Responses to winter dormancy,temperature, and plant hormones share gene networks

Gene networks modulated in winter dormancy (WD) in relation to temperature and hormone responses were analyzed in tea [Camellia sinensis (L.) O. Kuntze]. Analysis of subtracted cDNA libraries prepared using the RNA isolated from the apical bud and the associated two leaves (two and a bud, TAB) of actively growing (AG) and winter dormant plant showed the downregulation of genes involved in cell cycle/cell division and upregulation of stress-inducible genes including those encoding chaperons during WD. Low temperature (4°C) modulated gene expression in AG cut-shoots in similar fashion as observed in TAB during WD. In tissue harvested during WD, growth temperature (25°C) modulated gene expression in the similar way as observed during the period of active growth (PAG). Abscisic acid (ABA) and gibberellic acid (GA₃) modulated expression of selected genes, depending upon if the tissue was harvested during PAG or WD. Tissue preparedness was critical for ABA- and GA₃-mediated response, particularly for stress-responsive genes/chaperons. Data identified the common gene networks for winter dormancy, temperature, and plant hormone responses.     

Early low-temperature responsive mitogen activated protein kinases <Emphasis Type="Italic">RaMPK1</Emphasis> and <Emphasis Type="Italic">RaMPK2</Emphasis> from <Emphasis Type="Italic">Rheum australe</Emphasis> D. Don respond differentially to diverse stresses

Rheum grows luxuriantly in a niche of low temperature (LT) at high altitudes in Himalayan belt. The plant is expected to harbor novel genes particularly for tolerance to LT. Using differential display, two cDNAs RaMPK1 and RaMPK2, showing homology to mitogen-activated protein kinases (MAPKs) were isolated. As compared to RaMPK1, RaMPK2 exhibited strong up-regulation in response to LT. RaMPK1 was novel in terms of possessing a small glutamine and proline rich region at the N-terminal end. Secondly, though RaMPK1 showed homology with salicylic acid (SA) responsive MAPKs, the gene was down-regulated by SA but activated by jasmonate (JA). Abscisic acid (ABA) and polyethylene glycol (PEG) also down-regulated RaMPK1. RaMPK2 showed down-regulation within 5 min of exposure to JA and SA treatments, followed by gradual increase in expression. Expression of RaMPK2 was wavy in response to ABA and PEG treatment. Results are discussed in light of the novelty of these MAPKs.     

Somatic embryogenesis and plant regeneration in yakutanegoyou, <Emphasis Type="Italic">Pinus armandii</Emphasis> Franch. var. <Emphasis Type="Italic">amamiana</Emphasis> (Koidz.) Hatusima,an endemic and endangered species in Japan

Induction of somatic embryogenesis in Pinus armandii var. amamiana, an endemic and endangered species in Japan, was initiated from megagametophytes containing immature zygotic embryos on both media with and without plant growth regulators. Across nine open-pollinated families initiation frequency ranged from 0 to 20%, with an average of 1.5%. Embryogenic cultures were maintained and proliferated on a medium supplemented with 2,4-dichlorophenoxyacetic acid (3 μM) and 6-benzylaminopurine (1 μM). Maturation of somatic embryos occurred on medium containing maltose (50 g l⁻¹), activated charcoal (2 g l⁻¹), abscisic acid (100 μM), and polyethylene glycol (100 g l⁻¹). The frequencies of germination and plant conversion of somatic embryos differed among the embryogenic lines from 16 to 51% and from 12 to 40%, respectively. Growth of regenerated somatic plants has been monitored in the field.     

Dehydrin2 is a stress-inducible, whereas Dehydrin1 is constitutively expressed but up-regulated gene under varied cues in tea [Camellia sinensis (L.) O. Kuntze]

Two expressed sequence tags with similarity to dehydrin were cloned to full-length (CsDHN1 and CsDHN2) through rapid amplification of cDNA ends. CsDHN1 and CsDHN2 were 1,027 and 992 base pair long and encoded for predicted polypeptides of 260 and 201 amino acids, respectively. Deduced CsDHN1 protein had a S-segment and three lysine-rich consensus motifs (K-segments). The Y-segment was, however, absent in the deduced CsDHN1. CsDHN2 had three Y motifs, one S-segment and two K-segments. Expression of CsDHN1 was visible at all the time points of study, though up-regulation was observed in response to winter dormancy (WD) as well as abiotic stresses [low temperature, sodium chloride, polyethylene glycol, and hydrogen peroxide]. Expression of CsDHN2 was strongly up-regulated within 1 h of exposure to abiotic stresses as well in the tissue harvested during WD in contrast to the respective “control” for abiotic stresses and in tissue during the period of active growth, when the expression was not detectable.     

