Genome-wide association study of stem structural characteristics that extracted by a high-throughput phenotypic analysis "LabelmeP rice" in rice.

Stem is important for assimilating transport and plant strength; however, less is known about the genetic basis of its structural characteristics. In this study, a high-throughput method, "LabelmeP rice" was developed to generate 14 traits related to stem regions and vascular bundles, which allows the establishment of a stem cross-section phenotype dataset containing anatomical information of 1738 images from hand-cut transections of stems collected from 387 rice germplasm accessions grown over two successive seasons. Then, the phenotypic diversity of the rice accessions was evaluated. Genome-wide association studies identified 94, 83, and 66 significant single nucleotide polymorphisms (SNPs) for the assayed traits in 2 years and their best linear unbiased estimates, respectively. These SNPs can be integrated into 29 quantitative trait loci (QTL), and 11 of them were common in 2 years, while correlated traits shared 19. In addition, 173 candidate genes were identified, and six located at significant SNPs were repeatedly detected and annotated with a potential function in stem development. By using three introgression lines (chromosome segment substitution lines), four of the 29 QTLs were validated. LOC_Os01g70200, located on the QTL uq1.4, is detected for the area of small vascular bundles (SVB) and the rate of large vascular bundles number to SVB number. Besides, the CRISPR/Cas9 editing approach has elucidated the function of the candidate gene LOC_Os06g46340 in stem development. In conclusion, the results present a time- and cost-effective method that provides convenience for extracting rice stem anatomical traits and the candidate genes/QTL, which would help improve rice.

Effect of postoperative exercise training on physical function and quality of life of lung cancer patients with chronic obstructive pulmonary disease: A randomized controlled trial.

BACKGROUND: Postoperative rehabilitation programs consisting of exercise training are considered effective for unselected lung cancer patients. However, whether postoperative exercise is beneficial to lung cancer patients comorbid with chronic obstructive pulmonary disease remains unknown. METHODS: Eighty-four patients diagnosed with both lung cancer and chronic obstructive pulmonary disease were randomized into the exercise group and control group. Both groups were given standard postoperative rehabilitation for 1 week. After that, oxygen therapy (if needed) and nebulization were given to the control group, while patients in the exercise group started to participate in exercise programs on the basis of receiving oxygen therapy and nebulization as in the control group. The exercise programs consisted of 24 training sessions. RESULTS: In both groups, the functional status and the results of the pulmonary function test decreased from baseline to the endpoint. However, after surgery and the intervention program, both the maximal oxygen consumption in the cardiopulmonary exercise test and walking distance in the 6-minute walk test in the exercise group were significantly better than those in the control group [15.5 (±1.4) mL/kg/min vs 13.1 (±1.3) mL/kg/min, P = 0.016; 437.4 (±48.6) m vs 381.7 (±40.5) m, P = 0.040]. Force vital capacity and forced expiratory volume in the first second in the exercise group were better than those in the control group, but the differences were not statistically significant [1798.1 (±298.9) mL vs 1664.0 (±329.7) mL, P = 0.254; 1155.7 (±174.3) mL vs 967.4 (±219.4) mL, P = 0.497]. The decline in the standard score of the QLQ-C30 (V3.0) was smaller in the exercise group, but the difference did not meet a statistically significant level [61.7 (±5.7) vs 58.4 (±9.3), P = 0.318]. CONCLUSION: This study demonstrates that a short-term postoperative exercise training program can facilitate the recovery of functional capacity in lung cancer patients with comorbidities of chronic obstructive pulmonary disease.

CALMODULIN-LIKE16 and PIN-LIKES7a cooperatively regulate rice seedling primary root elongation under chilling.

