Ascorbic acid (AsA) is naturally occurring compound with antioxidant activity and plays a pivotal role in plant cell adaptation to salinity stress. The objective of this work was to assess the influence of exogenous AsA on the embryogenic callus of indica rice (Oryza sativa L.) cv. MRQ74 cultivated under saline conditions. NaCl (200 mM) decreased callus fresh and dry masses, relative growth rate, and K⁺ and Ca⁺² content, and increased Na⁺ content and Na⁺/K⁺ ratio. Application of AsA (0.5 or 1 mM) alleviated these effects of salinity. Activities of peroxidase, catalase, superoxide dismutase, as well as content of proline increased due to the NaCl treatment, and these parameters were mostly further increased by 0.5 mM AsA. Thus, AsA can increase callus tolerance to NaCl stress.

Na⁺/H⁺ exchanger (NHX)-mediated Na⁺ and H⁺ antiport is an important mechanism for salt tolerance in plants. In this study, an Na⁺/H⁺ antiporter gene, referred to as NsNHX1, was isolated from the halophyte Nitraria sibirica Pall. using degenerate polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE). The resulting 2 182 bp NsNHX1 cDNA contained a 1 635 bp open reading frame (ORF) that encoded 544 amino acids and showed striking sequence similarity to tonoplast-localized NHXs from other plants. Subcellular localization analysis confirmed NsNHX1 to be a tonoplast-localized protein. Cis-elements described as being responsive to biotic and abiotic stresses were present in the NsNHX1 promoter region, and reverse transcription (RT)-PCR analysis confirmed that NsNHX1 expression was induced by exogenous abscisic acid (ABA), cold, and NaCl. Transcription of NsNHX1 increased sharply 3 h after treatment with 200 mM NaCl revealing that NsNHX1 responded rapidly to the salt stress. Overexpression of NsNHX1 enhanced salt tolerance in transgenic Arabidopsis thalliana L. suggesting that NsNHX1-mediated Na⁺ compartmentalization played an important role in enhancing plant salt tolerance.

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.

The nuclear factor Y (NF-Y) is one of the largest transcription factor families in plants consisting of NF-YA, NF-YB, and NF-YC subunits. It could play important roles in various processes such as flowering time, seed development, and response to drought. In this study, 6 NF-YA, 13 NF-YB, and 8 NF-YC proteins were identified and characterized in Prunus mume. Analyses of a conserved domain indicated that the PmNF-Y subunits shared an elevated degree of homology with the corresponding Arabidopsis NF-Y ones. Phylogenetic analysis showed that each NF-Y subunit family from Prunus mume and Arabidopsis could be divided into 4 or 2 clades based on their full-length proteins. The gene expression patterns of all 27 PmNF-Y genes were examined under abscisic acid (ABA), osmotic, salt, and H₂O₂ treatments using real-time quantitative PCR analyses. PmNF-YA1/2/4/5/6, PmNF-YB3/4/8/10/11/13, and PmNF-YC1/2/4/5/6/8 were found to be up-regulated under the ABA and osmotic treatments. PmNF-YA1/2/3/4/5/6, PmNF-YB1/3/8/10/11/13, and PmNF-YC1/2/5/6/8 were obviously induced by the H₂O₂. In addition, only PmNF-YA2 and PmNF-YB3 expressions were enhanced under the salt stress. These findings could provide an entry point to investigating the roles of PmNF-Y genes during abiotic stress responses.

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.

