Chilling tolerance in rice: Past and present.

Rice is generally sensitive to chilling stress, which seriously affects growth and yield. Since early in the last century, considerable efforts have been made to understand the physiological and molecular mechanisms underlying the response to chilling stress and improve rice chilling tolerance. Here, we review the research trends and advances in this field. The phenotypic and biochemical changes caused by cold stress and the physiological explanations are briefly summarized. Using published data from the past 20 years, we reviewed the past progress and important techniques in the identification of quantitative trait loci (QTL), novel genes, and cellular pathways involved in rice chilling tolerance. The advent of novel technologies has significantly advanced studies of cold tolerance, and the characterization of QTLs, key genes, and molecular modules have sped up molecular design breeding for cold tolerance in rice varieties. In addition to gene function studies based on overexpression or artificially generated mutants, elucidating natural allelic variation in specific backgrounds is emerging as a novel approach for the study of cold tolerance in rice, and the superior alleles identified using this approach can directly facilitate breeding.

OsGRF6 interacts with SLR1 to regulate OsGA2ox1 expression for coordinating chilling tolerance and growth in rice.

Low temperature is one of the abiotic stressors that affect growth and productivity of rice. The plant hormone gibberellin not only regulates growth and development but is also involved in stress defense. Our rice seedling experiments demonstrated that overexpression of SLR1, a gene that encodes the rice DELLA protein, enhanced chilling tolerance. In contrast, overexpression of the active GA synthesis gene OsGA20ox1 reduced chilling tolerance, indicating that weakening GA signaling promoted plant defense against cold stress. CoIP-MS and BiFC assays showed that SLR1 physically interacted with OsGRF6. After cold treatment and recovery, the survival rates of OsGRF6-overexpression lines and an osgrf6 mutant and its complementary lines indicated that OsGRF6 is a negative regulator of chilling tolerance in rice. The yeast one-hybrid, qRT-PCR, and transactivation assays showed that both SLR1 and OsGRF6 can bind to the promoter of the active GA catabolic gene OsGA2ox1, where SLR1 promoted and OsGRF6 suppressed OsGA2ox1 expression. At normal temperature, OsGRF6 was responsible for maintaining active GA levels by inhibiting OsGA2ox1. When rice seedlings were subjected to chilling stress, the repressive effect of OsGRF6 on OsGA2ox1 was released by cold-induced SLR1, which activated OsGA2ox1 expression to decrease the active GA levels, enhancing chilling tolerance. These results suggest that OsGRF6 is an important regulator in the balance between growth and chilling tolerance in rice.

A C2H2 zinc-finger protein OsZFP213 interacts with OsMAPK3 to enhance salt tolerance in rice.

Improvement of salt tolerance is one of the major targets in rice breeding. Here, we report that the zinc-finger protein (ZFP) OsZFP213 functions in enhancing salt tolerance in rice. OsZFP213 is localized in the nucleus and has transactivation activity. Transgenic rice overexpressing OsZFP213 showed enhanced salt tolerance compared with wild type and OsZFP213 RNAi plants. Furthermore, OsZFP213 overexpression plants showed higher transcription levels of antioxidant system genes and higher catalytic activity of scavenging enzymes of reactive oxygen, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR), and a lower level of ROS accumulation than that in wild type and OsZFP213 RNAi plants under salt treatment. Yeast two-hybrid, pull-down, and BiFC analysis showed that OsMAPK3 is a direct partner of OsZFP213, and this interaction enhanced the transactivation activity of OsZFP213. Taken together, these results suggest that OsZFP213 cooperates with OsMAPK3 in the regulation of rice salt stress tolerance by enhancing the ability of scavenging reactive oxygen.

OsMADS57 together with OsTB1 coordinates transcription of its target OsWRKY94 and D14 to switch its organogenesis to defense for cold adaptation in rice.

Plants modify their development to adapt to their environment, protecting themselves from detrimental conditions such as chilling stress by triggering a variety of signaling pathways; however, little is known about how plants coordinate developmental patterns and stress responses at the molecular level. Here, we demonstrate that interacting transcription factors OsMADS57 and OsTB1 directly target the defense gene OsWRKY94 and the organogenesis gene D14 to trade off the functions controlling/moderating rice tolerance to cold. Overexpression of OsMADS57 maintains rice tiller growth under chilling stress. OsMADS57 binds directly to the promoter of OsWRKY94, activating its transcription for the cold stress response, while suppressing its activity under normal temperatures. In addition, OsWRKY94 was directly targeted and suppressed by OsTB1 under both normal and chilling temperatures. However, D14 transcription was directly promoted by OsMADS57 for suppressing tillering under the chilling treatment, whereas D14 was repressed for enhancing tillering under normal condition.We demonstrated that OsMADS57 and OsTB1 conversely affect rice chilling tolerance via targeting OsWRKY94. Our findings highlight a molecular genetic mechanism coordinating organogenesis and chilling tolerance in rice, which supports and extends recent work suggesting that chilling stress environments influence organ differentiation.

