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Cuticular wax plays crucial roles in protecting plants from environmental stresses, particularly drought stress. Many enzymeencoding genes and transcription factors involved in wax biosynthesis have been identified, but the underlying posttranslational regulatory mechanisms are poorly understood. Here, we demonstrate that DROUGHT HYPERSENSITIVE (DHS), encoding a Really Interesting New Gene (RING)-type protein, is a critical regulator of wax biosynthesis in rice (Oryza sativa). The cuticular wax contents were significantly reduced in DHS overexpression plants but increased in dhs mutants compared with the wild type, which resulted in a response opposite that of drought stress. DHS exhibited E3 ubiquitin ligase activity and interacted with the homeodomain-leucine zipper IV protein ROC4. Analysis of ROC4 overexpression plants and roc4 mutants indicated that ROC4 positively regulates cuticular wax biosynthesis and the drought stress response. ROC4 is ubiquitinated in vivo and subjected to ubiquitin/26S proteasome-mediated degradation. ROC4 degradation was promoted by DHS but delayed in dhs mutants. ROC4 acts downstream of DHS, and Os-BDG is a direct downstream target of the DHS-ROC4 cascade. These results suggest a mechanism whereby DHS negatively regulates wax biosynthesis by promoting the degradation of ROC4, and they suggest that DHS and ROC4 are valuable targets for the engineering of drought-tolerant rice cultivars.

Grain size is a key agronomic trait that affects grain yield and quality in rice (Oryza sativa L.). The transcription factor OsWRKY53 positively regulates grain size through brassinosteroid (BR) signaling and Mitogen-Activated Protein Kinase (MAPK) cascades. However, whether the OsWRKY53 homologs OsWRKY24 and OsWRKY70 also contribute to grain size which remains unknown. Here, we report that grain size in OsWRKY24 overexpression lines and oswrky24 mutants is similar to that of the wild type. However, the oswrky24 oswrky53 double mutant produced smaller grains than the oswrky53 single mutant, indicating functional redundancy between OsWRKY24 and OsWRKY53. In addition, OsWRKY70 overexpression lines displayed an enlarged leaf angle, reduced plant height, longer grains, and higher BR sensitivity, phenotypes similar to those of OsWRKY53 overexpression lines. Importantly, a systematic characterization of seed length in the oswrky70 single, the oswrky53 oswrky70 double and the oswrky24 oswrky53 oswrky70 triple mutant indicated that loss of OsWRKY70 also leads to increased seed length, suggesting that OsWRKY70 might play a role distinct from that of OsWRKY53 in regulating grain size. Taken together, these findings suggest that OsWRKY24 and OsWRKY70 regulate rice grain size redundantly and independently from OsWRKY53.

The genetic diversity and phenotypic variability of crop agronomic traits is valued by breeders for their benefits in crop breeding but are limited for most target traits. The rice Waxy (Wx) gene (LOC_Os06g04200) encodes granule‐bound starch synthase I (GBSSI), which determines the amylose content (AC) of endosperm by controlling amylose synthesis. The number of altered bases in each line (coloured in red) is indicated by the letter S followed by a number. (c) A structural model of Wxb constructed using the PROTEIN DATA BANK server; mutated residues contributing to the changes of AC are shown as spheres and are coloured (P124 in apricot, R125 in blue, R158 in red violet, G159 in white, V160 in green, D161 in red, T178 in orange and Y191 in purple). (d) Analysis of potential off‐target sites in the seven T1 edited lines. To determine the effect of these mutations on AC, we measured the apparent amylose contents (AACs) of grains from the seven mutant lines (Wxm5‐Wxm11), NIP (Wxb) and a 'soft rice' control Nangeng9108 (NG9108) (Wxmp) (Figure 1e).

Acyl-CoA-binding proteins (ACBPs) are divided into class I–IV in plants. Class IV ACBPs are kelch motif containing proteins that are specific to plants. The currently known subcellular localizations of plant class IV ACBPs are either in the cytosol (Arabidopsis) or in the peroxisomes (rice). However, it is not clear whether peroxisomal localization of class IV ACBP is a shared character that distinguishes eudicots and monocots. Here, the phylogeny of class IV ACBPs from 73 plant species and subcellular localization of class IV ACBPs from six monocots and eudicots were conducted. Phylogenetic analysis of 112 orthologues revealed that monocot class IV ACBPs were basal to the monophyletic clade formed by eudicots and basal angiosperm. Transient expression of GFP fusions in onion epidermal cells demonstrated that monocot maize (Zea mays), wheat (Triticum aestivum), and sorghum (Sorghum bicolor) and eudicot poplar (Populus trichocarpa) all contained at least one peroxisomal localized class IV ACBP, while orthologues from cucumber (Cucumis sativus L.) and soybean (Glycine max) were all cytosolic. Combining the location of Arabidopsis and rice class IV ACBPs, it indicates that maintaining at least one peroxisomal class IV ACBP could be a shared feature within the tested monocots, while cytosolic class IV ACBPs would be preferred in the tested eudicots. Furthermore, the interaction between OsACBP6 and peroxisomal ATP-binding cassette (ABC) transporter provided clues for the functional mechanism of OsACBP6.

