Mutations of genes in synthesis of the carotenoid precursors of ABA lead to pre-harvest sprouting and photo-oxidation in rice

Pre-harvest sprouting (PHS) or vivipary in cereals is an important agronomic trait that results in significant economic loss. A considerable number of mutations that cause PHS have been identified in several species. However, relatively few viviparous mutants in rice ( Oryza sativa L.) have been reported. To explore the mechanism of PHS in rice, we carried out an extensive genetic screening and identified 12 PHS mutants ( phs ). Based on their phenotypes, these phs mutants were classified into three groups. Here we characterize in detail one of these groups, which contains mutations in genes encoding major enzymes of the carotenoid biosynthesis pathway, including phytoene desaturase (OsPDS), ζ-carotene desaturase (OsZDS), carotenoid isomerase (OsCRTISO) and lycopene β -cyclase (β-OsLCY), which are essential for the biosynthesis of carotenoid precursors of ABA. As expected, the amount of ABA was reduced in all four phs mutants compared with that in the wild type. Chlorophyll fluorescence analysis revealed the occurrence of photoinhibition in the photosystem and decreased capacity for eliminating excess energy by thermal dissipation. The greatly increased activities of reactive oxygen species (ROS) scavenging enzymes, and reduced photosystem (PS) II core proteins CP43, CP47 and D1 in leaves of the Oscrtiso / phs3-1 mutant and OsLCY RNAi transgenic rice indicated that photo-oxidative damage occurred in PS II, consistent with the accumulation of ROS in these plants. These results suggest that the impairment of carotenoid biosynthesis causes photo-oxidation and ABA-deficiency phenotypes, of which the latter is a major factor controlling the PHS trait in rice.     

Polymorphic Homoeolog of Key Gene of RdDM Pathway,ARGONAUTE4_9 class Is Associated with Pre-Harvest Sprouting in Wheat (Triticum aestivum L.)

Resistance to pre-harvest sprouting (PHS) is an important objective for the genetic improvement of many cereal crops, including wheat. Resistance, or susceptibility, to PHS is mainly influenced by seed dormancy, a complex trait. Reduced seed dormancy is the most important aspect of seed germination on a spike prior to harvesting, but it is influenced by various environmental factors including light, temperature and abiotic stresses. The basic genetic framework of seed dormancy depends on the antagonistic action of abscisic acid (ABA) and gibberellic acid (GA) to promote dormancy and germination. Recent studies have revealed a role for epigenetic changes, predominantly histone modifications, in controlling seed dormancy. To investigate the role of DNA methylation in seed dormancy, we explored the role of ARGONAUTE4_9 class genes in seed development and dormancy in wheat. Our results indicate that the two wheat AGO4_9 class genes i.e. AGO802 and AGO804 map to chromosomes 3S and 1S are preferentially expressed in the embryos of developing seeds. Differential expressions of AGO802-B in the embryos of PHS resistant and susceptible varieties also relates with DNA polymorphism in various wheat varieties due to an insertion of a SINE-like element into this gene. DNA methylation patterns of the embryonic tissue from six PHS resistant and susceptible varieties demonstrate a correlation with this polymorphism. These results suggest a possible role for AGO802-B in seed dormancy and PHS resistance through the modulation of DNA methylation.     

水稻不同品种的穗发芽及其对外源激素的反应

选择8份早籼稻品种在开花后不同天数取穗子进行保湿培养,观察其抗穗发芽能力的差异。结果显示不同品种之间的抗穗发芽能力存在较大差异;处理以开花后22d左右的穗子保湿培养6d考查穗发芽,就能较好地比较出品种之间的差异。以3种不同浓度的赤霉素(GA3)和脱落酸(ABA)进行溶液培养,30mg／kg的赤霉素有明显促进穗发芽的效果,脱落酸抑制穗发芽的效果在不同品种之间表现出比较大的差异。     

Barley mutants with low rates of endosperm starch synthesis have low grain dormancy and high susceptibility to preharvest sprouting

