Jun-Xiang Shan

The novel quantitative trait locus GL3.1 controls rice grain size and yield by regulating Cyclin-T1;3

Increased crop yields are required to support rapid population growth worldwide. Grain weight is a key component of rice yield, but the underlying molecular mechanisms that control it remain elusive. Here, we report the cloning and characterization of a new quantitative trait locus (QTL) for the control of rice grain length, weight and yield. This locus, GL3.1, encodes a protein phosphatase kelch (PPKL) family — Ser/Thr phosphatase. GL3.1 is a member of the large grain WY3 variety, which is associated with weaker dephosphorylation activity than the small grain FAZ1 variety. GL3.1-WY3 influences protein phosphorylation in the spikelet to accelerate cell division, thereby resulting in longer grains and higher yields. Further studies have shown that GL3.1 directly dephosphorylates its substrate, Cyclin-T1;3, which has only been rarely studied in plants. The downregulation of Cyclin-T1;3 in rice resulted in a shorter grain, which indicates a novel function for Cyclin-T in cell cycle regulation. Our findings suggest a new mechanism for the regulation of grain size and yield that is driven through a novel phosphatase-mediated process that affects the phosphorylation of Cyclin-T1;3 during cell cycle progression, and thus provide new insight into the mechanisms underlying crop seed development. We bred a new variety containing the natural GL3.1 allele that demonstrated increased grain yield, which indicates that GL3.1 is a powerful tool for breeding high-yield crops.

Natural alleles of a proteasome α2 subunit gene contribute to thermotolerance and adaptation of African rice

Global warming threatens many aspects of human life, for example, by reducing crop yields. Breeding heat-tolerant crops using genes conferring thermotolerance is a fundamental way to help deal with this challenge. Here we identify a major quantitative trait locus (QTL) for thermotolerance in African rice (Oryza glaberrima), Thermo-tolerance 1 (TT1), which encodes an α2 subunit of the 26S proteasome involved in the degradation of ubiquitinated proteins. Ubiquitylome analysis indicated that OgTT1 protects cells from heat stress through more efficient elimination of cytotoxic denatured proteins and more effective maintenance of heat-response processes than achieved with OsTT1. Variation in TT1 has been selected for on the basis of climatic temperature and has had an important role in local adaptation during rice evolution. In addition, we found that overexpression of OgTT1 was associated with markedly enhanced thermotolerance in rice, Arabidopsis and Festuca elata. This discovery may lead to an increase in crop security in the face of the ongoing threat of global warming.

Identification of quantitative trait Loci for lipid metabolism in rice seeds.

Plant seed oil is important for human dietary consumption and industrial application. The oil trait is controlled by quantitative trait loci (QTLs), but no QTLs for fatty acid composition are known in rice, the monocot model plant. QTL analysis was performed using F(2) and F(2:3) progeny from a cross of an indica variety and a japonica variety. Gas chromatography-mass spectrometry (GC-MS) analysis revealed significant differences between parental lines in fatty acid composition of brown rice oil, and 29 associated QTLs in F(2) and/or F(2:3) populations were identified throughout the rice genome, except chromosomes 9 and 10. Eight QTLs were repeatedly identified in both populations across different environments. Five loci pleiotropically controlled different traits, contributing to complex interactions of oil with fatty acids and between fatty acids. Nine rice orthologs of Arabidopsis genes encoding key enzymes in lipid metabolism co-localized with 11 mapped QTLs. A strong QTL for oleic (18:1) and linoleic (18:2) acid were associated with a rice ortholog of a gene encoding acyl-CoA:diacylglycerol acyltransferase (DGAT), and another for palmitic acid (16:0) mapped similarly to the acyl-ACP thioesterase (FatB) gene ortholog. Our approach rapidly and efficiently identified candidate genes for mapped QTLs controlling fatty acid composition and oil concentration, providing information for improving rice grain quality by marker assisted selection.

A two-locus interaction causes interspecific hybrid weakness in rice

Reproductive barriers perform a vital role during speciation. Hybrid weakness, the poorer development of hybrids compared with their parents, hinders gene exchange between different species at the postzygotic stage. Here we show that two incompatible dominant loci (Hwi1 and Hwi2) involving three genes are likely to determine the high temperature-dependent expression of hybrid weakness in interspecific hybrids of rice. Hwi1 comprises two leucine-rich repeat receptor-like kinase (LRR–RLK) genes, 25L1 and 25L2, which are specific to wild rice (Oryza rufipogon) and induce hybrid weakness. Hwi2, a rare allele that is predominantly distributed in indica rice (Oryza sativa), encodes a secreted putative subtilisin-like protease. Functional analysis indicated that pyramiding of Hwi1 and Hwi2 activates the autoimmune response in the basal nodes of hybrids, interrupting root formation and then impairing shoot growth. These findings bring new insights into our understanding of reproductive isolation and may benefit rice breeding.

