UV-B stress-induced ABA production in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> mutants defective in ethylene signal transduction pathway

In this study, we examined the influence of UV-B radiation (280–320 nm) on ABA accumulation in 14-day-old Arabidopsis thaliana (L.) Heynh plants of wild type (WT), ethylene receptor mutant (etr1-1), and mutant with a constitutively active ethylene signal transduction pathway (ctr1-1). ABA content in nonirradiated WT plants was twice higher than in each mutant. UV-B irradiation caused dose-dependent ABA accumulation in WT plants. In the etr1-1 mutant, the amount of accumulated ABA was significantly less. In the ctr1-1 mutant, ABA content didn’t increase after UV-B irradiation. These data suggest that start of stress-induced ABA formation requires the adjustable ethylene signal pathway. In the ctr1-1 mutant, a constitutively active (nonadjustable) ethylene signal pathway blocks stress-induced ABA accumulation.     

A role of ETR1 in regulating leaf petiole elongation mediated by elevated temperature in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Temperature fluctuation profoundly affects the plant growth and development. In this study, we show that ethylene receptor ETR1 is involved in regulating leaf petiole elongation mediated by higher temperatures (at 32 °C in this study). ETR1 loss-of-function mutant etr1-7 cannot elongate the leaf petiole at 32 °C as much as wild-type seedlings (WT). Overexpression of ETR1 in etr1-7 not only fully rescued the deficient in petiole elongation under higher temperature conditions but also caused longer petiole length under normal temperature conditions (22 °C). Plants with different mutant ETR1 alleles including etr1-7 etr1-1, and etr1-9 but not etr1-3 impair the petiole elongation mediated by elevated temperature. RNA-Seq analysis showed that hundreds of genes induced by elevated temperature in WT were not differentially expressed in etr1-7. Gene ontology enrichment analysis reveals that the molecular functions of these genes primarily relate to photosynthesis and protein degradation. Furthermore, genes involved in regulating organ elongation (such as BRI1-EMS-SUPPRESSOR 1, BES1), are significantly up-regulated in WT rather than in etr1-7 after the treatment of higher temperature. The results from this study suggest ETR1 is involved in regulating Arabidopsis response to elevated ambient temperature in both molecular and morphological levels.     

Effects of medium-wave ultraviolet radiation on levels and spectrum of polyamines in leaves and roots of wild-growing plants

The work continues serial studies on short-term effects of medium-wave ultraviolet radiation (UV-B) at 12.5 kJ/m² on plants. Special attention is paid to the rapid response of the antioxidant system. Free and conjugated forms of putrescine polyamines (putrescine, spermine, and spermidine), as well as those of cadaverine, are recognized to be constituents of the antioxidant system. These compounds were analyzed in plants 24 h after UV-B irradiation. Thellungiella salsuginea (Pallas) O.E.Schulz, Salvia officinalis L, Plantago major L., and Geum urbanum L. grown in aquatic culture under phytotron conditions were examined. The results support the hypothesis that putrescine plays the chief role in the plant defense response against medium-wave ultraviolet irradiation. Three of four plants manifested an increase in the content of this polyamine in leaves. It is the change that determines the enhanced total level of free polyamines. We failed to reveal a general tendency in dynamics of levels of conjugated forms of spermine, spermidine, and cadaverine; only conjugates of putrescine demonstrated a distinct increase. This study allows a conclusion that contributions of particular polyamines to the protective response primarily depend on the species to which the investigated plant belongs. It is likely that conjugated polyamines can be reserved as a pool necessary for rapid recovery of free polyamine levels.     

Interaction between ethylene and ABA in the regulation of polyamine level in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> during UV-B stress

The effects of treatment with ethylene (0.01–100 μl/l) on ABA and polyamine contents and treatment with ABA on ethylene synthesis, polyamines content, and the resistance to UV-B radiation of two-week-old Arabidopsis thaliana (L.) Heynh, Columbia ecotype plants grown u?er sterile conditions were studied. Ethylene stimulated the accumulation of polyamines only at concentrations of 0.1–10 μl/l, which could activate ABA synthesis. Treatment with ABA (50–5000 μM, 1 μl per plant) decreased the UV-B-induced ethylene synthesis and a spermine and spermidine loss, increasing the content of putrescine, the precursor of these polyamines. ABA inhibited fresh weight accumulation in irradiated and nonirradiated plants but prevented them from severe damage and death at the high (18 kJ/m²) and lethal (27 kJ/m²) UV-B dose, respectively. The data obtained demonstrated a mutual regulation of ethylene and ABA syntheses and the participation of these hormones in the control of the polyamine level during adaptation of A. thaliana to UV-B stress.     

