ABSTRACT
MAIN CONCLUSION: Excess of KRP4 in the developing kernels in rice causes poor filling of the grains possibly through inhibition of CDKA;2 and CDKB;1 activity mediated by its interaction with CDKF;3. The potential yield of the rice varieties producing compact and heavy panicles bearing numerous spikelets is compromised because a high percentage of spikelets remain poorly filled, reportedly because of a high expression of KRPs that causes suppression of endosperm cell proliferation. To test the stated negative relationship between KRP expression and grain filling, Orysa;KRP4 was overexpressed under the control of seed-specific glutelin promoter in IR-64 rice variety that shows good grain filling. The transgenic lines showed more than 15-fold increase in expression of KRP4 in the spikelets concomitant with nearly 50% reduction in grain filling compared with the wild type without producing any significant changes on the other yield-related parameters like panicle length and the spikelets numbers that were respectively 30.23 ± 0.89 cm and 229.25 ± 33.72 per panicle in the wild type, suggesting a highly organ-targeted effect of the genetic transformation. Yeast two-hybrid test revealed CDKF;3 as the interacting partner of KRP4, and CDKF;3 was found to interact with CDKA;2, CDKB;1 and CDKD;1. Significant decrease in grain filling in the transgenic lines compared with the wild type due to overexpression of KRP4 could be because of suppression of the activity of CDKB;1 and CDKA;2 by inhibition of their phosphorylation directly by CDKF;3, or mediated through inhibition of phosphorylation of CDKD;1 by CDKF;3. The study thus indicated that suppression of expression of KRP(s) by genetic manipulation of their promoters could be an important way of improving the yield of the rice varieties bearing compact and heavy panicles.
Subject(s)
Edible Grain , Gene Expression Regulation, Plant , Oryza , Plant Proteins , Plants, Genetically Modified , Seeds , Oryza/genetics , Oryza/metabolism , Oryza/growth & development , Plant Proteins/genetics , Plant Proteins/metabolism , Seeds/genetics , Seeds/growth & development , Seeds/metabolism , Edible Grain/genetics , Edible Grain/growth & development , Edible Grain/metabolism , Promoter Regions, Genetic/genetics , Two-Hybrid System Techniques , Cyclin-Dependent Kinases/metabolism , Cyclin-Dependent Kinases/geneticsABSTRACT
The demand for rice is likely to increase approximately 1.5 times by the year 2050. In contrast, the rice production is stagnant since the past decade as the ongoing rice breeding program is unable to increase the production further, primarily because of the problem in grain filling. Investigations have revealed several reasons for poor filling of the grains in the inferior spikelets of the compact panicle, which are otherwise genetically competent to develop into well-filled grains. Among these, the important reasons are 1) poor activities of the starch biosynthesizing enzymes, 2) high ethylene production leading to inhibition in expressions of the starch biosynthesizing enzymes, 3) insufficient division of the endosperm cells and endoreduplication of their nuclei, 4) low accumulation of cytokinins and indole-3-acetic acid (IAA) that promote grain filling, and 5) altered expressions of the miRNAs unfavorable for grain filling. At the genetic level, several genes/QTLs linked to the yield traits have been identified, but the information so far has not been put into perspective toward increasing the rice production. Keeping in view the genetic competency of the inferior spikelets to develop into well-filled grains and based on the findings from the recent research studies, improving grain filling in these spikelets seems plausible through the following biotechnological interventions: 1) spikelet-specific knockdown of the genes involved in ethylene synthesis and overexpression of ß-CAS (ß-cyanoalanine) for enhanced scavenging of CN- formed as a byproduct of ethylene biosynthesis; 2) designing molecular means for increased accumulation of cytokinins, abscisic acid (ABA), and IAA in the caryopses; 3) manipulation of expression of the transcription factors like MYC and OsbZIP58 to drive the expression of the starch biosynthesizing enzymes; 4) spikelet-specific overexpression of the cyclins like CycB;1 and CycH;1 for promoting endosperm cell division; and 5) the targeted increase in accumulation of ABA in the straw during the grain filling stage for increased carbon resource remobilization to the grains. Identification of genes determining panicle compactness could also lead to an increase in rice yield through conversion of a compact-panicle into a lax/open one. These efforts have the ability to increase rice production by as much as 30%, which could be more than the set production target by the year 2050.
