Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism.

In flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling, an inducer of the synergid nuclear disorganization. Therefore, two female gametes (the egg and the central cell) control independent pathways yet coordinately accomplish the elimination of the persistent synergid cell by double fertilization.

Rice LIKE EARLY STARVATION1 cooperates with FLOURY ENDOSPERM6 to modulate starch biosynthesis and endosperm development.

In cereal grains, starch is synthesized by the concerted actions of multiple enzymes on the surface of starch granules within the amyloplast. However, little is known about how starch-synthesizing enzymes access starch granules, especially for amylopectin biosynthesis. Here, we show that the rice (Oryza sativa) floury endosperm9 (flo9) mutant is defective in amylopectin biosynthesis, leading to grains exhibiting a floury endosperm with a hollow core. Molecular cloning revealed that FLO9 encodes a plant-specific protein homologous to Arabidopsis (Arabidopsis thaliana) LIKE EARLY STARVATION1 (LESV). Unlike Arabidopsis LESV, which is involved in starch metabolism in leaves, OsLESV is required for starch granule initiation in the endosperm. OsLESV can directly bind to starch by its C-terminal tryptophan (Trp)-rich region. Cellular and biochemical evidence suggests that OsLESV interacts with the starch-binding protein FLO6, and loss-of-function mutations of either gene impair ISOAMYLASE1 (ISA1) targeting to starch granules. Genetically, OsLESV acts synergistically with FLO6 to regulate starch biosynthesis and endosperm development. Together, our results identify OsLESV-FLO6 as a non-enzymatic molecular module responsible for ISA1 localization on starch granules, and present a target gene for use in biotechnology to control starch content and composition in rice endosperm.

The transcription factors ZmNAC128 and ZmNAC130 coordinate with Opaque2 to promote endosperm filling in maize.

Endosperm filling in maize (Zea mays), which involves nutrient uptake and biosynthesis of storage reserves, largely determines grain yield and quality. However, much remains unclear about the synchronization of these processes. Here, we comprehensively investigated the functions of duplicate NAM, ATAF1/2, and CUC2 (NAC)-type transcription factors, namely, ZmNAC128 and ZmNAC130, in endosperm filling. The gene-edited double mutant zmnac128 zmnac130 exhibits a poorly filled kernel phenotype such that the kernels have an inner cavity. RNA sequencing and protein abundance analysis revealed that the expression of many genes involved in the biosynthesis of zein and starch is reduced in the filling endosperm of zmnac128 zmnac130. Further, DNA affinity purification and sequencing combined with chromatin-immunoprecipitation quantitative PCR and promoter transactivation assays demonstrated that ZmNAC128 and ZmNAC130 are direct regulators of 3 (16-, 27-, and 50-kD) γ-zein genes and 6 important starch metabolism genes (Brittle2 [Bt2], pullulanase-type starch debranching enzyme [Zpu1], granule-bound starch synthase 1 [GBSS1], starch synthase 1 [SS1], starch synthase IIa [SSIIa], and sucrose synthase 1 [Sus1]). ZmNAC128 and ZmNAC130 recognize an additional cis-element in the Opaque2 (O2) promoter to regulate its expression. The triple mutant zmnac128 zmnac130 o2 exhibits extremely poor endosperm filling, which results in more than 70% of kernel weight loss. ZmNAC128 and ZmNAC130 regulate the expression of the transporter genes sugars that will eventually be exported transporter 4c (ZmSWEET4c), sucrose and glucose carrier 1 (ZmSUGCAR1), and yellow stripe-like2 (ZmYSL2) and in turn facilitate nutrient uptake, while O2 plays a supporting role. In conclusion, ZmNAC128 and ZmNAC130 cooperate with O2 to facilitate endosperm filling, which involves nutrient uptake in the basal endosperm transfer layer (BETL) and the synthesis of zeins and starch in the starchy endosperm (SE).

Initiation of B-type starch granules in wheat endosperm requires the plastidial α-glucan phosphorylase PHS1.

