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.

Eragrostis curvula (Schrad) Nees (weeping lovegrass) represents important cultivated forage in semiarid regions, and the most useful cultivars are tetraploid and reproduce by pseudogamous diplosporous apomixis. We previously produced a series of genetically related E. curvula lines that provide a suitable system for the identification of gene(s) involved in diplosporous apomixis and ploidy, including a natural apomictic tetraploid (T), a diploid sexual line (D), and a tetraploid sexual plant (C). A collection of expressed sequence tags (ESTs) was generated from cDNA libraries obtained from panicles of the D, T, and C, and leaves of the T. The present study aimed to analyze the repetitive content of these four cDNA libraries and further identify and characterize transposable element (TE)-related ESTs. Repetitive sequences were identified through the interface RepeatMasker (RM) using the database Repbase Update and further classification of TEs was performed manually from the RM output. The different contribution of ESTs with identity to TEs among libraries was further evaluated, and such differences were validated through RT-qPCR. We found that the percentage of repetitive content in the leaf cDNA library was almost double than in inflorescence libraries, with retrotransposons contributing mostly in all libraries. The expression of TE-related ESTs was compared in cDNA samples extracted from D, T, and C leaves or inflorescences revealing that seven mRNAs containing MuDR-like DNA transposons, Gypsy-like, and Copia-like retrotransposons were differentially represented according to tissue, reproductive mode, or ploidy. The euploid series of Eragrostis curvula is a useful model to the study of epigenomic changes produced after changes in ploidy. The present work constitutes the first detailed report on repetitive sequences of Eragrostis curvula at the transcriptome level.

In vitro orchid micropropagation is efficient biotechnological strategy for conservation and commercial plantlet production. However, micropropagated plantlets generally need to adapt to survive severe changes in humidity, irradiance, and growing medium that accompany the transfer to ex vitro conditions. Such adaptive cellular changes would give insights into the phenotypic plasticity of the model plant Cattleya xanthina (L.) Van den Berg. Therefore, we aimed to evaluate structural changes in the leaves of C. xanthina cultivated in vitro and acclimatized ex vitro using qualitative and quantitative analyses. During acclimatization, we observed a higher accumulation of dry mass, a greater convexity of the outer surface of epidermal cells, an increased deposition of epicuticular waxes, a greater elongation of mesophyll parenchymatic cells, and finally, the presence of chloroplasts with organized thylakoids and well-developed grana. Stomatal density was not changed. Furthermore, a gradual acclimatization allows this species the best adaptation to a new environment.

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.

The roles of ethylene (ET) or salicylic acid (SA) in plant response to low temperature (LT, 5 °C) have been implicated. However, the combined effect of ET- and SA-signaling on plant growth and metabolism under LT remains to be evaluated. In this study, we comparatively analyzed the response of Arabidopsis ethylene insensitive (ein) 2-1 (an ET insensitive mutant), nonexprressor of pathogenesis relative (npr)1-1 (an SA insensitive mutant) and double mutant ein2-1/npr1-1 plants to LT. The results show that a LT of 5 °C induced plant growth retardation to a less degree in ein2-1, an intermediate degree in npr1-1, but a much larger in ein2-1/npr1-1 compared to the wild-type (WT) plants. The LT susceptibility of the ein2-1/npr1-1 plants was correlated to a lower net photosynthetic rate and proline content, and a higher content of H₂O₂ and malondialdehyde and electrolyte leakage relative to the WT plants. Lower activities of superoxide dismutase, peroxidase, and catalase, as well as a lower glutathione content and a ratio of its reduced form to its oxidized form were also observed in the double mutant plants as compared with the WT plants. However, at normal conditions (23 °C), all the tested physiological and biochemical parameters were comparable between the ein2-1/npr1-1 and WT plants, and plant growth was even better in the double mutant than in the WT plants. On the contrary, most of the above-mentioned parameters were advantageous in the ein2-1 and npr1-1 plants over the WT plants under the LT conditions. These data suggest that a parallel function or physiological redundancy of nonexpressor of pathogenesis relative 1 and ethylene insensitive 2 existed in the Arabidopsis plant response to the LT. On the other hand, an interaction between ET- and SA-signaling occurred during this process.

