Molecular Characterization of a Date Palm Vascular Highway 1-Interacting Kinase (PdVIK) Under Abiotic Stresses.

The date palm (Khalas) is an extremophile plant that can adapt to various abiotic stresses including drought and salinity. Salinity tolerance is a complex trait controlled by numerous genes. Identification and functional characterization of salt-responsive genes from the date palm is fundamental to understand salinity tolerance at the molecular level in this plant species. In this study, a salt-inducible vascular highway 1-interacting kinase (PdVIK) that is a MAP kinase kinase kinase (MAPKKK) gene from the date palm, was functionally characterized using in vitro and in vivo strategies. PdVIK, one of the 597 kinases encoded by the date palm genome possesses an ankyrin repeat domain and a kinase domain. The recombinant PdVIK protein exhibited phosphotyrosine activity against myelin basic protein (MBP) substrate. Overexpression of PdVIK in yeast significantly improved its tolerance to salinity, LiCl, and oxidative stresses. Transgenic Arabidopsis seedlings overexpressing PdVIK displayed improved tolerance to salinity, osmotic, and oxidative stresses as assessed by root growth assay. The transgenic lines grown in the soil also displayed modulated salt response, compared to wild-type controls as evaluated by the overall plant growth and proline levels. Likewise, the transgenic lines exhibited drought tolerance by maintaining better relative water content (RWC) compared to non-transgenic control plants. Collectively, these results implicate the involvement of PdVIK in modulating the abiotic stress response of 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.

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.

Functional Characterization of Date Palm Aquaporin Gene PdPIP1;2 Confers Drought and Salinity Tolerance to Yeast and Arabidopsis.

Recent studies on salinity tolerance in date palm revealed the discovery of salt-responsive genes including PdPIP1;2, a highly conserved aquaporin gene in plants, which was functionally characterized in this study to investigate its precise role in drought and salinity tolerance. Immunoblot assay showed a high level of PIP1 protein accumulation only in the leaves of date palm plants when grown under drought, an observation which may imply the involvement of PIP1;2 in CO2 uptake. Heterologous overexpression of PdPIP1;2 in yeast (Saccharomyces cerevisiae) improved tolerance to salinity and oxidative stress. While, heterologous overexpression of PdPIP1;2 in Arabidopsis had significantly (p < 0.05) increased biomass, chlorophyll content, and root length under drought and salinity. In addition, a significantly (p < 0.05) higher percentage of transgenic plants could be recovered by rewatering after drought stress, indicating the ability of the transgenic plants to maintain water and viability under drought. Transgenic plants under drought and salinity maintained significantly (p < 0.05) higher K+/Na+ ratios than wild type (WT) plants, an observation which may represent an efficient tolerance mechanism controlled by the transgene. Collectively, this study provided an insight on the mechanism by which PdPIP1;2 conferred tolerance to salt and drought stresses in date palm.

Identification of Candidate Genes Involved in the Salt Tolerance of Date Palm (Phoenix dactylifera L.) Based on a Yeast Functional Bioassay.

Although date palm is a relatively salt-tolerant plant, the molecular basis of this tolerance is complex and poorly understood. Therefore, this study aimed to identify the genes involved in salinity tolerance using a basic yeast functional bioassay. To achieve this, a date palm cDNA library was overexpressed in Saccharomyces cerevisiae cells. The expression levels of selected genes that make yeast cells tolerant to salt were subsequently validated in the leaf and root tissues of date palm seedlings using a quantitative PCR method. About 6000 yeast transformant cells were replica printed and screened on a synthetic minimal medium containing 1.0 M of NaCl. The screening results showed the presence of 62 salt-tolerant transformant colonies. Sequence analysis of the recombinant yeast plasmids revealed the presence of a group of genes with potential salt-tolerance functions, such as aquaporins (PIP), serine/threonine protein kinases (STKs), ethylene-responsive transcription factor 1 (ERF1), and peroxidases (PRX). The expression pattern of the selected genes endorsed the hypothesis that these genes may be involved in salinity tolerance, as they showed a significant (p < 0.05) overexpression trend in both the leaf and root tissues in response to salinity. The genes identified in this project are suitable candidates for the further functional characterization of date palms.

Genome-wide DNA Methylation analysis in response to salinity in the model plant caliph medic (Medicago truncatula).