This study aimed to investigate sucrose-metabolizing enzymes and their genes in fruits of two longan (Dimocarpus longan Lour) cultivars Cihezhong (CHZ) and Lidongben (LDB). Content of sucrose, glucose, and fructose were measured by high-performance liquid chromatography. The genes of sucrose-metabolizing enzymes were cloned by combining reverse transcription polymerase chain reaction and rapid amplification of cDNA ends, and enzyme activities were analyzed at various points in the fruiting cycle. The total soluble solid (TSS) content of longan arils rose and was positively correlated with sucrose content during maturation and then declined as the fruit senesced. Cihezhong showed a more rapid decrease in sucrose content than LDB. The activities of both sucrose phosphate synthase (SPS) and sucrose synthase (SS) were lower in CHZ, whereas the activities of soluble acid invertase (SAI) and neutral invertase (NI) were higher. The full-length cDNA of the genes of the sucrose-metabolizing enzymes were cloned successfully. The patterns of changes of sucrose synthase-2 (DlSS-2), sucrose synthase-3 (DlSS-3), and neutral invertase-3 (DlNI-3) gene expressions corresponded to those of SS and NI activities. The rate of sucrose decline in the longan fruits was related to sugar receding, sucrose metabolizing enzyme activities, and corresponding gene expressions.

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.

Plant microRNAs (miRNAs) play important roles in regulating plant growth, development, and responses to abiotic stresses. In this study, 38 miRNAs (TaMIRs) from wheat (Triticum aestivum L.), 36 from the miRBase database, and two from our previous work were characterized and subjected to an expression pattern analysis under normal conditions and a drought stress. A semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), real-time quantitative PCR (qPCR), and small RNA blot analyses revealed that two TaMIRs (TaMIR1120 and TaMIR1123) were root-predominant and two TaMIRs (TaMIR1121 and TaMIR1134) were leaf-predominant. Seven TaMIR precursors showed altered expressions after the drought; of these, TaMIR1136 was upregulated, whereas TaMIR156, TaMIR408, TaMIR1119, TaMIR1129, TaMIR1133, and TaMIR1139 were downregulated. These seven drought-responsive TaMIRs showed dose-dependent and typical temporal expression patterns during drought induction, and they gradually returned back under the normal growth conditions. The drought-responsive and the tissue-predominant TaMIRs had varying numbers of target genes. Randomly selected target genes exhibited opposite expression patterns to their corresponding TaMIRs suggesting that they were regulated by distinct TaMIRs through a post-transcriptional cleavage. The target genes regulated by drought-responsive and tissue-predominant TaMIRs are involved in various cellular processes, such as signal transduction, transcriptional regulation, primary and secondary metabolisms, development, and defense responses. These results provide a novel insight into the miRNA-mediated responses of wheat to drought stress.

The effects of short-term osmotic stress [a polyethylene glycol (PEG) treatment] on photosystem II (PS II) of upland and lowland rice seedlings were investigated using chlorophyll (Chl) fluorescence imaging. Spatial heterogeneity in the top fully expanded leaf for all Chl fluorescence parameters was found under stress conditions. After exposure to PEG, a decrease in the effective quantum yield of PS II photochemistry (φPS II) and photochemical quenching (qP), and an increase in non-photochemical quenching (NPQ) proceeded from the upper section to the base of the leaf. The most sensitive position in the leaf was different between the two ecotypes. Chl fluorescence parameters, net photosynthetic rates (P_N), and stomatal conductance (g_s) were more sensitive to the PEG stress in upland rice than in lowland rice. These results also indicate different leaf anatomy and development in the two rice ecotypes. Additionally, the findings suggest a more rapid stress response in upland rice.

In this study, the anthocyanin content and real-time quantitative expression of the anthocyanin biosynthesis-related genes were investigated in leaves, stems, and tubers of purple yam (Dioscorea alata L.). The anthocyanin content, its accumulation, and the expression of genes encoding phenylalanine ammonia lyase (PAL), flavanone-3-hydroxylase (F3H), anthocyanidin synthase (ANS), and UDP-glycose flavonoid glycosyl transferase (UFGT) were studied. The anthocyanin content in the leaves and stems was high at early stages of growth, but it decreased and remained at a similar level from the 35^th day onward. The anthocyanin content in the tubers firstly increased, reached a high peak at the 110^th day of growth, after which decreased. Anthocyanin accumulation rates and the expressions of the anthocyanin biosynthesis genes were high at the early stages of growth in the leaves and stems, but in tubers, two peaks were observed: at days 80 and 140 for the gene expression and at days 125 and 170 for the anthocyanin accumulation rate. Thus, there was coordination between the gene expressions and the anthocyanin accumulation rates in the various organs as well as in the entire plants.