The plastidial α-glucan phosphorylase (PHS1) can elongate and degrade maltooligosaccharides (MOSs), but its exact physiological role in plants is poorly understood. Here, we discover a specialized role of PHS1 in establishing the unique bimodal characteristic of starch granules in wheat (Triticum spp.) endosperm. Wheat endosperm contains large A-type granules that initiate at early grain development and small B-type granules that initiate in later grain development. We demonstrate that PHS1 interacts with B-GRANULE CONTENT1 (BGC1), a carbohydrate-binding protein essential for normal B-type granule initiation. Mutants of tetraploid durum wheat (Triticum turgidum) deficient in all homoeologs of PHS1 had normal A-type granules but fewer and larger B-type granules. Grain size and starch content were not affected by the mutations. Further, by assessing granule numbers during grain development in the phs1 mutant and using a double mutant defective in both PHS1 and BGC1, we demonstrate that PHS1 is exclusively involved in B-type granule initiation. The total starch content and number of starch granules per chloroplast in leaves were not affected by loss of PHS1, suggesting that its role in granule initiation in wheat is limited to the endosperm. We therefore propose that the initiation of A- and B-type granules occurs via distinct biochemical mechanisms, where PHS1 plays an exclusive role in B-type granule initiation.

Endosperm cellularization failure induces a dehydration-stress response leading to embryo arrest.

The endosperm is a nutritive tissue supporting embryo growth in flowering plants. Most commonly, the endosperm initially develops as a coenocyte (multinucleate cell) and then cellularizes. This process of cellularization is frequently disrupted in hybrid seeds generated by crosses between different flowering plant species or plants that differ in ploidy, resulting in embryo arrest and seed lethality. The reason for embryo arrest upon cellularization failure remains unclear. In this study, we show that triploid Arabidopsis thaliana embryos surrounded by uncellularized endosperm mount an osmotic stress response that is connected to increased levels of abscisic acid (ABA) and enhanced ABA responses. Impairing ABA biosynthesis and signaling aggravated triploid seed abortion, while increasing endogenous ABA levels as well as the exogenous application of ABA-induced endosperm cellularization and suppressed embryo growth arrest. Taking these results together, we propose that endosperm cellularization is required to establish dehydration tolerance in the developing embryo, ensuring its survival during seed maturation.

NAKED ENDOSPERM1, NAKED ENDOSPERM2, and OPAQUE2 interact to regulate gene networks in maize endosperm development.

NAKED ENDOSPERM1 (NKD1), NKD2, and OPAQUE2 (O2) are transcription factors important for cell patterning and nutrient storage in maize (Zea mays) endosperm. To study the complex regulatory interrelationships among these 3 factors in coregulating gene networks, we developed a set of nkd1, nkd2, and o2 homozygous lines, including all combinations of mutant and wild-type genes. Among the 8 genotypes tested, we observed diverse phenotypes and gene interactions affecting cell patterning, starch content, and storage proteins. From ∼8 to ∼16 d after pollination, maize endosperm undergoes a transition from cellular development to nutrient accumulation for grain filling. Gene network analysis showed that NKD1, NKD2, and O2 dynamically regulate a hierarchical gene network during this period, directing cellular development early and then transitioning to constrain cellular development while promoting the biosynthesis and storage of starch, proteins, and lipids. Genetic interactions regulating this network are also dynamic. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) showed that O2 influences the global regulatory landscape, decreasing NKD1 and NKD2 target site accessibility, while NKD1 and NKD2 increase O2 target site accessibility. In summary, interactions of NKD1, NKD2, and O2 dynamically affect the hierarchical gene network and regulatory landscape during the transition from cellular development to grain filling in maize endosperm.

Nitrogen-dependent binding of the transcription factor PBF1 contributes to the balance of protein and carbohydrate storage in maize endosperm.

Fluctuations in nitrogen (N) availability influence protein and starch levels in maize (Zea mays) seeds, yet the underlying mechanism is not well understood. Here, we report that N limitation impacted the expression of many key genes in N and carbon (C) metabolism in the developing endosperm of maize. Notably, the promoter regions of those genes were enriched for P-box sequences, the binding motif of the transcription factor prolamin-box binding factor 1 (PBF1). Loss of PBF1 altered accumulation of starch and proteins in endosperm. Under different N conditions, PBF1 protein levels remained stable but PBF1 bound different sets of target genes, especially genes related to the biosynthesis and accumulation of N and C storage products. Upon N-starvation, the absence of PBF1 from the promoters of some zein genes coincided with their reduced expression, suggesting that PBF1 promotes zein accumulation in the endosperm. In addition, PBF1 repressed the expression of sugary1 (Su1) and starch branching enzyme 2b (Sbe2b) under normal N supply, suggesting that, under N-deficiency, PBF1 redirects the flow of C skeletons for zein toward the formation of C compounds. Overall, our study demonstrates that PBF1 modulates C and N metabolism during endosperm development in an N-dependent manner.

Auxin: a molecular trigger of seed development.

