S. R. Guo

Effects of salt stress on the structure and function of the photosynthetic apparatus in Cucumis sativus and its protection by exogenous putrescine

With the objective to clarify the physiological significance of polyamines (PAs) in the photosynthetic apparatus, the present study investigated the effects of salt stress with and without foliar application of putrescine (Put) on the structure and function of the photosynthetic apparatus in cucumber. Salt stress at 75 mM NaCl for 7 days resulted in a severe reduction of photosynthesis. The fast chlorophyll afluorescence transient analysis showed that salt stress inhibited the maximum quantum yield of PSII photochemistry (F(v)/F(m)), mainly due to damage at the receptor side of PSII. In addition, salt stress decreased the density of active reaction centers and the structure performance. The microscopic analysis revealed that salt stress-induced destruction of the chloroplast envelope and increased the number of plastoglobuli along with aberrations in thylakoid membranes. Besides, salt stress caused a decrease in the content of endogenous PAs, conjugated and bound forms of spermidine and spermine in particular, in thylakoid membranes. However, applications of 8 mM Put alleviated the salt stress-mediated decrease in net photosynthetic rates (Pn) and actual efficiency of PSII(Φ(PSII)). Put increased PAs in thylakoid membranes and overcame the damaging effects of salt stress on the structure and function of the photosynthetic apparatus in salt-stressed plant leaves. Put application to control plants neither increased PAs in thylakoid membranes nor affected photosynthesis. These results indicate that PAs in chloroplasts play crucial roles in protecting the thylakoid membranes against the deleterious influences of salt stress. In addition, the present results point to the probability that the salt-induced dysfunction of photosynthesis is largely attributable to the loss of PAs in the photosynthetic apparatus.

Proteomic analysis of cucumber seedling roots subjected to salt stress.

To understand metabolic modifications in plants under salt stress, the physiological and biochemical responses of cucumber (Cucumis sativus L. cv. Jinchun No. 2) seedlings to salt stress was investigated. The dry weight and fresh weight of cucumber seedling roots were significantly reduced by treatment with NaCl; Na(+) and Cl(-) were increased, while K(+) and K(+)/Na(+) ratio were decreased. To identify components of salt stress signaling, we compared the high resolution two-dimensional gel electrophoresis (2-DE) protein profiles of control and NaCl-treated roots, and the intensity of 34 protein spots varied. Of these spots, the identities of 29 (21 up-regulated and 8 down-regulated protein spots induced after salt stress) were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography electro-spray ionization tandem mass spectrometry (LC-ESI-MS/MS). The majority of the proteins had functions related to metabolism, energy and transport, and are involved in regulating reactions and defending against stress. A semi-quantitative reverse transcriptional-polymerase chain reaction (PCR) approach based on peptide sequences was used to compare transcript and protein accumulation patterns for 10 candidate proteins. Of these proteins, 8 patterns of induced transcript accumulation were consistent with those of induced protein accumulation. It is therefore likely that the response of the plants proteome to NaCl stress is complex, and that the identified proteins may play an important role in regulating adaptation activities following exposure to NaCl stress in order to facilitate ion homeostasis.

Proteomic study participating the enhancement of growth and salt tolerance of bottle gourd rootstock-grafted watermelon seedlings.

An insertion grafting technique to do research on salt tolerance was applied using watermelon (Citrullus lanatus [Thunb.] Mansf. cv. Xiuli) as a scion and bottle gourd (Lagenaria siceraria Standl. cv. Chaofeng Kangshengwang) as a rootstock. Rootstock-grafting significantly relieved the inhibition of growth and photosynthesis induced by salt stress in watermelon plants. Proteomic analysis revealed 40 different expressed proteins in response to rootstock-grafting and/or salt stress. These proteins were involved in Calvin cycle, amino acids biosynthesis, carbohydrate and energy metabolism, ROS defense, hormonal biosynthesis and signal transduction. Most of these proteins were up-regulated by rootstock-grafting and/or susceptible to salt stress. The enhancement of the metabolic activities of Calvin cycle, biosynthesis of amino acids, carotenoids and peroxisomes, glycolytic pathway and tricarboxylic acid cycle will probably contribute to intensify the biomass and photosynthetic capacity in rootstock-grafted seedlings under condition without salt. The accumulation of key enzymes included in these biological processes described above seems to play an important role in the enhancement of salt tolerance of rootstock-grafted seedlings. Furthermore, leucine-rich repeat transmembrane protein kinase and phospholipase may be involved in transmitting the internal and external stimuli induced by grafting and/or salt stress.

