The effects of acclimation to cold (4 °C) and heat (36/38/40 °C) on corresponding freezing and heat tolerances of one-year-old Trigonobalanus doichangensis seedlings were studied. In addition, the effects of abscisic acid (ABA) and salicylic acid (SA) pretreatments on the tolerance of this species to temperature extremes were tested. The results show that the content of soluble sugars increased with the duration of acclimation to cold (4 °C), and the relative electrical conductivity and malondialdehyde content increased significantly after 7 d; however, the content of proline did not vary significantly. After acclimation to cold for 3 and 7 d, the semilethal low temperature (LLT₅₀) was 0.8 and 1.1 °C lower, respectively, compared with that of the control. The maximum quantum yield of photosystem II (measured as variable to maximum fluorescence ratio, F_v/F_m) decreased significantly after freezing treatments (-4 to -8 °C), however, less when the plants were pretreated with 1-100 mg dm^-3 ABA. Acclimation to heat did not increase the semilethal high temperature (LHT₅₀). A low concentration (1 mg dm^-3) of SA increased LHT₅₀, but medium and high concentrations (10 and 100 mg dm^-3) decreased it. F_v/F_m decreased significantly after a heat shock (45-54 °C). The pretreatment with 1-50 mg dm^-3 SA ameliorated a subsequent heat (48 °C) stress.

Pepper (Capsicum annum L.) fruit colours vary from green, ivory, or yellow at the juvenile stage to red, orange, and yellow at the mature stage. Carotenoid accumulation causes fruit colour formation in ripe Capsicum fruits, and capsanthin is a main carotenoid in ripe red pepper fruits. In this study, pepper cultivars with red, yellow, and orange fruits were employed and four key genes named phytoene synthase (Psy), lycopene-β-cyclase (Lcyb), β-carotene hydroxylase (Crtz), and capsanthin/capsorubin synthase (Ccs) in the capsanthin biosynthetic pathway were analyzed. Results show that a clear expression of the Ccs gene in cv. CK7 (yellow) was detected, but the expressions of the Psy and Lcyb genes were low comparing with the other tested cultivars. In addition, the expression of the Ccs gene was not observed in cv. R37-1 (orange). Furthermore, though the four key genes (Psy, Lcyb, Crtz, and Ccs) were detected in red cultivar R15, the expressions of them were low. Sequence analysis further indicated that the Ccs genes in cv. CK7 and cv. R37-1 had a high sequence identity (97.36 %). Interestingly, amino acid sequences coded by the Ccs genes from cv. CK7, cv. R37-1, and cv. R15 were the same. The above results suggest that the genes Psy, Lcyb, Crtz, and Ccs were required for capsanthin synthesis in pepper, and their varied expression patterns resulted in the variety of pepper fruit colours.

The JNK motif is repeated 4 000 times in an additional heterochromatin band. Flanking sequences of a JNK region, which form extra heterochromatin in 2R rye (Secale vavilovii Grossh.) chromosomes, were studied using a genome walking technique. The results clearly indicate that there were blocks of JNK sequences adjacent to the R173 family of repeated sequences. Moreover, it appears that the R173 are sequences flanking in both directions, i.e., upstream and downstream. Downstream, the R173 is adjacent to the JNKs in an anti-parallel orientation, whereas upstream it is adjacent in a parallel orientation. In order to confirm the presence of the R173 sequence, fluorescence in situ hybridization was carried out. Using both JNK and R173 molecular probes, overlapping hybridization signals in the 2RL pair of chromosomes were observed, indicating an identical location of the two sequence elements.

Jatropha curcas L. is a perennial, drought-resistant, and non-food oilseed crop. The fatty acid composition of seed oil, especially the ratio of 16- to 18-carbon fatty acids, has a direct impact on the biodiesel quality. In plants, fatty acid chain lengths are mainly determined by the plastidial fatty acid synthase complex which includes three β-ketoacyl-acyl carrier protein synthases (KASs), KASI, KASII, and KASIII. The KASIII is thought to play a rate-limiting role in fatty acid synthesis. Here, we report the functional characterization of a putative JcKASIII gene from Jatropha curcas using Arabidopsis thaliana L. as model system. The transcripts of JcKASIII were detected in all tissues examined and increased in seeds. Overexpression of JcKASIII in Arabidopsis led to an increased content of palmitic acid and a higher ratio of 16- to 18-carbon fatty acids. Moreover, functional analysis of JcAKSIII in kasI or kasII knock down Arabidopsis mutants revealed that the composition of seed oil changed. Taken together, these results suggest that heterologous JcKASIII could function as one of the major regulators of fatty acid composition.

