Monosaccharide transporter OsMST6 is activated by transcription factor OsERF120 to enhance chilling tolerance in rice seedlings.

Chilling stress caused by extreme weather is threatening global rice (Oryza sativa L.) production. Identifying components of the signal transduction pathways underlying chilling tolerance in rice would advance molecular breeding. Here, we report that OsMST6, which encodes a monosaccharide transporter, positively regulates the chilling tolerance of rice seedlings. The mst6 mutants showed hypersensitivity to chilling, while the OsMST6 overexpression lines were tolerant. During chilling stress, OsMST6 transported more glucose into cells to modulate sugar and ABA signal pathways. We showed that the transcription factor OsERF120 could bind to the DRE/CRT element of the OsMST6 promoter and activate the expression of OsMST6 to positively regulate chilling tolerance. Genetically, OsERF120 was functionally dependent on OsMST6 when promoting chilling tolerance. In summary, OsERF120 and OsMST6 form a new downstream chilling regulatory pathway in rice in response to chilling stress, providing valuable findings for molecular breeding aimed at achieving global food security.

COG3 confers the chilling tolerance to mediate OsFtsH2-D1 module in rice.

The chilling stress induced by the global climate change harms rice production, especially at seedling and booting stage, which feed half the population of the world. Although there are key quantitative trait locus genes identified in the individual stage, few genes have been reported and functioned at both stages. Utilizing chromosome segment substitution lines (CSSLs) and a combination of map-based cloning and phenotypes of the mutants and overexpression lines, we identified the major gene Chilling-tolerance in Geng/japonica rice 3 (COG3) of q chilling-tolerance at the booting and seedling stage 11 (qCTBS11) conferred chilling tolerance at both seedling and booting stages. COG3 was significantly upregulated in Nipponbare under chilling treatment compared with its expression in 93-11. The loss-of-function mutants cog3 showed a reduced chilling tolerance. On the contrary, overexpression enhanced chilling tolerance. Genome evolution and genetic analysis suggested that COG3 may have undergone strong selection in temperate japonica during domestication. COG3, a putative calmodulin-binding protein, physically interacted with OsFtsH2 at chloroplast. In cog3-1, OsFtsH2-mediated D1 degradation was impaired under chilling treatment compared with wild-type. Our results suggest that COG3 is necessary for maintaining OsFtsH2 protease activity to regulate chilling tolerance at the booting and seedling stage.

The COG1-OsSERL2 complex senses cold to trigger signaling network for chilling tolerance in japonica rice.

Improvement of chilling tolerance is a key strategy to face potential menace from abnormal temperature in rice production, which depends on the signaling network triggered by receptors. However, little is known about the QTL genes encoding membrane complexes for sensing cold. Here, Chilling-tolerance in Gengdao/japonica rice 1 (COG1) is isolated from a chromosome segment substitution line containing a QTL (qCS11-jap) for chilling sensitivity. The major gene COG1 is found to confer chilling tolerance in japonica rice. In natural rice populations, only the haplogroup1 encodes a functional COG1. Evolutionary analysis show that COG1 originates from Chinese O. Rufipogon and is fixed in japonica rice during domestication. COG1, a membrane-localized LRR-RLP, targets and activates the kinase OsSERL2 in a cold-induced manner, promoting chilling tolerance. Furthermore, the cold signal transmitted by COG1-OsSERL2 activates OsMAPK3 in the cytoplasm. Our findings reveal a cold-sensing complex, which mediates signaling network for the chilling defense in rice.

COG2 negatively regulates chilling tolerance through cell wall components altered in rice.

KEY MESSAGE: Chilling-tolerant QTL gene COG2 encoded an extensin and repressed chilling tolerance by affecting the compositions of cell wall. Rice as a major crop is susceptible to chilling stress. Chilling tolerance is a complex trait controlled by multiple quantitative trait loci (QTLs). Here, we identify a QTL gene, COG2, that negatively regulates cold tolerance at seedling stage in rice. COG2 overexpression transgenic plants are sensitive to cold, whereas knockout transgenic lines enhance chilling tolerance. Natural variation analysis shows that Hap1 is a specific haplotype in japonica/Geng rice and correlates with chilling tolerance. The SNP1 in COG2 promoter is a specific divergency and leads to the difference in the expression level of COG2 between japonica/Geng and indica/Xian cultivars. COG2 encodes a cell wall-localized extensin and affects the compositions of cell wall, including pectin and cellulose, to defense the chilling stress. The results extend the understanding of the adaptation to the environment and provide an editing target for molecular design breeding of cold tolerance in rice.

