Unlocking fertility in the female gametophyte: a DEAD-box RNA helicase is essential for embryo sac development and seed setting

This article comments on: Huang J, Qiao Z, Yu H, Lu Z, Chen W, Lu J, Wu J, Bao Y, Shahid MQ, Liu X. 2024. OsRH52A, a DEAD-box protein, regulates functional megaspore specification and is required for embryo sac development in rice. Journal of Experimental Botany 75, 4802–4821. https://doi.org/10.1093/jxb/erae180

A flowering plant has two phases in its life cycle, diploid sporophytic generation and haploid gametophytic generation.Generally, both male and female gametophytes are present in the same plant and are essential for sexual reproduction.A female gametophyte is required to guide the pollen tube to the ovary for fertilization and induction of seed development (Yadegari and Drews, 2004).Development of the female gametophyte is a highly organized process that occurs within an ovule and has been well reviewed in terms of several aspects, such as zygote activation (Xie et al., 2022), zygote embryogenesis (Dresselhaus and Jürgens, 2021), and embryo-endosperm interaction (Doll and Ingram, 2022).However, how defects of the female gametophyte lead to sterility and low yield remained elusive.
Embryo sac formation begins with megasporogenesis, where a megaspore mother cell (a diploid cell) undergoes meiosis, resulting in four haploid megaspores (Fig. 1).Typically, three of these megaspores degenerate, and the remaining one undergoes three rounds of mitotic division, leading to an eight-nucleate structure called the mature embryo sac.A mature embryo sac consists of three antipodal cells at the chalazal end (opposite the egg cell), two synergids and an egg cell near the micropylar end (closest to the opening for pollen tube entry), and two polar nuclei in the central cell.The fusion of the central cell (2n) with a sperm cell (n) during double fertilization forms the triploid endosperm, which provides nourishment for the developing embryo.The formation of an egg apparatus, comprising an egg cell and two synergids, is essential for fertilization.The antipodal cells, whose function is not entirely clear, are believed to play a role in nutrient transfer or hormone production.This intricate process ensures successful sexual reproduction in angiosperms by preparing the female gametophyte for fusion of the egg cell with the male gamete (sperm cell).
Male and female gametes are prone to degeneration, also known as abortion, due to several factors, for example programmed cell death (PCD), excessive reactive oxygen species (ROS), lack of proper nutrition, abnormal mitosis and meiosis, and extreme environmental conditions (Yadegari and Drews, 2004;Ali et al., 2019;Sankaranarayanan et al., 2020).Similarly, cytological aberrations of chromosomes, cytoskeleton organization, spindle assembly, and an abnormal number of sporocytes result in the failure of normal gamete development (Zhao et al., 2008;Hafidh and Honys, 2021;Shi et al., 2022;Fábián et al., 2024).In a recent study, EMBRYO SAC DEVELOPMENT 1 (ESD1) was found to encode a protein of an ovate family that regulates seed setting by controlling the development of functional megaspores (Wang et al., 2021).Similarly, DEFECTIVE EMBRYO SAC1 (DES1) that encodes a nuclear envelope membrane protein is required for embryo sac development, and its absence leads to low seed setting (Hu et al., 2023).However, detailed physiological and mechanistic insights into fertility are as yet unavailable.Seed setting is susceptible to many physiological and environmental conditions, and we outline a potential mechanism in Box 1.
OsRH52A encodes a DEAD-box helicase and is essential for maintaining embryo sac fertility RNA helicases of the DEAD-box family are highly conserved enzymes and are reported to perform diverse biological functions including suppression of PCD by regulating the expression of downstream genes (Linder and Jankowsky, 2011).In a previous study, Yu et al. (2020) discovered that a mutation in OsRH52A is responsible for low seed setting in neo-tetraploid rice; however, its biological function and the molecular mechanism were not explored.The expression spectrum showed that OsRH52A is preferentially expressed in anther and ovary development.Huang et al. (2024) used CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] to target two sites in the genome to knock out OsRH52A and developed two mutants, rh52a-m1 and rh52a-m2, in which nucleotide insertions or deletions led to a premature stop codon and truncated proteins lacking the DEXDc and HELICs domains which show significant reductions of up to ~35% and ~29%, respectively, in seed yield.Phenotypic analysis revealed that there were no apparent defects in the morphology of floral organs.
To investigate the reason for low seed setting, whole-mount eosin B-staining confocal laser scanning microscopy (WE-CLSM) was performed to search for defects associated with embryo sac fertility.In a recent study, Hu et al. (2023) also found that a mutation in DES1, which interacts with LONELY GUY (LOG), caused defective development of pistils provided evidence that low seed setting in rh52 mutants is caused by a degenerated embryo sac.Indeed, cytological observations revealed that in contrast to the wild type (WT) with only 5% abnormal embryo sacs, rh52a-m1 and rh52a-m2 had 35% and 41% abnormal embryo sacs, respectively (Huang et al., 2024).Further analyses revealed that four different types of embryo sacs, namely the double female gametophyte (12%), degenerated embryo sacs (21%), embryo sacs without the female germline (6%), and embryo sacs without an egg and synergids (4%), were present in rh52a-m1 and rh52a-m2.The results of Huang et al. (2024) revealed the following (but not limited to) types of aberrations in embryo sacs of rh52a.
(i) Double functional megaspores: under normal (WT) conditions, usually one megasporocyte divides into two cells and then into four cells following mitosis.Among these four cells, three of them undergo degeneration and only one survives, forming only one functional megaspore (Fig. 1).However, in rh52a, two cells were present, forming a double functional megaspore.(ii) Double mono-nucleate embryo sacs: in the WT, one cell survives near the chalazal end; however, in rh52a, two cells survived, one near the chalazal end and the other near the micropylar end, and each of them developed into mononucleate embryo sacs forming double mono-nucleate embryo sacs.(iii) Double bi-, tetra-, and eight-nucleate embryo sacs: in WT, the functional megaspore near the chalazal end is mono-nucleate.However, in rh52a, double mononucleate embryo sacs undergo one, two, or three mitotic cell divisions, forming bi-, tetra-, and octa-nucleate embryo sacs, respectively.
Box 1. Physiological mechanism of low seed setting caused by degeneration of male and female gametophytes Normal development of male and female gametophytes is essential for successful fertilization and ultimately seed setting.Many physiological and molecular factors have been reported to cause the degeneration or defects in male and female germlines that produce a significant reduction in seed yield (Fig. 2; Zhou et al., 2017;Ali et al., 2019).Previous studies reported that anthers and spikelets are prone to abortion due to increased ROS and abnormal PCD (Ali et al., 2022;Zhao et al., 2023), which lead to DNA fragmentation in reproductive tissues.Moreover, defects in tapetum cell death and cell division may also produce less viable pollen and cause a decrease in seed setting rate (Yan et al., 2020).Similarly, other defects, such as lack of wax and cuticle formation, and defects in the morphology of male gametes, also cause excessive loss of water from the surface of anthers, which results in sterility and reduction in seed yield (Jung et al., 2006).Chromosomal aberrations, complications associated with pollen tube growth, cell division, abnormal functional megaspore, and defective embryo development also result in a reduction in seed setting due to degeneration of the embryo sac (Hafidh and Honys, 2021;Fábián et al., 2024).As a result of these cytological aberrations, the fertilized embryo two sacs (one near the chalazal end and one near the micropylar end).Among them, the one near the chalazal end could not undergo cellularization on 1 day after fertilization (DAF) and 3 DAF.Mutants defective in female gametophyte development are helpful tools for studying the complexity of sexual reproduction, as it is affected by many factors, stages, and the position of the egg cell (Yadegari and Drews, 2004;Sun et al., 2021).Cytological aberrations observed in rh52a can be used to gain further insights into how exactly the degenerated embryo sac fails fertilization, and whether the activation, recognition, and delivery of the sperm cell were normal in rh52a.One possible reason for low fertility can be attributed to the fact that rh52a has an abnormal egg and synergids, which are essential for guiding the pollen tube.Moreover, it remains to be shown whether there are any maternal effects associated with its functional role in reproductive development and whether its overexpression can increase the seed setting.Additional studies could aim to obtain more insights into the practical implications of hybrid rice development.
Interplay of OsRH52A with fertilityassociated genes OsMFS1 and ZIP4 Genetic analysis confirmed that OsRH52A is essential for functional megaspore and embryo sac development.Yeast two-hybrid (Y2H) screening was conducted to find potential interactions with other proteins involved in reproductive development.Y2H and bimolecular fluorescence complementation results revealed that OsRH52A interacts with MALE AND FEMALE STERILITY 1 (OsMFS1) and ZMM protein ZIP4.
OsMFS1 encodes a coiled-coil domain-containing protein, and dysregulation of its protein causes complete sterility in male and female organs due to abnormal meiotic recombination (Lu et al., 2020).ZIP4 also plays a role in crossover formation, and its knockdown may reduce the frequency of chiasma formation that leads to sterility (Shen et al., 2012).The study of Huang et al. (2024) not only highlights the molecular pathway of embryo sac development but also advocates for more detailed investigations of meiotic recombination and synopsis for a better understanding of the ZIP4-OsRH52A-OsMFS1 complex.

