Effects of plant growth regulators on transient expression of foreign gene in Nicotiana benthamiana L. leaves.

BACKGROUND: In the last decades, replicating expression vectors based on plant geminivirus have been widely used for enhancing the efficiency of plant transient expression. By using the replicating expression vector derived from bean yellow dwarf virus and green fluorescent protein as a reporter, we investigated the effects of α-naphthalene acetic acid, gibberellins3, and 6-benzyladenine, as three common plant growth regulators, on the plant biomass and efficiency of transient expression during the process of transient expression in Nicotiana benthamiana L. leaves. RESULTS: With the increase of the concentration of α-naphthalene acetic acid, gibberellins3, and 6-benzyladenine (from 0.1 to 1.6 mg/L), the fresh weight, dry weight, and leaf area of the seedlings increased first and then returned to the levels similar to the controls (without chemical treatment). The treatment with α-naphthalene acetic acid at 0.2 and 0.4 mg/L can enhance the level of transient expression of green fluorescent protein, which peaked at 0.4 mg/L α-naphthalene acetic acid and was increased about by 19%, compared to the controls. Gibberellins3 at 0.1-0.4 mg/L can enhance the level of transient expression of green fluorescent protein, which peaked at 0.2 mg/L gibberellins3 and was increased by 25%. However, the application of 6-benzyladenine led to decrease in the level of transient expression of green fluorescent protein. CONCLUSIONS: The appropriate plant growth regulators at moderate concentration could be beneficial to the expression of foreign genes from the Agrobacterium-mediated transient expression system in plants. Thus, appropriate plant growth regulators could be considered as exogenous components that are applied for the production of recombinant protein by plant-based transient expression systems.

[Regulation of extracellular ATP onchlorophyll content and fluorescence characteristics of Angelica sinensis seedlings under drought and low temperature stress].

As an important signal molecule, extracellular ATP(eATP) can regulate many physiological and biochemical responses to plant stress. In this study, the regulation of extracellular ATP(eATP) on chlorophyll content and chlorophyll fluorescence parameters of Angelica sinensis seedlings were studied under drought and low temperature stress. The results showed that all the chlorophyll content, the actual photochemical efficiency [Y(Ⅱ)], the electron transfer rate(ETR), the photochemical quenching coefficient(qP and qL) of A. sinensis leaves were significantly decreased under drought and low temperature stress, respectively. At the same time, non-photochemical quenching(NPQ and qN) were also all significantly increased, respectively. The application of eATP alleviated the decrease of chlorophyll content, Y(Ⅱ), ETR, qP and qL of A. sinensis leaves under drought and low temperature stress, and eliminated the increase of qN and NPQ. The results indicated that eATP could effectively increase the open ratio of PSⅡ reaction centers, and improve the electron transfer rate and light energy conversion efficiency of PSⅡ of A. sinensis leaves under drought and low temperature stress. It is beneficial to enhance the chlorophyll synthesis and the adaptability of PSⅡ about A. sinensis seedlings to drought and low temperature stress.

Extracellular ATP released by copper stress could act as diffusible signal in alleviating the copper stress-induced cell death.

In the present work, by using tobacco cell suspension and wheat seedlings, we studied that eATP (extracellular ATP) released by copper (Cu) stress could act as diffusible signal in alleviating the Cu stress-induced cell death. A semipermeable membrane was fixed in the middle of a plastic box to divide the box into two equal compartments (A and B, respectively). This semipermeable membrane can prevent direct cell-to-cell (or seedling-to-seedling) contact and the diffusion of the macromolecules [such as ATPase (adenosine 5'-triphosphatase)] between these two compartments. The cell suspension directly stressed with CuCl2 was placed in compartment A and was incubated with the untreated cell suspension in compartment B. Such treatment significantly increased the levels of cell death and eATP content of the cell suspension in these two compartments. In contrast, addition of ATPase into the cell suspension directly stressed with CuCl2 decreased the eATP level in these two compartments but further increased the level of cell death in compartment B, compared to no addition of ATPase. Similar results were obtained when tobacco cell suspension was replaced by wheat seedlings. These observations indicate that when Cu stress from compartment A induced the plant cell death in compartment B, ATP transferred from compartment A could play a role in alleviating this cell death. Thus, it is suggested that eATP released by copper stress could act as diffusible signal in alleviating the Cu stress-induced cell death.