The evolution of seeds defines a remarkable landmark in the history of land plants. A developing seed contains three genetically distinct structures: the embryo, the nourishing tissue, and the seed coat. While fertilization is necessary to initiate seed development in most plant species, apomicts have evolved mechanisms allowing seed formation independently of fertilization. Despite their socio-economical relevance, the molecular mechanisms driving seed development have only recently begun to be understood. Here we review the current knowledge on the role of the hormone auxin for the initial development of the three seed structures and as a trigger of fertilization-independent seed development.

Dynamic transcriptome landscape of maize pericarp development.

As a maternal tissue, the pericarp supports and protects for other components of seed, such as embryo and endosperm. Despite the importance of maize pericarp in seed, the genome-wide transcriptome pattern throughout maize pericarp development has not been well characterized. Here, we developed RNA-seq transcriptome atlas of B73 maize pericarp development based on 21 samples from 5 days before fertilization (DBP5) to 32 days after fertilization (DAP32). A total of 25 346 genes were detected in programming pericarp development, including 1887 transcription factors (TFs). Together with pericarp morphological changes, the global clustering of gene expression revealed four developmental stages: undeveloped, thickening, expansion and strengthening. Coexpression analysis provided further insights on key regulators in functional transition of four developmental stages. Combined with non-seed, embryo, endosperm, and nucellus transcriptome data, we identified 598 pericarp-specific genes, including 75 TFs, which could elucidate key mechanisms and regulatory networks of pericarp development. Cell wall related genes were identified that reflected their crucial role in the maize pericarp structure building. In addition, key maternal proteases or TFs related with programmed cell death (PCD) were proposed, suggesting PCD in the maize pericarp was mediated by vacuolar processing enzymes (VPE), and jasmonic acid (JA) and ethylene-related pathways. The dynamic transcriptome atlas provides a valuable resource for unraveling the genetic control of maize pericarp development.

Triacylglycerol lipase, OsSG34, plays an important role in grain shape and appearance quality in rice.

Optimal grain-appearance quality is largely determined by grain size. To date, dozens of grain size-related genes have been identified. However, the regulatory mechanism of slender grain formation is not fully clear. We identified the OsSG34 gene by map-based cloning. A 9-bp deletion on 5'-untranslated region of OsSG34, which resulted in the expression difference between the wild-type and sg34 mutant, led to the slender grains and good transparency in sg34 mutant. OsSG34 as an α/ß fold triacylglycerol lipase affected the triglyceride content directly, and the components of cell wall indirectly, especially the lignin between the inner and outer lemmas in rice grains, which could affect the change in grain size by altering cell proliferation and expansion, while the change in starch content and starch granule arrangement in endosperm could affect the grain-appearance quality. Moreover, the OsERF71 was identified to directly bind to cis-element on the mutant site, thereby regulating the OsSG34 expression. Knockout of three OsSG34 homologous genes resulted in slender grains as well. The study demonstrated OsSG34, involved in lipid metabolism, affected grain size and quality. Our findings suggest that the OsSG34 gene could be used in rice breeding for high yield and good grain-appearance quality via marker-assisted selection and gene-editing approaches.

Toward unveiling transcriptome dynamics and regulatory modules at the maternal/filial interface of developing maize kernel.

The basal region of maize (Zea mays) kernels, which includes the pedicel, placenta-chalazal, and basal endosperm transfer layers, serves as the maternal/filial interface for nutrient transfer from the mother plant to the developing seed. However, transcriptome dynamics of this maternal/filial interface remain largely unexplored. To address this gap, we conducted high-temporal-resolution RNA sequencing of the basal and upper kernel regions between 4 and 32 days after pollination and deeply analyzed transcriptome dynamics of the maternal/filial interface. Utilizing 790 specifically and highly expressed genes in the basal region, we performed the gene ontology (GO) term and weighted gene co-expression network analyses. In the early-stage basal region, we identified five MADS-box transcription factors (TFs) as hubs. Their homologs have been demonstrated as pivotal regulators at the maternal/filial interface of rice or Arabidopsis, suggesting their potential roles in maize kernel development. In the filling-stage basal region, numerous GO terms associated with transcriptional regulation and transporters are significantly enriched. Furthermore, we investigated the molecular function of three hub TFs. Through genome-wide DNA affinity purification sequencing combined with promoter transactivation assays, we suggested that these three TFs act as regulators of 10 basal-specific transporter genes involved in the transfer of sugars, amino acids, and ions. This study provides insights into transcriptomic dynamic and regulatory modules of the maternal/filial interface. In the future, genetic investigation of these hub regulators must advance our understanding of maternal/filial interface development and function.