In plant cells, anion channels and transporters are essential for key functions. Members of the chloride channel (CLC) family located in intracellular organelles are required for anion accumulation, pH adjustment, and salt tolerance. Here, we cloned a maize (Zea mays L.) CLC gene, named ZmCLC-d, and found that its transcription was up-regulated under cold, drought, salt, and heat stresses, and after hydrogen peroxide (H₂O₂) and abscisic acid (ABA) treatments. The overexpression of ZmCLC-d in Arabidopsis conferred tolerance to cold, drought, and salt stresses; this tolerance was primarily displayed by an increased germination rate, root length, plant survival rate, antioxidant enzyme (catalase, peroxidase, and superoxide dismutase) activities, and a reduced accumulation of Cl^- in transgenic plants as compared with wild type (WT) plants. The accumulation of H₂O₂ and superoxide anion in leaves of the ZmCLC-d-overexpressing plants is much less than that of the WT plants. The expressions of some stress related genes, such as CBF1, CBF2, CBF3, DREB2A, and RCI2A, increased to a greater extent in the ZmCLC-d-overexpressing plants than in the WT. Our results strongly suggest that ZmCLC-d played an important role in stress tolerance.

We compared photosynthetic performance between cucumber (Cucumis sativus L.) leaves acclimated to saturating irradiances with high red: far red (R:FR = 10) and normal R:FR (= 1.4) ratios. The net photosynthetic rate (P_N) and stomatal conductance (g_s) of the leaves acclimated to high R:FR were greater than those of the leaves acclimated to normal R:FR; the greater g_s partly explains the greater P_N. The greater g_s of the high-R:FR-leaves probably resulted from a higher stomatal density and/or a greater size. P_N of the high R:FR leaves was still greater than that of the normal R:FR leaves at the same intercellular CO₂ concentration (c_i). This indicates that non-stomatal factors also increased the photosynthetic capacity of the high R:FR leaves. The maximum Rubisco carboxylase activity estimated from a P_N-c_i curve analysis was also greater in the high R:FR leaves, however, the intrinsic water-use efficiency (WUE_i = P_N/g_s) of the high R:FR leaves was lower than that of the normal R:FR leaves.

Roles of an altered porphyrin biosynthesis and antioxidants in protection against chilling and heat stresses were evaluated in rice (Oryza sativa L.). When exposed to the same exposure time (6 or 30 h), heat-stressed (45 °C) plants exhibited a less oxidative stress as indicated by a lower dehydration, ion leakage, and H₂O₂ production compared to chilling-stressed (4 °C) plants. Malondialdehyde production also increased after a mild chilling stress, whereas it increased only after a long-term heat stress. The content of protoporphyrin IX, Mg-protoporphyrin IX and its methyl ester, and protochlorophyllide drastically declined under both the stresses, particularly under the long-term heat stress. Greater increases in catalase and peroxidase activities in heat-stressed plants indicate more cofactors supplied for hemoproteins compared to those of chilling-stressed and untreated control plants. Intermediates of carotenoid biosynthesis, zeaxanthin and antheraxanthin, also increased under the chilling and heat stresses. In comparison to chilling-stressed plants, heat-stressed plants were more efficient in porphyrin scavenging and antioxidant enzyme responses, which may play crucial roles in plant protection under temperature stress, thereby suffering less from oxidative stress.

The secretory (Sec) pathway is one of the most important systems for transporting proteins across the thylakoid membrane into the lumen. Two Arabidopsis genes encoding SecY translocon proteins, designated SCY1 and SCY2, were characterized in this study. Semi-quantitative RT-PCR and histochemical staining β-glucuronidase (GUS) activity reveal that both SCY1 and SCY2 promoters were active in germinating seeds, etiolated cotyledons, and flowers, but not in roots. In particular, the expression of GUS gene driven by the SCY1 promoter was almost undetectable in green leaves, whereas GUS staining controlled by the SCY2 promoter was clearly detected. Moreover, homozygous scy1-1 plants could grow heterotrophically but appeared sensitive to radiation. Further studies show that chloroplasts of scy1-1 were arrested in early developmental stages with fewer thylakoid membranes. Real-time quantitative RT-PCR reveals that a number of nuclear-encoded genes involved in chlorophyll biosynthesis and photosynthesis were substantially down-regulated in the scy1-1 mutant. All these results indicate that the SCY1/2 genes were regulated developmentally and spatially, and a loss-of-function mutation in SCY1 triggered chloroplast-to-nucleus retrograde signaling in Arabidopsis thaliana.

