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.

胚胎和胚乳发育是拟南芥种子发育过程中的关键生物学事件，其受多种基因表达动态变化所调控。尽管如此，胚乳细胞化和胚胎分化调控机制尚不明确。在本研究中，我们针对naa15突变体进行了早期胚胎和胚乳发育的表征，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整的胚乳细胞化。我们利用RNA测序和qPCR检测方法，比较研究了野生型拟南芥种子在授粉后3、4、5天（3W、4W、5W）以及白化纯合胚胎缺失的naa15种子在授粉后5、6、7天（5M、6M、7M）的基因表达变化。转录组分析显示，在4W阶段（胚乳细胞化启动阶段和心形胚胎阶段）与3W阶段（原生质阶段和球形胚胎阶段）相比，存在2040个差异表达基因（DEGs），在5W阶段（胚乳细胞化结束阶段和梭形胚胎阶段）与4W阶段相比，存在3630个DEGs。KEGG和GO分析表明，与naa15相比，野生型中脂质代谢过程、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的基因显著富集。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）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被识别为这些DEGs。此外，通过免疫荧光实验，我们发现naa15的胚胎和胚乳中的纤维素荧光信号显著降低，naa15胚乳中低甲基化和高甲基化果胶的信号也明显降低。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的细胞壁特征，这为揭示其调控机制提供了重要的转录和基因表达信息。