Soil salinity leads to a reduction in plant growth, germination, relative water content, and production of wheat plants worldwide. Chitosan showed a positive effect on plant growth and development and improved plant stress tolerance. The current study aimed to examine the effect of different chitosan concentrations on the gamma-aminobutyric acid (GABA) shunt pathway in germinating seeds of wheat (Triticum durum L.) under salt stress (25 - 200 mM NaCl). We determined the seed germination pattern, seed moisture content, GABA shunt metabolites (GABA, glutamate, and alanine), oxidative damage in terms of malondialdehyde (MDA) accumulation, and the glutamate decarboxylase (GAD) mRNA transcription. Pre-treatment of wheat seeds with chitosan improved germination by enhancing germination percentage, seedling length, and seedling fresh and dry masses under salt stress. Data showed an increase in GABA shunt and their metabolites (alanine and glutamate), MDA content, and GAD mRNA transcription, and a decrease in germination percentage, seedling length, seedling fresh and dry masses for both untreated and chitosan-treated seeds under salt stress. Our results suggest that the elevation of GABA in chitosan-treated seeds was able to maintain metabolic stability under salt stress. The MDA content increased in chitosan-treated seeds as NaCl concentration increased, however, the increase was slightly lower than the MDA content in untreated seeds which confirmed that chitosan activates GAD mRNA expression that leads to activate GABA shunt to involve in the reduction of membrane damage and activation of reactive oxygen species scavenging systems under salt stress. Consequently, this study demonstrated that chitosan significantly enhanced the accumulation of GABA and amino acids metabolism to maintain the C:N balance and improved salt tolerance in wheat seeds during seed germination.

Involving the natural host-resistance mechanisms to pathogens are essential and one of the most promising approaches in development of first-line defenses against viral plant diseases. Polysaccharides isolated from natural sources are considered the most active resistance inducers. The biological activity of polysaccharides depends on the nature and chemical structure of the constituent components of complex preparations. In this view, the objective of our study was to evaluate the biological activity of complex preparations composed of glycans, rhamnolipids, and thiosulfonates as inducers of natural plant resistance and inhibitors of tobacco mosaic virus (TMV). Complex preparations were obtained using the following components: biogenic glycolipids - rhamnolipids of the Pseudomonas sp. strain PS-17, glycans - Ganoderma adspersum glucan and Candida maltosa mannan, as well as synthetic biocides - thiosulfonates (methylthiosulfanilate). The biological activity of the preparations was investigated in the host-virus model system Nicotiana tabacum L. and TMV. It was shown that preparations at concentrations of 10 and 100 μg mL^-1 were active plant resistance inducers in N. tabacum cv. Immune 580, hypersensitive to TMV. At the same concentrations, complex preparations also reduced infectivity of TMV on Datura metel L. acting as viral infection inhibitors. The inducing activity of the complex preparations is sensitive to well-known transcription inhibitor actinomycin D (10 μg mL^-1). This fact may indicate the important role of RNA synthesis in the activation of plant virus resistance by the studied preparations.

Gibberellins (GAs) are a large family of tetracyclic diterpenoids, controlling important aspects of growth and development throughout the plant life cycle. To explore the possibility that gibberellin A₃ (GA₃) signalling induces epigenetic alteration(s), we carried out a field experiment study using Nicotiana tabacum as a model system. The GA₃ application on leaves resulted in increased plant-height, foliage density, leaf cell area, and trichome density. The plants exposed to GA₃ also exhibited: 1) increased chromatin de-condensation, 2) reduced global DNA methylation, 3) reduced DNA methyltransferases (NtDNMTs) activities accompanied by decreased amounts of NtMET1 and NtCMT3 transcripts, and 4) partial restoration of phenotype and expression of epigenetically silenced reporter transgene. Based on these observations, we propose that GA₃ application induces complex epigenetic re-programming, which may lead to distinct developmental phenotypes. These results could provide an important insight for future studies on epigenetic mechanism(s) in other important crops.

Ent-kaurene oxidase (EKO) catalyze three sequential oxidations in the early steps of gibberellin biosynthesis pathway. In this research, a cDNA sequence of InEKO1 gene in the model short-day plant Ipomoea nil was identified. Our studies revealed that inductive conditions for flowering caused an increase in the transcriptional activity of the examined gene in the cotyledons-the main organs for the perception of the photoperiodic stimulus. In contrast, in the second half of the 16 h long inductive night and after that, a decreased amount of InEKO1 mRNA in the apexes was detected. What is more, ethylene, the key inhibitor of flower induction in I. nil, elevated the InEKO1 expression exclusively in the cotyledons between 10 and 14 h of the inductive night.