Low-temperature germination (LTG) is an important agronomic trait for rice ( Oryza sativa ). Japonica rice generally has greater capacity for germination at low temperatures than the indica subpopulation. However, the genetic basis and molecular mechanisms underlying this complex trait are poorly understood. Here, we report that OsUBC12 , encoding an E2 ubiquitin-conjugating enzyme, increases low-temperature germinability in japonica , owing to a transposon insertion in its promoter enhancing its expression. Natural variation analysis reveals that transposon insertion in the OsUBC12 promoter mainly occurs in the japonica lineage. The variation detected in eight representative two-line male sterile lines suggests the existence of this allele introgression by indica - japonica hybridization breeding, and varieties carrying the japonica OsUBC12 locus (transposon insertion) have higher low-temperature germinability than varieties without the locus. Further molecular analysis shows that OsUBC12 negatively regulate ABA signaling. OsUBC12-regulated seed germination and ABA signaling mainly depend on a conserved active site required for ubiquitin-conjugating enzyme activity. Furthermore, OsUBC12 directly associates with rice SUCROSE NON-FERMENTING 1-RELATED PROTEIN KINASE 1.1 (OsSnRK1.1), promoting its degradation. OsSnRK1.1 inhibits LTG by enhancing ABA signaling and acts downstream of OsUBC12. These findings shed light on the underlying mechanisms of UBC12 regulating LTG and provide genetic reference points for improving LTG in indica rice. Japonica rice generally has greater capacity for low temperatures germination (LTG) than indica rice. Here, the authors report an E2 ubiquitin-conjugating enzyme-encoding gene OsUBC12 can increase LTG in japonica rice by negatively regulate ABA signaling via promoting the proteasomal degradation of OsSnRK1.1.

Flowering time of rice (Oryza sativa L.) is among the most important agronomic traits for region adaptation and grain yield. In the process of rice breeding, efficient and slightly modulating the flowering time of an elite cultivar would be more popular with breeder. Hence, we are interested in slightly increasing the expression of flowering repressors by CRISPR/Cas9 genome editing system. It was predicated there were three uORFs in 5’ leader sequence of Hd2. In this study, through editing Hd2 uORFs, we got four homozygous mutant lines. Phenotypic analysis showed that the hd2 urf edited lines flowered later by 4.6–11.2 days relative to wild type SJ2. Supporting the later flowering phenotype, the expression of Ehd1, Hd3a, and RFT1 is significantly decreased in hd2 urf than that in wild type. Moreover, we found that the transcription level of Hd2 is not affected, whereas the Hd2 protein level was increased in hd2 urf compared with wild type, which indicated that Hd2 uORFs indeed affect the translation of a downstream Hd2 pORF. In summary, we developed a efficient approach for delaying rice heading date based on editing uORF region of flowering repressor, which is time and labor saving compared to traditional breeding. In future, uORF of other flowering time related genes, including flowering promoter and flowering repressor genes, can also be used as targets to fine-tune the flowering time of varieties.

Background Abscisic acid (ABA) plays crucial roles in regulating plant growth and development, especially in responding to abiotic stress. The pyrabactin resistance-like (PYL) abscisic acid receptor family has been identified and widely characterized in Arabidopsis . However, PYL families in rice were largely unknown. In the present study, 10 out of 13 PYL orthologs in rice ( OsPYL ) were isolated and investigated. Results Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that expression of OsPYL genes is tissue-specific and display differential response to ABA treatment, implying their functional diversity. The interaction between 10 OsPYL members and 5 protein phosphatase 2C in rice (OsPP2C) members was investigated in yeast two-hybrid and tobacco transient expression assays, and an overall interaction map was generated, which was suggestive of the diversity and complexity of ABA-sensing signaling in rice. To study the biological function of OsPYLs , two OsPYL genes ( OsPYL3 and OsPYL9 ) were overexpressed in rice. Phenotypic analysis of OsPYL3 and OsPYL9 transgenic rice showed that OsPYLs positively regulated the ABA response during the seed germination. More importantly, the overexpression of OsPYL3 and OsPYL9 substantially improved drought and cold stress tolerance in rice. Conclusion Taken together, we comprehensively uncovered the properties of OsPYLs , which may be good candidates for the improvement of abiotic stress tolerance in rice.

