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Previous studies have shown that TaNHX2 transgenic alfalfa (Medicago sativa L.) accumulated more K(+) and less Na(+) in leaves than did the wild-type plants. To investigate whether the increased K(+) accumulation in transgenic plants is attributed to TaNHX2 gene expression and whether the compartmentalization of Na(+) into vacuoles or the intracellular compartmentalization of potassium is the critical mechanism for TaNHX2-dependent salt tolerance in transgenic alfalfa, aerated hydroponic culture was performed under three different stress conditions: control condition (0.1 mM Na(+) and 6 mM K(+) inside culture solution), K(+)-sufficient salt stress (100 mM NaCl and 6 mM K(+)) and K(+)-insufficient salt stress (100 mM NaCl and 0.1 mM K(+)). The transgenic alfalfa plants had lower K(+) efflux through specific K(+) channels and higher K(+) absorption through high-affinity K(+) transporters than did the wild-type plants. Therefore, the transgenic plants had greater K(+) contents and [K(+)]/[Na(+)] ratios in leaf tissue and cell sap. The intracellular compartmentalization of potassium is critical for TaNHX2-induced salt tolerance in transgenic alfalfa.

Soybean is a difficult crop to manipulate through Agrobacteriumtumefaciens-mediated genetic transformation. Plant–bacterium interaction plays an important role in the transformation process. Being rich with isoflavones, soybean may have an adverse effect on the A. tumefaciens-mediated genetic transformation. To investigate whether high content of endogenous isoflavones in soybean is a serious obstacle in achieving high efficient Agrobacterium-mediated transformation in soybean, a series of experiments on inhibition of isoflavone biosynthesis were conducted to improve upon soybean transformation efficiency. Results indicated that soybean isoflavones inhibited A. tumefaciens growth and respiration, the transformation efficiency [β-glucuronidase (GUS) transient expression] was negatively correlated with the phenylalanine ammonia-lyase activity and isoflavones content. The biosynthesis of soybean isoflavones was partially inhibited by sonication treatment and applying antagonists in co-culture medium and thereby decreased the adverse effects of isoflavones on Agrobacterium infection. A discernible improvement in transformation efficiency was achieved when sonication at 40 kHz for 3 min was applied along with Agro-infection and the explants were cultured on co-culture medium containing 20 μM α-aminooxyacetic acid (AOA), with the percentage of GUS transient expression as 41.4 %, being 3.6 times higher than that not sonicated and co-cultured on medium without AOA. Sonication was found not only to simply make micro-wounds for Agrobacterium to penetrate or releasing phenolic compounds for induced Agrobacterium vir gene expression; it disturbed the biosynthesis of isoflavones at the transcription level and decreased the adverse effects of isoflavones on soybean transformation, and thereby improving soybean transformation efficiency.

Hybrid millet Zhang and its parental cultivars were studied for their potassium (K) uptake and accumulation characteristics and related physiological mechanisms. Hydroponic culture was performed with two K levels (i.e., high and low) set up. At high K level, hybrid millet showed heterobeltiosis in K accumulation and leaf K content, and it also had higher H+-ATPase activity, respiration rate, root oxidant activity and root K+ influx rate than its parental cultivars. All these lay the physiological foundation of heterosis for potassium uptake of hybrid millet. At low K level, the hybrid millet had a lower H+-ATPase activity and a higher K efflux rate than its parental cultivars, thus heterobeltiosis in K accumulation or leaf K content was not observed. Therefore, high level K fertilizer application is recommended for hybrid millet cultivation as it is favorable for hybrid millet to display heterosis in K uptake and K accumulation.

Gene BADH was transformed into alfalfa (Medicago sativa L.) through Agrobacterium-mediated transformation method, and salt tolerance of the transgenic progenies was assessed. Among 247 plants obtained after transformation, 43 were positive by polymerase chain reaction (PCR) detection using the primers specific for BADH gene. Reverse transcription polymerase chain reaction (RT-PCR) analysis indicated that gene BADH was expressed in 42 plants at transcriptional level. Southern blotting analysis also showed that the target was integrated into Medicago genome. The transgenic plants grew vigorous in salt stress condition, whereas the wild type plants was retarded and did not survive after cradle. The relative electrical conductivity and malondialdehyde (MDA) contents in the T1 transgenic plants were lower, but peroxidase (POD) and superoxide dismutase (SOD) activities were higher than those of the wild type plants. These results demonstrated that the expression of foreign BADH gene enhanced the salt tolerance in transgenic alfalfa.