Germin and germin-like proteins (GLPs) are a broad and diverse family of developmentally regulated proteins widely distributed in plants. Oryza sativa L. harbours a large family of GLPs and serves as a good model for their study. In the present study, a germin-like protein gene (OsRGLP1) of rice origin was characterized by its heterologous expression in tobacco. The real-time PCR established almost a uniform expression of OsRGLP1 in leaves, stem, and roots of T₁ Nicotiana tabacum cv. Samsun. Although no morphological difference was apparent between T₀ transgenic and wild-type plants, leaves of mature transgenic plants showed necrotic lesions associated with an elevated content of H₂O₂, which was evidenced by in situ 3,3'-diaminobenzidine staining. A significantly higher activity of heat resistant superoxide dismutase (SOD) was observed in the transgenic plants as compared to the wild-type. The SOD activity in the transgenic plants was insensitive to potassium cyanide and sensitive to H₂O₂.

Phospholipase D (PLD) and its product phosphatidic acid are now considered to be one of the key elements of numerous physiological processes in plants including the salicylic acid signalling pathway. The presented study investigates the transcriptional regulation of Brassica napus PLDs following treatments with defense-related stimuli. We cloned eight B. napus genes encoding members of PLDβ, γ, and δ isoforms and performed phylogenetic analysis with its ancestor species Brassica rapa and Brassica oleracea, and with the model plant Arabidopsis thaliana. Transcription of the identified genes was monitored after treatment with benzothiadiazole (BTH), methyl jasmonate (MeJA), bacterial elicitor flg22, wounding, and after infection with fungal pathogens Sclerotinia sclerotiorum and Leptosphaeria maculans. Most of the genes responded specifically to a particular treatment. Remarkably the genes encoding the PLDγ and PLDβ isoforms were up-regulated by stimuli associated with the salicylic acid signalling pathway. The generality of this finding was confirmed by the analysis of public transcriptional data from Arabidopsis thaliana.

Analysis of structural changes of octoploid triticale genomes was conducted in F₂ and F₃ generations. The plants were derived from crosses of five cultivars and breeding lines of hexaploid wheat (Triticum aestivum L.) with one cultivar of rye (Secale cereale L). The study used four marker systems: inter-simple sequence repeat (ISSR), inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP), and a technique named inter-transposon amplified polymorphism (ITAP) developed by the authors. Most frequently, elimination of specific bands was observed, especially of rye bands. Depending on the cross combination, the percentage of eliminated rye bands ranged from 73.6 to 80.6 %. A lower percentage of wheat bands was eliminated, i.e., from 57.6 to 76.48 %, depending on the combination of crosses. The emergence of new types of bands in hybrids absent in the parental forms was the rarest phenomenon (14.5-17.9 %). The results indicate the ongoing process of genome rearrangements at the molecular level in the early generations of plant crosses that also involve repeated nucleotide sequences of DNA.

