SlEIN2调控番茄果实大小的机制研究

    果实大小直接决定作物的最终产量，是果实作物驯化过程的主要变量，研究果实大小的调控对基础科学和农业都具有重要意义。番茄（Solanum lycopersicum）是全球第四大经济作物，同时遗传转化体系稳定，基因组信息完整，生命周期较短，是典型的呼吸跃变型果实，因此被用作研究果实发育的模式物种。番茄果实大小受多种内源激素的影响。过去研究认为赤霉素及生长素是影响番茄果实大小的主要内源激素。乙烯作为一种重要的内源激素，已知其能影响果实成熟。然而，乙烯是否参与果实大小的调控仍然不是很清楚。本研究通过分析番茄中乙烯信号关键正调控组分SlEIN2的基因敲除突变体果实的表型发现，slein2突变体果实与野生型相比明显变小，这表明内源乙烯也能调控番茄果实大小发育。在此基础上，本研究以SlEIN2为切入点，旨在揭示乙烯调控果实大小的分子机制。

    本研究通过酵母双杂交文库筛选获得SlEIN2的互作蛋白SlCNR8（Cell Number Regulator 8），SlCNR8在番茄及玉米中的多个同源基因参与果实或器官大小的调控，暗示SlEIN2有可能协同SlCNR8一起影响果实大小。本研究进一步通过酵母双杂交验证了SlEIN2和SlCNR8的相互作用。另外，本研究在细菌中表达了SlEIN2羧基端和SlCNR8，通过pull-down实验进一步证明SlEIN2和SlCNR8在体外能直接互作。本论文进一步利用拟南芥原生质体双分子荧光互补（BiFC）实验证明SlEIN2和SlCNR8在植物体内也能发生互作。此外，本研究系统分析了SlEIN2介导的果实发育不同时期的基因表达变化，发现生长素转运、玉米素及油菜素内酯合成以及细胞分裂素的响应途径很有可能介导了SlEIN2对果实大小的调控；并进一步通过共表达网络分析找到SlEIN2调控果实大小的关键基因。本论文进一步构建了SlCNR8的基因敲除和过表达株系等遗传材料，未来将进一步结合这些遗传材料的表型深入探讨SlEIN2和SlCNR8对果实大小的调控。

    综上，本研究首次揭示了植物激素乙烯对果实大小调控具有重要的作用，系统建立了SlEIN2介导的果实大小调控的分子网络，为研究果实大小调控提供新的思路。

[1] FRARY A, NESBITT T C, GRANDILLO S. Fw2.2: A Quantitative Trait Locus Key to the Evolution of Tomato Fruit Size.[J]. Science （New York, N.Y.）, 2000, 289（5476）: 85–88.
[2] CHEVALIER C, BOURDON M, PIRRELLO J. Endoreduplication and fruit growth in tomato: Evidence in favour of the karyoplasmic ratio theory[J]. Journal of Experimental Botany, 2014, 65（10）: 2731–2746.
[3] AZZI L, DELUCHE C, GÉVAUDANT F. Fruit Growth-Related Genes in Tomato.[J]. Journal of Experimental Botany, 2015, 66（4）: 1075–1086.
[4] DE JONG M, MARIANI C, VRIEZEN W H. The Role of Auxin and Gibberellin in Tomato Fruit Set.[J]. Journal of Experimental Botany, 2009, 60（5）: 1523–1532.
[5] CZEREDNIK A, BUSSCHER M, BIELEN B A M. Regulation of tomato fruit pericarp development by an interplay between CDKB and CDKA1 cell cycle genes[J]. Journal of Experimental Botany, 2012, 63（7）: 2605–2617.
[6] SU L, BASSA C, AUDRAN C. The Auxin Sl-IAA17 Transcriptional Repressor Controls Fruit Size via the Regulation of Endoreduplication-Related Cell Expansion.[J]. Plant & Cell Physiology, 2014, 55（11）: 1969–1976.
[7] RENAU-MORATA B, CARRILLO L, CEBOLLA-CORNEJO J. The Targeted Overexpression of SlCDF4 in the Fruit Enhances Tomato Size and Yield Involving Gibberellin Signalling.[J]. Scientific Reports, 2020, 10（1）: 10645.
[8] NITSCH L, KOHLEN W, OPLAAT C. ABA-deficiency results in reduced plant and fruit size in tomato[J/OL]. Journal of Plant Physiology, 2012, 169（9）: 878–883.