Low-temperature sensitivity at the germination stage is a challenge for direct seeding of rice in Asian countries. How Ca2+ and auxin (IAA) signaling regulate primary root growth under chilling remains unexplored. Here, we showed that OsCML16 interacted specifically with OsPILS7a to improve primary root elongation of early rice seedlings under chilling. OsCML16, a subgroup 6c member of the OsCML family, interacted with multiple cytosolic loop regions of OsPILS7a in a Ca2+-dependent manner. OsPILS7a localized to the endoplasmic reticulum membranes and functioned as an auxin efflux carrier in a yeast growth assay. Transgenics showed that presence of OsCML16 enhanced primary root elongation under chilling, whereas the ospils7a knockout mutant lines showed the opposite phenotype. Moreover, under chilling conditions, OsCML16 and OsPILS7a-mediated Ca2+ and IAA signaling and regulated the transcription of IAA signaling-associated genes (OsIAA11, OsIAA23, and OsARF16) and cell division marker genes (OsRAN1, OsRAN2, and OsLTG1) in primary roots. These results show that OsCML16 and OsPILS7a cooperatively regulate primary root elongation of early rice seedlings under chilling. These findings enhance our understanding of the crosstalk between Ca2+ and IAA signaling and reveal insights into the mechanisms underlying cold-stress response during rice germination.

The OsTIL1 lipocalin protects cell membranes from reactive oxygen species damage and maintains the 18:3-containing glycerolipid biosynthesis under cold stress in rice.

Lipocalins constitute a conserved protein family that binds to and transports a variety of lipids while fatty acid desaturases (FADs) are required for maintaining the cell membrane fluidity under cold stress. Nevertheless, it remains unclear whether plant lipocalins promote FADs for the cell membrane integrity under cold stress. Here, we identified the role of OsTIL1 lipocalin in FADs-mediated glycerolipid remodeling under cold stress. Overexpression and CRISPR/Cas9 mediated gene edition experiments demonstrated that OsTIL1 positively regulated cold stress tolerance by protecting the cell membrane integrity from reactive oxygen species damage and enhancing the activities of peroxidase and ascorbate peroxidase, which was confirmed by combined cold stress with a membrane rigidifier dimethyl sulfoxide or a H2 O2 scavenger dimethyl thiourea. OsTIL1 overexpression induced higher 18:3 content, and higher 18:3/18:2 and (18:2 + 18:3)/18:1 ratios than the wild type under cold stress whereas the gene edition mutant showed the opposite. Furthermore, the lipidomic analysis showed that OsTIL1 overexpression led to higher contents of 18:3-mediated glycerolipids, including galactolipids (monoglactosyldiacylglycerol and digalactosyldiacylglycerol) and phospholipids (phosphatidyl glycerol, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and phosphatidyl inositol) under cold stress. RNA-seq and enzyme linked immunosorbent assay analyses indicated that OsTIL1 overexpression enhanced the transcription and enzyme abundance of four ω-3 FADs (OsFAD3-1/3-2, 7, and 8) under cold stress. These results reveal an important role of OsTIL1 in maintaining the cell membrane integrity from oxidative damage under cold stress, providing a good candidate gene for improving cold tolerance in rice.

OsFBN7-OsKAS I module promotes formation of plastoglobules clusters in rice chloroplasts.

Plastoglobules (PGs) contiguous with the outer leaflets of thylakoid membranes regulate lipid metabolism, plastid developmental transitions, and responses to environmental stimuli. However, the function of OsFBN7, a PG-core fibrillin gene in rice, has not been elucidated. Using molecular genetics and physiobiochemical approaches, we observed that OsFBN7 overexpression promoted PG clustering in rice chloroplasts. OsFBN7 interacted with two KAS I enzymes, namely OsKAS Ia and OsKAS Ib, in rice chloroplasts. Lipidomic analysis of chloroplast subcompartments, including PGs in the OsFBN7 overexpression lines, confirmed that levels of diacylglycerol (DAG), a chloroplast lipid precursor and monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), the main chloroplast membrane lipids, were increased in PGs and chloroplasts. Furthermore, OsFBN7 enhanced the abundances of OsKAS Ia/Ib in planta and their stability under oxidative and heat stresses. In addition, RNA sequencing and real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analyses showed that the expression of the DAG synthetase gene PAP1 and MGDG synthase gene MDG2 was upregulated by OsFBN7. In conclusion, this study proposes a new model in which OsFBN7 binds to OsKAS Ia/Ib in chloroplast and enhances their abundance and stability, thereby regulating the chloroplast and PG membrane lipids involved in the formation of PG clusters.

