OsATL38 mediates mono-ubiquitination of the 14-3-3 protein OsGF14d and negatively regulates the cold stress response in rice.

One of the major regulatory pathways that permits plants to convert an external stimulus into an internal cellular response within a short period of time is the ubiquitination pathway. In this study, OsATL38 was identified as a low temperature-induced gene that encodes a rice homolog of Arabidopsis Tóxicos en Levadura RING-type E3 ubiquitin (Ub) ligase, which was predominantly localized to the plasma membrane. OsATL38-overexpressing transgenic rice plants exhibited decreased tolerance to cold stress as compared with wild-type rice plants. In contrast, RNAi-mediated OsATL38 knockdown transgenic progeny exhibited markedly increased tolerance to cold stress relative to that of wild-type plants, which indicated a negative role of OsATL38 in response to cold stress. Yeast two-hybrid, in vitro pull-down, and co-immunoprecipitation assays revealed that OsATL38 physically interacted with OsGF14d, a rice 14-3-3 protein. An in vivo target ubiquitination assay indicated that OsGF14d was mono-ubiquitinated by OsATL38. osgf14d knockout mutant plants were more sensitive to cold stress than wild-type rice plants, indicating that OsGF14d is a positive factor in the response to cold stress. These results provide evidence that the RING E3 Ub ligase OsATL38 negatively regulates the cold stress response in rice via mono-ubiquitination of OsGF14d 14-3-3 protein.

Abiotic Stress-Induced Actin-Depolymerizing Factor 3 From Deschampsia antarctica Enhanced Cold Tolerance When Constitutively Expressed in Rice.

The Antarctic flowering plant Deschampsia antarctica is highly sensitive to climate change and has shown rapid population increases during regional warming of the Antarctic Peninsula. Several studies have examined the physiological and biochemical changes related to environmental stress tolerance that allow D. antarctica to colonize harsh Antarctic environments; however, the molecular mechanisms of its responses to environmental changes remain poorly understood. To elucidate the survival strategies of D. antarctica in Antarctic environments, we investigated the functions of actin depolymerizing factor (ADF) in this species. We identified eight ADF genes in the transcriptome that were clustered into five subgroups by phylogenetic analysis. DaADF3, which belongs to a monocot-specific clade together with cold-responsive ADF in wheat, showed significant transcriptional induction in response to dehydration and cold, as well as under Antarctic field conditions. Multiple drought and low-temperature responsive elements were identified as possible binding sites of C-repeat-binding factors in the promoter region of DaADF3, indicating a close relationship between DaADF3 transcription control and abiotic stress responses. To investigate the functions of DaADF3 related to abiotic stresses in vivo, we generated transgenic rice plants overexpressing DaADF3. These transgenic plants showed greater tolerance to low-temperature stress than the wild-type in terms of survival rate, leaf chlorophyll content, and electrolyte leakage, accompanied by changes in actin filament organization in the root tips. Together, our results imply that DaADF3 played an important role in the enhancement of cold tolerance in transgenic rice plants and in the adaptation of D. antarctica to its extreme environment.

Improvement of moss photosynthesis by humic acids from Antarctic tundra soil.

There have been several published reports regarding the growth promoting effect of humic acids (HA) on vascular plants; however, the effect of HA on bryophytes is still unknown. Due to the ecological importance of mosses, which dominate the Antarctic flora, we assessed the effectiveness of HA as a biostimulant using three moss species: Antarctic Ceratodon purpureus KMA5038, Arctic Bryum sp. KMR5045, and Physcomitrella patens which inhabits temperate regions. Natural HA (KS1-3_HA) were extracted through acidic precipitation of alkaline extracts from Antarctic tundra soil. Spectroscopic structural properties of KS1-3_HA were characterized and determined to possess several functional groups such as hydroxyl (R-OH) and carboxyl (R-COOH), implying they could have a growth-related biological function. For two polar mosses, increasing HA concentrations correlated with increased growth and photosynthesis. The efficiency for temperate moss increased at lower concentrations tested, but rather began to reduce at the highest HA concentration, indicating that effective concentrations of HA vary depending on the moss species and habitat. Based on these results, Antarctic HA may have ecological role in enhancing the growth and photosynthesis of Antarctic mosses. We believe this is the first study to establish a positive physiological effect of HA on mosses and hope it may serve as a basis for studying the role of HA in preserving the terrestrial ecosystem of Antarctica.

