OsMYB58 Negatively Regulates Plant Growth and Development by Regulating Phosphate Homeostasis.

Phosphate (Pi) starvation is a critical factor limiting crop growth, development, and productivity. Rice (Oryza sativa) R2R3-MYB transcription factors function in the transcriptional regulation of plant responses to various abiotic stresses and micronutrient deprivation, but little is known about their roles in Pi starvation signaling and Pi homeostasis. Here, we identified the R2R3-MYB transcription factor gene OsMYB58, which shares high sequence similarity with AtMYB58. OsMYB58 expression was induced more strongly by Pi starvation than by other micronutrient deficiencies. Overexpressing OsMYB58 in Arabidopsis thaliana and rice inhibited plant growth and development under Pi-deficient conditions. In addition, the overexpression of OsMYB58 in plants exposed to Pi deficiency strongly affected root development, including seminal root, lateral root, and root hair formation. Overexpressing OsMYB58 strongly decreased the expression of the rice microRNAs OsmiR399a and OsmiR399j. By contrast, overexpressing OsMYB58 strongly increased the expression of rice PHOSPHATE 2 (OsPHO2), whose expression is repressed by miR399 during Pi starvation signaling. OsMYB58 functions as a transcriptional repressor of the expression of its target genes, as determined by a transcriptional activity assay. These results demonstrate that OsMYB58 negatively regulates OsmiR399-dependent Pi starvation signaling by enhancing OsmiR399s expression.

Identification of a Novel Gene, Osbht, in Response to High Temperature Tolerance at Booting Stage in Rice.

Rice is one of the world's leading food crops, and over 90% of the world's rice production stems from Asia. In particular, an increase of 1 °C in the minimum temperature reduces the quantity of rice by 10%. Therefore, the development of rice varieties that can stably maintain the yield and quality of the rice even under these rapid climate changes is indispensable. In this study, we performed quantitative trait loci (QTL) mapping after treatment with heat stress during the booting stage in rice. We performed a QTL analysis using the Cheongcheong/Nagdong double haploid (CNDH) line and identified 19 QTLs during the 2 year analysis. Of these QTL regions, the 2.2 cM region of RM3709-RM11694 on chromosome 1 was shared among the six traits (heading date; culm length; panicle length; number of tiller; 1000 grain weight; and content of chlorophyll) examined. Rice Microsatellite (RM) 3709-RM11694 contained 27 high-temperature-tolerance candidate genes. Among the candidate genes, OsBHT showed a different gene expression level between CNDH75, which is a high-temperature tolerant line, and CNDH11 which is a susceptible line. Although some existing high-temperature-tolerant genes have been reported, OsBHT can be used more effectively for the development of heat tolerance in rice.

The MYB-CC Transcription Factor PHOSPHATE STARVATION RESPONSE-LIKE 7 (PHL7) Functions in Phosphate Homeostasis and Affects Salt Stress Tolerance in Rice.

Inorganic phosphate (Pi) homeostasis plays an important role in plant growth and abiotic stress tolerance. Several MYB-CC transcription factors involved in Pi homeostasis have been identified in rice (Oryza sativa). PHOSPHATE STARVATION RESPONSE-LIKE 7 (PHL7) is a class II MYC-CC protein, in which the MYC-CC domain is located at the N terminus. In this study, we established that OsPHL7 is localized to the nucleus and that the encoding gene is induced by Pi deficiency. The Pi-responsive genes and Pi transporter genes are positively regulated by OsPHL7. The overexpression of OsPHL7 enhanced the tolerance of rice plants to Pi starvation, whereas the RNA interference-based knockdown of this gene resulted in increased sensitivity to Pi deficiency. Transgenic rice plants overexpressing OsPHL7 produced more roots than wild-type plants under both Pi-sufficient and Pi-deficient conditions and accumulated more Pi in the shoots and roots. In addition, the overexpression of OsPHL7 enhanced rice tolerance to salt stress. Together, these results demonstrate that OsPHL7 is involved in the maintenance of Pi homeostasis and enhances tolerance to Pi deficiency and salt stress in rice.

Gibberellic acid sensitive dwarf encodes an ARPC2 subunit that mediates gibberellic acid biosynthesis, effects to grain yield in rice.

