<Emphasis Type="Italic">Piriformospora indica</Emphasis>-mediated salinity tolerance in <Emphasis Type="Italic">Aloe vera</Emphasis> plantlets

Piriformospora indica, a root endophytic fungus, has been reported to promote growth of many plants under normal condition and allow the plants to survive under stress conditions. However, its impact on an important medicinal plant Aloe vera L. has not been well studied. Therefore, this study was undertaken to investigate the effect of P. indica on salinity stress tolerance of A. vera plant. P. indica inoculated and non-inoculated A. vera plantlets were subjected to four levels of salinity treatment- 0, 100, 200 and 300 mM NaCl. The salinity stress decreased the ability of the fungus to colonize roots of A. vera but the interaction of A. vera with P. indica resulted in an overall increase in plant biomass and greater shoot and root length as well as number of shoots and roots. The photosynthetic pigment (Chl a, Chl b and total Chl) and gel content were significantly higher for the fungus inoculated A. vera plantlets, at respective salinity concentrations. Furthermore, the inoculated plantlets had higher phenol, flavonoid, flavonol, aloin contents and radical scavenging activity at all salinity concentrations. The higher phenolic and flavonoid content may help the plants ameliorate oxidative stress resulting from high salinity.     

Peanut RNA Helicase <Emphasis Type="Italic">AhRH47</Emphasis> Sustains Protein Synthesis Under Stress and Improves Stress Adaptation in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Stress responsive RNA helicases are involved in translation initiation sustain protein synthesis. In this study, a stress responsive DEAD box RNA helicase, AhRH47 from peanut cDNA library was identified and characterised during stress. In silico analysis of AhRH47 showed the nine conserved motifs characteristic of an RNA helicase. The phylogenetic and amino acid sequence alignment analyses revealed that AhRH47 is highly homologous to an important DEAD box RNA helicase (eIF4A), which is involved in translation initiation. AhRH47 is stress responsive, being highly expressed under salinity and moisture stress, which is induced to a lesser extent under PEG and ABA treatments. Constitutive overexpression of AhRH47 in Arabidopsis conferred enhanced tolerance to salinity and mannitol-induced stresses. In addition, the transgenic plants showed improved tolerance under moisture stress and exhibited improved recovery growth on stress alleviation. Overexpressing plants showed increased ¹⁴C-labelled amino acids incorporation in to protein especially under stress condition. The results suggest AhRH47 transgenic lines maintained higher protein synthesis under stress and thus improved adaptation to osmotic and desiccation stresses.     

Growth,ionic response,and gene expression of shoots and roots of perennial ryegrass under salinity stress

Growth, ionic responses, and expression of candidate genes to salinity stress were examined in two perennial ryegrass accessions differing in salinity tolerance. The salinity tolerant (PI265349) and sensitive accessions (PI231595) were subjected to 75-mM NaCl for 14 days in a growth chamber. Across two accessions, salinity stress increased shoot dry weight and concentrations of malondialdehyde (MDA) and Na⁺ in the shoots and roots, but decreased shoot Ca²⁺ and root K⁺ concentrations. Salinity stress also increased root expressions of SOS1, PIP1, and TIP1. Plant height and chlorophyll content were unaffected by salinity stress in the tolerant accession but significantly decreased in the sensitive accession. Shoot MDA content did not change in the tolerant accession but increased in the sensitive accession. A more dramatic increase in Na⁺ was found in the roots of the sensitive accession. Relative to the control, salinity stress reduced expression of SOS1, NHX1, PIP1, and TIP1 in the shoots but increased expression of these genes in the roots of the tolerant accession. Expression levels of SOS1 increased in the roots and expression of NHX1 increased in the shoots but decreased in the roots of the sensitive accession under salinity stress. A decline in PIP1 expression in the shoots and dramatic increases in TIP expression in both shoots and roots were found in the sensitive accession under salinity stress. The results suggested maintenance of plant growth and leaf chlorophyll content, lesser Na⁺ accumulation in the roots, and lower lipid peroxidation in the shoots which could be associated with salinity tolerance. The decreased expressions of SOS1, NHX1, and TIP1 in the shoots, and increased expressions of NHX1 and PIP1 in the roots might also be related to salinity tolerance in perennial ryegrass.     