The aim of this study was to investigate the effects of salinity on germination, seedling growth, free polyamines (putrescine, spermidine, and spermine), and ethylene metabolism of two species (Atriplex prostrata Bouchér and Plantago coronopus L.) with different salt sensitivities. Seeds collected from Barranco Hondo (salt marshes, Jaén, southern Spain) were germinated at 0, 50, 100, and 200 mM NaCl in a growth chamber. The germination of P. coronopus seeds decreased considerably with an increasing NaCl concentration, however, seeds of A. prostrata showed high germination percentages (84, 87, and 80 %) at 0 (control), 50, and 100 mM NaCl, respectively, and only at 200 mM NaCl, the germination was reduced to 25 %. In the early phase of vegetative growth (8-d-old seedlings), the fresh mass increased in A. prostrata at 50 and 100 mM NaCl but the fresh mass of P. coronopus showed no significant differences. With respect to polyamines, there was a decrease of the putrescine and spermidine content at all the NaCl treatments, however, the spermine content increased and was much higher in P. coronopus than in A. prostrata. The ethylene, 1-aminocyclopropane-1-carboxylic acid content, and the 1-amino-cyclopropane-1-carboxylic acid synthase activity increased with the increasing NaCl concentration in A. prostrata, and only the ethylene content in P. coronopus. These results indicate that P. coronopus increased the free spermine content, whereas A. prostrata increased the ethylene biosynthetic pathway in order to survive in the saline conditions.

The role of chilling in bud dormancy release and biochemical changes in different organs were evaluated in stem cuttings of pear (Pyrus pyrifolia) cv. Cuiguan selected at the leaf fall. The cuttings were exposed to 5 °C for 0, 100, 200, 300, 400, 500, 600, and 700 chilling hours (named positive chill units; PCU). A 50 % bud break was observed in floral and vegetative bud cuttings at 300 and 600 PCU, respectively. A mean time to bud break was inversely proportional to the chilling treatment. The low-temperature stimulated starch hydrolysis accompanied with sucrose accumulation in all organs. Sucrose and sorbitol content increased substantially peaking at 100, 400, and 100 PCU in floral buds, vegetative buds, and bark, respectively, thereafter decreased when buds approached chilling satisfaction (300 and 600 PCU for the floral and vegetative buds, respectively), and then increased again up to 700 PCU. Hexoses (glucose and fructose) accumulated constantly in the buds from 0 to 700 PCU. In bark, glucose and fructose content increased up to 400 PCU, and then gradually decreased. Total amylolytic and α-amylase activities increased in all organs, especially in the floral and vegetative buds up to 100 PCU and then decreased in the floral and vegetative buds before increasing again after endo-dormancy release. Invertase activity remained high in the buds during chilling satisfaction possibly because of translocation of sucrose to the buds which functioned as a strong sink. The results suggest that a low availability of hexoses may be the cause of limited bud breaks due to lack of chilling. Chilling satisfaction of the buds may increase the content of soluble sugars and acid invertase activity, and decrease the starch content, which may correlate with improved bud breaks.

The objective of the current study was to develop an efficient and reproducible protocol for plant regeneration using nodal (1.0-1.5 cm) explants excised from a field grown mature plant of Cassia occidentalis L. The highest shoot regeneration frequency (80 %) with a maximum number of shoots (11.66) and shoot length (3.83 cm) after eight weeks of culture were observed on a Murashige and Skoog (MS) medium amended with 5.0 μM 6-benzyladenine, 100 μM citric acid, and 1.0 μM α-naphthalene acetic acid. A half-strength MS medium supplemented with 1.5 μM indole-3-butyric acid proved best for the induction of maximum roots (8.33) per shoot. Plantlets with well-developed shoots and roots were successfully acclimatized in plastic pots containing sterile Soilrite under two irradiances of 50 and 300 μmol m^-2 s^-1 (LI and HI, respectively) in a culture room, and after transfer to the field, the survival rate was 70 %. A significant increase in chlorophyll, carotenoid, and malondialdehyde content was found during acclimatization under both the irradiances but higher under HI. Similarly, the activities of superoxide dismutase, catalase, glutathione reductase, and ascorbate peroxidase increased more under HI. Plantlets acclimatized under HI exhibited a better growth than those under LI.