Effects of 24-epibrassinolide on nitrogen metabolism in cucumber seedlings under Ca(NO3)2 stress

Ca(NO(3))(2) accumulation is a major factor that limits greenhouse production in China. The present investigation was carried out to study the effect of 24-epibrassinolide (EBL) on nitrogen metabolism (including contents of NO(3)(-), NH(4)(+) and amino acids and related enzymes activities) in cucumber seedlings (Cucumis sativus L. cv. Jinyou No. 4) under 80 mM Ca(NO(3))(2) stress. This study found that exogenous EBL significantly reduced the accumulation of NO(3)(-) and NH(4)(+) by Ca(NO(3))(2), and enhanced the inactivated enzymes activities involved in the nitrogen metabolism. In addition, EBL alleviated the inhibition of photosynthesis nitrogen-use efficiency by Ca(NO(3))(2). Increased total amino acids by EBL under stress increased the precursor of proteins biosynthesis, thus promoting the biosynthesis nitrogen containing compounds. The presence of Ca(NO(3))(2) increased polyamines level, which might result from the increased content of free putrescine that is harmful to plant growth. However, exogenous EBL induced a further increase in total polyamines. The increase is likely caused by the elevated contents of conjugated and bound forms of polyamines. In summary, exogenously EBL compensated for the damage/losses by Ca(NO(3))(2) stress to some extent through the regulation of nitrogen metabolism and metabolites.

Effects of 24-epibrassinolide on ascorbate–glutathione cycle and polyamine levels in cucumber roots under Ca(NO3)2 stress

The effects of 24-epibrassinolide (EBL) applied by spraying the leaves of Cucumis sativus L (cv. Jinyou No.4) on the roots of ascorbate–glutathione (AsA–GSH) cycle and levels and forms of polyamines were investigated under Ca(NO3)2 stress. Ca(NO3)2 stress caused significant decreases in ascorbate acid (AsA) and glutathione (GSH) levels and the activities of antioxidant enzymes involved in the AsA–GSH cycle, including ascorbate peroxidase (APX, EC 1.11.1.11), glutathione reductase (GR, EC 1.6.4.2), monodehydroascorbate reductase (MDAR, EC 1.6.5.4) and dehydroascorbate reductase (DHAR, EC 1.6.5.4). In contrast, exogenous EBL in NB combination increased AsA and GSH levels through enhancing activities of APX, GR, MDAR and DHAR. This alleviated the declines of AsA/DHA and GSH/GSSG ratios, which could maintain cellular homeostasis. In addition, EBL application reduced the accumulation of putrescine (Put) induced by excess Ca(NO3)2, and promoted the conversion of Put into spermidine (Spd) and/or spermine (Spm) able to bind to cell membrane structures. These results showed that EBL could alleviate the damage from oxidative stress by up-regulating the capacity of the AsA–GSH cycle and altering polyamine levels and forms.

Effects of exogenous spermidine on antioxidant system of tomato seedlings exposed to high temperature stress

The effects of foliar spraying with spermidine (Spd) on antioxidant system in tomato (Lycopersicon esculentum Mill.) seedlings were investigated under high temperature stress. The high temperature stress significantly inhibited plant growth and reduced chlorophyll (Chl) content. Application of exogenous 1 mM Spd alleviated the inhibition of growth induced by the high temperature stress. Malondialdehyde (MDA), hydrogen peroxide (H2O2) content and superoxide anion (O2) generation rate were significantly increased by the high temperature stress, but Spd significantly reduced the accumulation of reactive oxygen species (ROS) and MDA content under the stress. The high temperature stress significantly decreased glutathione (GSH) content and activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR), but increased contents of dehydroascorbic acid (DHA), ascorbic acid (AsA), and oxidized glutathione (GSSG) in tomato leaves. However, Spd significantly increased the activities of antioxidant enzymes, levels of antioxidants and endogenous polyamines in tomato leaves under the high temperature stress. In addition, to varying degrees, Spd regulated expression of MnSOD, POD, APX2, APX6, GR, MDHAR, DHAR1, and DHAR2 genes in tomato leaves exposed to the high temperature stress. These results suggest that Spd could change endogenous polyamine levels and alleviate the damage by oxidative stress enhancing the non-enzymatic and enzymatic antioxidant system and the related gene expression.

