广东高州普通野生稻生殖特性的研究Ⅱ.胚囊育性、胚囊发育、胚胎发生和胚乳发育

利用整体染色激光扫描共聚焦显微镜术（WCLSM）,对采自广东省高州市6个地点共141个编号的高州普通野生稻的成熟胚囊育性和胚囊形成发育特点等进行研究。结果表明,供试的绝大多数高州普通野生稻材料成熟胚囊均存在不同程度的育性异常现象,包括雌性生殖单位退化、极核位置异常、极核数目异常、胚囊退化等。这些异常结构的胚囊由于没有正常的卵细胞,不能正常受精,影响子粒结实。141个编号平均异常胚囊频率为11．11％,最高异常率为67．86％。高州普通野生稻胚囊发育过程与正常栽培稻一致,属寥型。对一些结实率偏低材料的研究,发现在胚囊发育过程的不同时期存在一些异常现象,包括功能大孢子退化,二至八核胚囊发育异常等。对柱头上的花粉量调查,发现观察的69个编号中,多数编号柱头上花粉量偏少。研究表明,花粉量偏少影响受精是导致结实率偏低的最主要原因之一。本文对导致结实率偏低的综合因素进行了讨论。     

EARLY IN SHORT DAYS 7 (ESD7) encodes the catalytic subunit of DNA polymerase epsilon and is required for flowering repression through a mechanism involving epigenetic gene silencing

We have characterized a mutation affecting the Arabidopsis EARLY IN SHORT DAYS 7 (ESD7) gene encoding the catalytic subunit of DNA polymerase epsilon (ε), AtPOL2a. The esd7‐1 mutation causes early flowering independently of photoperiod, shortened inflorescence internodes and altered leaf and root development. esd7‐1 is a hypomorphic allele whereas knockout alleles displayed an embryo‐lethal phenotype. The esd7 early flowering phenotype requires functional FT and SOC1 proteins and might also be related to the misregulation of AG and AG‐like gene expression found in esd7. Genes involved in the modulation of chromatin structural dynamics, such as LHP1/TFL2 and EBS, which negatively regulate FT expression, were found to interact genetically with ESD7. In fact a molecular interaction between the carboxy terminus of ESD7 and TFL2 was demonstrated in vitro. Besides, fas2 mutations suppressed the esd7 early flowering phenotype and ICU2 was found to interact with ESD7. Discrete regions of the chromatin of FT and AG loci were enriched in activating epigenetic marks in the esd7‐1 mutant. We concluded that ESD7 might be participating in processes involved in chromatin‐mediated cellular memory.     

EARLY IN SHORT DAYS 1 (ESD1) encodes ACTIN-RELATED PROTEIN 6 (AtARP6), a putative component of chromatin remodelling complexes that positively regulates FLC accumulation in Arabidopsis

We have characterized Arabidopsis esd1 mutations, which cause early flowering independently of photoperiod, moderate increase of hypocotyl length, shortened inflorescence internodes, and altered leaf and flower development. Phenotypic analyses of double mutants with mutations at different loci of the flowering inductive pathways suggest that esd1 abolishes the FLC-mediated late flowering phenotype of plants carrying active alleles of FRI and of mutants of the autonomous pathway. We found that ESD1 is required for the expression of the FLC repressor to levels that inhibit flowering. However, the effect of esd1 in a flc-3 null genetic background and the downregulation of other members of the FLC-like/MAF gene family in esd1 mutants suggest that flowering inhibition mediated by ESD1 occurs through both FLC-and FLC-like gene-dependent pathways. The ESD1 locus was identified through a map-based cloning approach. ESD1 encodes ARP6, a homolog of the actin-related protein family that shares moderate sequence homology with conventional actins. Using chromatin immunoprecipitation (ChIP) experiments, we have determined that ARP6 is required for both histone acetylation and methylation of the FLC chromatin in Arabidopsis.     

