Isopentylamine is a novel defence compound induced by insect feeding in rice

Plants produce a broad variety of defensive metabolites to protect themselves against herbivorous insects. Although polyamines have been implicated in various responses to abiotic and biotic stress, there have been no studies focused on amines in response to insect herbivory. By screening for bioactive amines, we identified isopentylamine as a novel type of herbivory‐induced compound in rice leaves, which was derived from the amino acid leucine in stable isotope labelling experiments. Accumulation of isopentylamine increased during herbivory by the brown planthopper (Nilaparvata lugens, BPH) and the rice‐feeding armyworm (Mythimna loreyi), as well as in response to treatment with the plant hormone, jasmonic acid. Likewise, isopentylamine accumulation was compromised in rice jasmonate biosynthesis mutants, hebiba and Osjar1. In bio‐assays, BPH insects feeding on rice seedlings submerged in 50 mg/L isopentylamine solution had a higher mortality compared with BPH feeding on seedlings submerged in water. Notably, the rice leaves submerged in 50 mg/L solution showed the endogenous concentrations of isopentylamine similar to that induced by BPHs. These results suggest that isopentylamine functions as a new type of plant defence metabolite that is rapidly induced by herbivore attack and deters insect herbivores in rice.     

OsARG encodes an arginase that plays critical roles in panicle development and grain production in rice

Nitrogen is a crucial nutrient for plant growth and development. Arginine is considered to be an important amino acid for nitrogen transport and storage, playing a crucial role during plant seedling development. However, little is known about the role of arginine in nitrogen remobilization at the reproductive stage. We isolated a rice mutant nglf‐1 with reduced plant height, small panicle and grain size, and low seed‐setting rate (10% in nglf‐1 compared to 93% in wild‐type). Map‐based cloning revealed that the mutant phenotype was caused by loss of function of a gene (OsARG) encoding an arginine hydrolysis enzyme, which is consistent with arginine accumulation in the mutant. The phenotype was partially corrected supplying exogenous nitrogen, and fully corrected by expression of a wild‐type OsARG transgene. Over‐expression of OsARG in rice (cv. Kitaake) increased grain number per plant under nitrogen‐limited conditions. OsARG was ubiquitously expressed in various organs, but most strongly in developing panicles. The OsARG protein was localized in the mitochondria, consistent with other arginases. Our results suggest that the arginase encoded by OsARG, a key enzyme in Arg catabolism, plays a critical role during panicle development, especially under conditions of insufficient exogenous nitrogen. OsARG is a potential target for crop improvement.     

A rice gene encoding glycosyl hydrolase plays contrasting roles in immunity depending on the type of pathogens

Because pathogens use diverse infection strategies, plants cannot use one-size-fits-all defence and modulate defence responses based on the nature of pathogens and pathogenicity mechanism. Here, we report that a rice glycoside hydrolase (GH) plays contrasting roles in defence depending on whether a pathogen is hemibiotrophic or necrotrophic. The Arabidopsis thaliana MORE1 (M agnaporthe o ryzae re sistance 1) gene, encoding a member of the GH10 family, is needed for resistance against M. oryzae and Alternaria brassicicola, a fungal pathogen infecting A. thaliana as a necrotroph. Among 13 rice genes homologous to MORE1, 11 genes were induced during the biotrophic or necrotrophic stage of infection by M. oryzae. CRISPR/Cas9-assisted disruption of one of them (OsMORE1a) enhanced resistance against hemibiotrophic pathogens M. oryzae and Xanthomonas oryzae pv. oryzae but increased susceptibility to Cochliobolus miyabeanus, a necrotrophic fungus, suggesting that OsMORE1a acts as a double-edged sword depending on the mode of infection (hemibiotrophic vs. necrotrophic). We characterized molecular and cellular changes caused by the loss of MORE1 and OsMORE1a to understand how these genes participate in modulating defence responses. Although the underlying mechanism of action remains unknown, both genes appear to affect the expression of many defence-related genes. Expression patterns of the GH10 family genes in A. thaliana and rice suggest that other members also participate in pathogen defence.     