OsRH52A affects the expression of downstream genes involved in megaspore development
To obtain a deeper understanding of downstream genes involved in megaspore development, a comparative transcriptome analysis of the WT and rh52a was carried out at meiosis and functional megaspore development stages.A total of 594 and 787 differentially expressed genes (DEGs) were up-and down-regulated at each developmental stage.Gene Ontology (GO) enrichment analysis indicated that the most enriched GO term was 'microtubule-related processes', which suggests that down-regulated DEGs were mainly responsible for defective embryo sac development.Notably, among the downregulated DEGs, MULTIPLE SPOROCYTE1 (OsMSP1) encodes a LEUCINE-RICH PROTEIN KINASE that controls the number of sporocytes in ovule development (Zhao et al., 2008).Similarly, HYBRID STERILITY-A1 (HSA1) encodes a protein of DOMAIN OF UNKNOWN FUNCTION (DUF1618) and controls the sterility of both F 1 and F 2 hybrids (Kubo et al., 2016).Further studies on single and double mutants of rh52a with hsa1 and msp1 will help to understand why there is a sudden loss in the seed setting in F 2 compared with F 1 , which remains open to study.In addition, it is thus far not known whether OsRH52A has the potential to be utilized for induction of synthetic apospory (development of an embryo from the nucellus without meiosis), which is considered an alternative mechanism to avoid heavy losses in F 2 during hybrid seed production.

Fig. 1 .
Fig. 1.Male and female gametophyte development in flowering plants (angiosperm); see text for details.

Fig. 2 .
Fig. 2. Proposed mechanism of low seed setting caused by degeneration of male and female gametophytes.ROS, reactive oxygen species; PCD, programmed cell death.See Box 1 for details.