Effects of extracellular ATP on local and systemic responses of bean (Phaseolus vulgaris L) leaves to wounding.

Wounding increased the extracellular Adenosine 5'-triphosphate (eATP) level of kidney bean leaves. Treatment with wounding or exogenous ATP increased the hydrogen peroxide (H2O2) content, activities of catalase and polyphenol oxidase, and malondialdehyde content in both the treated and systemic leaves. Pre-treatment with ATP-degrading enzyme, apyrase, to the wounded leaves reduced the wound-induced local and systemic increases in H2O2 content, activities of catalase and polyphenol oxidase, and malondialdehyde content. Application of dimethylthiourea (DMTU) and diphenylene iodonium (DPI) to the wounded and ATP-treated leaves, respectively, reduced the wound- and ATP-induced local and systemic increases in H2O2 content, activities of catalase and polyphenol oxidase, and malondialdehyde content. Moreover, the wound- and ATP-induced systemic increases of these physiological parameters were suppressed when DMTU or DPI applied to leaf petiole of the wounded and ATP-treated leaves. These results suggest that eATP at wounded sites could mediate the wound-induced local and systemic responses by H2O2-dependent signal transduction.

[Physiological effects of cadmium stress on Astragalus membranaceus seedlings and alleviative effects of attapulgite clay on cadmium stress].

In this study we investigate the effects of cadmium stress on Astragalus membranaceus seedlings and the alleviative effects of attapulgite clay in growth substrate on cadmium stress to A. membranaceus seedlings. The results showed that the Y (Ⅱ) (effective photochemical quantum yield of PSⅡ photosynthetic), qP(photochemical quenching coefficient), ETR(the rate of non-cyclic electrontransport through PSⅡ), and chlorophyll content of the leaves were significantly decreased with the increase of cadmium concentrations, while the cadmium content, non-photochemical quenching(NPQ, qN) of the leaves and cadmium content, MDA content, plasma membrane permeability, and the damage degree of root apical membrane of the roots were significantly increased. Simultaneously, the activities of APX(ascorbate peroxidase), SOD(superoxide dismutase), POD(peroxidase), CAT(catalase), soluble protein content, and soluble sugar content of roots were increased first but then decreased with the increasing cadmium concentration. Under the condition of without Cd stress, the attapulgite clay into the growth substrate did not significantly affect above physiological indexes of leaves, but significantly increased SOD activity and soluble sugar content of roots and decreased the MDA content, damage degree of root apical membrane of roots, while other physiological indexes did not significantly change. Under cadmium stress, the presence of attapulgite clay in the growth substrate significantly alleviated the cadmium-induced decreases Y (Ⅱ), qP, ETR and chlorophyll content of leaves, and the CAT activity, soluble protein content, and soluble sugar content of roots. Under condition with cadmium stress, the presence of attapulgite clay significantly alleviated the cadmium-induced increases of leaves cadmium content, qN and NPQ, and the cadmium content, MDA content, plasma membrane permeability, damage degree of root apical membrane, SOD, POD, and APX activity of the roots. And, the alleviative effects of attapulgite clay on cadmium stress to A. membranaceus roots were more obvious with the increase of cadmium stress time. The above results showed that the addition of attapulgite clay into the growth substrate has certain alleviative effect on the cadmium stress to A. membranaceus seedlings.

The responses of photosystem II and intracellular ATP production of Arabidopsis leaves to salt stress are affected by extracellular ATP.

Hypertonic salt stress with different concentrations of NaCl increased the levels of extracellular ATP of Arabidopsis leaves. And, hypertonic salt stress decreased the levels of F v /F m (the maximal efficiency of photosystem II), Φ PSII (the photosystem II operating efficiency), qP (photochemical quenching), and intracellular ATP (iATP) production. The treatment with ß,γ-methyleneadenosine 5'-triphosphate (AMP-PCP), which can exclude extracellular ATP from its binding sites of extracellular ATP receptors, caused a further decrease in the levels of F v /F m , Φ PSII, qP, and iATP production of the salt-stressed Arabidopsis leaves, while the addition of exogenous ATP rescued the inhibitory effects of AMP-PCP on Φ PSII , qP, and iATP production under hypertonic salt stress. Under hypertonic salt stress, the values of F v /F m , Φ PSII , qP, and iATP production were lower in the dorn 1-3 mutant than in the wild-type plants. These results indicate that the responses of photosystem II and intracellular ATP production to salt stress could be affected by extracellular ATP.