Poly ADP-ribose polymerase-1 promotes seed-setting rate by facilitating gametophyte development and meiosis in rice (Oryza sativa L.)

Poly(ADP-ribose) polymerases (PARPs), which transfer either monomer or polymer of ADP-ribose from nicotinamide adenine dinucleotide (NAD⁺) onto target proteins, are required for multiple processes in DNA damage repair, cell cycle, development, and abiotic stress in animals and plants. Here, the uncharacterized rice (Oryza sativa) OsPARP1, which has been predicted to have two alternative OsPARP1 mRNA splicing variants, OsPARP1.1 and OsPARP1.2, was investigated. However, bimolecular fluorescence complementation showed that only OsPARP1.1 interacted with OsPARP3 paralog, suggesting that OsPARP1.1 is a functional protein in rice. OsPARP1 was preferentially expressed in the stamen primordial and pollen grain of mature stamen during flower development. The osparp1 mutant and CRISPR plants were delayed in germination, indicating that defective DNA repair machinery impairs early seed germination. The mutant displayed a normal phenotype during vegetative growth but had a lower seed-setting rate than wild-type plants under normal conditions. Chromosome bridges and DNA fragmentations were detected in male meiocytes at anaphase I to prophase II. After meiosis II, malformed tetrads or tetrads with micronuclei were formed. Meanwhile, the abnormality was also found in embryo sac development. Collectively, these results suggest that OsPARP1 plays an important role in mediating response to DNA damage and gametophyte development, crucial for rice yield in the natural environment.     

An-1 Encodes a Basic Helix-Loop-Helix Protein That Regulates Awn Development,Grain Size,and Grain Number in Rice

Long awns are important for seed dispersal in wild rice (Oryza rufipogon), but are absent in cultivated rice (Oryza sativa). The genetic mechanism involved in loss-of-awn in cultivated rice remains unknown. We report here the molecular cloning of a major quantitative trait locus, An-1, which regulates long awn formation in O. rufipogon. An-1 encodes a basic helix-loop-helix protein, which regulates cell division. The nearly-isogenic line (NIL-An-1) carrying a wild allele An-1 in the genetic background of the awnless indica Guangluai4 produces long awns and longer grains, but significantly fewer grains per panicle compared with Guangluai4. Transgenic studies confirmed that An-1 positively regulates awn elongation, but negatively regulates grain number per panicle. Genetic variations in the An-1 locus were found to be associated with awn loss in cultivated rice. Population genetic analysis of wild and cultivated rice showed a significant reduction in nucleotide diversity of the An-1 locus in rice cultivars, suggesting that the An-1 locus was a major target for artificial selection. Thus, we propose that awn loss was favored and strongly selected by humans, as genetic variations at the An-1 locus that cause awn loss would increase grain numbers and subsequently improve grain yield in cultivated rice.     

CLUSTERED PRIMARY BRANCH 1, a new allele of DWARF11, controls panicle architecture and seed size in rice

Panicle architecture and seed size are important agronomic traits that directly determine grain yield in rice (Oryza sativa L.). Although a number of key genes controlling panicle architecture and seed size have been cloned and characterized in recent years, their genetic and molecular mechanisms remain unclear. In this study, we identified a mutant that produced panicles with fascicled primary branching and reduced seeds in size. We isolated the underlying CLUSTERED PRIMARY BRANCH 1 (CPB1) gene, a new allele of DWARF11 (D11) encoding a cytochrome P450 protein involved in brassinosteroid (BR) biosynthesis pathway. Genetic transformation experiments confirmed that a His360Leu amino acid substitution residing in the highly conserved region of CPB1/D11 was responsible for the panicle architecture and seed size changes in the cpb1 mutants. Overexpression of CPB1/D11 under the background of cpb1 mutant not only rescued normal panicle architecture and plant height, but also had a larger leaf angle and seed size than the controls. Furthermore, the CPB1/D11 transgenic plants driven by panicle‐specific promoters can enlarge seed size and enhance grain yield without affecting other favourable agronomic traits. These results demonstrated that the specific mutation in CPB1/D11 influenced development of panicle architecture and seed size, and manipulation of CPB1/D11 expression using the panicle‐specific promoter could be used to increase seed size, leading to grain yield improvement in rice.     

