植物种子大小直接影响着植物的进化和农作物的产量，是人们最为关注的农艺性状之一，而种皮颜色是十字花科芸薹属作物育种的一个重要性状指标，通常黄色种子较棕色（褐色）种子具有更高的油含量和品质。因此，发掘在种子形成和发育过程中调控种子大小、种皮颜色和脂肪酸含量的基因资源，解析其发挥功能的调控机制，有利于创制高产量、高品质的农作物和油料作物种质资源及优良品种。本课题组前期实验结果显示，作为糖酵解过程中烯醇化酶的编码基因，拟南芥ENO2（Arabidopsis enolase2, AtENO2）在角果中的含量显著高于根、叶和花等组织，且在发育种子中的表达量显著高于角果皮。AtENO2的突变能够导致植物变矮、花粉萌发率下降、角果变短、花粉粒畸形等表型，但是AtENO2对种子生长发育的影响有待研究。因此，本研究旨在利用遗传学、生物化学与分子生物学、生物信息学等技术与方法，研究ENO2对拟南芥种子性状的影响及其调控机制，为进一步研究该基因翻译的两种蛋白（ENO2和MBP-1（c-Myc Binding-promoter Protein））所具有的生物学功能及其分子调控机理奠定基础。同时，本课题的研究成果将为作物遗传育种和农艺性状改良提供优质的基因资源和科学的理论依据。本课题的主要研究结果如下：

1）AtENO2对种子性状的影响

经电镜观察及数据统计分析显示：ENO2突变体（eno2^-）种子的长和宽、种子表面积、子叶面积和胚均小于野生型（WT），但子叶细胞的面积无差异；eno2^-种子的重量也显著低于WT。在突变体中回补ENO2，种子的表型恢复到了WT状态。同时，相比于WT，eno2^-的种皮颜色发黄，香兰素染色结果也显示eno2^-种皮着色程度明显弱于WT，这表明eno2^-种子中的原花青素含量低于WT。因此，AtENO2突变改变了种子的大小和色泽。

2）AtENO2对糖代谢及黄酮类物质合成的影响

物质含量检测结果表明：eno2^-种子中的葡萄糖含量显著高于WT（P < 0.05），蔗糖和果糖含量略高于WT，而淀粉含量却显著低于WT。同时，类黄酮和原花青素的含量也显著低于WT，且eno2^-和WT种子中的黄酮类物质成分发生了改变。钌红染色结果显示eno2^-种子中多糖物质粘液质的含量也明显少于WT。代谢组数据结果显示在eno2^-中，与氨基酸合成有关的差异代谢物L-苏氨酸、L-谷氨酸和L-谷氨酰胺等的含量均高于WT，而三羧酸循环中的柠檬酸、顺式乌头酸和α-酮戊二酸的含量以及类黄酮物质合成通路中表儿茶素、槲皮素和槲皮素-3-半乳糖苷的含量均低于WT。WT和eno2^-角果的转录组数据分析表明，在开花后发育4天（4 DAF）的角果中，类黄酮合成途径和苯丙烷类物质合成途径均达到了显著富集水平，淀粉和蔗糖代谢通路在9 DAF时得到了显著富集。苯丙烷合成途径中的PAL1（PHE AMMONIA LYASE 1）、4CL3（4-COUMARATE:COA LIGASE 3）和4CL4（4-COUMARATE:COA LIGASE 4）以及类黄酮合成途径中的CHI1（CHALCONE ISOMERASE 1）、CHI3（CHALCONE ISOMERASE 1）、CHS（CHALCONE SYNTHASE）、LDOX（LEUCOANTHOCYANIDIN DIOXYGENASE）和BAN（BANYULS）均在4 DAF的eno2^-角果中发生了显著下调表达。因此，AtENO2突变减少了表儿茶素、槲皮素和槲皮素-3-半乳糖苷等类黄酮物质含量，引起原花青素的合成受阻，进而导致种皮颜色变黄。

3）AtENO2对种子中脂肪酸含量的影响

GC-MS检测结果显示：在eno2^-种子中，油酸（C18:1）、亚油酸（C18:2）和亚麻酸（C18:3）含量得到增加，总脂肪酸含量也显著高于WT。成熟种子的超薄切片观察结果表明eno2^-种子中的油体明显大于WT。转录组数据显示：脂肪酸合成途径在14 DAF的WT和eno2^-角果中得到了显著富集，且有8个LACSs（LONG-CHAIN ACYL-COA SYNTHETASEs）、4个KASs（3-KETOACYL-ACYL CARRIER PROTEIN SYNTHASEs）、2个BCCPs（BIOTIN CARBOXYL CARRIER PROTEINs）和2个ACCs（ACETYL-COA CARBOXYLASEs）基因在eno2^-角果中发生了显著上调表达。由此可以判断，AtENO2突变上调了脂肪酸合成途径中关键基因的表达，促进了种子中不饱和脂肪酸含量的上升。