Genome-Wide Association Mapping Identifies New Candidate Genes for Cold Stress and Chilling Acclimation at Seedling Stage in Rice (Oryza sativa L.).

Rice (Oryza sativa L.) is a chilling-sensitive staple food crop, and thus, low temperature significantly affects rice growth and yield. Many studies have focused on the cold shock of rice although chilling acclimation is more likely to happen in the field. In this paper, a genome-wide association study (GWAS) was used to identify the genes that participated in cold stress and chilling accumulation. A total of 235 significantly associated single-nucleotide polymorphisms (SNPs) were identified. Among them, we detected 120 and 88 SNPs for the relative shoot fresh weight under cold stress and chilling acclimation, respectively. Furthermore, 11 and 12 quantitative trait loci (QTLs) were identified for cold stress and chilling acclimation, respectively, by integrating the co-localized SNPs. Interestingly, we identified 10 and 15 candidate genes in 11 and 12 QTLs involved in cold stress and chilling acclimation, respectively, and two new candidate genes (LOC_Os01g62410, LOC_Os12g24490) were obviously up-regulated under chilling acclimation. Furthermore, OsMYB3R-2 (LOC_Os01g62410) that encodes a R1R2R3 MYB gene was associated with cold tolerance, while a new C3HC4-type zinc finger protein-encoding gene LOC_Os12g24490 was found to function as a putative E3 ubiquitin-protein ligase in rice. Moreover, haplotype, distribution, and Wright's fixation index (FST) of both genes showed that haplotype 3 of LOC_Os12g24490 is more stable in chilling acclimation, and the SNP (A > T) showed a difference in latitudinal distribution. FST analysis of SNPs in OsMYB3R-2 (LOC_Os01g62410) and LOC_Os12g24490 indicated that several SNPs were under selection in rice indica and japonica subspecies. This study provided new candidate genes in genetic improvement of chilling acclimation response in rice.

Global identification of quantitative trait loci and candidate genes for cold stress and chilling acclimation in rice through GWAS and RNA-seq.

MAIN CONCLUSION: Associated analysis of GWAS with RNA-seq had detected candidate genes responsible for cold stress and chilling acclimation in rice. Haplotypes of two candidate genes and geographic distribution were analyzed. To explore new candidate genes and genetic resources for cold tolerance improvement in rice, genome-wide association study (GWAS) mapping experiments with 351 rice core germplasms was performed for three traits (survival rate, shoot length and chlorophyll content) under three temperature conditions (normal temperature, cold stress and chilling acclimation), yielding a total of 134 QTLs, of which 54, 59 and 21 QTLs were responsible for normal temperature, cold stress and chilling acclimation conditions, respectively. Integrated analysis of significant SNPs in 134 QTLs further identified 116 QTLs for three temperature treatments, 53, 43 and 18 QTLs responsible for normal temperature, cold stress and chilling acclimation, respectively, and 2 QTLs were responsible for both cold stress and chilling acclimation. Matching differentially expressed genes from RNA-seq to 43 and 18 QTLs for cold stress and chilling acclimation, we identified 69 and 44 trait-associated candidate genes, respectively, to be classified into six and five groups, particularly involved in metabolisms, reactive oxygen species scavenging and hormone signaling. Interestingly, two candidate genes LOC_Os01g04814, encoding a vacuolar protein sorting-associating protein 4B, and LOC_Os01g48440, encoding glycosyltransferase family 43 protein, showed the highest expression levels under chilling acclimation. Haplotype analysis revealed that both genes had a distinctive differentiation with subpopulation. Haplotypes of both genes with more japonica accessions have higher latitude distribution and higher chilling tolerance than the chilling sensitive indica accessions. These findings reveal the new insight into the molecular mechanism and candidate genes for cold stress and chilling acclimation in rice.