The plant hormone gibberellic acid (GA) is important for plant growth and productivity. Actin-related proteins (ARPs) also play central roles in plant growth, including cell elongation and development. However, the relationships between ARPs and GA signaling and biosynthesis are not fully understood. Here, we isolated OsGASD, encoding an ARP subunit from rice (Oryza sativa), using the Ac/Ds knockout system. The osgasd knockout (Ko) mutation reduced GA3 content in shoots as well as plant growth and height. However, GA application restored the plant height of the osgasd Ko mutant to a height similar to that of the wild type (WT). Rice plants overexpressing OsGASD (Ox) showed increased plant height and grain yield compared to the WT. Transcriptome analysis of flag leaves of OsGASD Ox and osgasd Ko plants revealed that OsGASD regulates cell development and the expression of elongation-related genes. These observations suggest that OsGASD is involved in maintaining GA homeostasis to regulate plant development, thereby affecting rice growth and productivity.

Characterization of dwarf and narrow leaf (dnl-4) mutant in rice.

Height and leaf morphology are important agronomic traits of the major crop plant rice (Oryza sativa). In previous studies, the dwarf and narrow leaf genes (dnl1, dnl2 and dnl3) have identified in rice. Using the Ac/Ds knockout system, we found a new dwarf and narrow leaf (dnl) mutant and identified mutated gene. The dnl-4 mutant showed reduced plant height and leaf blade width compared to the wild type, and increased leaf inclination. The morphological defects of the mutant were caused by the suppressed expression of the DNL-4 gene, which encodes a pfkB carbohydrate kinase protein. These results suggest that DNL-4 expression is involved in modulating plant height and leaf growth. Furthermore, DNL-4 expression also affects productivity in rice: the dnl-4 mutant exhibited reduced panicle length and grain width compared with the wild type. To understand DNL-4 function in rice, we analyzed the expression levels of leaf growth-related genes, such as NAL1, NAL7, and CSLD4, in the dnl-4 mutant. Expression of NAL1 and NAL7 was downregulated in the dnl-4 mutant compared to the wild type. The observation that DNL-4 expression corresponded with that of NAL1 and NAL7 is consistent with the narrow leaf phenotype of the dnl-4 mutant. These results suggest that DNL-4 regulates plant height and leaf structure in rice.

Comparative analysis of isoflavone aglycones using microwave-assisted acid hydrolysis from soybean organs at different growth times and screening for their digestive enzyme inhibition and antioxidant properties.

The objectives of this research were to demonstrate the changes in isoflavone-aglycones, total phenolics, and biological properties (digestive enzyme inhibition; antioxidant) from six organs including leaves, leafstalks, roots, stems, seeds, and pods at different growth times of soybean plant. Three isoflavone-aglycones in microwave-assisted acid hydrolysis extracts were elucidated using UHPLC-ESI-Q-TOF-MS/MS and their contents exhibited remarkable differences in leaves (245.93-2239.33 µg/g), roots (854.96-4425.34 µg/g), and seeds (ND-2339.62 µg/g). Specifically, the collected samples on 15-Oct (leaves: 2239.33; seeds: 2339.62 µg/g) and 31-Aug (roots: 4425.34 µg/g) showed the highest isoflavone-aglycones, and daidzein was observed the most abundant component, comprising approximately 70%. Moreover, the inhibitions against α-glucosidase and α-amylase displayed the predominant effects in roots (89;91%) and leaves (81;85%) of samples on 31-Aug and 15-Oct at 300 µg/ml. The antioxidant activities on ABTS, DPPH, and hydroxyl radicals increased considerably with the increases of growth times in leaves and seeds, especially, ABTS showed the highest scavenging abilities: leaves (15-Oct;83%) > roots (31-Aug;75%) > seeds (15-Oct;68%). Therefore, our results suggest that soybean leaves, roots and seeds may be considered as excellent natural sources for nutraceuticals.

Spider Silk Fibroin Protein Heterologously Produced in Rice Seeds Reduce Diabetes and Hypercholesterolemia in Mice.

