Genome-wide identification of the plant homeodomain-finger family in rye and ScPHD5 functions in cold tolerance and flowering time.

KEY MESSAGE: 111 PHD genes were newly identified in rye genome and ScPHD5's role in regulating cold tolerance and flowering time was suggested. Plant homeodomain (PHD)-finger proteins regulate the physical properties of chromatin and control plant development and stress tolerance. Although rye (Secale cereale L.) is a major winter crop, PHD-finger proteins in rye have not been studied. Here, we identified 111 PHD genes in the rye genome that exhibited diverse gene and protein sequence structures. Phylogenetic tree analysis revealed that PHDs were genetically close in monocots and diverged from those in dicots. Duplication and synteny analyses demonstrated that ScPHDs have undergone several duplications during evolution and that high synteny is conserved among the Triticeae species. Tissue-specific and abiotic stress-responsive gene expression analyses indicated that ScPHDs were highly expressed in spikelets and developing seeds and were responsive to cold and drought stress. One of these genes, ScPHD5, was selected for further functional characterization. ScPHD5 was highly expressed in the spike tissues and was localized in the nuclei of rye protoplasts and tobacco leaves. ScPHD5-overexpressing Brachypodium was more tolerant to freezing stress than wild-type (WT), with increased CBF and COR gene expression. Additionally, these transgenic plants displayed an extremely early flowering phenotype that flowered more than two weeks earlier than the WT, and vernalization genes, rather than photoperiod genes, were increased in the WT. RNA-seq analysis revealed that diverse stress response genes, including HSPs, HSFs, LEAs, and MADS-box genes, were also upregulated in transgenic plants. Our study will help elucidate the roles of PHD genes in plant development and abiotic stress tolerance in rye.

Genome-wide identification and characterization of the lettuce GASA family in response to abiotic stresses.

BACKGROUND: Lettuce is one of the most extensively farmed vegetables in the world, and it prefers cool growing conditions. High temperatures promote premature bolt formation, reducing quality and yield. The gibberellic acid-stimulated Arabidopsis (GASA) family genes play critical roles in plant growth, development, and stress responses. However, the biological functions of GASA proteins in lettuce have yet to be thoroughly investigated. RESULTS: Using genome-wide analysis, 20 GASAs were identified in lettuce including, three groups of LsGASA proteins based on the phylogenetic analysis. Except for one, all GASA proteins included a conserved GASA domain with 12 cysteine residues. Cis-element analysis showed that LsGASAs were closely associated with light, phytohormones, and stress resistance. Five segmental and three tandem duplication events were observed in the LsGASA family based on duplication analysis. GASA synteny analysis among lettuce, Arabidopsis, tobacco, and rice revealed that LsGASA5 is highly collinear with all species. Six of the 20 LsGASA showed increased expression patterns at specific time points in the shoot apical meristem when subjected to heat stress. According to gene expression analysis, the majority of GASA were highly expressed in flowers compared to other organs, and six GASA exhibited highly increased expression levels in response to NaCl, abscisic acid, and gibberellin treatment. Furthermore, LsGASA proteins are predominantly found in the plasma membrane and/or the cytosol. CONCLUSIONS: This study provides a comprehensive characterization of LsGASA genes for their diversity and biological functions. Moreover, our results will be useful for further studies on the function of lettuce GASA in abiotic stress- and heat-induced bolting signaling.

Molecular characterization of wheat floret development-related F-box protein (TaF-box2): Possible involvement in regulation of Arabidopsis flowering.

In wheat (Triticum aestivum L.), the floret development stage is an important step in determining grain yield per spike; however, the molecular mechanisms underlying floret development remain unclear. In this study, we elucidated the role of TaF-box2, a member of the F-box-containing E3 ubiquitin protein ligases, which is involved in floret development and anthesis of wheat. TaF-box2 was transiently expressed in the plasma membrane and cytoplasm of both tobacco and wheat. We also found that the SCFF-box2 (Skp1-Cul1-Rbx1-TaF-box2) ubiquitin ligase complex mediated self-ubiquitination activity. Transgenic Arabidopsis plants that constitutively overexpressed TaF-box2 showed markedly greater hypocotyl and root length than wild-type plants, and produced early flowering phenotypes. Flowering-related genes were significantly upregulated in TaF-box2-overexpressing Arabidopsis plants. Further protein interaction analyses such as yeast two-hybrid, in vitro pull-down, and bimolecular fluorescence complementation assays confirmed that TaF-box2 physically interacted with TaCYCL1 (Triticum aestivum cyclin-L1-1). Ubiquitination and degradation assays demonstrated that TaCYCL1 was ubiquitinated by SCFF-box2 and degraded through the 26S proteasome complex. The physiological functions of the TaF-box2 protein remain unclear; however, we discuss several potential routes of involvement in various physiological mechanisms which counteract flowering in transgenic Arabidopsis plants.