[Study on the Benefit of Postoperative Exercise Rehabilitation in Patients with 
Lung Cancer Complicated with Chronic Obstructive Pulmonary Disease].

BACKGROUND: Chronic obstructive pulmonary diseases (COPD) affects 45%-63% of lung cancer patients worldwide. Lung cancer patients complicated with COPD have decreased cardiopulmonary function and increased perioperative risk, and their postoperative exercise endurance and lung function are significantly lower than those with conventional lung cancer. Previous studies have shown that postoperative exercise training can improve the exercise endurance of unselected lung cancer patients, but it is unclear whether lung cancer patients with COPD can also benefit from postoperative exercise training. This study intends to explore the effects of postoperative exercise training on exercise endurance, daily activity and lung function of lung cancer patients with COPD. METHODS: Seventy-four patients with non-small cell lung cancer (NSCLC) complicated with COPD who underwent pneumonectomy in the lung cancer center of West China Hospital of Sichuan University from August 5, 2020 to August 25, 2021 were prospectively analyzed. They were randomly divided into exercise group and control group; The patients in the two groups received routine postoperative rehabilitation in the first week after operation, and the control group was given routine nursing from the second week. On this basis, the exercise group received postoperative exercise rehabilitation training for two weeks. Baseline evaluation was performed 3 days before operation and endpoint evaluation was performed 3 weeks after operation. RESULTS: The exercise endurance, daily activity and pulmonary function test results of the two groups decreased from baseline to the end point. However, after the operation and intervention program, the maximum oxygen consumption of Cardiopulmonary Exercise Test and the walking distance of 6-Minute Walking Test in the exercise group were significantly better than those in the control group [(13.09±1.46) mL/kg/min vs (11.89±1.38) mL/kg/min, P=0.033; (297±46) m vs (243±43) m, P=0.041]. The average number of we-chat steps in the exercise group was also significantly better than that in the control group (4,381±397 vs 3,478±342, P=0.035). Forced vital capacity (FVC) and forced expiratory volume in one second (FEV1) in the exercise group were better than those in the control group, but the difference did not reach a statistically significant level [(1.76±0.19) L vs (1.60±0.28) L, P=0.084; (1.01±0.17) L vs (0.96±0.21) L, P=0.467]. CONCLUSIONS: Postoperative exercise rehabilitation training can improve exercise endurance and daily activity ability of patients with lung cancer complicated with COPD and promote postoperative rehabilitation.

OsVDE, a xanthophyll cycle key enzyme, mediates abscisic acid biosynthesis and negatively regulates salinity tolerance in rice.

MAIN CONCLUSION: OsVDE, a lipocalin-like protein in chloroplasts, negatively regulated the ABA biosynthesis and stomatal closure under salt stress in rice seedlings. Violaxanthin de-epoxidase (VDE) is a key enzyme of xanthophyll cycle. It plays a critical role in abscisic acid (ABA) biosynthesis, growth and stress responses in plants. Although functions of several VDE genes have been characterized, it is largely unknown whether OsVDE regulates the ABA biosynthesis and salt stress tolerance in rice. In this study, we generated the OsVDE overexpressing and CRISPR-Cas9-mediated gene-editing transgenic lines, and identified that the gene-editing mutant lines showed the dwarfism, shorter panicle and lower seed-setting rate than the wild type whereas the overexpression lines did not exhibit the difference from the wild type. In addition, the gene-editing transgenic lines were hypersensitive to exogenous ABA during germination. Under salt stress, the gene-editing transgenic seedlings had a higher ABA level, higher stomatal closure percentage and higher survival rate than the wild type. The qRT-PCR analysis confirmed that OsVDE negatively regulated the OsNECD2/4/5 expressions, ABA biosynthesis and salt stress tolerance in rice seedlings. These results provide new evidence that VDE plays an essential role in ABA biosynthesis and salt stress tolerance in plants.

