Transcriptome analysis of embryonic domains in Norway spruce reveals potential regulators of suspensor cell death

The terminal differentiation and elimination of the embryo-suspensor is the earliest manifestation of programmed cell death (PCD) during plant ontogenesis. Molecular regulation of suspensor PCD remains poorly understood. Norway spruce (Picea abies) embryos provide a powerful model for studying embryo development because of their large size, sequenced genome, and the possibility to obtain a large number of embryos at a specific developmental stage through somatic embryogenesis. Here, we have carried out global gene expression analysis of the Norway spruce embryo-suspensor versus embryonal mass (a gymnosperm analogue of embryo proper) using RNA sequencing. We have identified that suspensors have enhanced expression of the NAC domain-containing transcription factors, XND1 and ANAC075, previously shown to be involved in the initiation of developmental PCD in Arabidiopsis. The analysis has also revealed enhanced expression of Norway spruce homologues of the known executioners of both developmental and stress-induced cell deaths, such as metacaspase 9 (MC9), cysteine endopeptidase-1 (CEP1) and ribonuclease 3 (RNS3). Interestingly, a spruce homologue of bax inhibitor-1 (PaBI-1, for Picea abies BI-1), an evolutionarily conserved cell death suppressor, was likewise up-regulated in the embryo-suspensor. Since Arabidopsis BI-1 so far has been implicated only in the endoplasmic reticulum (ER)-stress induced cell death, we investigated its role in embryogenesis and suspensor PCD using RNA interference (RNAi). We have found that PaBI-1-deficient lines formed a large number of abnormal embryos with suppressed suspensor elongation and disturbed polarity. Cytochemical staining of suspensor cells has revealed that PaBI-1 deficiency suppresses vacuolar cell death and induces necrotic type of cell death previously shown to compromise embryo development. This study demonstrates that a large number of cell-death components are conserved between angiosperms and gymnosperms and establishes a new role for BI-1 in the progression of vacuolar cell death.胚柄的终端分化与消亡是植物个体发育过程中程序性细胞死亡（programmed cell death, PCD）最早的表现形式。目前对于胚柄程序性细胞死亡的分子调控机制仍知之甚少。挪威云杉（Picea abies）的胚胎因其体积庞大、基因组已完成测序，且可通过体细胞胚胎发生技术获取大量特定发育阶段的胚胎，成为研究胚胎发育的理想模型。本研究利用RNA测序（RNA sequencing, RNA-seq）技术，对挪威云杉的胚柄与胚性团块（裸子植物中对应被子植物胚本体的结构）进行了全局基因表达分析。研究鉴定发现，胚柄中含NAC结构域的转录因子XND1与ANAC075的表达显著上调，此前已有研究表明这两个因子参与拟南芥发育性程序性细胞死亡的启动过程。本分析还揭示，挪威云杉的多个同源基因呈现表达上调，这些基因是已知的发育性与胁迫诱导性细胞死亡效应因子，例如半胱氨酸蛋白酶9（metacaspase 9, MC9）、半胱氨酸内肽酶1（cysteine endopeptidase 1, CEP1）以及核糖核酸酶3（ribonuclease 3, RNS3）。有趣的是，bax抑制剂-1（bax inhibitor-1, BI-1）的云杉同源基因PaBI-1（即挪威云杉BI-1）作为一类进化保守的细胞死亡抑制因子，在胚柄中同样呈现表达上调。此前拟南芥中的BI-1仅被证实与内质网（endoplasmic reticulum, ER）胁迫诱导的细胞死亡相关，本研究通过RNA干扰（RNA interference, RNAi）技术探究了其在胚胎发生与胚柄程序性细胞死亡中的功能。研究发现，PaBI-1缺陷型株系会产生大量异常胚胎，表现为胚柄伸长受抑制且极性发育紊乱。对胚柄细胞的细胞化学染色结果显示，PaBI-1的缺失会抑制液泡型细胞死亡，并诱导此前已证实会损害胚胎发育的坏死型细胞死亡。本研究证实，被子植物与裸子植物之间存在大量保守的细胞死亡相关组分，并首次确立了BI-1在液泡型细胞死亡进程中的新功能。