Transient expression in Arabidopsis thaliana protoplasts derived from rapidly established cell suspension cultures

Arabidopsis thaliana has emerged as a model species for the analysis of genes controlling plant development. However, its small size has impaired biochemical analyses, and the absence of a transient expression system has hampered promoter analysis. Here, we report a method for rapidly establishing A. thaliana suspension cultures that yield protoplasts that can be readily transfected. We have optimized transient expression conditions using a modified polyethylene glycol / calcium nitrate transformation protocol and a Cauliflower Mosaic Virus 35S promoter-luciferase reporter gene construct. Our methods permit isolation of large quantities of rapidly growing cells and analysis of Arabidopsis promoters in vivo in a homologous system.Abbreviations CaMV Cauliflower Mosaic Virus - 2,4D 2,4-dichlorophenoxyacetic acid - MES 2-(N-morpholino)ethanesulfonic acid - PEG polyethylene glycol     

Characterization of gene expression of <Emphasis Type="Italic">QM</Emphasis> from <Emphasis Type="Italic">Caragana jubata</Emphasis>, a plant species that grows under extreme cold

Caragana [Caragana jubata (Pall.) Poir] is a temperate plant that thrives well under extremes of cold in high altitude of Himalaya and hence the plant is expected to be a source of genes that might play an important role in tolerance to low temperature (LT). In order to identify LT inducible gene(s), differential display of mRNA (DD) was performed using the apical buds growing under snow as well as growing in the near vicinity without snow, and a LT inducible QM gene (CjQM) homologue was identified. Realizing the importance of QM gene (which encodes human Wilms’ tumor suppressor QM protein) in aggregation of 40 and 60S ribosomal subunit and that not much has been reported on this gene in plant systems in relation to its relationship with LT, full length cDNA of CjQM was cloned through rapid amplification of cDNA ends. The gene (977 bp), encoded by small gene family, had an open reading frame of 651 bp and was found to be intronless. The gene exhibited up-regulation within 20 min of exposure to LT and abscisic acid (ABA), but no significant change in gene expression was observed in response to drought stress (DS), salicylic acid (SA) and methyl jasmonate (MJ) application. Up-regulation of CjQM was obtained in the tissues growing in situ under snow. Non-responsiveness of CjQM towards DS, SA and MJ, but up-regulation in response to LT and ABA suggested a specific regulation of the gene in Caragana under varied cues.     

Stress-induced curcin-L promoter in leaves of <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. and characterization in transgenic tobacco

Ribosome-inactivating proteins (RIPs) represent a type of protein that universally inactivates the ribosome thus inhibiting protein biosynthesis. Curcin-L was a type I RIP found in Jatropha curcas L.. Its expression could be activated in leaves by treatments with abscisic acid, salicylic acid, polyethylene glycol, temperature 4, 45°C and ultraviolet light. A 654 bp fragment of a 5′ flanking region preceding the curcin-L gene, designated CP2, was cloned from the J. curcas genome and its expression pattern was studied via the expression of the β-glucuronidase (GUS) gene in transgenic tobacco. Analysis of GUS activities showed that the CP2 was leaf specific, and was able to drive the expression of the reporter gene under stress-induction conditions. Analysis of a series of 5′-deletions of the CP2 suggested that several promoter motifs were necessary to respond to environmental stresses.     

Physiological evaluation of drought stress tolerance and recovery in Verbascum sinuatum plants treated with methyl jasmonate,salicylic acid and titanium dioxide nanoparticles