ABSTRACT
The world's increase in rice (Oryza sativa L.) production is not keeping up with the increase in its population. To boost the introduction of new high-yielding cultivars, knowledge is being gained on the genes and quantitative trait loci (QTLs) determining the panicle phenotype. The important are those determining yield of the crop, such as grain numbers per panicle and size and weight of the grains. Biochemical and molecular functions of many of them are understood in some details. Among these, OsCKX2 and OsSPL14 have been shown to increase panicle branching and grain numbers when overexpressed. Furthermore, miRNAs appear to play an important role in determining the panicle morphology by regulating the expressions of the genes like OsSPL14 and GRF4 involved in panicle branching and grain numbers and length. Mutations also greatly influence the grain shape and size. However, the information gained so far on the genetic regulation of grain filling and panicle morphology has not been successfully put into commercial application. Furthermore, the identification of the gene(s)/QTLs regulating panicle compactness is still lacking, which may enable the researchers to convert a compact-panicle cultivar into a lax/open one, and thereby increasing the chances of enhancing the yield of a desired compact-panicle cultivar obtained by the breeding effort.
Subject(s)
Oryza , Edible Grain/genetics , Gene Expression Regulation, Plant/genetics , Oryza/genetics , Plant Breeding , Quantitative Trait Loci/geneticsABSTRACT
The increase in much required rice production through breeding programmes is on decline. The primary reason being poor filling of grains in the basal spikelets of the heavy and compact panicle rice developed. These spikelets are genetically competent to develop into well filled grains, but fail to do so because the carbohydrate assimilates available to them remain unutilized, reportedly due to poor activities of the starch biosynthesizing enzymes, high production of ethylene leading to enhanced synthesis of the downstream signaling component RSR1 protein that inhibits GBSS1 activity, poor endosperm cell division and endoreduplication of the endosperm nuclei, altered expression of the transcription factors influencing grain filling, enhanced expression and phosphorylation of 14-3-3 proteins, poor expression of the seed storage proteins, reduced synthesis of the hormones like cytokinins and IAA that promote grain filling, and altered expression of miRNAs preventing their normal role in grain filling. Since the basal spikelets are genetically competent to develop into well filled mature grains, biotechnological interventions in terms of spikelet-specific overexpression of the genes encoding enzymes involved in grain filling and/or knockdown/overexpression of the genes influencing the activities of the starch biosynthesizing enzymes, various cell cycle events and hormone biosynthesis could increase rice production by as much as 30%, much more than the set production target of 800 mmt. Application of these biotechnological interventions in the heavy and compact panicle cultivars producing grains of desired quality would also maintain the quality of the grains having demand in market besides increasing the rice production per se.
Subject(s)
Oryza , Edible Grain/metabolism , Endosperm , Oryza/genetics , Oryza/metabolism , Plant Breeding , Starch/metabolismABSTRACT
KEY MESSAGE: Drought stress response studies and overexpression of vun-miR408 proved it to be essential for abiotic stress tolerance in cowpea. Small RNA and transcriptome sequencing of an elite high-yielding drought-tolerant Indian cowpea cultivar, Pusa Komal revealed a differential expression of 198 highly conserved, 21 legume-specific, 14 less-conserved, and 10 novel drought-responsive microRNAs (miRNAs) along with 3391 (up-regulated) and 3799 (down-regulated) genes, respectively, in the leaf and root libraries. Among the differentially expressed miRNAs, vun-miR408-3p, showed an up-regulation of 3.53-log2-fold change under drought stress. Furthermore, laccase 12 (LAC 12) was identified as the potential target of vun-miR408-3p using 5' RNA ligase-mediated rapid amplification of cDNA ends. The stable transgenic cowpea lines overexpressing artificial vun-miR408-3p (OX-amiR408) displayed enhanced drought and salinity tolerance as compared to the wild-type plants. An average increase of 30.17% in chlorophyll, 26.57% in proline, and 27.62% in relative water content along with lesser cellular H2O2 level was observed in the transgenic lines in comparison with the wild-type plants under drought stress. Additionally, the scanning electron microscopic study revealed a decrease in the stomatal aperture and an increase in the trichome density in the transgenic lines. The expression levels of laccase 3 and laccase 12, the potential targets of miR408, related to lipid catabolic processes showed a significant reduction in the wild-type plants under drought stress and the transgenic lines, indicating the regulation of lignin content as a plausibly essential trait related to the drought tolerance in cowpea. Taken together, this study primarily focused on identification of drought-responsive miRNAs and genes in cowpea, and functional validation of role of miR408 towards drought stress response in cowpea.