? Studies of embryo dormancy in relation to preharvest sprouting (PHS) in cereals have focused on ABA and other hormones. The relationship between these phenomena and the rate of grain filling has not been investigated. ? A collection of barley mutants impaired in starch synthesis was assessed for preharvest sprouting in the field. In subsequent glasshouse experiments, developing grains were assayed for germination index, sugars, abscisic acid (ABA) and the effects of temperature and exogenous ABA on germination. ? Mutant lines displayed greater preharvest sprouting in the field than parental lines. In the glasshouse, nondeep physiological dormancy was reduced in developing grains of five lines with mutations affecting proteins involved in endosperm starch synthesis. Inhibition of germination by exogenous ABA and elevated temperature was decreased in developing mutant grains. Sugar concentrations were high but embryo and endosperm ABA contents were unaltered. ? We reveal a direct connection between grain filling and the extent of grain dormancy. Impaired endosperm starch synthesis directly influences the acquisition of embryo dormancy, perhaps because endosperm sugar concentrations modulate the ABA responsiveness of the embryo. Thus environmental or genetic factors that reduce grain filling are likely to reduce dormancy and enhance susceptibility to PHS.     

Control of rice pre‐harvest sprouting by glutaredoxin‐mediated abscisic acid signaling

Pre‐harvest sprouting (PHS) is one of the major problems in cereal production worldwide, which causes significant losses of both yield and quality; however, the molecular mechanism underlying PHS remains largely unknown. Here, we identified a dominant PHS mutant phs9‐D. The corresponding gene PHS9 encodes a higher plant unique CC‐type glutaredoxin and is specifically expressed in the embryo at the late embryogenesis stage, implying that PHS9 plays some roles in the late stage of seed development. Yeast two‐hybrid screening showed that PHS9 could interact with OsGAP, which is an interaction partner of the abscicic acid (ABA) receptor OsRCAR1. PHS9‐ or OsGAP overexpression plants showed reduced ABA sensitivity in seed germination, whereas PHS9 or OsGAP knock‐out mutant plants showed increased ABA sensitivity in seed germination, suggesting that PHS9 and OsGAP acted as negative regulators in ABA signaling during seed germination. Interestingly, the germination of PHS9 and OsGAP overexpression or knock‐out plant seeds was weakly promoted by H₂O₂, implying that PHS9 and OsGAP could affect reactive oxygen species (ROS) signaling during seed germination. These results indicate that PHS9 plays an important role in the regulation of rice PHS through the integration of ROS signaling and ABA signaling.     

Comparative genetic analysis of a wheat seed dormancy QTL with rice and <Emphasis Type="Italic">Brachypodium</Emphasis> identifies candidate genes for ABA perception and calcium signaling

Wheat preharvest sprouting (PHS) occurs when seed germinates on the plant before harvest resulting in reduced grain quality. In wheat, PHS susceptibility is correlated with low levels of seed dormancy. A previous mapping of quantitative trait loci (QTL) revealed a major PHS/seed dormancy QTL, QPhs.cnl-2B.1, located on wheat chromosome 2B. A comparative genetic study with the related grass species rice (Oryza sativa L.) and Brachypodium distachyon at the homologous region to the QPhs.cnl-2B.1 interval was used to identify the candidate genes for marker development and subsequent fine mapping. Expressed sequence tags and a comparative mapping were used to design 278 primer pairs, of which 22 produced polymorphic amplicons that mapped to the group 2 chromosomes. Fourteen mapped to chromosome 2B, and ten were located in the QTL interval. A comparative analysis revealed good macrocollinearity between the PHS interval and 3 million base pair (mb) region on rice chromosomes 7 and 3, and a 2.7-mb region on Brachypodium Bd1. The comparative intervals in rice were found to contain three previously identified rice seed dormancy QTL. Further analyses of the interval in rice identified genes that are known to play a role in seed dormancy, including a homologue for the putative Arabidopsis ABA receptor ABAR/GUN5. Additional candidate genes involved in calcium signaling were identified and were placed in a functional protein association network that includes additional proteins critical for ABA signaling and germination. This study provides promising candidate genes for seed dormancy in both wheat and rice as well as excellent molecular markers for further comparative and fine mapping.     