Effects of NaCl stress on nitrogen metabolism of cucumber seedlings

To investigate the effects of NaCl stress on plant growth, nitrogen assimilation, proline and soluble protein contents, and gene expression of enzymes of nitrogen metabolism, a hydroponic experiment using cucumber (Cucumis sativus L.) seedlings was performed. The seedlings were grown in nutrient solution supplemented with 0 or 84 mM NaCl for up to 9 days. Plant biomass, especially root biomass, was significantly decreased under NaCl stress. Salinity significantly increased ammonium content, but decreased nitrate and soluble protein contents in leaves and roots. Salt stress caused a significant increase in proline content, which peaked on day 3 of NaCl treatment. Moreover, salt stress significantly decreased activities of nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) in the roots and leaves on the 3rd, 6th, and 9th day of NaCl treatment. A semiquantitative RT-PCR approach showed that changes in NR, GS and GOGAT gene expression were consistent with the salt-induced changes in enzyme activities. These results suggest that salt stress-induced growth inhibition in cucumber seedlings may involve disruption of nitrogen absorption and decreased activities of enzymes associated with nitrogen assimilation.

Resistance of spinach plants to seawater stress is correlated with higher activity of xanthophyll cycle and better maintenance of chlorophyll metabolism.

The relationship between the activity of xanthophyll cycle and chlorophyll (Chl) metabolism was investigated using two cultivars, Helan No. 3 (seawater-tolerant cultivar) and Yuanye (seawater-sensitive cultivar), of spinach (Spinacia oleracea L.) plants cultured in Hoagland’s nutrient solution, with or without seawater (40%). The results showed that, in plants of two cultivars with seawater, the xanthophyll cycle seems to show a principal protection mechanism against photoinhibition under seawater stress. Furthermore, accumulation of reactive oxygen species (ROS) in chloroplasts of two cultivars was enhanced by seawater to lower the activity of porphobilinogen deaminase. Namely, the conversion of porphobilinogen into uroporphyrinogen III involved in Chl biosynthetic processes was inhibited by seawater. In Helan No. 3 spinach plants with seawater, higher activity of xanthophyll cycle in the leaves dissipated more excess light energy, which appeared to lower the levels of ROS in chloroplasts. As a consequence, the Chl biosynthesis in Helan No. 3 leaves with seawater showed only a weak inhibition and the activity of chlorophyllase (Chlase) was not affected by seawater stress. In contrast, a more pronounced accumulation of ROS in chloroplasts of Yuanye leaves, which possess lower xanthophyll cycle activity, severely inhibited Chl biosynthesis and remarkably enhanced the activity of Chlase, which aggravates the decomposition of Chl. These results suggest that higher activity of xanthophyll cycle in seawater-tolerant spinach plays a role in maintaining Chl metabolic processes, probably by decreasing the levels of ROS, when the plants are cultured in the nutrient solution with seawater (40%).

The positive roles of exogenous putrescine on chlorophyll metabolism and xanthophyll cycle in salt-stressed cucumber seedlings

The effects of foliar spray of putrescine (Put; 8 mM) on chlorophyll (Chl) metabolism and xanthophyll cycle in cucumber seedlings were investigated under saline conditions of 75 mM NaCl. Exogenous Put promoted the conversion of uroporhyrinogen III to protoporphyrin IX and alleviated decreases in Chl contents and in a size of the xanthophyll cycle pool under salt stress. Moreover, the Put treatment reduced the activities of uroporphyrinogen III synthase, chlorophyllase, and Mg-dechelatase and downregulated the transcriptional levels of glutamyl-tRNA reductase, 5-aminolevulinate dehydratase, uroporphyrinogen III synthase, uroporphyrinogen III decarboxylase, and chlorophyllide a oxygenase, but significantly increased the expression levels of non-yellow coloring 1-like, pheide a oxygenase, red chlorophyll catabolite reductase, and violaxanthin de-epoxidase. Taken together, these results suggest that Put might improve Chl metabolism and xanthophyll cycle by regulating enzyme activities and mRNA transcription levels in a way that improved the salt tolerance of cucumber plants.