Brassinosteroids (BRs) are plant hormones that were isolated for the first time in the 1970s. This group currently includes more than 70 compounds that differ in their structure and physiological activity. BRs are present in plants in a free form or in the form of conjugates. BRs are known as plant growth regulators, but they also play a role in the plant response to environmental stresses. In the case of plants that are exposed to low/high temperature, exogenous BRs can counteract growth inhibition and reduce biomass losses as well as increase plant survival. BRs show a multidirectional activity in regulating the metabolism of plants exposed to extreme temperatures. The following BRs actions can be distinguished: changes in membrane physicochemical properties, regulation of the expression of selected genes (including stress-responsive genes), as well as indirect effects on metabolism through other hormones or signalling molecules (such as hydrogen peroxide). This review summarizes the current knowledge about the effects of BRs on the physiological and biochemical processes that occur in plants during exposure to low or high temperatures.

Masson pine (Pinus massoniana Lamb.) is an important tree species of high economic value in southern China, but osmotic stress threatens its growth and development. In this study, physiological measurements and RNA-Seq analysis were used to clarify the physiological and molecular responses of P. massoniana under osmotic stress. Osmotic treatment caused cell membrane damage and reactive oxygen species (ROS) accumulation in the tree seedlings, but it also increased their antioxidant enzyme (superoxide dismutase, peroxidase, and catalase) activities and osmotic substances (soluble sugars, proline, and trehalose) content so as to adjust to osmotic stress conditions. A total of 1 789 differentially expressed genes (DEGs) were identified by transcriptome sequencing, of which 962 were up-regulated and 827 genes down-regulated. A series of stress-induced genes associated with signal transduction, ROS-scavenging, osmotic regulation, late embryogenesis abundant (LEA) protein, pentatricopeptide repeat-containing protein, and transcription factors' regulation were distinguishable. This detailed investigation of the stress-responsive genes and pathways provides new insight into molecular mechanism of abiotic stress response in P. massoniana. Further, this study's data can contribute to genetic engineering or molecular breeding efforts to enhance osmotic resistance in P. massoniana stands.

Late embryogenesis abundant (LEA) proteins are important for promoting the growth and stress tolerance of plants. They are widely involved in plant growth regulation and responses to hormones and environmental factors. However, knowledge of the functions of the LEA gene in ginseng species remains limited. In this study, a Panax ginseng LEA gene (PgLEA) expression vector was constructed, and stable transgenic Arabidopsis lines were established. The PgLEA protein was classified in the LEA-2 subgroup. Reverse-transcription quantitative PCR analysis showed that the expression of PgLEA increased under 300 mM NaCl or 10 % (m/v) polyethylene glycol treatments. Under salt and osmotic stresses, overexpression of PgLEA in transgenic Arabidopsis plants improved germination rate, root length, and survival rate compared to wild-type plants. In response to drought or salt stress, transgenic plants increased proline accumulation, decreased malonaldehyde content and ion leakage. Furthermore, the transgenic plants exhibited significantly increased activity of superoxide dismutase, peroxidase, and catalase, and reduced accumulation of hydrogen peroxide and superoxide. Moreover, overexpression of PgLEA affected the expression of genes related to salt/drought stress. Taken together, PgLEA is a positive regulator of drought and salinity stress, and positively functioned in pleiotropic effects through regulating osmotic balance, reactive oxygen species scavenging and inducing transcription of stress-related genes. PgLEA may enable ginseng plants to adapt to adverse environments. The data presented herein imply that PgLEA may be useful for breeding new stress-tolerant ginseng cultivars.

Proline, an amino acid, plays an important role in plants, and it is involved in stress resistance and development. Earlier, to study the proline role in maintaining stress resistance in plants, we obtained genetically modified transgenic lines of tobacco (Nicotiana tabacum L.) with reduced activity of proline dehydrogenase (PDH, the proline degradation gene) and increased content of proline. Transgenic tobacco plants demonstrated greater resistance to high concentrations of NaCl, drought, low temperatures, and heavy metals vs. control plants. The visual assessment showed that the leaf pubescence in transgenic plants varied noticeably. Here we apply automated analysis of the tobacco leaf folds to estimate quantitative characteristics of pubescence in genetically modified tobacco plants and the control SR1 line under non-stress conditions. Our results showed differences in the number of trichomes and their length between transgenic and control plants. The trichome number significantly increased in transgenic plants (from 1.5 to 3 times). The largest differences in the trichome numbers were observed for trichomes with lengths from 0 to 380 µm. When assessing the trichome length, the opposite was observed. In all three transgenic lines, the trichome length was significantly lower than that of the control SR1 line. The data obtained indicate the effect of proline as an important metabolome component affecting the plant phenotype. Our results demonstrate perspectives of tobacco transgenic lines as promising genetic models for studying the proline role in plant morphogenesis.

