Overexpression of 4-hydroxyphenylpyruvate dioxygenase (IbHPPD) increases abiotic stress tolerance in transgenic sweetpotato plants.

Tocopherols are lipid-soluble compounds regarded as vitamin E compounds and they function as antioxidants in scavenging lipid peroxyl radicals and quenching reactive oxygen species (ROS). In our previous studies, we isolated five tocopherol biosynthesis genes from sweetpotato (Ipomoea batatas [L.] Lam) plants including 4-hydroxyphenylpyruvate dioxygenase (IbHPPD). HPPD is the first regulatory enzyme in vitamin E biosynthesis and serves to catalyze in the first steps α-tocopherol and plastoquinone biosynthesis by converting 4-hydroxyphenylpyruvate (HPP) to homogentisic acid (HGA). In this study, we generated transgenic sweetpotato plants overexpressing IbHPPD under the control of cauliflower mosaic virus (CaMV) 35S promoter (referred to as HP plants) via Agrobacterium-mediated transformation to understand the function of IbHPPD in sweetpotato. Three transgenic lines (HP3, HP14 and HP15) with high transcript levels of IbHPPD were selected for further characterization. Compared with non-transgenic (NT) plants, HP plants exhibited enhanced tolerance to multiple environmental stresses, including salt, drought, and oxidative stresses. In addition, HP plants showed increased tolerance to the herbicide sulcotrione, which is involved in the inhibition of the HPPD. Interestingly, after stress treatments, HP plants also showed higher abscisic acid (ABA) contents than NT plants. Under dehydrated condition, HP plants displayed an elevated α-tocopherol content to 19-27% in leaves compared with NT plants. These results indicate that increased abiotic stress tolerance in HP plants is related to inducing enhancement of α-tocopherol and ABA contents.

Arabidopsis galactinol synthases 1 (AtGOLS1) negatively regulates seed germination.

Seed germination begins the growth phases of plants and its rate is affected not only by plant hormones, including abscisic acid (ABA), gibberellin (GA) and brassinosteroids (BRs), but also by environmental factors. In this study, we searched for additional chemical reagents that affect seed germination, using the det2-1 and ga1-3 mutants that showed reduced seed germination due to defective BR- or GA- biosynthesis, respectively. We found that the reducing reagent dithiothreitol (DTT) specifically enhanced seed germination of det2-1 compared with that of ga1-3. To further investigate the underlying molecular mechanism for this phenomenon, we identified AtGOLS1 as a differentially expressed gene in germinating seeds treated with DTT by GeneFishing analysis. AtGOLS1 encodes a galactinol synthase, critical for the first step in raffinose family oligosaccharides synthesis during seed maturation. We observed that expression of AtGOLS1 decreased when conditions were favorable for seed germination. We also determined that the seed germination rate was faster in T-DNA knockout atgols1 mutant and transgenic plants transformed with an RNA interference construct targeting AtGOLS1 compared with wild type plants. The double mutant of det2-1 and atgols1 also suppressed the reduced seed germination of the det2-1. Taken together, our results suggest that AtGOLS1 acts as a negative regulator in seed germination.

Overexpressing IbCBF3 increases low temperature and drought stress tolerance in transgenic sweetpotato.

Dehydration-responsive element-binding/C-repeat-binding factor (DREB/CBF) proteins regulate the transcription of genes involved in cold acclimation in several species. However, little is known about the physiological functions of CBF proteins in the low temperature-sensitive crop sweetpotato. We previously reported that the DREB1/CBF-like sweetpotato gene SwDREB1/IbCBF3 is involved in responses to diverse abiotic stresses. In this study, we confirmed that IbCBF3 is localized to the nucleus and binds to the C-repeat/dehydration-responsive elements (CRT/DRE) in the promoters of cold-regulated (COR) genes. We generated transgenic sweetpotato plants overexpressing IbCBF3 under the control of the CaMV 35S promoter (referred to as SC plants) and evaluated their responses to various abiotic stresses. IbCBF3 expression was dramatically induced by cold and drought but much less strongly induced by high salinity and ABA. We further characterized two SC lines (SC3 and SC6) with high levels of IbCBF3 transcript. The SC plants displayed enhanced tolerance to cold, drought, and oxidative stress on the whole-plant level. Under cold stress treatment (4 °C for 48 h), severe wilting and chilling injury were observed in the leaves of wild-type (WT) plants, whereas SC plants were not affected by cold stress. In addition, the COR genes were significantly upregulated in SC plants compared with the WT. The SC plants also showed significantly higher tolerance to drought stress than the WT, which was associated with higher photosynthesis efficiency and lower hydrogen peroxide levels. These results indicate that IbCBF3 is a functional transcription factor involved in the responses to various abiotic stresses in sweetpotato.

Overexpression of miR172 suppresses the brassinosteroid signaling defects of bak1 in Arabidopsis.

