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Double fertilization is an innovative phenomenon in angiosperms, in which one sperm cell first fuses with the egg cell to produce the embryo, and then the other sperm fuses with the central cell to produce the endosperm. However, the molecular mechanism of the preferential fertilization of egg cells is poorly understood. In this study, we report that two egg cell-secreted aspartic proteases, ECS1 and ECS2, play an important role in promoting preferential fertilization of egg cells in Arabidopsis. We show that simultaneous loss of ECS1 and ECS2 function resulted in an approximately 20% reduction in fertility, which can be complemented by the full-length ECS1/2 but not by corresponding active site mutants or by secretion-defective versions of ECS1/2. Detailed phenotypic analysis revealed that the egg cell-sperm cell attachment was compromised in ecs1 ecs2 siliques. Limited pollination assays with cyclin-dependent kinase a1 (cdka;1) pollen showed that preferential egg cell fertilization was impaired in the ecs1 ecs2 mutant. Taken together, these results demonstrate that egg cells secret two aspartic proteases, ECS1 and ECS2, to facilitate the attachment of sperm cells to egg cells so that preferential fertilization of egg cells is achieved. This study reveals the molecular mechanism of preferential fertilization in Arabidopsis thaliana.

Timing a switch in tissue integrity In plants, sperm cells travel through the pollen tube as it grows toward the ovule. Successful fertilization depends on the pollen tube rupturing to release the sperm cells (see the Perspective by Stegmann and Zipfel). Ge et al. and Mecchia et al. elucidated the intercellular cross-talk that maintains pollen tube integrity during growth but destroys it at just the right moment. The signaling peptides RALF4 and RALF19, derived from the pollen tube, maintain its integrity as it grows. Once in reach of the ovule, a related signaling peptide, RALF34, which derives from female tissues, takes over and causes rupture of the pollen tube. Science , this issue p. 1596 , p. 1600 ; see also p. 1544

In angiosperms, two sperm cells are transported and delivered by the pollen tube to the ovule to achieve double fertilization. Extensive communication takes place between the pollen tube and the female tissues until the sperm cell cargo is ultimately released. During this process, a pollen tube surface-located receptor complex composed of ANXUR1/2 (ANX1/2) and Buddha's Paper Seal 1/2 (BUPS1/2) was reported to control the maintenance of pollen tube integrity by perceiving the autocrine peptide ligands rapid alkalinization factor 4 and 19 (RALF4/19). It was further hypothesized that pollen-tube rupture to release sperm is caused by the paracrine RALF34 peptide from the ovule interfering with this signaling pathway. In this study, we identified two Arabidopsis pollen-tube-expressed glycosylphosphatidylinositol-anchored proteins (GPI-APs), LORELEI-like-GPI-anchored protein 2 (LLG2) and LLG3, as co-receptors in the BUPS-ANX receptor complex. llg2 llg3 double mutants exhibit severe fertility defects. Mutant pollen tubes rupture early during the pollination process. Furthermore, LLG2 and LLG3 interact with ectodomains of both BUPSs and ANXURs, and this interaction is remarkably enhanced by the presence of RALF4/19 peptides. We further demonstrate that the N terminus (including a YISY motif) of the RALF4 peptide ligand interacts strongly with BUPS-ANX receptors but weakly with LLGs and is essential for its biological function, and its C-terminal region is sufficient for LLG binding. In conclusion, we propose that LLG2/3 serve as co-receptors during BUPS/ANX-RALF signaling and thereby further establish the importance of GPI-APs as key regulators in plant reproduction processes.

Racing to fertilization Pollen tubes, which carry plant sperm, need to grow from where they land in the flower to where the ovule is. Zhong et al. now show how pollen from related plant species race to reach the ovule first. One set of fast-evolving peptide signals is tuned to speed up growth of conspecific pollen tubes. A related set of evolutionarily ancient peptides is tuned to attract all pollen tubes. Thus, fertilization is more likely to happen through conspecific pollen tubes, but a fail-safe system encourages even the laggards to get where they need to go. Science , this issue p. eaau9564