Modern elite rice varieties of the 'Green Revolution' have retained a large introgression from wild rice around the Pi33 rice blast resistance locus

During the breeding process of cultivated crops, resistance genes to pests and diseases are commonly introgressed from wild species. The size of these introgressions is predicted by theoretical models but has rarely been measured in cultivated varieties. By combining resistance tests with isogenic strains, genotyping and sequencing of different rice accessions, it was shown that, in the elite rice variety IR64, the resistance conferring allele of the rice blast resistance gene Pi33 was introgressed from the wild rice Oryza rufipogon (accession IRGC101508). Further characterization of this introgression revealed a large introgression at this locus in IR64 and the related variety IR36. The introgressed fragment represents approximately half of the short arm of rice chromosome 8. This is the first report of a large introgression in a cultivated variety of rice. Such a large introgression is likely to have been maintained during backcrossing only if a selection pressure was exerted on this genomic region. The possible traits that were selected are discussed.     

Involvement of abscisic acid and ethylene in the responses of rice grains to water stress during filling

This study was to test the hypothesis that the interaction between abscisic acid (ABA) and ethylene may be involved in mediating the effects of water stress on grain filling. Two high lodging‐resistant rice (Oryza sativa L.) cultivars were pot‐grown. Three treatments, well‐watered, moderate water‐stressed (MD), and severe water‐stressed (SD), were imposed from 9 d post‐anthesis until maturity. Grain filling rate and grain weight were significantly increased under MD but decreased under SD. The two cultivars behaved the same. ABA concentration in the grains was very low during the grain filling stage, reaching a maximum when the grain filling rate was highest. Both the grain filling rate and ABA concentration were substantially enhanced by water stress. In contrast to ABA, concentrations of ethylene and 1‐aminocylopropane ‐1‐carboxylic acid (ACC) in the grains were very high at early grain filling stage and sharply decreased during the linear period of grain growth. MD reduced, whereas SD remarkably increased, their accumulation. The ratio of ABA to ACC was increased in MD grains but decreased in SD grains, indicating that there was a greater enhancement of ABA concentration than ethylene production in the MD treatment only. Application of cobalt ion (inhibitor of ethylene synthesis) or ABA at the early grain filling stage significantly increased grain filling rate. Spraying with ethephon (ethylene‐releasing agent) or fluridone (inhibitor of ABA synthesis) had the opposite effect. The results suggest that antagonistic interactions between ABA and ethylene mediate the grain filling rate, and a high ratio of ABA to ethylene enhances grain filling rate.     

Developmental modification and hybridization of allelic acid phosphatase isozymes in homo- and heterozygotes for the Acp-1 locus in rice

A number of alleles each specified a set of three major and three minor bands of acid phosphatase (E.C. 3.1.3.2) in wild and cultivated rice strains. Relative intensity of the major bands was found to differ significantly according to the developmental stages of the leaves, suggesting the presence of protein modification genes. In heterozygotes, six parental and three hybrid major bands were clearly observed in most of the heterozygotes, but the intensities of the hybrid bands were found to be generally lower than those theoretically expected due to random association of enzyme subunits. The cause of this phenomenon is discussed.Contribution No. 1342 from the National Institute of Genetics.     

QTL analysis to study the association between leaf size and abscisic acid accumulation in droughted rice leaves and comparisons across cereals

Plants accumulate abscisic acid (ABA) under droughted conditions. Genetic variation in the accumulation of ABA in detached and partially dehydrated leaves of rice has previously been reported, and this was found to be associated with variation in leaf size (smaller leaves made more ABA). Correlation analysis failed to distinguish clearly between a causal relationship between the two traits and close genetic linkage between loci controlling the traits. Here we present a detailed genetic analysis of ABA accumulation in detached and partially dehydrated rice leaves, using a population of F₂ plants generated from the lowland × upland cross IR20 (high-ABA) × 63-83 (low-ABA) which was mapped with RFLP and AFLP markers. Several highly significant quantitative trait loci (QTLs) for ABA accumulation and leaf weight were identified. Only one of the minor QTLs for ABA accumulation (accounting for only 4% of the phenotypic variance) was coincident with any QTLs for leaf size such that the high-ABA allele was associated with smaller leaves. This analysis, therefore, showed that the association previously found between ABA accumulation and leaf size was probably largely due to genetic linkage and not to a direct effect of leaf size on ABA accumulation or vice versa. Because of the importance of ABA accumulation in regulating responses of plants to drought stress and the effects of plant size on the rate of development of stress, QTLs for drought-induced ABA accumulation, leaf size and tiller number were compared between rice and wheat. In particular, a possible location in rice was sought for a homoeologue of the major wheat vernalization responsive gene, Vrn1, as this gene is also associated with major effects on leaf size, tiller number and ABA accumulation in wheat. The likelihood of homoeologous loci regulating ABA accumulation, leaf size and tiller number in the two crops is discussed.     