Transfection of Arctic Bryum sp. KMR5045 as a Model for Genetic Engineering of Cold-Tolerant Mosses.

Mosses number about 13,000 species and are an important resource for the study of the plant evolution that occurred during terrestrial colonization by plants. Recently, the physiological and metabolic characteristics that distinguish mosses from terrestrial plants have received attention. In the Arctic, in particular, mosses developed their own distinct physiological features to adapt to the harsh environment. However, little is known about the molecular mechanisms by which Arctic mosses survive in extreme environments due to the lack of basic knowledge and tools such as genome sequences and genetic transfection methods. In this study, we report the axenic cultivation and transfection of Arctic Bryum sp. KMR5045, as a model for bioengineering of Arctic mosses. We also found that the inherent low-temperature tolerance of KMR5045 permitted it to maintain slow growth even at 2°C, while the model moss species Physcomitrium patens failed to grow at all, implying that KMR5045 is suitable for studies of cold-tolerance mechanisms. To achieve genetic transfection of KMR5045, some steps of the existing protocol for P. patens were modified. First, protoplasts were isolated using 1% driselase solution. Second, the appropriate antibiotic was identified and its concentration was optimized for the selection of transfectants. Third, the cell regeneration period before transfer to selection medium was extended to 9 days. As a result, KMR5045 transfectants were successfully obtained and confirmed transfection by detection of intracellular Citrine fluorescence derived from expression of a pAct5:Citrine transgene construct. This is the first report regarding the establishment of a genetic transfection method for an Arctic moss species belonging to the Bryaceae. The results of this study will contribute to understanding the function of genes involved in environmental adaptation and to application for production of useful metabolites derived from stress-tolerant mosses.

Poaceae Type II Galactinol Synthase 2 from Antarctic Flowering Plant Deschampsia antarctica and Rice Improves Cold and Drought Tolerance by Accumulation of Raffinose Family Oligosaccharides in Transgenic Rice Plants.

Deschampsia antarctica is a Poaceae grass that has adapted to and colonized Antarctica. When D. antarctica plants were subjected to cold and dehydration stress both in the Antarctic field and in laboratory experiments, galactinol, a precursor of raffinose family oligosaccharides (RFOs) and raffinose were highly accumulated, which was accompanied by upregulation of galactinol synthase (GolS). The Poaceae monocots have a small family of GolS genes, which are divided into two distinct groups called types I and II. Type II GolSs are highly expanded in cold-adapted monocot plants. Transgenic rice plants, in which type II D. antarctica GolS2 (DaGolS2) and rice GolS2 (OsGolS2) were constitutively expressed, were markedly tolerant to cold and drought stress as compared to the wild-type rice plants. The RFO contents and GolS enzyme activities were higher in the DaGolS2- and OsGolS2-overexpressing progeny than in the wild-type plants under both normal and stress conditions. DaGolS2 and OsGolS2 overexpressors contained reduced levels of reactive oxygen species (ROS) relative to the wild-type plants after cold and drought treatments. Overall, these results suggest that Poaceae type II GolS2s play a conserved role in D. antarctica and rice in response to drought and cold stress by inducing the accumulation of RFO and decreasing ROS levels.

The complete chloroplast genome of an Antarctic moss Syntrichia filaris (Müll.Hal.) R.H. Zander.