Hydroponics experiments were conducted to study the effects of sodium nitroprusside (SNP, a donor of NO) on lead toxicity in ryegrass (Lolium perenne L.) seedlings. When the ryegrass seedlings were grown in a nutrient solution containing 500 μM Pb²⁺ for two weeks, the plant biomass as well as net photosynthetic rate, transpiration rate, chlorophyll and carotenoid content of leaves decreased. The Pb stress also induced the production of superoxide anion (O₂ ^.-) and hydrogen peroxide (H₂O₂), leading to malondialdehyde (MDA) accumulation. Furthermore, the activities of superoxide dismutase (SOD), peroxidase (POD), and ascorbate peroxidase (APX) decreased in the Pb-treated seedlings, but the catalase (CAT) activity increased. Additionally, the content of Cu in shoots and the content of K, Mg, Fe, and Zn in both shoots and roots decreased, but the content of Ca in shoots and roots increased under the Pb stress. Moreover, Pb accumulated mostly in roots, whereas a small quantity was translocated to shoots. However, the addition of 50, 100, and 200 μM SNP into the solution containing Pb increased the chlorophyll content and net photosynthetic rate, reduced Pb-induced oxidative damages, improved antioxidant enzyme activities, and inhibited translocation of Pb from roots to shoots. In particular, 100 μM SNP had the best effect on promoting growth of the ryegrass seedlings under the Pb toxicity. However, the application of 400 μM SNP had no obvious alleviating effect on Pb toxicity in the ryegrass seedlings.

Pogostemon cablin (Blanco) Benth is a medicinal plant rich in terpenoids and flavonoids. Currently, the molecular mechanism of flavonoid biosynthesis in P. cablin remains unclear. In this study, three copies of the phenylalanine ammonia lyase (PAL) gene designated as PcPAL1 - PcPAL3, one copy of the chalcone synthase (CHS) gene referred to as PcCHS, and four copies of the CHS-like gene referred to as PcCHSL1 - PcCHSL4 were isolated from P. cablin. PcPAL1, PcPAL2, and PcPAL3 comprised 2 136, 2 136, and 2 148 bp full-length open reading frames (ORFs) encoding 711, 711, and 715 amino acids, respectively. PcCHS, PcCHSL1, PcCHSL2, PcCHSL3, and PcCHSL4 contained 1 173, 1 176, 1 179, 1 173, and 1 170 bp ORFs encoding 390, 391, 392, 390, and 389 amino acids, respectively. A phylogenetic analysis indicates that PcPAL2 was closer to PcPAL3 than PcPAL1, and that PcCHS was clustered with other plant CHS genes but independently to the PcCHSLs subgroup. A sequence analysis shows that PcCHSLs diverged in functional residues when compared to plant CHSs including PcCHS, suggesting that PcCHSLs may be divergent in tertiary structures. A quantitative reverse-transcription polymerase chain reaction analysis indicates that the expression profiles of PcPALs, PcCHS, and PcCHSLs in leaves and stems differed among four cultivars. Our results suggest that divergence in sequence and expression profiles of the tested genes may contribute to the diversity in flavonoids among the four cultivars.

We previously reported that LeMYB1 might be a crucial transcription factor in regulating shikonin formation in Lithospermum erythrorhizon. In this study, by overexpressing LeMYB1 under the control of CaMV35S promoter in L. erythrorhizon hairy roots, we further clarified the role of LeMYB1 in the shikonin formation and its regulation. The LeMYB1-overexpressing transgenic hairy roots were successfully induced by infecting seedling nodes with Agrobacterium rhizogenes strain ATCC15834 that carried the pBI121-LeMYB1 vector. The LeMYB1 transcripts were significantly up-regulated in the transgenic hairy root lines compared with the wild type lines, and the total content of shikonin and its derivatives was dramatically enhanced by the LeMYB1 overexpression. Real-time PCR results reveal that the enhanced shikonin biosynthesis in the overexpressing lines were mainly caused by a highly up-regulated expression of genes coding key enzymes (PAL, HMGR, and PGT) and key regulators (LeDI-2 and LePS-2) involved in the shikonin biosynthesis. Overall, our results suggest that LeMYB1 plays a positive role in regulating the shikonin biosynthesis in L. erythrorhizon.