Growth of Transgenic Tobacco Plants with Changed Expression of Genes Encoding Expansins under the Action of Stress Factors

The peculiarities of root growth and stress tolerance of transgenic tobacco plants with constitutive expression of NtEXPA1 and NtEXPA5 genes, as well as plants with reduced expression of NtEXPA4 gene encoding α-expansins of Nicotiana tabacum, were studied during prolonged cultivation under conditions of drought, salinity, and low positive temperatures. Increased expression of expansin genes led to an increase in the growth rate and root length both under normal plant growth conditions and at 12°C and 50 mM NaCl. Increased expression of expansin genes influenced the changes in the fresh and dry mass of a shoot, leading to an increase in their exposure to hypothermia. Transgenic plants with a reduced level of NtEXPA4 expansin gene expression were characterized by a reduction in the fresh and dry weight of a shoot due to drought and low positive temperatures. The totality of the data obtained may indicate the involvement of NtEXPA1, NtEXPA4, and NtEXPA5 tobacco expansin genes in the regulation of growth under hypothermia, drought, and salinity.     

Antifungal Activity of <Emphasis Type="Italic">Lactobacillus pentosus</Emphasis> ŁOCK 0979 in the Presence of Polyols and Galactosyl-Polyols

The antifungal activity of Lactobacillus pentosus ?OCK 0979 depends both on the culture medium and on the fungal species. In the control medium, the strain exhibited limited antagonistic activity against indicator food-borne molds and yeasts. However, the supplementation of the bacterial culture medium with polyols (erythritol, lactitol, maltitol, mannitol, sorbitol, xylitol) or their galactosyl derivatives (gal-erythritol, gal-sorbitol, gal-xylitol) enhanced the antifungal properties of Lactobacillus pentosus ?OCK 0979. Its metabolites were identified and quantified by enzymatic methods, HPLC, UHPLC-MS coupled with QuEChERS, and GC-MS. The presence of polyols and gal-polyols significantly affected the acid metabolite profile of the bacterial culture supernatant. In addition, lactitol and mannitol were used by bacteria as alternative carbon sources. A number of compounds with potential antifungal properties were identified, such as phenyllactic acid, hydroxyphenyllactic acid, and benzoic acid. Lactobacillus bacteria cultivated with mannitol synthesized hydroxy-fatty acids, including 2-hydroxy-4-methylpentanoic acid, a well-described antifungal agent. Scanning electron microscopy (SEM) and light microscopy confirmed a strong antifungal effect of L. pentosus ?OCK 0979.     

<Emphasis Type="Italic">Rhizophagus irregularis</Emphasis> as an elicitor of rosmarinic acid and antioxidant production by transformed roots of <Emphasis Type="Italic">Ocimum basilicum</Emphasis> in an in vitro co-culture system

Arbuscular mycorrhiza is a symbiotic association formed between plant roots and soil borne fungi that alter and at times improve the production of secondary metabolites. Detailed information is available on mycorrhizal development and its influence on plants grown under various edapho-climatic conditions, however, very little is known about their influence on transformed roots that are rich reserves of secondary metabolites. This raises the question of how mycorrhizal colonization progresses in transformed roots grown in vitro and whether the mycorrhizal fungus presence influences the production of secondary metabolites. To fully understand mycorrhizal ontogenesis and its effect on root morphology, root biomass, total phenolics, rosmarinic acid, caffeic acid and antioxidant production under in vitro conditions, a co-culture was developed between three Agrobacterium rhizogenes-derived, elite-transformed root lines of Ocimum basilicum and Rhizophagus irregularis. We found that mycorrhizal ontogenesis in transformed roots was similar to mycorrhizal roots obtained from an in planta system. Mycorrhizal establishment was also found to be transformed root line-specific. Colonization of transformed roots increased the concentration of rosmarinic acid, caffeic acid and antioxidant production while no effect was observed on root morphological traits and biomass. Enhancement of total phenolics and rosmarinic acid in the three mycorrhizal transformed root lines was found to be transformed root line-specific and age dependent. We reveal the potential of R. irregularis as a biotic elicitor in vitro and propose its incorporation into commercial in vitro secondary metabolite production via transformed roots.     

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.     

Overexpression of <Emphasis Type="Italic">MuHSP70</Emphasis> gene from <Emphasis Type="Italic">Macrotyloma uniflorum</Emphasis> confers multiple abiotic stress tolerance in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A 70-KD heat shock protein (HSP70) is one of the most conserved chaperones. It is involved in de novo protein folding and prevents the aggregation of unfolded proteins under lethal environmental factors. The purpose of this study is to characterise a MuHSP70 from horsegram (Macrotyloma uniflorum) and elucidating its role in stress tolerance of plants. A MuHSP70 was cloned and characterised from a natural drought stress tolerant HPK4 variety of horsegram (M. uniflorum). For functional characterization, MuHSP70 was overexpressed in transgenic Arabidopsis. Overexpression of MuHSP70 was found to provide tolerance to the transgenic Arabidopsis against various stresses such as heat, cold, drought, salinity and oxidative stress. MuHSP70 transgenics were observed to maintain the shoot biomass, root length, relative water content, and chlorophyll content during exposure to multi-stresses relative to non-transgenic control. Transgenic lines have further shown the reduced levels of MDA, H₂O₂, and proteolytic activity. Together, these findings suggest that overexpression of MuHSP70 plays an important role in improving abiotic stress tolerance and could be a crucial candidate gene for exploration in crop improvement program.     