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.

Effects of four amino acids, L-asparagine, L-cysteine, L-citrulline, and L-glutamine in different concentrations (0, 0.5, 1, and 2 mg dm^-3) combined with 2 mg dm^-3 indole-3-butyric acid, on in vitro rooting and biochemical constituents of cherry rootstocks CAB-6P (Prunus cerasus L.) and Gisela 6 (P. canescens × P. cerasus) were investigated. In CAB-6P, root number and root fresh mass (FM) were maximum at 0.5 mg dm^-3 cysteine. All amino acids reduced root length in CAB-6P and root number as well as root FM in Gisela 6. In Gisela 6, 0.5 mg dm^-3 asparagine or 2 mg dm^-3 glutamine reduced root length. In CAB-6P, 100 % rooting was achieved in the control and with 1 and 2 mg dm^-3 cysteine or 1 mg dm^-3 citrulline. In Gisela 6, the rooting percentage was maximum (76.92 %) with 0.5 mg dm^-3 asparagine. Callus FM in CAB-6P was the greatest at 1 mg dm^-3 and in Gisela 6 at 2 mg dm^-3 citrulline. Callusing was 100 % in the majority of treatments for CAB-6P and 92.31 % for Gisela 6 with 0.5 or 2 mg dm^-3 citrulline. Cysteine, citrulline, and glutamine diminished chlorophyll content in Gisela 6 whereas in CAB-6P all four amino acids hardly affected it. Carotenoid and porphyrin content in CAB-6P was decreased due to asparagine (0.5 or 1 mg dm^-3). Porphyrin content in CAB-6P was also reduced by adding 0.5 or 1 mg dm^-3 cysteine or 2 mg dm^-3 citrulline. In Gisela 6, all amino acids decreased carotenoid and porphyrin content. In CAB-6P, all treatments except 0.5 mg dm^-3 glutamine or 2 mg dm^-3 asparagine increased leaf sucrose content. In roots, both sucrose and proline content were increased only at 1 mg dm^-3 cysteine whereas in leaves only 0.5 mg dm^-3 asparagine caused a 3-fold increase in proline content. A decrease in root proline in CAB-6P was observed due to asparagine, citrulline, or glutamine. In Gisela 6, decreased leaf sucrose and proline content was recorded at 2 mg dm^-3 cysteine. All amino acids did not alter root sugar content remarkably whereas root proline content was raised by adding 0.5 mg dm^-3 glutamine or 1 mg dm^-3 cysteine.

To investigate the antioxidative response of glutathione metabolism in Urtica dioica L. to a cadmium induced oxidative stress, activities of glutathione reductase (GR), glutathione-S-transferase (GST), and glutathione peroxidase (GSH-Px), content of reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation (LPO), and also accumulation of Fe, Zn, Mn, Cu besides Cd were determined in the roots, stems, and leaves of plants exposed to 0 (control), 0.045, and 0.09 mM CdCl₂ for 58 h. Whereas the Cd content continuously increased in all organs, the Fe, Zn, Mn, and Cu content decreased in dependence on the applied Cd concentration and incubation time. The Cd treatment resulted in increased GR and GST activities in all organs, however, GSH-Px activity was dependent on Cd concentration and plant organ. The GSH/GSSG ratio maintained above the control level in the stems at both Cd concentrations. The LPO was generally close to the control values in the roots and stems but it increased in the leaves especially at 0.09 mM Cd.

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.

