Introduction

The Plant HomeoDomain (PHD) motif defines a family of proteins that can recognise and bind histones depending on covalent modification status of the histone tails (Mouriz et al., 2015). By recruitment and regulation of chromatin remodelling factors and transcriptional regulators, PHD proteins can thereby control chromatin compaction and gene expression in a histone modification-governed manner.

Phylogenetic analysis places angiosperm PHD proteins into five main clades and 16 subfamilies (Cao et al., 2018). Among these, clade IIa comprises members from both mono- and dicotyledonous species including the Arabidopsis genes MALE STERILITY 1 (AtMS1) and MALE MEIOCYTE DEATH 1 (AtMMD1). AtMS1 and AtMMD1 encode similar protein products that are both essential for pollen production in anthers (Wilson et al., 2001; Reddy et al., 2003; Yang et al., 2003). Still, the two genes exert their functions in distinct anther cell types. Therefore, AtMMD1 controls gene expression and chromosome condensation needed for completion of meiosis in microsporocytes (Reddy et al., 2003; Yang et al., 2003) while AtMS1 controls gene expression and function of tapetal cells surrounding and nursing the microsporocytes and microspores on their route towards functional pollen (Wilson et al., 2001; Ito & Shinozaki, 2002; Alves-Ferreira et al., 2007; Yang et al., 2007; Reimegård et al., 2017; Lu et al., 2020). Both genes exert their functions through modification of chromatin structure. Therefore, AtMS1 activates genes organised in clusters by the relaxation of chromatin condensation (Reimegård et al., 2017) and AtMMD1 can bind histone tails in a modification-dependent manner (Andreuzza et al., 2015; Wang et al., 2016). A detailed mode of action was recently proposed for AtMMD1 in meiotic cells where the protein is recruited to H3K4me3 marks allowing it to modulate target specificity of nearby JUMONJI 16 (JMJ16) histone demethylases through a physical interaction dependent on its central MMD domain (Wang et al., 2020).

The monophyletic group of land plants is characterised by a huge variation in reproductive solutions that is the result of successive key evolutionary innovations (Renzaglia et al., 2000; Hackenberg & Twell, 2019; Hisanaga et al., 2019; Rensing & Weijers, 2021; Sharma et al., 2021). The developmental process controlled by AtMS1 and AtMMD1, that is tapetum-assisted microspore and pollen formation facilitating downstream male gametogenesis, is for example a derived trait of angiosperms (Hackenberg & Twell, 2019). Gametogenesis in the common ancestors of all extant land plants is instead likely to have been reminiscent of that in bryophytes, ferns and lycophytes of today, where eggs and flagellated sperms are produced by female archegonia and male antheridia formed by a dominant haploid gametophyte generation (Renzaglia et al., 2000; Hackenberg & Twell, 2019). These gametophytic reproductive organs were eventually lost from the angiosperm lineage as part of a drastic reduction of the haploid gametophyte generation accompanied by increased complexity of the diploid sporophyte generation (Harrison, 2017). As part of this transition, pollen is hypothesised to have evolved from walled spores reminiscent of those in extant bryophytes thanks to two key evolutionary adaptations (Hackenberg & Twell, 2019). First, divisions in the male gametophyte generation were almost completely abolished to arrive at the situation in present-day angiosperms where only two sequential specialised divisions produce a pair of male gametes inside a vegetative cell from the primary meiotic product (the microspore). Second, breakage of the spore wall was deferred so that the cell divisions producing the two gametes could be completed within a still intact wall, today recognised as the angiosperm pollen wall. The proposed evolutionary origin of pollen indicates that tapetum-derived pollen production in angiosperms is related by descent to tapetum-dependent spore formation in bryophytes (Lopez-Obando et al., 2022).