INTRODUCTION: Reproductive isolation is the inability of a species to breed with a related species and thus is key to the formation and evolution of a new plant species. Within the genus Arabidopsis, pistils of A. thaliana can be fertilized by pollen of related species, such as A. lyrata, but conspecific (self) pollen is preferred to maintain reproductive isolation. The molecular mechanisms by which the plant’s own pollen tubes outperform heterospecific (alien) pollen tubes is largely unknown. In A. thaliana, maternal cysteine-rich peptides AtLURE1.1 to −1.5, secreted from the ovule, were reported to function through the male receptor PRK6 to mediate pollen tube attraction. RATIONALE: Although AtLURE1s have been identified as species-specific pollen tube attractants in A. thaliana, down-regulation of AtLURE1 genes and knockout of their receptor PRK6 did not disturb fertilization and seed set, indicating that the biological functions of AtLURE1s are not fully understood. We initially aimed to fully knock out genes for AtLURE1.1 to −1.5, as well as additional related AtLURE1 genes, to understand their contribution to fertilization success and reproductive isolation. RESULTS: In addition to the five reported AtLURE1 genes, we identified two further A. thaliana–specific AtLURE1 genes and generated a loss-of-function atlure1 null septuple mutant by knocking out the whole gene family. Although atlure1 null mutants, resembling prk6 receptor mutants, exhibited normal fertility, pollen tubes in atlure1 null pistils, like prk6 pollen tubes, displayed delayed emergence at the septum. These data collectively suggest that AtLURE1-PRK6 signaling accelerates pollen tubes to penetrate the septum and to grow toward ovules. We further tested the function of AtLURE1-PRK6 signaling in reproductive isolation by depositing alien A. lyrata pollen onto A. thaliana pistils and showed that A. lyrata pollen tubes emerged much more slowly out of the septum. However, the emergence of A. lyrata pollen tubes in A. thaliana pistils was not further impaired in atlure1 null pistils, suggesting that AtLURE1s promote reproductive isolation by accelerating their own pollen tubes, which thus outperform alien tubes. This hypothesis requires the existence of more general, genus- and/or family-specific ovular guidance molecules. Therefore, we next investigated the functions of four AtLURE1-related Brassicaceae-conserved cysteine-rich peptides named XIUQIU1 to −4. We found that XIUQIU peptides attract pollen tubes in a non–species-specific manner and independently of the PRK6 receptor. This finding suggests that evolutionarily ancient XIUQIUs function as general pollen tube attractants in the Brassicaceae. Finally, after combining atlure1 null with xiuqiu loss-of-function mutations, fertility in A. thaliana was reduced, supporting the biological importance of these cysteine-rich peptides in plant reproduction. CONCLUSION: In A. thaliana, species-specific female AtLURE1 peptides and their male receptor PRK6 promote and maintain reproductive isolation by accelerating conspecific pollen tube growth to penetrate the septum. The AtLURE1-related cysteine-rich XIUQIU peptides are evolutionarily ancient and conserved attractants in the Brassicaceae and attract pollen tubes in a non–species-specific manner. XIUQIUs contribute to reproductive success and heterospecific fertilization. Their activity may lead to new genome combinations and thus to the formation of new species. Reproductive isolation is a prerequisite for speciation. Failure of communication between female tissues of the pistil and paternal pollen tubes imposes hybridization barriers in flowering plants. Arabidopsis thaliana LURE1 (AtLURE1) peptides and their male receptor PRK6 aid attraction of the growing pollen tube to the ovule. Here, we report that the knockout of the entire AtLURE1 gene family did not affect fertility, indicating that AtLURE1-PRK6–mediated signaling is not required for successful fertilization within one Arabidopsis species. AtLURE1s instead function as pollen tube emergence accelerators that favor conspecific pollen over pollen from other species and thus promote reproductive isolation. We also identified maternal peptides XIUQIU1 to −4, which attract pollen tubes regardless of species. Cooperation between ovule attraction and pollen tube growth acceleration favors conspecific fertilization and promotes reproductive isolation.

