Presoaking with hemin improves salinity tolerance during wheat seed germination

The aim of this study was to investigate whether presoaking with hemin, an inducer of heme oxygenase-1 (HO-1), could alleviate salinity damage during wheat seed germination in comparison with the pretreatment of a well-known nitric oxide (NO) donor sodium nitroprusside (SNP). The results showed that, compared with the samples upon 150 mM NaCl salt stress alone, both 10 ??M hemin and 200 ??M SNP pretreatments could (1) significantly attenuate the inhibition of seed germination and thereafter seedling growth; (2) induce HO expression; (3) enhance amylase activity, thus accelerating the formation of reducing sugar and total soluble sugar; and (4) increase the potassium (K) to sodium (Na) ratio, particularly in the shoot parts. Hemin and SNP could also increase antioxidant enzyme activities, including superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), and ascorbate peroxidase (APX), thus resulting in the alleviation of oxidative damage, as indicated by the decrease of thiobarbituric acid reactive substances (TBARS) content. Moreover, semi-quantitative RT-PCR and isozymatic analysis illustrated that hemin and SNP pretreatment were able to up-regulate the expression of Mn-SOD (especially) and Cu/Zn-SOD gene, and activate SOD isozymatic activities. Since the addition of the NO scavenger methylene blue (MB) differentially reversed the above effects, the protective roles of hemin might be related to the induction of endogenous NO signal. Meanwhile, hemin-driven NO production was confirmed. Together, these results indicated that hemin exerted an advantageous effect on enhancing salinity tolerance during wheat seed germination, which might interact with NO.     

Isolation and Characterization of a Heme Oxygenase-1 Gene from Chinese Cabbage

Heme oxygenase-1 (HO1) is a heme-catabolizing enzyme induced by a variety of stress conditions. This article described the cloning and characterization of BrHO1 gene which codes for a putative HO1 from Chinese cabbage (Brassica rapa subsp. pekinensis). BrHO1 consists of three exons and encodes a protein precursor of 32.3 kD with a putative N-terminal plastid transit peptide. The amino acid sequence of BrHO1 was 84% similar to Arabidopsis counterpart HY1. The three-dimensional structure of BrHO1 showed a high degree of structural conservation compared with the known HO1 crystal structures. Phylogenetic analysis revealed that BrHO1 clearly grouped with the HO1-like sequences. The recombinant BrHO1 protein expressed in Escherichia coli was active in the conversion of heme to biliverdin IXα (BV). Furthermore, the results of subcellular localization of BrHO1 demonstrated that BrHO1 gene product was most likely localized in the chloroplasts. BrHO1 was differently expressed in all tested tissues and could be induced upon osmotic and salinity stresses, cadmium (Cd) exposure, hydrogen peroxide (H₂O₂), and hemin treatments. Together, the results suggested that BrHO1 plays an important role in abiotic stress responses.     

Overexpression of MsALMT1, from the Aluminum-Sensitive Medicago sativa, Enhances Malate Exudation and Aluminum Resistance in Tobacco

Aluminum (Al) toxicity is a major limiting factor for plant growth and crop production in acidic soils. Al-induced organic acid (OA) exudation plays an important role in plant Al resistance. The exudation of OAs is mediated by membrane-localized OA transporters. In our previous study, a gene encoding the Al-induced malate transporter (MsALMT1) was identified in the roots of the Al-sensitive plant Medicago sativa L. cv. Yumu no. 1 (YM1). To further validate the function of MsALMT1, transgenic plants that overexpressed MsALMT1 under the control of the CaMV 35S (35S) promoter were generated. This transgenic tobacco showed an enhanced capacity for malate efflux and better Al resistance than wild type (WT) plants after exposure to 30 μM Al for 24 h. The Al content in the transgenic plant roots decreased to 40–52 % of that in WT plant roots. These results demonstrate that MsALMT1 is an Al-resistant gene in YM1 and encodes a malate transporter, the overexpression of which effectively enhances the Al resistance of transgenic tobacco plants.     