BRI1-Associated Receptor Kinase 1 (BAK1) is a leucine-rich repeat serine/threonine receptor-like kinase (LRR-RLK) that is involved in multiple developmental pathways, such as brassinosteroid (BR) signaling, plant immunity and cell death control in plants. Because the roundish and compact rosette leaves of bak1 mutant plants are characteristic phenotypes for deficient BR signaling, we screened genetic suppressors of bak1 according to changes in leaf shape to identify new components that may be involved in BAK1-mediated BR signaling using the activation-tagging method. Here, we report bak1-SUP1, which exhibited longer and narrower rosette leaves and an increased BR sensitivity compared with those of bak1. Analyses of the T-DNA insertional site and the gene expression that was affected by the T-DNA insertion revealed that a microRNA, namely, miR172, over-accumulates in bak1-SUP1. Detailed phenotypic analyses of bak1-SUP1 and a single mutant in which the bak1 mutation was segregated out (miR172-D) revealed that the overexpression of miR172 promotes leaf length elongation in adult plants and increases the root and hypocotyl growth during the seedling stage compared with that of wild type plants. Taken together with its increased BR sensitivity, these results suggest that miR172 regulates vegetative growth patterns by modulating BR sensitivity as well as by the previously identified developmental phase transition.

Assessing the diverse functions of BAK1 and its homologs in arabidopsis, beyond BR signaling and PTI responses.

Plants possess a variety of extracellular leucine-rich repeats receptor-like kinases (LRR-RLKs) to coordinate developmental programs with responses to environmental changes. Out of sixteen families of LRR-RLKs in Arabidopsis, the LRR-RLKII family consists of fourteen individual members, including five Arabidopsis thaliana somatic embryogenesis receptor kinases (AtSERKs). BAK1/AtSERK3 was first identified as a dual co-receptor of BRI1 and FLS2, mediating BR signaling and pathogen-associated molecular pattern (PAMP) triggered immunity (PTI), respectively. Since its identification, many researchers have attempted to elucidate the phosphorylation mechanisms between receptor complexes and identify additional components that interact with receptor complexes to transduce the signaling downstream. Relatively detailed early events in complex formation, phosphorylation sites on the BRI1/BAK1 complex and BAK1-interacting proteins, such as BIK1 and PUB13, have been identified. Small receptor complexes consisting of BAK1 and BIR1 or BAK1 and AtSERK4 regulate cell death during steady state conditions. Moreover, the redundant and distinct functions of AtSERK proteins and other members of the LRR-RLKII family have been revealed. This review focuses on the integration of the information from the most recent studies concerning BAK1 and its homologs.

Genes encoding plant-specific class III peroxidases are responsible for increased cold tolerance of the brassinosteroid-insensitive 1 mutant.

We previously reported that one of the brassinosteroidinsensitive mutants, bri1-9, showed increased cold tolerance compared with both wild type and BRI1-overexpressing transgenic plants, despite its severe growth retardation. This increased tolerance in bri1-9 resulted from the constitutively high expression of stress-inducible genes under normal conditions. In this report, we focused on the genes encoding class III plant peroxidases (AtPrxs) because we found that, compared with wild type, bri1-9 plants contain higher levels of reactive oxygen species (ROS) that are not involved with the activation of NADPH oxidase and show an increased level of expression of a subset of genes encoding class III plant peroxidases. Treatment with a peroxidase inhibitor, salicylhydroxamic acid (SHAM), led to the reduction of cold resistance in bri1-9. Among 73 genes that encode AtPrxs in Arabidopsis, we selected four (AtPrx1, AtPrx22, AtPrx39, and AtPrx69) for further functional analyses in response to cold temperatures. T-DNA insertional knockout mutants showed increased sensitivity to cold stress as measured by leaf damage and ion leakage. In contrast, the overexpression of AtPrx22, AtPrx39, and AtPrx69 increased cold tolerance in the BRI1-GFP plants. Taken together, these results indicate that the appropriate expression of a particular subset of AtPrx genes and the resulting higher levels of ROS production are required for the cold tolerance.

A subset of OsSERK genes, including OsBAK1, affects normal growth and leaf development of rice.

Since the identification of BRI1-Associated receptor Kinase 1 (BAK1), a member of the Somatic Embryogenesis Receptor Kinase (SERK) family, the dual functions of BAK1 in BR signaling and innate immunity in Arabidopsis have attracted considerable attention as clues for understanding developmental processes that must be balanced between growth and defense over the life of plants. Here, we extended our research to study cellular functions of OsSERKs in rice. As it was difficult to identify an authentic ortholog of AtBAK1 in rice, we generated transgenic rice in which the expression of multiple OsSERK genes, including OsBAK1, was reduced by OsBAK1 RNA interference. Resulting transgenic rice showed reduced levels of Os-BAK1 and decreased sensitivity to BL, leading to semidwarfism in overall growth. Moreover, they resulted in abnormal growth patterns, especially in leaf development. Most of the OsBAK1RNAi transgenic rice plants were defective in the development of bulliform cells in the leaf epidermal layer. They also showed increased expression level of pathogenesis-related gene and enhanced susceptibility to a rice blast-causing fungal pathogen, Magnaporthe oryzae. These results indicate that OsSERK genes, such as OsBAK1, play versatile roles in rice growth and development.