Effect of chilling acclimation on germination and seedlings response to cold in different seed coat colored wheat (Triticum aestivum L.).

BACKGROUND: Flavonoids can protect plants against extreme temperatures and ROS due to their antioxidant activities. We found that deep-purple seed coat color was controlled by two gene interaction (12:3:1) from the cross between yellow and deep-purple seed coat colored inbreds. F2:3 seeds were grouped in 3 by seed coat color and germinated under chilling (4 °C) and non-acclimated conditions (18 °C) for a week, followed by normal conditions (18 °C) for three weeks and a subsequent chilling stress (4 °C) induction. We analyzed mean daily germination in each group. Additionally, to study the acclimation in relationship to the different seed coat colors on the germination ability and seedling performances under the cold temperatures, we measured the chlorophyll content, ROS scavenging activity, and expression levels of genes involved in ROS scavenging, flavonoid biosynthetic pathway, and cold response in seedlings. RESULTS: The results of seed color segregation between yellow and deep purple suggested a two-gene model. In the germination study, normal environmental conditions induced the germination of yellow-seed, while under chilling conditions, the germination ratio of deep purple-seed was higher than that of yellow-colored seeds. We also found that the darker seed coat colors were highly responsive to cold acclimation based on the ROS scavenging enzymes activity and gene expression of ROS scavenging enzymes, flavonoid biosynthetic pathway and cold responsive genes. CONCLUSIONS: We suggest that deep purple colored seed might be in a state of innate pre-acquired stress response state under normal conditions to counteract stresses in a more effective way. Whereas, after the acclimation, another stress should enhance the cold genes expression response, which might result in a more efficient chilling stress response in deep purple seed seedlings. Low temperature has a large impact on the yield of crops. Thus, understanding the benefit of seed coat color response to chilling stress and the identification of limiting factors are useful for developing breeding strategies in order to improve the yield of wheat under chilling stress.

Identification of genetic loci associated with major agronomic traits of wheat (Triticum aestivum L.) based on genome-wide association analysis.

BACKGROUND: Bread wheat (Triticum aestivum L.) is one of the most widely consumed cereal crops, but its complex genome makes it difficult to investigate the genetic effect on important agronomic traits. Genome-wide association (GWA) analysis is a useful method to identify genetic loci controlling complex phenotypic traits. With the RNA-sequencing based gene expression analysis, putative candidate genes governing important agronomic trait can be suggested and also molecular markers can be developed. RESULTS: We observed major quantitative agronomic traits of wheat; the winter survival rate (WSR), days to heading (DTH), days to maturity (DTM), stem length (SL), spike length (SPL), awn length (AL), liter weight (LW), thousand kernel weight (TKW), and the number of seeds per spike (SPS), of 287 wheat accessions from diverse country origins. A significant correlation was observed between the observed traits, and the wheat genotypes were divided into three subpopulations according to the population structure analysis. The best linear unbiased prediction (BLUP) values of the genotypic effect for each trait under different environments were predicted, and these were used for GWA analysis based on a mixed linear model (MLM). A total of 254 highly significant marker-trait associations (MTAs) were identified, and 28 candidate genes closely located to the significant markers were predicted by searching the wheat reference genome and RNAseq data. Further, it was shown that the phenotypic traits were significantly affected by the accumulation of favorable or unfavorable alleles. CONCLUSIONS: From this study, newly identified MTA and putative agronomically useful genes will help to study molecular mechanism of each phenotypic trait. Further, the agronomically favorable alleles found in this study can be used to develop wheats with superior agronomic traits.

Identification and analysis of a differentially expressed wheat RING-type E3 ligase in spike primordia development during post-vernalization.