Brassinosteroids regulate grain filling in rice

Genes controlling hormone levels have been used to increase grain yields in wheat (Triticum aestivum) and rice (Oryza sativa). We created transgenic rice plants expressing maize (Zea mays), rice, or Arabidopsis thaliana genes encoding sterol C-22 hydroxylases that control brassinosteroid (BR) hormone levels using a promoter that is active in only the stems, leaves, and roots. The transgenic plants produced more tillers and more seed than wild-type plants. The seed were heavier as well, especially the seed at the bases of the spikes that fill the least. These phenotypic changes brought about 15 to 44% increases in grain yield per plant relative to wild-type plants in greenhouse and field trials. Expression of the Arabidopsis C-22 hydroxylase in the embryos or endosperms themselves had no apparent effect on seed weight. These results suggested that BRs stimulate the flow of assimilate from the source to the sink. Microarray and photosynthesis analysis of transgenic plants revealed evidence of enhanced CO₂ assimilation, enlarged glucose pools in the flag leaves, and increased assimilation of glucose to starch in the seed. These results further suggested that BRs stimulate the flow of assimilate. Plants have not been bred directly for seed filling traits, suggesting that genes that control seed filling could be used to further increase grain yield in crop plants.     

A nuclear protease required for flowering-time regulation in Arabidopsis reduces the abundance of SMALL UBIQUITIN-RELATED MODIFIER conjugates

The Arabidopsis mutant early in short days4 (esd4) shows extreme early flowering and alterations in shoot development. We have identified ESD4 and demonstrate that it encodes a nuclear protein located predominantly at the periphery of the nucleus. ESD4 contains a segment of >200 amino acids with strong similarity to yeast and animal proteases that are specific for the protein modifier SMALL UBIQUITIN-RELATED MODIFIER (SUMO). ESD4 shows a similar function to these proteases in vitro and processes the precursor of Arabidopsis SUMO (AtSUMO) to generate the mature form. This activity of ESD4 is prevented by mutations that affect the predicted active site of the protease or the cleavage site of the AtSUMO precursor. In yeast, these proteases also recycle SUMO from conjugates, and this appears to be the major role of ESD4 in vivo. This is suggested because esd4 mutants contain less free AtSUMO and more SUMO conjugates than wild-type plants, and a transgene expressing mature SUMO at high levels enhanced aspects of the esd4 phenotype. ESD4 defines an important role for protein modification by AtSUMO in the regulation of flowering.     

Effects of APETALA2 on embryo, endosperm, and seed coat development determine seed size in Arabidopsis

Arabidopsis APETALA2 (AP2) controls seed mass maternally, with ap2 mutants producing larger seeds than wild type. Here, we show that AP2 influences development of the three major seed compartments: embryo, endosperm, and seed coat. AP2 appears to have a significant effect on endosperm development. ap2 mutant seeds undergo an extended period of rapid endosperm growth early in development relative to wild type. This early expanded growth period in ap2 seeds is associated with delayed endosperm cellularization and overgrowth of the endosperm central vacuole. The subsequent period of moderate endosperm growth is also extended in ap2 seeds largely due to persistent cell divisions at the endosperm periphery. The effect of AP2 on endosperm development is mediated by different mechanisms than parent-of-origin effects on seed size observed in interploidy crosses. Seed coat development is affected; integument cells of ap2 mutants are more elongated than wild type. We conclude that endosperm overgrowth and/or integument cell elongation create a larger postfertilization embryo sac into which the ap2 embryo can grow. Morphological development of the embryo is initially delayed in ap2 compared with wild-type seeds, but ap2 embryos become larger than wild type after the bent-cotyledon stage of development. ap2 embryos are able to fill the enlarged postfertilization embryo sac, because they undergo extended periods of cell proliferation and seed filling. We discuss potential mechanisms by which maternally acting AP2 influences development of the zygotic embryo and endosperm to repress seed size.     