The changes of cytosolic Ca2+ fluorescence intensity and the activities of calcium channel of primary maize root tip cells induced by PEG6000 or abscisic acid(ABA) were studied by both confocal techniques and the whole-cell patch clamping in this study. The Ca2+ fluorescence intensity increased while treated with PEG or ABA within 10 min, illuminating that Ca2+ participated in the process of ABA signal transduction. For further proving the mechanism and origin of cytosolic Ca2+ increase induced by PEG treatments, N,N,N',N'-tetraacetic acid (EGTA), Verapamil (VP) and Trifluoperazine (TFP) were added to the PEG solution in the experiments separately. The results showed that Ca2+ fluorescence intensity induced by PEG was suppressed by both EGTA and VP obviously in the root tip cells. The Ca2+ fluorescence intensity of plants changed after the addition of CaM inhibitor TFP while subjected to osmotic stress, which seemed to show that CaM participated in the process of signal transduction of osmotic stress too. The mechanism about it is unknown today. Further, a hyperpolarization-activated calcium permeable channel was recorded in plasma membrane of maize root tip cells. The Ca2+ current (I-Ca) intensity increased remarkably after PEG treatment, and the open voltage of the calcium conductance increased. Similar changes could be observed after ABA treatment, but the channel opened earlier and the current intensity was stronger than that of PEG treatment. The activation of calcium channel initiated by PEG strongly was inhibited by EGTA, VP or TFP respectively. The results revealed that Ca2+ participated in the signals transduction process of osmotic stress, and the cytosolic free Ca2+ increase by osmotic stress mainly came from the extracellular, and some came from the release of cytoplasmic calcium pool. (C) 2010 Elsevier Masson SAS. All rights reserved.

Maize cultivar zhengdan958 was selected as materials. The sub-cellular distribution of soluble calcium at different phases was shown by the potassium-pyroantinonate-precipitation method and transmission electron microscopy. The results showed that the deposits of calcium antimonate as the indicator for Ca(2+) localization were mainly concentrated within the vacuoles and intercellular spaces without PEG treatment. Firstly, when the leaf was treated with PEG, the Ca(2+) level increased remarkably in the cytoplasm, but considerably decreased in vacuoles and intercellular gaps. Meanwhile, the level of Ca(2+) also increased in chloroplast and nucleus. When the treatment continued, the level of Ca(2+) in chloroplasts and nucleus continued to increase and some cells and chloroplasts finally disintegrated, showing that there is a relationship between the distribution of Ca(2+) and the super-microstructure of cells. Ca(2+) plays a role in the plant drought resistance. The changes of cytosolic Ca(2+) localization in cells treated by ABA, EGTA, Verapamil and TFP were investigated too. The increase of cytosolic calcium induced by ABA was mainly caused by calcium influx. Calmodulin participated in ABA signal transduction, which was indicated by the variation of cytosolic Ca(2+)/CaM concentration change induced by ABA. The above results provided a direct evidence for calcium ion as an important signal at the experimental cellular level.

In this study, the direct effects of exterior abscisic acid (ABA) on both calcium distribution of mesophyll cells and cytosolic calcium concentration of guard cells were examined. The distribution of Ca2+ localization were observed with calcium antimonate precipitate-electromicroscopic-cyto-chemical methods after treated with ABA and pretreated with ethylene glycol-bis-(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), verapamil (Vp), and trifluoperazine (TFP). The laser scanning confocal microscopy was used to measure the cytosolic calcium concentrations of guard cells under different treatments. The results showed that the cytosolic Ca2+ concentration of mesophyll cells was induced to increase by ABA, but to decrease in both outside cell and the vacuoles within 10 min after treatments. The cytosolic calcium concentration of guard cells was increased gradually with the lag in treatment time. However, both EGTA and TFP could inverse those effects, indicating that the increase of cytosolic calcium induced by exterior ABA was mainly caused by calcium influx. The results also showed that calmodulin could influence both the calcium distribution of mesophyll cells and calcium concentration of guard cells. It shows that calmodulin participates in the process of ABA signal transduction, but the mechanism is not known as yet. The changes both calcium distribution of mesophyll cells and calcium concentration of guard cells further proved that the variations of cytosolic Ca2+ concentration induced by ABA were involved in the stomatal movements of maize seedlings.