Cloning and expression of a sorghum gene with homology to maize vp1. Its potential involvement in pre-harvest sprouting resistance

Pre-harvest sprouting (PHS) in sorghum is related to the lack of a normal dormancy level during seed development and maturation. Based on previous evidence that seed dormancy in maize is controlled by the vp1 gene, we used a PCR-based approach to isolate two Sorghum bicolor genomic and cDNA clones from two genotypes exhibiting different PHS behaviour and sensitivity to abscisic acid (ABA). The two 699 amino acid predicted protein sequences differ in two residues at positions 341 (Gly or Cys within the repression domain) and 448 (Pro or Ser) and show over 80, 70 and 60% homology to maize, rice and oat VP1 proteins respectively.Expression analysis of the sorghum vp1 gene in the two lines shows a slightly higher level of vp1 mRNA in the embryos susceptible to PHS than in those resistant to PHS during embryogenesis. However, timing of expression was different between these genotypes during this developmental process. Whereas for the former the main peak of expression was observed at 20 days after pollination (DAP), the peak in the latter was found at later developmental stages when seed maturation was almost complete.Under favourable germination conditions and in the presence of fluridone (an inhibitor of ABA biosynthesis), sorghum vp1 mRNA showed to be consistently correlated with sensitivity to ABA but not with ABA content and dormancy.     

QTL mapping of pre-harvest sprouting resistance in a white wheat cultivar Danby

Key message

One major and three minor QTLs for resistance to pre-harvest sprouting (PHS) were identified from a white wheat variety “Danby.” The major QTL on chromosome 3A is TaPHS1, and the sequence variation in its promoter region was responsible for the PHS resistance. Additive?×?additive effects were detected between two minor QTLs on chromosomes 3B and 5A, which can greatly enhance the PHS resistance.

Abstract

Pre-harvest sprouting (PHS) causes significant losses in yield and quality in wheat. White wheat is usually more susceptible to PHS than red wheat. Therefore, the use of none grain color-related PHS resistance quantitative trait loci (QTLs) is essential for the improvement in PHS resistance in white wheat. To identify PHS resistance QTLs in the white wheat cultivar “Danby” and determine their effects, a doubled haploid population derived from a cross of Danby?×?“Tiger” was genotyped using genotyping-by-sequencing markers and phenotyped for PHS resistance in two greenhouse and one field experiments. One major QTL corresponding to a previously cloned gene, TaPHS1, was consistently detected on the chromosome arm 3AS in all three experiments and explained 21.6–41.0% of the phenotypic variations. A SNP (SNP?222) in the promoter of TaPHS1 co-segregated with PHS in this mapping population and was also significantly associated with PHS in an association panel. Gene sequence comparison and gene expression analysis further confirmed that SNP?222 is most likely the causal mutation in TaPHS1 for PHS resistance in Danby in this study. In addition, two stable minor QTLs on chromosome arms 3BS and 5AL were detected in two experiments with allele effects consistently contributed by Danby, while one minor QTL on 2AS was detected in two environments with contradicted allelic effects. The two stable minor QTLs showed significant additive?×?additive effects. The results demonstrated that pyramiding those three QTLs using breeder-friendly KASP markers developed in this study could greatly improve PHS resistance in white wheat.

     

Discovery of loci determining pre-harvest sprouting and dormancy in wheat and barley applying segregation and association mapping

Three wheat and two barley populations were studied in order to find loci responsible for dormancy and pre-harvest sprouting. A classical quantitative trait loci analysis was combined with an association mapping approach. Many quantitative trait loci and marker trait associations could be detected on all seven chromosome groups of wheat and on the chromosomes 2H, 3H, 5H, 6H, and 7H of barley. Especially, the known regions on chromosomes 3A and 4A for wheat and 5H for barley were confirmed. Putative functions could be found via a candidate homologues search and via expressed sequence tag annotation. On chromosome 3A, the viviparous1 gene is located which is associated to preharvest sprouting and dormancy. On chromosome 4A, a protein is detected which belongs to the aquaporin family. In barley, an association with the aleurain gene on chromosome 5H was found. The expression of aleurain is regulated by abscisic acid and gibberelic acid. An influence of both hormones on dormancy and pre-harvest sprouting is known. It can be concluded that dormancy and pre-harvest sprouting are very complex traits regulated by multigenes and/or quantitative trait loci.     