Lilium species with ornamental, edible, and medicinal values are distributed all over the world. Little is known about the responses of Lilium genotypes to waterlogging stress. Lilium hybrid 'Brindisi' was used to study the physiological responses of roots, bulbs, and leaves to 1, 2, 4, 8, and 13 d of waterlogging stress. Results showed that waterlogging stress seriously hindered the transport of nutrients from bulbs to stems and leaves. The physiological indicators could be divided into two categories. The first category was the physiological parameters indicating plant damage. The dry and fresh masses of stems and leaves, chlorophyll (Chl) a, Chl b, Chl a+b, and carotenoid content decreased, the dry and fresh masses of bulbs, malondialdehyde and H₂O₂ content increased under waterlogging stress. The other category was the physiological indicators that regulate the plant adaptability to waterlogging stress. Among them, superoxide dismutase and pyruvate decarboxylase activity changed little, proline content increased significantly, soluble sugar and protein content, and ascorbate peroxidase (APX), catalase (CAT), alcohol dehydrogenase, and lactate dehydrogenase activities increased in the early stage, and decreased in the later stage of waterlogging stress. The turning point of these physiological parameters was 4 - 8 d after waterlogging stress. Bulbs played an important role in alleviating flooding stress in the early stage of waterlogging. APX and CAT also played an important role in eliminating ROS in the early stage. This research lays foundation for the research on the mechanism of waterlogging tolerance and breeding of waterlogging-tolerant cultivars of Lilium spp.

Glandular trichomes (GTs) are one of the epidermal tissues of medicinal plants which function in the synthesis, storage, and secretion of secondary metabolites. The active ingredients of Chinese medicinal materials are mostly secondary metabolites of plants. Accordingly, it is of great research value to explore the quality of medicinal materials using the GTs of medicinal plants as the starting point. However, most of the current studies on GTs of medicinal plants are still at the simple morphological identification stage, and there are few studies on the compounds secreted by GTs and secondary metabolic processes. Here, we reviewed the literature, summarized the morphological types of medicinal plant GTs, separation and purification technology, analysis technology, and biological activities of secondary metabolites, and established a research approach to medicinal plant GTs. We hope to provide a reference for future research on GT inclusions and secondary metabolism.

A plasmid and two isocaudamer systems, namely, NotI/Bsp120I and SpeI/XbaI/NheI, were used to construct a new type of multi-gene plant transformation vector system. This system included a transformation vector containing the restriction enzyme cutting sites Bsp120I and XbaI as well as a cloning vector containing the restriction enzyme cutting sites NotI, Bsp120I, SpeI, and NheI. The open reading frame of the new target genes was connected to the transformation vector. The original restriction enzyme cutting site disappeared after connecting to the isocaudamer. The plant transformation vector p096871, which contained Bacillus thuringiensis (Bt) genes Cry1Ac and Cry3A as well as p09X6, which contained mtlD, strD, betA, nhaA, and ostAB, were constructed using this vector system. Resistant plants were obtained after tobacco was transformed by two vectors via the Agrobacterium-mediated method. Detection by PCR revealed that all exogenous genes were inserted into the genome of tobacco. Real-time fluorescence quantification PCR, reverse transcription PCR, and ELISA detections were performed on five transgenic lines transformed by two Bt genes. Cry1Ac and Cry3A were inserted into the genome with a single copy to transcribe and express Bt toxins. The proposed vector system reduced the number of operational procedures and minimized the difficulty of the experiment.

High temperatures have become a major threat that seriously affects crop growth and yield. The present work aimed to investigate the acclimation process in adjusting plant responses to high root temperatures. Tomato (Solanum lycopersicum L., cv. Micro-Tom) during the flowering time was subjected to heat treatments (day/night temperatures at the root level of 40 or 45 °C for 4 d) while control plants were maintained at 25 °C, and the heat-stress treatment effects were analysed in the tomato leaves. The results showed a reduction in the content of chlorophylls a and b as well as chlorophyll a/b ratio at both high temperatures. Further, the increase in the amount of malondialdehyde as an indicator of lipid peroxidation was greater at 45 °C. The leaf content of hydrogen peroxide was induced in tomato plants subjected to 45 °C whereas it was markedly decreased in plants maintained at 40 °C as compared to control plants. Antioxidant enzymes showed higher activity in tomatoes treated at 45 °C compared to those treated at 40 °C. Moreover, the highest amount of antioxidants such as carotenoids and ascorbate in tomato plants were found at a temperature of 45 °C. Collectively, we provide evidence that physiological and biochemical components can be altered depending on the heat level, exposure time, and developmental stage. The interaction of root and shoot under high temperatures must be further characterized in terms of understanding the challenging climate changes.

