Marker-free transgenic cucumber (Cucumis sativus L.) cv. Poinsett 76 SR plants were produced by Agrobacterium mediated transformation. A transformation efficiency of 1.62 was observed on using Agrobacterium tumefaciens strain LBA4404 harbouring Arabidopsis cbf1 gene driven by the inducible promoter RD29A in a binary vector system pCAMBIA. Transgene integration and single copy insert in transgenic cucumber was confirmed by polymerase chain reaction (PCR) and Southern blot analysis in T₀ lines and also confirmed marker-free status in T₁ generation. Transgene expression was confirmed by reverse transcription (RT)-PCR in T₁ generation transgenic cucumber and advanced to T₂ generation. Upon exposure to chilling stress (4 °C), the T₂ generation transgenic plants survived up to 36 h; however, wild-type plants could not survive and gradually died. A significant decrease in membrane injury index (MII), increase in activities of antioxidant enzymes (SOD and CAT), free proline content and relative water content (RWC) in the leaves were observed in transgenic cucumber as compared to wild-type under chilling stress. Thus, the transgenic cucumber plants expressing Arabidopsis cbf1 gene conferred protection against chilling stress.

Somatic embryogenesis (SE) in Arabidopsis was induced using various systems, including auxin treatment of in vitro cultured explants (immature zygotic embryos, IZEs) and transgenic plants overexpressing embryogenesis-related transcription factors, e.g. LEC2 together with the GUS reporter gene under control of the auxin-induced DR5 promoter. The study indicated that the SE-systems used gave different embryogenic capacities for the production of true embryos. The highest ratio of true embryos (75 %) was found among embryo-like structures in transgenic seedlings overexpressing LEC2. Analysis of in vitro induced SE systems indicated that in somatic embryos produced in response to exogenous auxin treatment the formation of root poles is frequently disturbed. A lack of a properly formed root meristem was observed in 35-80 % of in vitro induced somatic embryos, in dependence on auxin concentration and duration of treatment.

The coat protein (CP) gene of Tobacco streak virus (TSV) from sunflower (Helianthus annuus L.) was amplified, cloned and sequenced. A 421 bp fragment of the TSV coat protein gene was amplified and a gene construct encoding the hairpin RNA (hpRNA) of the TSV-CP sequence was made in the plasmid pHANNIBAL. The construct contains sense and antisense CP sequences flanking a 742 bp spacer sequence (Pdk intron) under the control of the constitutive CaMV35S promoter. A 3.6 kb Not I fragment containing the hpRNA cassette (TSV-CP) was isolated from pHANNIBAL and sub-cloned into the binary vector pART27. This chimeric gene construct was then mobilized into Agrobacterium tumefaciens strain LBA4404 via triparental mating using pRK2013 as a helper. Sunflower (cv. Co 4) and tobacco (cv. Petit Havana) plants were transformed with A. tumefaciens strain LBA4404 harbouring the hpRNA cassette and in vitro selection was performed with kanamycin. The integration of the transgene into the genome of the transgenic lines was confirmed by PCR analysis. Infectivity assays with TSV by mechanical sap inoculation demonstrated that both the sunflower and tobacco transgenic lines exhibited resistance to TSV infection and accumulated lower levels of TSV compared with non-transformed controls.

Arabidopsis shoots regenerate from root explants through a two-step process consisting of pre-incubation on an auxin-rich callus induction medium (CIM), followed by transfer to a cytokinin-rich shoot induction medium (SIM). The auxin receptor gene TIR1 was up-regulated when explants were transferred to SIM. The CIM pre-incubation is required for its up-regulation. The tir1-1, TIR1 knockdown mutant, reduced the efficiency of shoot regeneration in tissue culture, while its over-expression mutant significantly improved efficiency. TIR1 promoter::GUS fusion analysis demonstrated that TIR1 expression was in the shoot and the newly emerging leaves. After 10 d on SIM, several cytokinin related genes (CDKB1;1, CKS1, IPT4 and ARR15), which associate with shoot regeneration, were up-regulated in plants over-expressing TIR1 and some of these were down-regulated in the tir1-1 mutant. Thus, TIR1 appears to be involved in regulating shoot regeneration.