Effects of salinity and clipping on biomass and competition between a halophyte and a glycophyte

Global climate change will likely result in the reduction of water levels in intermountain wetlands and ponds, and the vegetation communities associated with these wetlands are an important forage source for livestock. Lowered water levels will not only constrict wetland plant communities, it will potentially change aquatic and soil salt concentrations. Such an increase in salinity can reduce plant growth and potentially affect competitive interactions between plants. A greenhouse experiment examined the effects of salinity and competition on the growth of two wet meadow grass species, Poa pratensis (a glycophyte) and Puccinellia nuttalliana (a halophyte). The following hypotheses based on published data were tested: (1) Biomass of both species will decrease with increasing concentration of salt; (2) root:shoot (R:S) ratio of P. pratensis will decrease with increasing salt concentration while R:S ratio of P. pratensis and P. nuttalliana will increase with clipping; (3) competitive importance will decrease for P. pratensis and P. nuttalliana with increasing salt concentration because salt induces a stress response and competitive importance is reduced in stressed environments. A factorial design included 3 plant treatments (P. nuttalliana alone, P. pratentsis alone, P. nuttalliana + P. pratensis) × 4 salinity rates (control; 5, 10, 15 g/L NaCl) × 2 clipping intensities (plants clipped or not clipped) for a total of 24 combinations replicated 6 times over a period of 90 days. We found a reduction in dry biomass as salinity increased, and this effect was greatest for P. pratensis. (1.94 g (SE 0.13) at 0 g/L NaCl to 0.22 g (SE 0.11) at 15 g/L NaCl). The R:S ratio of P. pratensis was reduced by salinity, but not for P. nuttalliana. Competitive importance of both species was reduced by clipping and by salinity, but the effect was greater and more consistent for P. pratensis. We conclude that salt concentration reduces plant growth and the effect of competition.     

Evaluation of heme oxygenase 1 (HO 1) in Cd and Ni induced cytotoxicity and crosstalk with ROS quenching enzymes in two to four leaf stage seedlings of <Emphasis Type="Italic">Vigna radiata</Emphasis>

Research on heme oxygenase in plants has received consideration in recent years due to its several roles in development, defense, and metabolism during various environmental stresses. In the current investigation, the role of heme oxygenase (HO) 1 was evaluated in reducing heavy metal (Cd and Ni) uptake and alleviating Cd and Ni toxicity effects in the hydroponically grown seedlings of Vigna radiata var. PDM 54. Seedlings were subjected to Cd- and Ni-induced oxidative stress independently at different concentrations ranging from 10 to 100 μM. After 96 h (fourth day) of treatment, the stressed plants were harvested to study the cellular homeostasis and detoxification mechanism by examining the growth, stress parameters (LPX, H₂O₂ content), and non-enzymatic and enzymatic parameters (ascorbate peroxidase (APX), guaicol peroxidase (GPX), and catalase (CAT)) including HO 1. At 50 μM CdCl₂ and 60 μM NiSO₄, HO 1 activity was found to be highest in leaves which were 1.39 and 1.16-fold, respectively. The greatest HO 1 activity was reflected from the reduction of H₂O₂ content at these metal concentrations (50 μM CdCl₂ and 60 μM NiSO₄) which is correlated with the increasing activity of other antioxidant enzymes (CAT, APX). Thus, HO 1 works within a group that generates the defense machinery for the plant’s survival by scavenging ROS which is confirmed by a time-dependent study. Hence, it is concluded that seedlings of V. radiata were more tolerant towards metal-induced oxidative stress in which HO 1 is localized in its residential area (plastids).     

β-Cyclodextrin–hemin enhances tolerance against salinity in tobacco seedlings by reestablishment of ion and redox homeostasis

β-Cyclodextrin–hemin (β-CDH) is a complex combining hemin with β-cyclodextrin (β-CD), which could improve hemin solubility. Our previous results showed that β-CDH, was able to enhance alfalfa tolerance against cadmium stress. However, whether or how β-CDH influences salinity tolerance is still elusive. In this report, we observed that similar to the beneficial responses of hemin rather than β-CD, the addition of β-CDH not only alleviated salinity-induced seedling growth inhibition (in particular), but also arrested chlorophyll degradation in tobacco seedlings. The efficiency of β-CDH against salinity stress compared to that of hemin, was confirmed, since the maximum beneficial responses against NaCl stress was obtained with 0.1 μM β-CDH and 10 μM hemin, respectively. Subsequent work showed that the redox imbalance caused by salinity stress could be improved by β-CDH. This was suggested by the reduced lipid peroxidation and hydrogen peroxide accumulation, as well as the induction of representative antioxidant genes, encoding superoxide dismutase, guaiacol peroxidase, and ascorbate peroxidase. Meanwhile, compared to control conditions, the ratio of K⁺ to Na⁺ was relatively low in NaCl-stressed tobacco seedlings. By contrast, the administration of β-CDH not only significantly blocked the increase of Na⁺, but also obviously increased K⁺, thus resulting in a high K⁺ to Na⁺ ratio in both shoot and root parts. Ion homeostasis is therefore reestablished. Together, our results suggested that β-CDH was able to improve salinity tolerance via the reestablishment of redox and ion homeostasis.     