Syntrichia filaris is one of the common mosses in the northern maritime Antarctic. In this study, we determined the complete chloroplast genome of S. filaris (GenBank accession number MK852705) to provide a genetic resource for phylogenetic study on Bryophytes. It is of 136,227 bp length, containing 8 ribosomal RNA (rRNA), 37 transfer RNA, and 85 protein-coding genes. The chloroplast genome structure and gene order were similar to other bryophytes. Phylogenetic tree based on combined amino acids sequences of 72 chloroplast genes common in S. filaris, 7 Bryophyta, 1 Anthocerotophyta, and 2 Marchantiophyta, was congruent with the traditional position of Pottiales in Bryophytes.

OsDIRP1, a Putative RING E3 Ligase, Plays an Opposite Role in Drought and Cold Stress Responses as a Negative and Positive Factor, Respectively, in Rice (Oryza sativa L.).

As higher plants are sessile organisms, they are unable to move to more favorable places; thus, they have developed the ability to survive under potentially detrimental conditions. Ubiquitination is a crucial post-translational protein modification and participates in abiotic stress responses in higher plants. In this study, we identified and characterized OsDIRP1 (Oryza sativa Drought-Induced RING Protein 1), a nuclear-localized putative RING E3 ubiquitin (Ub) ligase in rice (Oryza sativa L.). OsDIRP1 expression was induced by drought, high salinity, and abscisic acid (ABA) treatment, but not by low temperature (4°C) stress, suggesting that OsDIRP1 is differentially regulated by different abiotic stresses. To investigate its possible role in abiotic stress responses, OsDIRP1-overexpressing transgenic rice plants (Ubi:OsDIRP1-sGFP) were generated, and their phenotypes were analyzed. The T4 Ubi:OsDIRP1-sGFP lines showed decreased tolerance to drought and salt stress as compared to wild-type rice plants. Moreover, Ubi:OsDIRP1-sGFP progeny were less sensitive to ABA than the wild-type during both germination and post-germination growth. In contrast, Ubi:OsDIRP1-sGFP plants exhibited markedly higher tolerance to prolonged cold (4°C) treatment. These results suggest that OsDIRP1 acts as a negative regulator during drought and salt stress, whereas it functions as a positive factor during the cold stress response in rice.

Telomere association of Oryza sativa telomere repeat-binding factor like 1 and its roles in telomere maintenance and development in rice, Oryza sativa L.

Telomeres are specialized nucleoprotein complexes that function to protect eukaryotic chromosomes from recombination and erosion. Several telomere binding proteins (TBPs) have been characterized in higher plants, but their detailed in vivo functions at the plant level are largely unknown. In this study, we identified and characterized OsTRFL1 (Oryza sativa Telomere Repeat-binding Factor Like 1) in rice, a monocot model crop. Although OsTRFL1 did not directly bind to telomere repeats (TTTAGGG)4 in vitro, it was associated with telomeric sequences in planta. OsTRFL1 interacted with rice TBPs, such as OsTRBF1 and RTBP1, in yeast and plant cells as well as in vitro. Thus, it seems likely that the association of OsTRFL1 with other TBPs enables OsTRFL1 to bind to telomeres indirectly. T-DNA inserted OsTRFL1 knock-out mutant rice plants displayed significantly longer telomeres (6-25 kb) than those (5-12 kb) in wild-type plants, indicating that OsTRFL1 is a negative factor for telomere lengthening. The reduced levels of OsTRFL1 caused serious developmental defects in both vegetative and reproductive organs of rice plants. These results suggest that OsTRFL1 is an essential factor for the proper maintenance of telomeres and normal development of rice. [BMB Reports 2018; 51(11): 578-583].

Identification of Rice Genes Associated With Enhanced Cold Tolerance by Comparative Transcriptome Analysis With Two Transgenic Rice Plants Overexpressing DaCBF4 or DaCBF7, Isolated From Antarctic Flowering Plant Deschampsia antarctica.