Dependence of root biomass accumulation on the content and metabolism of cytokinins in ethylene-insensitive plants

Diverse functions of ethylene in plants may depend on its ability to interact with other hormones. We studied the participation of ethylene in the regulation of accumulation and metabolism of cytokinins comparing ethylene-insensitive mutant plants of arabidopsis (Arabidopsis thaliana [L.] Heynh., etr1-1) with the plants of original ecotype Columbia (Col-0). Because cytokinins can regulate growth of both leaves and roots, we determined the weights of these organs and the ratio between them. The content of zeatin and its riboside in the roots of etr1-1 plants was two times greater than in Col-0 plants, which could be accounted for by inhibition of conversion of these forms of cytokinins into 9-N-glucosides. In the leaves of mutant plants, expression of IPT3 gene responsible for the synthesis of cytokinins was more intense than in Col-0 plants, which could also contribute to a rise in the content of cytokinins. In this case, the weight of roots in etr1-1 mutants was lower than in the plants of original ecotype. Because high concentrations of cytokinins can inhibit root growth, suppression of accumulation of their biomass in mutant plants may be related to a greater content of cytokinins therein. The obtained results suggest that ethylene can suppress accumulation of cytokinins and, thereby, maintain redistribution of biomass in favor of the roots, which is important for plant adaptation to a shortage of water and ions.     

Ethylene evolution and ABA and polyamine contents in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> during UV-B stress

Changes in plant growth, membrane integrity, ethylene evolution, ABA content, and the content of free polyamines were examined in 14-day-old Arabidopsis thaliana (L.) Heynh., strain Columbia (Col-0) plants after a single UV-B irradiation with low (3 kJ/m²), moderate (6–9 kJ/m²), high (18 kJ/m²), and lethal (27 kJ/m²) doses. The UV-B treatment caused dose-dependent suppression of plant growth. One hour after irradiation, the membrane damage was evident from the increased leakage of electrolytes. The low-dose and moderate-dose irradiation caused a transient increase in evolution of ethylene and in the content of putrescine (spermidine and spermine precursor) with the peaks of these parameters attained at 5 and 24 h, respectively. The high-and lethaldose irradiation induced a smaller rise in ethylene evolution, with a slight trend to its decrease, especially, after the exposure to the lethal dose. The high and lethal doses of UV-B suppressed putrescine accumulation, depleted spermidine and spermine pools, and caused severe injuries and plant death. During the first day after irradiation, the ABA content increased in proportion to the irradiation dose. On the second day, the accumulation of ABA was observed in plants irradiated with moderate doses. The accumulation was arrested after a high-dose irradiation and was diminished by 45% after a lethal dose treatment. The results provide evidence for the involvement of ethylene, ABA, and polyamines in plant responses induced by UV-B irradiation.     

Improved Postharvest Quality of Inflorescences of <Emphasis Type="Italic">fbp1::etr1-1</Emphasis> Transgenic <Emphasis Type="Italic">Burrageara</Emphasis> ‘Stefan Isler Lava Flow’

Flowers of the complex orchid hybrid Burrageara ‘Stefan Isler Lava Flow’ had been shown previously to react sensitively to ethylene. Via Agrobacterium tumefaciens, the mutant ethylene receptor ETHYLENE RESPONSE 1 (etr1-1) from Arabidopsis thaliana under the control of the flower-specific promoter FLOWER BINDING PROTEIN 1 (fbp1) from Petunia hybrida was transferred to Burrageara. One single-copy event was analyzed in this study aiming to investigate the expression of the fbp1::etr1-1 transgene in different plant and flower organs by quantitative RT-PCR and the reaction of flowers and inflorescences to ethylene. It was shown that the heterologous promoter led to high expression levels in the perianth of the orchid flowers compared to low levels in leaves and roots. The expression shift to the first whorl (sepals) described here corresponds to extended expression of endogenous B class MADS box homeotic genes in orchids in general. The transgenic plants grew and developed similar to the wild-type plants, except for slightly faster rooting in vitro and smaller flowers. Flower longevity was improved by 7 days in 10 ppm ethylene. Moreover, bud drop starting at day 5 of incubation of inflorescences in 10 ppm ethylene in the wild-type was efficiently prevented for at least 19 days in the fbp1::etr1-1 transgenic plants. The function of the tissue-specific promoter fbp1 and the mutant receptor etr1-1 was shown for the first time in a monocotyledonous plant.     

