Heat stress often results in the generation of reactive oxygen species, such as hydrogen peroxide, which plays a vital role as a secondary messenger in the process of abscisic acid (ABA)-mediated stomatal closure. Here, we characterized the rice (Oryza sativa) HEAT TOLERANCE AT SEEDLING STAGE (OsHTAS) gene, which plays a positive role in heat tolerance at the seedling stage. OsHTAS encodes a ubiquitin ligase localized to the nucleus and cytoplasm. OsHTAS expression was detected in all tissues surveyed and peaked in leaf blade, in which the expression was concentrated in mesophyll cells. OsHTAS was responsive to multiple stresses and was strongly induced by exogenous ABA. In yeast two-hybrid assays, OsHTAS interacted with components of the ubiquitin/26S proteasome system and an isoform of rice ascorbate peroxidase. OsHTAS modulated hydrogen peroxide accumulation in shoots, altered the stomatal aperture status of rice leaves, and promoted ABA biosynthesis. The results suggested that the RING finger ubiquitin E3 ligase OsHTAS functions in leaf blade to enhance heat tolerance through modulation of hydrogen peroxide-induced stomatal closure and is involved in both ABA-dependent and DROUGHT AND SALT TOLERANCE-mediated pathways.

Lysine (Lys) is considered to be the first limiting essential amino acid in rice. Although there have been extensive efforts to improve the Lys content of rice through traditional breeding and genetic engineering, no satisfactory products have been achieved to date. We expressed a LYSINE-RICH PROTEIN gene (LRP) from Psophocarpus tetragonolobus (L.) DC using an endosperm-specific GLUTELIN1 promoter (GT1) in Peiai64S (PA64S), an elite photoperiod-thermo sensitive male sterility (PTSMS) line. The expression of the foreign LRP protein was confirmed by Western blot analysis. The Lys level in the transgenic rice seeds increased more than 30 %, the total amount of other amino acids also increased compared to wild-type. Persistent investigation of amino acids in 3 generations showed that the Lys content was significantly increased in seeds of transgenic rice. Furthermore, Lys content in the hybrid of the transgenic plants also had an approximate 20 % increase compared to hybrid control. At the grain-filling stage, we monitored the transcript abundance of many genes encoding key enzymes involved in amino acid metabolism, and the results suggested that reduced amino acid catabolism led to the accumulation of amino acids in the transgenic plants. The genetically engineered rice showed unfavorable grain phenotypes compared to wild-type, however, its hybrid displayed little negative effects on grain. Endosperm-specific expression of foreign LRP significantly increased the Lys content in the seeds of transgenic plant, and the the Lys increase was stably heritable with 3 generation investigation. The hybrid of the transgenic plants also showed significant increases of Lys content in the seeds. These results indicated that expression of LRP in rice seeds may have promising applications in improving Lys levels in rice.

Although several site-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas, have emerged as powerful tools for targeted gene editing in many organisms, to date, gene targeting (GT) in plants remains a formidable challenge. In the present study, we attempted to substitute a single base in situ on the rice OsEPSPS gene by co-transformation of TALEN with chimeric RNA/DNA oligonucleotides (COs), including different strand composition such as RNA/DNA (C1) or DNA/RNA (C2) but contained the same target base to be substituted. In contrast to zero GT event obtained by the co-transformation of TALEN with homologous recombination plasmid (HRP), we obtained one mutant showing target base substitution although accompanied by undesired deletion of 12 bases downstream the target site from the co-transformation of TALEN and C1. In addition to this typical event, we also obtained 16 mutants with different length of base deletions around the target site among 105 calli lines derived from transformation of TALEN alone (4/19) as well as co-transformation of TELAN with either HRP (5/30) or C1 (2/25) or C2 (5/31). Further analysis demonstrated that the homozygous gene-edited mutants without foreign gene insertion could be obtained in one generation. The induced mutations in transgenic generation were also capable to pass to the next generation stably. However, the genotypes of mutants did not segregate normally in T1 population, probably due to lethal mutations. Phenotypic assessments in T1 generation showed that the heterozygous plants with either one or three bases deletion on target sequence, called d₁ and d₃, were more sensitive to glyphosate and the heterozygous d1 plants had significantly lower seed-setting rate than wild-type.

Plants have a family of proteins referred to as ICKs (inhibitors of cyclin-dependent kinase, CDK) or KRPs (Kip-related proteins) that function to regulate the activities of CDK. Knowledge of these plant CDK inhibitors has been gained mostly from studies of selected members in dicotyledonous plants, particularly Arabidopsis. Much remains to be learned regarding the differences among various members of the ICK/KRP family, and regarding the function and regulation of these proteins in monocotyledonous plants. We analysed ICK-related sequences in the rice (Orysa sativa L. subsp. indica) genome and determined that there are six members with the conserved C-terminal signature region for ICK/KRP proteins. They are referred to as OsiICKs and further analyses were performed. The interactions with CDKs and cyclins were determined by a yeast two-hybrid assay, and cellular localization by fusion with the enhanced green fluorescence protein (EGFP). The expression of OsiICK6 in different tissues and in response to several treatments was analysed by reverse transcriptase-mediated polymerase chain reaction (RT-PCR) and real-time PCR. Furthermore, OsiICK6 was over-expressed in transgenic rice plants and significant phenotypes were observed. Based on putative protein sequences, the six OsiICKs are grouped into two classes, with OsiICK1 and OsiICK6 in each of the two classes, respectively. Results showed that OsiICK1 and OsiICK6 interacted with OsCYCD, but differed in their interactions with CDKA. Both EGFP:OsiICK1 and EGFP:OsiICK6 were localized in the nucleus. Whereas EGFP:OsiICK6 showed a punctuate subnuclear distribution, OsiICK1 had a homogeneous pattern. Over-expression of OsiICK6 resulted in multiple phenotypic effects on plant growth, morphology, pollen viability and seed setting. In OsiICK6-over-expressing plants, leaves rolled toward the abaxial side, suggesting that cell proliferation is critical in maintaining an even growth along the dorsal–ventral plane of leaf blades.

The initiation stage of protein biosynthesis is a sophisticated process tightly regulated by numerous initiation factors and their associated components. However, the mechanism underlying translation initiation has not been completely understood in rice. Here, we showed knock-out mutation of the rice eukaryotic translation initiation factor 3 subunit h (OseIF3h) resulted in plant growth retardation and seed-setting rate reduction as compared to the wild type. Further investigation demonstrated an interaction between OseIF3h and OsMTA2 (mRNA adenosine methylase 2), a rice homolog of METTL3 (methyltransferase-like 3) in mammals, which provided new insight into how N6-methyladenosine (m6A) modification of messenger RNA (mRNA) is engaged in the translation initiation process in monocot species. Moreover, the RIP-seq (RNA immunoprecipitation sequencing) data suggested that OseIF3h was involved in multiple biological processes, including photosynthesis, cellular metabolic process, precursor metabolites, and energy generation. Therefore, we infer that OseIF3h interacts with OsMTA2 to target a particular subset of genes at translational level, regulating plant growth and pollen development.