The effect of a rare earth element europium (Eu) on the long-distance transport of a plant defence compound quercetin (Q) was investigated. The complex Q/Eu³⁺ was synthesized in a HEPES buffer and tested for its transport ability through Arabidopsis thaliana and its ability to interact with target molecules in plant cells. Our results show that complexation with Eu³⁺ enhanced the transport of Q through Arabidopsis roots. During the transport, the complex degraded and released a free Q to tissues where Q was originally not available. Thus, the plant became better supplied with the defensive compound Q. The obtained spectrophotometric data imply that one of the reasons for the Q/Eu³⁺ degradation was the interaction of the complex with double stranded RNAs (dsRNAs) present in Arabidopsis. Since dsRNAs are replicative forms of plant RNA viruses, the ability of Q/Eu³⁺ to release a free Q in their presence suggests further investigation of this complex as a potential antiviral agent.

Whereas microsatellite markers are well described, there are few studies investigating minisatellites. Therefore, this study aimed to identify, characterize, and validate minisatellite loci for papaya (Carica papaya L.). The entire papaya genome, which covers 330 Mb, was used to mine minisatellites with motifs ranging from 6 to 500 bp, and at least six replicates and 82 loci were validated in a set of 24 accessions. A total of 1 730 minisatellite loci were identified, located in 695 sequences, with an average of one minisatellite every 156 kb. Variation in GC content ranged from 0.00 to 83.84 % with an average of 28.84 % indicating that these papaya minisatellites are AT rich. Motifs of up to 20 bases represented 71.45 % of the markers. In addition, the observed variation in the number of motif repeats was from 6 to 186 with an average of 9.27 per minisatellite. Independent of the classification, the frequency of minisatellites decreased with an increase in the number of repeating units. Among the validated loci, 48.78 % were found to be polymorphic, and the number of alleles (N_A) ranged from two to seven with a mean of 3.10. The average polymorphic information content (PIC), expected heterozygosity (He), and observed heterozygosity (Ho) were 0.38, 0.43, and 0.11, respectively. According to genetic diversity parameters, such as NA, He, and PIC, no significant correlations were found among the size of motifs, the number of repeats, and the GC content of these minisatellites. This study clearly demonstrates the polymorphism of minisatellites and their potential use in detection of intraspecific genetic variations in C. papaya. The results will be useful when managing genetic resources and plant breeding.

A wheat stay-green mutant, named tasg1, was generated using the mutagen ethyl methane sulphonate applied to wheat (Triticum aestivum L.) cv. HS2. A drought stress was imposed by controlling irrigation and sheltering plants from rain. The antioxidant defence was characterized in the flag leaves of the tasg1 and wild-type (WT). Compared with WT, tasg1 had higher reduced ascorbate/oxidized ascorbate ratio, reduced glutathione/oxidized glutathione ratio, and antioxidant enzyme activities during senescence under both normal and drought stress conditions. The DHAR gene expression remained higher in tasg1 than in WT during the drought stress and tasg1 had a higher antioxidant defence competence which may contribute towards the delayed leaf senescence. The different transcriptional responses of some wheat senescence-associated genes to the drought stress between tasg1 and WT were observed. These results suggest that the competent antioxidative capacity might play an important role in the enhanced drought tolerance in tasg1.

Natural accessions of Arabidopsis thaliana exhibit a clinal variation in freezing tolerance following temperature changes across the natural habitat. Here we performed molecular evolution and population genetic analyses of homologous INDUCER OF CBF EXPRESSION1 (ICE1) and ICE2 genes, the master regulators of plant cold response. A study of ICE genes polymorphism was performed using 60 A. thaliana ecotypes grouped according to their geographic origin. The genetic diversity of ICE2 was characterized by a high number of haplotypes and an overall high diversity. The levels of nonsynonymous nucleotide polymorphism increased from a northern group southward. On the contrary, the ICE1 gene sequence was less diverse and there was no clinal variation in the sequence polymorphism. Thus, different selection forces acting on the ICE2 gene might be one of the reasons of clinal variation in freezing tolerance. This clinal variation also indicates that ICE2 is more important for a cold response than ICE1. The study of the ratio of numbers of nonsynonymous to synonymous substitutions (K_a/K_s) between A. thaliana paralogs shows that the sequence diversification and emergence of two new ICE2-specific motifs could contribute to the functional diversification of the duplicates. The K_a/K_s for ICE2 of A. thaliana and A. lyrata orthologs was an order of magnitude greater than that for the ICE1 orthologs, which suggests that the protein sequence of ICE2, an early duplicate, evolved under a weaker selective constraint. A relaxed selection on ICE2 in southern populations and more stringent in northern populations also confirmed its role in a cold resistance. The selection pressure on ICE1 might be caused by its role in the control of more essential than cold response functions.