The Divergent Roles of the Rice bcl-2 Associated Athanogene (BAG) Genes in Plant Development and Environmental Responses.

Bcl-2-associated athanogene (BAG), a group of proteins evolutionarily conserved and functioned as co-chaperones in plants and animals, is involved in various cell activities and diverse physiological processes. However, the biological functions of this gene family in rice are largely unknown. In this study, we identified a total of six BAG members in rice. These genes were classified into two groups, OsBAG1, -2, -3, and -4 are in group I with a conserved ubiquitin-like structure and OsBAG5 and -6 are in group Ⅱ with a calmodulin-binding domain, in addition to a common BAG domain. The BAG genes exhibited diverse expression patterns, with OsBAG4 showing the highest expression level, followed by OsBAG1 and OsBAG3, and OsBAG6 preferentially expressed in the panicle, endosperm, and calli. The co-expression analysis and the hierarchical cluster analysis indicated that the OsBAG1 and OsBAG3 were co-expressed with primary cell wall-biosynthesizing genes, OsBAG4 was co-expressed with phytohormone and transcriptional factors, and OsBAG6 was co-expressed with disease and shock-associated genes. ß-glucuronidase (GUS) staining further indicated that OsBAG3 is mainly involved in primary young tissues under both primary and secondary growth. In addition, the expression of the BAG genes under brown planthopper (BPH) feeding, N, P, and K deficiency, heat, drought and plant hormones treatments was investigated. Our results clearly showed that OsBAGs are multifunctional molecules as inferred by their protein structures, subcellular localizations, and expression profiles. BAGs in group I are mainly involved in plant development, whereas BAGs in group II are reactive in gene regulations and stress responses. Our results provide a solid basis for the further elucidation of the biological functions of plant BAG genes.

Convergence and Divergence: Signal Perception and Transduction Mechanisms of Cold Stress in Arabidopsis and Rice.

Cold stress, including freezing stress and chilling stress, is one of the major environmental factors that limit the growth and productivity of plants. As a temperate dicot model plant species, Arabidopsis develops a capability to freezing tolerance through cold acclimation. The past decades have witnessed a deep understanding of mechanisms underlying cold stress signal perception, transduction, and freezing tolerance in Arabidopsis. In contrast, a monocot cereal model plant species derived from tropical and subtropical origins, rice, is very sensitive to chilling stress and has evolved a different mechanism for chilling stress signaling and response. In this review, the authors summarized the recent progress in our understanding of cold stress response mechanisms, highlighted the convergent and divergent mechanisms between Arabidopsis and rice plasma membrane cold stress perceptions, calcium signaling, phospholipid signaling, MAPK cascade signaling, ROS signaling, and ICE-CBF regulatory network, as well as light-regulated signal transduction system. Genetic engineering approaches of developing freezing tolerant Arabidopsis and chilling tolerant rice were also reviewed. Finally, the future perspective of cold stress signaling and tolerance in rice was proposed.

Genome-Wide Association Study in Rice Revealed a Novel Gene in Determining Plant Height and Stem Development, by Encoding a WRKY Transcription Factor.

Semi-dwarfism is a main agronomic trait in crop breeding. In this study, we performed genome-wide association study (GWAS) and identified a new quantitative trait nucleotide (QTN) for rice shoot length. The peak QTN (C/T) was located in the first coding region of a group III WRKY transcription factor OsWRKY21 (LOC_Os01g60640). Interestingly, further haplotype analysis showed that C/T difference only existed in the indica group but not in the japonica group, resulting in significant differences in plant height among the different indica rice varieties. OsWRKY21 was expressed in embryo, radicle, shoots, leaves, and stems. Most notably, overexpressing OsWRKY21 resulted in the semi-dwarf phenotype, early heading date and short internodes compared to the wild type, while the knockout mutant plants by CRISPR/Cas9 technology yielded the opposite. The overexpressing lines exhibited the decreased length of the cells near sclerenchyma epidermis, accompanied with the lower levels of indole-3-acetic acid (IAA) and gibberellin 3 (GA3), but increased levels of the abscisic acid (ABA) and salicylic acid (SA) in the internodes at heading stage. Moreover, the semi-dwarf phenotype could be fully rescued by exogenous GA3 application at seedling stage. The RNA-seq and qRT-PCR analysis confirmed the differential expression levels of genes in development and the stress responses in rice, including GA metabolism (GA20ox2, GA2ox6, and YABY1) and cell wall biosynthesis (CesA4, 7, and 9) and regulation (MYB103L). These data suggest the essential role of OsWRKY21 in regulation of internode elongation and plant height in rice.