Natural variation of codon repeats in COLD11 endows rice with chilling resilience.

Abnormal temperature caused by global climate change threatens the rice production. Defense signaling network for chilling has been uncovered in plants. However, less is known about repairing DNA damage produced from overwhelmed defense and its evolution during domestication. Here, we genetically identified a major QTL, COLD11, using the data-merging genome-wide association study based on an algorithm combining polarized data from two subspecies, indica and japonica, into one system. Rice loss-of-function mutations of COLD11 caused reduced chilling tolerance. Genome evolution analysis of representative rice germplasms suggested that numbers of GCG sequence repeats in the first exon of COLD11 were subjected to strong domestication selection during the northern expansion of rice planting. The repeat numbers affected the biochemical activity of DNA repair protein COLD11/RAD51A1 in renovating DNA damage under chilling stress. Our findings highlight a potential way to finely manipulate key genes in rice genome and effectively improve chilling tolerance through molecular designing.

Cold-induced calreticulin OsCRT3 conformational changes promote OsCIPK7 binding and temperature sensing in rice.

Unusually low temperatures caused by global climate change adversely affect rice production. Sensing cold to trigger signal network is a key base for improvement of chilling tolerance trait.  Here, we report that Oryza sativa Calreticulin 3 (OsCRT3) localized at the endoplasmic reticulum (ER) exhibits conformational changes under cold stress, thereby enhancing its interaction with CBL-interacting protein kinase 7 (OsCIPK7) to sense cold. Phenotypic analyses of OsCRT3 knock-out mutants and transgenic overexpression lines demonstrate that OsCRT3 is a positive regulator in chilling tolerance. OsCRT3 localizes at the ER and mediates increases in cytosolic calcium levels under cold stress. Notably, cold stress triggers secondary structural changes of OsCRT3 and enhances its binding affinity with OsCIPK7, which finally boosts its kinase activity. Moreover, Calcineurin B-like protein 7 (OsCBL7) and OsCBL8 interact with OsCIPK7 specifically on the plasma membrane. Taken together, our results thus identify a cold-sensing mechanism that simultaneously conveys cold-induced protein conformational change, enhances kinase activity, and Ca2+ signal generation to facilitate chilling tolerance in rice.

The methyl-CpG-binding domain family member PEM1 is essential for Ubisch body formation and pollen exine development in rice.

Pollen exine is composed of finely-organized nexine, bacula and tectum, and is crucial for pollen viability and function. Pollen exine development involves a complicated molecular network that coordinates the interaction between pollen and tapetal cells, as well as the biosynthesis, transport and assembly of sporopollenin precursors; however, our understanding of this network is very limited. Here, we report the roles of PEM1, a member of methyl-CpG-binding domain family, in rice pollen development. PEM1 expressed constitutively and, in anthers, its expression was detectable in tapetal cells and pollen. This predicted PEM1 protein of 240 kDa had multiple epigenetic-related domains. pem1 mutants exhibited abnormal Ubisch bodies, delayed exine occurrence and, finally, defective exine, including invisible bacula, amorphous and thickened nexine and tectum layer structures, and also had the phenotype of increased anther cuticle. The mutation in PEM1 did not affect the timely degradation of tapetum. Lipidomics revealed much higher wax and cutin contents in mutant anthers than in wild-type. Accordingly, this mutation up-regulated the expression of a set of genes implicated in transcriptional repression, signaling and diverse metabolic pathways. These results indicate that PEM1 mediates Ubisch body formation and pollen exine development mainly by negatively modulating the expression of genes. Thus, the PEM1-mediated molecular network represents a route for insights into mechanisms underlying pollen development. PEM1 may be a master regulator of pollen exine development.

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.

The RING E3 ligase CLG1 targets GS3 for degradation via the endosome pathway to determine grain size in rice.

