Table_1_Transcriptome characteristics during cell wall formation of endosperm cellularization and embryo differentiation in Arabidopsis.xlsx

Embryonic and endosperm development are important biological events during Arabidopsis seed development, and are controlled by dynamic changes in a range of gene expression. Nevertheless, the regulatory mechanisms of endosperm cellularization and embryo differentiation remain unclear. Here, we characterized the early embryo and endosperm development of the naa15 mutant that had abnormal embryo differentiation and incomplete endosperm cellularization compared to WT of Arabidopsis, and comparatively investigated the changes of gene expressions in WT seeds at 3, 4, and 5 days after pollination (3W, 4W, and 5W) and the white homozygous aborted naa15 seeds at 5, 6, and 7 DAP (5M, 6M, and 7M) from naa15-1/+ siliques using RNA sequencing and qPCR assays. The transcriptome analyses showed that there were 2040 and 3630 differentially expressed genes (DEGs) in 4W (at endosperm cellularization initiation stage and heart embryo stage) vs 3W (at syncytium stage and globular embryo stage), and 5W (at end of endosperm cellularization stage and torpedo embryo stage) vs 4W, respectively. The KEGG and GO analyses showed that lipid metabolic processes and transmembrane transport related to cell wall biogenesis, cell division and differentiation, the plant hormone signaling pathway, photosynthesis, and transcription regulator activity were evidently enriched in WT and naa15. The heatmap and qPCR analyses showed that auxin response genes (ARFs), auxin transport genes (PINs) cytokinin synthesis genes (LOGs), cytokinin dehydrogenase genes (CKXs), cytokinin receptor, transcription factors (MYB, bHLH, MADS-box, and ERF) were significantly downregulated in naa15 compared to WT. A series of cell wall genes annotated to xyloglucan endotransglycosylase/hydrolase, pectin methyl esterase, and pectin methyl esterase inhibitor were also identified in these DEGs. Moreover, using an immunofluorescent assay, the features of cell walls displayed that cellulose fluorescence signals in the embryo and endosperm of naa15 were significantly decreased, and the signals of low- and high- methyl esterification of pectin were also obviously decreased in the endosperm of naa15. In summary, we identified a large number of DEGs and investigated the features of cell walls during endosperm cellularization and embryonic differentiation, which provided important information on transcription and gene expression to reveal their regulatory mechanisms.

胚胎与胚乳发育是拟南芥（Arabidopsis）种子发育过程中的重要生物学事件，受一系列基因表达的动态变化调控。然而，目前关于胚乳细胞化与胚胎分化的调控机制仍不明确。本研究对拟南芥naa15突变体的早期胚胎与胚乳发育进行了表征，该突变体相较于野生型（Wild Type, WT）存在胚胎分化异常、胚乳细胞化不完全的表型；同时利用RNA测序（RNA sequencing）与实时定量PCR（quantitative PCR, qPCR）技术，对比分析了来自naa15-1/+杂合角果的野生型种子（授粉后3、4、5天，分别记为3W、4W、5W）以及纯合败育的白色naa15种子（授粉后5、6、7天，分别记为5M、6M、7M）的基因表达变化。转录组分析显示，在4W（处于胚乳细胞化起始阶段与心形胚阶段）vs 3W（处于合胞体阶段与球形胚阶段）、5W（处于胚乳细胞化结束阶段与鱼雷形胚阶段）vs 4W的对比组中，分别筛选到2040个和3630个差异表达基因（differentially expressed genes, DEGs）。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）与基因本体（Gene Ontology, GO）富集分析显示，在野生型与naa15突变体中，与细胞壁生物发生、细胞分裂与分化相关的脂质代谢过程、跨膜运输，植物激素信号通路、光合作用以及转录调控因子活性均显著富集。热图与qPCR分析显示，相较于野生型，naa15突变体中的生长素响应因子（ARFs）、生长素转运蛋白基因（PINs）、细胞分裂素合成基因（LOGs）、细胞分裂素脱氢酶基因（CKXs）、细胞分裂素受体以及转录因子（MYB、bHLH、MADS-box及ERF家族）均显著下调表达。本研究在这些差异表达基因中还鉴定到一系列注释为木葡聚糖内转糖苷酶/水解酶、果胶甲酯酶以及果胶甲酯酶抑制剂的细胞壁相关基因。此外，通过免疫荧光（immunofluorescence）实验分析细胞壁特征发现，naa15突变体胚胎与胚乳中的纤维素荧光信号显著降低，且其胚乳中低甲基酯化与高甲基酯化果胶的信号也明显减弱。综上，本研究鉴定了大量差异表达基因，并解析了胚乳细胞化与胚胎分化过程中的细胞壁特征，为揭示二者的调控机制提供了重要的转录与基因表达层面的研究依据。