Functional characterization of the Glyoxalase-I (PdGLX1) gene family in date palm under abiotic stresses.

Methylglyoxal (MG), a cytotoxic oxygenated short aldehyde, is a by-product of various metabolic reactions in plants, including glycolysis. The basal level of MG in plants is low, whereby it acts as an essential signaling molecule regulating multiple cellular processes. However, hyperaccumulation of MG under stress conditions is detrimental for plants as it inhibits multiple developmental processes, including seed germination, photosynthesis, and root growth. The evolutionarily conserved glyoxalase system is critical for MG detoxification, and it comprises of two-enzymes, the glyoxalase-I and glyoxalase-II. Here, we report the functional characterization of six putative glyoxalase-I genes from date palm (Phoenix dactylifera L.) (PdGLX1), by studying their gene expression under various environmental stress conditions and investigating their function in bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) mutant cells. The putative PdGLX1 genes were initially identified using computational methods and cloned using molecular tools. The PdGLX1 gene expression analysis using quantitative PCR (qPCR) revealed differential expression under various stress conditions such as salinity, oxidative stress, and exogenous MG stress in a tissue-specific manner. Further, in vivo functional characterization indicated that overexpression of the putative PdGLX1 genes in E. coli enhanced their growth and MG detoxification ability. The putative PdGLX1 genes were also able to complement the loss-of-function MG hypersensitive GLO1 (YML004C) yeast mutants and promote growth by enhancing MG detoxification and reducing the accumulation of reactive oxygen species (ROS) under stress conditions as indicated by flow cytometry. These findings denote the potential importance of PdGLX1 genes in MG detoxification under stress conditions in the date palm.

A novel tonoplast Na+/H+ antiporter gene from date palm (PdNHX6) confers enhanced salt tolerance response in Arabidopsis.

KEY MESSAGE: A sodium hydrogen exchanger (NHX) gene from the date palm enhances tolerance to salinity in Arabidopsis plants. Plant sodium hydrogen exchangers/antiporters (NHXs) are pivotal regulators of intracellular Na+/K+ and pH homeostasis, which is essential for salt stress adaptation. In this study, a novel orthologue of Na+/H+ antiporter was isolated from date palm (PdNHX6) and functionally characterized in mutant yeast cells and Arabidopsis plants to assess the behavior of the transgenic organisms in response to salinity. Genetically transformed yeast cells with PdNHX6 were sensitive to salt stress when compared to the empty vector (EV) yeast cells. Besides, the acidity value of the vacuoles of the transformant yeast cells has significantly (p ≤ 0.05) increased, as indicated by the calibrated fluorescence intensity measurements and the fluorescence imagining analyses. This observation supports the notion that PdNHX6 might regulate proton pumping into the vacuole, a crucial salt tolerance mechanism in the plants. Consistently, the transient overexpression and subcellular localization revealed the accumulation of PdNHX6 in the tonoplast surrounding the central vacuole of Nicotiana benthamiana leaf epidermal cells. Stable overexpression of PdNHX6 in Arabidopsis plants enhanced tolerance to salt stress and retained significantly higher chlorophyll, water contents, and increased seed germination under salinity when compared to the wild-type plants. Despite the significant increase of Na+, transgenic Arabidopsis lines maintained a balanced Na+/K+ ratio under salt stress conditions. Together, the results obtained from this study imply that PdNHX6 is involved in the salt tolerance mechanism in plants by controlling K+ and pH homeostasis of the vacuoles.

Metabolomic analysis of date palm seedlings exposed to salinity and silicon treatments.

Silicon is known to promote plant growth as well as stress tolerance of plants. The current study was undertaken to assess the growth promoting effect of silicon on date palm seedling development as well as its ability to abate some of the negative effects of salinity. In this study, date palm seedlings were treated with silicon and sodium chloride salts, and the effect of these salts on some physiological parameters of the plants was determined. In addition, a global nontargeted metabolomics analysis was performed for the leaf and root tissues using liquid chromatography-mass spectrometry (LC-MS). The results showed that under non-stress conditions, silicon treatment enhanced the growth of the date palm seedlings, however, under salinity, silicon slightly mitigates the negative effects of salt stress on the date palm seedlings although it enhances the potassium accumulation under this condition. The global metabolomics analysis has identified a total of 1,101 significant differentially accumulated (p, q ≤ 0.05) metabolites in leaves and roots under silicon, salinity or their combination. A differential pairwise metabolic profile comparison revealed the accumulation of distinct metabolites in response to silicon and salinity treatments such as antioxidant compounds pyridoxine, cepharanthine, allithiamine, myristic acid and boldine; osmoregulators such as mucic acid; along with the accumulation of detoxification intermediates such as S-D-lactoylglutathione, beta-cyano-L-alanine and gamma-glutamyl-conjugates. In addition, histochemical analyses revealed that application of silicon significantly (p ≤ 0.05) enhanced the formation of the Casparian strip. Identification of the differentially accumulated metabolites could offer an insight into how silicon is able to promote growth and salinity tolerance in date palms.