The QTL GNP1 Encodes GA20ox1, Which Increases Grain Number and Yield by Increasing Cytokinin Activity in Rice Panicle Meristems.

Cytokinins and gibberellins (GAs) play antagonistic roles in regulating reproductive meristem activity. Cytokinins have positive effects on meristem activity and maintenance. During inflorescence meristem development, cytokinin biosynthesis is activated via a KNOX-mediated pathway. Increased cytokinin activity leads to higher grain number, whereas GAs negatively affect meristem activity. The GA biosynthesis genes GA20oxs are negatively regulated by KNOX proteins. KNOX proteins function as modulators, balancing cytokinin and GA activity in the meristem. However, little is known about the crosstalk among cytokinin and GA regulators together with KNOX proteins and how KNOX-mediated dynamic balancing of hormonal activity functions. Through map-based cloning of QTLs, we cloned a GA biosynthesis gene, Grain Number per Panicle1 (GNP1), which encodes rice GA20ox1. The grain number and yield of NIL-GNP1TQ were significantly higher than those of isogenic control (Lemont). Sequence variations in its promoter region increased the levels of GNP1 transcripts, which were enriched in the apical regions of inflorescence meristems in NIL-GNP1TQ. We propose that cytokinin activity increased due to a KNOX-mediated transcriptional feedback loop resulting from the higher GNP1 transcript levels, in turn leading to increased expression of the GA catabolism genes GA2oxs and reduced GA1 and GA3 accumulation. This rebalancing process increased cytokinin activity, thereby increasing grain number and grain yield in rice. These findings uncover important, novel roles of GAs in rice florescence meristem development and provide new insights into the crosstalk between cytokinin and GA underlying development process.

Rice Carotenoid β-ring Hydroxylase CYP97A4 is Involved in Lutein Biosynthesis

Lutein is the most abundant plant carotenoid and plays essential roles in photosystem assembly and stabilization, as well as protection against photostress. To date, only a few lutein biosynthesis genes have been identified in crop plants. In this study, the rice Cyt P450 gene CYP97A4 encoding a carotenoid β-ring hydroxylase was shown to be involved in lutein biosynthesis. The results revealed that CYP97A4 was preferentially expressed in leaf compared with spikelet, sheath, stalk and root, and encoded a protein localized at the subcellular level to the chloroplasts. Compared with the wild type, the three allelic mutants of CYP97A4 displayed lutein reductions of 12-24% with substantially increased α-carotene, while Chl a/b levels were unaltered. The increased α-carotene in the mutants led to greater sensitivity under high light stress. Similarly, reactive oxygen species (ROS) imaging of leaves treated with intense light showed that the mutants generally accumulated greater levels of ROS compared with wild-type plants, which probably caused detrimental effects to the plant photosystem. In conclusion, this study demonstrated the important role of CYP97A4 in α-carotene hydroxylation in rice, and knock-out of the gene reduced lutein and increased α-carotene, contributing to sensitivity to intense light.

DCA1 Acts as a Transcriptional Co-activator of DST and Contributes to Drought and Salt Tolerance in Rice.

Natural disasters, including drought and salt stress, seriously threaten food security. In previous work we cloned a key zinc finger transcription factor gene, Drought and Salt Tolerance (DST), a negative regulator of drought and salt tolerance that controls stomatal aperture in rice. However, the exact mechanism by which DST regulates the expression of target genes remains unknown. In the present study, we demonstrated that DST Co-activator 1 (DCA1), a previously unknown CHY zinc finger protein, acts as an interacting co-activator of DST. DST was found to physically interact with itself and to form a heterologous tetramer with DCA1. This transcriptional complex appears to regulate the expression of peroxidase 24 precursor (Prx 24), a gene encoding an H2O2 scavenger that is more highly expressed in guard cells. Downregulation of DCA1 significantly enhanced drought and salt tolerance in rice, and overexpression of DCA1 increased sensitivity to stress treatment. These phenotypes were mainly influenced by DCA1 and negatively regulated stomatal closure through the direct modulation of genes associated with H2O2 homeostasis. Our findings establish a framework for plant drought and salt stress tolerance through the DCA1-DST-Prx24 pathway. Moreover, due to the evolutionary and functional conservation of DCA1 and DST in plants, engineering of this pathway has the potential to improve tolerance to abiotic stress in other important crop species.