To induce virus resistance in tobacco and rice we constructed hairpin RNA expression system harbouring inverted repeat fragments of coat protein cDNA of Potato virus Y (PVY) or Rice stripe virus (RSV). These structures were driven by three promoters [cauliflower mosaic virus 35S (CaMV 35S), polyubiqutin gene of maize (Ubi), and Pharbitis nil leucine zipper gene (PNZIP)] which have different tissue-specific activity. PVY resistance ratios were 65.18, 24.33 and 83.54 % in transgenic tobacco plants harboring p35S-PVY, pUbi-PVY and pPNZIP-PVY. RSV resistance was 16.21, 28.61 and 29.33 % in transgenic rice plants harboring p35S-RSV, pUbi-RSV and pPNZIP-RSV. Northern blotting and GUS assay demonstrated that virus resistance levels were related to promoter activity. Therefore, choice of the more effective and tissue-specific promoter to reinforce transcription of hpRNAs will favour the cultivation of highly virusresistant transgenic plants.

The human papillomavirus type 16 (HPV 16) oncogene E7 fused with the gene for β-glucuronidase (gus) was used in plant transformation experiments. The E7 gene modified for lower cancerogenicity and fused with the 5' end of the gus in cassettes with cauliflower mosaic virus 35S promoter and transcription terminator produced high contents of fusion proteins in potato protoplasts. Expression vectors harbouring E7 fusion cassettes were used for Agrobacterium tumefaciens LBA4404 mediated transformation of either potato (Solanum tuberosum L. cv. Bintje) or tomato (Lycopersicon esculentum Mill. cv. Moneymaker). A fusion gene was found in all rooted regenerants using polymerase chain reaction with primers providing amplified fragments from E7 and gus genes. GUS activity was revealed in all regenerants obtained. Nevertheless, the level of GUS expression in different constructs varied much more than in transient expression experiments with potato protoplasts. Especially, expression level in plants carrying vectors with the whole E7 gene fused with gus was lowered by 2-3 orders of magnitude comparing with fusion of the first 41 codons of E7 and gus. Southern hybridisation of 18 tomato and 23 potato regenerants revealed mostly multiple tandem integration of T-DNA into the plant genome and Western blot proved the presence of the fusion protein in 9 tomato and 11 potato plants out of 41 tested individuals.

Formins (FH2 proteins) are implicated in F-actin nucleation and other aspects of cytoskeletal organization. Plants possess two formin clades, relatively well-described Class I formins and so far poorly characterized Class II formins. Comparison of Class II formin genes of two Arabidopsis species, A. thaliana and A. lyrata, indicates dynamic evolution within the Class II formin clade. Disruption of an outlier A. thaliana Class II formin gene, AtFH12 (At1g42980), whose expression is induced by NaCl, produced only negligible phenotypic effects under a variety of conditions, including salt stress, suggesting functional redundancy among Class II formins. However, the same mutation massively aggravated toxic effects of the expression of a fluorescent actin marker, GFP-tagged mouse talin (GFP-mTalin), known to interfere with normal actin dynamics. Abnormal actin structures were observed in atfh12 mutants expressing GFP-mTalin as compared to wild type. This not only demonstrates an actin-associated function for AtFH12, but also documents the feasibility of using the heterologous actin marker to "stress-test" the actin cytoskeleton in phenotyping "weak" actin related mutant alleles.

Glycinin is one of the abundant storage proteins in soybean seeds. A modified Gy1 (A1aB1b) proglycinin gene with a synthetic DNA encoding four continuous methionines (V3-1) was connected between the hpt gene and the modified green fluorescent protein sGFP(S65T) gene, and a resultant plasmid was introduced into soybean by particle bombardment in order to improve nutritional value of its seeds. After the selection with hygromycin, the efficiency of gene introduction was evaluated. More than 60 % of the regenerated plants tolerant to hygromycin yielded the hpt and V3-1 fragment by polymerase chain reaction (PCR) analysis, and the expression of sGFP was detected in about 50 % of putative transgenic soybeans. Southern hybridization confirmed the presence of transgenes in T0 plants and the transgenic soybeans hybridized with the hpt and V3-1 genes were analyzed showed different banding patterns. Most of the transgenic plants were growing, flowering normally and produced seeds. Analysis of seed obtained from transgenic soybean plants expressing hpt and V3-1 genes showed higher accumulation of glycinin compared with non-transgenic plants. In addition, protein expression in transgenic soybean plants was observed by using 2D-electrophoresis.

