, has been functionally characterised up to date, being described as a COP1-repressed positive regulator of chloroplast biogenesis, whose constitutive overexpression leads to dwarf plants bearing ripe fruits with increased carotenoid content37. Thus, it remains to be explored in a broader manner the role of BBX proteins in light-regulated physiological processes in tomato.Here, in structure group IV and V, which encompasses most of the light-regulated BBX proteins described in A. thaliana, 15 tomato sequences were identified (Fig. 1). Then, they were transcriptionally profiled in source and sink leaves, seedling de-etiolation, and along fruit development and ripening (Fig. 3). The comparison of the mRNA accumulation level among the different profiled organs revealed that SlBBX transcripts accumulate most in source leaves or cotyledons (Supplementary Fig. S3), which is mostly in line with the profile previously reported in tomato36. The vast majority of SlBBXs displayed higher amounts of mRNA in source than in sink leaves hinting a correlation with chloroplast number and activity (Fig. 3a). The pattern of mRNA accumulation during seedlings skoto- and photomorphogenesis showed that out of the 15 analysed genes, 8 showed to be induced by light (SlBBX16, SlBBX17, SlBBX18, SlBBX24, SlBBX28, SlBBX29, SlBBX30 and SlBBX31); while only four showed to be light-downregulated (SlBBX19, SlBBX20, SlBBX22 and SlBBX25) in at least hypocotyl or cotyledon. Two genes showed inversed pattern in response to light in both organs (SlBBX21 and SlBBX26) and one was invariable (SlBBX23). These results indicate that tomato BBX genes that belong to structure group IV and V are light responsive, like observed in A. thaliana4, and most are light-induced. The expression pattern of BBX encoding genes in Solanum tuberosum during de-etiolation was also addressed and the expression of most of the genes belonging to structural groups IV and V was modulated upon illumination of etiolated leaves51. This profile provides further evidences about a link between mRNA levels of BBX proteins from structure groups IV and V and plastid biogenesis and differentiation, revealing that they are affected, to some extent, by the light signalling cascade.Regarding fruit development and ripening (Fig. 3c), six genes stood out as their transcripts were gradually reduced from green stages towards ripening (SlBBX16, SlBBX28 and SlBBX29) or sharply induced upon this process triggering (SlBBX19, SlBBX20 and SlBBX26), indicating that their expression is also modulated by the plastid developmental stage, i.e. chloroplast to chromoplast transition. Interestingly, with the exception of SlBBX19 and SlBBX26, the mRNA accumulation profile observed here was in agreement with that reported by36.Led by the particular pattern found in fruits for SlBBX16, SlBBX19, SlBBX20, SlBBX26, SlBBX28 and SlBBX29, together with the occurrence in their promoter regions of binding motifs for TFs involved in the light signalling cascade (i.e. PIF, HY5 and GLK, Supplementary Fig. S4), their transcripts were profiled in genotypes with altered fruit light perception or without proper fruit chloroplast differentiation (Fig. 4). The three SlBBX genes downregulated from immature towards ripe stages showed induction by chloroplast maturation and light (Fig. 3a,b) and, except for SlBBX28 that did not show alterations of its transcript abundance, SlBBX16 and SlBBX29 were induced in a SlGLK2- and SlPHY-dependent manner at green stages. SlGLK2, directly and/or indirectly, i.e. inducing chloroplasts biogenesis and maintenance33,55, promoted the mRNA accumulation of SlBBX16 and SlBBX29 at green stages of fruit development (Fig. 4). Interestingly, it was shown that SlPHYB2 represses SlGLK2 mRNA accumulation30 thus, explaining the inducible effect of SlPHYB2 deficiency on the expression of these genes at green stages (Fig. 4). Finally, SlPHYA-silenced fruits displayed reduced number of chloroplasts with limited differentiation of its intermembranous structure30, which may be associated with the SlBBX16 and SlBBX29 downregulation detected in this genotype at MG stage.The disruption of PHY-mediated light signalling or chloroplast differentiation by the lack of active SlGLK2 attenuated the ripening-associated transcript accumulation of SlBBX19, SlBBX20 and SlBBX26. The minor effects observed in early stages indicate that these genes are rather induced along ripening than repressed during green stages of tomato fruit development. Since the mRNA amount of SlGLK2 is almost undetectable from breaker towards fully ripe stage33,55, the observed reduction in mRNA level in Slglk2 mutant for these three genes at ripening stages might be an indirect effect of the fewer and not fully differentiated chloroplasts in this genotype33,55, which are further converted into chromoplasts as ripening proceeds73. In a similar way, SlPHYA-silenced fruits also displayed poorly developed chloroplasts in the green stages30 that, as aforementioned, might lead to the observed reduction in the transcription of the three SlBBX genes. Interestingly, the observed downregulation of SlBBX19 in the lack of PHYA or PHYB2 was also reported for its A. thaliana ortholog, AtBBX19, in AtphyA and AtphyB mutant seedlings12. As chlorophyll degrades, the chlorophyll self-shading effect is reduced allowing the pass of sunlight through the flesh of green fruit. Light shifts the photoequilibrium of PHYs to the active form promoting the inactivation of their downstream negative effectors SlPIFs and leading to the upregulation of light-dependent ripening associated genes31,72. As PIF-binding motifs were identified in SlBBX19, SlBBX20 and SlBBX26 promoters (Supplementary Fig. S4), these TFs that are altered in SlphyA and SlphyB230 might downregulate the accumulation of these BBX transcripts in the PHY deficient lines.Moreover, the ripening-associated mRNA accumulation of SlBBX19, SlBBX20 and SlBBX26 raised the hypothesis of the involvement of the master regulator of tomato fruit ripening SlRIN61 in the regulation of these genes. Indeed, in the promoter region of all three genes, RIN-binding motifs were found (Fig. 5a) and, by ChIP-qPCR, the direct binding of SlRIN was confirmed (Fig. 5b). This is in line with the previously reported ChIP-Seq results that showed the direct interaction between SlRIN and SlBBX20 promoter63, and also with the reduced mRNA amount of this gene in SlRIN-silenced fruits74. Altogether, these results indicate that SlBBX19, SlBBX20 and SlBBX26 are light- and SlRIN-regulated, playing a role in tomato fruit ripening.Collectively, data obtained here provided a robust phylogenetic analysis of BBX proteins, giving a new perspective of the events that led to the diversification of these proteins in six structure groups. A comprehensive transcriptional profile of 15 SlBBXs revealed a correlation of mRNA amounts with the state of chloroplast development, as well as their regulation by the light signalling cascade. Additionally, a more detailed profiling in fruits led to the identification of three putative SlRIN-regulated ripening-associated SlBBX genes and other three loci associated with the early fruit development (Fig. 6). These results give insights on putative roles of SlBBX proteins in other light-regulated physiological process aside seedling photomorphogenesis and allow the identification of putative candidates for further characterization that may affect tomato fruit development and/or ripening.Figure 6Proposed regulatory network for the control of fruit development- and ripening-associated SlBBX genes. During early tomato fruit development, SlGLK2 induces the expression of several genes leading to chloroplast differentiation. SlPHYs have an inverse effect over plastidial development at green stages. While SlPHYB2 inhibits SlGLK2 transcript accumulation, SlPHYA positively controls chloroplast division regulators30. Chloroplast biogenesis and maturation positively influence SlBBX16 and SlBBX29 transcript accumulation. As the fruit matures, the transcript abundance of both these SlBBX genes decreases. Once ripening initiates, the conversion of chloroplast to chromoplast begins and SlRIN accumulates, activating the expression of several ripening associated genes, including SlBBX19, SlBBX20 and SlBBX26. During ripening, these three SlBBX genes are also positively regulated by SlPHYs, probably, through the repression of several light signalling negative regulators, such as COP1 and PIFs. The absence of properly differentiated chloroplast due to SlGLK2 deficiency attenuates the upregulation of SlBBX19, SlBBX20 and SlBBX26 during ripening. Continuous lines indicate direct effect; dotted lines indicate that the effects may not be due to direct interaction. Arrow-ended lines indicate induction; bar-ended lines indicate repression.Full size image

