Genome-wide identification and expression analysis of Na+/H+antiporter (NHX) genes in tomato under salt stress.

Plant Na +/H + antiporter (NHX) genes enhance salt tolerance by preventing excessive Na+ accumulation in the cytosol through partitioning of Na+ ions into vacuoles or extracellular transport across the plasma membrane. However, there is limited detailed information regarding the salt stress responsive SlNHXs in the most recent tomato genome. We investigated the role of this gene family's expression patterns in the open flower tissues under salt shock in Solanum lycopersicum using a genome-wide approach. A total of seven putative SlNHX genes located on chromosomes 1, 4, 6, and 10 were identified, but no ortholog of the NHX5 gene was identified in the tomato genome. Phylogenetic analysis revealed that these genes are divided into three different groups. SlNHX proteins with 10-12 transmembrane domains were hypothetically localized in vacuoles or cell membranes. Promoter analysis revealed that SlNHX6 and SlNHX8 are involved with the stress-related MeJA hormone in response to salt stress signaling. The structural motif analysis of SlNHX1, -2, -3, -4, and -6 proteins showed that they have highly conserved amiloride binding sites. The protein-protein network revealed that SlNHX7 and SlNHX8 interact physically with Salt Overly Sensitive (SOS) pathway proteins. Transcriptome analysis demonstrated that the SlNHX2 and SlNHX6 genes were substantially expressed in the open flower tissues. Moreover, quantitative PCR analysis indicated that all SlNHX genes, particularly SlNHX6 and SlNHX8, are significantly upregulated by salt shock in the open flower tissues. Our results provide an updated framework for future genetic research and development of breeding strategies against salt stress in the tomato.

Improved drought tolerance of EMS mutagenized Alfalfa (Medicago sativa L.) mutants by in vitro screening at germination stage.

The objectives of this study were to determine drought tolerant novel mutant of alfalfa (Medicago sativa L.) genotypes by screening EMS mutagenized 340675 M3 seeds at germination stages in the presence of osmotic stress of 35% PEG6000. Root growth assay provided several drought tolerant candidate mutants. Of those, 4 mutants were further evaluated at water deficit conditions applied for 24 days after the first cutting at flowering bud stage. The results revealed that mutants determined as drought tolerant at germination stage were also tolerant to water deficit conditions. Protein content and superoxide dismutase values were found to be higher in all mutants than controls. Ascorbate peroxides, glutton reductase and lipid peroxidase values varied based on the mutant genotype and duration of drought stress. Drought stress significantly changed transcriptional levels of MtP5CS, MtDehyd, MseIF-2, MtRD2 and MsNAC genes. These results indicated that in vitro screening of alfalfa mutant seeds for osmatic tolerance at germination and early seedling growth stages was successfully able to determine the drought tolerant alfalfa mutants which were also tolerant to water deficit conditions after the first cutting at flowering bud stage.

Genetic variation among einkorn genotypes based on gene targeted functional markers and its possible relationship with drought tolerance at seed germination stage.

BACKGROUND: Einkorn wheat is one of the first primary genetic resources for discovery of new alleles related to biotic and abiotic stress tolerances for further genetic improvements while it became more popular especially for its native grain status as healthy food resources. Therefore, this study aims to determine germination parameters of 32 local einkorn (Triticum monococcum) genotypes collected from Kastamonu and its vicinity, Turkey under drought stress conditions, and to reveal the genetic relationship of those genotypes based on drought related gene targeted functional markers (GTFMs). METHODS AND RESULTS: Germination test was conducted at 20 ± 0.5 °C in darkness with randomized complete block design with 4 replications. Seeds placed on double filter papers in a covered glass petri dishes (80 × 15 mm) were treated with polyethylene glycol (PEG6000) with a -0.23 MPa. The final germination percentages (FGPs), speed and span of germination parameters were determined. Drought stress severely worsened all germination parameters measured. The genotypes SG24 and SG12 were the most drought tolerant and sensitive genotypes based on 21.1% and 64.8% of reduction rates in FGPs, respectively. Twelve GTFMs produced a total of 32 bands and 26 of them were polymorphic. The mean polymorphism ratio of the markers and average band per marker were determined as 86.31% and 2.66, respectively. The highest polymorphism information content (PIC) was obtained from AIB1 gene marker (0.875). CONCLUSIONS: This study revealed that there was a significant genetic variation for drought tolerance levels of local einkorn wheat genotypes at germination stage and drought related GTFMs can be used not only to reveal genetic variation but also to distinguish the drought tolerant genotypes.