To investigate whether exogenous sucrose can protect cucumber from water stress, cucumber (Cucumis sativus L.) seedlings were pretreated with 90 mM sucrose or 90 mM mannitol for 1 d and then were dehydrated with 10 % (m/v) polyethylene glycol (PEG) 6000 for further 1 d. Dehydration inhibited plant growth and decreased osmotic potential and relative water content (RWC) in leaves. The pretreatment with 90 mM sucrose further reduced the osmotic potential but increased the RWC and alleviated the growth inhibition. Compared with the PEG treatment alone, the combination of sucrose + PEG increased the activities of superoxide dismutase, guaiacol peroxidase, glutathione reductase, dehydroascorbate reductase, monodehydroascorbate reductase, ascorbate peroxidase, and glutathione peroxidase, and elevated the content of endogenous sucrose, glucose, and fructose together with the activities of soluble acid invertase and neutral invertase. This was in accordance with the enhanced transcription of genes encoding copper/zinc superoxide dismutase, guaiacol peroxidase, and glutathione reductase. Furthermore, the sucrose pretreatment decreased the content of malondialdehyde and hydrogen peroxide and increased the content of ascorbate, reduced glutathione, and proline under the dehydration. Taken together, the pretreatment with 90 mM sucrose, but much less with mannitol, induced antioxidants, proline, and soluble sugars and thus reduced dehydration-caused damage to the cucumber seedlings.

Barley (Hordeum vulgare L.), an important food and fodder crop, is potentially tolerant to salinity. To identify quantitative trait loci (QTLs) controlling salt tolerance, the population of 162 recombinant inbred lines (RILs) derived from F8 generation of Arigashar (an extremely salt tolerant Iranian six-rowed barley landrace) crossed with Igri (a salt semi-sensitive two-rowed cultivar) were evaluated. The growth of shoots, roots, and coleoptiles, and root numbers are four important growth characteristics severely affected by salt stress at seedling growth stages. A linkage map was constructed using 106 AFLP and SSR markers spanning six barley chromosomes including 2(2H), 3(3H), 4(4H), 7(5H), 6(6H), and 1(7H). Out of totally 26 detected QTLs, 17 QTLs were found effective for salt tolerance at 250 and 350 mM NaCl which localized on chromosomes 2H, 3H, 4H, 6H, 7H, and linkage group L1, whereas considering equivalent overlapped QTLs with a pleiotropic effect led to detection of totally 9 distinctive QTLs (QClgH2.1b, QSdgH2.1b, QSlgH2.1c, QNrgH2.1b, QTwgH2.2c, QSdg3Hb, QSlg4Hb1, QClg4Hb, and QSlg6Hc2) effective for salinity tolerance. 2(2H), 4(4H), and 6(6H) were major chromosomes harboring QTLs which effectively controlled salt tolerance in the Igri×Arigashar population. An interesting QTL, QTwg4Hc, was localized on chromosome 4H in the XE41-M61 marker distance that controls several traits including shoot and coleoptile lengths and shoot fresh mass under salt stress. A dense marker cluster around a resistance gene could offer a starting point for positional cloning.

Protein synthesis is a ubiquitous and essential process in all organisms, including plants. It is primarily regulated at translation initiation stage which is mediated through a number of translation initiation factors (eIFs). It is now becoming more apparent that in addition to synthesis of proteins, eIFs also regulate various aspects of plant development and their interaction with environment. Translation initiation factors, such as eIF3, eIF4A, eIF4E, eIF4G, and eIF5A affect different processes during vegetative and reproductive growth like embryogenesis, xylogenesis, flowering, sporogenesis, pollen germination, etc. On the contrary, eIF1A, eIF2, eIF4, and eIF5A are associated with interaction of plants with different abiotic stresses, such as high temperature, salinity, oxidative stress, etc. Similarly, eIF4E and eIF4G have roles in interaction with many viruses. Therefore, the translation initiation factors are important candidates for improving plant performance and adaptation. A large number of genes encoding eIFs can functionally be validated and utilized through genetic engineering approaches for better adaptability and performance of plants by inhibiting/minimizing or increasing expression of desired eIF(s).

