Plant WRKY genes encode a complex and ancient class of zinc-finger transcription factors that are involved in multiple biological processes, especially in regulating defense against abiotic stresses. Despite a growing number of studies on the genomic organization of the WRKY gene family in various species, little information is available about this family in hemp (Cannabis sativa L.). In this study, based on the hemp genome sequence, 40 hemp WRKY (CsWRKY) genes were classified into three main groups and five subgroups according to their orthologs in Arabidopsis. Among these, 23, 15, and 14 CsWRKY genes were responsive to drought, NaCl, and Cd stress, respectively. Interestingly, the expressions of all of the 23 drought stress-responsive genes were up-regulated. Moreover, 18 CsWRKY genes were induced by abscisic acid (ABA) treatment. A total of six up-regulated genes related to all three stresses were identified. Among these, five were up-regulated, and one was down-regulated by ABA. These results indicate a diverse function of the CsWRKY genes, which provides a basis for future clarification of their function in hemp tolerance to abiotic stresses.

Vetiver grass [Vetiveria zizanioides (L.) Nash] displays comprehensive abiotic stress tolerance closely related to fine maintenance of plant water relation mediated by plasma membrane intrinsic proteins (PIPs). Two open reading frame sequences of PIPs (867 and 873 bp) were cloned from vetiver grass and named as VzPIP1;1 and VzPIP2;1, respectively. Expression of green fluorescent protein revealed only subcellular localization of VzPIP2;1 in the plasma membrane. Agrobacterium tumefaciens mediated transgenic (VzPIP2;1) soybean plants had a higher water content in above-ground parts under sufficient water supply through enhancing transpiration as compared to the non-transgenic plants but displayed a more severe drought injury because of a lower photosynthesis and a higher transpiration rate. However, A. rhizogenes mediated transgenic soybean plants kept a higher water content in above-ground parts by improving root water transport and kept a more effective photosynthesis under normal and drought conditions.

Polyamines play important roles in plant responses to various environmental stresses. In this study, changes in free polyamine content and transcription of genes participating in polyamine metabolism were investigated in sweet orange (Citrus sinensis Osbeck cv. Jincheng) seedlings under drought (DR), high-temperature (HT), and combined DR and HT (DRHT) stresses. Content of putrescine (Put) and spermidine (Spd) was the highest and that of spermine (Spm) was the lowest under the DR stress, whereas Put and Spd content was the lowest, and Spm content was the highest under the HT stress. Content of the three polyamines under the DRHT stress ranged between the values detected under the DR and HT at most time points. The fluctuation of Put content during the three stresses was irregular; significant amounts of Spd under the DRHT and Spm under all the three stresses were accumulated. Expression of genes encoding arginine decarboxylase, Spd synthase, and Spm synthase was significantly induced under the DR but not under the HT. The relief from all the stresses did not cause an expected decrease or increase in polyamine content and expression of related genes.

In the present study, three Arabidopsis thaliana pop2 mutant lines with different T-DNA insertions in a gene coding γ-aminobutyric acid transaminase (GABA-TA) were screened for seed germination percentage, stress-induced oxidative damage, and GABA content and metabolism under various abiotic stresses including high temperature (42 °C), low temperature (4 °C), salinity (NaCl), and osmotic stress (mannitol). All mutant lines showed a decreased germination under all the stress treatments with a significant reduction in the pop2-1 and pop2-3 mutant lines. Content of GABA and MDA increased significantly in all pop2 mutants and wild type (WT) seedlings in response to all the treatments. However, content of GABA and MDA was lower in all pop2 mutants comparing to the WT under the same treatments. GABA increased already after 30 min and increased significantly after 2 h at 42 °C especially in the pop2-3 and WT seedlings. In response to the cold treatment, GABA content increased up to 4-fold compared to the control in all pop2 mutants and WT seedlings. In response to the NaCl treatment, GABA accumulated slightly in the WT and all pop2 mutants. On the contrary, GABA content increased significantly in the pop2, pop2-1, and pop2-3 mutants and WT under all mannitol treatments.

As result of a close evolutionary relationship between Triticeae B, G, and S genomes, the exchange of genetic material between them is possible and may be beneficial for broadening the genetic diversity of cultivated bread wheat. However, the extent to which regulatory networks are conserved remains poorly researched. Here, the structural organization and transcriptional activity of the B, S, and G genome copies of a gene encoding flavonoid biosynthesis enzyme chalcone-flavanone isomerase (CHI) were explored using introgression lines which differ from the wild type by carrying a non-bread wheat Chi-1 gene. Chi-S1, Chi-G1, and Chi-B1 all mapped to a comparable region of chromosomes 5S, 5G, and 5B, respectively. Nucleotide sequences of Aegilops speltoides Chi-S1 and Triticum timopheevii Chi-G1 were determined and compared with T. aestivum Chi-B1 sequences. The enzymes encoded by these three genes shared the same predicted tertiary structure and active sites. However, the replacement of Chi-B1 by Chi-S1 or Chi-G1 in a wheat background resulted in a significant decrease in the global amount of the Chi-1 transcript present in the seedling shoot indicating divergence in regulation of expression of the orthologous Chi-1 genes among Triticeae ssp.