Glutathione-S-transferases (GSTs) are ubiquitous enzymes that play a key role in stress tolerance and cellular detoxification. The GST gene GsGST14 selected from the gene expression profiles of Glycine soja under alkaline stress was transformed into alfalfa (Medicago sativa L.). Transgenic alfalfa plants showed 1.73-1.99 times higher GST activity than wild-type plants. Transgenic alfalfa grew well in the presence of 100 mM NaHCO₃, while wild-type plants exhibited chlorosis and stunted growth, even death. There were marked changes in malondialdehyde content and relative membrane permeability caused by alkaline stress in non-transgenic lines compared to transgenic lines. The results indicate that the gene GsGST14 could enhance alkaline resistance in transgenic alfalfa.

Harpin, an elicitor molecule of bacterial origin induces hypersensitive response (HR) in non-host plants. In an attempt to induce male sterility, harpin was tagged with a signal peptide and expressed downstream to tapetum-specific TA29 promoter resulting in extracellular secretion, subsequent degeneration of tapetum and development of male sterility in tobacco. Putative transgenics were analyzed by PCR amplification of transgene, semiquantitative RT-PCR analysis from total RNA extracts from anther tissue with transgene specific probe, Western blotting using polyclonal antibody raised against harpin, by transmission and scanning electron microscopy, and by confocal microscopy of anthers and pollen at various stages of development. Varying degrees of male sterility (30-100 %) was observed with plants showing complete and partial male sterility as well as several morphological variations were seen especially in leaves and flowers. Further, some of the transgenics showed un-induced of HR-like local lesions in the vegetative tissues. Harpin_Pss got deposited on the pollen grains upon tapetal degeneration resulting in significant alterations in the morphology of pollen cell wall. However, megagametogenesis was not affected in complete and partial male sterile plants and female gametes were completely fertile. The complete male sterility was attributed to premature tapetal cell death due to sufficient extracellular harpin_Pss accumulation whereas insufficient protein content might be the reason for partial male sterility. These findings indicate the possible use of cytotoxic harpin_Pss for the development of male sterile plants.

We report here the development of transgenic tobacco plants with thaumatin gene of Thaumatococcus daniellii under the control of a strong constitutive promoter-CaMV 35S. Both polymerase chain reaction and genomic Southern analysis confirmed the integration of transgene. Transgenic plants exhibited enhanced resistance with delayed disease symptoms against fungal diseases caused by Pythium aphanidermatum and Rhizoctonia solani. The leaf extract from transgenic plants effectively inhibited the mycelial growth of these pathogenic fungi in vitro. The transgenic seeds exhibited higher germination percentage and seedling survival under salinity and PEG-mediated drought stress as compared to the untransformed controls. These observations suggest that thaumatin gene can confer tolerance to both fungal pathogens and abiotic stresses.

Limited information is available on the role of brassinosteroids (BRs) in response of plants to nutrient deficiency. To understand the functions of BRs in response to iron deficiency, we investigated the effect of 24-epibrassinolide (EBR) on activities of ferric-chelate reductase (FCR), H⁺-ATPase, Ca²⁺-ATPase, nitrate reductase (NR), antioxidant enzymes, Fe and other minerals content and distribution, chlorophylls, soluble protein, free proline, reactive oxygen species, and malondialdehyde in peanut (Arachis hypogea L.) plants subjected to Fe deficiency (10^-5 M Fe(III)-EDTA) with foliar application of EBR (0, 10^-8, 5.0×10^-8, 10^-7, 5.0×10^-7, and10^-6 M). Results show that EBR increased Fe translocation from roots to shoots and increased Fe content in cell organelles. Activities of antioxidant enzymes increased and so the ability of resistance to oxidative stress was enhanced. As result of enhancement of H⁺-ATPase and Ca²⁺-ATPase activities, the inhibition of Fe, Ca, Mg, and Zn uptake and distribution was ameliorated. Chlorophyll, soluble protein, and free proline content also increased and consequently, chlorosis induced by Fe deficiency was alleviated. The results demonstrate that EBR had a positive role in regulating peanut growth and development under Fe deficiency and an optimal concentration appeared to be 10^-7 M.

Phenylalanine ammonia lyase (PAL) is a specific branch point enzyme of primary and secondary metabolism. The Citrus reticulata Blanco PAL gene was cloned and designated as CrPAL1. The cDNA sequence of CrPAL1 was 2 166 bp, encoding 721 amino acid residues. Sequence alignment indicates that CrPAL1 shared a high identity with PAL genes found in other plants. Both the dominant and catalytic active sites of CrPAL1 were similar to PAL proteins observed in Petroselinum crispum. Phylogenetic tree analysis indicates that CrPAL1 was more closely related to PALs in Citrus clementina × C. reticulata and Poncirus trifoliata than to those from other plants. Subcellular localization reveals that CrPAL1-green fluorescent protein fusion protein was specifically localized in the plasma membrane. Activity of PAL as well as CrPAL1 expression increased under Fe deficiency. A similar result was noted for total phenolic content. The root exudates of C. reticulata strongly promoted reutilization of apoplastic Fe in roots. Furthermore, Fe was more desorbed from the cell wall under Fe deficiency than in sufficient Fe supply.

