Gene expression related to seed shattering and the cell wall in cultivated and weedy rice

Seed shattering is an evolutionary trait that is essential to the survival of wild and weedy rice. Discovery of the qSH1 gene in rice subspecies Japonica and Sh4 in the rice subspecies Indica indicated the possibility that seed shattering is governed by major genes in a qualitative manner. However, observation of the large variability of seed shattering in weedy rice has led us to hypothesise that other genes related to abscission layer integrity could also be important in the regulation of seed shattering in rice. Gene expression 10 days after pollination and nucleotide composition revealed that qSH1 and Sh4 that are described as major players in seed shattering were not important in weedy rice. High expression of the gene OsCPL1 was positively associated with the occurrence of high seed shattering in weedy rice, which did not concur in previous studies of cultivated rice. This result is related to the absence of four SNPs and an indel in the OsCPL1 gene in weedy rice that are related to seed shattering in previous studies. Analysis of the expression of six genes related to cell wall synthesis/degradation revealed the importance of the genes OsXTH8 and OsCel9D in seed shattering in weedy rice. Therefore, in addition to qSH1 and Sh4, the genes OsCPL1, OsXTH8 and OsCel9D should be considered in studies of rice evolution and in the development of mitigation approaches of gene flow in transgenic rice.     

On the role of abscisic acid in seed dormancy of red rice

Abscisic acid (ABA) is commonly assumed to be the primary effector of seed dormancy, but conclusive evidence for this role is lacking. This paper reports on the relationships occurring in red rice between ABA and seed dormancy. Content of free ABA in dry and imbibed caryopses, both dormant and after-ripened, the effects of inhibitors, and the ability of applied ABA to revert dormancy breakage were considered. The results indicate: (i) no direct correlation of ABA content with the dormancy status of the seed, either dry or imbibed; (ii) different sensitivity to ABA of non-dormant seed and seed that was forced to germinate by fluridone; and (iii) an inability of exogenous ABA to reinstate dormancy in fluridone-treated seed, even though applied at a pH which favoured high ABA accumulation. These considerations suggest that ABA is involved in regulating the first steps of germination, but unidentified developmental effectors that are specific to dormancy appear to stimulate ABA synthesis and to enforce the responsiveness to this phytohormone. These primary effectors appear physiologically to modulate dormancy and via ABA they effect the growth of the embryo. Therefore, it is suggested that ABA plays a key role in integrating the dormancy-specific developmental signals with the control of growth.     

Nucleotide variability and gene expression reveal new putative genes related to seed shattering in weedy rice

Seed shattering is one of the main traits related with the domestication of cultivated rice and with the invasiveness and persistence of weedy rice. Two independent studies in 2006 have indicated that qSH1 in Japonica and Sh4 in Indica rice are major genes governing this trait. However, a wide variation of seed shattering occurs in weedy rice ecotypes from the same geographic region and even within the same ecotype. The aim of this study was to evaluate the nucleotide variability of known and putative genes related to seed shattering in cultivated rice and to identify and validate new genes related to this trait in weedy rice. The qSH1 gene was not associated with seed shattering in the evaluated genotypes. The nucleotide variability of the genes Os01g0849100 and Os08g0512400, previously identified based on a genome‐wide resequencing study, was related to seed shattering in rice. The nucleotide variability of three single nucleotide polymorphisms (SNPs) of the OsXTH8 gene, which is related to cell wall biosynthesis, was not associated with seed shattering. However, the high expression of this gene was related to the occurrence of this trait. This study evaluated jointly a series of genes involved in rice seed shattering and indicated that the genes OsXTH8, Os08g0512400 and Os01g0849100 are important for the regulation of this trait in weedy rice in addition to previously described genes. Seed shattering in weedy rice has a more complex regulation than in cultivated rice where few major genes were identified.     