Involvement of DNA polymerase (pol) enzymes in meiotic DNA repair has not been clearly understood in plants. DNA polymerase λ is involved in short patch DNA synthesis and base excision repair in both plants and animals. The presence and activity of the pol λ enzyme was studied in a protein isolated from spikelets during flower development stages of rice (Oryza sativa L.) and maize (Zea mays L.). Western blot analysis showed a 2- to 2.5-fold higher accumulation of pol λ in spikelets than in shoots. Assays of pol and in-gel activity showed the dideoxynucleotide triphosphate sensitive pol λ enzyme in spikelets of both the plants. An enhanced presence of the enzyme and its high activity suggests an active role of pol λ in meiotic recombination during microspore development.

The halophyte Thellungiella salsuginea is a new model plants due to its small genome size, short life cycle, and copious seed production. Although T. salsuginea shares a high sequence identity with its close relative Arabidopsis thaliana, it shows a greater tolerance to salinity, drought, freezing, heat, and cold. To elucidate the mechanism of abiotic stress resistance in T. salsuginea, we characterized its cytosolic Apx1 gene (TsApx1) and established A. thaliana transgenic lines overexpressing TsApx1. Under 300 mM NaCl, the content of H₂O₂, malondialdehyde, and proline were lower and the activities of superoxide dismutase, catalase, glutathione peroxidase, and ascorbate peroxidase were all higher in the transgenic plants overexpressing TsApx1 (35S:TsApx1-GFP) than in the wild-type plants. The atapx1 mutant plants of A. thaliana had a NaCl/mannitol-sensitive phenotype. The ectopic expression of TsApx1 in the atapx1 mutant effectively remedied the phenotype. These results suggest that TsApx1 plays an important role in scavenging reactive oxygen species in the cytoplasm under salinity or drought. Although TsApx1 in T. salsuginea was constantly expressed at a high level, this gene was clearly inducible. In summary, the high constitutive expression and rapid induction of TsApx1 may contribute to the tolerance to abiotic stresses in T. salsuginea.

Gamma-aminobutyric acid (GABA) is a non-protein amino acid that accumulates in a number of plant species under various environmental stresses. In this paper, the ability of applied GABA for the alleviation of NaCl stress was investigated in view of growth parameters, gas exchange, photosynthetic pigments, chlorophyll fluorescence, activities of antioxidant enzymes, malondialdehyde (MDA) content, and electrolyte conductivity (REC) in wheat seedlings. Germination rate and shoot dry mass decreased with an increasing NaCl concentration and this decrease was less pronounced when 0.5 mM GABA was applied. In the NaCl-treated seedlings, exogenous GABA partially enhanced photosynthetic capacity and antioxidant enzyme activities and decreased MDA content and REC. Therefore, GABA reduced the impact of salinity on the wheat seedlings.

Soybean is stress-sensitive crop that exhibits markedly reduced growth under flooding and drought conditions. Three S-adenosylmethionine synthetases (SAMs) proteins were identified as flooding and drought responsive proteins in soybean using a proteomic technique. To better understand the role of these SAMs proteins in soybean under flooding and drought stresses, temporal, organ, and stress specificities were examined at mRNA and enzyme activity levels. The activity of SAMs decreased in response to the flooding, however, it was not significantly changed by NaCl, cold, gibberellic acid, and calcium in soybean roots. The activity of SAMs was induced in roots and hypocotyls under drought. The mRNA expression of the S-adenosylmethionine synthetase (SAMs) family was down-regulated in root tips and roots under the flooding and the drought, and SAMs 1 and SAMs 2 were down-regulated in roots under both stresses. A gene 1-aminocyclopropane-1-carboxylate synthase was up-regulated in root tips, roots, and hypocotyls under drought, however, it was not changed in root tips and roots under the flooding. In addition, 1-aminocyclopropane-1-carboxylate oxidase was induced in root tips under flooding and drought. These results suggest that SAMs was involved in the response to the flooding and drought and it might affect ethylene biosynthesis in soybean.

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).