BACKGROUND: DNA methylation has a potential role in controlling gene expression and may, therefore, contribute to salinity adaptation in plants. Caliph medic (Medicago truncatula) is a model legume of moderate salinity tolerance capacity; however, a base-resolution DNA methylome map is not yet available for this plant. RESULTS: In this report, a differential whole-genome bisulfite sequencing (WGBS) was carried out using DNA samples extracted from root tissues exposed to either control or saline conditions. Around 50 million differentially methylated sites (DMSs) were recognized, 7% of which were significantly (p < 0.05, FDR < 0.05) altered in response to salinity. This analysis showed that 77.0% of the contexts of DMSs were mCHH, while only 9.1% and 13.9% were mCHG and mCG, respectively. The average change in methylation level was increased in all sequence contexts, ranging from 3.8 to 10.2% due to salinity stress. However, collectively, the level of the DNA methylation in the gene body slightly decreased in response to salinity treatment. The global increase in DNA methylation due to salinity was confirmed by mass spectrometry analysis. Gene expression analysis using qPCR did not reveal a constant relationship between the level of mCG methylation and the transcription abundance of some genes of potential importance in salinity tolerance, such as the potassium channel KAT3, the vacuolar H+-pyrophosphatase (V-PPase), and the AP2/ERF and bZIP transcription factors, implying the involvement of other epigenetic gene expression controllers. Computational functional prediction of the annotated genes that embrace DMSs revealed the presence of enzymes with potential cellular functions in biological processes associated with salinity tolerance mechanisms. CONCLUSIONS: The information obtained from this study illustrates the effect of salinity on DNA methylation and shows how plants can remodel the landscape of 5-methylcytosine nucleotide (5-mC) in the DNA across gene structures, in response to salinity. This remodeling varies between gene regions and between 5-mC sequence contexts. The mCG has a vague impact on the expression levels of a few selected potentially important genes in salt tolerant mechanisms.

Differential DNA methylation and transcription profiles in date palm roots exposed to salinity.

As a salt-adaptive plant, the date palm (Phoenix dactylifera L.) requires a suitable mechanism to adapt to the stress of saline soils. There is growing evidence that DNA methylation plays an important role in regulating gene expression in response to abiotic stresses, including salinity. Thus, the present study sought to examine the differential methylation status that occurs in the date palm genome when plants are exposed to salinity, and to identify salinity responsive genes that are regulated by DNA methylation. To achieve these, whole-genome bisulfite sequencing (WGBS) was employed and mRNA was sequenced from salinity-treated and untreated roots. The WGBS analysis included 324,987,795 and 317,056,091 total reads of the control and the salinity-treated samples, respectively. The analysis covered about 81% of the total genomic DNA with about 40% of mapping efficiency of the sequenced reads and an average read depth of 17-fold coverage per DNA strand, and with a bisulfite conversion rate of around 99%. The level of methylation within the differentially methylated regions (DMRs) was significantly (p < 0.05, FDR ≤ 0.05) increased in response to salinity specifically at the mCHG and mCHH sequence contexts. Consistently, the mass spectrometry and the enzyme-linked immunosorbent assay (ELISA) showed that there was a significant (p < 0.05) increase in the global DNA methylation in response to salinity. mRNA sequencing revealed the presence of 6,405 differentially regulated genes with a significant value (p < 0.001, FDR ≤ 0.05) in response to salinity. Integration of high-resolution methylome and transcriptome analyses revealed a negative correlation between mCG methylation located within the promoters and the gene expression, while a positive correlation was noticed between mCHG/mCHH methylation rations and gene expression specifically when plants grew under control conditions. Therefore, the methylome and transcriptome relationships vary based on the methylated sequence context, the methylated region within the gene, the protein-coding ability of the gene, and the salinity treatment. These results provide insights into interplay among DNA methylation and gene expression, and highlight the effect of salinity on the nature of this relationship, which may involve other genetic and epigenetic players under salt stress conditions. The results obtained from this project provide the first draft map of the differential methylome and transcriptome of date palm when exposed to an abiotic stress.

Detection of Differential DNA Methylation Under Stress Conditions Using Bisulfite Sequence Analysis.

DNA methylation is the most important epigenetic change affecting gene expression in plants grown under normal as well as under stress conditions. Therefore, researchers study differential DNA methylation under distinct environmental conditions and their relationship with transcriptome abundance. Up to date, more than 25 methods and techniques are available to detect DNA methylation based on different principles. Bisulfite sequencing method is considered as a gold standard since it is able to distinguish 5-methylcytosine from cytosine using the bisulfite treatment. Therefore, it is useful for qualitative and semiquantitative measurement of DNA methylation. However, the reliability of data obtaining from this technique is mainly depending on the efficiency of bisulfite conversion and number of sequencing clones representing the target-converted sequence. Therefore, it is labor intensive and time-consuming. Revolution of next generation DNA sequencing (NGS) has allowed researches to combine conventional bisulfite sequencing methods with high-throughput Illumina sequencing in a technique called whole genome bisulfite sequencing (WGBS). This technique allows a single nucleotide resolution of 5-methylcytosine on a genome scale. WGBS technique workflow involves DNA fragmentation, processing through end blunting, terminal A(s) addition at 3' end and adaptor ligation, bisulfite treatment, PCR amplification, sequencing libraries and assembling, and finally alignment with the reference genome and data analysis. Despite the fact that WGBS is more reliable than the conventional clone-based bisulfite sequencing, it is costly, requires large amount of DNA and its output data is not easily handled.