Abstract

The genus Verbascum L. (Scrophulariaceae) includes medicinal plants, which have several bioactive compounds especially saponins. The possible recovery ability of Verbascum sinuatum from drought stress conditions was assessed by using salicylic acid (SA), methyl jasmonate (MJA) and titanium dioxide nanoparticles (TiO₂NPs) as plant growth regulators (PGRs) in liquid culture media. Thirty days-old plants were exposed to different concentrations of polyethylene glycol (PEG-6000) for creating artificial drought conditions (0, ?0.3, and ?0.6?MPa osmotic potential) and also treated with 200?µM methyl jasmonate (MJA), 100?µM salicylic acid (SA) and 20?ppm TiO₂ nanoparticles (TiO₂NPs). Results showed that the growth parameters and the content of photosynthetic pigments decreased at higher drought level (?0.6?MPa). However, SA and TiO₂NPs alleviated the adverse effects of drought stress by increasing water stress tolerance through promotion of enzymatic and nonenzymatic antioxidant defense systems. MJA negatively affected the growth parameters and increased the content of malondialdehyde (MDA), hydrogen peroxide (H₂O₂) and total saponin and also the activity of peroxidase (POD) and polyphenol oxidase (PPO). Based on the results obtained from this study, the recovery treatments mainly affected the defense-related metabolism in Verbasum sinuatum plants.     

Enhancement of stress tolerance in cucumber seedlings by proanthocyanidins

Proanthocyanidins (PAs) are the main products of the flavonoid biosynthetic pathway in many plants. However, their biological function during environmental stresses in plants is rarely reported. In the present study, the effects of pretreatment with PAs on the response of cucumber (Cucumis sativus L.) seedlings to high irradiance (HI), polyethylene glycol (PEG), and cold stress were investigated. The PAs pretreament alleviated stress-induced oxidative damage in plant cells and increased the activity of alternative oxidase (AOX) and content of abscisic acid (ABA). Furthermore, PAs-pretreated seedlings suffered less damage by the stress conditions, maintained higher content of chlorophyll a+b and AOX proteins in comparison with the control. Therefore, our findings suggest that PAs might contribute to plant tolerance to environmental stresses.     

A simple and efficient protocol for cryopreservation of embryogenic calli of the medicinal plant <Emphasis Type="Italic">Anemarrhena asphodeloides</Emphasis> Bunge by vitrification

Somatic embryogenesis and whole plant regeneration was achieved in callus cultures derived from immature zygotic embryos of Prosopis laevigata (Humb. & Bonpl. ex Willd.) M.C. Johnst., recently identified as chromium (Cr), cadmium (Cd), lead (Pb) and nickel (Ni) accumulator. Embryogenic calli were induced on Murashige and Skoog (MS) medium added with a mixture of organic components plus N-6 benzyladenine (BA) (6.62 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (2.26 μM) or thidiazuron (4.54–9.08 μM) and indole-3-acetic acid (1.42 μM). Embryogenic calli transferred onto half-strength MS medium without plant growth regulators developed globular embryos, of which 20% matured when treated with 3.75% (w/v) polyethylene glycol (PEG), and of these 50% fully differentiated into plantlet embryo. Regenerated plants were successfully acclimatized (90%), while in vitro seedlings transferred to MS medium containing 0.5 mM Cd, Cr, Ni or Pb, exhibited high heavy metals accumulation (627 mg Cr kg⁻¹, 5,688 mg Cd kg⁻¹, 1,148 mg Ni kg⁻¹, and 3,037 mg Pb kg⁻¹ dry weight) and efficient roots to shoots translocation (42–73%).     

Characterization of the LhcSR Gene Under Light and Temperature Stress in the Green Alga Ulva linza

As a green-tide-forming macroalga, Ulva linza is distributed worldwide and therefore subject to various environmental stresses. The LHCSR (also known as LI818 in green alga and LHCX in diatoms) protein is a stress-related member of the LHC family that plays an important role in photo-protective mechanism, which has been only found in algae. In this study, we cloned full-length cDNA sequence encoding the LhcSR gene from U. linza and analyzed its expression in response to different temperature and illumination gradients. The results showed that high light (HL) could enhance expression of LhcSR and that the expression level peaked at 3 h under HL. Similarly, the expression of LhcSR could also be induced by low temperature (LT). However, the expression patterns of LhcSR were quite different in response to LT and HL treatment. Specifically, the maximum gene expression under LT was much higher (11.8-fold) than under HL (5.4-fold) when compared to the expression under normal conditions. The upregulated expression of LhcSR lasted for 12 h under LT, but 3 h under HL. These data suggest that the LhcSR gene is involved in photoprotection in U. linza, and the results suggest a stronger link to LT. In addition, the discrepancy in expression under HL and LT was consistent with the ecological features of this alga, which only thrives during the cold season (featured as LT and low light).     