Turnip (Brassica rapa L.) plants were grown in Perlite with low (< 2.5 μM) or adequate (25 μM) boron supply under well-watered and drought conditions for 12 weeks. Dry mass of leaves and roots was reduced under drought by about 61 and 56 % in plants supplied adequately with B, while up to 84 and 74 % under B starvation. Drought reduced B content by about 70 and 82 % for B-sufficient and B-deficient plants, respectively. According to the chlorophyll fluorescence parameters, the photosynthesis processes conserved their normal activities under low B supply in well-watered plants, while a serious damage to photosystem 2 occurred under drought stress. Stomatal limitation was the most important cause for a 17 % lower net photosynthetic rate (P_N) of drought stressed B-sufficient plants. In B-deficient plants, however, both stomatal and non-stomatal limitations were involved in 53 % reduction of P_N. Low B supply reduced strongly leaf water potential.

Physiological changes associated with senescence of flowers and abscission of floral parts in Oncidesa (formerly Oncidium) cv. Gower Ramsey are caused by a plant hormone ethylene which is produced by pollinia cap dislodgment during postharvest handling and transportation. The ethylene receptor gene OgERS1 of Oncidesa has been previously cloned and characterized. To analyze promoter activity of OgERS1, transgenic Arabidopsis thaliana plants were generated to express the ß-glucuronidase (GUS) reporter gene under the control of 5'-upstream sequence of OgERS1 from Oncidesa. The expression pattern of the OgERS1 promoter at the cellular level was investigated by analysis of GUS activity. This promoter can activate gene expression in both actively dividing young tissues and abscission-related aging tissues. Expression of GUS was detected in the shoot meristem uniquely in 10 to 30 d-old-plants and was found in flower buds, axillary buds, flower stems, and abscission layers during later development. In 2- to 3-week-old transgenic Arabidopsis, exogenous ethylene, glucose, lactose, and maltose enhanced promoter activity implying that crosstalk between sugar and an ethylene receptor may exist. However, indole-3-acetic acid, benzylaminopurine, abscisic acid, heat, wounding, salinity, drought, and flooding slightly suppressed promoter activity. These results demonstrate that the promoter of OgERS1 was developmentally and environmentally regulated, and imply a potential for application of this bi-functional promoter to increase branching or enhanced dwarfing.

MicroRNAs (miRNAs) are small non-coding RNAs that play crucial regulatory roles in diverse developmental processes via cleavage or translational inhibition of their target mRNAs. Although a growing number of miRNAs and their targets have been predicted and discovered via experimentation in many plants, little is known about conserved miRNAs and their target genes in Pinus densata. In the present study, the conserved miRNAs, miR171 and miR482, from Pinus densata were characterized. Analysis of miR171 and miR482 reveal that these miRNAs were highly conserved in other plant species. In addition, the precursors of miR171 and miR482 were validated by real time-PCR and sequencing. Using real-time quantitative PCR, miR171 and miR482 as well as their corresponding targets were found to be differentially expressed in needles, stems, and roots of Pinus densata. Furthermore two target genes, one GRAS family transcription factor protein gene and one nucleotide-binding site leucine-rich repeat (NBS-LRR) resistance protein gene, were experimentally verified to be the targets of pde-miR171 and pde-miR482, respectively, using RNA ligase-mediated 5'-rapid amplification of cDNA ends (RLM-RACE).

Moth orchids (Phalaenopsis) are among the top-traded blooming potted plants in the world. To explore mitochondrial DNA (mtDNA) markers for species identification, we located simple sequence repeats in the mtDNA of Phalaenopsis aphrodite subsp. formosana and then pre-screened them for polymorphic markers by their comparison with corresponding mtDNA regions of P. equestris. The combination of 13 selected markers located in intergenic spacers could unambiguously distinguish 15 endemic moth orchids. Five most variable markers with polymorphic information content (PIC) ≥ 0.7 could be combined to classify 18 of 19 endemic moth orchids including parental strains most commonly used in breeding programs. The sequences of four selected mtDNA regions were highly variable, and one region (MT2) could be used to completely distinguish 19 endemic moth orchids. Though mitochondrial introns were highly conserved among moth orchids, evolutionary hotspots, such as variable simple sequence repeats and minisatellite repeats, were identified as useful markers. Furthermore, a marker technology was applied to reveal the maternal inheritance mode of mtDNA in the moth orchids. Moreover, phylogenetic analysis indicates that the mtDNA was nonmonophyletic below the Phalaenopsis genus. In summary, we have revealed a set of mtDNA markers that could be used for identification and phylogenetic study of Phalaenopsis orchids.