Understanding the basis of the genetic variations responsible for the complex traits found in Eucalyptus cladocalyx under arid environmental conditions is crucial for designing genetic architecture studies. Forty-five half-sib families from Australia were used to identify inter-simple sequence repeat (ISSR) markers that are associated with growth (height, diameter at breast height, and stem straightness), flowering traits (flowering intensity, flowering precocity, reproductive capacity, and late flowering) and tree survival under arid conditions in southern Atacama Desert, Chile. Each DNA pellet consisted of a pool of five trees from each family. ISSR markers were associated with all the traits studied and accounted for 9.8 to 23.4 % of the phenotypic variation. Several loci were associated with more than one trait. For example, UBC_{810(450-500 bp)}, ISO_{1(600-610 bp)}, and TGT_{9(780-800 bp)} were associated with three of the traits studied. These identified genomic regions may contribute to the increase of the efficiency of the conventional tree breeding program for E. cladocalyx.

The present work examined seven Hypericum species (H. perforatum, H. humifusum, H. kalmianum, H. annulatum, H. tomentosum, H. pulchrum, and H. rumeliacum) produced in vitro and regenerated after cryopreservation. The aim of the study was to assess, by means of light microscopy (LM) and transmission electron microscopy (TEM), the effect of freezing temperature on leaf histological organization and mesophyll chloroplast ultrastructure. Histological analysis showed a negative effect of ultralow temperatures on leaf tissue structure in H. pulchrum and a positive effect in H. perforatum. The TEM analysis showed that chloroplasts from ultralow temperature treated H. annulatum, H. tomentosum, and H. rumeliacum had a typically structured internal membrane system without destruction of thylakoid membranes, however, those of H. humifusum had very high grana, and in H. perforatum, chloroplast thylakoid destruction occurred. The chloroplast internal membrane system of in vitro cultured control plants and in vitro cultured cryopreserved plants of H. kalmianum and H. pulchrum had a specific spatial orientation without any destruction of the membranes. The results show a specific response of each species to these experimental conditions.

Chloride channels (CLCs) play pivotal roles in plant development and anion transport. However, little research has been conducted about the CLC in fruit-bearing plants. Here we provide an insight into the evolution and expression patterns of CLC gene family members in various tissues of trifoliate orange [Poncirus trifoliata (L.) Raf.] and their responses to several treatments. Genome-wide analysis identified six PtrCLC genes. The predicted proteins had similar numbers of amino acids, but shared a low sequence identity. Phylogenetic analysis revealed that PtrCLC were classified into two separate subgroups, and PtrCLC4 and PtrCLC6 in subgroup II were more closely related to bacterial CLCs. Sequence comparison with EcCLCA from Escherichia coli reveals that PtrCLC showed amino acid divergence in anion selectivity of CLC proteins. Real time qPCR analysis shows that PtrCLC genes, particularly PtrCLC6, preferentially expressed in leaves. Nitrogen deficiency irreversibly inhibited expression of PtrCLC genes except for PtrCLC1. In contrast, NaCl stress profoundly induced expression of PtrCLC genes, particularly PtrCLC2 and PtrCLC4, both of which were also upregulated by ABA treatment. The results presented here provide a solid foundation for a future functional research on citrus CLC genes.

The aim of this work was to elucidate the molecular and biochemical mechanisms that control L-ascorbic acid (AsA) content variation in Myrciaria dubia. The AsA was quantified by high-performance liquid chromatography, gene expression by real-time quantitative PCR, and enzyme activities by spectrophotometric methods from leaves and immature fruits of two genotypes (Md-60,06 and Md-02,04) with pronounced (about 2 times) differences in the AsA content. In either genotype, the fruit peel had ∼ 1.5 times more AsA than the fruit pulp and ∼ 15.0 times more than the leaf. All tissues examined demonstrated the capability for AsA biosynthesis through the D-mannose/L-galactose pathway because mRNAs of the six key genes [GDP-D-mannose pyrophosphorylase (GMP), GDP-D-mannose-3',5'-epimerase (GME), GDP-L-galactose phosphorylase (GGP), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH), and L-galactono-1-4-lactone dehydrogenase (GLDH)] and catalytic activities of the corresponding enzymes (GMP, GDH, and GLDH) were detected. The differential expressions of genes and enzyme activities mostly correlated with the respective AsA content. Thus, the expression of several genes of the D-mannose/L-galactose pathway determined the AsA content variation in tissues of M. dubia.