OsMAPK3 Phosphorylates OsbHLH002/OsICE1 and Inhibits Its Ubiquitination to Activate OsTPP1 and Enhances Rice Chilling Tolerance.

Improvement of chilling tolerance is a major target in rice breeding. The signaling pathways regulating chilling consist of complex networks, including key transcription factors and their targets. However, it remains largely unknown how transcription factors are activated by chilling stress. Here, we report that the transcription factor OsbHLH002/OsICE1 is phosphorylated by OsMAPK3 under chilling stress. The osbhlh002-1 knockout mutant and antisense transgenic plants showed chilling hypersensitivity, whereas OsbHLH002-overexpressing plants exhibited enhanced chilling tolerance. OsbHLH002 can directly target OsTPP1, which encodes a key enzyme for trehalose biosynthesis. OsMAPK3 interacts with OsbHLH002 to prevent its ubiquitination by the E3 ligase OsHOS1. Under chilling stress, active OsMAPK3 phosphorylates OsbHLH002, leading to accumulation of phospho-OsbHLH002, which promotes OsTPP1 expression and increases trehalose content and resistance to chilling damage. Taken together, these results indicate that OsbHLH002 is phosphorylated by OsMAPK3, which enhances OsbHLH002 activation to its target OsTPP1 during chilling stress.

OsmiR396d-regulated OsGRFs function in floral organogenesis in rice through binding to their targets OsJMJ706 and OsCR4.

Inflorescence and spikelet development determine grain yields in cereals. Although multiple genes are known to be involved in the regulation of floral organogenesis, the underlying molecular network remains unclear in cereals. Here, we report that the rice (Oryza sativa) microRNA396d (OsmiR396d) and its Os Growth Regulating Factor (OsGRF) targets, together with Os Growth Regulating Factor-Interacting Factor1 (OsGIF1), are involved in the regulation of floral organ development through the rice JMJD2 family jmjC gene 706 (OsJMJ706) and crinkly4 receptor-like kinase (OsCR4). Transgenic knockdown lines of OsGRF6, a predicted target of OsmiR396d, and overexpression lines of OsmiR396d showed similar defects in floral organ development, including open husks, long sterile lemmas, and altered floral organ morphology. These defects were almost completely rescued by overexpression of OsGRF6. OsGRF6 and its ortholog OsGRF10 were the most highly expressed OsGRF family members in young inflorescences, and the grf6/grf10 double mutant displayed abnormal florets. OsGRF6/OsGRF10 localized to the nucleus, and electrophoretic mobility shift assays revealed that both OsGRF6 and OsGRF10 bind the GA response element in the promoters of OsJMJ706 and OsCR4, which were reported to participate in the regulation of floral organ development. In addition, OsGRF6 and OsGRF10 could transactivate OsJMJ706 and OsCR4, an activity that was enhanced in the presence of OsGIF1, which can bind both OsGRF6 and OsGRF10. Together, our results suggest that OsmiR396d regulates the expression of OsGRF genes, which function with OsGIF1 in floret development through targeting of JMJ706 and OsCR4. This work thus reveals a microRNA-mediated regulation module for controlling spikelet development in rice.

Nitric oxide enhances aluminum tolerance by affecting cell wall polysaccharides in rice roots.

Nitric oxide (NO) is a key signal molecule involved in many physiological processes in plants. To study the mechanisms of exogenous NO contribution to alleviate the aluminum (Al) toxicity, roots of rice (Oryza sativa) seedlings pre-treated with sodium nitroprusside (SNP, a NO donor) were used to investigate the effect of Al in this study. Results indicated that NO alleviated the lipid peroxidation induced by Al and promoted the root elongation, whereas butylated hydroxyanisole (BHA), an efficient lipophilic antioxidant, alleviated the lipid peroxidation only. Rice seedling roots pre-treated with SNP followed by Al treatment had lower contents of pectin and hemicellulose, lower Al accumulation in root tips and cell walls, higher degree of methylation of pectin and lower wall Al-binding capacity than the roots with Al treatment only. Therefore, the decreased Al accumulation in the cell walls of rice roots is likely to be the reason for the NO-induced increase of Al tolerance in rice, and it seems that exogenous NO enhanced Al tolerance in rice roots by decreasing the contents of pectin and hemicellulose, increasing the degree of methylation of pectin, and decreasing Al accumulation in root cell walls.