[9] GUO M, RUPE M A, DIETER J A. Cell Number Regulator1 Affects Plant and Organ Size in Maize: Implications for Crop Yield Enhancement and Heterosis.[J]. The Plant Cell, 2010, 22（4）: 1057–1073.
[10] ROSA M, ABRAHAM-JUÁREZ M J, LEWIS M W. The Maize MID-COMPLEMENTING ACTIVITY Homolog CELL NUMBER REGULATOR13/NARROW ODD DWARF Coordinates Organ Growth and Tissue Patterning.[J]. The Plant Cell, 2017, 29（3）: 474–490.
[11] LINKIES A, MÜLLER K, MORRIS K. Ethylene Interacts with Abscisic Acid to Regulate Endosperm Rupture during Germination: A Comparative Approach Using Lepidium Sativum and Arabidopsis Thaliana.[J]. The Plant Cell, 2009, 21（12）: 3803–3822.
[12] JOHNSON P R, ECKER J R. The Ethylene Gas Signal Transduction Pathway: A Molecular Perspective.[J]. Annual Review of Genetics, 1998, 32: 227–254.
[13] BUCHANAN _BB_, GRUISSEM W. Biochemistry and Molecular Biology of Plants[J]. harshman lg hoffmann fg claudianos c.jhe in gryllus assimilis cloning, 2000.
[14] RAVANEL S, GAKIÈRE B, JOB D. The Specific Features of Methionine Biosynthesis and Metabolism in Plants.[J]. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95（13）: 7805–7812.
[15] WANG K L-C, LI H, ECKER J R. Ethylene Biosynthesis and Signaling Networks.[J]. The Plant Cell, 2002, 14 Suppl（Suppl）: S131-51.
[16] FUJISAWA M, NAKANO T, SHIMA Y. A Large-Scale Identification of Direct Targets of the Tomato MADS Box Transcription Factor RIPENING INHIBITOR Reveals the Regulation of Fruit Ripening.[J]. The Plant Cell, 2013, 25（2）: 371–386.
[17] RAZ V, ECKER J R. Regulation of Differential Growth in the Apical Hook of Arabidopsis.[J]. Development （Cambridge, England）, 1999, 126（16）: 3661–3668.
[18] GUZMÁN P, ECKER J R. Exploiting the Triple Response of Arabidopsis to Identify Ethylene-Related Mutants.[J]. The Plant Cell, 1990, 2（6）: 513–523.
[19] BLEECKER A B, ESTELLE M A, SOMERVILLE C. Insensitivity to Ethylene Conferred by a Dominant Mutation in Arabidopsis Thaliana.[J]. Science （New York, N.Y.）, 1988, 241（4869）: 1086–1089.
[20] HAO D, SUN X, MA B. 6 - Ethylene[M/OL]. LI J, LI C, SMITH S M, Hormone Metabolism and Signaling in Plants. Academic Press, 2017: 203–241.
[21] GUO H, ECKER J R. The Ethylene Signaling Pathway: New Insights.[J]. Current Opinion in Plant Biology, 2004, 7（1）: 40–49.
[22] HUA J, MEYEROWITZ E M. Ethylene Responses Are Negatively Regulated by a Receptor Gene Family in Arabidopsis Thaliana.[J]. Cell, 1998, 94（2）: 261–271.
[23] KIEBER J J, ROTHENBERG M, ROMAN G. CTR1, a Negative Regulator of the Ethylene Response Pathway in Arabidopsis, Encodes a Member of the Raf Family of Protein Kinases.[J]. Cell, 1993, 72（3）: 427–441.
[24] HUANG Y, LI H, HUTCHISON C E. Biochemical and Functional Analysis of CTR1, a Protein Kinase That Negatively Regulates Ethylene Signaling in Arabidopsis.[J]. The Plant Journal : For Cell and Molecular Biology, 2003, 33（2）: 221–233.
[25] ALONSO J M, HIRAYAMA T, ROMAN G. EIN2, a Bifunctional Transducer of Ethylene and Stress Responses in Arabidopsis.[J]. Science （New York, N.Y.）, 1999, 284（5423）: 2148–2152.
[26] LIN J, HU J. SeqNLS: Nuclear Localization Signal Prediction Based on Frequent Pattern Mining and Linear Motif Scoring.[J]. PloS One, 2013, 8（10）: e76864.
[27] BISSON M M A, BLECKMANN A, ALLEKOTTE S. EIN2, the Central Regulator of Ethylene Signalling, Is Localized at the ER Membrane Where It Interacts with the Ethylene Receptor ETR1.[J]. The Biochemical Journal, 2009, 424（1）: 1–6.