Heat shock transcription factors (Hsfs) participate in a variety of plant physiological processes including the regulation of transcription factors associated with thermotolerance. Here, a Hsf gene DcHsfA1d was identified from carnation (Dianthus caryophyllus L.). The open reading frame (ORF) of DcHsfA1d was 1 368 bp and encoded a protein of 455 amino acids with a molecular mass of 51.039 kDa and an isoelectric point of 4.94. Sequence domain prediction revealed that DcHsfA1d protein exhibited five typical functional features and motifs. The transcription of DcHsfA1d was significantly up-regulated under heat stress or ABA treatment. Yeast two-hybrid experiment indicated that DcHsfA1d and DcHsp70 physically interact with each other. Overexpression of DcHsfA1d in Arabidopsis ecotype Columbia enhanced seedling thermotolerance by increasing the activities of catalase, peroxidase, and superoxide dismutase while reducing relative electrolyte leakage, malondialdehyde content, accumulation of O₂^- and H₂O₂ and by initiating transcriptional regulation of thermal protective gene expression under heat stress. Furthermore, under salt stress, the root length and fresh mass of Arabidopsis ectopically expressing DcHsfA1d were significantly higher than those of wild type, which indicated that the salt tolerance of transgenic Arabidopsis was improved to a certain extent. In summary, DcHsfA1d was demonstrated to play a positive regulatory role in heat stress response and it might be a candidate gene for salt tolerance using genetic modification.

Biochemical and transcriptional approaches can provide crucial evidence about the physiological changes which can occur in organic and conventional cultivated common orange [Citrus sinensis (L.) Osbeck]. This study aimed to investigate the change in physicochemical parameters, the concentrations of free amino acids and other N-containing compounds, and the expressions of key genes coding for enzymes linked to N assimilation in fruits of common orange cv. "Valencia Late". Two enzymes involved in different ways in N assimilation were considered: nitrate reductase (NR), catalyzing the conversion of nitrate into nitrite, and glutamate dehydrogenase (GDH), operating in the assimilation of ammonium (interacting with glutamate synthase), and in ammonium re-assimilation through glutamate deamination. Results showed that the different fertilizers did not affect the physicochemical characteristics of fruits but induced the different accumulation of free amino acids, with higher concentrations of proline and contemporarily lower concentrations of glutamate, in addition to upregulated the expression of GDH gene in fruits from organically managed tress. This study identified a possible adaptive response of common orange plants to organic or conventional fertilizers. The present work is intended as a first step to make the mechanisms underlying plant responses to N supply clearer by comparing organic and conventional cultivation. It also can support breeders to select the best citrus cultivars and agronomists to improve crop fertilization and production management.

Flavonoids are secondary metabolites widely distributed in plants. They not only confer a wide spectrum of pigmentation to plant flowers but also protect plants from various biotic and abiotic stresses. Simultaneously, these compounds also offer health benefits to humans. Significant efforts have been made to correlate specific flavonoid production with biosynthetic pathway gene expression. Some structure genes and transcription factors that regulate the biosynthetic pathway have been identified. However, the diverse and complex control of flavonoid accumulation is still not well understood. In this mini-review, we summarized the improvement of flavonoids by genetic engineering from the aspects of flower colour, plant resistance, and benefits on the human diet. A perspective on flavonoid research in plants is provided.

Midvein is an important structure of the upright leaf of rice, and its normal development is essential to the formation of a common plant type of rice (Oryza sativa L.). To reveal the effect of midvein deficiency on photosynthesis-related characteristics, leaf microstructure, and vein characteristics, the photosynthetic features between the midvein-deficient mutant dl-14 and wild-type Huanghuazhan plants were analyzed. The results indicated that the midvein area of the dl-4 mutant lacked large intercellular space and instead it was filled with mesophyll cells. Moreover, the vein density of the dl-14 mutant was significantly higher than that in cv. Huanghuazhan. Chlorophyll (Chl) a, Chl b, and carotenoid content were markedly elevated in dl-14. In terms of photosynthetic characteristics, we observed that under high irradiance and high CO₂ concentration, the net photosynthetic rate of dl-14 plants was significantly higher than that of Huanghuazhan plants, but its water use efficiency was significantly lower. In addition, several major photosynthetic parameters, including characteristics of chlorophyll fluorescence (the efficiency of excitation capture of open PS II center, photochemical quenching, effective quantum yield of PS II photochemistry, and electron transfer rate) were significantly higher in dl-14 plants compared to Huanghuazhan plants, but the nonphotochemical quenching of dl-14 mutant was significantly lower than that of Huanghuazhan. These findings indicate that the dl-14 mutant has higher vein density, stronger photon conversion ability, and weaker radiation dissipation ability. This study can provide theoretical support for breeders to use the midvein-deficient mutant.