Plant shoot branching is pivotal for developmental plasticity and crop yield. The formation of branch meristems is regulated by several key transcription factors including REGULATOR OF AXILLARY MERISTEMS1 (RAX1), RAX2, and RAX3. However, the regulatory network of shoot branching is still largely unknown. Here, we report the identification of EXCESSIVE BRANCHES1 (EXB1), which affects axillary meristem (AM) initiation and bud activity. Overexpression of EXB1 in the gain-of-function mutant exb1-D leads to severe bushy and dwarf phenotypes, which result from excessive AM initiation and elevated bud activities. EXB1 encodes the WRKY transcription factor WRKY71, which has demonstrated transactivation activities. Disruption of WRKY71/EXB1 by chimeric repressor silencing technology leads to fewer branches, indicating that EXB1 plays important roles in the control of shoot branching. We demonstrate that EXB1 controls AM initiation by positively regulating the transcription of RAX1, RAX2, and RAX3. Disruption of the RAX genes partially rescues the branching phenotype caused by EXB1 overexpression. We further show that EXB1 also regulates auxin homeostasis in control of shoot branching. Our data demonstrate that EXB1 plays pivotal roles in shoot branching by regulating both transcription of RAX genes and auxin pathways.

Sperm cells of seed plants have lost their motility and are transported by the vegetative pollen tube cell for fertilization, but the extent to which they regulate their own transportation is a long-standing debate. Here we show that Arabidopsis lacking two bHLH transcription factors produces pollen without sperm cells. This abnormal pollen mostly behaves like the wild type and demonstrates that sperm cells are dispensable for normal pollen tube development.

Summary Successful sexual reproduction in animals and plants requires communication between male and female gametes. In flowering plants, unlike in animals, eggs and sperm cells are enclosed in multicellular embryo sacs and pollen grains, respectively [1]; guided growth of the pollen tube into the ovule is necessary for fertilization [2]. Pollen tube guidance requires accurate perception of ovule-emitted guidance cues by the receptors in pollen tubes [2–4]. Although several ovule-secreted peptides controlling pollen tube guidance have recently been identified, i.e., maize EGG APPARATUS1 (EA1) [5], Torenia LURE1/LURE2 [6], and Arabidopsis CRP810_1/AtLURE1 [7], little is known about the receptors. Here, we identified two receptor-like kinase (RLK) genes preferentially expressed in Arabidopsis pollen tubes, Lost In Pollen tube guidance 1 ( LIP1 ) and 2 ( LIP2 ), which are involved in guidance control of pollen tubes. LIP1 and LIP2 were anchored to the membrane in the pollen tube tip region via palmitoylation, which was essential for their guidance control. Simultaneous inactivation of LIP1 and LIP2 led to impaired pollen tube guidance into micropyle and significantly reduced attraction of pollen tubes toward AtLURE1 [7]. Our results suggest that LIP1 and LIP2 represent essential components of the pollen tube receptor complex to perceive the female signal AtLURE1 for micropylar pollen tube guidance.

The cuticle is a hydrophobic lipid layer covering the epidermal cells of terrestrial plants. Although many genes involved in Arabidopsis cuticle development have been identified, the transcriptional regulation of these genes is largely unknown. Previously, we demonstrated that AtCFL1 negatively regulates cuticle development by interacting with the HD-ZIP IV transcription factor HDG1. Here, we report that two bHLH transcription factors, AtCFL1 associated protein 1 (CFLAP1) and CFLAP2, are also involved in AtCFL1-mediated regulation of cuticle development. CFLAP1 and CFLAP2 interact with AtCFL1 both in vitro and in vivo. Overexpression of either CFLAP1 or CFLAP2 led to expressional changes of genes involved in fatty acids, cutin and wax biosynthesis pathways and caused multiple cuticle defective phenotypes such as organ fusion, breakage of the cuticle layer and decreased epicuticular wax crystal loading. Functional inactivation of CFLAP1 and CFLAP2 by chimeric repression technology caused opposite phenotypes to the CFLAP1 overexpressor plants. Interestingly, we find that, similar to the transcription factor HDG1, the function of CFLAP1 in cuticle development is dependent on the presence of AtCFL1. Furthermore, both HDG1 and CFLAP1/2 interact with the same C-terminal C4 zinc finger domain of AtCFL1, a domain that is essential for AtCFL1 function. These results suggest that AtCFL1 may serve as a master regulator in the transcriptional regulation of cuticle development, and that CFLAP1 and CFLAP2 are involved in the AtCFL1-mediated regulation pathway, probably through competing with HDG1 to bind to AtCFL1., Author Summary The cuticle is a continuous lipid layer covering the aerial parts of land plants. It is very important for the plants, especially for those in the drought area. The biosynthesis of cuticle have been studied well in past decades, however, the transcriptional regulation is still largely unknown. Here we found two new bHLH transcription factors, AtCFL1 associated protein 1 (CFLAP1) and its homolog CFLAP2, which could interact with AtCFL1, a previously identified negative regulator of Arabidopsis cuticle formation. Overexpression of CFLAP1 and CFLAP2 caused cuticle developmental defects, which are similar to the phenotypes of AtCFL1 overexpression plants. Functional inactivation of CFLAP1 in Arabidopsis presents opposite phenotypes to those of its overexpressor. Interestingly, the function of CFLAP1 is dependent on the presence of AtCFL1. These results suggest that CFLAP1 and CFLAP2 regulate cuticle development by interacting with AtCFL1, and that AtCFL1 may work as a master regulator in the transcriptional regulation network.