KEY MESSAGE: We identified a RING-type E3 ligase (TaBAH1) protein in winter wheat that targets TaSAHH1 for degradation and might be involved in primordia development by regulating targeted protein degradation. Grain yield per spike in wheat (Triticum aestivum), is mainly determined prior to flowering during mature primordia development; however, the genes involved in primordia development have yet to be characterized. In this study, we demonstrated that, after vernalization for 50 days at 4 °C, there was a rapid acceleration in primordia development to the mature stages in the winter wheat cultivars Keumgang and Yeongkwang compared with the Chinese Spring cultivar. Although Yeongkwang flowers later than Keumgang under normal condition, it has the same heading time and reaches the WS9 stage of floral development after vernalization for 50 days. Using RNA sequencing, we identified candidate genes associated with primordia development in cvs. Keumgang and Yeongkwang, that are differentially expressed during wheat reproductive stages. Among these, the RING-type E3 ligase TaBAH1 (TraesCS5B01G373000) was transcriptionally upregulated between the double-ridge (WS2.5) stage and later stages of floret primordia development (WS10) after vernalization. Transient expression analysis indicated that TaBAH1 was localized to the plasma membrane and nucleus and was characterized by self-ubiquitination activity. Furthermore, we found that TaBAH1 interacts with TaSAHH1 to mediate its polyubiquitination and degradation through a 26S proteasomal pathway. Collectively, the findings of this study indicate that TaBAH1 might play a prominent role in post-vernalization floret primordia development.

Molecular Characterization of U-box E3 Ubiquitin Ligases (TaPUB2 and TaPUB3) Involved in the Positive Regulation of Drought Stress Response in Arabidopsis.

Plant U-box E3 ubiquitin ligase (PUB) is involved in various environmental stress conditions. However, the molecular mechanism of U-box proteins in response to abiotic stress in wheat remains unknown. In this study, two U-box E3 ligase genes (TaPUB2 and TaPUB3), which are highly expressed in response to adverse abiotic stresses, were isolated from common wheat, and their cellular functions were characterized under drought stress. Transient expression assay revealed that TaPUB2 was localized in the cytoplasm and Golgi apparatus, whereas TaPUB3 was expressed only in the Golgi apparatus in wheat protoplasts. Additionally, TaPUB2 and TaPUB3 underwent self-ubiquitination. Moreover, TaPUB2/TaPUB3 heterodimer was identified in yeast and the cytoplasm of wheat protoplasts using a pull-down assay and bimolecular fluorescence complementation analysis. Heterogeneous overexpression of TaPUB2 and TaPUB3 conferred tolerance to drought stress. Taken together, these results implied that the heterodimeric form of U-box E3 ubiquitin ligases (TaPUB2/TaPUB3) responded to abiotic stress and roles as a positive regulator of drought stress tolerance.

Genome Wide Analysis of U-Box E3 Ubiquitin Ligases in Wheat (Triticum aestivum L.).

U-box E3 ligase genes play specific roles in protein degradation by post-translational modification in plant signaling pathways, developmental stages, and stress responses; however, little is known about U-box E3 genes in wheat. We identified 213 U-box E3 genes in wheat based on U-box and other functional domains in their genome sequences. The U-box E3 genes were distributed among 21 chromosomes and most showed high sequence homology with homoeologous U-box E3 genes. Synteny analysis of wheat U-box E3 genes was conducted with other plant species such as Brachypodium distachyon, barley, rice, Triricum uratu, and Aegilops tauschii. A total of 209 RNA-seq samples representing 22 tissue types, from grain, root, leaf, and spike samples across multiple time points, were analyzed for clustering of U-box E3 gene expression during developmental stages, and the genes responded differently in various tissues and developmental stages. In addition, expression analysis of U-box E3 genes under abiotic stress, including drought, heat, and both heat and drought, and cold conditions, was conducted to provide information on U-box E3 gene expression under specific stress conditions. This analysis of U-box E3 genes could provide valuable information to elucidate biological functions for a better understanding of U-box E3 genes in wheat.

Isolation and characterization of kelch repeat-containing F-box proteins from colored wheat.