Genomic regions affecting seed shattering and seed dormancy in rice

Non-shattering of the seeds and reduced seed dormancy were selected consciously and unconsciously during the domestication of rice, as in other cereals. Both traits are quantitative and their genetic bases are not fully elucidated, though several genes with relatively large effects have been identified. In the present study, we attempted to detect genomic regions associated with shattering and dormancy using 125 recombinant inbred lines obtained from a cross between cultivated and wild rice strains. A total of 147 markers were mapped on 12 rice chromosomes, and QTL analysis was performed by simple interval mapping and composite interval mapping. For seed shattering, two methods revealed the same four QTLs. On the other hand, for seed dormancy a number of QTLs were estimated by the two methods. Based on the results obtained with the intact and de-hulled seeds, QTLs affecting hull-imposed dormancy and kernel dormancy, respectively, were estimated. Some QTLs detected by simple interval mapping were not significant by composite interval mapping, which reduces the effects of residual variation due to the genetic background. Several chromosomal regions where shattering QTLs and dormancy QTLs are linked with each other were found. This redundancy of QTL associations was explained by ”multifactorial linkages” followed by natural selection favoring these two co-adapted traits. Received: 23 November 1998 / Accepted: 27 August 1999     

Molecular evolution of shattering loci in U.S. weedy rice

Cultivated rice fields worldwide are plagued with weedy rice, a conspecific weed of cultivated rice (Oryza sativa L.). The persistence of weedy rice has been attributed, in part, to its ability to shatter (disperse) seed prior to crop harvesting. In the United States, separately evolved weedy rice groups have been shown to share genomic identity with exotic domesticated cultivars. Here, we investigate the shattering phenotype in a collection of U.S. weedy rice accessions, as well as wild and cultivated relatives. We find that all U.S. weedy rice groups shatter seeds easily, despite multiple origins, and in contrast to a decrease in shattering ability seen in cultivated groups. We assessed allelic identity and diversity at the major shattering locus, sh4, in weedy rice; we find that all cultivated and weedy rice, regardless of population, share similar haplotypes at sh4, and all contain a single derived mutation associated with decreased seed shattering. Our data constitute the strongest evidence to date of an evolution of weeds from domesticated backgrounds. The combination of a shared cultivar sh4 allele and a highly shattering phenotype, suggests that U.S. weedy rice have re‐acquired the shattering trait after divergence from their progenitors through alternative genetic mechanisms.     

Sequence polymorphisms in wild,weedy, and cultivated rice suggest seed‐shattering locus sh4 played a minor role in Asian rice domestication

The predominant view regarding Asian rice domestication is that the initial origin of nonshattering involved a single gene of large effect, specifically, the sh4 locus via the evolutionary replacement of a dominant allele for shattering with a recessive allele for reduced shattering. Data have accumulated to challenge this hypothesis. Specifically, a few studies have reported occasional seed‐shattering plants from populations of the wild progenitor of cultivated rice (Oryza rufipogon complex) being homozygous for the putative “nonshattering” sh4 alleles. We tested the sh4 hypothesis for the domestication of cultivated rice by obtaining genotypes and phenotypes for a diverse set of samples of wild, weedy, and cultivated rice accessions. The cultivars were fixed for the putative “nonshattering” allele and nonshattering phenotype, but wild rice accessions are highly polymorphic for the putative “nonshattering” allele (frequency ~26%) with shattering phenotype. All weedy rice accessions are the “nonshattering” genotype at the sh4 locus but with shattering phenotype. These data challenge the widely accepted hypothesis that a single nucleotide mutation (“G”/“T”) of the sh4 locus is the major driving force for rice domestication. Instead, we hypothesize that unidentified shattering loci are responsible for the initial domestication of cultivated rice through reduced seed shattering.     

Antagonism between abscisic acid and gibberellins is partially mediated by ascorbic acid during seed germination in rice

The antagonism between abscisic acid (ABA) and gibberellin (GA) plays a key role in controlling seed germination,^1,2 but the mechanism of antagonism during this process is not known. In the associated study,³ we investigated the relationship among ABA, reactive oxygen species (ROS), ascorbic acid (ASC) and GA during rice seed germination. ROS production is reduced by ABA, which hence results in decreasing ASC accumulation during imbibition. GA accumulation was also suppressed by a reduced ROS and ASC level, whereas application of exogenous ASC can partially rescue seed germination from ABA treatment. Further results show that production of ASC, which acts as a substrate in GA biosynthesis, was significantly inhibited by lycorine which thus suppressed the accumulation of GA. Consequently, expression of GA biosynthesis genes was suppressed by the low levels of ROS and ASC in ABA-treated seeds. These studies reveal a new role for ASC in mediating the antagonism between ABA and GA during seed germination in rice.     