Few plant species can survive in Antarctica, the harshest environment for living organisms. Deschampsia antarctica is the only natural grass species to have adapted to and colonized the maritime Antarctic. To investigate the molecular mechanism of the Antarctic adaptation of this plant, we identified and characterized D. antarctica C-repeat binding factor 4 (DaCBF4), which belongs to monocot CBF group IV. The transcript level of DaCBF4 in D. antarctica was markedly increased by cold and dehydration stress. To assess the roles of DaCBF4 in plants, we generated a DaCBF4-overexpressing transgenic rice plant (Ubi:DaCBF4) and analyzed its abiotic stress response phenotype. Ubi:DaCBF4 displayed enhanced tolerance to cold stress without growth retardation under any condition compared to wild-type plants. Because the cold-specific phenotype of Ubi:DaCBF4 was similar to that of Ubi:DaCBF7 (Byun et al., 2015), we screened for the genes responsible for the improved cold tolerance in rice by selecting differentially regulated genes in both transgenic rice lines. By comparative transcriptome analysis using RNA-seq, we identified 9 and 15 genes under normal and cold-stress conditions, respectively, as putative downstream targets of the two D. antarctica CBFs. Overall, our results suggest that Antarctic hairgrass DaCBF4 mediates the cold-stress response of transgenic rice plants by adjusting the expression levels of a set of stress-responsive genes in transgenic rice plants. Moreover, selected downstream target genes will be useful for genetic engineering to enhance the cold tolerance of cereal plants, including rice.

Homologous U-box E3 Ubiquitin Ligases OsPUB2 and OsPUB3 Are Involved in the Positive Regulation of Low Temperature Stress Response in Rice (Oryza sativa L.).

Rice U-box E3 Ub ligases (OsPUBs) are implicated in biotic stress responses. However, their cellular roles in response to abiotic stress are poorly understood. In this study, we performed functional analyses of two homologous OsPUB2 and OsPUB3 in response to cold stress (4°C). OsPUB2 was up-regulated by high salinity, drought, and cold, whereas OsPUB3 was constitutively expressed. A subcellular localization assay revealed that OsPUB2 and OsPUB3 were localized to the exocyst positive organelle (EXPO)-like punctate structures. OsPUB2 was also localized to the nuclei. OsPUB2 and OsPUB3 formed a hetero-dimeric complex as well as homo-dimers in yeast cells and in vitro. OsPUB2/OsPUB3 exhibited self-ubiquitination activities in vitro and were rapidly degraded in the cell-free extracts with apparent half-lives of 150-160 min. This rapid degradation of OsPUB2/OsPUB3 was delayed in the presence of the crude extracts of cold-treated seedlings (apparent half-lives of 200-280 min). Moreover, a hetero-dimeric form of OsPUB2/OsPUB3 was more stable than the homo-dimers. These results suggested that OsPUB2 and OsPUB3 function coordinately in response to cold stress. OsPUB2- and OsPUB3-overexpressing transgenic rice plants showed markedly better tolerance to cold stress than did the wild-type plants in terms of survival rates, chlorophyll content, ion leakage, and expression levels of cold stress-inducible marker genes. Taken together, these results suggested that the two homologous rice U-box E3 Ub ligases OsPUB2 and OsPUB3 are positive regulators of the response to cold stress.

Suppression of OsKu80 results in defects in developmental growth and increased telomere length in rice (Oryza sativa L.).

The Ku70-Ku80 heterodimer plays a critical role in the maintenance of genomic stability in humans and yeasts. In this report, we identified and characterized OsKu80 in rice, a model monocot crop. OsKu80 forms a heterodimer with OsKu70 in yeast and plant cells, as demonstrated by yeast two-hybrid, in vivo co-immunoprecipitation, and bimolecular fluorescence complementation assays. RNAi-mediated knock-down T3 transgenic rice plants (Ubi:RNAi-OsKu80) displayed a retarded growth phenotype at the post-germination stage. In addition, the Ubi:RNAi-OsKu80 knock-down progeny exhibited noticeably increased telomere length as compared to wild-type rice. These results are discussed with the idea that OsKu80 plays a role in developmental growth and telomere length regulation in rice plants.