F-box proteins play important roles in the regulation of various developmental processes in plants. Approximately 1796 F-box genes have been identified in the wheat genome, but details of their functions remain unknown. Moreover, not much was known about the roles of kelch repeat domain-containing F-box genes (TaKFBs) in wheat. In the present study, we isolated five TaKFBs to investigate the roles of KFBs at different stages of colored wheat grain development. The cDNAs encoding TaKFB1, TaKFB2, TaKFB3, TaKFB4, and TaKFB5 contained 363, 449, 353, 382, and 456 bp open reading frames, respectively. All deduced TaKFBs contained an F-box domain (IPR001810) and a kelch repeat type 1 domain (IPR006652), except TaKFB2. Expression of TaKFBs was elevated during the pigmentation stages of grain development. To clarify how TaKFB and SKP interact in wheat, we investigated whether five TaKFB proteins showed specificity for six SKP proteins using a yeast two-hybrid (Y2H) assay. An Y2H screen was performed to search for proteins capable of binding the TaKFBs and interaction was identified between TaKFB1 and aquaporin PIP1. To examine the subcellular localization of TaKFBs, we transiently expressed TaKFB-green fluorescent protein (GFP) fusions in tobacco leaves; the TaKFB-GFP fusions were detected in the nucleus and the cytoplasm. Y2H and bimolecular fluorescence complementation (BiFC) assays revealed that TaKFB1 specifically interacts with aquaporin PIP1. These results will provide useful information for further functional studies on wheat F-box proteins and their possible roles.

Genome-wide transcript analysis of inflorescence development in wheat.

The process of inflorescence development is directly related to yield components that determine the final grain yield in most cereal crops. Here, microarray analysis was conducted for four different developmental stages of inflorescence to identify genes expressed specifically during inflorescence development. To select inflorescence-specific expressed genes, we conducted meta-analysis using 1245 Affymetrix GeneChip array sets obtained from various development stages, organs, and tissues of members of Poaceae. The early stage of inflorescence development was accompanied by a significant upregulation of a large number of cell differentiation genes, such as those associated with the cell cycle, cell division, DNA repair, and DNA synthesis. Moreover, key regulatory genes, including the MADS-box gene, KNOTTED-1-like homeobox genes, GROWTH-REGULATING FACTOR 1 gene, and the histone methyltransferase gene, were highly expressed in the early inflorescence development stage. In contrast, fewer genes were expressed in the later stage of inflorescence development, and played roles in hormone biosynthesis and meiosis-associated genes. Our work provides novel information regarding the gene regulatory network of cell division, key genes involved in the differentiation of inflorescence in wheat, and regulation mechanism of inflorescence development that are crucial stages for determining final grain number per spike and the yield potential of wheat.

Over-expression of the Brachypodium ASR gene, BdASR4, enhances drought tolerance in Brachypodium distachyon.

KEY MESSAGE: BdASR4 expression was up-regulated during abiotic stress and hormone treatments. Plants over-expressing BdASR4 improved drought tolerant. BdASR4 may regulate antioxidant activities and transcript levels of stress-related and abscisic acid-responsive genes. Abiotic stress conditions negatively affect plant growth and developmental processes, causing a reduction in crop productivity. The abscisic acid-, stress-, ripening-induced (ASR) proteins play important roles in the protection of plants from abiotic stress. Brachypodium distachyon L. is a well-studied monocot model plant. However, ASR proteins of Brachypodium have not been widely studied. In this study, five ASR genes of Brachypodium plant were cloned and characterized. The BdASR genes were expressed in response to various abiotic stresses and hormones. In particular, BdASR4 was shown to encode a protein containing a nuclear localization signal in its C-terminal region, which enabled protein localization in the nucleus. To further examine functions of BdASR4, transgenic Brachypodium plants harboring BdASR4 were generated. Over-expression of BdASR4 was associated with strong drought tolerance, and plants over-expressing BdASR4 preserved more water and displayed higher antioxidant enzyme activities than did the wild-type plants. The transcript levels of stress-responsive genes, reactive oxygen species scavenger-associated genes, and abscisic acid-responsive genes tended to be higher in transgenic plants than in WT plants. Moreover, plants over-expressing BdASR4 were hypersensitive to exogenous abscisic acid at the germination stage. Taken together, these findings suggest multiple roles for BdASR4 in the plant response to drought stress by regulating antioxidant enzymes and the transcription of stress- and abscisic acid-responsive genes.