Genetic relationship and nuclear dna content variation in Tef [Eragrostis tef (Zucc.) Trotter] accessions.

This study was initiated to reveal genetic relationship of 25 tef (Eragrostis tef (Zucc.) accessions by using 10 SSR markers and to determine DNA content variation by using flow cytometer analysis. Ten markers produced a total of 18 alleles and 11 of those were polymorphic. The mean polymorphism rate was 66.6%. The highest polymorphism information content value was obtained from marker CNLTs370 with 0.69 while markers CNTLs11 and CNTLs133 produced monomorphic bands only. UPGMA analysis divided 25 tef genotypes into three main clades. The accessions PI193511 and PI195934 were distinctly separated from the others. No ploidy differences were determined among the 25 tef accessions. 2C mean nuclear DNA content ranged from 1.406 pg to 1.510 with mean of 1.460 pg. The results of this study indicated that SSR markers successfully determined genetic relationship of 25 tef accession although they had a low rate of polymorphism. This study also revealed that available tef related SSR markers should be optimized before use and their efficiency may vary based on tef genotypes or accessions used.

Reversal of the inhibitory effect of light and high temperature on germination of Phacelia tanacetifolia seeds by melatonin.

Possible role of melatonin in the germination of negatively photoblastic and thermosensitive seeds of Phacelia tanacetifolia Benth was studied. Final germination percentage (FGP) was determined in the presence or absence of light at various temperatures, ranging from 0 to 40°C. The highest FGP was determined as 48.7% and 92% at temperature of 15°C in the presence and absence of light, respectively. Seeds were primed with 1% KNO(3) containing various concentrations (0.3, 1, 6, 12, 30, 60, or 90 µM) of melatonin for 2 days at 15°C in darkness. Primed seeds were germinated at an inhibitory temperature of 30°C, and results were compared to those occurring at the optimum temperature of 15°C under both light and no light conditions. Melatonin incorporated into priming medium significantly reversed the inhibitory effects of light and high temperature. Germination was elevated from 2.5% to 52% of FGP for seeds primed in the presence of 6 µM melatonin in darkness at 30°C, while 1 µM melatonin had the highest FGP (21.0%) in the presence of light at 30°C. The highest FGP (47.5%) was obtained from seeds primed in the presence of 0.3 µM melatonin under the light condition at 15°C, while untreated seeds had 1.5% of FGP. The fastest seed germination was determined from seeds primed in the presence of 0.3 µM melatonin (G(50) = 0.56 days) at 15°C in darkness. The possible roles of melatonin in promoting germination parameters of photo- and thermosensitive seed germination are discussed.

Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid.

Substantial evidence indicates that amino acid conjugates of indole-3-acetic acid (IAA) function in auxin homeostasis, yet the plant enzymes involved in their biosynthesis have not been identified. We tested whether several Arabidopsis thaliana enzymes that are related to the auxin-induced soybean (Glycine max) GH3 gene product synthesize IAA-amino acid conjugates. In vitro reactions with six recombinant GH3 enzymes produced IAA conjugates with several amino acids, based on thin layer chromatography. The identity of the Ala, Asp, Phe, and Trp conjugates was verified by gas chromatography-mass spectrometry. Insertional mutations in GH3.1, GH3.2, GH3.5, and GH3.17 resulted in modestly increased sensitivity to IAA in seedling root. Overexpression of GH3.6 in the activation-tagged mutant dfl1-D did not significantly alter IAA level but resulted in 3.2- and 4.5-fold more IAA-Asp than in wild-type seedlings and mature leaves, respectively. In addition to IAA, dfl1-D was less sensitive to indole-3-butyric acid and naphthaleneacetic acid, consistent with the fact that GH3.6 was active on each of these auxins. By contrast, GH3.6 and the other five enzymes tested were inactive on halogenated auxins, and dfl1-D was not resistant to these. This evidence establishes that several GH3 genes encode IAA-amido synthetases, which help to maintain auxin homeostasis by conjugating excess IAA to amino acids.

The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine in Arabidopsis.