Antagonists and sonication treatment relieved the structural barriers of Agrobacterium entering into cells; hindered signal perception and transmission; alleviated defense responses and increased cell susceptibility to Agrobacterium infection. Soybean gene expression analysis was performed to elucidate the general response of soybean plant to Agrobacterium at an early stage of infection. Agrobacterium infection stimulated the PAMPs-triggered immunity (BRI1, BAK1, BZR1, FLS2 and EFR) and effector-triggered immunity (RPM1, RPS2, RPS5, RIN4, and PBS1); up-regulated the transcript factors (WRKY25, WRKY29, MEKK1P, MKK4/5P and MYC2) in MAPK pathway; strengthened the biosynthesis of flavonoid and isoflavonoid in the second metabolism; finally led to a fierce defense response of soybean to Agrobacterium infection and thereby lower transformation efficiency. To overcome it, antagonist α-aminooxyacetic acid (AOA) and sonication treatment along with Agrobacterium infection were applied. This novel method dramatically decreased the expression of genes coding for F3'H, HCT, β-glucosidase and IF7GT, etc., which are important for isoflavone biosynthesis or the interconversion of aglycones and glycon; genes coding for peroxidase, FLS2, PBS1 and transcription factor MYC2, etc., which are important components in plant-pathogen interaction; and genes coding for GPAT and α-L-fucosidase, which are important in polyesters formation in cell membrane and the degradation of fucose-containing glycoproteins and glycolipids on the external surface of cell membrane, respectively. This analysis implied that AOA and sonication treatment not only relieved the structural membrane barriers of Agrobacterium entering into cells, but also hindered the perception of 'invasion' signal on cell membrane and intercellular signal transmission, thus effectively alleviated the defense responses and increased the cell susceptibility to Agrobacterium infection. All these factors benefit the transformation process; other measures should also be further explored to improve soybean transformation.

Based on the information of 25 heat shock transcription factor (Hsf) homologues in maize according to a genome-wide analysis, ZmHsf06 was cloned from maize leaves and transformed into Arabidopsis thaliana (L. Heynh.) (ecotype, Col-0). Three transgenic positive lines were selected to assess the basic and acquired thermotolerance and drought-stress tolerance under stresses and for some physiological assays. The sequence analysis indicates that ZmHsf06 contained the characteristic domains of class A type plant Hsfs. The results of qRT–PCR showed that the expression levels of ZmHsf06 were elevated by heat shock and drought stress to different extents in three transgenic lines. Phenotypic observation shows that compared with the Wt (wild-type) controls, the overexpressing ZmHsf06 of Arabidopsis plants have enhanced basal and acquired thermotolerance, stronger drought-stress tolerance and growth advantages under mild heat stress conditions. These results are further confirmed by physiological and biochemical evidence that transgenic Arabidopsis plants exhibit higher seed germination rate, longer axial-root length, higher activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), higher leaf chlorophyll content, but lower relative electrical conductivity (REC), malondialdehyde (MDA) and osmotic potential (OP) than the Wt controls after heat shock and drought treatments. ZmHsf06 may be a central representative of maize Hsfs and could be useful in molecular breeding of maize or other crops for enhanced tolerances, particularly during terminal heat and drought stresses.

Resistance to maize southern leaf spot disease was induced by the low-concentration filtrate of Bipolaris maydis race T cultivation in an experiment. The nuclear neterogeny corn C103 was used as the test material. The lesion area on the leaves was significant difference by connalysis. The lesion areas on pretreated leaves were (0.3 ± 0.05)–(0.9 ± 0.5) mm 2 , but those on the control were (23.1 ± 8.7) mm 2 . At the same time, the changes in peroxidase, phenylalanine, ammonialyase, and malondialdehvde activities were determined. During 0–96 h of inspection, phenylalanine and ammonialyase (PAL) activities increased by 64.2%, peroxidase (POD) activities increased by 41.2%, but the malondialdenvde (MDA) content decreased by 29.7% compared with the control. It seems that the low-concentration filtrate of Bipolaris maydis race T cultivation itself can be used as an elicitor to enhance the induced resistance.

TaNHX2, a vacuolar Na+-H+ antiport gene from wheat (Triticum aestivum L.), was transformed into alfalfa (Medicago sativa L.) via Agrobacterium-mediated transformation to evaluate the role of vacuolar energy providers in plant salt stress responses. PCR and Southern blotting analysis showed that the target gene was integrated into the Medicago genome. Reverse transcription-PCR indicated that gene TaNHX2 was expressed at the transcriptional level. The relative electrical conductivity in the T2 transgenic plants was lower and the osmotic potential was higher compared to the wild-type plants under salt stress conditions. The tonoplast H+-ATPase, H+-pyrophosphatase (PPase) hydrolysis activities and ATP-dependent proton pump activities in transgenic plants were all higher than those of wild-type plants, and the enzyme activities could be induced by salt stress. The PPi-dependent proton pump activities decreased when NaCl concentrations increased from 100mM to 200mM, especially in transgenic plants. The vacuolar Na+-H+ antiport activities of transgenic plants were 2-3 times higher than those of the wild -type plants under 0mM and 100mM NaCl stress. Na+-H+ antiport activity was not detectable for wild-type plants under 200mM NaCl, but for transgenic plants, it was further increased with an increment in salt stress intensity. These results demonstrated that expression of the foreign TaNHX2 gene enhanced salt tolerance in transgenic alfalfa.