One of the largest families of transcriptional regulators contains WRKY proteins. They play important roles in plant defense responses. In this study, a novel WRKY gene, CsWRKY2, was isolated from the tea [Camellia sinensis (L.) O. Kuntze] plant. The full-length cDNA of CsWRKY2 was 2 050 bp in length and encoded a 522-amino acid peptide chain containing two typical WRKY domains and two zinc finger motifs, suggesting that CsWRKY2 was member of the WRKY group I family. A subcellular localization assay shows that CsWRKY2 was localized to the nucleus. Real time qPCR analysis shows that CsWRKY2 expression was higher in leaves than in other organs and was induced by cold (4 °C), drought stress, and exogenous abscisic acid (ABA). Additionally, ABA content was enhanced after the cold or drought stress and the effects were relieved by an ABA biosynthesis inhibitor. Furthermore, the expression of CsWRKY2 was up-regulated by exogenous ABA under the cold and drought stresses and down-regulated by an ABA biosynthesis inhibitor. Our findings indicate that CsWRKY2 played an important role in plant defense responses to the cold and drought stresses by participating in the ABA signaling pathway, downstream to ABA.

Nitric oxide (NO) and 5-aminolevulinic acid (5ALA) play fundamental roles in plant responses to environmental stresses, but their cross-talk in antioxidant defense in cold-stressed Elymus nutans Griseb. have not been investigated. We herein report that 5ALA and NO donor, sodium nitroprusside (SNP), alleviated cold stress-induced plant growth inhibition and lipid peroxidation in roots of two E. nutans ecotypes (Damxung, DX and Zhengdao, ZD). However, application of an NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (PTIO) differentially blocked these protective effects indicating that an inhibition of NO accumulation reduced 5ALA-enhanced cold resistance. Application of exogenous 5ALA or NO markedly up-regulated activities of superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase, enhanced reduced glutathione accumulation and reduced glutathione to oxidized glutathione ratio, activated plasma membrane (PM) H⁺-ATPase, and reduced Na⁺/K⁺ ratio in roots of the two E. nutans ecotypes. Moreover, in the presence of 5ALA, nitric oxide synthase (NOS) activity and NO release in cold-resistant DX were higher than those in cold-sensitive ZD. Conversely, both NO treatment and inhibition of endogenous NO accumulation by PTIO or NOS inhibitor N^ω-nitro-L-arginine did not induce 5ALA production. These results suggest that NO might be acting as a downstream signal to mediate 5ALA-induced cold resistance by activating antioxidant defense and PM H⁺-ATPase and maintaining Na⁺ and K⁺ homeostasis.

Screening salt-sensitive mutants is a powerful method to identify genes associated with salt tolerance. We used forward genetic screening with sodium azide-mutated rice (Oryza sativa L. cv. Tainung 67) to identify mutants showing hypersensitivity to salt stress. A new mutant line, named salt hypersensitive 1 (shs1) and exhibiting a severe salt-sensitivity when grown under a high NaCl concentration, was identified; the salt hypersensitivity was caused by duplicate recessive epistasis with mutations likely in two different loci. The shs1 salt sensitive phenotypes included a decreased seed germination rate, reduced shoot height and root length, severe and quick wilting, and overaccumulation of sodium ions in shoots as compared with wild-type plants. In addition, shs1 showed a decreased photosynthetic efficiency and enhanced hydrogen peroxide (H₂O₂) production under the salt stress. An increased superoxide dismutase activity and decreased catalase activity were responsible for the hyperaccumulation of H₂O₂ in shs1. The hypersensitivity of shs1 to the salt stress might be caused by an impaired antioxidant machinery and cellular Na⁺ homeostasis.

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

The amount and composition of phytohormones, sugars, and some other leaf characteristics depending on a crop load were evaluated in apple (Malus domestica Borkh. cv. Ligol grafted on P 60 rootstock) trees in order to prevent biennial bearing. The crop load was adjusted to 12 (control, unthinned), 8, 4, and 0 (non-fruiting) inflorescences (or fruits) per cm² of trunk cross-sectional area (TCSA). Inflorescences were removed in May before flowering. Phytohormones were analyzed in axillary buds and leaves in September. Results show that, in contrast to the unthinned trees, thinning to 4 fruits cm^-2(TCSA) resulted in a significant decrease of yield per tree, but a significant increase of fruit mass, return bloom, and leaf area. The heavy crop load resulted in suppressed bloom in the following year. Composition and content of phytohormones was changed considerably. Moreover, thinning resulted in an increased hexose accumulation. Such data suggest that flowering inhibition depended on the phytohormones that were exported to buds and on sugar-hormone signalling cross-talk.