The existence of PHD clade IIa homologues in gametophyte-dominant bryophytes (Higo et al., 2016; Sanchez-Vera et al., 2022), separated from angiosperms c. 450 million years ago (Ma) (Morris et al., 2018), suggests that clade IIa-related genes were present already in the common ancestors of all extant land plants. Recent transcriptome data from the bryophyte model moss Physcomitrium patens report the expression of two clade IIa homologues in sporophytes at stages during which tapetal-like cells are active and spores and their precursors develop (Perroud et al., 2018; Lopez-Obando et al., 2022). Moreover, the expression of the two genes was also detected in antheridia (male reproductive organs) and in the egg cell in archegonia (female reproductive organs), both produced by the haploid gametophytic generation (Meyberg et al., 2020; Sanchez-Vera et al., 2022 and references therein). Similarly, a putative clade IIa homologue of the model liverwort Marchantia polymorpha is also active in reproductive organs, at least in the antheridia (Higo et al., 2016). This points towards a function for bryophyte clade IIa genes during gametogenesis. Further dissection of this function may add to our understanding about the mechanisms, regulation and evolution of gametogenesis in land plants (Berger & Twell, 2011; Hisanaga et al., 2019).

Here we describe the functional characterisation of the clade IIa PHD homologues PpMS1A and PpMS1B in the moss P. patens. PpMS1A and PpMS1B are together required for male and female fertility by providing functions essential for the development of the gamete-producing inner cells of both antheridia and archegonia. The expression domains of the two genes furthermore suggest functions in sporogenous cells and in foot transfer cells of the diploid sporophyte generation. Based on these findings, we discuss a possible ancestral function for clade IIa PHD proteins and elaborate on how this may have evolved into the functions evident in present-day bryophytes and angiosperms.

中文翻译：

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苔藓中的 MS1/MMD1 同系物 Physcomitrium patens 是雄性和雌性配子发生所必需的

介绍

植物同源结构域 (PHD) 基序定义了一个蛋白质家族，可以根据组蛋白尾部的共价修饰状态识别和结合组蛋白（Mouriz等人，  2015 年）。通过招募和调节染色质重塑因子和转录调节因子，PHD 蛋白可以以组蛋白修饰控制的方式控制染色质压缩和基因表达。

系统发育分析将被子植物 PHD 蛋白分为五个主要进化枝和 16 个亚科 (Cao et al .,  2018 )。其中，进化枝 IIa 包含来自单子叶植物和双子叶植物物种的成员，包括拟南芥基因MALE STERILITY 1 ( AtMS1 ) 和MALE MEIOCYTE DEATH 1 ( AtMMD1 )。AtMS1和AtMMD1编码相似的蛋白质产物，它们都是花药产生花粉所必需的（Wilson等人，  2001 年；Reddy等人，  2003 年；Yang等人，  2003 年）). 尽管如此，这两个基因在不同的花药细胞类型中发挥其功能。因此，AtMMD1控制小孢子母细胞减数分裂完成所需的基因表达和染色体浓缩（Reddy等人，  2003 年；Yang等人，  2003 年），而AtMS1控制小孢子母细胞和小孢子周围的绒毡层细胞的基因表达和功能。功能性花粉的途径（Wilson等人，  2001 年；Ito 和 Shinozaki，  2002 年；Alves-Ferreira等人，  2007 年；Yang等人，  2007 年; Reimegård等人，  2017 年；卢等，  2020）。这两种基因都通过染色质结构的修饰发挥其功能。因此，AtMS1 通过放松染色质凝聚来激活成簇组织的基因 (Reimegård等人，  2017 年)，而 AtMMD1 可以以修饰依赖的方式结合组蛋白尾部 (Andreuzza等人，  2015 年；Wang等人，  2016 年)). 最近针对减数分裂细胞中的 AtMMD1 提出了一种详细的作用模式，其中蛋白质被募集到 H3K4me3 标记，允许它通过依赖于其中央 MMD 结构域的物理相互作用调节附近 JUMONJI 16 (JMJ16) 组蛋白去甲基化酶的靶标特异性（Wang等人.,  2020 年）。