A new member of the APETALA2/ethylene responsive element binding protein (AP2/EREBP) transcription factor family, HhDREB2, was isolated from Halimodendron halodendron. Based on the similarity of the AP2/ERF domain, HhDREB2 was classified into A-5 group of the DREB subfamily. The expression of HhDREB2 gene was induced by drought, high salinity, and low temperature, but not by exogenous plant hormones. Trans-activity assay demonstrated that HhDREB2 gene encodes a transcription activator. Furthermore, over-expression of HhDREB2 gene under the stress-inducible rd29A promotor in Arabidopsis resulted in enhanced tolerance to salt and drought stresses. The overall results reveal that HhDREB2 functioned as important transcription factor in regulation of stress-responsive signaling in plants and may be used for improving plant tolerance to abiotic stresses.

An essential step in the carotenoid biosynthesis pathway is the formation of phytoene by phytoene synthase (PSY). In this study, three new genes coding PSYs (CpPSYA, CpPSYB, and CpPSYC) were cloned from Cucurbita pepo and their expression patterns analysed in three cultivars of summer squash which had a different carotenoid content. The gene sequences had a high similarity with those from other plant species, and their predicted proteins were significantly different from each other. A phylogenetic analysis indicates that CpPSYA and CpPSYB shared a high homology and were also homologous with PSYs from others cucurbits, whereas CpPSYC was more closely related to orthologues from strawberry and carrot. An expression analysis revealed that CpPSYA had a higher expression in flowers compared to leaves and showed a differential expression during fruit development. The amount of CpPSYA transcript was higher in fruits with a higher carotenoid content than in those with a lower carotenoid content. However, CpPSYB and CpPSYC showed a relatively high expression in leaves, and their expression in fruits varied among the different cultivars and fruit tissues. These results suggest that the CpPSY genes were under different regulatory mechanisms and they may have different roles in C. pepo.

The effects of foliar application of 24-epibrassinolide (EBR) on chlorophyll content, oxidative damage, and antioxidant capacity in eggplant (Solanum melongena L.) seedlings during a short-term chilling stress and subsequent recovery were investigated. The eggplant seedlings pretreated with 0.1 μM EBR were exposed to 5 °C for 12 h in the dark and then recovered under a normal temperature for another 12 h. The results show that EBR alleviated a chilling-induced oxidative stress reflected by a decrease in malondialdehyde and H₂O₂ content, O₂ ^.- production rate, and an increase in chlorophyll content and activities of superoxide dismutase, guaicol peroxidase, and catalase. The EBR pretreatment also promoted activities of ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, as well as the accumulation of ascorbic acid (AsA) and glutathione (GSH) involved in the AsA-GSH cycle. Moreover, after the12 h recovery, EBR plus chilling treated leaves showed no significant changes in content of MDA and chlorophyll a, lower reactive oxygen species (ROS) content, and greater activities of AsA-GSH cycle enzymes than in seedlings only chilled. The results suggest that EBR alleviated an oxidative damage caused by the dark chilling stress and accelerated a recovery rate mainly through increasing the ROS scavenging system including the AsA-GSH cycle.

The effects of AT3-gene silencing on the expression of genes involved in capsaicinoid biosynthesis was investigated in chili pepper (Capsicum annuum L.) cv. Tampiqueño 74 fruits. Seeds were germinated and seedlings were grown in a greenhouse until they produced fruits. Capsaicinoids (capsaicin and dihydrocapsaicin) content and AT3 gene expression were determined in placenta tissue from fruits at 10, 20, 30, 40, 50, and 60 days post-anthesis (DPA). Capsaicin was more abundant than dihydrocapsaicin and both exhibited a similar accumulation pattern at different developmental stages starting at 20 DPA, reaching maximum values at 30-40 DPA before decreasing. The AT3 gene expression, as measured by quantitative RT-PCR, was positively correlated with capsaicinoid accumulation; AT3 transcripts were detected at 20 DPA, achieved a maximum at 30-40 DPA and then decreased. The Tampiqueño 74 seedlings were infected with Agrobacterium tumefaciens bearing a pTRV2-AT3 construct to induce virus-mediated silencing. Fruits were harvested at 40 DPA, and capsaicinoid content and AT3 gene expression were carried out in placenta tissue. A reduction of 81.1 % in AT3 expression and also in capsaicin (89.6 %) and dihydrocapsaicin (87.7 %) content was recorded in the AT3-gene silenced chili pepper plants. Furthermore, fruits from the AT3-silenced plants compared to the non-infected control plants showed a statistically significant reduction in the expression of genes involved in capsaicinoid biosynthesis [pAmt (89.4 %), BCAT (68.8 %), Kas (90.4 %) and Acl (58.6 %)]. These data indicate that AT3 silencing had a negative effect on the transcription of genes involved mainly in the branched-chain fatty acid pathway of capsaicinoid biosynthesis.