Signal cross talk in Arabidopsis exposed to cadmium, silicon, and Botrytis cinerea

The role of defence gene expression triggered by Cd toxicity in the plant’s response to Botrytis cinerea was investigated in Arabidopsis thaliana Columbia 0. Silicon (0 or 1.5 mM) and Cd (0, 1 or 10 μM) were supplied to 3-month-old solution-cultured plants. After 3 days, half of the plants of each treatment were inoculated with Botrytis. Supplied Cd concentrations were below the toxicity threshold and did not cause shoot growth inhibition or evidence of oxidative stress, while Botrytis infection severely decreased plant growth in all treatments. The expression of marker genes PR1 and BGL2 for the salicylic acid (SA) and the PDF1.2 for the jasmonic acid–ethylene (JA–ET) signalling pathways was enhanced in 10 μM Cd-treated non-infected plants. Twenty hours after inoculation, PDF1.2 expression showed a strong increase in all treatments, while enhanced PR1, BGL2, and CHIB expression was only found 7 days after infection. A great synergistic effect of Cd and Botrytis on PDF1.2 expression was found in 10 μM Cd-treated plants. Silicon decreased PR1, BGL2, and CHIB, while increasing PDF1.2 expression, which indicates its role as a modulator of the signalling pathways involved in the plant’s response to fungal infection. Botrytis growth decreased in 10 μM Cd-treated plants, which could be due to the combined effects of Cd and Botrytis activating the SA and JA–ET-mediated signalling pathways. Taken together, our results provide support for the view that Cd concentrations close to the toxicity threshold induce defence signalling pathways which potentiate the plant’s response against fungal infection.     

Functional characterization of plasma membrane-localized organic acid transporter (CsALMT1) involved in aluminum tolerance in <Emphasis Type="Italic">Camelina sativa</Emphasis> L.

Aluminum (Al) toxicity is a major limiting factor that inhibits root elongation and decreases crop production in acidic soils. The symptoms of inhibited root growth include a reduced uptake of nutrients because the roots become stubby and brittle. The release of organic anions from roots can protect a plant from Al toxicity. The mechanism relies on the efflux of organic anions, such as malate or citrate, which protect roots by chelating the Al³⁺. In this study, homologs of TaALMT1, a Camelina gene that encodes an aluminum-activated malate transporter, were investigated. The expression of this gene was induced by Al in the root, but not in the shoots. Using green fluorescent protein (GFP) fusion constructs and Western-blot analysis, we observed that CsALMT1 was localized in the plasma membrane. Also, to determine the degree to which Al tolerance was affected by malate secretion in Camelina root, we generated CsALMT1 overexpressing plants. CsALMT1 overexpressing transgenic plants showed a higher root elongation rate than the wild-type plant. Damaged cell staining analysis by hematoxylin under 25 µM Al treatment for 2, 4, and 6 h showed a pattern of less damage in CsALMT1 transgenic plants than in wild-type plant, especially in the root elongation zone. Furthermore, the rate of increase of secretion of organic acid in overexpressed plants after Al treatment was higher than that in the wild-type plant. In addition, in the Al-specific dye morin staining on root protoplast under 50 µM Al treatment, less Al accumulation was observed in the CsALMT1 transgenic plants than in the wild-type plant. The Al contents in the roots of the transgenic plants were at a lower level than those in the wild-type plant. These results show that the overexpression of CsALMT1 improves Al tolerance by increasing the release of malate from the root to the soil and, thereby, detoxifies the Al³⁺.     