Prunella vulgaris L. have high medicinal and ornamental values. It blooms in summer and then quickly withers, and there is no research on the regulatory mechanisms of flowering-related genes. Therefore, in this study, the flowering (leaves and spicas) and nonflowering (leaves) parts of P. vulgaris were sequenced with an Illumina HiSeq 4000, and 187 387 transcripts were obtained using Trinity software package. A total of 10 158 differentially expressed genes (DEGs) were found in the leaves of flowering and nonflowering P. vulgaris, with 6 294 upregulated genes and 3 864 downregulated genes. DEGs in leaves of flowering and nonflowering P. vulgaris were mainly annotated for metabolic processes (4 207) in Gene Ontology (GO) and ribosomal pathways (416) in Kyoto Encyclopedia of Genes and Genomes (KEGG). Screening of this set of genes yielded 50 flowering-related unigenes homologous to Arabidopsis genes involved in multiple regulatory pathways related to plant flowering, including autonomous, vernalization, gibberellin, and age pathways. In addition, there are significant differences in the expressions of genes related to flowering, such as PvFLC, PvSOC1, and PvFY, as well as genes involved in plant hormone signal transduction and sugar metabolism. The accuracy and reliability of the transcriptome results were verified by quantitative real-time RT-qPCR analysis of PvGA20OX, PvSVP, PvELF3, PvCRY1, and PvSOC1. Thus, we speculate that the flowering of P. vulgaris is regulated by certain genes related to the flowering regulation pathway and sugar and hormone metabolism pathways. Analysis of the P. vulgaris transcriptome will provide a basis for revealing its flowering mechanism.

Drought and salinity, which can alter the water balance, disrupt the ionic equilibrium, and create reactive oxygen species (ROS), are capable of destroying plant tissues. In this study, transcriptomics, proteomics, and metabolomics have been used to elucidate various abiotic stress responses. In transcriptional signaling pathways, abscisic acid (ABA) is one of the plant phytohormones that regulate the stress response. On the other hand, several regulons and factors of transcription contributed in the reaction to osmotic stresses, as well as in ABA-dependent/independent signaling pathways. However, the findings display that intricate molecular reaction of plants under stress conditions may be controlled by complicated regulative networks of gene expression and signal transduction, as well as by the interaction between them. From the point of view of proteomics, protein modifications in response to stress can be considered as a molecular tool to improve the resistance of plants to environmental stresses. These studies have provided new information about the significance of several gene and protein networks involved in the response of plants to salinity and drought, and the induction of tolerance. Moreover, identifying the crucial pathways which are involved in salinity and drought resistance can open doors for the establishment of commercial-resistant crop cultivars, and might be very useful in the next-generation crop breeding strategies to produce plants with salinity and drought-resistant traits.

Paspalum fasciculatum Willd. ex Flüggé grows in mining soils which are Cd- and Pb-contaminated where it exhibits tolerance to Pb and the ability to extract Pb from these soils. To elucidate tolerance mechanisms to Pb-stress, liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to quantify changes in the accumulation of proteins in leaves. We identified 323 proteins involved in primary metabolism and response to biotic or abiotic stresses. Although proteins involved in the processes of photosynthesis and saccharide and energy metabolism presented the greatest amount of down-regulated proteins, the plant was able to maintain photosynthetic functions and obtain energy to sustain the vital balance. P. fasciculatum based their tolerance on increased antioxidant defenses, improving the protection and repair of proteins and transduction signals to coordinate physiological response to Pb-stress. Our results provide important information to understand the tolerance mechanisms in P. fasciculatum and could be important in future molecular studies on the resistance and accumulation of Pb in plants.

The potential toxicity of nanoparticles (NPs) is under debate. Information about TiO₂ NPs phytotoxicity is still limited partly due to the different TiO₂ NP forms that may be found in the environment. The present work investigated the impact of different TiO₂ NPs forms (rutile and anatase) on germination, growth, cell cycle profile, ploidy level, and micronucleus formation in Lactuca sativa (lettuce) and Ocimum basilicum (basil). Seeds were exposed to anatase (ana) or rutile + anatase (rut+ana) at concentrations 5 - 150 mg dm^-3 for 5 d and after that different parameters were analyzed. Rut+ana showed high potential to impair germination and growth. On the other hand, ana alone showed a positive influence on seedling growth. Despite that, ana induced severe alterations in cell cycle dynamics. Regarding species, basil was more sensitive to TiO₂ NPs cytostatic effects (delay/arrest in G₀/G₁ phase), whereas in lettuce, TiO₂ NPs were more genotoxic (micronucleus formation increase). Finally, we propose that, besides germination and plant growth, cell cycle dynamics and micronucleus formation can be sensitive biomarkers of these NPs.