A new PCR-based marker on chromosome 4AL for resistance to pre-harvest sprouting in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Pre-harvest sprouting (PHS) is a complex trait controlled by multiple genes with strong interaction between environment and genotype that makes it difficult to select breeding materials by phenotypic assessment. One of the most important genes for pre-harvest sprouting resistance is consistently identified on the long arm of chromosome 4A. The 4AL PHS tolerance gene has therefore been targeted by Australian white-grained wheat breeders. A new robust PCR marker for the PHS QTL on wheat chromosome 4AL based on candidate genes search was developed in this study. The new marker was mapped on 4AL deletion bin 13-0.59-0.66 using 4AL deletion lines derived from Chinese Spring. This marker is located on 4AL between molecular markers Xbarc170 and Xwg622 in the doubled-haploid wheat population Cranbrook × Halberd. It was mapped between molecular markers Xbarc170 and Xgwm269 that have been previously shown to be closely linked to grain dormancy in the doubled haploid wheat population SW95-50213 × Cunningham and was co-located with Xgwm269 in population Janz × AUS1408. This marker offers an additional efficient tool for marker-assisted selection of dormancy for white-grained wheat breeding. Comparative analysis indicated that the wheat chromosome 4AL QTL for seed dormancy and PHS resistance is homologous with the barley QTL on chromosome 5HL controlling seed dormancy and PHS resistance. This marker will facilitate identification of the gene associated with the 4A QTL that controls a major component of grain dormancy and PHS resistance.     

Carotenoid deficiency impairs ABA and IAA biosynthesis and differentially affects drought and cold tolerance in rice

Plant responses to abiotic stresses are coordinated by arrays of growth and developmental programs. Phytohormones such as abscisic acid (ABA) and indole-3-acetic acid (IAA) play critical roles in developmental progresses and environmental responses through complex signalling networks. However, crosstalk between the two hormones at the biosynthesis level remains largely unknown. Here, we report that carotenoid-deficient mutants (phs1, phs2, phs3-1, phs4, and PDS-RNAi transgenic rice) were impaired in the biosynthesis of ABA and IAA. Under drought conditions, phs3-1 and PDS-RNAi transgenic rice showed larger stomata aperture and earlier wilting compared to the wild type at both seedling and panicle developmental stage. Interestingly, these carotenoid-deficient lines showed increased cold resistance, which was likely due to the combined effects of reduced IAA content, alleviated oxidative damage and decreased membrane penetrability. Furthermore, we found that IAA content was significantly declined in rice treated with fluridone (a carotenoid and ABA biosynthesis inhibitor), and expression of auxin synthesis and metabolism-related genes were altered in the fluridone-treated rice similar to that in the carotenoid-deficient mutants. In addition, exogenous IAA, but not ABA, could restore the dwarf phenotype of phs3-1 and PDS-RNAi transgenic rice. These results support a crosstalk between ABA and IAA at the biosynthesis level, and this crosstalk is involved in development and differentially affects drought and cold tolerance in rice.     

A major QTL controlling seed dormancy and pre-harvest sprouting resistance on chromosome 4A in a Chinese wheat landrace

Wheat pre-harvest sprouting (PHS) can cause significant reduction in yield and end-use quality of wheat grains in many wheat-growing areas worldwide. To identify a quantitative trait locus (QTL) for PHS resistance in wheat, seed dormancy and sprouting of matured spikes were investigated in a population of 162 recombinant inbred lines (RILs) derived from a cross between the white PHS-resistant Chinese landrace Totoumai A and the white PHS-susceptible cultivar Siyang 936. Following screening of 1,125 SSR primers, 236 were found to be polymorphic between parents, and were used to screen the mapping population. Both seed dormancy and PHS of matured spikes were evaluated by the percentage of germinated kernels under controlled moist conditions. Twelve SSR markers associated with both PHS and seed dormancy were located on the long arm of chromosome 4A. One QTL for both seed dormancy and PHS resistance was detected on chromosome 4AL. Two SSR markers, Xbarc 170 and Xgwm 397, are 9.14 cM apart, and flanked the QTL that explained 28.3% of the phenotypic variation for seed dormancy and 30.6% for PHS resistance. This QTL most likely contributed to both long seed dormancy period and enhanced PHS resistance. Therefore, this QTL is most likely responsible for both seed dormancy and PHS resistance. The SSR markers linked to the QTL can be used for marker-assisted selection of PHS-resistant white wheat cultivars. Shi-Bin Cai and Cui-Xia Chen contributed equally to this work.     