[28] QIAO H, SHEN Z, HUANG S C. Processing and Subcellular Trafficking of ER-Tethered EIN2 Control Response to Ethylene Gas.[J]. Science （New York, N.Y.）, 2012, 338（6105）: 390–393.
[29] QIAO H, CHANG K N, YAZAKI J. Interplay between Ethylene, ETP1/ETP2 F-Box Proteins, and Degradation of EIN2 Triggers Ethylene Responses in Arabidopsis.[J]. Genes & Development, 2009, 23（4）: 512–521.
[30] WEN X, ZHANG C, JI Y. Activation of Ethylene Signaling Is Mediated by Nuclear Translocation of the Cleaved EIN2 Carboxyl Terminus.[Z]（2012–11）.
[31] LI W, MA M, FENG Y. EIN2-Directed Translational Regulation of Ethylene Signaling in Arabidopsis.[J]. Cell, 2015, 163（3）: 670–683.
[32] MERCHANTE C, BRUMOS J, YUN J. Gene-Specific Translation Regulation Mediated by the Hormone-Signaling Molecule EIN2.[J]. Cell, 2015, 163（3）: 684–697.
[33] ZHANG F, QI B, WANG L. EIN2-Dependent Regulation of Acetylation of Histone H3K14 and Non-Canonical Histone H3K23 in Ethylene Signalling.[J]. Nature Communications, 2016, 7: 13018.
[34] CHAO Q, ROTHENBERG M, SOLANO R. Activation of the Ethylene Gas Response Pathway in Arabidopsis by the Nuclear Protein ETHYLENE-INSENSITIVE3 and Related Proteins.[J]. Cell, 1997, 89（7）: 1133–1144.
[35] BINDER B M, WALKER J M, GAGNE J M. The Arabidopsis EIN3 Binding F-Box Proteins EBF1 and EBF2 Have Distinct but Overlapping Roles in Ethylene Signaling.[J]. The Plant Cell, 2007, 19（2）: 509–523.
[36] GUO H, ECKER J R. Plant Responses to Ethylene Gas Are Mediated by SCF（EBF1/EBF2）-Dependent Proteolysis of EIN3 Transcription Factor.[J]. Cell, 2003, 115（6）: 667–677.
[37] CARY A J, LIU W, HOWELL S H. Cytokinin Action Is Coupled to Ethylene in Its Effects on the Inhibition of Root and Hypocotyl Elongation in Arabidopsis Thaliana Seedlings.[J]. Plant Physiology, 1995, 107（4）: 1075–1082.
[38] GIOVANNONI J. MOLECULAR BIOLOGY OF FRUIT MATURATION AND RIPENING.[J]. Annual Review of Plant Physiology and Plant Molecular Biology, 2001, 52: 725–749.
[39] LIU M, PIRRELLO J, CHERVIN C. Ethylene control of fruit ripening: Revisiting the complex network of transcriptional regulation[J]. Plant Physiology, 2015, 169（4）: 2380–2390.
[40] SHINOZAKI Y, HAO S, KOJIMA M. Ethylene suppresses tomato （Solanum lycopersicum） fruit set through modification of gibberellin metabolism[J]. Plant Journal, 2015, 83（2）: 237–251.
[41] LI S, CHEN K, GRIERSON D. A Critical Evaluation of the Role of Ethylene and MADS Transcription Factors in the Network Controlling Fleshy Fruit Ripening.[J]. The New Phytologist, 2019, 221（4）: 1724–1741.
[42] YU Y, MENG X, GUO D. Grapevine U-Box E3 Ubiquitin Ligase VlPUB38 Negatively Regulates Fruit Ripening by Facilitating Abscisic-Aldehyde Oxidase Degradation.[J]. Plant & Cell Physiology, 2021, 61（12）: 2043–2054.
[43] SU W, SHAO Z, WANG M. EjBZR1 Represses Fruit Enlargement by Binding to the EjCYP90 Promoter in Loquat.[J]. Horticulture Research, 2021, 8（1）: 152.
[44] ANTHONEY N, FOLDI I, HIDALGO A. Toll and Toll-like Receptor Signalling in Development.[J]. Development （Cambridge, England）, 2018, 145（9）.
[45] BRISOU G, PIQUEREZ S J M, MINOIA S. Induced Mutations in SlE8 and SlACO1 Control Tomato Fruit Maturation and Shelf-Life.[J]. Journal of Experimental Botany, 2021, 72（20）: 6920–6932.