G-protein signaling and ubiquitin-dependent degradation are both involved in grain development in rice, but how these pathways are coordinated in regulating this process is unknown. Here, we show that Chang Li Geng 1 (CLG1), which encodes an E3 ligase, regulates grain size by targeting the Gγ protein GS3, a negative regulator of grain length, for degradation. Overexpression of CLG1 led to increased grain length, while overexpression of mutated CLG1 with changes in three conserved amino acids decreased grain length. We found that CLG1 physically interacts with and ubiquitinats GS3which is subsequently degraded through the endosome degradation pathway, leading to increased grain size. Furthermore, we identified a critical SNP in the exon 3 of CLG1 that is significantly associated with grain size variation in a core collection of cultivated rice. This SNP results in an amino acid substitution from Arg to Ser at position 163 of CLG1 that enhances the E3 ligase activity of CLG1 and thus increases rice grain size. Both the expression level of CLG1 and the SNP CLG1163S may be useful variations for manipulating grain size in rice.

Integrated global analysis reveals a vitamin E-vitamin K1 sub-network, downstream of COLD1, underlying rice chilling tolerance divergence.

Rice, a staple food with tropical/subtropical origination, is susceptible to cold stress, one of the major constraints on its yield and distribution. Asian cultivated rice consists of two subspecies with diverged chilling tolerance to adapt to different environments. The mechanism underlying this divergence remains obscure with a few known factors, including membrane protein CHILLING-TOLERANCE DIVERGENCE 1 (COLD1). Here, we reveal a vitamin E-vitamin K1 sub-network responsible for chilling tolerance divergence through global analyses. Rice genome regions responsible for tolerance divergence are identified with chromosome segment substitution lines (CSSLs). Comparative transcriptomic and metabolomic analysis of chilling-tolerant CSSL4-1 and parent lines uncovered a vitamin E-vitamin K1 sub-network in chloroplast with tocopherol (vitamin E) mediating chloroplast-to-nucleus signaling. COLD1, located in the substitution segment in CSSL4-1, is confirmed as its upstream regulator by transgenic material analysis. Our work uncovers a pathway downstream of COLD1, through which rice modulates chilling tolerance for thermal adaptation, with potential utility in crop improvement.

The vernalization-induced long non-coding RNA VAS functions with the transcription factor TaRF2b to promote TaVRN1 expression for flowering in hexaploid wheat.

Vernalization is a physiological process in which prolonged cold exposure establishes flowering competence in winter plants. In hexaploid wheat, TaVRN1 is a cold-induced key regulator that accelerates floral transition. However, the molecular mechanism underlying the gradual activation of TaVRN1 during the vernalization process remains unknown. In this study, we identified the novel transcript VAS (TaVRN1 alternative splicing) as a non-coding RNA derived from the sense strand of the TaVRN1 gene only in winter wheat, which regulates TaVRN1 transcription for flowering. VAS was induced during the early period of vernalization, and its overexpression promoted TaVRN1 expression to accelerate flowering in winter wheat. VAS physically associates with TaRF2b and facilitates docking of the TaRF2b-TaRF2a complex at the TaVRN1 promoter during the middle period of vernalization. TaRF2b recognizes the Sp1 motif within the TaVRN1 proximal promoter region, which is gradually exposed along with the disruption of a loop structure at the TaVRN1 locus during vernalization, to activate the transcription of TaVRN1. The tarf2b mutants exhibited delayed flowering, whereas transgenic wheat lines overexpressing TaRF2b showed earlier flowering. Taken together, our data reveal a distinct regulatory mechanism by which a long non-coding RNA facilitates the transcription factor targeting to regulate wheat flowering, providing novel insights into the vernalization process and a potential target for wheat genetic improvement.

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.

Phosphatase OsPP2C27 directly dephosphorylates OsMAPK3 and OsbHLH002 to negatively regulate cold tolerance in rice.