Characterization of novel mutants of hexaploid wheat (Triticum aestivum L.) with various depths of purple grain color and antioxidant capacity.

BACKGROUND: Wheat grain is recognized as a rich source of nutrients, including proteins, vitamins, minerals, fibers and antioxidants. In recent years, the focus of wheat breeding has been to increase the content of bioactive compounds to improve human health and prevent diseases. RESULTS: Five novel wheat mutant lines with variable seed color were developed using gamma irradiation of hexaploid wheat inbred line K4191 (purple seed color). The total anthocyanin contents of three mutant lines (L47, L167 and L925) were significantly higher than those of wild-type lines, including K4191 and 'Keumkang' (white seed color). L925 showed the highest total anthocyanin content, and cyanidin-3-glucoside was presented as the most predominant anthocyanin. Compared with 'Keumkang', the expression of anthocyanin biosynthesis genes was significantly up-regulated in purple seed mutant lines. The highest antioxidant activity was observed in L925 extracts. The expression of a few antioxidant-related genes and total anthocyanin content were positively correlated with antioxidant capacity. These data suggest that anthocyanins and phenolic compounds in wheat grains contribute to the antioxidant potential. CONCLUSION: Purple grain color is associated with higher anthocyanin accumulation and antioxidant capacity in wheat. Wheat mutants developed in this study may serve as a valuable source of antioxidants. © 2018 Society of Chemical Industry.

Comparison of radiosensitivity response to acute and chronic gamma irradiation in colored wheat.

We aimed to investigate the biological responses induced by acute and chronic gamma irradiation in colored wheat seeds rich in natural antioxidants. After acute and chronic irradiation, the phenotypic effects on plant growth, germination rate, seedling height, and root length were examined, and the biochemical changes were investigated by analyzing the expression of antioxidant enzyme-related genes, antioxidant enzyme activities, and total antioxidant capacity. High dosages of chronic radiation reduced plant growth compared with the controls. Electron spin resonance measurement and 2,2-diphenyl-1-picrylhydrazyl activity analysis showed lower amount of free radicals in colored wheat seeds on chronic irradiation with low dosage of gamma rays compared to seeds subjected to acute irradiation. Expression levels of anthocyanin biosynthesis genes, antioxidant-related genes, and antioxidant enzyme activity in seeds and young leaves of seedling showed diverse effects in response to different dosages and types of gamma irradiation. This suggests that phenotype is affected by the dosage and type of gamma radiation, and the phytochemicals in colored wheat seeds involved in antioxidant activity to scavenge free radicals respond differently to irradiation types. This provides evidence that acute and chronic exposure to radiation have different effects on seeds and young leaves after germination.

Identification of candidate genes for the seed coat colour change in a Brachypodium distachyon mutant induced by gamma radiation using whole-genome re-sequencing.

Brachypodium distachyon has been proposed as a model plant for agriculturally important cereal crops such as wheat and barley. Seed coat colour change from brown-red to yellow was observed in a mutant line (142-3) of B. distachyon, which was induced by chronic gamma radiation. In addition, dwarf phenotypes were observed in each of the lines 142-3, 421-2, and 1376-1. To identify causal mutations for the seed coat colour change, the three mutant lines and the wild type were subjected to whole-genome re-sequencing. After removing natural variations, 906, 1057, and 978 DNA polymorphisms were detected in 142-3, 421-2, and 1376-1, respectively. A total of 13 high-risk DNA polymorphisms were identified in mutant 142-3. Based on a comparison with DNA polymorphisms in 421-2 and 1376-1, candidate causal mutations for the seed coat colour change in 142-3 were selected. In the two independent Arabidopsis thaliana lines carrying T-DNA insertions in the AtCHI, seed colour change was observed. We propose a frameshift mutation in BdCHI1 as a causal mutation responsible for seed colour change in 142-3. The DNA polymorphism information for these mutant lines can be utilized for functional genomics in B. distachyon and cereal crops.

Identification of lignin-deficient Brachypodium distachyon (L.) Beauv. mutants induced by gamma radiation.