Secretion of organic acids (OAs) by roots has been suggested to be an important mechanism of Al resistance in many species. In Stylosanthes, the participation of OAs in the mechanism of Al resistance is poorly understood. We aimed to study the production and secretion of OAs by two Brazilian Stylosanthes species with different Al resistance. Stylosanthes capitata and S. guianensis were treated with Al at different concentrations in 0.5 mM CaCl₂ (pH 4.0), and then root elongation, Al and OA content, OA secretion into the external solution, and the activity of citrate synthase (CS) were measured. Al-induced secretion of citric acid was also evaluated in the presence of protein synthesis and anion channel inhibitors. S. guianensis accumulated lower amounts of Al in its roots and displayed less inhibition of root elongation compared to S. capitata. Citric and malic acids were the most abundant OAs in the roots, and their content decreased with the Al treatment, except for citric acid in S. guianensis. Citric acid was the only OA secreted into the nutrient solution by the Al-treated plants of both species, but more by S. guianensis. Citrase synthase activity decreased in S. capitata but increased in S. guianensis with the Al treatment, and it may have a crucial role in the maintenance of citric acid content in the roots of S. guianensis. The use of anion channel and protein synthesis inhibitors reveal that anion channels were likely involved in the secretion of citric acid, and channel protein transcription was up-regulated by exposure to Al in Stylosanthes.

We tested the capability of plants to utilize a mixture of amino acids and peptides as the exclusive source of nitrogen. The aim of this study was to find out how such a nutrition affected plantlet growth, photosynthetic performance, and N assimilation metabolism in tobacco (Nicotiana tabacum L., cv. Petit Havana SR1) grown in vitro. Plantlets grown in a casein hydrolysate-supplemented (CA) medium were compared to plantlets grown in a complete Murashige-Skoog (MS) medium, plantlets grown in an ammonium-deficient medium (N1), or plantlets grown in a nitrate-reduced medium (N2). In addition, the plantlets were grown in the presence or absence of 1.5 % (m/v) saccharose as an additional carbon source. Casein hydrolysate, similarly as inorganic N limitation, reduced generally plantlet growth, whereas no significant effects were observed on photosynthetic parameters evaluated by chlorophyll a fluorescence. Although addition of saccharose stimulated the plantlet growth particularly in the MS, it showed a rather negative influence both on the growth and on the photochemical efficiency of photosystem II in the plantlets grown in the CA and N1. The activities of enzymes involved in N assimilation, such as nitrate reductase (NR) and glutamine synthetase (GS), were lower in the plantlets grown in the CA, N1, and N2, both in leaves and in roots. On the other hand, glutamate synthase and glutamate dehydrogenase were employed by the plantlets grown in the CA. The presence of saccharose in the growth medium stimulated mainly NR and GS activities in the MS grown plantlets, whereas enzyme activities of the plantlets grown on the N1, N2, and CA were not significantly influenced. We proved that the tobacco plantlets can utilize casein hydrolysate as the sole source of N particularly during their photoautotrophic cultivation. Contrary to positive effects of photomixotrophic nutrition for the MS grown plantlets, exogenous sugar seemed to diminish the ability of the casein hydrolysate-supplemented plantlets to utilize efficiently the additional C source.