Despite its importance in a variety of plant defense responses, our understanding of how jasmonic acid (JA) functions at the biochemical level is limited. Several amino acid conjugates of JA were tested for their ability to complement the JA-insensitive Arabidopsis thaliana mutant jar1-1. Unlike free JA, JA-Ile inhibited root growth in jar1-1 to the same extent as in the wild type, whereas JA-Val, JA-Leu, and JA-Phe were ineffective inhibitors in both genotypes. Thin-layer chromatography and gas chromatography-mass spectrometry (GC-MS) analysis of products produced in vitro by recombinant JAR1 demonstrated that this enzyme forms JA-amido conjugates with several amino acids, including JA-Ile. JA-Val, -Leu, -Ile, and -Phe were each quantified in Arabidopsis seedlings by GC-MS. JA-Ile was found at 29.6 pmole g(-1) fresh weight (FW) in the wild type but was more than sevenfold lower in two jar1 alleles. JA-Leu, -Val, and -Phe were present at only low levels in both genotypes. Expression of wild-type JAR1 in transgenic jar1-1 plants restored sensitivity to JA and elevated JA-Ile to the same level as in the wild type. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) conjugated to JA was also found in plant tissue at 18.4 pmole g(-1) FW. JA-ACC was determined not be an effective jasmonate root inhibitor, and surprisingly, was twofold higher in the mutants than in the wild type. This suggests that another JA-conjugating enzyme(s) is present in Arabidopsis. Synthesis of JA-ACC might provide a mechanism to coregulate the availability of JA and ACC for conversion to the active hormones JA-Ile and ethylene, respectively. We conclude that JAR1 is a JA-amino synthetase that is required to activate JA for optimal signaling in Arabidopsis. Plant hormone activation by conjugation to amino acids and the enzymes involved in their formation were previously unknown.

An Arabidopsis mutant defective in jasmonate response is allelic to the auxin-signaling mutant axr1.

A screen for Arabidopsis mutants that were insensitive to methyl jasmonate (MeJA) in an assay for seedling root growth yielded only alleles of previously isolated mutants jar1 and coi1, with one exception. Mapping of the locus and morphological characterization of the new mutant suggested it might be allelic to axr1, which had not previously been reported to show resistance to MeJA. The F(1) from a cross of the new mutant with axr1-3 did not show complementation, confirming that these are the same genes. The new allele is called axr1-24. In addition to MeJA and indole-3-acetic acid (IAA), axr1-24 had decreased sensitivity to 1-aminocyclopropane-1-carboxylic acid, 6-benzylamino-purine, epi-brassinolide, and abscisic acid. Both axr1-24 and the previously characterized axr1-3 allele were shown to be susceptible to the opportunistic pathogen Pythium irregulare, a trait found in other jasmonate response mutants, including jar1-1. The double mutant jar1-1/axr1-3 was more resistant to inhibition of root growth by MeJA and was more susceptible to P. irregulare infection than either single mutant, suggesting these genes might act in independent response pathways. In contrast, resistance to IAA in the double mutant was not different from axr1-3. Northern-blot analysis showed that IAA induced the jasmonate-responsive lipoxygenase 2, AOS, and AtVSP gene transcripts and induction was strongly impaired in axr1-3. However, transcript induction by MeJA was only minimally affected in axr1-3. This study demonstrates that in addition to auxin signaling, the AXR1 locus is involved in MeJA response, providing a mechanistic link between jasmonate and auxin-signaling pathways.

Jasmonate response locus JAR1 and several related Arabidopsis genes encode enzymes of the firefly luciferase superfamily that show activity on jasmonic, salicylic, and indole-3-acetic acids in an assay for adenylation.

Jasmonic acid (JA) and related cyclopentanones are critical plant signaling molecules, but their mode of action at the molecular level is unclear. A map-based approach was used to identify the defective gene in the Arabidopsis JA response mutant jar1-1. JAR1 is 1 of 19 closely related Arabidopsis genes that are similar to the auxin-induced soybean GH3 gene. Analysis of fold predictions for this protein family suggested that JAR1 might belong to the acyl adenylate-forming firefly luciferase superfamily. These enzymes activate the carboxyl groups of a variety of substrates for their subsequent biochemical modification. An ATP-PPi isotope exchange assay was used to demonstrate adenylation activity in a glutathione S-transferase-JAR1 fusion protein. Activity was specific for JA, suggesting that covalent modification of JA is important for its function. Six other Arabidopsis genes were specifically active on indole-3-acetic acid (IAA), and one was active on both IAA and salicylic acid. These findings suggest that the JAR1 gene family is involved in multiple important plant signaling pathways.