The effects of a short (30 min) heat shock (HS) on plants subsequently grown under a salinity stress (SS, 200 mM NaCl) for 10 d were investigated in barley (Hordeum vulgare L.) cv. Tokak 157/37. The maximum temperature for HS allowing plant survival was 45 °C. The root length was significantly decreased by SS, whereas HS alone did not affect root growth. Interestingly, HS stimulated root elongation under SS. An osmotic adjustment was promoted in leaves by SS. On the contrary, HS increased the osmotic potential in leaves in the absence of SS, and partly counteracted the effect of SS in the HS+SS treatment. Cu/Zn-SOD, HvAPX, HvCAT2, HSP17, HSP18, and HSP90 were transcribed in leaves of HS-treated plants, but not in control plants. The HSP70 was constitutively transcribed in both the SS and control plants, but after HS, a shorter amplicon was also observed. The genes coding antioxidants, Cu/Zn-SOD, HvCAT2 and HvAPX, were differentially influenced by SS or HS+SS in the roots and leaves. In the roots, the mRNA content of BAS1, HvDRF1, HvMT2, and HvNHX1 increased after the HS treatment. In a recovery experiment in which plants were grown to maturity after HS and HS+SS stress exposure, the plant height increased and the time to maturity was reduced in comparison with SS. Our results show that HS could stimulate plant growth and reduce some of the negative effects of SS, and that it affected the transcription of several stress-related genes.

Cytosine methylation (C meth) is a ubiquitous regulator of gene transcription. The maize gene ZmGRP3 is a notable marker for root initiation, but how its root-specific transcription is regulated is not understood. Here, bisulfite sequencing and a C meth sensitive Southern blot assay were used to show that the transcription of ZmGRP3 was promoted by a reduction in the extent of C meth both in its promoter and in the vicinity of its translation start site. The result was validated by revealing the effect of 5-aza-2-deoxycytidine on the C meth status in various seedling organs. An analysis of methylation patterns indicates that the C meth of cytosine-guanine dinucleotides was the most important determinant of transcription, however, the C meth of CHG or CHH trinucleotides had little influence.

Two barley (Hordeum vulgare L.) cultivars, Czech spring cv. Amulet and Syrian landrace Tadmor, were subject to different salinity treatments: 1) the NaCl concentration was gradually increased from 0 (the control) to either 100 (a moderate salt stress) or 300 mM NaCl (a high salt stress), 2) the NaCl concentration was increased directly either from 0 to 300 mM NaCl or from 100 to 300 mM NaCl, and 3) a recovery when all variants were transferred back to control conditions and cultivated for seven additional days before sampling. The following parameters were determined: water saturation deficit (WSD), osmotic potential (ψ_s), leaf proline content, maximum quantum yield of photosystem (PS) II photochemistry (measured as variable to maximum chlorophyll a fluorescence ratio, F_v/F_m), and relative accumulation of dehydrins (DHN). Both quantitative and qualitative differences in dehydrins were found between NaCl-treated Amulet and Tadmor. A principal component analysis (PCA) of all experiment data revealed a differential ability of Amulet and Tadmor to recover after the 300 mM NaCl treatments indicating better salt tolerance in Tadmor. Correlation analyses have shown statistically significant correlations between WSD, ψ_s, proline, and DHN.

Lipase class 3 is part of the triacylglycerol lipase family involved in lipid degradation, esterification, and transesterification processes in plants. In this study, a lipase class 3 gene and promoter from oil palm (Elaeis guineensis Jacq.) were isolated and characterized by Northern blot, Southern blot, oil palm genome sequence, and transient expression GUS assay. The full-length lipase class 3 (FLL1) deduced polypeptide encoded 483 amino acids and was identical to that deduced from lipase (EgLip1) cDNA (GI: 409994625). It contained the lipase consensus sequence, GxSxG motif, and a putative catalytic triad and had a 3-dimensional protein model similar to that of a lipase from Giberella zeae with a 50 % identity. The Northern blot and reverse transcription polymerase chain reaction (RT-PCR) show that FLL1 was predominantly expressed in the mesocarp and the expression increased as fruits reached maturity. A lower expression was detected in germinated seedlings and especially in roots. The expression of FLL1 was also enhanced in the mesocarp of cold treated fruits. A high oil accumulation in the mesocarp during fruit development makes this tissue a suitable target for a genetic modification, hence the isolation of the FLL1 promoter. The transient expression of the β-glucuronidase (GUS) gene driven by the FLL1 promoter detected the GUS expression in mesocarp slices, especially in vascular bundles. This suggests the potential role of using the promoter as tool to direct the expression of a transgene to the mesocarp of transgenic oil palm.