Calcium ion (Ca²⁺) is essential secondary messenger in plant signaling networks. In this study, the effect of Ca²⁺ on oxidative damage caused by a high irradiance (HI) was investigated in the leaves of two cultivars of tall fescue (Arid3 and Houndog5). Pretreatment of the tall fescue leaves with a CaCl₂ solution significantly increased Ca²⁺ content and intrinsic HI tolerance due to a decreased ion leakage and content of malondialdehyde, hydrogen peroxide, and superoxide radicals. Moreover, the activities of superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase increased in both the cultivars in the presence of Ca²⁺ under the HI stress. In contrast, treatments with a Ca²⁺ chelator ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) or a plasma membrane Ca²⁺ channel blocker LaCl₃ reversed these effects. On the other hand, a pronounced increase in nitric oxide synthase-like activity and NO release by exogenous Ca²⁺ treatment was observed in the tolerant Arid3 plants after exposure to the HI, whereas only a small increase was observed in more sensitive Houndog5. Moreover, the inhibition of NO production by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or N ^ω-nitro-L-arginine blocked the protective effect of exogenous Ca²⁺, whereas the inhibition of Ca²⁺ by EGTA or LaCl₃ had no influence on the protective effect of NO. The results indicate that NO might be involved in the Ca²⁺-induced activities of antioxidant enzymes further protecting against HI-induced oxidative damage. This protective mechanism was found to be more efficient in Arid3 than in Houndog5.

Alternative respiration pathway (AP) is an important pathway which can be induced by environment stresses in plants. In the present study, we show a new mechanism involving the AP in nitrogen deprivation-induced tolerance of Poa annua callus to salt stress. The AP capacity markedly increased under a 600 mM NaCl treatment or nitrogen deprivation pretreatment and reached a maximum under the nitrogen deprivation pretreatment combined with the NaCl treatment (-N+NaCl). Malondialdehyde (MDA) and H₂O₂ content and Na⁺/K⁺ ratio significantly increased under the 600 mM NaCl treatment but less under the-N+NaCl treatment. Moreover, both the nitrogen deprivation and the NaCl stress stimulated the plasma membrane (PM) H⁺-ATPase activity and increased pyruvate content. The maximal stimulating effect was found under the-N+NaCl treatment. When the AP capacity was reduced by salicylhydroxamic acid (SHAM, an inhibitor of AP), content of MDA and H₂O₂ and Na⁺/K⁺ ratio dramatically increased, whereas PM H⁺-ATPase activity decreased. Moreover, exogenous application of pyruvate produced a similar effect as the nitrogen deprivation pretreatment. The effects of SHAM on the Poa annua callus were counteracted by catalase (a H₂O₂ scavenger) and diphenylene iodonium (a plasma membrane NADPH oxidase inhibitor). Taken together, our results suggest that the nitrogen deprivation enhanced the capacity of AP by increasing pyruvate content, which in turn prevented the Poa annua callus from salt-induced oxidative damages and Na⁺ over-uptake.

Plants are subjected to several abiotic stresses that adversely affect growth, metabolism and yield. The dynamic research in plant genetics complemented by genome sequencing has opened up avenues to address multiple problems caused by abiotic stresses. Though many drought-induced genes have been phytoengineered in a wide range of plants, the drought signal transduction pathways, and the alteration of plant sensing and signaling systems to adverse environments still remain an intriguing subject for comprehensive investigation. To impart enhanced drought tolerance in plants, a thorough perception of physiological, biochemical and gene regulatory networks is essential. Recent functional genomics tools have facilitated the progress in our understanding of stress signaling and of the linked molecular regulatory networks. This has revealed several stress-inducible genes and various transcription and signaling factors that regulate the drought stress-inducible systems. Translational genomics of these drought specific genes using model plants have provided encouraging outcomes, but the in-depth knowledge of the specific roles of various metabolites in plant stress tolerance will lead to evolvement of strategies for the phytoengineering of drought tolerance in plants in future.

Histone deacetylases (HDACs) catalyze histone deacetylation and play an important role in suppression of gene transcription in multiple cellular processes. HDACs are widely distributed in eukaryotes, however, detailed characterrization of HDACs in woody plants is not available. In this study, the sequences of reduced potassium dependency 3/histone deacetylase 1 (RPD3/HDA1) family proteins in black cottonwood (Populus trichocarpa Torr. & A. Gray) were characterized and their expression patterns in response to cold and salt stresses were determined. The RPD3/HDA1 proteins had conserved HDAC domains and can be divided into three classes based on sequence similarity and phylogenetic analysis. The transcripts of the HDAC genes were detected in different amounts in leaves, stems, and roots. The expressions of HDAC genes HDA902, HDA903, HDA904, HDA909, and HDA912 were up-regulated in a cold stress. Interestingly, in a salt stress, most of the HDAC genes were down-regulated. These results indicate that the poplar HDAC genes were regulated by the cold and salt stresses, and the members of the RPD3/HDA1 family play a role in stress responses.