The CaM1-associated CCaMK-MKK1/6 cascade positively affects lateral root growth via auxin signaling under salt stress in rice.

Ca2+/calmodulin (CaM)-dependent protein kinases (CCaMKs) and mitogen-activated protein kinase kinases (MAPKKs) are two types of kinases that regulate salt stress response in plants. It remains unclear, however, how they cooperatively affect lateral root growth under salt stress. Here, two conserved phosphorylation sites (S102 and T118) of OsCaM1 were identified, and found to affect the ability to bind to Ca2+in vitro and the kinase activity of OsCCaMK in vivo. OsCCaMK specifically interacted with OsMKK1/6 in a Ca2+/CaM-dependent manner. In vitro kinase and in vivo dual-luciferase assays revealed that OsCCaMK phosphorylated OsMKK6 while OsMKK1 phosphorylated OsCCaMK. Overexpression and antisense-RNA repression expression of OsCaM1-1, and CRISPR/Cas9-mediated gene editing mutations of OsMKK1, OsMKK6, and OsMKK1/6 proved that OsCaM1-1, OsMKK1, and OsMKK6 enhanced the auxin content in roots and lateral root growth under salt stress. Consistently, OsCaM1-1, OsMKK1, and OsMKK6 regulated the transcript levels of the genes of this cascade, and salt stress-related and lateral root growth-related auxin signaling under salt stress in rice roots. These findings demonstrate that the OsCaM1-associated OsCCaMK-OsMKK1/6 cascade plays a critical role in recruiting auxin signaling in rice roots. These results also provide new insight into the regulatory mechanism of the CaM-mediated phosphorylation relay cascade to auxin signaling in lateral root growth under salt stress in plants.

Genome-Wide Association Study Reveals the QTLs for Seed Storability in World Rice Core Collections.

Seed storability is a main agronomically important trait to assure storage safety of grain and seeds in rice. Although many quantitative trait loci (QTLs) and associated genes for rice seed storability have been identified, the detailed genetic mechanisms of seed storability remain unclear in rice. In this study, a genome-wide association study (GWAS) was performed in 456 diverse rice core collections from the 3K rice genome. We discovered the new nine QTLs designated as qSS1-1, qSS1-2, qSS2-1, qSS3-1, qSS5-1, qSS5-2, qSS7-1, qSS8-1, and qSS11-1. According to the analysis of the new nine QTLs, our results could well explain the reason why seed storability of indica subspecies was superior to japonica subspecies in rice. Among them, qSS1-2 and qSS8-1 were potentially co-localized with a known associated qSS1/OsGH3-2 and OsPIMT1, respectively. Our results also suggest that pyramiding breeding of superior alleles of these associated genes will lead to new varieties with improved seed storability in the future.

Uncovering genomic regions controlling plant architectural traits in hexaploid wheat using different GWAS models.