4）AtENO2对种子中植物激素水平的影响

分析转录组数据可知：在WT和eno2^-的三个不同发育时期角果中，分别有359、390和1090个差异表达基因（DEGs）涉及到植物激素合成及其激活的信号通路中。各个时期中，与脱落酸和生长素相关的DEGs最多，其次是乙烯和水杨酸。统计结果显示，与植物激素相关且影响种子次级代谢的关键差异表达基因一共有65个，其中参与生长素和茉莉酸合成代谢且影响种子次级代谢的DEGs最多，均有14个，并且前者包含的DEGs主要影响黄酮类物质的合成，后者包含的DEGs主要参与脂肪酸的合成。利用LC-MS技术，本研究检测了WT和eno2^-种子中植物激素的含量。结果表明在eno2^-种子中，生长素和脱落酸的含量显著高于WT，但是细胞分裂素、乙烯前体ACC和茉莉酸的含量均显著低于野生型，没有检测到赤霉素的含量。因此，AtENO2的缺失也改变了植物激素的水平。

5）AtENO2对角果中水杨酸合成的影响

检测不同发育时期（4 DAF、9 DAF和14 DAF）角果中的水杨酸含量，结果显示eno2^-角果中的水杨酸含量始终高于WT角果，但随着角果（种子）的发育，均呈现下降趋势。在eno2^-角果中，合成水杨酸的主要途径——异分支酸合成通路中的PBS3（avrPPHB SUSCEPTIBLE 3）、ICS2（ISOCHORISMATE SYNTHASE 2）和EPS1（ENHANCED PSEUDOMONAS SUSCEPTIBILTY 1）均在14 DAF时期显著高表达于WT，且ICS2在9 DAF时的表达量也显著高于WT，而次要途径——苯丙氨酸合成通路中的PAL则在4 DAF时发生了显著下调表达。由此推断，AtENO2突变促进了质体中异分支酸合成途径中调节基因的表达，进而增加了植物体内水杨酸的含量。

6）ENO2/MBP-1互作蛋白的筛选与验证

利用酵母双杂交实验，本研究筛选到ENO2和MBP-1均可以与bZIP75（BASIC LEUCINE-ZIPPER 75）或TGA5（TGACG MOTIF-BINDING FACTOR 5）存在相互作用，而Pull-down实验、荧光素酶互补（LUC）实验和双分子荧光互补（BiFC）实验表明：仅ENO2能够与bZIP75或TGA5存在互作关系。

综上所述，拟南芥ENO2通过调控糖代谢、黄酮类物质的合成以及植物激素的含量，影响了种子的大小、重量、色泽和脂肪酸含量。

In plants, seed size is one of the most concerned agronomic traits which directly affects the evolution of plants and the yield of crops. The seed coat color is an important trait indicator for the breeding of Cruciferous Brassica crops. Compared with brown-seeds, yellow-seeds have higher oil content and quality. Thus, it is essential to discover genetic resources that regulate seed size, seed coat color and fatty acid content during seed formation and development, and analyze the regulatory mechanism which is conducive to creating high-yield, high-quality germplasm resources of crops and excellent varieties. Our previous results showed that the expression level of AtENO2 was significantly higher in silique than that in roots, leaves, flowers and other tissues. As a gene encoding enolase, the mutation of Arabidopsis ENO2 (AtENO2) caused the defective growth and reproduction, such as smaller plant, reduced germination rate of pollen, shorter silique and malformed pollen. It is worth noting that the expression level of AtENO2 in ovule was significantly higher than that in pericarp. The regulatory mechanism of ENO2 on the seed properties are still not clear. In this study, genetics, biochemistry, molecular biology and bioinformatics will be used to further investigate the effects of ENO2 on the seed size and fatty acid biosynthesis in Arabidopsis thaliana and its regulatory mechanism. The results will help to understand the function of ENO2 more deeply, and lay a foundation for further research on the biological functions and regulatory mechanisms of ENO2 and MBP-1 (c-Myc Binding-promoter Protein) which were alternative-translated by ENO2. At the same time, our study also provided high-quality genetic resources and theoretical basis for the genetic breeding of crops and the improvement of agronomic traits. The main results are as follows:

1) The effects of AtENO2 on seed traits

The results of electron microscope and statistical analysis of data showed that the length and width of seeds, seed area, cotyledon area and embryos in eno2^- were smaller than those in WT, but the cotyledon cell area of eno2^- did not show significant difference from that of WT. The expression of a 35S promoter-driven AtENO2 construct into the ENO2 mutant (eno2^-/35S:AtENO2) complemented the phenotype of seeds in eno2^- plants. At the same time, the eno2^- seeds were yellow compared to WT. Vanillin staining also showed that the seed coat color of eno2^- was significantly lighter than that of WT, which indicated that the content of proanthocyanidins in eno2^- seeds were lower than that of WT. Therefore, the mutation of AtENO2 changes the size and color of seeds.