Subject(s)
Droughts , MicroRNAs/genetics , RNA, Plant/genetics , Transcriptome/genetics , Vigna/physiology , Gene Expression Profiling , MicroRNAs/metabolism , RNA, Plant/metabolism , Sequence Analysis, RNAABSTRACT
The Teosinte branched 1/Cycloidea/Proliferating cell factor (TCP) transcription factors are potent growth and developmental regulators in plants, also responsive to various hormonal and environmental stimuli. In this study, we primarily focused on the functional role of TCP9, a nuclear-localised Class-I TCP transcription factor in a drought and heat-tolerant legume crop, cowpea (Vigna unguiculata). Under drought stress, a higher protein expression level of TCP9 was observed in the leaves of the drought-tolerant cowpea cultivar Pusa Komal as compared to the drought-sensitive cultivar TVu-7778. Further, overexpression of VuTCP9 resulted in reduced cell and stomata size, aperture length and width while cell and overall stomatal density in the 35S::VuTCP9 transgenic cowpea lines increased. Phenotypic alterations, such as reduced leaf size and vigour, altered seed coats displaying extension pattern similar to the 'Watson pattern' and delayed senescence were prominent in the transgenic lines. Under normal conditions, the gas exchange and fluorescence measurements indicated reduction in transpiration rate (E), stomatal conductance (gs ) and photosynthetic efficiency (Φ PSII). However, water usage efficiency (WUE) remained unaltered in the transgenic lines as compared to the wild-type (WT) plants. Furthermore, the transgenic lines displayed higher tolerance to oxidative, drought and salinity stress, maintained relatively higher relative water content and lower occurrence of H2 O2 , as compared to the WT plants. Genes related to the jasmonic acid biosynthesis, stomatal development and abiotic stress responsiveness, such as TTG1, NAC25, SPCH and GRP1, increased and LOX2 decreased significantly in the transgenic lines.
Subject(s)
Droughts , Vigna , Gene Expression Regulation, Plant , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified/genetics , Salt Tolerance/physiology , Stress, Physiological , Vigna/genetics , Vigna/metabolismABSTRACT
Development of rice cultivars bearing numerous spikelets by breeding approach to increase the yearly production of rice to approximately 800 million metric tons to feed the ever increasing population of the world accompanies poor grain filling in the inferior spikelets preventing achievement of the yield potential. As the initial stages of caryopses development are of much importance for grain filling, spatio-temporal expressions of the miRNAs were studied during these periods in the spikelets of a compact-panicle rice cultivar, Oryza sativa cv. Mahalaxmi, bearing numerous spikelets per panicle to understand the reason of poor grain filling at the level of the initial biochemical events. Differential expression of several known miRNAs between the superior and inferior spikelets suggested great difference in metabolism related to grain filling in the spikelets based on their spatial location on compact panicle. Expressions of five known and four novel miRNAs were validated by Northern. Their targets included the enzymes directly involved in starch biosynthesis like sucrose synthase, starch synthase and pullulanase, besides others. Spatio-temporal expression studies of these miRNAs in the spikelets of Mahalaxmi revealed a pattern of mostly a greater expression in the inferior spikelets compared with the superior ones concomitant with an inverse expression of the target genes, which was not observed in the lax-panicle cultivar Upahar. The study thus revealed that the grain filling in rice is greatly regulated by miRNAs, and these miRNAs or their target genes could be considered for biotechnological interventions for improving grain filling in the rice cultivars of interest.
Subject(s)
Edible Grain , Gene Expression Regulation, Plant , MicroRNAs , Oryza , Plant Proteins , Edible Grain/genetics , Edible Grain/growth & development , Edible Grain/metabolism , Gene Expression Profiling , MicroRNAs/genetics , Oryza/genetics , Oryza/growth & development , Plant Breeding , Plant Proteins/genetics , Plant Proteins/metabolismABSTRACT
Rice being a staple crop for human, its production is required to be increased significantly, particularly keeping in view the expected world's population of 9.6 billion by the year 2050. In this context, although the rice breeding programs have been successful in increasing the number of spikelets per panicle, the basal spikelets remain poorly filled, undermining the yield potential. The present study also found the grain filling to bear negative correlation with the panicle grain density. The poorly filled basal spikelets of the compact-panicle cultivars showed a lower endosperm cell division rate and ploidy status of the endosperm nuclei coupled with no significant greater expression of CYCB;1 and CYCH;1 compared with the apical spikelets, unlike that observed in the lax-panicle cultivars, which might have prevented them from overcoming apical dominance. Significantly greater expression of CYCB2;2 in the basal spikelets than in the apical spikelets might also have prevented the former to enter into endoreduplication. Furthermore, expression studies of KRPs in the caryopses revealed that a higher expression of KRP;1 and KRP;4 in the basal spikelets than in the apical spikelets of the compact-panicle cultivars could also be detrimental to grain filling in the former, as KRPs form complex primarily with CDKA-CYCD that promotes S-phase activity and G1/S transition, and thus inhibits endosperm cell division. The study indicates that targeted manipulation of expression of CYCB1;1, CYCB2;2, CYCH1;1, KRP;1 and KRP4 in the basal spikelets of the compact-panicle cultivars may significantly improve their yield performance.