The growth of the Cd-hyperaccumulator Solanum nigrum L. and its physiological responses to a short-term (7 d) Cd stress and to exogenous methyl jasmonate (MeJA) were investigated. Compared with the leaves of S. nigrum, the roots were more liable to Cd and showed a significantly decreased dry mass and increased malondialdehyde content. Cd accumulation in the shoots and roots of S. nigrum were proportional to the Cd concentration in the hydroponic solution. The application of a low concentration of MeJA (0.01 μM) significantly reduced the translocation/accumulation of Cd in both the shoots and roots compared with a 40 mg dm^-3 Cd treatment only. Moreover, 40 mg dm^-3 Cd significantly decreased the activity of leaf superoxide dismutase, but 0.01 μM MeJA restored it. MeJA also enhanced the activity of catalase in the leaves but showed no significant effect on peroxidase activity. The content of both endogenous jasmonic acid (JA) and MeJA in the leaves of S. nigrum increased with the increase of exogenous MeJA concentration.

The GhMYB9 encodes a R2R3 MYB transcription factor in the upland cotton (Gossypium hirsutum L.) genome. Our studies show that GhMYB9 predominantly expressed in flowers and fibers. To gain a better understanding of its regulatory mechanism, we isolated the 5'-flanking region of GhMYB9 which was 1 487 bp in length. The cis-acting element prediction shows that this region contained the basic structure of the core promoter elements (TATA-box, CAAT-box) and the transcription start site (TSS). Other motifs, such as defense and stress responsiveness (TC-rich repeats), anaerobic induction (ARE), and MYB binding sites involved in drought-inducibility (MBS), were also found. Histochemical assay shows that the GhMYB9 promoter governed β-glucuronidase (GUS) expression mainly in seeds, fibers, and flowers of transgenic cotton. Also, the activity of the promoter was induced by auxin in fibers of transgenic cotton. This is consistent with its transcript abundance in different tissues. A further deletion analysis confirms that a promoter region from -1 231 to -860 was required for auxin response. Our findings provide a useful reference for the understanding of the transcriptional regulation mechanism of the GhMYB9 gene.

Actinidia kolomikta (Rupr. & Maxim.) Maxim. leaves showed dramatic colour changes during plant growth phases, and we studied structure and optical properties of variegated leaves. Leaf surface cells were smooth, and there were no surface appendages (wax or trichomes) observed in variegated leaves. Palisade tissue cells in white and pink leaves were looser and contained relatively fewer chloroplasts. White leaves contained many intercellular spaces between the epidermal and mesophyll cells or within the palisade cell layer. Variegated leaves had three distinct radiation reflection patterns: a bright white area, a spotted pattern, and a polygonal pattern. Reflectance at 450-1100 nm from the adaxial surface of white leaves was greater than that of green leaves, but anthocyanin accumulation in pink leaves decreased the reflectance at 500-600 nm. When variegated leaves turned green, the reflectance at 500-600 nm increased. On abaxial surfaces, the reflectance of variegated leaves was similar to green leaves at 450-700 nm. In conclusion, reflection patterns and the formation of variegated leaves of A. kolomikta were significantly correlated with the leaf anatomy. The white and pink colours of leaves were a result of an internal reflection between air spaces and cells in the leaves, chlorophyll deficiency in palisade tissue, and anthocyanin accumulation. Variegated leaves turned green when the chlorophyll content in palisade tissue increased.