Molecular basis and evolutionary drivers of endosperm-based hybridization barriers.

The endosperm, a transient seed tissue, plays a pivotal role in supporting embryo growth and germination. This unique feature sets flowering plants apart from gymnosperms, marking an evolutionary innovation in the world of seed-bearing plants. Nevertheless, the importance of the endosperm extends beyond its role in providing nutrients to the developing embryo by acting as a versatile protector, preventing hybridization events between distinct species and between individuals with different ploidy. This phenomenon centers on growth and differentiation of the endosperm and the speed at which both processes unfold. Emerging studies underscore the important role played by type I MADS-box transcription factors, including the paternally expressed gene PHERES1. These factors, along with downstream signaling pathways involving auxin and abscisic acid, are instrumental in regulating endosperm development and, consequently, the establishment of hybridization barriers. Moreover, mutations in various epigenetic regulators mitigate these barriers, unveiling a complex interplay of pathways involved in their formation. In this review, we discuss the molecular underpinnings of endosperm-based hybridization barriers and their evolutionary drivers.

DEFECTIVE KERNEL 56 functions in mitochondrial RNA editing and maize seed development.

Proper seed development is essential for achieving grain production, successful seed germination, and seedling establishment in maize (Zea mays). In the past few decades, pentatricopeptide repeat (PPR) proteins have been proven to play an essential role in regulating the development of maize kernels through posttranscriptional RNA modification of mitochondrial genes. However, the underlying mechanisms remain largely unknown. Here, we characterized a mutant of DEFECTIVE KERNEL 56 (DEK56) with defective kernels that exhibited arrested development of both the embryo and endosperm. Accordingly, we isolated DEK56 through a map-based cloning strategy and found that it encoded an E subgroup PPR protein located in the mitochondria. Dysfunction of DEK56 resulted in altered cytidine (C)-to-uridine (U) editing efficiency at 48 editing sites across 21 mitochondrial transcripts. Notably, the editing efficiency of the maturase-related (matR)-1124 site was substantially reduced or abolished in the dek56 mutant. Furthermore, we found that the splicing efficiency of NADH dehydrogenase subunit 4 (nad4) Introns 1 and 3 was substantially reduced in dek56 kernels, which might be a consequence of the defective MatR function. Through a protein-protein interaction test, we hypothesized that DEK56 carries out its function by recruiting the PPR-DYW protein PPR motif, coiled-coil, and DYW domain-containing protein 1 (PCW1). This interaction is facilitated by Multiple Organellar RNA Editing Factors (ZmMORFs) and Glutamine-Rich Protein 23 (ZmGRP23). Based on these findings, we developed a working model of PPR-mediated mitochondrial processing that plays an essential role in the development of maize kernels. The present study will further broaden our understanding of PPR-mediated seed development and provide a theoretical basis for maize improvement.

Regulatory loops between rice transcription factors OsNAC25 and OsNAC20/26 balance starch synthesis.

Several starch synthesis regulators have been identified, but these regulators are situated in the terminus of the regulatory network. Their upstream regulators and the complex regulatory network formed between these regulators remain largely unknown. A previous study demonstrated that NAM, ATAF, and CUC (NAC) transcription factors, OsNAC20 and OsNAC26 (OsNAC20/26), redundantly and positively regulate the accumulation of storage material in rice (Oryza sativa) endosperm. In this study, we detected OsNAC25 as an upstream regulator and interacting protein of OsNAC20/26. Both OsNAC25 mutation and OE resulted in a chalky seed phenotype, decreased starch content, and reduced expression of starch synthesis-related genes, but the mechanisms were different. In the osnac25 mutant, decreased expression of OsNAC20/26 resulted in reduced starch synthesis; however, in OsNAC25-overexpressing plants, the OsNAC25-OsNAC20/26 complex inhibited OsNAC20/26 binding to the promoter of starch synthesis-related genes. In addition, OsNAC20/26 positively regulated OsNAC25. Therefore, the mutual regulation between OsNAC25 and OsNAC20/26 forms a positive regulatory loop to stimulate the expression of starch synthesis-related genes and meet the great demand for starch accumulation in the grain filling stage. Simultaneously, a negative regulatory loop forms among the 3 proteins to avoid the excessive expression of starch synthesis-related genes. Collectively, our findings demonstrate that both promotion and inhibition mechanisms between OsNAC25 and OsNAC20/26 are essential for maintaining stable expression of starch synthesis-related genes and normal starch accumulation.