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Download referencesFundingB.S.L. and D.R. were recipients of FAPESP fellowships (2017/14953-0, 2015/14658-3). M.R., L.F., M.J.O. and L.S. were funded by a fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). A.C.D.L. was recipient of CAPES fellowship. This work was partially supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 2016/01128-9 (Brazil), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Finance Code 001 (Brazil) and Universidade de São Paulo, Brazil.Author informationAuthor notesDaniele RosadoPresent address: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY, 11724, USAThese authors contributed equally: Maria José Oliveira and Lumi Shiose.Authors and AffiliationsDepartamento de Botânica, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, 277, São Paulo, 05508-090, BrasilBruno Silvestre Lira, Maria José Oliveira, Lumi Shiose, Raquel Tsu Ay Wu, Daniele Rosado, Alessandra Cavalcanti Duarte Lupi, Luciano Freschi & Magdalena RossiAuthorsBruno Silvestre LiraView author publicationsYou can also search for this author in

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PubMed Google ScholarContributionsB.S.L. designed and performed most of the experiments, analysed the data and wrote the article with contributions of all the authors; M.J.O., L.S., R.T.A.W., A.C.D.L. and D.R. performed the experiments. L.F. designed the experiments, contributed to data analysis and complemented the writing; M.R. designed the experiments, contributed to data analysis and wrote the article with contributions of all the authors.Corresponding authorCorrespondence to

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The authors declare no competing interests.

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Reprints and permissionsAbout this articleCite this articleLira, B.S., Oliveira, M.J., Shiose, L. et al. Light and ripening-regulated BBX protein-encoding genes in Solanum lycopersicum.

Sci Rep 10, 19235 (2020). https://doi.org/10.1038/s41598-020-76131-0Download citationReceived: 05 June 2020Accepted: 20 October 2020Published: 06 November 2020DOI: https://doi.org/10.1038/s41598-020-76131-0Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard

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