A photoperiod-sensitive soybean [Glycine max (L.) Merr] cv. ZhongDou 24 (ZD24) exhibiting delayed flowering when grown under long-days (LD, a 16-h photoperiod) was used to identify the genetic control of flowering delay. A differential expression profiling technique enabled identification of a gene fragment that was up-regulated under LD. This fragment was homologous to a gene encoding methionine synthase (MS) in soybean and was named GmMS. The RNA content confirmed that GmMS was expressed in roots, stems, and leaves of soybean grown under LD. The highest expression was in stems. Full length GmMS, encoding 763 amino acids, was transferred into tobacco plants. The ectopic expression of GmMS in tobacco resulted in delayed flowering. Other effects included stunting, an increased MS activity and methionine content, a higher content of alcohol-soluble proteins and of chlorophylls, and a lower content of anthocyanins.

Extracellular ATP (eATP) has been considered as signalling compound to mediate several physiological processes. Here we show that eATP played a role in alleviating the salicylic acid (SA)-induced inhibition of cell viability and respiration in tobacco (Nicotiana tabacum L. cv. Bright Yellow-2) suspension cells. Exogenous SA at higher concentrations (0.05, 0.1, 0.3, 0.5, or 0.7 mM) caused a significant reduction in respiratory O₂ uptake or cell viability. The addition of exogenous ATP alleviated the SA-induced reductions in cell viability and respiration, but the effect was dependent on the concentrations of either exogenous SA or ATP. Further study shows that the alleviative effects of exogenous ATP were abolished by the addition of GdCl₃ (an inhibitor of Ca²⁺ channels) or EGTA (a Ca²⁺ chelator). These results suggest that this role of extracellular ATP could be related to a Ca²⁺ signalling pathway.

In this study, proteome and activities of glutathione (GSH)-related enzymes were investigated in detached leaves of Arabidopsis thaliana treated with ochratoxin A (OTA) alone or supplemented with buthionine sulfoximine (BSO, a specific inhibitor of the first step in GSH biosynthesis). A comparative proteomic study using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS) identified 12 differentially expressed proteins mainly involved in GSH metabolism, energy metabolism, sugar metabolism, and photosynthesis. The treatment with OTA significantly enhanced the activities of glutathione-S-transferase (GST) and glutathione reductase (GR) through up-regulating the corresponding genes (GSTF7, GR1), an the diminishing effect of BSO on them counteracted the results. However, both OTA and BSO decreased the activity of ascorbate peroxidase (APX), and OTA also decreased the monodehydroascorbate reductase (MDHAR) and glutathione peroxidase (GPX) activities. Briefly, the OTA-induced phytotoxicity to the A. thaliana detached leaves was increased slightly by addition of BSO, and the fluctuation in GSH synthesis, GSH metabolism and disorder of cellular metabolism happened.

Coleus forskohlii (syn. Plectranthus barbatus) is a widely used medicinal plant and its main bioactive constituents are diterpenes forskolin and isoforskolin. The present study aimed to construct a cDNA library to identify expressed sequence tags related to terpene biosynthesis in C. forskohlii. We constructed a high quality normalized full-length cDNA library which reached the requirements (abundance, integrity, and library content) for isolating full-length genes. A total of 4 224 cDNA clones were sequenced and 2 394 unigenes were assembled with an average unigene size of 753 bp. A total of 2 100 (87.7 %) unigenes were functionally classified using gene ontologies, and 1 716 (71.7 %) unigenes were assigned to establish pathway associations in KEGG mapings. Notably, 64 unigenes putatively participated in the biosynthesis of secondary metabolites, in which 17 unigenes were identified that might be involved in the biosynthesis of the terpenoid backbone and monoterpenes, diterpenes, and triterpenes.