Tillering and panicle branching genes in rice

Rice (Oryza sativa L.) is one of the most important staple food crops in the world, and rice tillering and panicle branching are important traits determining grain yield. Since the gene MONOCULM 1 (MOC 1) was first characterized as a key regulator in controlling rice tillering and branching, great progress has been achieved in identifying important genes associated with grain yield, elucidating the genetic basis of yield-related traits. Some of these important genes were shown to be applicable for molecular breeding of high-yielding rice. This review focuses on recent advances, with emphasis on rice tillering and panicle branching genes, and their regulatory networks.     

The Ubiquitin Receptor DA1 Interacts with the E3 Ubiquitin Ligase DA2 to Regulate Seed and Organ Size in Arabidopsis

Seed size in higher plants is determined by the coordinated growth of the embryo, endosperm, and maternal tissue. Several factors that act maternally to regulate seed size have been identified, such as AUXIN RESPONSE FACTOR2, APETALA2, KLUH, and DA1, but the genetic and molecular mechanisms of these factors in seed size control are almost totally unknown. We previously demonstrated that the ubiquitin receptor DA1 acts synergistically with the E3 ubiquitin ligase ENHANCER1 OF DA1 (EOD1)/BIG BROTHER to regulate the final size of seeds in Arabidopsis thaliana. Here, we describe another RING-type protein with E3 ubiquitin ligase activity, encoded by DA2, which regulates seed size by restricting cell proliferation in the maternal integuments of developing seeds. The da2-1 mutant forms large seeds, while overexpression of DA2 decreases seed size of wild-type plants. Overexpression of rice (Oryza sativa) GRAIN WIDTH AND WEIGHT2, a homolog of DA2, restricts seed growth in Arabidopsis. Genetic analyses show that DA2 functions synergistically with DA1 to regulate seed size, but does so independently of EOD1. Further results reveal that DA2 interacts physically with DA1 in vitro and in vivo. Therefore, our findings define the genetic and molecular mechanisms of three ubiquitin-related proteins DA1, DA2, and EOD1 in seed size control and indicate that they are promising targets for crop improvement.     

Characterization of the Arabidopsis Nitrate Transporter NRT1.6 Reveals a Role of Nitrate in Early Embryo Development

This study of the Arabidopsis thaliana nitrate transporter NRT1.6 indicated that nitrate is important for early embryo development. Functional analysis of cDNA-injected Xenopus laevis oocytes showed that NRT1.6 is a low-affinity nitrate transporter and does not transport dipeptides. RT-PCR, in situ hybridization, and β-glucuronidase reporter gene analysis showed that expression of NRT1.6 is only detectable in reproductive tissue (the vascular tissue of the silique and funiculus) and that expression increases immediately after pollination, suggesting that NRT1.6 is involved in delivering nitrate from maternal tissue to the developing embryo. In nrt1.6 mutants, the amount of nitrate accumulated in mature seeds was reduced and the seed abortion rate increased. In the mutants, abnormalities (i.e., excessive cell division and loss of turgidity), were found mainly in the suspensor cells at the one- or two-cell stages of embryo development. The phenotype of the nrt1.6 mutants revealed a novel role of nitrate in early embryo development. Interestingly, the seed abortion rate of the mutant was reduced when grown under N-deficient conditions, suggesting that nitrate requirements in early embryo development can be modulated in response to external nitrogen changes.     