The over-expression of Chrysanthemum crassum CcSOS1 improves the salinity tolerance of chrysanthemum

Soil salinity represents a major constraint on plant growth. Here, we report that the over-expression of the Chrysanthemum crassum plasma membrane Na⁺/H⁺ antiporter gene CcSOS1, driven by the CaMV 35S promoter, improved the salinity tolerance of chrysanthemum ‘Jinba’. In salinity-stressed transgenic plants, both the proportion of the leaf area suffering damage and the electrical conductivity of the leaf were lower in the transgenic lines than in salinity-stressed wild type plants. After a 6 day exposure to 200 mM NaCl, the leaf content of both chlorophyll (a+b) and proline was higher in the transgenic than in the wild type plants. The activity of both superoxide dismutase and peroxidase was higher in the transgenic than in the wild type plants throughout the period of NaCl stress. The transgenic plants had a stronger control over the ingress of Na⁺ into the plant, particularly with respect to the youngest leaves, and so maintained a more favorable K⁺/Na⁺ ratio. The result suggests that a possible strategy for improving the salinity tolerance of chrysanthemum could target the restriction of Na⁺ accumulation. This study is the first to report the transgenic expression of a Na⁺ efflux carrier in chrysanthemum.     

Silicon Application to Rice Root Zone Influenced the Phytohormonal and Antioxidant Responses Under Salinity Stress

Silicon (Si) application shows beneficial effects on plant growth; however, its effects on the phytohormone and enzymatic antioxidant regulation have not been fully understood. We studied the effects of short-term (6, 12, and 24 h) silicon (0.5, 1.0, and 2.0 mM) application on salinity (NaCl)-induced phytohormonal [abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA)] and antioxidant regulation in Oryza sativa. The results showed that Si treatments significantly increased rice plant growth compared to controls under salinity stress. Si treatments reduced the sodium accumulation resulting in low electrolytic leakage and lipid peroxidation compared to control plants under salinity stress. Enzymatic antioxidant (catalase, peroxidase and polyphenol oxidase) responses were more pronounced in control plants than in Si-treated plants under salinity stress. Stress- and defense-related phytohormones like JA were significantly downregulated and SA was irregular after short-term Si applications under salinity stress compared to control. Conversely, ABA was significantly higher after 6 and 12 h but insignificant after 24 h in Si-treated plants under salinity stress. After 6 and 12 h, Si and salinity stress resulted in upregulation of zeaxanthin epoxidase and 9-cis-epoxycarotenoid dioxygenase 1 and 4 (NCED1 and 4), whereas 24-h treatments significantly downregulated the expressions of these genes compared to those in the control. NCED3 expression increased after 6 and 24 h but it was insignificant after 12 h of Si application compared to control. The current findings indicate that increasing the Si concentrations for longer periods of time can regulate the salinity-induced stress by modulating phytohormonal and enzymatic antioxidants’ responses.     

Molecular cloning and characterization of the <Emphasis Type="Italic">MsHSP17.7</Emphasis> gene from <Emphasis Type="Italic">Medicago sativa</Emphasis> L.

Heat shock proteins (HSPs) are ubiquitous protective proteins that play crucial roles in plant development and adaptation to stress, and the aim of this study is to characterize the HSP gene in alfalfa. Here we isolated a small heat shock protein gene (MsHSP17.7) from alfalfa by homology-based cloning. MsHSP17.7 contains a 477-bp open reading frame and encodes a protein of 17.70-kDa. The amino acid sequence shares high identity with MtHSP (93.98 %), PsHSP17.1 (83.13 %), GmHSP17.9 (74.10 %) and SlHSP17.6 (79.25 %). Phylogenetic analysis revealed that MsHSP17.7 belongs to the group of cytosolic class II small heat shock proteins (sHSP), and likely localizes to the cytoplasm. Quantitative RT-PCR indicated that MsHSP17.7 was induced by heat shock, high salinity, peroxide and drought stress. Prokaryotic expression indicated that the salt and peroxide tolerance of Escherichia coli was remarkably enhanced. Transgenic Arabidopsis plants overexpressing MsHSP17.7 exhibited increased root length of transgenic Arabidopsis lines under salt stress compared to the wild-type line. The malondialdehyde (MDA) levels in the transgenic lines were significantly lower than in wild-type, although proline levels were similar between transgenic and wild-type lines. MsHSP17.7 was induced by heat shock, high salinity, oxidative stress and drought stress. Overexpression analysis suggests that MsHSP17.7 might play a key role in response to high salinity stress.     