The effects of copper oxide nanoparticles (CuONPs) on germinating seedlings of green pea (Pisum sativum L.) were studied. The seedlings were grown in a half-strength Murashige and Skoog semisolid medium containing 0, 50, 100, 200, 400, and 500 mg dm^-3 CuONPs for 14 d under controlled growth chamber conditions. Exposures to 100, 200, 400, and 500 mg dm^-3 CuONPs significantly reduced plant growth (shoot and root lengths) and increased reactive oxygen species (ROS) generation and lipid peroxidation. Gene expression study using real-time polymerase chain reaction showed no significant change in the expression of genes coding CuZn-superoxide dismutase (CuZnSOD), catalase (CAT), and ascorbate peroxidase (APX) in shoots. However in roots, a significant increase in the expression of the CuZnSOD gene was observed under the exposures to 100, 200, 400, and 500 mg dm^-3 CuONPs, in the expression of the CAT gene under 100 and 200 mg dm^-3 CuONPs, and in the expression of APX under 200 and 400 mg dm^-3 CuONPs.

Tospoviruses are devastating plant viruses causing severe economic losses in a diverse range of crops worldwide. Here, we describe the development and evaluation of an RNA interference (RNAi) broad-spectrum virus resistance strategy based on a unique and short hairpin-RNA-generating construct (pNhpRNA). This construct was designed from a region of the nucleocapsid gene (N) of Tomato spotted wilt virus (TSWV) that showed a high sequence identity to the corresponding region in the related species Groundnut ringspot virus (GRSV) and Tomato chlorotic spot virus (TCSV). To test the effectiveness of the pNhpRNA construct, we developed a silencing reporter assay based on three fusion proteins in which the complete viral N gene sequence from each of the three tospoviruses was fused in frame to the green fluorescent protein (GFP) sequence. Co-agroinoculation of these constructs with pNhpRNA into leaves of Nicotiana benthamiana resulted in a strong silencing phenotype determined by GFP decay and suppression of the three N genes at the RNA and protein levels. To test the potential of the pNhpRNA construct to generate virus-resistant plants, we infiltrated the whole shoots of N. benthamiana with pNhpRNA. When these infiltrated plants were mechanically inoculated with the mentioned viruses 100, 70, and 60 % resistance phenotypes to TSWV, GRSV, and TCSV, respectively, were observed. The induction of a broad tospovirus resistance with a simple construct and a minimized off-target effect are the main contributions of pNhpRNA.

In shaded wheat (Triticum aestivum L.) leaves, the suppression of blue radiation (BR) triggers senescence. This phenomenon is correlated to an increase in oxidative stress symptoms and a decrease of catalase (CAT) activity, among other traits. Previous data suggest that the radiation signal transduction pathway may involve changes in Ca²⁺ and H₂O₂ homeostasis. For better a understanding of the interaction among the spectral composition of radiation, Ca²⁺ availability, and the antioxidant metabolism in the regulation of shade-induced senescence, detached wheat leaves were placed in a growth chamber and exposed to either blue (B, high BR transmittance) and/or green (G, very low BR transmittance) Lee® filters in the absence or presence of 0.8 mM verapamil (a Ca²⁺ channels blocker), 4.0 mM EGTA (a Ca²⁺chelator), or 8.0 mM 3-amino-1,2,4-triazole (a CAT inhibitor). At defined time points, the leaf samples were analyzed for changes in chlorophyll content, specific activities of CAT, ascorbate peroxidase (APX), and guaiacol peroxidase (POX), CAT isozymes, and gene expression of CAT1, CAT2, and two senescence markers (TaSAG1 and TaSAG3). BR transmittance decreased the chlorophyll degradation rate and SAG genes expression either in leaves continuously exposed under the B filter, as well as in leaves previously exposed under the G filter. The effect of BR was associated with the maintenance of a high CAT (but not APX and POX) activity, and it was suppressed either in the presence of 3-AT or when Ca²⁺ availability was decreased. BR altered the CAT activity both at the transcriptional and at the posttranscriptional level. Nevertheless, different responses of CAT isozymes and CAT genes expression profiles to specific treatment combinations indicate that they differed in their regulatory pathways.