Silk fibroin proteins are biomaterials with diverse applications. These spider and silkworm proteins have specific biological effects when consumed by mammals; in addition to reducing blood pressure and blood glucose and cholesterol levels, they have anti-human immunodeficiency virus activity. In the present study, rice (Oryza sativa) was engineered to produce the C-terminus of the major ampullate spidroin protein from the spider Araneus ventricosus under the control of a Prolamin promoter. Homozygous transgenic rice lines were identified, and the therapeutic effect of this spider silk fibroin protein on the lipid and glucose metabolism was analyzed in a mouse model. Feeding fat-fed mice, the transgenic rice seeds for four weeks reduced serum concentrations of triglycerides, total cholesterol, low-density lipoprotein cholesterol, glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase, and lowered blood glucose levels. This is the first study to investigate the effects of consumption of rice seeds heterologously expressing spider silk fibroin protein in a mammalian model. Our findings suggest that functional foods containing spider silk fibroin protein might be useful as potential pharmaceutical materials for preventing and treating diabetes, hyperlipidemia, and hypercholesterolemia.

Hepatic Steatosis Alleviated in Diabetic Mice upon Dietary Exposure to Fibroin via Transgenic Rice: Potential STAMP2 Involvement in Hepatocytes.

Many benefits of silk protein fibroin (SPF) have been suggested in biomedical applications; and notably, significant SPF effects have been observed for metabolic syndromes that are directly linked to insulin resistance, such as type 2 diabetes mellitus (T2DM). Based on our previous findings, we believe that SPF from spiders exhibits outstanding glucose-lowering effects in diabetic BKS.Cg-m+/+Leprdb mice. In order to evaluate the dietary effects of SPF in diabetic animals, we generated several lines of transgenic rice (TR) that expresses SPF, and the feeding of TR-SPF to diabetic animals decreased blood glucose levels, but did not change insulin levels. Western blot analyses of hepatic proteins showed that AMP-activated protein kinase (AMPK) expression and phosphorylation both decreased in TR-SPF-fed groups, compared with controls. This finding suggests that the glucose-lowering effects in this diabetic animal model might be AMPK-independent. In contrast, six-transmembrane protein of prostate 2 (STAMP2) was upregulated after TR-SPF exposure. Together with STAMP2, the Akt protein phosphorylation increased after TR-SPF exposure, which indicates that STAMP2 leads to Akt phosphorylation and thus increases insulin sensitivity in hepatocytes. Importantly, the hepatic steatosis that was seen in the liver of diabetic mice was remarkably alleviated in TR-SPF-fed mice. Hepatocytes that were immunopositive for STAMP2 were overwhelmingly observed in hepatic tissues from TR-SPF-fed mice compared to the control. Taken together, these results suggest that feeding diabetic mice with TR-SPF upregulates STAMP2 expression and increases Akt phosphorylation in hepatic tissues and thus potentially alleviates insulin resistance and hepatic steatosis.

Assessment of the Genetic Diversity of Rice Germplasms Characterized by Black-Purple and Red Pericarp Color Using Simple Sequence Repeat Markers.

The assessment of the genetic diversity within germplasm collections can be accomplished using simple sequence repeat (SSR) markers and association mapping techniques. The present study was conducted to evaluate the genetic diversity of a colored rice germplasm collection containing 376 black-purple rice samples and 172 red pericarp samples, conserved by Dong-A University. There were 600 pairs of SSR primers screened against 11 rice varieties. Sixteen informative primer pairs were selected, having high polymorphism information content (PIC) values, which were then used to assess the genetic diversity within the collection. A total of 409 polymorphic amplified fragments were obtained using the 16 SSR markers. The number of alleles per locus ranged from 11 to 47, with an average of 25.6. The average PIC value was 0.913, ranging from 0.855 to 0.964. Four hundred and nine SSR loci were used to calculate Jaccard's distance coefficients, using the unweighted pair-group method with arithmetic mean cluster analysis. These accessions were separated into several distinctive groups corresponding to their morphology. The results provided valuable information for the colored rice breeding program and showed the importance of protecting germplasm resources and the molecular markers that can be derived from them.

Dietary Exposure to Transgenic Rice Expressing the Spider Silk Protein Fibroin Reduces Blood Glucose Levels in Diabetic Mice: The Potential Role of Insulin Receptor Substrate-1 Phosphorylation in Adipocytes.

Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance (IR). T2DM is correlated with obesity and most T2DM medications have been developed for enhancing insulin sensitivity. Silk protein fibroin (SPF) from spiders has been suggested as an attractive biomaterial for medical purposes. We generated transgenic rice (TR) expressing SPF and fed it to diabetic BKS.Cg-m+/+Leprdb mice to monitor the changes in blood glucose levels and adipose tissue proteins associated with energy metabolism and insulin signaling. In the present study, the adipocyte size in abdominal fat in TR-SPF-fed mice was remarkably smaller than that of the control. Whereas the adenosine monophosphate-activated protein kinase (AMPK)-activated protein kinase and insulin receptor substrate 1 (IRS1) protein levels were increased in abdominal adipose tissues after TR-SPF feeding, levels of six-transmembrane protein of prostate 2 (STAMP2) proteins decreased. Phosphorylation of AMPK at threonine 172 and IRS1 at serine 307 and tyrosine 632 were both increased in adipose tissues from TR-SPF-fed mice. Increased expression and phosphorylation of IRS1 at both serine 307 and tyrosine 632 in adipose tissues indicated that adipocytes obtained from abdominal fat in TR-SPF-fed mice were more susceptible to insulin signaling than that of the control. STAMP2 protein levels decreased in adipose tissues from TR-SPF-fed mice, indicating that STAMP2 proteins were reducing adipocytes that were undergoing lipolysis. Taken together, this study showed that TR-SPF was effective in reducing blood glucose levels in diabetic mice and that concurrent lipolysis in abdominal adipocytes was associated with alterations of AMPK, IRS1, and STAMP2. Increased IRS1 expression and its phosphorylation by TR-SFP were considered to be particularly important in the induction of lipolysis in adipocytes, as well as in reducing blood glucose levels in this animal model.

Rice OsMYB5P improves plant phosphate acquisition by regulation of phosphate transporter.

Myeloblastosis (MYB) transcription factors play central roles in plant developmental processes and in responses to nutrient deficiency. In this study, OsMYB5P, an R2R3-MYB transcription factor, was isolated and identified from rice (Oryza sativa L. 'Dongjin') under inorganic phosphate (Pi)-deficient conditions. OsMYB5P protein is localized to the nucleus and functions as a transcription activator in plant development. Overexpression of OsMYB5P in rice and Arabidopsis (Arabidopsis thaliana Col-0) increases tolerance to phosphate starvation, whereas OsMYB5P knock-out through RNA interference increases sensitivity to Pi depletion in rice. Furthermore, shoots and roots of transgenic rice plants overexpressing OsMYB5P were longer than those of wild plants under both normal and Pi-deficient conditions. These results indicate that OsMYB5P is associated with the regulation of shoot development and root- system architecture. Overexpression of OsMYB5P led to increased Pi accumulation in shoots and roots. Interestingly, OsMYB5P directly bound to MBS (MYB binding site) motifs on the OsPT5 promoter and induced transcription of OsPT5 in rice. In addition, overexpression of OsMYB5P in Arabidopsis triggered increased expression of AtPht1;3, an Arabidopsis Pi transporter, in shoots and roots under normal and Pi-deficient conditions. Together, these results demonstrate that overexpression of OsMYB5P increases tolerance to Pi deficiency in plants by modulating Pi transporters at the transcriptional level in monocots and dicots.

Overexpression of OsMYB4P, an R2R3-type MYB transcriptional activator, increases phosphate acquisition in rice.

R2R3 MYB transcription factors play regulatory roles in plant responses to various environmental stresses and nutrient deficiency. In this study, we isolated and designated OsMYB4P, an R2R3 MYB transcription factor, from rice (Oryza sativa L. 'Dongjin') under phosphate-deficient conditions. OsMYB4P was localized in the nucleus and acted as a transcriptional activator. Transcriptional levels of OsMYB4P in cell suspension, shoots, and roots of rice increased under phosphate-deficient conditions. Shoots and roots of OsMYB4P-overexpressing plants grew well in high- and phosphate-deficient conditions. In addition, root system architecture was altered considerably as a result of OsMYB4P overexpression. Under both phosphate-sufficient and -deficient conditions, more Pi accumulated in shoots and roots of OsMYB4P-overexpressing plants than in the wild type. Overexpression of OsMYB4P led to greater expression of Pi transporter-family proteins OsPT1, OsPT2, OsPT4, OsPT7, and OsPT8 in shoots, and to decreased or unchanged expression of these proteins in roots, with the exception of OsPT8. These results demonstrate that OsMYB4P may be associated with efficient utilization of Pi in rice through transcriptional activation of Pi homeostasis-related genes.