Genetic and Physiological Analysis of a Novel Type of Interspecific Hybrid Weakness in Rice

Hybrid weakness is an important reproductive barrier that hinders genetic exchange between different species at the post-zygotic stage. However, our understanding of the molecular mechanisms underlying hybrid weakness is limited. In this study, we report discovery of a novel interspecific hybrid weakness in a rice chromosome segment substitution line (CSSL) library derived from a cross between the indica variety Teqing (Oryza sativa) and common wild rice (O. rufipogon). The dominant Hybrid weakness i1 (Hwi1) gene from wild rice is genetically incompatible with Teqing and induced a set of weakness symptoms, including growth suppression, yield decrease, impaired nutrient absorption, and the retardation of crown root initiation. Phytohormone treatment showed that salicylic acid (SA) could restore the height of plants expressing hybrid weakness, while other phytohormones appear to have little effect. Fine mapping indicated that Hwi1 is located in a tandem leucine-rich repeat receptor-like kinase (LRR-RLK) gene cluster. Within the 13.2-kb candidate region on the short arm of chromosome 11, there are two annotated LRR-RLK genes, LOC_Os11g07230 and LOC_Os11g07240. The Teqing allele of LOC_Os11g07230 and the wild rice allele of LOC_Os11g07240 encode predicted functional proteins. Based on the genetic inheritance of hybrid weakness, LOC_Os11g07240 is implicated as the candidate gene for Hwi1. Functional analysis of Hwi1 will expand our knowledge of the regulation of hybrid weakness in rice.

OsHAL3, a Blue Light-Responsive Protein, Interacts with the Floral Regulator Hd1 to Activate Flowering in Rice

In flowering plants, photoperiodic flowering is controlled by a complicated network. Light is one of the most important environmental stimuli that control the timing of the transition from vegetative growth to reproductive development. Several photoreceptors, including PHYA, PHYB, CRY2, and FKF1 in Arabidopsis and their homologs (OsPHYA, OsPHYB, OsPHYC, and OsCRY2) in rice, have been identified to be related to flowering. Our previous study suggests that OsHAL3, a flavin mononucleotide-binding protein, may function as a blue-light sensor. Here, we report the identification of OsHAL3 as a positive regulator of flowering in rice. OsHAL3 overexpression lines exhibited an early flowering phenotype, whereas downregulation of OsHAL3 expression by RNA interference delayed flowering under an inductive photoperiod (short-day conditions). The change in flowering time was not accompanied by altered Hd1 expression but rather by reduced accumulation of Hd3a and MADS14 transcripts. OsHAL3 and Hd1 colocalized in the nucleus and physically interacted in vivo under the dark, whereas their interaction was inhibited by white or blue light. Moreover, OsHAL3 directly bound to the promoter of Hd3a, especially before dawn. We conclude that OsHAL3, a novel light-responsive protein, plays an essential role in photoperiodic control of flowering time in rice, which is probably mediated by forming a complex with Hd1. Our findings open up new perspectives on the photoperiodic flowering pathway.

GRAIN SIZE AND NUMBER1 Negatively Regulates the OsMKKK10-OsMKK4-OsMPK6 Cascade to Coordinate the Trade-off between Grain Number per Panicle and Grain Size in Rice

The GRAIN SIZE AND NUMBER1-mitogen-activated protein kinase module coordinates the trade-off between grain number and size in rice by integrating localized cell differentiation and proliferation. Grain number and size are interactive agronomic traits that determine grain yield. However, the molecular mechanisms responsible for coordinating the trade-off between these traits remain elusive. Here, we characterized the rice (Oryza sativa) grain size and number1 (gsn1) mutant, which has larger grains but sparser panicles than the wild type due to disordered localized cell differentiation and proliferation. GSN1 encodes the mitogen-activated protein kinase phosphatase OsMKP1, a dual-specificity phosphatase of unknown function. Reduced expression of GSN1 resulted in larger and fewer grains, whereas increased expression resulted in more grains but reduced grain size. GSN1 directly interacts with and inactivates the mitogen-activated protein kinase OsMPK6 via dephosphorylation. Consistent with this finding, the suppression of mitogen-activated protein kinase genes OsMPK6, OsMKK4, and OsMKKK10 separately resulted in denser panicles and smaller grains, which rescued the mutant gsn1 phenotypes. Therefore, OsMKKK10-OsMKK4-OsMPK6 participates in panicle morphogenesis and acts on a common pathway in rice. We confirmed that GSN1 is a negative regulator of the OsMKKK10-OsMKK4-OsMPK6 cascade that determines panicle architecture. The GSN1-MAPK module coordinates the trade-off between grain number and grain size by integrating localized cell differentiation and proliferation. These findings provide important insights into the developmental plasticity of the panicle and a potential means to improve crop yields.