Analysis of metabolic responses of <Emphasis Type="Italic">Dunaliella salina</Emphasis> to phosphorus deprivation

It has been reported that phosphorus deprivation can induce β-carotene and triacylglycerol accumulation in Dunaliella salina cells. In this study, we aimed to elucidate the metabolic responses of D. salina to phosphorus deprivation, using gas chromatography-mass spectrometry as analytical tool. A total of 79 metabolites were identified in cells cultured in either phosphorus-deprived or replete media, including 18 amino acids, 28 other acids, 16 sugars, 12 alcohols, and 5 amino compounds. Hierarchical clustering was used to sort these metabolites into three groups with different change trends. Most amino acids and sugars, including the abiotic stress-related metabolites lysine, proline, trehalose, talose, and tagatose, increased, whereas N,N-dimethylglycine, L-serine, D-erythro-pentose, and D-ribose remained constant upon phosphorus deprivation. Multivariate statistical partial least squares and principal component analyses indicated that metabolite profiles were significantly changed upon phosphorus deprivation, and 18 biomarkers which can be used to distinguish the two culture conditions were identified. Stress-related polyamines such as cadaverine, antioxidants such as L-ascorbic acid, and L-methionine, as well as the osmolytes proline, mannitol, and arabitol, also increased. Furthermore, phosphorus deprivation resulted in increases of both saturated and unsaturated fatty acids in D. salina cells. These results suggest that phosphorus deprivation triggers comprehensive metabolic responses in D. salina which may be useful for future bioprocesses.     

The abundance of certain metabolites responds to drought stress in the highly drought tolerant plant <Emphasis Type="Italic">Caragana korshinskii</Emphasis>

Metabolomics offers opportunities for studying the systematic response of an organism to a genetic and/or an environmental change. Here, the metabolic consequences of drought stress were characterized in the highly drought tolerant plant Caragana korshinskii. The time-of-flight mass spectrometry platform employed identified several hundred metabolites in extracts of the leaf, stem, root collar, and root of plants which had been either subjected to drought stress or were well-watered. Each of the four organs harbored a number of potential metabolite markers for the drought response. An increased abundance of various small carbohydrates and soluble amino acids in each of the four organs was induced by the stress; these compounds may act as compatible solutes or antioxidants. Across the whole plant, there was a fall in the content of several Krebs cycle and glycolysis intermediates, as well as in that of the amino acids glutamic acid and aspartic acid. Pathway analysis suggested that most of the potential metabolite markers were involved in energy metabolism and amino-acid metabolism. The implication was that energy metabolism and photosynthesis are compromised during the adaptation of C. korshinskii to drought stress. Given the different spectrum of metabolites associated with the drought response in the four organs, it was concluded that each organ employs a distinct strategy to cope with drought stress.     

Evaluation of Barley lncRNAs Expression Analysis in Salinity Stress

Long noncoding RNAs (lncRNAs) act important roles in a wide range of biological processes. The regulatory roles of lncRNAs are still poorly understood. One of the major problems of limiting plant productivity is the salinity in the worldwide that barley (Hordeum vulgare L.) seems to be relatively well adapted to salinity environments. The aim of this study is the investigation of lncRNAs’ expression levels on four barley genotypes (Hasat, Beysehir 99, Konevi 98 and Tarm 92) to 150 mM salt stress application during 3 days germination. Grains were placed randomly in petri dishes containing filter paper soaked in (a) only H₂O (control), (b) 150 mM NaCl for 72 h. RNA extraction were carried out using TriPure® reagent from root and shoot samples obtained after 150 mM salt treatment. Expression levels of CNT0018772 and CNT0031477 were determined by qPCR. Expression analysis demonstrated salinity effected expression levels of CNT0018772 and CNT0031477 on roots and shoots during germination. The expression levels of CNT0018772 for 150 mM salt applied groups were down-regulated raged between (log2–0.52 and–35.65) compared controls on roots and shoot. The expression levels of CNT0031477 in 150 mM salt applied groups were also down-regulated ranged between (log₂–10.40 and 33.59) compared controls on roots and shoot except for Tarm 92 variety. On the contrary, expression levels of CNT0031477 were up-regulated on root and shoot of Tarm 92. Comparison of CNT0018772 and CNT0031477 expression levels on roots, there was no significant difference between barley varieties compared to controls (p > 0.05). However, it was found there was statistically significant difference between 150 mM salt treatment and control groups for CNT0031477 expression levels (p < 0.05). It was determined Konevi 98 shoot control expression level was statistically higher than Tarm 92 shoot control. This is the first report about the lncRNAs expression levels of barley under salinity.     