中国杂草稻种群的发芽期耐冷性研究

以中国75个杂草稻种群及其对应采样田的水稻品种为试验材料,研究了不同处理(破除休眠与不破除休眠、常温25℃与低温15℃、7 d测定和14 d测定)对种子发芽率的影响。结果表明,杂草稻的发芽率与对应采样点水稻品种的发芽率呈现极显著相关性。杂草稻破除休眠处理的发芽率显著或极显著高于不破除休眠处理。破除休眠与不破除休眠下,15℃处理的杂草稻种子发芽率均显著高于对应采样田的水稻品种,证明杂草稻相对于采样田水稻品种具有更强的耐冷性,可能进化出了新的耐冷性机制。25℃处理下杂草稻和对应水稻品种的发芽率均与纬度呈显著或极显著负相关,表明休眠性有随着纬度的降低而减弱的趋势。     

Ascorbic acid and reactive oxygen species are involved in the inhibition of seed germination by abscisic acid in rice seeds

The antagonism between abscisic acid (ABA) and gibberellin (GA) plays a key role in controlling seed germination, but the mechanism of antagonism during this process is not known. The possible links among ABA, reactive oxygen species (ROS), ascorbic acid (ASC), and GA during rice seed germination were investigated. Unlike in non-seed tissues where ROS production is increased by ABA, ABA reduced ROS production in imbibed rice seeds, especially in the embryo region. Such reduced ROS also led to an inhibition of ASC production. GA accumulation was also suppressed by a reduced ROS and ASC level, which was indicated by the inhibited expression of GA biosynthesis genes, amylase genes, and enzyme activity. Application of exogenous ASC can partially rescue seed germination from ABA treatment. Production of ASC, which acts as a substrate in GA biosynthesis, was significantly inhibited by lycorine which thus suppressed the accumulation of GA. Consequently, expression of GA biosynthesis genes was suppressed by the low levels of ROS and ASC in ABA-treated seeds. It can be concluded that ABA regulates seed germination in multiple dimensions. ROS and ASC are involved in its inhibition of GA biosynthesis.     

Proteomic analysis of phosphoproteins regulated by abscisic acid in rice leaves

Abscisic acid (ABA) is a hormone that regulates plant development and adaptation to environmental stresses. Protein phosphorylation has been recognized as an important mechanism for ABA signaling. However, the target phosphoproteins regulated by ABA are still largely unknown. Here, we report the identification of ABA-regulated phosphoproteins in rice using proteomic approaches. Six ABA-regulated phosphoproteins were identified as G protein beta subunit-like protein, ascorbate peroxidase, manganese superoxide dismutase, triosephosphate isomerase, putative Ca²⁺/H⁺ antiporter regulator protein, and glyoxysomal malate dehydrogenase. These results provide new insight into the regulatory mechanism for some ABA signaling proteins and implicate several previously unrecognized proteins in ABA action.     

Genetic analysis of adaptive syndromes interrelated with seed dormancy in weedy rice (Oryza sativa)

Seed dormancy in rice interrelates to the weedy characteristics shattering, awn, black hull color, and red pericarp color. A cross between the weedy strain SS18-2 and the breeding line EM93-1 was developed to investigate the genetic basis and adaptive significance of these interrelationships. These characteristics or their components differed in average degree of dominance from –0.8 to 1.5, in heritability from 0.5 to 0.96, and in their contribution to phenotypic or genotypic variation in dormancy by up to 25%. Five dormancy, four shattering, and three awn-length quantitative trait loci (QTLs) were detected in the BC₁ population replicated in 2 years. Two QTLs for hull color were identified, and the SS18-2-derived and EM93-1-derived alleles increased the intensity of black, and red or yellow pigmentations, respectively. The only QTL for pericarp color co-located with the red pericarp gene Rc, with the SS18-2-derived allele increasing the intensity of black and red pigmentations. Four of the five dormancy QTLs were flanked or bracketed by one to four QTLs for the interrelated characteristics. The QTL organization pattern indicates the central role of seed dormancy in adaptive syndromes for non-domesticated plants, implies that the elimination of dormancy from cultivars could arise from the selections against multiple interrelated characteristics, and challenges the use of dormancy genes at these loci in breeding varieties for resistance to pre-harvest sprouting (PHS). However, another QTL (qSD12) provides candidate gene(s) for PHS resistance because it has a large effect in the population and it is independent of the loci for interrelated characteristics.     