Although fullerene (C₆₀) has attracted great interest as a carbon-based nanomaterial with unique properties, today, little is known about the interaction of its water-soluble derivates, including fullerenol with higher plants. Here, we investigated how fullerenol [C₆₀(OH)_22-24] affects Zea mays, as a Strategy II plant, depending on its iron status. Iron deficiency chlorosis is a common nutritional disorder affecting plants. Maize plants were grown hydroponically, either with [+Fe^II (ferrous) or +Fe^III (ferric)] or in Fe-free (-Fe^II and -Fe^III) nutrient solution and with or without a fullerenol supply. Fullerenol affected plants differently depending on their Fe status. The beneficial effects of fullerenol were observed in the Fe^II-deprived plants, including successful suppression of plant Fe-deficiency chlorosis mainly in the younger (basal and middle) region of the leaf blade. This region expressed more severe chlorosis as compared with the older (apical) region of the leaf blade. These changes were accompanied by a significant increase in leaf active Fe and lowering the root apoplastic Fe, suggesting that fullerenol may enhance Fe mobilization in the roots, helping to alleviate Fe deficiency chlorosis. By contrast, there were no observable effects in the Fe^III-deprived plants being significantly lower in the root apoplastic Fe as compared with the Fe^II-deficient plants. Additionally, fullerenol did not affect the Fe-sufficient plants, irrespective of the Fe species (Fe^III-EDTA or Fe^II-EDTA) used as Fe-sources. Our results provide new evidence for the beneficial role of Fe-fullerenol interactions in the enhancement of gramineous plant tolerance to Fe deficiency conditions, which are one of the major limiting factors for crop production all over the world.

Trehalose is a nonreducing disaccharide that is involved in the regulation of plant responses to a variety of environmental stresses. Trehalose 6-phosphate synthase (TPS) and trehalose 6-phosphate phosphatase (TPP) are two key enzymes in trehalose synthesis and they are widely distributed in higher plants. At present, TPS family genes have been systematically identified and analyzed in many plant species, but the TPP family genes have been rarely studied. In this study, ten TPS and six TPP genes in cannabis (Cannabis sativa L.) were identified at the genomic level. The phylogenetic tree of TPS and TPP family members in cannabis, Arabidopsis, and rice was constructed, and all the genes were divided into three subgroups: Class I, Class II, and Class III. The number of exons and motif types among Class I members was exactly the same, as were Class II members, but the gene structure and motif types of Class III members were slightly different. There were four pairs of CsTPSs and CsTPPs that had gene duplication, indicating that gene duplication events played an important role in the amplification of TPS and TPP families in cannabis. The results of expression analysis under abiotic stresses showed that 68.75 % of CsTPS and CsTPP genes were significantly induced by at least one abiotic stress. Among these genes, the expression of CsTPS1, CsTPS9, and CsTPPA was highest under at least one abiotic stress. These three genes may play a key role in abiotic stress responses. Most of the CsTPS and CsTPP genes that are closely located in the evolutionary tree have the same or similar functions. To our knowledge, this is the first paper that systematically reports the TPS and TPP gene families in cannabis.

Long-term low temperatures restrict the regrowth of winter wheat (Triticum aestivum L.), thus decreasing agricultural output. Non-enzymatic expansins, which are related to plant growth, have been reported to respond to drought, salinity, and low-temperature stress. We obtained an expansin 3 gene, TaEXPA9. It is located in winter wheat cv. Dongnong with high cold hardiness. We analyzed the expression patterns of TaEXPA9-A/B/D in this cultivar and conducted a subcellular localization analysis of TaEXPA9-A/B/D in the onion epidermis. Transgenic Arabidopsis thaliana line with EXPA9-A/B/D overexpression was obtained to examine the effects of the orthologous genes of these expansins on plant growth and low-temperature stress resistance. The results showed that EXPA9-A/B/D expression significantly increased at 4 °C, it was higher in the roots than in shoots, and EXPA9-A/B/D was localized in the cell wall. The roots were well-developed in the transgenic A. thaliana, and the growth-related markers and setting rate were better than in the wild-type. Recovery was stronger in the transgenic plants after freezing stress. At low-temperature stress, the antioxidant enzyme activities and content of osmoregulatory substances in the TaEXPA9-A/B/D-overexpressing A. thaliana plants were significantly higher than in the wild-type plants, and the degree of membrane lipid peroxidation was lower. In summary, TaEXPA9 orthologous genes participate in the low-temperature stress response, and they might be of great importance in molecular breeding.