Improving chilling tolerance is a major target of rice breeding. The OsMAPK3-OsbHLH002-OsTPP1 signalling pathway enhances chilling tolerance in rice: the kinase is activated by cold stress, and subsequently the transcription factor is phosphorylated by the activated kinase, triggering the expression of cold response genes. However, it is largely unknown how this pathway is suppressed in time to avoid it being in a continuously activated state. We found that a novel type 2C protein phosphatase, OsPP2C27, functions as a negative regulator of the OsMAPK3-OsbHLH002-OsTPP1 pathway. A dynamic change in OsMAPK3 activity was found during cold treatment. We show that OsPP2C27 interacts physically with and dephosphorylates OsMAPK3 in vitro and in vivo. Interestingly, OsPP2C27 can also directly dephosphorylate OsbHLH002, the target of OsMAPK3. After cold treatment, survival rates were higher in OsPP2C27-RNAi lines and a T-DNA insertion mutant, and lower in OsPP2C27-overexpression lines, compared to wild type. Moreover, expression of the OsTPP1 and OsDREBs were increased in OsPP2C27-RNAi lines and decreased in OsPP2C27-overexpression lines. These results indicate that cold-induced OsPP2C27 negatively regulates the OsMAPK3-OsbHLH002-OsTPP1 signalling pathway by directly dephosphorylating both phospho-OsMAPK3 and phospho-OsbHLH002, preventing the sustained activation of a positive pathway for cold stress and maintaining normal growth under chilling conditions.

A Cyclophilin OsCYP20-2 Interacts with OsSYF2 to Regulate Grain Length by Pre-mRNA Splicing.

BACKGROUND: Grain size is one of the key agronomic traits that impact grain yield. Several regulatory pathways had been reported to participate in grain size determination via cell expansion or proliferation in rice. However, little is known about cyclophilin and spliceosome participation in grain shape regulation. RESULTS: Here, we identified OsCYP20-2, a cyclophilin that influences spliceosome assembly to determine grain length. oscyp20-2 t1, a knock out mutant of OsCYP20-2 caused by T-DNA insertion, produced shorter grains with deficient cell elongation. Through yeast two-hybrid screening and pull-down assays, OsSYF2, a pre-mRNA splicing factor, was identified as an interacting protein of OsCYP20-2. The phenotypes of transgenic lines indicated that OsSYF2 positively regulates grain length via its influence on cell expansion. Transcriptomic analysis showed that OsSYF2 controls the expression and pre-mRNA alternative splicing of genes involved in sugar metabolism. In addition, these two genes have similar effects on panicle architecture. CONCLUSIONS: Taken together, OsSYF2, an interacting protein of OsCYP20-2, controls grain length and panicle architecture by regulating the alternative splicing of pre-mRNA involved in cell elongation and sugar metabolism.

OsNSUN2-Mediated 5-Methylcytosine mRNA Modification Enhances Rice Adaptation to High Temperature.

Extreme weather events can cause heat stress that decreases crop production. Recent studies have demonstrated that protein degradation and rRNA homeostasis as well as transcription factors are involved in the thermoresponse in plants. However, how RNA modifications contribute to temperature stress response in plant remains largely unknown. Herein, we identified OsNSUN2 as an RNA 5-methylcytosine (m5C) methyltransferase in rice. osnsun2 mutant displayed severe temperature- and light-dependent lesion-mimic phenotypes and heat-stress hypersensitivity. Heat stress enhanced the OsNSUN2-dependent m5C modification of mRNAs involved in photosynthesis and detoxification systems, such as ß-OsLCY, OsHO2, OsPAL1, and OsGLYI4, which increased protein synthesis. Furthermore, the photosystem of osnsun2 mutant was vulnerable to high ambient temperature and failed to undergo repair under tolerable heat stress. Thus, OsNSUN2 mutation reduced photosynthesis efficiency and accumulated excessive reactive oxygen species upon heat treatment. Our findings demonstrate an important mechanism of mRNA m5C-dependent heat acclimation in rice.

Cyclophilin OsCYP20-2 with a novel variant integrates defense and cell elongation for chilling response in rice.

Coordinating stress defense and plant growth is a survival strategy for adaptation to different environments that contains a series of processes, such as, cell growth, division and differentiation. However, little is known about the coordination mechanism for protein conformation change. A cyclophilin OsCYP20-2 with a variant interacts with SLENDER RICE1 (SLR1) and OsFSD2 in the nucleus and chloroplasts, respectively, to integrate chilling tolerance and cell elongation in rice (Oryza sativa) (FSD2, Fe-superoxide dismutase 2). Mass spectrum assay showed that OsNuCYP20-2 localized at the nucleus (nuclear located OsCYP20-2) was a new variant of OsCYP20-2 that truncated 71 amino-acid residues in N-terminal. The loss-of function OsCYP20-2 mutant showed sensitivity to chilling stress with accumulation of extra reactive oxygen species (ROS). In chloroplasts, the full-length OsCYP20-2 promotes OsFSD2 forming homodimers which enhance its activity, eliminating the accumulation of ROS under chilling stress. However, the mutant had shorter epidermal cells in comparison with wild-type Hwayoung (HY). In the nucleus, OsCYP20-2 caused conformation change of SLR1 to promote its degradation for cell elongation. Our data reveal a cyclophilin with a variant with dual-localization in chloroplasts and the nucleus, which mediate chilling tolerance and cell elongation.