Effect of growth regulators on maturation of geranium (Pelargonium × hortorum) somatic embryos

Interactions of growth regulators and polyethylene glycol on maturation of geranium somatic embryos were investigated. Somatic embryos were induced on medium with 20 M thidiazuron for 3 days. The growth regulators used were 1 µM abscisic acid, jasmonic acid, napthaleneacetic acid and benzylaminopurine at 21 days from the start of induction. Benzylaminopurine and napthaleneacetic acid did not enhance abscisic acid effects on maturation frequency but only improved maturation frequency in the presence of polyethylene glycol. Abscisic acid significantly improved protein content in the presence of polyethylene glycol. Benzylaminopurine and napthalene acetic acid in combination with abscisic acid and jasmonic acid improved protein types in somatic embryos only in the absence of polyethylene glycol. Osmoticum effected by polyethylene glycol seems the main component required for protein synthesis. This study showed significant improvement of somatic embryo quality for artificial seed production.     

Theanine transporters identified in tea plants (Camellia sinensis L.)

Theanine, a unique non‐proteinogenic amino acid, is an important component of tea, as it confers the umami taste and relaxation effect of tea as a beverage. Theanine is primarily synthesized in tea roots and is subsequently transported to young shoots, which are harvested for tea production. Currently, the mechanism for theanine transport in the tea plant remains unknown. Here, by screening a yeast mutant library, followed by functional analyses, we identified the glutamine permease, GNP1 as a specific transporter for theanine in yeast. Although there is no GNP1 homolog in the tea plant, we assessed the theanine transport ability of nine tea plant amino acid permease (AAP) family members, with six exhibiting transport activity. We further determined that CsAAP1, CsAAP2, CsAAP4, CsAAP5, CsAAP6, and CsAAP8 exhibited moderate theanine affinities and transport was H⁺‐dependent. The tissue‐specific expression of these six CsAAPs in leaves, vascular tissues, and the root suggested their broad roles in theanine loading and unloading from the vascular system, and in targeting to sink tissues. Furthermore, expression of these CsAAPs was shown to be seasonally regulated, coincident with theanine transport within the tea plant. Finally, CsAAP1 expression in the root was highly correlated with root‐to‐bud transport of theanine, in seven tea plant cultivars. Taken together, these findings support the hypothesis that members of the CsAAP family transport theanine and participate in its root‐to‐shoot delivery in the tea plant.     

The interactive effect of water deficit and UV-B radiation on salicylic acid accumulation in barley roots and leaves

The aim of the paper was to determine the effect of water deficit (WD) and UV-B radiation acting individually and in combination on salicylic acid (SA) accumulation as well as on the activity of phenylalanine ammonia-lyase (PAL) and benzoic acid hydroxylase (BA2H) that control its biosynthetic route from phenylalanine. An additional aim was to test whether the interaction of these stresses limits the negative effect of a single stress on tissue hydration and membrane injury. Two-week-old seedlings were subjected to water deficit (WD), UV-B irradiation (UV-B) and three different combinations of WD and UV-B: (I) WD and UV-B applied at the same time, (II) UV-B applied before WD, and (III) WD applied before UV-B. Water deficit was imposed by immersing the root system in aerated nutrient solution with polyethylene glycol (PEG 6000) of water potential – 0.5 MPa. UV-B dosage was 24 kJ m⁻² day⁻¹ (0.84 W m⁻²) at the canopy level. UV-B and WD imposed individually and jointly, caused, in a time-dependent manner, an increase in SA content in both organs. Increased levels of SA in WD stressed plants were accompanied by an increase in the activity of PAL and BA2H. However, in plants exposed to UV-B were accompanied only by an increase in the activity of BA2H. Under WD conditions, an earlier increase of SA content was observed in roots than in leaves, which may indicate the involvement of SA in the signal transduction between roots and leaves. In plants exposed to sequential action of WD and UV-B, regardless of the order of its imposition, the effect of each single factor on SA accumulation in leaves was strengthened. WD had a greater effect on water loss and membrane injury than UV-B radiation. In plants exposed to WD after pre-treatment with UV-B radiation, a cross-tolerance mechanism was observed. Leaves of these plants did not show increased lipid peroxidation, measured in terms of malondialdehyde content, and a decrease in water content. This protective action was probably caused by the increase of the SA level in leaves of the UV-B treated plants prior to WD imposition.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot culture and antimicrobial activity of <Emphasis Type="Italic">Berberis buxifolia</Emphasis> Lam

