Arabidopsis PRC1 core component AtRING1 regulates stem cell-determining carpel development mainly through repression of class I KNOX genes

The development of both animals and plants relies on stem cells, which are defined by their ability to renew themselves and give rise to daughter cells that differentiate and contribute to tissue and organ formation. In higher plants, stem cells reside in meristems, and cell lineage is easily traceable due to the immobility of cells. The shoot apical meristem (SAM) initiates at the embryo stage, and continuously produces the aerial part of the plant during post-embryonic growth. Upon transition to the reproductive phase, SAM usually shifts to the fate of inflorescence meristem (IM) and subsequently generates floral meristems (FMs) from the IM flanks [1–4]. Distinct from the indeterminacy of SAM and IM, the determinate FM produces a fixed number of peripheral floral organs around a central population of stem cells that are consumed in the formation of carpels. The ovules emerge from the meristematic placenta within the carpel, undergo the production of the embryo sac (ES, the female gametophyte/megagametophyte), and upon double fertilization ultimately give rise to seeds [5, 6]. For female gametophyte development, firstly, a single and enlarged megasporocyte (also called megaspore mother cell, MMC) differentiates from the archesporial cell at the tip of the ovule primordium and undergoes meiosis to develop a tetrad of four haploid megaspores (developmental stage FG1). Normally the chalazal-proximal one survives and becomes the functional megaspore. This megaspore undergoes three rounds of mitotic division and cellularization to give rise to an eight-nucleate/seven-celled female gametophyte, which comprises three antipodal cells, two synergids, one central cell containing two unfused polar nuclei, and one egg cell (developmental stage FG5) [7].

The class I KNOX (KNOX-I) family gene SHOOT MERISTEMLESS (STM) and the feedback loop formed by CLAVATA (CLV) and WUSCHEL (WUS) have independent but complementary functions in shoot stem cell maintenance. For instance, STM prevents stem cell differentiation, while WUS specifies stem cell identity (reviewed in [8]). The knockdown mutants stm and wus display very similar flower phenotypes, such as the absence of carpels and a reduced number of other floral organs. In addition to STM, the other KNOX-I family genes KNAT1/BREVIPEDICELLUS (BP), KNAT2, and KNAT6 may also have a role in carpel development because overexpression of either STM or KNAT2 can induce ectopic carpel formation and ovule-to-carpel homeotic conversion within the gynoecium [9]. Very importantly, AGAMOUS (AG) plays a key role in the termination of floral stem cell maintenance. At flower developmental stage 3, WUS together with LEAFY (LFY) activate AG, which in turn shuts off WUS expression at stage 6, leading to the termination of stem cell maintenance and the initiation of carpel primordia [10–14]. Either ag, displaying spatially restricted but delayed WUS extinction, or clv, displaying an enlarged WUS expression domain, is sufficient to induce FM indeterminacy [13–18]. Thus, AG combined with the CLV–WUS feedback loop regulates carpel development, conveniently named the WUS–AG pathway. Recent studies demonstrate that some Polycomb group (PcG) proteins play an essential role within the WUS–AG pathway to terminate floral stem cell fate [19, 20].

PcG proteins constitute two major types of complexes: Polycomb repressive complex 2 (PRC2), which catalyzes histone H3 lysine 27 trimethylation (H3K27me3) on target chromatin, and PRC1, which acts as both the H3K27me3 reader and the histone H2A lysine 119 monoubiquitination (H2AK119ub1) writer. Arabidopsis PRC2 components are able to form at least three different complexes involved in somatic cell fate determinacy, vegetative development maintenance, vernalization, flower timing regulation, and seed development (reviewed in [21]). Arabidopsis PRC1 core components, including LIKE HETEROCHROMOTIN PROTEIN1 (LHP1), AtBMI1, and AtRING1, display different evolutionary conservation [22]. Though LHP1 can interact with AtRING1 and AtBMI1 in vitro [23], the mutant phenotype of lhp1 shows some degree of difference from that of the atring1a;atring1b or atbmi1a;atbmi1b double mutant. Furthermore, LHP1 was recently reported to co-purify with the PRC2 complex in vivo [24, 25], indicating that LHP1 is more closely associated with PRC2 in this specific context than PRC1. Arabidopsis PRC1 RING finger proteins AtRING1 and AtBMI1 act as the most conserved components involved in preventing seed germination and development of somatic embryo traits [23, 26, 27], maintaining stem cell identity [28], and promoting floral transition [29]. Intriguingly, atring1a;atring1b mutants display abnormal flower developmental phenotypes, yet the underlying mechanisms remain to be investigated.

In this study, we show that AtRING1a and AtRING1b play an essential role in Arabidopsis floral stem cell maintenance and carpel development, primarily via repression of the KNOX-I family genes. Both AtRING1a and AtRING1b genes display very similar expression patterns throughout the whole plant life cycle, except for the imprinting expression of AtRING1b, but not AtRING1a, in the endosperm. Indeterminate carpel growth in the atring1a;atring1b mutant is associated with homeotic replum-to-carpel and ovule-to-carpel conversions. Further molecular and genetic analyses demonstrate that AtRING1a/b modulate floral stem cell activity and carpel development, mainly through repression of the KNOX-I pathway. Lastly, our analyses indicate that defective ovule development in the atring1a;atring1b mutant is essentially due to survival of non-functional megaspores, growth arrest of integuments, and overproliferation of the nucellus.