Constitutive expression of DaCBF7, an Antarctic vascular plant Deschampsia antarctica CBF homolog, resulted in improved cold tolerance in transgenic rice plants.

Deschampsia antarctica is an Antarctic hairgrass that grows on the west coast of the Antarctic peninsula. In this report, we have identified and characterized a transcription factor, D. antarctica C-repeat binding factor 7 (DaCBF7), that is a member of the monocot group V CBF homologs. The protein contains a single AP2 domain, a putative nuclear localization signal, and the typical CBF signature. DaCBF7, like other monocot group V homologs, contains a distinct polypeptide stretch composed of 43 amino acids in front of the AP2 motif. DaCBF7 was predominantly localized to nuclei and interacted with the C-repeat/dehydration responsive element (CRT/DRE) core sequence (ACCGAC) in vitro. DaCBF7 was induced by abiotic stresses, including drought, cold, and salinity. To investigate its possible cellular role in cold tolerance, a transgenic rice system was employed. DaCBF7-overexpressing transgenic rice plants (Ubi:DaCBF7) exhibited markedly increased tolerance to cold stress compared to wild-type plants without growth defects; however, overexpression of DaCBF7 exerted little effect on tolerance to drought or salt stress. Transcriptome analysis of a Ubi:DaCBF7 transgenic line revealed 13 genes that were up-regulated in DaCBF7-overexpressing plants compared to wild-type plants in the absence of cold stress and in short- or long-term cold stress. Five of these genes, dehydrin, remorin, Os03g63870, Os11g34790, and Os10g22630, contained putative CRT/DRE or low-temperature responsive elements in their promoter regions. These results suggest that overexpression of DaCBF7 directly and indirectly induces diverse genes in transgenic rice plants and confers enhanced tolerance to cold stress.

Suppression of OsRAD51D results in defects in reproductive development in rice (Oryza sativa L.).

The cellular roles of RAD51 paralogs in somatic and reproductive growth have been extensively described in a wide range of animal systems and, to a lesser extent, in Arabidopsis, a dicot model plant. Here, the OsRAD51D gene was identified and characterized in rice (Oryza sativa L.), a monocot model crop. In the rice genome, three alternative OsRAD51D mRNA splicing variants, OsRAD51D.1, OsRAD51D.2, and OsRAD51D.3, were predicted. Yeast two-hybrid studies, however, showed that only OsRAD51D.1 interacted with OsRAD51B and OsRAD51C paralogs, suggesting that OsRAD51D.1 is a functional OsRAD51D protein in rice. Loss-of-function osrad51d mutant rice plants displayed normal vegetative growth. However, the mutant plants were defective in reproductive growth, resulting in sterile flowers. Homozygous osrad51d mutant flowers exhibited impaired development of lemma and palea and contained unusual numbers of stamens and stigmas. During early meiosis, osrad51d pollen mother cells (PMCs) failed to form normal homologous chromosome pairings. In subsequent meiotic progression, mutant PMCs represented fragmented chromosomes. The osrad51d pollen cells contained numerous abnormal micro-nuclei that resulted in malfunctioning pollen. The abnormalities of heterozygous mutant and T2 Ubi:RNAi-OsRAD51D RNAi-knock-down transgenic plants were intermediate between those of wild type and homozygous mutant plants. The osrad51d and Ubi:RNAi-OsRAD51D plants contained longer telomeres compared with wild type plants, indicating that OsRAD51D is a negative factor for telomere lengthening. Overall, these results suggest that OsRAD51D plays a critical role in reproductive growth in rice. This essential function of OsRAD51D is distinct from Arabidopsis, in which AtRAD51D is not an essential factor for meiosis or reproductive development.

OsKu70 is associated with developmental growth and genome stability in rice.