Overexpression of a Metallothionein 2A Gene from Date Palm Confers Abiotic Stress Tolerance to Yeast and Arabidopsis thaliana.

Although the date palm tree is an extremophile with tolerance to drought and certain levels of salinity, the damage caused by extreme salt concentrations in the soil, has created a need to explore stress-responsive traits and decode their mechanisms. Metallothioneins (MTs) are low-molecular-weight cysteine-rich proteins that are known to play a role in decreasing oxidative damage during abiotic stress conditions. Our previous study identified date palm metallothionein 2A (PdMT2A) as a salt-responsive gene, which has been functionally characterized in yeast and Arabidopsis in this study. The recombinant PdMT2A protein produced in Escherichia coli showed high reactivity against the substrate 5'-dithiobis-2-nitrobenzoic acid (DTNB), implying that the protein has the property of scavenging reactive oxygen species (ROS). Heterologous overexpression of PdMT2A in yeast (Saccharomyces cerevisiae) conferred tolerance to drought, salinity and oxidative stresses. The PdMT2A gene was also overexpressed in Arabidopsis, to assess its stress protective function in planta. Compared to the wild-type control, the transgenic plants accumulated less Na+ and maintained a high K+/Na+ ratio, which could be attributed to the regulatory role of the transgene on transporters such as HKT, as demonstrated by qPCR assay. In addition, transgenic lines exhibited higher chlorophyll content, higher superoxide dismutase (SOD) activity and improved scavenging ability for reactive oxygen species (ROS), coupled with a better survival rate during salt stress conditions. Similarly, the transgenic plants also displayed better drought and oxidative stress tolerance. Collectively, both in vitro and in planta studies revealed a role for PdMT2A in salt, drought, and oxidative stress tolerance.

Genome Sequencing of Microbacterium sp. Yaish 1, a Bacterial Strain Isolated from the Rhizosphere of Date Palm Trees Affected by Salinity.

Microbacterium sp. strain Yaish 1 is a rhizospheric bacterium isolated from date palm orchards with high soil salinity. The genome was sequenced, and genes coding for growth-promoting 1-aminocyclopropane-1-carboxylate (ACC) deaminase, siderophore-producing proteins, and tryptophan biosynthesis proteins were identified. Here, we report the draft whole-genome sequencing of the strain.

Impact of Soil Salinity on the Structure of the Bacterial Endophytic Community Identified from the Roots of Caliph Medic (Medicago truncatula).

In addition to being a forage crop, Caliph medic (Medicago truncatula) is also a model legume plant and is used for research focusing on the molecular characterization of the interaction between rhizobia and plants. However, the endophytic microbiome in this plant is poorly defined. Endophytic bacteria play a role in supplying plants with the basic requirements necessary for growth and development. Moreover, these bacteria also play a role in the mechanism of salinity stress adaptation in plants. As a prelude to the isolation and utilization of these bacteria in Caliph medic farming, 41 bacterial OTUs were identified in this project from within the interior of the roots of this plant by pyrosequencing of the small ribosomal subunit gene (16S rDNA) using a cultivation-independent approach. In addition, the differential abundance of these bacteria was studied following exposure of the plants to salinity stress. About 29,064 high-quality reads were obtained from the sequencing of six libraries prepared from control and salinity-treated tissues. Statistical analysis revealed that the abundance of ~70% of the OTUs was significantly (p ≤ 0.05) altered in roots that were exposed to salinity stress. Sequence analysis showed a similarity between some of the identified species and other, known, growth-promoting bacteria, marine and salt-stressed soil-borne bacteria, and nitrogen-fixing bacterial isolates. Determination of the amendments to the bacterial community due to salinity stress in Caliph medic provides a crucial step toward developing an understanding of the association of these endophytes, under salt stress conditions, in this model plant. To provide direct evidence regarding their growth promoting activity, a group of endophytic bacteria were isolated from inside of plant roots using a cultivation-dependent approach. Several of these isolates were able to produce ACC-deaminase, ammonia and IAA; and to solubilize Zn+2 and PO4-3. This data is consistent with the predicted occurrence (based on cultivation-independent techniques) of these bacteria and provides some insight into the importance of the endophytic bacteria in Caliph medic when grown under normal and saline conditions.