Enhanced expression of alternative oxidase genes is involved in the tolerance of rice (Oryza sativa L.) seedlings to drought stress.

Drought stress significantly enhanced the capacity of the alternative respiratory pathway and induced AOX1a and AOX1b transcripts in rice seedling leaves. The drought-stressed seedlings pretreated with the inhibitor of the alternative respiratory pathway, 1 mM salicylhydroxamic acid, had a lower level of relative water content than the seedlings either subjected to drought or salicylhydroxamic acid treatment alone. This observation suggests that the alternative respiratory pathway could play a role in the tolerance of rice seedlings to drought stress. Pretreatment with exogenous hydrogen peroxide, salicylic acid, and abscisic acid alone mitigated the water loss of rice leaves exposed to drought stress. Exogenous application of hydrogen peroxide and salicylic acid increased the capacity of the alternative respiratory pathway and induced AOX1a and AOX1b transcripts, while exogenous abscisic acid failed to induce any expression of AOX1 genes. These observations suggest that rice AOX1a and AOX1b genes may be responsive especially to drought stress but not be induced by all of the stress signals related to drought.

[Analysis of nonenzymic source of superoxide anion in pathogenic Xanthomonas oryzae pv. oryzae].

With electron spin resonance and chemical assay, the production of superoxide anion in the Xanthomonas oryzae pv. oryzae strain OS-14 suspension is extracellularly localized. It was found that superoxide anion produced in the filtrate. It was confirmed that peptide or enzyme did not contribute to the production of superoxide anion in the filtrate of OS-14. Organic acids were shown to be the primary source of superoxide anion produced in the filtrate of OS-14. These results indicated that non-enzymic molecules might be another source of superoxide anion produced by Xanthomonas oryzae pv. oryzae, in addition to enzymes, which suggested the multiform generation of superoxide anion.

[Expression and function of AOX1 gene family during the greening of etiolated rice seedlings].

Under constant light, increases in V(t) (total respiration rate), V(alt) (capacity of alternative pathway), and V(alt)/V(t) ration values (Figs.1-3) in fully etiolated rice seedlings leaves were founded. Northern hybridization with special probes for rice AOX1a, AOX1b and AOX1c revealed that the AOX1c mRNA was mainly present under constant light (Fig.4). SHAM (salicylhydroxamic acid) decreased the relative oxygen evolution rate and chlorophyll content during the rapid greening of the etiolated rice seedlings (Figs.6, 7). We found that illumination caused bigger increase in V(alt) in fully etiolated rice seedlings than in any partially etiolated rice seedlings (Fig.8A), while the soluble sugar contents did not show any significant differences (Fig.8B). This revealed the connection between greening process and cyanide-resistant respiration in rice seedlings. We infer that one of the possible functions of the cyanide-resistant respiration is to maintain a high rate of respiratory electron transport for the essential biosynthesis processes at the early stage of chloroplast development. Cyanide-resistant respiration may provide a regulatory mechanism for the balance between the requirements of the energy and substances.

[Effects of drought stress on cyanide-resistant respiration and metabolism of reactive oxygen in wheat seedling].

The changes in cyanide-resistant respiration and metabolism of active oxygen species in drought-sensitive and drought-resistant wheat seedlings under water stress were studied. In general, drought stress decreased the level of cyanide-resistant respiration and AOX1 mRNA content in wheat seedling leaves. But the drought-resistant cultivars, which had much higher level of cyanide-resistant respiration and AOX1 mRNA content than the drought-sensitive ones, presented an ability to adapt to mild drought stress. Under drought stress, the drought-sensitive wheat seedling leaves had higher O(-.)(2) and H(2)O(2) contents, but lower SOD, POX and CAT activities than drought-resistant cultivars. We guessed that severe drought stress could break down the balance between active oxygen species and cyanide-resistant respiration. However, cyanide-resistant respiration still could participate in the drought-resistance by lowering the level of active oxygen species besides by other mechanism.