Activities of phenylalanine (Phe) biosynthetic enzymes chorismate mutase (CM) and arogenate dehydratase (ADT) and of phenylalanine ammonia lyase [PAL, an enzyme that directs Phe towards capsaicinoid (CAP) synthesis] were analyzed during Capsicum chinense Jacq. (habanero pepper) fruit development. A maximum CM activity coincided with a maximum CAP accumulation. However, ADT exhibited two activity peaks, one during the early phase (10 - 17 days post-anthesis, DPA) and another during the late phase (35 - 37 DPA); only the latter coincided with CAP. Interestingly, PAL activity was inversely related to CAP accumulation; lower activities coincided with a maximum CAP content. These results suggest the operation of a control mechanism that coordinated Phe synthesis and its channeling towards CAP synthesis during the course of fruit development.

Development of transgenic plants by introducing defense genes is one of the strategies to engineer disease resistance. Transgenic ASD16 rice plants harbouring rice chitinase chi11 gene, belonging to a PR-3 group of defense gene conferring sheath blight (Rhizoctonia solani Kuhn) resistance, were used in this study. Three T₂ homozygous lines (ASD16-4-1-1, 5-1-1, and 6-1-1) were identified from seven putative (T₀) transgenic lines expressing chi11 using Western blotting analysis. The inheritance of sheath blight resistance in those lines was studied over generations. The stability of chi11 expression up to T₄ generation in all the three homozygous lines was proved by Western blot and the stability of sheath blight resistance in the homozygous lines was proved up to T₄ generation using detached leaf and intact leaf sheath assays. Among the three homozygous lines tested, ASD16-4-1-1 showed consistent results in all the generations and gave a better protection against the sheath blight pathogen than the other two lines.

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.

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.

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.

Ttranscription factors WRKY play vital roles in response to biotic and abiotic stresses, and previous studies have predominantly focused on model plants and fairly limited research has been performed with tomato. In the present study, a novel pathogen-induced WRKY gene named SpWRKY6 was isolated from the late blight resistant tomato (Solanum pimpinellifolium) cultivar L3708 using in silico cloning and reverse transcription polymerase chain reaction (RT-PCR) methods. Multiple sequence alignment with other plant WRKYs indicates that SpWRKY6 contains two WRKY domains and belongs to group I WRKY transcription factors. Furthermore, some cis-acting elements associated with responses to environmental stresses were observed in the promoter region of this gene. Gene expression patterns were determined by analyzing microarray data of SpWRKY6 in tomato and of an orthologous gene from Arabidopsis thaliana using the Genevestigator tool. The results reveal a very strong biotic and abiotic stress responsive behaviour of this gene. Moreover, bioinformatics results were confirmed by real time quantitative polymerase chain reaction and show that SpWRKY6 expression was rapidly induced after infection with Phytophthora infestans and Botrytis cinerea, respectively. Expression of SpWRKY6 was up-regulated by application of various phytohormones including salicylic acid, methyl jasmonate, and abscisic acid. Likewise, the SpWRKY6 expression was induced by NaCl, drought, heat, cold, and HgCl₂ treatments.

Heat stress severely reduces rice yield and quality; however, differences between the superior, early-flowering and inferior, later-flowering spikelets of indica rice in response to high-temperature stress during grain filling remain unclear. This study investigated the effects of high temperature (HT, 33.6/20.7 °C day/night) on growth, endosperm structure, and hormone and polyamine content of superior and inferior spikelets of heat-sensitive (SG-1) and heat-tolerant (HHZ) indica cultivars. The HT decreased fertilization rate, caused earlier grain filling, and reduced duration of grain filling, thus resulting in decreased grain mass and a poor endosperm structure. In addition, soluble sugar and sucrose content increased, and starch synthesis decreased by HT at the early stage of grain filling. The HT increased polyamine [spermidine (Spd) and spermine (Spm)] and abscisic acid (ABA) content, but reduced zeatin (Z) + zeatin riboside (ZR) and indole-3-acetic acid (IAA) content in the grains. Such effects were more apparent in the inferior than superior spikelets; however, the inferior spikelets of SG-1 were more affected than those of HHZ. At the middle grain filling stage, HT produced little difference between the two cultivars. Our results suggest that the poor development of inferior spikelets of SG-1 under the HT could be attributed, at least in part, to the changed content and ratios of free polyamines [putrescine (Put), Spd, and Spm] and phytohormones (Z+ZR, IAA, and ABA) and the conversion efficiency of sucrose into starch.