Reduced expression of the genes encoding chloroplast-localized proteins in a cold-resistant bri1 (brassinosteroid-insensitive 1) mutant.

We showed that constitutive activation of the stress-inducible genes led to the endogenous difference to cold tolerance in the bri1-9 mutant and in the BRI1-GFP plants compared to the wild type. In order to get more insight into the balance between growth and stress resistance, we analyzed the cellular localization of the proteins encoded by the genes previously reported as up or downregulated in the bri1-9 and BRI1-GFP plants. We found that the genes responsible for the chloroplast-localized proteins are markedly downregulated in bri1-9, and the proteins encoded by them are involved with chloroplast development, metabolism, and the photosynthetic regulation that is an essential function of chloroplast in the plants cells during active growing periods. These imply that subsets of gene products that are yet uncharacterized modulate the metabolic and signaling processes that are occurring in the chloroplast, leading to the balanced growth and response to abiotic stresses including light stimulus.

Constitutive activation of stress-inducible genes in a brassinosteroid-insensitive 1 (bri1) mutant results in higher tolerance to cold.

Many plant hormones are involved in coordinating the growth responses of plants under stress. However, not many mechanistic studies have explored how plants maintain the balance between growth and stress responses. Brassinosteroids (BRs), plant-specific steroid hormones, affect many aspects of plant growth and development over a plant's lifetime. In this study we determined that exogenous treatment of BR helped the plant overcome the cold condition only when pretreated with less than 1 nM, and the brassinosteroid-insensitive 1 (bri1) mutation, which results in defective BR signaling and subsequent dwarfism, generates an increased tolerance to cold. In contrast, BRI1-overexpressing plants were more sensitive to the same stress than wild-type. We found that the bri1 mutant and BRI1-overexpressing transgenic plants contain higher basal level of expression of CBFs/DREB1s than wild-type. However, representative cold stress-related genes were regulated with the same pattern to cold in wild-type, bri1-9 and BRI1 overexpressing plants. To examine the global gene expression and compare the genes that show differential expression pattern in bri1-9 and BRI1-GFP plants other than CBFs/DREB1s, we analyzed differential mRNA expression using the cDNA microarray analysis in the absence of stress. Endogenous expression of both stress-inducible genes as well as genes encoding transcription factors that drive the expression of stress-inducible genes were maintained at higher levels in bri1-9 than either in wild-type or in BRI1 overexpressing plants. This suggests that the bri1-9 mutant could always be alert to stresses that might be exerted at any times by constitutive activation of subsets of defense.

BAK7 displays unequal genetic redundancy with BAK1 in brassinosteroid signaling and early senescence in Arabidopsis.

BRI1-Associated kinase 1 (BAK1), a five leucine-rich-repeat containing receptor-like serine/threonine kinase, has been shown to have dual functions: mediating brassinosteroid (BR) signaling and acting in the BR-independent plant defense response. Sequence analysis has revealed that BAK1 has two homologs, BAK7 and BAK8. Because BAK8 deviates from the canonical RD kinase motif, we focused on the functional analysis of BAK7. The expression pattern and tissues in which BAK7 appeared partially overlapped with those observed for BAK1. Expression levels of BAK7 increased in the bak1 mutant. Overexpression of BAK7 rescued the bri1 mutant phenotype, indicating that BAK7 can compensate for BAK1 in BR-mediated processes, especially in the absence of BAK1. However, root and hypocotyl elongation patterns of transgenic plants overexpressing BAK1 or BAK7 appeared to be different from the patterns observed in a BRI1 overexpressor. Furthermore, the sensitivity of transgenic plants overexpressing BAK7 to brassinazole, a biosynthetic inhibitor of brassinolide (BL), did not change compared to that of wild-type plants. In addition, we generated transgenic plants expressing BAK7 RNA interference constructs and found severe growth retardation and early senescence in these lines. Taken together, these results suggest that BAK7 is a component of the BR signaling pathway, with varying degrees of genetic redundancy with BAK1, and that it affects plant growth via BL-independent pathways in vivo.

Modulations of AtGSTF10 expression induce stress tolerance and BAK1-mediated cell death.

Glutathione-S-transferases are essential proteins involved in cellular detoxification. The expression of GSTs has been studied extensively under various environmental stressors including xenobiotics. Here, we have isolated AtGST10, one of the phi classes of AtGSTs on the basis of its interaction with BAK1 in a yeast two-hybrid screen. BAK1 is an LRR-RLK, acting in both brassinosteroid signaling and plant defense responses. We found that AtGSTF10 binds to BAK1 through its N-terminal domain. AtGSTF10 is expressed ubiquitously in plant tissues, and the endogenous transcript level of AtGSTF10 was not induced by plant growth regulators or abiotic stressors, except drought, unlike other GSTs. Overexpression of AtGSTF10 conferred higher tolerance to salt and disturbed redox status of transgenic plants. The down-regulation of AtGSTF10 produced by RNA interference caused reduced tolerance to abiotic stress and an accelerated senescence of transformants, indicating that AtGSTF10 is involved in stress tolerance and the BAK1-mediated spontaneous cell death signaling pathway in Arabidopsis.