RNAi-mediated silencing of spermidine synthase gene results in reduced reproductive potential in tobacco

Spermidine belongs to a class of polycationic compounds known as polyamines. Polyamines are known to be involved in a wide range of biological processes but the exact role and contribution of different polyamines to these processes are still not clear. In the present study, we have tried to understand the contribution of triamine spermidine to the growth and development of tobacco by downregulating spermidine synthase gene (SPDS) using RNA interference. Down-regulatioin of SPDS gene resulted in decreased spermidine levels and a slight increase in the levels of its precursor, the diamine putrescine and the molecule downstream of Spd, the tetraamine spermine. While the vegetative growth of the transgenics remained largely unaffected, SPDS down-regulation resulted in smaller size of flowers, decreased pollen viability and seed setting, and a reduced and delayed seed germination. When subjected to abiotic stress, the transgenics showed an increased tolerance to salinity and drought conditions owing to a steady intracellular pool of putrescine and spermine. The results not only highlight the importance of spermidine in determining reproductive potential in plants but have also help delineate its function from that of putrescine and spermine.     

Content of Osmolytes and Flavonoids under Salt Stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Plants Defective in Jasmonate Signaling

The effects of the salt stress (200 mM NaCl) and exogenous jasmonic acid (JA) on levels of osmolytes and flavonoids in leaves of four-week-old Arabidopsis thaliana L. plants of the wild-type (WT) Columbia-0 (Col-0) and the mutant jin1 (jasmonate insensitive 1) with impaired jasmonate signaling were studied. The increase in proline content caused by the salt stress was higher in the Col-0 plants than in the mutant jin1. This difference was especially marked if the plants had been pretreated with exogenous 0.1 μM JA. The sugar content increased in response to the salt stress in the JA-treated WT plants but decreased in the jin1 mutant. Treatment with JA of the WT plants but not mutant defective in jasmonate signaling also enhanced the levels of anthocyanins and flavonoids absorbed in UV-B range in leaves. The presence of JA increased salinity resistance of the Col-0 plants, since the accumulation of lipid peroxidation products and growth inhibition caused by NaCl were less pronounced. Under salt stress, JA almost did not render a positive effect on the jin1 plants. It is concluded that the protein JIN1/MYC2 is involved in control of protective systems under salt stress.     

Combined effect of ethylene- and salicylic acid-signaling insensitive mutation on Arabidopsis response to low temperature

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

Efficient direct plant regeneration from immature leaf roll explants of sugarcane (<Emphasis Type="Italic">Saccharum officinarum</Emphasis> L.) using polyamines and assessment of genetic fidelity by SCoT markers

A rapid and efficient method for in vitro direct plant regeneration from immature leaf roll explants of Saccharum officinarum L. (sugarcane) cv. Co 86032 was developed by the application of exogenous polyamines (PA). The effect of explant source from apical meristems and pre-culture of explants in the dark on shoot regeneration was studied. Adventitious shoot regeneration occurred on the proximal regions of immature leaf roll explants when pre-incubated in the dark for 2 wk and the regeneration response was decreased from the middle to distal end. A higher number of direct shoots (130 primary shoots explant^?1) and multiple shoots (657 secondary shoots explant^?1), were obtained with a combination of spermidine (103.27 μM), spermine (49.42 μM), and putrescine (31.04 μM) along with plant growth regulators. Shoot induction was increased up to twofold and multiplication was increased up to threefold in the medium supplemented with PA. Profuse rooting was observed in putrescine (93.12 μM), spermidine (68.84 μM), and spermine (24.71 μM), with mean number of 57 roots. A twofold increase in the number of roots was observed in medium supplemented with PA with respect to control cultures, which facilitated the successful transplantation and acclimatization process of in vitro propagated sugarcane plants. Histology and scanning electron microscopy analyses supported adventitious direct shoot regeneration from immature leaf roll explants. The genetic stability of in vitro regenerated plants was confirmed using start codon targeted polymorphism marker system.     