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

Jasmonate ZIM domain (JAZ) proteins are key regulators of the jasmonic acid (JA) signaling pathway. Repressors of JAZ remain bound to the myelocytomatosis 2 (MYC2) or MYC3/MYC4 transcription factors in the absence of JA and negatively regulate transcription of the JA responsive genes. In the presence of JA, JAZ proteins interact with coronative insensitive 1 (COI1), the recognition molecule of E3 ubiquitin ligase SCF^COI1 (COI1 stabilized by Skp, cullin, F-box containing complex), get ubiquitinated, and subsequently degraded by the 26S proteasome. However, there is a dearth of knowledge about this gene family in monocot cereals, specifically its role in finger millet is unknown till date. Here we present the isolation and characterization of a novel JAZ family repressor gene from nonsequenced Eleusine coracana (EcJAZ) utilizing available genome information of Oryza, Sorghum, and Setaria. The EcJAZ sequence showed the presence of a conserved ZIM domain, the Jas motif, and N-terminal motif 7 like other Group1 TIFY sequence containing proteins. We observed coronatine (an analog of JA-Ile) dependent and time dependent degradation of recombinant EcJAZ that thereby fulfilled the basic characteristic of the JAZ proteins. We found a proteasome inhibitor N-(phenylmethoxy) carbonyl-L-leucyl-N-[(1R)-1-formyl-3-methylbutyl]-L-leucinamide) (MG132) mediated degradation inhibition of EcJAZ that supported its 26S proteasome mediated degradation. Our study shows the nuclear localization of GFP-EcJAZ by Agrobacterium mediated transient transformation of onion scale epidermal cells. In Eleusine leaves, transcription of EcJAZ increased 4.2-fold by salt stress and 5.5-fold by coronatine application; thus ascertained its inducibility by the abiotic stress as well as by bioactive JA-Ile. Taken together, all these results contribute to our understanding of the JA signaling pathway in Eleusine coracana.

Floral initiation in apple (Malus domestica Borkh) was studied by a novel morphogenetic approach. Developmental stages of apices were evaluated based on the morphology of shoot apical meristem (SAM) from various collection dates. Besides, the frequency of each stage was calculated within apices populations after full blooming (DAFB). Prior to doming of apex, three marked phases were found based on SAM morphology: 1) narrow appearance (vegetative phase), 2) broadened form (transition phase), and 3) prominent shape (commitment phase). A furrow region was formed at the base of leaf primordium during the bract initiation, while significant broadening of SAM was observed. Cell division patterns manifested in modification of anisotropic clusters from isotropic cellular packets, as a result of which profound morphological changes of apices occurred. Based on these findings, we propose that the structural alterations prior to doming may be taken into account for determination of the initial development and reproduction signs in apple trees.

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.

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.

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.

Temperature and mineral nutrition are major environmental factors regulating plant growth and development. Yet, cold impact on mineral contents and the ability of the plants to perform changes in specific elements as a part of the acclimation process received little attention. Using five Coffea genotypes previously characterized concerning their cold sensitivity, a mineral analysis was performed considering macro (N, P, K, Ca, Mg, and S) and micro (Na, Fe, Mn, Zn, Cu, and B) nutrients in order to predict their importance in cold tolerance. The results showed a cold-induced dynamics of mineral nutrients in recently mature leaves. The less cold sensitive Icatu, and partially Catuaí, accumulated N, Ca, Mn, Cu, and Zn with potential implications in the maintenance of photosynthetic performance, the reinforcement of the antioxidative defense system, lipid metabolism, and the expression of cold regulated genes, thus constituting interesting traits to evaluate the cold acclimation ability. After a principal component analysis (PCA), N, Fe, Mn, and Cu were further confirmed as strong candidates for an early cold tolerance evaluation due to their dynamics and to specific roles in the activities of Cu/Zn-SOD (Cu), APX (Fe), and PSII (Mn).

Selenium (Se) is essential for health of humans, animals, and plants. Especially wheat is a major source of Se in the terrestrial food chain. In this study, an element analysis was optimized and the content of Ca, Mg, K, S, P, Fe, Se, Mn, Cu, Zn, and Mo in leaves, roots, and seeds were measured during growth of wheat (Triticum aestivum L. cv. Manu) in Hoagland nutrient solution with 5 and 15 μM Na₂SeO₄. Se was transported to all investigated tissues and accumulated in the seeds in proportion to used amounts. The supplementation of Se, independently of concentration, weakly modified the micro- and macro-elements content in the seedlings. In the flag-leaf stage, an increase of the Mo and S content in the shoots and the S and Cu content in the roots was found. Moreover, in the generative phase, a decrease in Ca and Fe in the roots was registered. Increased Se in the nutrient solution strongly stimulated the Se accumulation in the seeds.

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.

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.