Caffeine (1,3,7-trimethylxanthine) is purine alkaloid, and it is secondary metabolite produced naturally in plants. It plays a crucial role in defense and stress tolerance. A hydroponic experiment was carried out to study the morphological, biochemical, and ultrastructural effects of caffeine treatment on seedlings of tobacco (Nicotiana tabacum L. cv. Turkish). The plants were grown in a growth chamber for 14 d in Hoagland's nutrient solution supplemented with 0 (control), 25, 50, 100, 1000, and 5000 μM caffeine. Shoot heights as well as root lengths significantly decreased in the plants treated with 1000 and 5000 μM caffeine. Total protein and sugar content in leaves increased significantly in the 5000 μM caffeine-treated plants. Moreover, electron microscopic analysis shows that in plants exposed to 1000 and 5000 μM caffeine, mesophyll cells possessed enlarged chloroplasts with disrupted thylakoid membranes associated with large starch grains and plastoglobules. Scanning electron micrographs exhibit that the vast majority of stomata in the plants treated with 1000 and 5000 μM were closed. In contrast, the 25 and 50 μM caffeine-treated plants showed an enhanced growth and no signs of injury.

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.

Plant cell lines represent useful models in plant cell biology. They allow simple analysis of the effects of various factors including modulated gene expression at cellular and subcellular levels. The tobacco BY-2 cell line is a favoured model due to its high proliferation rate, capability of effective synchronization, and accessibility to transformation. A relatively high uniformity of BY-2 cultures allows morphological phenotyping and assessment of growth parameters like mitotic index, viability, or cell density. Here we review already published and newly introduced optimized guidelines to carry out reliable, reproducible and efficient characterization of BY-2 cultures from suggestions of appropriate methods to acquire primary data, proper statistical treatment, and biological interpretation. The presented experimental data demonstrate the extent of natural variability and the effect of initial cell density on various cell culture features. Supportive equations allow to estimate some derived phenotypic parameters like cell cycle duration or fresh biomass of the culture and to determine the size of data sets for reliable documentation of a certain phenotypic change. The optimized protocols and accompanying discussion of weak points of different approaches should serve as practical guide for both beginners and experienced researchers working on BY-2 cells.

Nitric oxide (NO) is an important molecule involved in the perception of stress induced by toxic compounds such as arsenic (As). The present study investigated the role of NO applied as sodium nitroprusside (SNP) in cell signalling and the ability of NO to attenuate the toxic effects of As (in the form of sodium arsenate) in water hyacinth (Eichhornia crassipes). Water hyacinth plants were collected and assigned to one of the following treatments: control; 100 μM SNP; 20 μM As; or 20 μM As + 100 μM SNP. The plants remained under these conditions for 0, 4, 12, and 24 h. After each time interval, the plants were collected and As absorption, production of reactive oxygen species (ROS), integrity of membranes, and antioxidant enzyme activities were evaluated. The plants were able to absorb and accumulate large amounts of As, even after only four hours of exposure to the pollutant. The absorption and bioaccumulation factor of As was even greater when plants were exposed to both As and SNP. The accumulation of As triggered increases in ROS production and cell membrane damage. In the presence of SNP, the tolerance index to As increased and damage was mitigated. Therefore, from the present work, it was possible to conclude that exogenous NO influenced the ability of plants to tolerate As; this finding has implications for phytoremediation in areas contaminated by As.