During the angiosperm (flowering-plant) life cycle, double fertilization represents the hallmark between diploid and haploid generations [1]. The success of double fertilization largely depends on compatible communication between the male gametophyte (pollen tube) and the maternal tissues of the flower, culminating in precise pollen tube guidance to the female gametophyte (embryo sac) and its rupture to release sperm cells. Several important factors involved in the pollen tube reception have been identified recently [2-6], but the underlying signaling pathways are far from being understood. Here, we report that a group of female-specific small proteins, early nodulin-like proteins (ENODLs, or ENs), are required for pollen tube reception. ENs are featured with a plastocyanin-like (PCNL) domain, an arabinogalactan (AG) glycomodule, and a predicted glycosylphosphatidylinositol (GPI) anchor motif. We show that ENs are asymmetrically distributed at the plasma membrane of the synergid cells and accumulate at the filiform apparatus, where arriving pollen tubes communicate with the embryo sac. EN14 strongly and specifically interacts with the extracellular domain of the receptor-like kinase FERONIA, localized at the synergid cell surface and known to critically control pollen tube reception [6]. Wild-type pollen tubes failed to arrest growth and to rupture after entering the ovules of quintuple loss-of-function EN mutants, indicating a central role of ENs in male-female communication and pollen tube reception. Moreover, overexpression of EN15 by the endogenous promoter caused disturbed pollen tube guidance and reduced fertility. These data suggest that female-derived GPI-anchored ENODLs play an essential role in male-female communication and fertilization.

Imprinted genes display biased expression of paternal and maternal alleles and are only found in mammals and flowering plants. Compared to several hundred imprinted genes that are functionally characterized in mammals, very few imprinted genes were confirmed in plants and even fewer of them have been functionally investigated. Here, we report a new imprinted gene, NUWA, in plants. NUWA is an essential gene, because loss of its function resulted in reduced transmission through the female gametophyte and defective cell/nuclear proliferation in early Arabidopsis embryo and endosperm. NUWA is a maternally expressed imprinted gene, as only the maternal allele of NUWA is transcribed and translated from gametogenesis to the 16-cell globular embryo stage after fertilization, and the de novo transcription of the maternal allele of NUWA starts from the zygote stage. Different from other identified plant imprinted genes whose encoded proteins are mostly localized to the nucleus, the NUWA protein was localized to the mitochondria and was essential for mitochondria function. Our work uncovers a novel imprinted gene of a previously unidentified type, namely, a maternal-specific expressed nuclear gene with its encoded protein localizing to and controlling the function of the maternally inherited mitochondria. This reveals a unique mechanism of maternal control of the mitochondria and adds an extra layer of complexity to the regulation of nucleus-organelle coordination during early plant development., Author Summary Imprinted genes are only found in mammals and flowering plants, and they express with parent-of-origin-specific patterns. Unlike in mammals, only a few imprinted genes was identified and functionally characterized in plants, and almost all of them encode nuclear proteins. Here, we identified NUWA as a new type of imprinted gene in Arabidopsis. NUWA has maternal-specific expression and controls the early seed development in Arabidopsis, with its encoded protein localizing to and functioning in the maternally inherited mitochondria. The discovery of NUWA reveals a unique mechanism of maternal control of mitochondrial function and adds an extra layer of complexity to nucleus-organelle coordination. It further indicates that, similar to mammals, imprinted genes in plants are also involved in various subcellular biological processes.