Berberis buxifolia Lam., known as “Calafate”, is a plant native to Argentina that exhibits antimicrobial activity. This biological activity is attributed to the isoquinoline alkaloid berberine. The aim of this research was to test the antimicrobial properties of different extracts of this species, taking berberine as the reference molecule, and to examine if the expression of bacterial multidrug resistance (MDR) efflux pumps could be responsible for possible resistance mechanisms. To this end, a wild-type and a mutant strain of Staphylococcus aureus with a defective MDR efflux pump were used and the minimum inhibitory concentrations of the extracts were determined. The studies were carried out with infusions of in vivo shoots and “Calafate” commercial tea, as well as with the media derived from shoot cultures incubated with different plant growth regulators (thidiazuron, picloram, and jasmonic acid). As far as antimicrobial activity is concerned, all the extracts tested were significantly more effective than berberine standard. “Calafate” commercial tea and shoot tea had inhibitory concentrations similar to the one observed for ampicillin standard. The media from the shoot cultures, however, were significantly more effective than all the others, particularly the one derived from jasmonic acid, suggesting the presence of compounds that could be acting synergistically with berberine. There were no differences in antimicrobial activity against the wild-type and the mutant S. aureus; no definite conclusions could be drawn concerning the relationship between MDR pumps and possible pathogen resistance to extracts of B. buxifolia.     

Bisulfite interacts with binding sites of the auxin-transport inhibitor N-1-naphthylphthalamic acid

The affinity of the auxin-transport inhibitor N-1-naphthylphthalamic acid (NPA) for membrane particles as well as for solubilized binding sites from Cucurbita pepo L. hypocotyls was reduced by low concentrations of bisulfite (half-maximal inhibition at 2·10^-3–3·10^-3 M). Two membrane fractions obtained by sedimentation aided with polyethylene glycol showed differential sensitivity to bisulfite. Other oxidizing or reducing substances tested at 1 mM had no effect, except for N-ethylmaleimide (80% inhibition) and iodine (complete inhibition), both of which reduced the number of binding sites but not their affinity. Addition of bisulfite to either the isoalloxane ring of flavoproteins or to pyridoxal phosphate or quinones is proposed as a possible mechanism of action. Sulfur dioxide, at concentrations measured in polluted air, can lead to bisulfite concentrations in plant tissue sufficient to interfere with NPA-binding sites and hence with auxin transport.Abbreviations DTE dithioerythritol - DTT dithiothreitol - IC₅₀ concentration of half-maximal inhibition - NAA 1-naphthylacetic acid - NEM N-ethylmaleimide - NPA N-1-naphthylphthalamic acid - PEG polyethylene glycol, 6000 molecular weight     

Effects of water deficit and selenium on common buckwheat (<Emphasis Type="Italic">Fagopyrum esculentum</Emphasis> Moench.) plants

Two cultivars of common buckwheat (Fagopyrum esculentum), Pyra and Siva, were exposed to three treatments: water deficit (WD), foliar spraying by selenium (as Na₂SeO₄) (Se), and the combination of both. In WD-plants the stomatal conductance (g _s) was significantly lower, while WD+Se-plants of Siva had significantly higher g _s. None of the treatments resulted in significant differences of potential photochemical efficiency of photosystem 2 (PS2). A significantly higher actual photochemical efficiency of PS2 was obtained in Siva WD-plants and in Pyra Se-and WD-plants which was possibly due to improvement of plant water management during treatment. A significant interaction was observed between the effects of WD and Se on respiratory potential in Pyra. WD, Se, and the WD+Se combination resulted in shorter Pyra and Siva plants, with a reduced number of nodes. WD slightly negatively affected the yield per plant. The yield was highest in plants exposed to Se only. In Siva the number of seeds was triple while the average seed mass remained unchanged.     

Growth regulators differentially affect photosensitivity induced by low temperature and osmotic stresses in germinating white clover seeds

Negative photoblastism induced in white clover seeds at 5°C or by lowered water potential (–0.3 MPa, polyethylene glycol) was affected by ethrel, gibberellin A₃, benzylaminopurine and kinetin treatments. The effects were different for water and temperature stressed seeds. The observed synergistic and additive effects of light and growth regulators confirm the earlier suggestion that there are two different mechanisms involved in the light inhibition of white clover seed germination induced by various adverse environmental conditions.Abbreviations ABA abscisic acid - BAP benzylaminopurine - GA₃ gibberellin A₃ - PEG polyethylene glycol