Genetic variation of Vp1 in Sichuan wheat accessions and its association with pre-harvest sprouting response

Pre-harvest sprouting (PHS) in bread wheat is a major abiotic constraint reducing yield and influencing the production of high quality grain. In China both spring and winter wheat regions are affected by PHS. Sichuan lies in southwest China, where the most of rainfall occurs during April to September when wheat is harvested. The present investigation was conducted to identify the allelic variability of Vp1, a gene that plays a role in maintenance and induction of dormancy, among Sichuan landraces and recent cultivars with different dormancy levels and to find potential sources of PHS resistance for breeding. Sichuan landrace and cultivar wheat accessions had a wide range of dormancy levels. The average germination index (GI) of Sichuan landrace accessions was 0.232, whereas at 0.674 it was much higher for cultivars. The different dormancy levels between landraces and cultivars indicated that pre-harvest sprouting resistance might have been neglected in recent Sichuan wheat breeding programs. The average GI of white grained accessions was higher than for red grained accessions. Particular Vp-1B gene fragments were specific in landraces or cultivars and in white or red grained accessions. The results indicated that Vp-1B markers could be used to distinguish cultivars and landraces. Significant relationships between certain Vp-1B allelesand GI of Sichuan wheat accessions were shown by Spearman’s rank correlation analysis.     

Disruption of the Homogentisate Solanesyltransferase Gene Results in Albino and Dwarf Phenotypes and Root,Trichome and Stomata Defects in Arabidopsis thaliana

Homogentisate solanesyltransferase (HST) plays an important role in plastoquinone (PQ) biosynthesis and acts as the electron acceptor in the carotenoids and abscisic acid (ABA) biosynthesis pathways. We isolated and identified a T-DNA insertion mutant of the HST gene that displayed the albino and dwarf phenotypes. PCR analyses and functional complementation also confirmed that the mutant phenotypes were caused by disruption of the HST gene. The mutants also had some developmental defects, including trichome development and stomata closure defects. Chloroplast development was also arrested and chlorophyll (Chl) was almost absent. Developmental defects in the chloroplasts were consistent with the SDS-PAGE result and the RNAi transgenic phenotype. Exogenous gibberellin (GA) could partially rescue the dwarf phenotype and the root development defects and exogenous ABA could rescue the stomata closure defects. Further analysis showed that ABA and GA levels were both very low in the pds2-1 mutants, which suggested that biosynthesis inhibition by GAs and ABA contributed to the pds2-1 mutants'' phenotypes. An early flowering phenotype was found in pds2-1 mutants, which showed that disruption of the HST gene promoted flowering by partially regulating plant hormones. RNA-sequencing showed that disruption of the HST gene resulted in expression changes to many of the genes involved in flowering time regulation and in the biosynthesis of PQ, Chl, GAs, ABA and carotenoids. These results suggest that HST is essential for chloroplast development, hormone biosynthesis, pigment accumulation and plant development.     

Physiological basis of pre-harvest sprouting resistance in Sorghum bicolor (L.) Moench. ABA levels and sensitivity in developing embryos of sprouting-resistant and -susceptible varieties

Pre–harvest sprouting is a major problem in Sorghum cropswhich leads to losses in seed viability and produces importantdecreases in grain weight. In this paper we aimed to have aninsight into the physiological basis of pre-harvest sproutingresistance in this crop by assessing germinability, ABA embryoniccontent and embryonic sensitivity to ABA during seed developmentin three varieties presenting contrasting sprouting behaviour:Redland B2 (very susceptible), SC 650 (moderately resistant)and IS 9530 (very resistant). Redland B2 caryopses were ableto germinate with high germination indices from early stagesof development, while caryopses from IS 9530 did not presentgermination indices different from 0 until near physiologicalmaturity. SC 650 (moderately resistant) grains presented anintermediate pattern of behaviour. In all three varieties isolatedembryos were able to germinate with maximum germination indicesfrom as early as 15 d after pollination (DAP). Differences ingrain dormancy level were not paralleled by a consistently differentendogenous ABA content throughout maturation. However, whenABA embryonic content was measured in incubated 35 DAP caryopses,ABA level in B2 embryos after 24 h of incubation was found tobe less than half that observed in IS 9530 embryo after thesame period of incubation. In addition, B2 embryos were foundto be 10–fold less sensitive to the inhibitory effectof ABA than embryos from the other two varieties. These resultsexplain to a considerable extent differences in germinabilitybetween sproutingresistant and –susceptible varietiesand are consistent with differences in sprouting behaviour. Key words: Sorghum bicolor, abscisic acid, pre-harvest sprouting, seed development, germination