BACKGROUND: Brachypodium distachyon (L.) Beauv. is a monocotyledonous model plant that has been studied to understand a range of biological phenomena for lignocellulosic bioethanol feedstocks and other cereal crops. The lignin makes its cell walls recalcitrant to saccharification, constituting the main barrier to lignocellulosic bioethanol production. In this study, lignin-deficient mutants of B. distachyon induced by chronic radiation were selected and the effects of the mutants on fermentable glucose production were identified. RESULTS: Brachypodium distachyon M2 mutants induced by chronically irradiated gamma radiation were screened by the Wiesner test. Lignin-deficient M2 mutants were further confirmed in subsequent M3 and M4 generations by determining acetyl bromide-soluble lignin. The lignin content was significantly reduced in mutant plants 135-2 (by 7.99%), 142-3 (by 13.8%) and 406-1 (by 8.13%) compared with the wild type. Moreover, fermentable glucose was significantly higher in 135-2 (by 23.91%) and 142-3 (by 36.72%) than in the wild type after 72 h of enzymatic hydrolysis. CONCLUSION: Three lignin-deficient B. distachyon mutants induced by chronically irradiated gamma radiation were obtained. This study will provide fundamental understanding of the B. distachyon cell wall and could contribute to increases in bioethanol production using bioenergy crops. © 2016 Society of Chemical Industry.

Effects of different depth of grain colour on antioxidant capacity during water imbibition in wheat (Triticum aestivum L.).

BACKGROUND: The importance of the effect of phytochemical accumulation in wheat grain on grain physiology has been recognised. In this study, we tracked phytochemical concentration in the seed coat of purple wheat during the water-imbibition phase and also hypothesised that the speed of germination was only relevant to its initial phytochemical concentration. RESULTS: The results indicate that the speed of germination was significantly reduced in the darker grain groups within the purple wheat. Total phenol content was slightly increased in all groups compared to their initial state, but the levels of other phytochemicals varied among groups. It is revealed that anthocyanin was significantly degraded during the water imbibition stage. Also, the activities of peroxidase, ascorbate peroxidase, catalase, glutathione S-transferase, glutathione reductase, and glutathione peroxidase in each grain colour group did not correlated with germination speed. Overall antioxidant activity was reduced as imbibition progressed in each group. Generally, darker grain groups showed higher total antioxidant activities than did lighter grain groups. CONCLUSION: These findings suggested that the reduced activity of reactive oxygen species, as controlled by internal antioxidant enzymes and phytochemicals, related with germination speed during the water imbibition stage in grains with greater depth of purple colouring. © 2016 Society of Chemical Industry.

Characterization of 4 TaGAST genes during spike development and seed germination and their response to exogenous phytohormones in common wheat.

Gibberellic acid (GA) is involved in the regulation of plant growth and development. We defined GA-stimulated transcript (GAST) gene family and characterized its four members (TaGAST1, 2, 3, and 4) in wheat spikes. Triticum aestivum whole spikes were collected at ten developmental stages and dehulled spikelets were obtained at various days after flowering. Expression of TaGAST1, 2, 3, and 4 was analyzed using RT-PCR at inflorescence development stages, in different tissues, and after phytohormones application. To identify proteins interacting with TaGAST1, yeast two-hybridization was performed and BiFC analysis was used for verification. TaGAST1 was expressed at the inflorescence stage and only expressed in seedlings under abscisic acid (ABA) treatment after phytohormone treatment. TaGAST2 and TaGAST3 showed moderate expression in the spike, vigorous transcript accumulation in the seedling, and up-regulation by exogenous GA in early germination stages. TaGAST4 was predominantly expressed in the seedling. Wheat cyclophilin A-1 (TaCypA1), identified as a TaGAST1-interacting protein, showed opposite expression pattern in the developing spike to TaGAST1. TaCypA1 transcript was slightly up-regulated by GA, slightly down-regulated by paclobutrazol, and was maintained after ABA treatment. The interaction of TaGAST1 with TaCypA1 is targeted to the plasma membrane. TaGAST1 was specifically expressed in the wheat spike and was stimulated by exogenous GA treatment. TaGAST2 and TaGAST3 expression in germinating seeds and seedlings was higher than that in the spike stage. TaGAST4 was not expressed in all developmental stages. TaGAST1 and TaCypA1 might be expressed antagonistically during wheat spike development.

Role of wheat trHb in nitric oxide scavenging.