OsMTOPVIB is required for meiotic bipolar spindle assembly.

The organization of microtubules into a bipolar spindle is essential for chromosome segregation. Both centrosome and chromatin-dependent spindle assembly mechanisms are well studied in mouse, Drosophila melanogaster, and Xenopus oocytes; however, the mechanism of bipolar spindle assembly in plant meiosis remains elusive. According to our observations of microtubule assembly in Oryza sativa, Zea mays, Arabidopsis thaliana, and Solanum lycopersicum, we propose that a key step of plant bipolar spindle assembly is the correction of the multipolar spindle into a bipolar spindle at metaphase I. The multipolar spindles failed to transition into bipolar ones in OsmtopVIB with the defect in double-strand break (DSB) formation. However, bipolar spindles were normally assembled in several other mutants lacking DSB formation, such as Osspo11-1, pair2, and crc1, indicating that bipolar spindle assembly is independent of DSB formation. We further revealed that the mono-orientation of sister kinetochores was prevalent in OsmtopVIB, whereas biorientation of sister kinetochores was frequently observed in Osspo11-1, pair2, and crc1 In addition, mutations of the cohesion subunit OsREC8 resulted in biorientation of sister kinetochores as well as bipolar spindles even in the background of OsmtopVIB Therefore, we propose that biorientation of the kinetochore is required for bipolar spindle assembly in the absence of homologous recombination.

The Protein Modifications of O-GlcNAcylation and Phosphorylation Mediate Vernalization Response for Flowering in Winter Wheat.

O-GlcNAcylation and phosphorylation are two posttranslational modifications that antagonistically regulate protein function. However, the regulation of and the cross talk between these two protein modifications are poorly understood in plants. Here we investigated the role of O-GlcNAcylation during vernalization, a process whereby prolonged cold exposure promotes flowering in winter wheat (Triticum aestivum), and analyzed the dynamic profile of O-GlcNAcylated and phosphorylated proteins in response to vernalization. Altering O-GlcNAc signaling by chemical inhibitors affected the vernalization response, modifying the expression of VRN genes and subsequently affecting flowering transition. Over a vernalization time-course, O-GlcNAcylated and phosphorylated peptides were enriched from winter wheat plumules by Lectin weak affinity chromatography and iTRAQ-TiO2, respectively. Subsequent mass spectrometry and gene ontology term enrichment analysis identified 168 O-GlcNAcylated proteins that are mainly involved in responses to abiotic stimulus and hormones, metabolic processing, and gene expression; and 124 differentially expressed phosphorylated proteins that participate in translation, transcription, and metabolic processing. Of note, 31 vernalization-associated proteins were identified that carried both phosphorylation and O-GlcNAcylation modifications, of which the majority (97%) exhibited the coexisting module and the remainder exhibited the potential competitive module. Among these, TaGRP2 was decorated with dynamic O-GlcNAcylation (S87) and phosphorylation (S152) modifications, and the mutation of S87 and S152 affected the binding of TaGRP2 to the RIP3 motif of TaVRN1 in vitro. Our data suggest that a dynamic network of O-GlcNAcylation and phosphorylation at key pathway nodes regulate the vernalization response and mediate flowering in wheat.

OsCIPK7 point-mutation leads to conformation and kinase-activity change for sensing cold response.

Calcineurin B-like interacting protein kinases (CIPKs) play important roles via environmental stress. However, less is known how to sense the stress in molecular structure conformation level. Here, an OsCIPK7 mutant via TILLING procedure with a point mutation in the kinase domain showed increased chilling tolerance, which could be potentially used in the molecular breeding. We found that this point mutation of OsCIPK7 led to a conformational change in the activation loop of the kinase domain, subsequently with an increase of protein kinase activity, thus conferred an increased tolerance to chilling stress.