A novel role of BPCs in the control of medial domain differentiation during gynoecium development in Arabidopsis thaliana

The gynoecium, a highly specialized structure in flowering plants, ensures their high reproductive success through the control of different crucial steps spanning from ovule protection to fertilization and seed maturation and dispersion. Multiple bpc mutants show reduced vigor, small fruit size and height, a reduced number of seeds and problems in septum fusion and formation. BPCs are known to be involved in the regulation of key factors involved in plant development, and they are thought to function both as activators and repressors of target gene expression. Here we showed that gynoecium development is affected in different multiple mutants of the Basic PentaCysteine (BPC) genes, where the septum fails to develop properly, and that BPCs of class I and II regulate the expression of different genes involved in carpel development and phytohormonal pathways regulation. Considering the fundamental role of the gynoecium, which affects the reproductive success of the plants, we focused on understanding which genes could be putative direct targets of BPCs and thus involved in gynoecium development. We demonstrated that SPATULA and NO TRANSMITTING TRACT (NTT), which play pivotal roles in carpel and transmitting tract development, are downregulated. As a consequence, bpc multiple mutants fail to properly develop the septum and the transmitting tract. Interestingly, among the downregulated genes, we also found PIN-LIKES3, whose promoter can be directly bound by BPCs, which is an auxin efflux carrier that regulates and controls cytoplasmatic availability of auxin and could contribute also to various growth processes. Overall design: RNAseq: Total RNA was extracted from 3 biological replicates from both wild type and bpcV mutant inflorescences apexes with flowers up to stage 12

雌蕊群（gynoecium）是开花植物中高度特化的结构，通过调控从胚珠保护、受精到种子成熟与散播的一系列关键步骤，保障植物的高繁殖成功率。多个bpc突变体表现出活力下降、果实与植株矮小、种子数量减少，以及隔膜融合与形成异常。已知BPC参与调控植物发育的关键因子，且被认为可同时作为靶基因表达的激活剂与阻遏物发挥功能。本研究发现，碱性五半胱氨酸蛋白（Basic PentaCysteine, BPC）基因的不同多重突变体中，雌蕊群发育受到影响，隔膜无法正常形成；且I类与II类BPC可调控参与心皮发育及植物激素通路调控的不同基因的表达。鉴于雌蕊群对植物繁殖成功率的核心作用，本研究聚焦于解析哪些基因可作为BPC的潜在直接靶标，进而参与雌蕊群发育过程。本研究证实，在心皮与引导组织发育中发挥关键作用的SPATULA及无引导组织基因（NO TRANSMITTING TRACT, NTT）表达被下调。因此，bpc多重突变体无法正常形成隔膜与引导组织。值得注意的是，在下调基因中，本研究还发现了PIN样蛋白3（PIN-LIKES3），其启动子可被BPC直接结合；该基因编码生长素输出载体，可调控细胞质内生长素的可用性，还可能参与多种生长发育过程。实验整体设计：RNA测序（RNAseq）：分别从野生型与bpcV突变体的花序顶端（包含发育至第12期及更早的花朵）的3个生物学重复样本中提取总RNA。