We examined the role of wheat truncated-hemoglobin (TatrHb) in nitric oxide (NO) scavenging in transgenic Arabidopsis plants by assessing the response to an NO donor/scavenger and salt stress. The degree of increase in Na(+) and decrease in K(+) levels in the transgenic plants were more than those in the wild-type plants, and the ratio of Na(+) to K(+) increased in the transgenic plants under salt stress. Endogenous NO increased dramatically in the salt-treated wild-type plants but not in the transgenic plants. Additionally, the maximum photosystem II quantum ratio of variable to maximum fluorescence (Fv/Fm) in transgenic plants decreased more significantly than that in the wild-type plants, indicating that the transgenic plants suffered more severe photosynthetic damage because of salt stress than that by the wild type. Similar results were observed in germination experiments by using Murashige and Skoog media containing 100 mM sodium chloride. The Fv/Fm decreased in the leaves of salt-treated transgenic plants, indicating that transgenic seeds were more sensitive to salt stress than that by the wild-type seeds. In addition, the negative effect on seed germination was more severe in transgenic plants than in the wild types under NaCl treatment conditions. The results support the hypothesis that plant trHb shares NO scavenging functions and characteristics with bacterial trHb.

Differentially expressed genes in response to gamma-irradiation during the vegetative stage in Arabidopsis thaliana.

Biochemical and physiological processes in plants are affected by gamma-irradiation, which causes significant changes in gene transcripts and expression. To identify the differentially expressed Arabidopsis genes in response to gamma-irradiation, we performed a microarray analysis with rosette leaves during the vegetative stage. Arabidopsis plants were exposed to a wide spectrum doses of gamma ray (100, 200, 300, 400, 800, 1200, 1600 or 2000 Gy) for 24 h. At the dose range from 100 to 400 Gy, irradiated plants were found to be shorter than controls after 8 days of irradiation, while doses over 800 Gy caused severe growth retardation. Therefore, 100 and 800 Gy were selected as adequate doses for microarray analysis to identify differentially expressed genes. Among the 20,993 genes used as microarray probes, a total number of 496 and 1,042 genes were up-regulated and down-regulated by gamma-irradiation, respectively (P < 0.05). We identified the characteristics of the genes that were up-and down-regulated fourfold higher genes by gamma irradiation according to The arabidopsis information resource gene ontology. To confirm the microarray results, we performed a northern blot and quantitative real-time PCR with several selected genes that had a large difference in expression after irradiation. In particular, genes associated with lipid transfer proteins, histones and transposons were down-regulated by 100 and/or 800 Gy of gamma irradiation. The expression patterns of selected genes were generally in agreement with the microarray results, although there were quantitative differences in the expression levels.

Physiological changes and anti-oxidative responses of Arabidopsis plants after acute and chronic γ-irradiation.

To identify the effects of acute and chronic γ-irradiation in Arabidopsis plants, physiological responses and antioxidant-related gene expression were investigated. Seedlings were exposed to 200 Gy of γ-irradiation in acute manner for 1 or 24 h (A1 and A24) or in chronic manner for 1, 2, or 3 weeks (C1 W, C2 W, and C3 W). Plant height, silique number, and silique length in A1 and A24 irradiated plants were significantly reduced when compared to non-irradiated plants. Silique number decreased in response to both acute and chronic irradiation, except with the C3 W treatment, and the number of trichomes dramatically increased in A1 and C1 W. Electron spin resonance signal intensities increased in A1 and in all chronically irradiated plants, but decreased in the A24-treated plant. To investigate the effects of acute and chronic γ-irradiation on antioxidant enzymes, we examined activity of four antioxidant enzymes: catalase (CAT), peroxidase (POD), ascorbate peroxidase, and superoxide dismutase. In general, POD and CAT activities decreased in response to acute and chronic γ-irradiation. Oligonucleotide microarrays were used to investigate transcriptional changes after irradiation. Several genes related to reactive oxygen species signaling were up-regulated after acute and chronic exposure, including genes encoding heat shock factors, zinc finger proteins, NADPH oxidase, WRKY DNA-binding proteins, and calcium binding proteins. Taken together, our data indicate that the responses and activation of antioxidant systems prompted by irradiation exposure are dependent upon the γ-ray dose rate.