Growth promotion and induced disease suppression of four vegetable crops by a selected plant growth-promoting rhizobacteria (PGPR) strain Bacillus subtilis 21-1 under two different soil conditions

This study aimed at investigating the efficacy of a PGPR strain, Bacillus subtilis 21-1 (BS21-1), under two different soil conditions for plant growth promotion and disease suppression. BS21-1 treatment significantly (P < 0.05) promoted plant growth as measured by plant height and leaf width and increased the seed germination rate in organic soil (OS) compared with seed bed soil (SBS). In Chinese cabbage and lettuce, soft rot disease was reduced to 45 and 23.5 %, respectively, by BS21-1, and to 33 and 52.5 %, respectively, by benzo-(1,2,3)-thidiazole-7-carbothioic acid S-methyl ester (BTH) treatments in OS compared with SBS. These levels of disease suppression were greater than those found in the water-treated control upon pathogen challenge. There was a greater reduction of anthracnose lesions on the leaves of cucumber plants treated with BS21-1 and BTH in OS when compared with SBS. Botrytis rot disease in tomato caused by Botrytis cinerea was drastically reduced to 2 % in OS and 4 % in SBS by BS21-1 treatment. In the four plants studied, there was increased disease suppression in OS compared with SBS. Upon treatment with BS21-1, there was an increased expression of the PR-1a gene by β-gulcuronidase (GUS) activity in tobacco (Nicotiana tabacum L. cv. Xanthi-nc) plants in OS compared with SBS, which indicates a possible role of the SA pathway in BS21-1 mediated plant protection. Thus, the isolate BS21-1 could effectively be used as one of the biocontrol agents for disease suppression in four vegetable crops through systemic resistance and for plant growth promotion.     

Genomic inversion caused by gamma irradiation contributes to downregulation of a WBC11 homolog in bloomless sorghum

Epicuticular wax (bloom) plays important roles in protecting the tissues of sorghum (Sorghum bicolor (L.) Moench) plants from abiotic stresses. However, reducing wax content provides resistance to greenbug and sheath blight—a useful trait in agricultural crops. We generated a sorghum bloomless (bm) mutant by gamma irradiation. One bm population segregated for individuals with and without epicuticular wax at a frequency of 72:22, suggesting that the bm mutation was under the control of a single recessive nuclear gene. Genes differentially expressed in the wild-type and the bm mutant were identified by RNA-seq technology. Of the 31 downregulated genes, Sb06g023280 was the most differentially expressed and was similar to WBC11, which encodes an ABC transporter responsible for wax secretion in Arabidopsis. An inversion of about 1.4 Mb was present in the region upstream of the Sb06g023280 gene in the bm mutant; it is likely that this inversion changed the promoter sequence of the Sb06g023280 gene. Using genomic PCR, we confirmed that six independent F₂ bm mutant-phenotype plants carried the same inversion. Therefore, we concluded that the inversion involving the Sb06g023280 gene inhibited wax secretion in the bloomless sorghum.     

NaCl-induced heme oxygenase in roots of rice seedlings is mediated through hydrogen peroxide

Recent findings have suggested that H₂O₂ is an important signaling molecule for regulating plant responses to abiotic stress. H₂O₂ plays a critical role in NaCl stress. Heme oxygenase (HO) is known to play a protective role against oxidative stress. In this study, we examined the possible involvement of H₂O₂ in regulating NaCl-promoted HO activity in rice roots. Treatment with NaCl increased HO activity and H₂O₂ content in rice roots. As well, NaCl could induce OsHO1 mRNA expression. NaCl (150 mM) and NaNO₃ (150 mM) were equally effective in inducing HO activity. However, mannitol at the concentration (276 mM) iso-osmotic with 150 mM NaCl had no effect on HO activity. NaCl-promoted HO activity and OsHO1 expression in rice roots was reduced by NADPH oxidase inhibitors i.e. dipehnyleneiodonium and imidazole. Moreover, exogenous application of H₂O₂ enhanced the activity of HO and the mRNA level of OsHO1. Our data suggest that H₂O₂ production plays a positive role in NaCl- induced HO activity by enhancing its mRNA level in rice roots.