Cold stress at the booting stage leads to a lower seed setting rate and seriously threatens the production of rice (Oryza sativa L.), which has become a major yield-limiting factor in higher-altitude and -latitude regions. Because cold tolerance at the booting stage (CTB) is a complex trait and is controlled by multiple loci, only a few genes have been reported so far. In this study, a function of OsMKKK70 (Mitogen Activated Protein Kinase Kinase Kinase 70) in response to CTB was characterized. OsMKKK70 expression was rapidly induced by cold stress at the booting stage. OsMKKK70 overexpression (OsMKKK70-OE) plants were more sensitive to cold stress at the booting stage with a lower seed setting and pollen fertility, but there was no significant difference between the osmkkk70 mutant and WT. Considering the effect of functional redundancy, we further tested the CTB response of osmkkk62/70 and osmkkk55/62/70, the double and triple mutants of OsMKKK70 with its closest homologs OsMKKK62 and OsMKKK55, and found that osmkkk62/70 and osmkkk55/62/70 displayed significantly increased CTB with a higher seed setting and pollen fertility, indicating that OsMKKK70 negatively regulates rice CTB. Moreover, under the low-temperature (LT) condition, the osmkkk62/70 mutant had slightly higher Gibberellin (GA) contents, increased expression of GA biosynthesis genes, and lower protein level of OsSLR1 in anthers than those in WT. By contrast, OsMKKK70-OE anther had a lower GA biosynthesis than that of WT. Together, these findings suggest that OsMKKK70 negatively regulates rice CTB by fine-tuning GA levels in anthers.

Embryoless rice is valuable for studying early seed development and has great breeding potential, however, related research remains limited. Here, we show that mutations in OsBZR4 , encoding brassinazole-resistance 4, lead to 60–100% embryoless seeds across different cultivars. OsBZR4 is specifically expressed at the scutellum-endosperm interface and regulates auxin levels and distribution during early embryo development. OsBZR4 represses the expression of YUC4 and PIN5b . Exogenous auxin and overexpression of YUC4 enhance the embryoless ratio, whereas the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) decrease it. Higher temperatures increase the embryoless ratio in bzr4 mutant lines. The expression of OsPIL13 is induced by high temperature, and its overexpression enhances the embryoless ratio in both wild type and bzr4 plants. Introducing bzr4 mutant allele into some elite cultivars can improve milled rice yield and storability. Collectively, manipulating OsBZR4 can lead to the development of thermosensitive embryoless rice varieties with increased energy reserves and improved storability, potentially enabling novel rice production technologies. Embryo development is critical to seed development and grain yield in rice. Here, the authors show that mutation of brassinazole-resistance 4 ( OsBZR4 ) cause embryoless phenotype with the possible involvement of auxin signaling and the penetration of the phenotype depending on genetic background and temperature.

CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is a RING/WD40 repeat-containing ubiquitin E3 ligase that is conserved from plants to humans. COP1 forms complexes with SUPPRESSOR OF PHYTOCHROME A (SPA) proteins, and these complexes degrade positively acting transcription factors in the dark to repress photomorphogenesis. Phytochrome-interacting basic helixloop-helix transcription factors (PIFs) also repress photomorphogenesis in the dark. In response to light, the phytochrome family of sensory photoreceptors simultaneously inactivates COP1-SPA complexes and induces the rapid degradation of PIFs to promote photomorphogenesis. However, the functional relationship between PIFs and COP1-SPA complexes is still unknown. Here, we present genetic evidence that the pif and cop1/spa Arabidopsis thaliana mutants synergistically promote photomorphogenesis in the dark. LONG HYPOCOTYL5 (HY5) is stabilized in the cop1 pif1, spa 123 pifq and pif double, triple, and quadruple mutants in the dark. Moreover, the hy5 mutant suppresses the constitutive photomorphogenic phenotypes of the pifq mutant in the dark. PIF1 forms complexes with COP1, HY5, and SPA1 and enhances the substrate recruitment and autoubiquitylation and transubiquitylation activities of COP1. These data uncover a novel function of PIFs as the potential cofactors of COP1 and provide a genetic and biochemical model of how PIFs and COP1-SPA complexes synergistically repress photomorphogenesis in the dark.