The cellular functions of Ku70 in repair of DNA double-stranded breaks and telomere regulation have been described in a wide range of organisms. In this study, we identified the rice (Oryza sativa) Ku70 homolog (OsKu70) from the rice genome database. OsKu70 transcript was detected constitutively in every tissue and developmental stage examined and also in undifferentiated callus cells in rice. Yeast two-hybrid and in vitro pull-down experiments revealed that OsKu70 physically interacts with OsKu80. We obtained loss-of-function osku70 T-DNA knockout mutant lines and constructed transgenic rice plants that overexpress the OsKu70 gene in the sense (35S:OsKu70) or antisense (35S:anti-OsKu70) orientation. The homozygous G2 osku70 mutant lines were more sensitive than wild-type plants to a DNA-damaging agent (0.01%-0.05% methyl-methane sulfonate), consistent with the notion that OsKu70 participates in the DNA repair mechanism. Terminal restriction fragment analysis revealed that telomeres in homozygous G2 osku70 mutants were markedly longer (10-20 kb) than those in wild-type plants (5-10 kb), whereas telomere length in heterozygous G2 osku70 mutant and T2 OsKu70-overexpressing transgenic (35S:OsKu70) rice resembled that of the wild-type plant. In contrast to what was observed in Arabidopsis (Arabidopsis thaliana) atku70 mutants, homozygous G2 osku70 rice plants displayed severe developmental defects in both vegetative and reproductive organs under normal growth conditions, resulting in sterile flowers. Analysis of meiotic progression in pollen mother cells demonstrated that up to 11.1% (seven of 63) of G2 mutant anaphase cells displayed one or more chromosomal fusions. These results suggest that OsKu70 is required for the maintenance of chromosome stability and normal developmental growth in rice plants.

Identification and characterization of three telomere repeat-binding factors in rice.

Telomeres consist of nucleoprotein complexes that protect chromosome end structures. Here, we describe three OsTRBF genes, encoding telomere repeat-binding factors of the single Myb histone family in rice. The predicted proteins contain a Myb DNA-binding motif and a linker histone H1/H5 domain in the N-terminal and central regions, respectively. The OsTRBF transcripts were constitutively detected in rice plants grown under greenhouse conditions. Gel retardation assays showed that these OsTRBF proteins bind specifically to the plant double-stranded telomeric sequence, TTTAGGG, with markedly different binding affinities as judged by their respective dissociation constants. Yeast two-hybrid and in vitro pull-down assays indicated that both OsTRBF1 and OsTRBF2 interact with one another to form homo- and hetero-complexes, while OsTRBF3 appeared to act as a monomer. Our results suggest that OsTRBFs play combinatory roles in the function and structure of telomeres in rice.

Suppression of RICE TELOMERE BINDING PROTEIN 1 results in severe and gradual developmental defects accompanied by genome instability in rice.

Although several potential telomere binding proteins have been identified in higher plants, their in vivo functions are still unknown at the plant level. Both knockout and antisense mutants of RICE TELOMERE BINDING PROTEIN1 (RTBP1) exhibited markedly longer telomeres relative to those of the wild type, indicating that the amount of functional RTBP1 is inversely correlated with telomere length. rtbp1 plants displayed progressive and severe developmental abnormalities in both germination and postgermination growth of vegetative organs over four generations (G1 to G4). Reproductive organ formation, including panicles, stamens, and spikelets, was also gradually and severely impaired in G1 to G4 mutants. Up to 11.4, 17.2, and 26.7% of anaphases in G2, G3, and G4 mutant pollen mother cells, respectively, exhibited one or more chromosomal fusions, and this progressively increasing aberrant morphology was correlated with an increased frequency of anaphase bridges containing telomeric repeat DNA. Furthermore, 35S:anti-RTBP1 plants expressing lower levels of RTBP1 mRNA exhibited developmental phenotypes intermediate between the wild type and mutants in all aspects examined, including telomere length, vegetative and reproductive growth, and degree of genomic anomaly. These results suggest that RTBP1 plays dual roles in rice (Oryza sativa), as both a negative regulator of telomere length and one of positive and functional components for proper architecture of telomeres.