2) The effect of AtENO2 on sugar metabolism and flavonoid synthesis

In our study, the levels of sucrose and fructose in eno2^- seeds were slightly elevated compared with other lines, while glucose content of eno2^- seeds was significantly higher than that of WT (P < 0.05). In contrast, starch content in eno2^- seeds was lower compared to WT. In addition, the contents of flavonoid and proanthocyanidin were also significantly lower than those of WT, and the compounds of flavonoid in eno2^- and WT seeds were different. The results of ruthenium red staining showed that the content of polysaccharide mucilage in eno2^- seeds was also significantly less than WT seeds. Metabolomic data displayed that the content of differential metabolites related to amino acid synthesis such as L-threonine, L-glutamic acid and L-glutamine in eno2^- were higher than those of WT, while the contents of citric acid, cis-aconitic acid and oxoglutaric in the tricarboxylic acid cycle and the contents of epicatechin, quercetin and quercetin-3-galactoside in the pathway of flavonoid synthesis were significantly reduced. Comparing the RNA-seq data of WT and eno2^- silique showed that the flavonoid synthesis pathway and the phenylpropane synthesis pathway were significantly enriched in silique at 4 days after flowering (4 DAF), the pathway of starch and sucrose metabolic was significantly enriched at 9DAF. The expression level of PAL1, 4CL3 and 4CL4 in the phenylpropane synthesis pathway and the expression level of CHI1, CHI3, CHS, LDOX and BAN in the flavonoid synthesis pathway were significantly down-regulated at 4 DAF silique of eno2^-. Therefore, the ENO2 mutation reduced the content of flavonoids such as epicatechin, quercetin, and quercetin-3-galactoside, inhibiting the synthesis of proanthocyanidins, which in turn caused the seed coat to appear yellow.

3) The effect of AtENO2 on the content of fatty acid

Relative to WT seeds, the seeds of eno2^- contained more fatty acid featured with enlarged proportions of oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3). Observing the ultra-thin sections of mature seeds, we found that the oil bodies in eno2^- seeds were apparent larger than WT. RNA-seq showed that the fatty acid synthesis pathway was significantly enriched at 14 DAF stage between WT and eno2^- silique, and 8 LCSs, 4 KASs, 2 BCCPs and 2 ACCs were significantly up-regulated in eno2^- silique. These results suggest that the loss of AtENO2 increases the contents of unsaturated fatty acids by up-regulating the expression levels of key genes in the fatty acid biosynthetic pathway.

4) The effect of AtENO2 on the level of plant hormones

As shown in the RNA-seq data, there are 359, 390 and 1, 090 differentially expressed genes (DEGs) involved in the signal pathways of plant hormone synthesis and hormone activation at the three developmental stages of WT and eno2^-, respectively. In each period, DEGs related to abscisic acid and auxin were the most, followed by ethylene and salicylic acid. A total of 65 key DEGs were identified which were involved in synthesis of plant hormones and seed secondary metabolism. Among them, the DEGs involved in auxin or jasmonic acid anabolism and seed secondary metabolism were the most, 14 of which were included. The DEGs contained in the former mainly affect the synthesis of flavonoids, and the DEGs contained in the latter were mainly involved in the synthesis of fatty acids. We detected the contents of plant hormones in WT and eno2^- seeds by using LC-MS. The levels of IAA (auxin) and ABA (abscisic acid) in the eno2^- seeds were significantly elevated than those in the WT seeds, but the contents of CTK (cytokinin), ACC (acetyl-coa carboxylase, a precursor of ethylene) and Jasmonic acid (JA) were significant reduced than those in the WT seeds. Gibberellin (GA) content was not detected. Therefore, the mutation of AtENO2 changes the levels of plant hormones.

5) The effect of AtENO2 on salicylic acid synthesis in silique

The content of salicylic acid in silique of different development days (4 DAF, 9 DAF and 14 DAF) was detected. The results showed that the level of salicylic acid in eno2^- silique was always higher than that in WT siliques, but was gradually decreased with the development of silique (seeds). Salicylic acid is mainly produced by the isochrisic acid synthesis pathway, whereas a small amount of salicylic acid is derived from phenylalanine ammonia lyase pathway. The RNA-seq analysis revealed that PBS3, ICS2 and EPS1 were significantly highly expressed in eno2^- at 14 DAF, and ICS2 was also significantly higher than WT at 9 DAF, while PAL was significantly down-regulated at 4 DAF. It was inferred that the loss of AtENO2 promotes the expression of regulatory genes in the isochrisic acid synthesis pathway of plastids, thereby increasing the salicylic acid content in plants.

6) The screening and verification of proteins interacting with ENO2/MBP-1

Using the yeast two-hybrid experiment, we found that ENO2 and MBP-1 interacted with bZIP75 or TGA5, but the pull-down assay, luciferase complementation (LUC) assay and bimolecular fluorescence complementation (BiFC) assay demonstrated that only ENO2 could interact with bZIP75 or TGA5.

In summary, AtENO2 affects the size, weight, color and fatty acid content of seeds by regulating sugar metabolism, flavonoid synthesis and plant hormone levels.