Putrescine biosynthesis and export genes are essential for normal growth of avian pathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background

Avian pathogenic Escherichia coli (APEC) is the infectious agent of a wide variety of avian diseases, which causes substantial economic losses to the poultry industry worldwide. Polyamines contribute to the optimal synthesis of nucleic acids and proteins in bacteria. The objectives of this study were to investigate; i) whether APEC E. coli encodes the same systems for biosynthesis and uptake as described for E. coli K12 and ii) the role of polyamines during in vitro growth of an avian pathogenic E. coli strain (WT-ST117- O83:H4T).

Results

Following whole genome sequencing, polyamine biosynthesis and export genes present in E. coli MG1655 (K-12) were found to be identical in WT-ST117. Defined mutants were constructed in putrescine and spermidine biosynthesis pathways (ΔspeB, ΔspeC, ΔspeF, ΔspeB/C and ΔspeD/E), and in polyamines transport systems (ΔpotE, ΔyeeF, ΔpotABCD and ΔpotFGHI). Contrary to what was observed for MG1655, the ΔpotE-ST117 mutant was growth attenuated, regardless of putrescine supplementation. The addition of spermidine or orthinine restored the growth to the level of WT-ST117. Growth attenuation after induction of membrane stress by SDS suggested that PotE is involved in protection against this stress. The ΔspeB/C-ST117 mutant was also growth attenuated in minimal medium. The addition of putrescine or spermidine to the media restored growth rate to the wild type level. The remaining biosynthesis and transport mutants showed a growth similar to that of WT-ST117. Analysis by Ultra-High Performance Liquid Chromatography revealed that the ΔspeB/C mutant was putrescine-deficient, despite that the gene speF, which is also involved in the synthesis of putrescine, was expressed.

Conclusions

Deletion of the putrescine transport system, PotE, or the putrescine biosynthesis pathway genes speB/C affected in vitro growth of APEC (ST117- O83:H4) strain, but not E. coli MG1655, despite the high similarity of the genetic make-up of biosynthesis and transport genes. Therefore, blocking these metabolic reactions may be a suitable way to prevent APEC growth in the host without disturbing the commensal E. coli population.

     

Responses of He-Ne laser on agronomic traits and the crosstalk between UVR8 signaling and phytochrome B signaling pathway in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> subjected to supplementary ultraviolet-B (UV-B) stress

UV-B acclimation effects and UV-B damage repair induced by a 632.8-nm He-Ne laser were investigated in Arabidopsis thaliana plants in response to supplementary UV-B stress. There was an increasing trend in growth parameters in the combination-treated plants with He-Ne laser and UV-B light compared to those stressed with enhanced UV-B light alone during different developmental stages of plants. The photosynthetic efficiency (Pn) and survival rates of seedlings were significantly higher in the combination treatments than UV-B stress alone. The expression of UVR8, phytochrome B (PhyB), and their mediated signal responsive genes such as COP1, HY5, and CHS were also significantly upregulated in plants with the laser irradiation compared with other groups without the laser. Levels of flavonol accumulation in leaves and capsule yield of He-Ne laser-treated plants were increased. The phyB-9 mutants were more sensitive to enhanced UV-B stress and had no obvious improvements in plant phenotypic development and physiological damage caused by enhanced UV-B stress after He-Ne laser irradiation. Our results suggested that UVR8 and its mediated signaling pathway via interaction with COP1 can be induced by He-Ne laser, and these processes were dependent on cytoplasmic PhyB levels in plant cells, which might be one of the most important mechanisms of He-Ne laser on UV-B protection and UV-B damage repair. These current data have also elucidated that the biostimulatory effects of He-Ne laser on Arabidopsis thaliana plants would happen not only during the early growth stage but also during the entire late developmental stage.     

