Table_10_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.

胚胎和胚乳发育是拟南芥种子发育过程中的重要生物学事件，其调控依赖于一系列基因表达的动态变化。尽管如此，胚乳细胞化和胚胎分化的调控机制仍不明确。在本研究中，我们对naa15突变体的早期胚胎和胚乳发育进行了特征化研究，该突变体与野生型拟南芥相比，表现出异常的胚胎分化和不完全的胚乳细胞化。通过RNA测序和qPCR实验，我们比较研究了野生型种子在授粉后第3、4、5天（3W、4W和5W）以及naa15-1/+角果中的白色同质子种子的第5、6、7天（5M、6M和7M）的基因表达变化。转录组分析显示，在4W（胚乳细胞化启动阶段和心形胚胎阶段）与3W（原生质阶段和球形胚胎阶段）之间，以及5W（胚乳细胞化结束阶段和梭形胚胎阶段）与4W之间，分别存在2040和3630个差异表达基因（DEGs）。KEGG和GO分析表明，与野生型相比，naa15在脂质代谢过程、与细胞壁生物合成、细胞分裂和分化、植物激素信号通路、光合作用以及转录调节活性相关方面明显富集。热图和qPCR分析显示，与野生型相比，naa15中生长素响应基因（ARFs）、生长素运输基因（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、转录因子（MYB、bHLH、MADS-box和ERF）的表达显著下调。一系列注释为木聚糖内糖基转移酶/水解酶、果胶甲酯酶和果胶甲酯酶抑制剂的细胞壁基因也在这组DEGs中被识别。此外，通过免疫荧光实验，胚乳和胚胎细胞壁的特征显示，naa15中的纤维素荧光信号显著降低，而果胶低甲基化和高甲基化信号的降低也明显。总之，我们鉴定了大量DEGs，并研究了胚乳细胞化和胚胎分化过程中细胞壁的特征，这为揭示其调控机制提供了重要的转录和基因表达信息。