Auxin receptor OsTIR1 mediates auxin signaling during seed filling in rice.

Cereal endosperm represents the most important source of the world's food. Nevertheless, the molecular mechanisms behind sugar import into rice (Oryza sativa) endosperm and their relationship with auxin signaling are poorly understood. Here, we report that auxin transport inhibitor response 1 (TIR1) plays an essential role in rice grain yield and quality via modulating sugar transport into endosperm. The fluctuations of OsTIR1 transcripts parallel to the early stage of grain expansion among those of the 5 TIR1/AFB (auxin-signaling F-box) auxin co-receptor proteins. OsTIR1 is abundantly expressed in ovular vascular trace, nucellar projection, nucellar epidermis, aleurone layer cells, and endosperm, providing a potential path for sugar into the endosperm. Compared to wild-type (WT) plants, starch accumulation is repressed by mutation of OsTIR1 and improved by overexpression of the gene, ultimately leading to reduced grain yield and quality in tir1 mutants but improvement in overexpression lines. Of the rice AUXIN RESPONSE FACTOR (ARF) genes, only the OsARF25 transcript is repressed in tir1 mutants and enhanced by overexpression of OsTIR1; its highest transcript is recorded at 10 d after fertilization, consistent with OsTIR1 expression. Also, OsARF25 can bind the promoter of the sugar transporter OsSWEET11 (SWEET, sugars will eventually be exported transporter) in vivo and in vitro. arf25 and arf25/sweet11 mutants exhibit reduced starch content and seed size (relative to the WTs), similar to tir1 mutants. Our data reveal that OsTIR1 mediates sugar import into endosperm via the auxin signaling component OsARF25 interacting with sugar transporter OsSWEET11. The results of this study are of great significance to further clarify the regulatory mechanism of auxin signaling on grain development in rice.

Imprinting but not cytonuclear interactions determines seed size heterosis in Arabidopsis hybrids.

The parent-of-origin effect on seeds can result from imprinting (unequal expression of paternal and maternal alleles) or combinational effects between cytoplasmic and nuclear genomes, but their relative contributions remain unknown. To discern these confounding factors, we produced cytoplasmic-nuclear substitution (CNS) lines using recurrent backcrossing in Arabidopsis (Arabidopsis thaliana) ecotypes Col-0 and C24. These CNS lines differed only in the nuclear genome (imprinting) or cytoplasm. The CNS reciprocal hybrids with the same cytoplasm displayed ∼20% seed size difference, whereas the seed size was similar between the reciprocal hybrids with fixed imprinting. Transcriptome analyses in the endosperm of CNS hybrids using laser-capture microdissection identified 104 maternally expressed genes (MEGs) and 90 paternally expressed genes (PEGs). These imprinted genes were involved in pectin catabolism and cell wall modification in the endosperm. Homeodomain Glabrous9 (HDG9), an epiallele and one of 11 cross-specific imprinted genes, affected seed size. In the embryo, there were a handful of imprinted genes in the CNS hybrids but only 1 was expressed at higher levels than in the endosperm. AT4G13495 was found to encode a long-noncoding RNA (lncRNA), but no obvious seed phenotype was observed in lncRNA knockout lines. Nuclear RNA Polymerase D1 (NRPD1), encoding the largest subunit of RNA Pol IV, was involved in the biogenesis of small interfering RNAs. Seed size and embryos were larger in the cross using nrpd1 as the maternal parent than in the reciprocal cross, supporting a role of the maternal NRPD1 allele in seed development. Although limited ecotypes were tested, these results suggest that imprinting and the maternal NRPD1-mediated small RNA pathway play roles in seed size heterosis in plant hybrids.

ENB1 encodes a cellulose synthase 5 that directs synthesis of cell wall ingrowths in maize basal endosperm transfer cells.

Development of the endosperm is strikingly different in monocots and dicots: it often manifests as a persistent tissue in the former and transient tissue in the latter. Little is known about the controlling mechanisms responsible for these different outcomes. Here we characterized a maize (Zea mays) mutant, endosperm breakdown1 (enb1), in which the typically persistent endosperm (PE) was drastically degraded during kernel development. ENB1 encodes a cellulose synthase 5 that is predominantly expressed in the basal endosperm transfer layer (BETL) of endosperm cells. Loss of ENB1 function caused a drastic reduction in formation of flange cell wall ingrowths (ingrowths) in BETL cells. Defective ingrowths impair nutrient uptake, leading to premature utilization of endosperm starch to nourish the embryo. Similarly, developing wild-type kernels cultured in vitro with a low level of sucrose manifested early endosperm breakdown. ENB1 expression is induced by sucrose via the BETL-specific Myb-Related Protein1 transcription factor. Overexpression of ENB1 enhanced development of flange ingrowths, facilitating sucrose transport into BETL cells and increasing kernel weight. The results demonstrated that ENB1 enhances sucrose supply to the endosperm and contributes to a PE in the kernel.