Dicer proteins belong to the RNase III family of proteins, which are key components in small RNA biogenesis. In Solanum lycopersicum, seven Dicer-like (DCL) genes have been identified and have been named SlDCL. In this study, we cloned the full-length sequence of the SlDCL genes including untranslated regions using RNA ligase-mediated rapid amplification of cDNA ends. Our analysis indicates that 7 SlDCLs were located on 5 tomato chromosomes (6, 7, 8, 10, and 11). The gene structure of the SlDCLs covered long genomic regions and contained more than 20 exons. Phylogenetic analysis divided the seven SlDCL members into four subgroups. In general, all seven SlDCLs were expressed in all organs but more in flowers and fruits than in the other parts. Moreover, the expressions of some genes changed slightly after treatment with ethylene or 1-methylcyclopropene suggesting their likely roles in plant responses to ethylene. Our findings provide essential information on SlDCL genes in tomato and will aid in the functional classification of DCL families in plants.

The life cycle of flowering plants is partially defined by environmental cues like day length and temperature. In the model plant Arabidopsis thaliana and temperate cereals, such as barley (Hordeum vulgare) and wheat (Triticum spp.), differences in life cycle control have been associated with a natural variation in FLOWERING LOCUS C (FLC) and VERNALIZATION 1-3 (VRN1-3). In sugar beet (Beta vulgaris L.), variation in vernalization requirement and life cycle is determined by a major gene at the B locus. This gene has recently been identified as a pseudo-response regulator (PRR) gene BOLTING TIME CONTROL 1 (BTC1). A second gene in beet with homology to BTC1 and ARABIDOPSIS PSEUDO RESPONSE REGULATOR 7 (APRR7) in Arabidopsis was identified and termed Beta vulgaris PSEUDO RESPONSE REGULATOR 7 (BvPRR7). We functionally characterized BvPRR7 by transgenic analysis in Arabidopsis and expression profiling during development in beet. We show that BvPRR7 was diurnally regulated and responded to cold. Constitutive expression of BvPRR7 distorted diurnal rhythms and caused late flowering in Arabidopsis suggesting a conserved function of BvPRR7 in clock regulation. Conceivably, the retention of a functional role of BvPRR7 in clock regulation may have facilitated the evolution of a distinct role as major floral regulator of the second PRR7 homolog in beet, BTC1.

Carbon monoxide (CO) is considered as a new emerging cell signal molecule which is involved in plant growth, development, and acquisition of stress tolerance. In recent years, hydrogen sulfide (H₂S) has been found to have similar functions, but crosstalk between CO and H₂S in the acquisition of heat tolerance in plants is not clear. In this study, pretreatment of tobacco (Nicotiana tabaccum L.) cells cultured in a suspension with a CO donor hematin significantly increased survival percentage of cells under a heat stress and regrowth ability after the heat stress, alleviated a decrease in cell vitality, and accumulation of malondialdehyde. In addition, treatment with hematin enhanced the activity of L-cysteine desulfhydrase, a key enzyme in H₂S biosynthesis, which in turn induced accumulation of endogenous H₂S in tobacco cells. Interestingly, hematin-induced heat tolerance was enhanced by addition of NaHS, a H₂S donor, but weakened by specific inhibitors of H₂S biosynthesis DL-propargylglycine or its scavenger hypotaurine. Furthermore, pretreatment with hemoglobin (a CO scavenger) and zinc protoporphyrin IX (a CO specific synthetic inhibitor) had no significant effect on NaHS-induced heat tolerance of tobacco cells. These results suggest that CO pretreatment could improve the heat tolerance of tobacco suspension cultured cells, and H₂S might exert its signal role downstream to CO-induced heat tolerance.

Water use efficiency (WUE) is an worth attempting trait to discover the genomic regions governing it, especially in view of the diminishing water resources for the crop plants in general and rice in particular. In order to address this, the present investigation was aimed at identification of genomic regions governing WUE employing a recombinant inbred line population derived from a cross between INRC10192, a high WUE landrace, and IR64, a high yielding cultivar. A total of 36 quantitative trait loci (QTLs) were detected under control as well as drought conditions on chromosomes 1, 2, 4, 8, 9, 10, and 11. Among all, the QTLs with the marker intervals RM486-RM6703, RM6703-RM11484, RM404-RM447, RM24879-RM171, and RM229-RM332 on chromosomes 1, 8, 10, and 11 were found to govern the water use efficiency related traits such as carbon isotope discrimination, specific leaf area, leaf width, and relative water content. Nine major QTL intervals were targeted for candidate gene identification using gene ontology (GO) and transcriptome-based analyses. Overrepresented GO terms in the targeted QTLs were found to be associated with the genes/pathways controlling stomatal regulatory mechanism, stress responsive genes or transcription factors, and saccharide biosynthesis pathways under stress situation. Hence, these genes or genomic regions are potential candidates for development of high WUE rice cultivars.