Trehalose, which plays important roles in resistance to abiotic stresses and preservation of biological activity in plants, is synthesized by two key enzymes, trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). Therefore, the expressions of the TPS and TPP genes directly affect trehalose synthesis and stress resistance of plants. In this study, CkTPS and CkTPP from Caragana korshinskii were identified, and the role of trehalose synthesis in the adaptation of this desert plant to adverse conditions was investigated. Higher CkTPS and CkTPP expressions were observed in the roots, whereas expressions were much lower in leaves and stems, and their expressions were upregulated under drought stress. Histochemical analyses showed that β-glucuronidase expression driven by the CkTPS and CkTPP promoters was strongly induced by abiotic stresses and phytohormones, such as abscisic acid, gibberellin, methyl jasmonate, and mannitol, which suggests that trehalose synthesis may be regulated by various signaling pathways. To determine the functional mechanism underlying the role of trehalose synthesis in regulating drought response in plants, CkTPS and CkTPP were introduced into Arabidopsis. Compared to wild-type (WT) plants, these transgenic plants showed higher germination rate, survival, less damage, better shoot growth, and longer roots under drought stress. Moreover, transgenic plants had a significantly higher content of proline, chlorophyll, trehalose, and activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and lower malondialdehyde (MDA) content than WT controls. Double-transgenic plants carrying CkTPS and CkTPP showed better growth and stronger drought tolerance than either single transgenic plant line. These results provide a theoretical and experimental basis for further understanding the function and regulatory mechanism of CkTPS and CkTPP, as well as the possibility of their application for improving drought tolerance in crops through genetic engineering.

Pathogenesis-related (PR) proteins play key roles in plant disease resistance. Here, we isolated and characterized pathogenesis-related PR2 gene encoding β-1,3-glucanase from Brassica juncea and named it BjPR2 (GenBank accession number DQ359125). The amino acid sequence of BjPR2 showed ~99 % similarity with β-1,3-glucanase of Brassica rapa, B. napus, and B. oleracea. BjPR2 transcription was rapidly increased after Alternaria brassicae infection, salicylic acid application, and wounding, but the induction was delayed in response to jasmonic acid. To investigate the transcriptional regulation of BjPR2 gene, its promoter was isolated. In silico analysis of BjPR2 promoter showed cis-regulatory elements upstream of TATA and CAAT boxes responsive to defense, hormones, wounding, and plant developmental stage. Homozygous Arabidopsis thaliana lines were developed with plasmid construct having β-glucuronidase (GUS) reporter gene driven by BjPR2 promoter. The analysis of GUS protein in Arabidopsis lines showed that BjPR2 promoter drived distinct pattern of pathogen inducible expression after fungal infection (Alternaria brassicae, Erysiphe orontii), phytohormones, and wounding. It also showed age dependent and organ specific expressions. BjPR2 promoter drove strong GUS activity in Arabidopsis seedlings and showed organ specific expression at the later growth stages (lateral organ junctions, leaf serrate, base of siliques, and receptacle). Due to stress-inducible and tissue specific nature, the BjPR2 promoter can serve as a potential candidate in genetic engineering.

The WRKY transcription factors (TFs) are integral parts of signaling pathways that regulate many processes, such as senescence, seed dormancy, seed germination, and resistance to abiotic and biotic stresses. Stress-related functions of WRKY6 have been characterized in Arabidopsis and other plant species, but its role has not been identified in apple. Here, we cloned WRKY6 genes from Malus prunifolia. Two homologues MpWRKY6a and MpWRKY6b found in this species were members of Group II WRKY6 TFs. They were localized to the cell nucleus. MpWRKY6a can bind to W-boxes. Compared with the untransformed wild type plants, MpWRKY6a-overexpressing Arabidopsis plants were more sensitive to methyl jasmonate (MeJA) and less sensitive to methyl viologen and abscisic acid (ABA), which suggests its role in responses to oxidative stress and MeJA or ABA signaling. The results fill a gap in the WRKY6 function in apple and provide basis for resistance improvement of Malus.