The maize gene maternal derepression of r1 encodes a DNA glycosylase that demethylates DNA and reduces siRNA expression in the endosperm.

Demethylation of transposons can activate the expression of nearby genes and cause imprinted gene expression in the endosperm; this demethylation is hypothesized to lead to expression of transposon small interfering RNAs (siRNAs) that reinforce silencing in the next generation through transfer either into egg or embryo. Here we describe maize (Zea mays) maternal derepression of r1 (mdr1), which encodes a DNA glycosylase with homology to Arabidopsis thaliana DEMETER and which is partially responsible for demethylation of thousands of regions in endosperm. Instead of promoting siRNA expression in endosperm, MDR1 activity inhibits it. Methylation of most repetitive DNA elements in endosperm is not significantly affected by MDR1, with an exception of Helitrons. While maternally-expressed imprinted genes preferentially overlap with MDR1 demethylated regions, the majority of genes that overlap demethylated regions are not imprinted. Double mutant megagametophytes lacking both MDR1 and its close homolog DNG102 result in early seed failure, and double mutant microgametophytes fail pre-fertilization. These data establish DNA demethylation by glycosylases as essential in maize endosperm and pollen and suggest that neither transposon repression nor genomic imprinting is its main function in endosperm.

ABA-induced phosphorylation of basic leucine zipper 29, ABSCISIC ACID INSENSITIVE 19, and Opaque2 by SnRK2.2 enhances gene transactivation for endosperm filling in maize.

Opaque2 (O2) functions as a central regulator of the synthesis of starch and storage proteins and the O2 gene is transcriptionally regulated by a hub coordinator of seed development and grain filling, ABSCISIC ACID INSENSITIVE 19 (ZmABI19), in maize (Zea mays). Here, we identified a second hub coordinator, basic Leucine Zipper 29 (ZmbZIP29) that interacts with ZmABI19 to regulate O2 expression. Like zmabi19, zmbzip29 mutations resulted in a dramatic decrease of transcript and protein levels of O2 and thus a significant reduction of starch and storage proteins. zmbzip29 seeds developed slower and had a smaller size at maturity than those of the wild type. The zmbzip29;zmabi19 double mutant displayed more severe seed phenotypes and a greater reduction of storage reserves compared to the single mutants, whereas overexpression of the two transcription factors enhanced O2 expression, storage-reserve accumulation, and kernel weight. ZmbZIP29, ZmABI19, and O2 expression was induced by abscisic acid (ABA). With ABA treatment, ZmbZIP29 and ZmABI19 synergistically transactivated the O2 promoter. Through liquid chromatography tandem-mass spectrometry analysis, we established that the residues threonine(T) 57 in ZmABI19, T75 in ZmbZIP29, and T387 in O2 were phosphorylated, and that SnRK2.2 was responsible for the phosphorylation. The ABA-induced phosphorylation at these sites was essential for maximum transactivation of downstream target genes for endosperm filling in maize.

RNA Pol IV induces antagonistic parent-of-origin effects on Arabidopsis endosperm.

Gene expression in endosperm-a seed tissue that mediates transfer of maternal resources to offspring-is under complex epigenetic control. We show here that plant-specific RNA polymerase IV (Pol IV) mediates parental control of endosperm gene expression. Pol IV is required for the production of small interfering RNAs that typically direct DNA methylation. We compared small RNAs (sRNAs), DNA methylation, and mRNAs in Arabidopsis thaliana endosperm from heterozygotes produced by reciprocally crossing wild-type (WT) plants to Pol IV mutants. We find that maternally and paternally acting Pol IV induce distinct effects on endosperm. Loss of maternal or paternal Pol IV impacts sRNAs and DNA methylation at different genomic sites. Strikingly, maternally and paternally acting Pol IV have antagonistic impacts on gene expression at some loci, divergently promoting or repressing endosperm gene expression. Antagonistic parent-of-origin effects have only rarely been described and are consistent with a gene regulatory system evolving under parental conflict.