Wheat is a major food crop worldwide. The plant architecture is a complex trait mostly influenced by plant height, tiller number, and leaf morphology. Plant height plays a crucial role in lodging and thus affects yield and grain quality. In this study, a wheat population was genotyped by using Illumina iSelect 90K single nucleotide polymorphism (SNP) assay and finally 22,905 high-quality SNPs were used to perform a genome-wide association study (GWAS) for plant architectural traits employing four multi-locus GWAS (ML-GWAS) and three single-locus GWAS (SL-GWAS) models. As a result, 174 and 97 significant SNPs controlling plant architectural traits were detected by ML-GWAS and SL-GWAS methods, respectively. Among these SNP makers, 43 SNPs were consistently detected, including seven across multiple environments and 36 across multiple methods. Interestingly, five SNPs (Kukri_c34553_89, RAC875_c8121_1490, wsnp_Ex_rep_c66315_64480362, Ku_c5191_340, and tplb0049a09_1302) consistently detected across multiple environments and methods, played a role in modulating both plant height and flag leaf length. Furthermore, candidate SNPs (BS00068592_51, Kukri_c4750_452 and BS00022127_51) constantly repeated in different years and methods associated with flag leaf width and number of tillers. We also detected several SNPs (Jagger_c6772_80, RAC875_c8121_1490, BS00089954_51, Excalibur_01167_1207, and Ku_c5191_340) having common associations with more than one trait across multiple environments. By further appraising these GWAS methods, the pLARmEB and FarmCPU models outperformed in SNP detection compared to the other ML-GWAS and SL-GWAS methods, respectively. Totally, 152 candidate genes were found to be likely involved in plant growth and development. These finding will be helpful for better understanding of the genetic mechanism of architectural traits in wheat.

OsClo5 functions as a transcriptional co-repressor by interacting with OsDi19-5 to negatively affect salt stress tolerance in rice seedlings.

Caleosins constitute a small protein family with one calcium-binding EF-hand motif. They are involved in the regulation of development and response to abiotic stress in plants. Nevertheless, how they impact salt stress tolerance in rice is largely unknown. Thereby, biochemical and molecular genetic experiments were carried out, and the results revealed that OsClo5 was able to bind calcium and phospholipids in vitro and localized in the nucleus and endoplasmic reticulum in rice protoplasts. At the germination and early seedlings stages, overexpression transgenic lines and T-DNA mutant lines exhibited reduced and increased tolerance to salt stress, respectively, compared with the wild-type. Yeast two-hybrid, bimolecular fluorescence complementation and in vitro pull-down assays demonstrated that the EF-hand motif of OsClo5 was essential for the interactions with itself and OsDi19-5. Yeast one-hybrid, electrophoretic migration shift and dual-luciferase reporter assays identified OsDi19-5 as a transcriptional repressor via the TACART cis-element in the promoters of two salt stress-related target genes, OsUSP and OsMST. In addition, OsClo5 enhanced the inhibitory effect of OsDi19-5 in the tobacco transient system, which was confirmed by qRT-PCR analysis in rice seedlings under salt stress. The collective results deepen the understanding of the molecular mechanism underlying the roles of caleosin in the salt stress response. These findings will also inform efforts to improve salt tolerance of rice.

Appraising the Genetic Architecture of Kernel Traits in Hexaploid Wheat Using GWAS.

Kernel morphology is one of the major yield traits of wheat, the genetic architecture of which is always important in crop breeding. In this study, we performed a genome-wide association study (GWAS) to appraise the genetic architecture of the kernel traits of 319 wheat accessions using 22,905 single nucleotide polymorphism (SNP) markers from a wheat 90K SNP array. As a result, 111 and 104 significant SNPs for Kernel traits were detected using four multi-locus GWAS models (mrMLM, FASTmrMLM, FASTmrEMMA, and pLARmEB) and three single-locus models (FarmCPU, MLM, and MLMM), respectively. Among the 111 SNPs detected by the multi-locus models, 24 SNPs were simultaneously detected across multiple models, including seven for kernel length, six for kernel width, six for kernels per spike, and five for thousand kernel weight. Interestingly, the five most stable SNPs (RAC875_29540_391, Kukri_07961_503, tplb0034e07_1581, BS00074341_51, and BobWhite_049_3064) were simultaneously detected by at least three multi-locus models. Integrating these newly developed multi-locus GWAS models to unravel the genetic architecture of kernel traits, the mrMLM approach detected the maximum number of SNPs. Furthermore, a total of 41 putative candidate genes were predicted to likely be involved in the genetic architecture underlining kernel traits. These findings can facilitate a better understanding of the complex genetic mechanisms of kernel traits and may lead to the genetic improvement of grain yield in wheat.