Abscisic acid (ABA) regulates various plant physiological processes, especially participates in the plant responses to harsh environments. The 9-cis-epoxycarotenoid dioxygenase (NCED) is a key enzyme in ABA biosynthesis pathway. Here, a TaNCED with an 1 887-bp open reading frame was cloned from wheat, which encodes a peptide of 628 amino acids. A chloroplast transit peptide sequence was found at the N-terminus of the TaNCED protein. Multiple sequence alignments indicate that the TaNCED protein shared high similarities with other NCEDs from different species. Real-time quantitative PCR analysis shows that expression of TaNCED was strongly up-regulated by treatments with ABA, polyethylene glycol, and drought stress, and it was down-regulated during germination of the wheat seeds. Ectopic overexpression of the TaNCED gene in Arabidopsis resulted in an increase of endogenous ABA and free proline content. A lower water loss rate and stomatal conductance of leaves were found in the transgenic plants in comparison with the wild type. Subsequently, the transgenic plants displayed an enhanced tolerance to drought stress but delayed seed germination. These data provide evidence that the TaNCED might play a primary role in regulation of ABA content during water stress and seed dormancy.

Glutathione transferases (GSTs) mainly catalyze the nucleophilic addition of glutathione to a large variety of hydrophobic molecules participating to the vacuole compartmentalization of many toxic compounds. In this work, the putative tolerance of transgenic tobacco plants over-expressing CsGSTU genes towards the chloroacetanilide herbicide alachlor was investigated. Our results show that the treatment with 0.0075 mg cm^-3 of alachlor strongly affects the growth of both wild type and transformed tobacco seedlings with the sole exception of the transgenic lines overexpressing CsGSTU2 isoform that are barely influenced by herbicide treatment. In order to correlate the in planta studies with enzyme properties, recombinant CsGSTs were in vitro expressed and tested for GST activity using alachlor as substrate. The recombinant GSTU2 enzyme was twice more active than GSTU1 in conjugating alachlor to GSH thus indicating that CsGSTU2 might play a crucial role in the plant defense against the herbicide. Moreover, as a consequence of the infiltration with a bacterial suspension of the P. syringae pv. tabaci, transgenic tobacco plants but not wild type plants bestowed the capability to limit toxic metabolite diffusion through plant tissues as indicated by the absence of chlorotic halos formation. Consequently, the transgenic tobacco plants described in the present study might be utilized for phytoremediation of residual xenobiotics in the environment and might represent a model for engineering plants that resist to pathogen attack.

The tonoplast and plasma membrane localized sodium (potassium)/proton antiporters have been shown to play an important role in plant resistance to salt stress. In this study, AtNHX1 and AtNHX3, two tonoplast Na⁺(K⁺)/H⁺ antiporter encoding genes from Arabidopsis thaliana, were expressed in poplar to investigate their biological functions in the resistance to abiotic stresses in woody plants. Transgenic poplar plants expressing either gene exhibited increased resistance to both salt and water-deficit stresses. Compared to the wild type (WT) plants, transgenic plants accumulated more sodium and potassium ions in the presence of 100 mM NaCl and showed reduced electrolyte leakage in the leaves under water stress. Furthermore, the proton-translocating and cation-dependent H⁺ (Na⁺/H⁺ or K⁺/H⁺) exchange activities in the tonoplast vesicles isolated from the leaves of transgenic plants were higher than in those isolated from WT plants. Therefore, constitutive expression of either AtNHX1 or AtNHX3 genetically modified the salt and water stress tolerance of transgenic poplar plants, providing a potential tool for engineering tree species with enhanced resistance to multiple abitotic stresses.

The trihelix genes encode plant-specific transcription factors, which play a vital role in plant morphological and developmental processes. However, information about the presence of trihelix genes in sunflower (Helianthus annuus L.) is scarce. Sunflower belongs to composite family and possesses strong drought and salt-alkali tolerance. In this study based on H. annuus genome data, we have identified and analyzed the trihelix genes with a complete description of their physical and chemical properties, phylogenetic relationships, motif composition, chromosome distribution, exon-intron structure, cis-acting elements, and chromosome collinearity. In H. annuus, 31 full-length trihelix genes were identified and categorized into six subgroups (SIP, GT1, SH4, Gδ, GT-γ, and GT2). Multiple Em for motif elicitation (MEME), used for conservative motif analysis, identified 10 distinct motifs unevenly distributed on 31 trihelix genes. In addition to that, chromosome localization analysis showed the number and distribution of these trihelix genes on 17 chromosomes of H. annuus. Transcriptional structure analysis revealed the structure of introns and exons of different gene members. Furthermore, cis-element analysis identified 19 different types of cis-elements mainly related to abiotic stress, hormones, and growth and development of plant. Results of this study manifested novel insights into phylogenetic relationships and possible functions of H. annuus trihelix genes. Moreover, these findings can assist in future studies regarding specific physiological effects of H. annuus trihelix transcription factors.