OsMPK4 promotes phosphorylation and degradation of IPA1 in response to salt stress to confer salt tolerance in rice

Salt stress adversely affects plant growth, development, and crop yield. Rice (Oryza sativa L.) is one of the most salt-sensitive cereal crops, especially at the early seedling stage. Mitogen-activated protein kinase (MAPK/MPK) cascades have been shown to play critical roles in salt response in Arabidopsis. However, the roles of the MPK cascade signaling in rice salt response and substrates of OsMPK remain largely unknown. Here, we report that the salt-induced OsMPK4-Ideal Plant Architecture 1 (IPA1) signaling pathway regulates the salt tolerance in rice. Under salt stress, OsMPK4 could interact with IPA1 and phosphorylate IPA1 at Thr180, leading to degradation of IPA1. Genetic evidence shows that IPA1 is a negative regulator of salt tolerance in rice, whereas OsMPK4 promotes salt response in an IPA1-dependent manner. Taken together, our results uncover an OsMPK4-IPA1 signal cascade that modulates the salt stress response in rice and sheds new light on the breeding of salt-tolerant rice varieties.     

VAP-RELATED SUPPRESSORS OF TOO MANY MOUTHS (VST) family proteins are regulators of root system architecture

Root system architecture (RSA) is a key factor in the efficiency of nutrient capture and water uptake in plants. Understanding the genetic control of RSA will be useful in minimizing fertilizer and water usage in agricultural cropping systems. Using a hydroponic screen and a gel-based imaging system, we identified a rice (Oryza sativa) gene, VAP-RELATED SUPPRESSOR OF TOO MANY MOUTHS1 (OsVST1), which plays a key role in controlling RSA. This gene encodes a homolog of the VAP-RELATED SUPPRESSORS OF TOO MANY MOUTHS (VST) proteins in Arabidopsis (Arabidopsis thaliana), which promote signaling in stomata by mediating plasma membrane–endoplasmic reticulum contacts. OsVST1 mutants have shorter primary roots, decreased root meristem size, and a more compact RSA. We show that the Arabidopsis VST triple mutants have similar phenotypes, with reduced primary root growth and smaller root meristems. Expression of OsVST1 largely complements the short root length and reduced plant height in the Arabidopsis triple mutant, supporting conservation of function between rice and Arabidopsis VST proteins. In a field trial, mutations in OsVST1 did not adversely affect grain yield, suggesting that modulation of this gene could be used as a way to optimize RSA without an inherent yield penalty.

Root meristem size and root system architecture in both rice and Arabidopsis are regulated by proteins related to mediators plasma membrane–endoplasmic reticulum contact.     

Truncation of a Protein Disulfide Isomerase,PDIL2-1, Delays Embryo Sac Maturation and Disrupts Pollen Tube Guidance in Arabidopsis thaliana

Pollen tubes must navigate through different female tissues to deliver sperm to the embryo sac for fertilization. Protein disulfide isomerases play important roles in the maturation of secreted or plasma membrane proteins. Here, we show that certain T-DNA insertions in Arabidopsis thaliana PDIL2-1, a protein disulfide isomerase (PDI), have reduced seed set, due to delays in embryo sac maturation. Reciprocal crosses indicate that these mutations acted sporophytically, and aniline blue staining and scanning electron microscopy showed that funicular and micropylar pollen tube guidance were disrupted. A PDIL2-1-yellow fluorescent protein fusion was mainly localized in the endoplasmic reticulum and was expressed in all tissues examined. In ovules, expression in integument tissues was much higher in the micropylar region in later developmental stages, but there was no expression in embryo sacs. We show that reduced seed set occurred when another copy of full-length PDIL2-1 or when enzymatically active truncated versions were expressed, but not when an enzymatically inactive version was expressed, indicating that these T-DNA insertion lines are gain-of-function mutants. Our results suggest that these truncated versions of PDIL2-1 function in sporophytic tissues to affect ovule structure and impede embryo sac development, thereby disrupting pollen tube guidance.