The changes of cell ultra structure as well as Ca2+ homeostasis involved in the drought-induced maize leaf senescence was investigated. Meanwhile, many indicatives of leaf senescence including thiobarbituric acid reactive substance (MDA), electrolyte leakage (EL), and chlorophyll along with soluble proteins were also detected during the process. The Polyethylene glycol6000(PEG6000)-incubated detached leaves showed a slight increase in the MDA content and electrolyte leakage during the first 30 min of our detection, which was corresponded to an unobvious alteration of the cell ultrastructure. Other typical senescence parameters measured in whole leaf exhibited a moderate elevation as well. Thereafter, however, the EL and MDA rose to a large extent, which was correlated with a dramatic damage to the cell ultrastructure with concomitant sharp decrease in the chlorophyll and soluble proteins content. The deposits of calcium antimonite, being an indicator for Ca2+ localization, were observed in the vacuoles as well as intercellular spaces in the leaves grown under normal condition. Nevertheless, after PEG treatment, it was revealed a distinct increment of Ca2+ in the cytoplasm as well as chloroplasts and nuclei. Moreover, with long-lasting treatment of PEG to the detached leaves, the concentration of Ca2+ as described above showed a continuous increment which was consist with the remarked alteration of physiological parameters and severe damage to the ultrastructure of cells, all of which indicated the leaf senescence. Such drought-induced leaf senescence might result from a loss of the cell's capability to extrude Ca2+. All above findings give us a good insight into the important role of Ca2+ homeostasis in the process of leaf senescence accelerated by the drought stress.

Serving as an important second messenger, calcium ion has unique properties and universal ability to transmit diverse signals that trigger primary physiological actions in cells in response to hormones, pathogens, light, gravity, and stress factors. Being a second messenger of paramount significance, calcium is required at almost all stages of plant growth and development, playing a fundamental role in regulating polar growth of cells and tissues and participating in plant adaptation to various stress factors. Many researches showed that calcium signals decoding elements are involved in ABA-induced stomatal closure and plant adaptation to drought, cold, salt and other abiotic stresses. Calcium channel proteins like AtTPC1 and TaTPC1 can regulate stomatal closure. Recently some new studies show that Ca(2+) is dissolved in water in the apoplast and transported primarily from root to shoot through the transpiration stream. The oscillating amplitudes of [Ca(2+)](o) and [Ca(2+)](i) are controlled by soil Ca(2+) concentrations and transpiration rates. Because leaf water use efficiency (WUE) is determined by stomatal closure and transpiration rate, so there may be a close relationship between Ca(2+) transporters and stomatal closure as well as WUE, which needs to be studied. The selection of varieties with better drought resistance and high WUE plays an increasing role in bio-watersaving in arid and semi-arid areas on the globe. The current paper reviews the relationship between calcium signals decoding elements and plant drought resistance as well as other abiotic stresses for further study.

TaNHX2, a vacuolar Na+–H+ antiport gene from wheat (Triticum aestivum L.), was transformed into alfalfa (Medicago sativa L.) via Agrobacterium-mediated transformation to evaluate the role of vacuolar energy providers in plant salt stress responses. PCR and Southern blotting analysis showed that the target gene was integrated into the Medicago genome. Reverse transcription–PCR indicated that gene TaNHX2 was expressed at the transcriptional level. The relative electrical conductivity in the T2 transgenic plants was lower and the osmotic potential was higher compared to the wild-type plants under salt stress conditions. The tonoplast H+-ATPase, H+-pyrophosphatase (PPase) hydrolysis activities and ATP-dependent proton pump activities in transgenic plants were all higher than those of wild-type plants, and the enzyme activities could be induced by salt stress. The PPi-dependent proton pump activities decreased when NaCl concentrations increased from 100 mM to 200 mM, especially in transgenic plants. The vacuolar Na+–H+ antiport activities of transgenic plants were 2–3 times higher than those of the wild -type plants under 0 mM and 100 mM NaCl stress. Na+–H+ antiport activity was not detectable for wild-type plants under 200 mM NaCl, but for transgenic plants, it was further increased with an increment in salt stress intensity. These results demonstrated that expression of the foreign TaNHX2 gene enhanced salt tolerance in transgenic alfalfa.