DCW11, down-regulated gene 11 in CW-type cytoplasmic male sterile rice, encoding mitochondrial protein phosphatase 2c is related to cytoplasmic male sterility

Causes of cytoplasmic male sterility (CMS) in plants have beenstudied for two decades, and mitochondrial chimeric genes havebeen predicted to induce CMS. However, it is unclear what happensafter CMS-associated proteins accumulate in mitochondria. Inour previous study of microarray analysis, we found that 140genes are aberrantly regulated in anthers of CW-type CMS ofrice (Oryza sativa L.). In the present study, we investigatedDCW11, one of the down-regulated genes in CW-CMS encoding aprotein phosphatase 2C (PP2C). DCW11 mRNA was preferentiallyexpressed in anthers, with the highest expression in maturepollen. As predicted by the N-terminal sequence, DCW11 signalpeptide–green fluorescent protein (GFP) fusion proteinwas localized in mitochondria. Knockdown of DCW11 in wild-typerice by RNA interference caused a major loss of seed-set fertility,without visible defect in pollen development. Since this knockdownphenotype resembled that of CW-CMS, we concluded that the down-regulationof DCW11 is correlated with CW-CMS. This idea was supportedby the up-regulation of alternative oxidase 1a (AOX1a), whichis known to be regulated by mitochondrial retrograde signaling,in DCW11 knockdown lines. Down-regulation of DCW11 and up-regulationof AOX1a were also observed in two other types of rice CMS.Our result indicates that DCW11 could play a role as a mitochondrialsignal transduction mediator in pollen germination.     

Identification, Expression and Functional Analysis of a Receptor-like Cytoplasmic Kinase, OsRLCK1, in Rice

Pollination involves a series of complex cellular interactions and signal transduction events. Numerous reports have suggested a central role for protein kinases in pollen germination and pollen tube growth and a large number of receptor-like kinases have been detected exclusively in pollen in higher plants. However, few are well characterized, especially for the receptor-like cytoplasmic kinases. Here we report a receptor-like kinase gene, OsRLCK1, which belongs to the receptor-like cytoplasmic kinase Ⅷ subfamily. Real-time quantitative polymerase chain reaction analysis and whole mount RNA in situ hybridization showed that OsRLCK1 is a pollen-specific gene and expressed only in the mature pollen. When expressed in the onion epidermal cells, the OsRLCK1-GFP fusion protein was diffused throughout the cell, indicating its cytoplasmic and nuclear localization. The Maltose Binding Protein-OsRLCK1 recombinant protein was found to be capable of autophosphorylation on threonine residue, showing that it encodes a functional kinase. These results suggest that OsRLCK1 is likely to play a role in a signaling pathway associated with pollen performance during pollination in rice.     