陆生植物单系群的特点是繁殖解决方案存在巨大差异，这是连续关键进化创新的结果（Renzaglia等人，  2000 年；Hackenberg 和 Twell，  2019 年；Hisanaga等人，  2019年；Rensing和 Weijers，  2021 年） ；夏尔马等人，  2021 年）。由AtMS1和AtMMD1控制的发育过程，即绒毡层辅助的小孢子和花粉形成，促进下游雄性配子发生，例如是被子植物的衍生性状（Hackenberg & Twell，  2019). 所有现存陆地植物的共同祖先的配子发生很可能让人想起今天的苔藓植物、蕨类植物和石松植物，其中卵和带鞭毛的精子由雌性古卵细胞产生，雄性花药由显性单倍体配子体世代形成（Renzaglia等人，  2000 年；Hackenberg & Twell，  2019 年）。这些配子体生殖器官最终从被子植物谱系中消失，这是单倍体配子体生成急剧减少的一部分，伴随着二倍体孢子体生成的复杂性增加（Harrison，  2017 年）). 作为这一转变的一部分，由于两个关键的进化适应，花粉被假设是从壁孢子进化而来的，让人联想到现存苔藓植物中的那些孢子（Hackenberg & Twell，  2019). 首先，雄性配子体世代的分裂几乎完全被取消，以达到当今被子植物的情况，其中只有两个连续的专门分裂在营养细胞内从初级减数分裂产物（小孢子）产生一对雄性配子。其次，孢子壁的破裂被推迟，因此产生两个配子的细胞分裂可以在仍然完好无损的壁内完成，今天被认为是被子植物的花粉壁。拟议的花粉进化起源表明，被子植物中绒毡层衍生花粉的产生与苔藓植物中绒毡层依赖性孢子形成的下降有关（Lopez-Obando等人，  2022 年）。

PHD 进化枝 IIa 同源物在配子体显性苔藓植物中的存在（Higo等人，  2016 年；Sanchez-Vera等人，  2022 年），与被子植物c分离。4.5 亿年前 (Ma)（Morris等人，  2018 年）表明，与进化枝 IIa 相关的基因已经存在于所有现存陆地植物的共同祖先中。来自苔藓植物模型苔藓Physcomitrium patens的最新转录组数据报告了两个进化枝 IIa 同系物在孢子体中的表达，在此期间绒毡层样细胞活跃且孢子及其前体发育（Perroud等人，  2018 年；Lopez-Obando等人，  2022 年）。此外，还在 antheridia（雄性生殖器官）和 archegonia（雌性生殖器官）的卵细胞中检测到这两个基因的表达，两者均由单倍体配子体世代产生（Meyberg等人，  2020 年；Sanchez-Vera等人）等人，  2022 年及其中的参考资料）。同样，模型地钱 Marchantia polymorpha的假定进化枝 IIa 同系物在生殖器官中也有活性，至少在花药中是这样（Higo等人，  2016 年）). 这指向苔藓植物进化枝 IIa 基因在配子发生过程中的功能。进一步剖析这一功能可能会增加我们对陆生植物配子发生的机制、调控和进化的理解（Berger & Twell，  2011 年；Hisanaga等人，  2019 年）。

在这里，我们描述了苔藓P. patens中进化枝 IIa PHD 同系物PpMS1A和PpMS1B的功能特征。PpMS1A和PpMS1B是雄性和雌性生育力所必需的，它们提供了花药和早卵器的产生配子的内部细胞发育所必需的功能。这两个基因的表达域进一步表明在二倍体孢子体世代的造孢细胞和足部转移细胞中的功能。基于这些发现，我们讨论了进化枝 IIa PHD 蛋白的一个可能的祖先功能，并详细说明了它是如何演变成现代苔藓植物和被子植物中明显的功能的。