OsGRF4 controls grain shape,panicle length and seed shattering in rice

Traits such as grain shape, panicle length and seed shattering, play important roles in grain yield and harvest. In this study, the cloning and functional analysis of PANICLE TRAITS 2 (PT2), a novel gene from the Indica rice Chuandali (CDL), is reported. PT2 is synonymous with Growth‐Regulating Factor 4 (OsGRF4), which encodes a growth‐regulating factor that positively regulates grain shape and panicle length and negatively regulates seed shattering. Higher expression of OsGRF4 is correlated with larger grain, longer panicle and lower seed shattering. A unique OsGRF4 mutation, which occurs at the OsmiRNA396 target site of OsGRF4, seems to be associated with high levels of OsGRF4 expression, and results in phenotypic difference. Further research showed that OsGRF4 regulated two cytokinin dehydrogenase precursor genes (CKX5 and CKX1) resulting in increased cytokinin levels, which might affect the panicle traits. High storage capacity and moderate seed shattering of OsGRF4 may be useful in high‐yield breeding and mechanized harvesting of rice. Our findings provide additional insight into the molecular basis of panicle growth.     

GC-MS identification of abscisic acid and abscisic acid metabolites in seed of Vigna unguiculata

Abscisic acid, phaseic acid and 4′-dihydrophaseic acid were identified by GC-MS of derivatized (Me, MeTMSi) extracts from immature fruits of Vigna unguiculata. The fruits also contained some other ABA-related compounds, one of which might be epi-4′-dihydrophaseic acid while another was tentatively identified as 6′-hydroxymethylabscisic acid.     

Seed dormancy and germination: the role of abscisic acid and gibberellins and the importance of hormone mutants

Over the past decades many studies have aimed at elucidating the regulation of seed dormancy and germination. Many hypotheses have been proposed and rejected but the regulatory principle behind changes in dormancy and induction of germination is still a black box. The majority of proposed mechanisms have a role for certain plant hormones in common. Abscisic acid and the gibberellins are the hormones most frequently suggested to control these processes. The development of hormone-deficient mutants made it possible to provide direct evidence for the involvement of hormones in germination and dormancy related processes.In the present paper an attempt is made to assess the role of abscisic acid and gibberellins in the transitions between dormant and non-dormant states and germination. First a conceptual framework is presented in which the different states of dormancy and germination are defined in order to contribute to a solution of the semantic confusion about these terms that has existed since the beginning of seed physiology.It is concluded that abscisic acid plays a pivotal role during the development of primary dormancy and gibberellins are involved in the induction of germination. Changes in sensitivity to these hormones occur during changes in dormancy. Both synthesis of and responsiveness to the hormones are controlled by natural environmental factors such as light, temperature and nitrate.     

Inactivation of the CTD phosphatase-like gene OsCPL1 enhances the development of the abscission layer and seed shattering in rice

Although susceptibility to seed shattering causes severe yield loss during cereal crop harvest, it is an adaptive trait for seed dispersal in wild plants. We previously identified a recessive shattering locus, sh-h , from the rice shattering mutant line Hsh that carries an enhanced abscission layer. Here, we further mapped sh-h to a 34-kb region on chromosome 7 by analyzing 240 F₂ plants and five F₃ lines from the cross between Hsh and Blue&Gundil. Hsh had a point mutation at the 3' splice site of the seventh intron within LOC_Os07g10690, causing a 15-bp deletion of its mRNA as a result of altered splicing. Two transferred DNA (T-DNA) insertion mutants and one point mutant exhibited the enhanced shattering phenotype, confirming that LOC_Os07g10690 is indeed the sh-h gene. RNA interference (RNAi) transgenic lines with suppressed expression of this gene exhibited greater shattering. This gene, which encodes a protein containing a conserved carboxy-terminal domain (CTD) phosphatase domain, was named Oryza sativa CTD phosphatase-like 1 ( OsCPL1 ). Subcellular localization and biochemical analysis revealed that the OsCPL1 protein is a nuclear phosphatase, a common characteristic of metazoan CTD phosphatases involved in cell differentiation. These results demonstrate that OsCPL1 represses differentiation of the abscission layer during panicle development.