水稻抗稻瘟病基因Pi-d（t）^1、Pi-b、Pi-tα^2的聚合及分子标记选择

冈46B(G46B)是水稻生产应用中的一个农艺性状十分优良的保持系,其主要的缺陷是稻瘟病抗性较弱,通过对地谷,BL-1,Pi-4号等三个分别含抗病基因Pi-d(t)^1、Pi-b、Pi-tα^2的稻瘟病抗性材料与G46B聚合杂交,并利用抗病基因连锁的分子标记对杂交后代进行辅助选择,在聚合杂交的F2代及B1C1代群体中共获得了15株含Pi-d(t)^1、Pi-b、Pi-tα^2等三个抗稻瘟病基因的材料,其可能的基因型分别为：三基因杂合体Pi-d(t)^1pi-d(t)^1,Pi-bpi-b／Pi-tα^2 pi-tα^2 4株,双基因杂合体10株,其中Pi-d(t)^1Pi-d(t)^1／Pi-bpi-b／Pi-tα^2pi-tα^2 6株,Pi-d(t)^1pi-d(t)^1／Pi-bpi-b／Pi-tα^2Pi-tα^2 3株,Pi-d(t)^1pi-d(t)^1,Pi-bPi-6,Pi-tα^2 pi-tα^2 1株,双基因纯合体Pi-d(t)^1Pi-d(t)^1/Pi-bpi-b/Pi-tα^2Pi-tα^2仅1株,这一研究结果为进一步改良G46B的稻瘟病抗性奠定了基础,同时这一研究结果表明利用分子标记可快速、有效地实现多个抗病基因的聚合,大大提高水稻抗病育种的效率。     

Roles of OsCKI1, a rice casein kinase I, in root development and plant hormone sensitivity

Casein kinases are critical in cell division and differentiation across species. A rice cDNA fragment encoding a putative casein kinase I (CKI) was identified via cDNA macroarray under brassinosteroid (BR) treatment, and a 1939-bp full-length cDNA, OsCKI1, was isolated and found to encode a putative 463-aa protein. RT-PCR and Northern blot analysis indicated that OsCKI1 was constitutively expressed in various rice tissues and upregulated by treatments with BR and abscisic acid (ABA). Enzymatic assay of recombinant OsCKI1 proteins expressed in Escherichia coli showed that the protein was capable of phosphorylating casein. The physiological roles of OsCKI1 were studied through antisense transgenic approaches, and homozygous transgenic plants showed abnormal root development, including fewer lateral and adventitious roots, and shortened primary roots as a result of reduced cell elongation. Treatment of wild-type plants with CKI-7, a specific inhibitor of CKI, also confirmed these functions of OsCKI1. Interestingly, in transgenic and CKI-7-treated plants, exogenously supplied IAA could restore normal root development, and measurement of free IAA content in CKI-deficient primary and adventitious roots revealed altered auxin content, indicating that OsCKI1 is involved in auxin metabolism or that it may affect auxin levels. Transgenic plants were less sensitive than control plants to ABA or BR treatment during germination, suggesting that OsCKI1 may be involved in various hormone-signaling pathways. OsCKI1-GFP fusion studies revealed the localization of OsCKI1 to the nucleus, suggesting a possible involvement in regulation of gene expression. In OsCKI1-deficient plants, differential gene expression was investigated using cDNA chip technology, and results indicated that genes related to signal transduction and hormone metabolism were indeed with altered expression.     

Positional cloning of rice semidwarfing gene, sd-1: rice "green revolution gene" encodes a mutant enzyme involved in gibberellin synthesis.

A rice semidwarfing gene, sd-1, known as the "green revolution gene," was isolated by positional cloning and revealed to encode gibberellin 20-oxidase, the key enzyme in the gibberellin biosynthesis pathway. Analysis of 3477 segregants using several PCR-based marker technologies, including cleaved amplified polymorphic sequence, derived-CAPS, and single nucleotide polymorphisms revealed 1 ORF in a 6-kb candidate interval. Normal-type rice cultivars have an identical sequence in this region, consisting of 3 exons (558, 318, and 291 bp) and 2 introns (105 and 1471 bp). Dee-Geo-Woo-Gen-type sd-1 mutants have a 383-bp deletion from the genome (278-bp deletion from the expressed sequence), from the middle of exon 1 to upstream of exon 2, including a 105-bp intron, resulting in a frame-shift that produces a termination codon after the deletion site. The radiation-induced sd-1 mutant Calrose 76 has a 1-bp substitution in exon 2, causing an amino acid substitution (Leu [CTC] to Phe [TTC]). Expression analysis suggests the existence of at least one more locus of gibberellin 20-oxidase which may prevent severe dwarfism from developing in sd-1 mutants.