Identification of novel OsCML16 target proteins and differential expression analysis under abiotic stresses in rice.

Calmodulin-like proteins (CMLs) have been shown to play key regulatory roles in calcium signaling in plants. However, few bona-fide CMLs binding proteins have been characterized in rice, a monocot model plant. Here, through large-scale screening of a yeast-two hybrid (Y2H) cDNA library with OsCML16 as a bait, six new putative interacting partners of OsCML16 were discovered and confirmed by both pairwise Y2H and bimolecular fluorescence complementation (BiFC) assays. Interestingly, the in vitro peptide-binding assays manifested that OsERD2 could bind both OsCaM1 and OsCML16 whereas other five target proteins could specifically bind OsCML16 but not OsCaM1. Furthermore, Ca2+ and TFP, a calmodulin (CaM) antagonist, were involved in the ABA-induced transcription of OsCML16 and its target genes, and they were also obviously induced by cold, drought, and salt stresses. Taken together, our new findings have provided the basis for the novel signaling pathways of OsCML16 in the abiotic stress response in rice.

Phylogeny, structural diversity and genome-wide expression analysis of fibrillin family genes in rice.

Fibrillins (FBNs) constitute a plastid-lipid-associated protein family that plays a role in chloroplast development, lipids metabolism and stress responses in plants. Until now, FBNs have been functionally characterized in stability of thylakoid and responses to the different stress stimuli. Consequently, phylogeny, domain composition and structural features of 121 FBNs family proteins from ten representative species have been identified. As results, phylogenetic analysis demonstrated that FBNs proteins were grouped into 24 clades and further subdivided into three groups, including terrestrial plant-specific, algae-specific, and intermediate group. These FBNs genes had different numbers of introns and exons but encoded the conserved N-terminal chloroplast transport peptide (CTP) domains and plastid lipid-associated protein (PAP) domains, which greatly contributed to the sub-functionalization and neo-functionalization. Meanwhile, the CTP domains of eleven OsFBN proteins except OsFBN8 could help them transport into chloroplasts. The PAP domains of OsFBN2 and OsFBN4 showed the in vitro specific binding activity to C12-C22 fatty acids that were affected by YxD motif. The qRT-PCR analysis showed that OsFBN genes were differentially induced by heat stress and cold stress in rice. Collectively, this study has provided the new insights into the evolution, structure, and functions of FBN gene family and will help to elucidate the molecular mechanisms of these proteins functioning in growth, development and adaptations in the global climate change.

A novel rice fragile culm 24 mutant encodes a UDP-glucose epimerase that affects cell wall properties and photosynthesis.

UDP-glucose epimerases (UGEs) are essential enzymes for catalysing the conversion of UDP-glucose (UDP-Glc) into UDP-galactose (UDP-Gal). Although UDP-Gal has been well studied as the substrate for the biosynthesis of carbohydrates, glycolipids, and glycoproteins, much remains unknown about the biological function of UGEs in plants. In this study, we selected a novel rice fragile culm 24 (Osfc24) mutant and identified it as a nonsense mutation of the FC24/OsUGE2 gene. The Osfc24 mutant shows a brittleness phenotype with significantly altered cell wall composition and disrupted orientation of the cellulose microfibrils. We found significantly reduced accumulation of arabinogalactan proteins in the cell walls of the mutant, which may consequently affect plant growth and cell wall deposition, and be responsible for the altered cellulose microfibril orientation. The mutant exhibits dwarfism and paler leaves with significantly decreased contents of galactolipids and chlorophyll, resulting in defects in plant photosynthesis. Based on our results, we propose a model for how OsUGE2 participates in two distinct metabolic pathways to co-modulate cellulose biosynthesis and cell wall assembly by dynamically providing UDP-Gal and UDP-Glc substrates.