Polyamines stimulate non-photochemical quenching of chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence in <Emphasis Type="Italic">Scenedesmus obliquus</Emphasis>

Polyamines (PAs) are small metabolites that are produced and oxidized in chloroplasts with an obscure mode of action. Recently, we showed that qE is stimulated by PAs in higher plants (Nicotiana tabacum) and in genetically modified plants with elevated thylakoid-associated PAs (Ioannidis and Kotzabasis Biochim Biophys Acta 1767:1371–1382, 2007; Ioannidis et al. Biochim Biophys Acta 1787:1215–1222, 2009). Here, we investigated further their quenching properties both in vivo in green algae and in vitro is isolated LHCII. In vivo spermine up-regulates NPQ in Scenedesums obliquus about 30%. In vitro putrescine—the obligatory metabolic precursor of PAs—has a marginal quenching effect, while spermidine and spermine exhibit strong quenching abilities in isolated LHCII up to 40%. Based on available 3D models of LHCII we report a special cavity of about 600 Å³ and a near-by larger pocket in the trimeric LHCII that could be of importance for the stimulation of qE by amines.     

Constitutive expression of a plant ferredoxin-like protein (pflp) enhances capacity of photosynthetic carbon assimilation in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>)

The plant ferredoxin-like protein (PFLP) gene, cloned from sweet peppers predicted as an electron carrier in photosynthesis, shows high homology to the Fd-I sequence of Arabidopsis thaliana, Lycopersicon esculentum, Oryza sativa and Spinacia oleracea. Most of pflp related studies focused on anti-pathogenic effects, while less understanding for the effects in photosynthesis with physiological aspects, such as photosynthesis rate, and levels of carbohydrate metabolites. This project focuses on the effects of pflp overexpression on photosynthesis by physiological evaluations of carbon assimilation with significant higher levels of carbohydrates with higher photosynthesis efficiency. In this report, two independent transgenic lines of rice plants (designated as pflp-1 and pflp-2) were generated from non-transgenic TNG67 rice plant (WT). Both transgenic pflp rice plants exhibited enhanced photosynthesis efficiency, and gas exchange rates of photosynthesis were 1.3- and 1.2-fold higher for pflp-1 and pflp-2 than WT respectively. Significantly higher electron transport rates of pflp rice plants were observed. Moreover, photosynthetic products, such as fructose, glucose, sucrose and starch contents of pflp transgenic lines were increased accordingly. Molecular evidences of carbohydrate metabolism related genes activities (osHXK5, osHXK6, osAGPL3, osAGPS2α, osSPS, ospFBPase, oscFBPase, and osSBPase) in transgenic lines were higher than those of WT. For performance of crop production, 1000-grain weight for pflp-1 and pflp-2 rice plants were 52.9 and 41.1 g that were both significantly higher than 31.6 g for WT, and panicles weights were 1.4- and 1.2-fold higher than WT. Panicle number, tiller number per plants for pflp rice plants were all significantly higher compared with those of WT where there was no significant difference observed between two pflp rice plants. Taken altogether; this study demonstrated that constitutive pflp expression can improve rice production by enhancing the capacity of photosynthetic carbon assimilation.     

Characterization of a maize ERF gene, <Emphasis Type="Italic">ZmERF1,</Emphasis> in hormone and stress responses

ERFs are downstream component in ethylene signaling pathway and involved in plant’s abiotic stress response. The specific role of ERFs under stress and the molecular mechanism underlying the signaling cross talk still need to be elucidated. This study describes the isolation and characterization of ZmERF1 promoter. There were many cis-regulatory elements related to stress responses in the ZmERF1 promoter sequence. ZmERF1 could be highly induced by ABA and ethylene treatment in maize, suggesting that it might be at the crossroads of multiple hormone signaling pathways. Furthermore, ZmERF1 transgenic Arabidopsis lines (35S::ZmERF1) showed higher salt-tolerant, drought- and heat resistance. Consistently, tolerance-related genes were up-regulated in 35S::ZmERF1 lines compared with the WT plants in Arabidopsis. Overall, ZmERF1 might play an important role in plant resistance to a coercive environment by mediating various physiological processes via ethylene and ABA signaling pathways.