A glucuronokinase gene in<Emphasis Type="Italic"> Arabidopsis</Emphasis>, <Emphasis Type="Italic">AtGlcAK</Emphasis>, is involved in drought tolerance by modulating sugar metabolism

Arabidopsis glucuronokinase (AtGlcAK), as a member of the GHMP kinases family, is implicated in the de novo synthesis of UDP-glucuronic acid (UDP-GlcA) by the myo-inositol oxygenation pathway. In this study, two T-DNA insertion homozygous mutants of AtGlcAK, atglcak-1 and atglcak-2, were identified. AtGlcAK was highly expressed in roots and flowers. There was reduced primary root elongation and lateral root formation in atglcak mutants under osmotic stress. The atglcak mutants displayed enhanced stomatal opening in response to abscisic acid (ABA), elevated water loss and impaired drought tolerance. Under water stress, the accumulation of reducing and soluble sugars was reduced in atglcak mutants, and the metabolism of glucose and sucrose was affected by the synthetic pathway of UDP-GlcA. Furthermore, a reduced level of starch in atglcak mutants was observed under normal conditions. The phylogenetic analysis suggested that GlcAK was conserved in numerous dicots and monocots plants. In short, AtGlcAK mutants displayed hypersensitivity to ABA and reduced root development under water stress, rendering the plants more susceptible to drought stress.     

Salt oversensitivity derived from mutation breeding improves salinity tolerance in barley <Emphasis Type="Italic">via</Emphasis> ion homeostasis

Salt stress imposes a major environmental threat to agriculture, therefore, understanding the basic physiology and genetics of cell under salt stress is crucial for developing any breeding strategy. In the present study, the expression profile of genes involved in ion homeostasis including salt overly sensitive (HvSOS1, HvSOS2, HvSOS3), vacuolar Na⁺/H⁺ antiporter (HvNHX1), and H⁺-ATPase (HVA) along with ion content measurement were investigated in two genotypes of Hordeum vulgare under 300 mM NaCl. The gene expressions were measured in the roots and shoots of a salt-tolerant mutant genotype M4-73-30 and in its wild-type cv. Zarjou by real-time qPCR technique. The critical differences between the salt-tolerant mutant and its wild-type were observed in the expressions of HvSOS1 (105-fold), HvSOS2 (24-fold), HvSOS3 (31-fold), and HVA (202-fold) genes in roots after 6-h exposure to NaCl. The parallel early up-regulation of these genes in root samples of the salt-tolerant mutant genotype indicated induction of Na⁺/H⁺ antiporters activity and Na+ exclusion into apoplast and vacuole. The earlier up-regulation of HvSOS1, HVA, and HvNHX1 genes in shoot of the wild-type genotype corresponded to the relative accumulation of Na⁺ which was not observed in salt-tolerant mutant genotype because of efficient inhibitory role of the root in Na+ transport to the shoot. In conclusion, the lack of similarity in gene expression patterns between the two genotypes with similar genetic background may confirm the hypothesis that mutation breeding could change the ability of salt-tolerant mutant genotype for efficient ion homeostasis via salinity oversensitivity response.     

The <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> methionine sulfoxide reductase gene family

The methionine sulfoxide reductases (MSRs) are a group of thiol-dependent enzymes able to catalyze the conversion of methionine sulfoxide to methionine. Although some plant MSRs are known to act as protectants against various abiotic stresses, their activity in the model grass species Brachypodium distachyon has not been characterized as yet. Here, six B. distachyon MSR (BdMSR) genes have been isolated; they generate eight distinct cDNAs, since two of them (BdMSRB1 and -B5) produce a pair of alternatively spliced messages. The genes were transcribed in the root, culm, leaf and during various stages of caryopsis development. Those induced by abiotic stress (salinity, drought, low temperature, CdCl₂, H₂O₂ and abscisic acid) harbored known stress-responsive cis elements in their promoter sequences. The heterologous expression of five of the BdMSRs (-A2, -A4, -B1.1, -B3 and -B5.1) in yeast revealed that their products gave a measure of protection against salinity, mannitol and oxidative stress. Substrate specificity analysis revealed that BdMSRB1.1 could reduce free Met-R-SO to Met. The enzymatic activities of BdMSRA4, -B1.1 and -B5.1 in transformed yeast under salt treatment have checked and increased obviously resulting in reducing more Met-SO to Met including the peptide and the free types under salt stress than those in control.