An excess of aluminum (Al) is a major factor limiting crop production in acidic soils. Secretion of organic acids (OAs) from the root apex of diverse plant species or genotypes via activation of anion channels has been recognized as the most important mechanism of Al exclusion. Citric, oxalic, and malic acids are the most effective OAs in detoxifying Al. In this review, we summarize biochemical properties of OAs secreted by plants. We also highlight the molecular mechanisms of Al signal perception, Al transport, signal regulators associated with OAs secretion, as well as interactions between Al and hormone signaling pathways. Based on a comprehensive understanding of the relationship between signal modulators and regulation of expression of relevant genes, a signal transduction model for Al-induced OAs secretion is proposed.

Roles for nitric oxide (NO) and reactive oxygen species (ROS) during pollen-tube growth have been well established in angiosperms, but there remains lack of information regarding their potential signalling roles in pollen tubes in gymnosperms. Here, the pollen-tube elongation of Arizona cypress (Cupressus arizonica Greene) was investigated. Nitric oxide, ROS, and actin were detected using their respective fluorescent probes. Both NO and ROS were observed in the nuclei of generative cells and pollen-tube cells, and in the cytoplasm in the tip region. An intracellular NO content in the pollen cells was lowered using an NO scavenger or an NO-synthase inhibitor. Similarly, an endogenous ROS content in the pollen cells was lowered using an NAD(P)H oxidase inhibitor. These treatments reduced pollen germination and pollen-tube growth, and induced severe morphological abnormalities. Inhibition of NO and ROS accumulation also severely disrupted the actin cytoskeleton in the pollen tubes. These data indicate that NO and ROS had signalling roles in pollen germination and pollen-tube formation in cypress.

The present study exemplifies morphogenic roles played by copper and zinc during micropropagation of Rauvolfia serpentina, an important medicinal shrub. Incorporation of 20 μM CuSO₄ or 25 μM ZnSO₄ to a Murashige and Skoog (MS) medium with optimized concentrations of auxins and cytokinins induced a maximum number of shoots per explant (40.67 ± 1.76 and 45.47 ± 0.24, respectively). However, higher concentrations of both the micronutrients negatively affected the morphogenic potential. The pigment content of the regenerants increased up to the optimal concentrations of both metals and thereafter decreased, whereas the maximum proline content was at the highest concentrations used. In vitro rooting of healthy shoots was accomplished using 0.5 μM IBA in a half strength liquid MS medium with 8.20 ± 0.37 roots, and root length of 5.50 ± 0.14 cm per microshoot. The plants survived a hardening procedure and were successfully acclimatized to field conditions with 95 % survival.

The interactive effects of salicylic acid (SA) and nitric oxide (NO) on alleviating cadmium (Cd) toxicity in peanut (Arachis hypogaea L.) were studied. Seedlings of two cultivars (Huayu 22 - a big seed type, and Xiaobaisha - a small seed type) were treated with 200 μM CdCl₂ without or with 0.1 mM SA or 0.25 mM sodium nitroprusside (SNP, an NO donor). Results show that the Cd exposure depressed the plant growth of both the cultivars but more of Huayu 22 than of Xiaobaisha. Exogenous SA and NO alleviated Cd toxicity in both the peanut cultivars: they improved growth, chlorophyll content, photosynthesis, and mineral nutrition. Furthermore, exogenous SA or NO decreased oxidative stress by increasing activities of antioxidant enzymes and content of non-enzymatic antioxidants. Besides, in roots and leaves of both the cultivars, exogenous SA and NO increased Cd accumulation in the cell wall and decreased Cd distribution to organelles. In particular, the effect of SA+SNP was most obvious.