The use of high throughput DNA sequence analysis to assess the endophytic microbiome of date palm roots grown under different levels of salt stress

Date palms are able to grow under diverse abiotic stress conditions including in saline soils, where microbial communities may be help in the plant’s salinity tolerance. These communities able to produce specific growth promoting substances can enhance date palm growth in a saline environment. However, these communities are poorly defined. In the work reported here, the date palm endophytic bacterial and fungal communities were identified using the pyrosequencing method, and the microbial differential abundance in the root upon exposure to salinity stress was estimated. Approximately 150,061 reads were produced from the analysis of six ribosomal DNA libraries, which were prepared from endophytic microorganisms colonizing date palm root tissues. DNA sequence analysis of these libraries predicted the presence of a variety of bacterial and fungal endophytic species, some known and others unknown. The microbial community compositions of 30% and 8% of the bacterial and fungal species, respectively, were significantly (p ≤ 0.05) altered in response to salinity stress. Differential enrichment analysis showed that microbe diversity indicated by the Chao, Shannon and Simpson indices were slightly reduced, however, the overall microbial community structures were not significantly affected as a consequence of salinity. This may reflect a buffering effect by the host plant on the internal environments that these communities are colonizing. Some of the endophytes identified in this study were strains that were previously isolated from saline and marine environments. This suggests possible interactions with the plant that are favorable to salinity tolerance in date palm (AU)

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The use of high throughput DNA sequence analysis to assess the endophytic microbiome of date palm roots grown under different levels of salt stress.

Date palms are able to grow under diverse abiotic stress conditions including in saline soils, where microbial communities may be help in the plant's salinity tolerance. These communities able to produce specific growth promoting substances can enhance date palm growth in a saline environment. However, these communities are poorly defined. In the work reported here, the date palm endophytic bacterial and fungal communities were identified using the pyrosequencing method, and the microbial differential abundance in the root upon exposure to salinity stress was estimated. Approximately 150,061 reads were produced from the analysis of six ribosomal DNA libraries, which were prepared from endophytic microorganisms colonizing date palm root tissues. DNA sequence analysis of these libraries predicted the presence of a variety of bacterial and fungal endophytic species, some known and others unknown. The microbial community compositions of 30% and 8% of the bacterial and fungal species, respectively, were significantly (p ≤ 0.05) altered in response to salinity stress. Differential enrichment analysis showed that microbe diversity indicated by the Chao, Shannon and Simpson indices were slightly reduced, however, the overall microbial community structures were not significantly affected as a consequence of salinity. This may reflect a buffering effect by the host plant on the internal environments that these communities are colonizing. Some of the endophytes identified in this study were strains that were previously isolated from saline and marine environments. This suggests possible interactions with the plant that are favorable to salinity tolerance in date palm. [Int Microbiol 19(3):143-155 (2016)].

Immunohistological insight into the correlation between neuropilin-1 and epithelial-mesenchymal transition markers in epithelial ovarian cancer.

The mechanism by which neuropilin-1 (NRP-1) induces malignancy in Epithelial Ovarian Cancer (EOC) is still unknown. This study is the first to demonstrate the relationship between NRP-1 expression and EMT markers vimentin, N-cadherin, E-cadherin and Slug. We used tissue microarrays containing the three main subtypes of EOC tumors: serous, mucinous cystadenocarcinoma and endometrioid adenocarcinoma and representative cases retrieved from our pathology archives. Immunohistochemistry was performed to detect the expression levels and location of NRP-1 and the aforementioned EMT proteins. NRP-1 was mainly expressed on cancer cells but not in normal ovarian surface epithelium (OSE). The Immunoreactive Scoring (IRS) values revealed that the expression of NRP-1, Slug and E-cadherin in the malignant subtypes of ovarian tissues was significantly higher (5.18 ± 0.64, 4.84 ± 0.7, 4.98 ± 0.68, respectively) than their expression in the normal and benign tissues (1.04 ± 0.29, 0.84 ± 0.68, 1.71 ± 0.66, respectively), with no significant differences among the studied subtypes. Vimentin was expressed in the cancer cell component of 43% of tumors and it was exclusively localized in the stroma of all mucinous tumors. The Spearman's rho value indicated that NRP-1 is positively related to the EMT markers E-cadherin and Slug. This notion might indicate that NRP-1 is a partner in the EMT process in EOC tumors.