Two genes encoding CAX2-like proteins were isolated from a Zn/Cd hyperaccumulating ecotype (HE) and nonhyperaccumulating ecotype (NHE) of Sedum alfredii Hance, and they were named SaCAX2h and SaCAX2n, respectively. Both SaCAX2h:eGFP and SaCAX2n:eGFP proteins were localized to the vacuolar membrane of tobacco epidermal cells and yeast mutants. Heterologous expression of SaCAX2h or SaCAX2n in the Δzrc1 yeast mutant increased Cd content in yeast cells. Yeast complementary assay also revealed that both the transporters could suppress Ca and Mn hypersensitivity and enhance Ca and Mn accumulation in a K667 yeast mutant. The expression patterns of the two genes were different under the Cd treatment. Transcription of SaCAX2h was down-regulated in roots and up-regulated in shoots whereas transcription of SaCAX2n was down-regulated in shoots after the exposition to Cd. Furthermore, over-expression of SaCAX2h enhanced metal accumulation in the tobacco plants. The Cd content increased by 17-19 % in shoots and 31-36 % in roots; the Ca content of the transgenic plants increased by 31-32 % in shoots, and the Mn content increased by 60-79 % in shoots and 22-29 % in roots. These results indicate that SaCAX2h was responsible for Ca and Mn sequestration into vacuoles, and over-expression of SaCAX2h enhanced Cd accumulation in the transgenic tobacco.

Several studies have been performed to elucidate the role of brassinosteroids (BRs) in plant growth and development. However, information on the role of BR signaling in nutrient uptake is limited. This study explores the relationship between BRs and ammonium transporter 1 (AMT1) expression in Arabidopsis roots. We found that BR treatment reduced the expression of AMT1 genes and that a BR receptor BRI1 mutant bri1-5 reversed its BR-repressed expression. Furthermore, the BR signaling transcription factor, BES1, regulates AMT1 expression in roots. NH₄ ⁺-mediated repression of AMT1;1, AMT1;2, and AMT1;3 was suppressed in a gain-of-function BES1 mutant (bes1-D). This mutant was more sensitive to methyl-ammonium and contained a higher ammonium content compared to wild-type plants. However, BES1 failed to bind E-box elements present in the promoter region of the AMT1 genes. Furthermore, NH₄ ⁺-mediated glutamine synthetase (GS) and glutamine oxoglutarate aminotransferase (GOGAT) gene expressions were partially inhibited, and GS activity was slightly lower in the bes1-D mutant relative to that observed in wild-type En2 roots. NH₄ ⁺-mediated AMT1 suppressions are known to be caused by N-metabolites rather than NH₄ ⁺ itself, and glutamine application inhibited AMT1 expression in both En2 and bes1-D indicating that BES1 activation inhibited NH₄ ⁺-mediated GS/GOGAT induction, which might in turn inhibit AMT1 repression. In conclusion, the present study demonstrates that BR regulated nitrogen uptake and assimilation via the BR signaling pathway.

The role of peroxisomes in the oxidative injury induced by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in leaves of pea (Pisum sativum L.) plants was studied. Applications of (2,4-D) on leaves or to root substrate increased the superoxide radical production in leaf peroxisomes. Foliar application also increased H₂O₂ contents in leaf peroxisomes. Reactive oxygen species (ROS) overproduction was accompanied by oxidative stress, as shown by the changes in lipid peroxidation, protein carbonyls, total and protein thiols, and by the up-regulation of the activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, catalase, glucose 6-phosphate dehydrogenase and NADP⁺-dependent isocitrate dehydrogenase. Foliar or root 2,4-D applications also induced senescence symptoms in pea leaf peroxisomes, as shown by the decrease of protein content and glycolate oxidase and hydroxypyruvate reductase activities, and by the increase of endopeptidase, xanthine oxidase, isocitrate lyase and acyl-CoA oxidase activities as well as of 3-ketoacyl-CoA thiolase and thiol-protease protein contents. 2,4-D did not induce proliferation of pea leaf peroxisomes but induced senescence-like morphological changes in these organelles. Results suggest that peroxisomes might contribute to 2,4-D toxicity in pea leaves by overproducing cell-damaging ROS and by participating actively in 2,4-D-induced leaf senescence.