Halophyte Thellungiella Halophila Research Articles

Thellungiella halophila ST5 improves salt tolerance in cotton

BackgroundSalinity is a major abiotic stress to global agriculture which hampers crop growth and development, and eventually reduces yield. Transgenic technology is an effective and efficient approach to improve crop salt tolerance but depending on the availability of effective genes. We previously isolated Salt Tolerance5 (ThST5) from the halophyte Thellungiella halophila, an ortholog of Arabidopsis SPT4-2 which encodes a transcription elongation factor. However, SPT4-2-confered salt tolerance has not been evaluated in crops yet. Here we report the evaluation of ThST5-conferred salt tolerance in cotton (Gossypium hirsutum L.).ResultsThe ThST5 overexpression transgenic cotton plants displayed enhanced tolerance to salt stress during seed germination and seedling stage compared with wild type. Particularly, the transgenic plants showed improved salinity tolerance as well as yield under saline field conditions. Comparative transcriptomic analysis showed that ThST5 improved salt tolerance of transgenic cotton mainly by maintaining ion homeostasis. In addition, ThST5 also orchestrated the expression of genes encoding antioxidants and salt-responsive transcription factors.ConclusionOur results demonstrate that ThST5 is a promising candidate to improve salt tolerance in cotton.

TsHD1 and TsNAC1 cooperatively play roles in plant growth and abiotic stress resistance of Thellungiella halophile.

T.HALOPHILA HOMEOBOX PROTEIN 1(TsHD1) cloned from the halophyte Thellungiella halophila is a homeodomain (HD) transcription factor gene and functions as a collaborator of TsNAC1. TsHD1 can form heterodimers with TsNAC1 via the interaction between its zinc finger (ZF) domain and the A subdomain of TsNAC1. The overexpression of TsHD1 improved the heat stress resistance of T.halophila and retarded its vegetative growth slightly. The co-overexpression of TsHD1 and TsNAC1 highly improved heat and drought stress resistance by increasing the accumulation of heat shock proteins and enhancing the expression levels of drought stress response genes, such as MYB DOMAIN PROTEIN 77 and MYB DOMAIN PROTEIN 96 (MYB77and MYB96) and SALT TOLERANCE ZINC FINGER 10 and SALT TOLERANCE ZINC FINGER 18 (ZAT10 and ZAT18), but seriously retarded the vegetative growth of T.halophila by restraining cell expansion. The heterodimer of TsHD1 and TsNAC1 has higher transcriptional activation activity and higher stability compared with the homodimer of TsHD1 or TsNAC1. The binding sites of the TsHD1 and TsNAC1 heterodimers were found to exist in the promoters of most upregulated genes in Cauliflower mosaic virus 35S promoter (P35S)::TsHD1 and P35S::TsNAC1 transgene lines compared with the wild type using RNA-seq and genomic data analyses. Moreover, the binding sites in the promoter region of the most downregulated genes were located in the vicinity of the TATA-box. This study reveals that TsNAC1 and TsHD1 play roles in plant growth and abiotic stress resistance synergistically, and the effects depend on the heterodimer binding to the specific target sites in the promoter region.

Cadmium Stress in Halophyte Thellungiella halophila: Consequences on Growth, Cadmium Accumulation, Reactive Oxygen Species and Antioxidative Systems

Cadmium (Cd) stress on the growth and antioxidant system of the halophyte Thellungiella halophila is studied to explore heavy metal tolerance and the role of antioxidants in Cd tolerance. Biomass production of both leaves and roots of four leaves T. halophila seedlings significantly decreased under 0, 0.3, 0.6, and 0.9 mmol L-1 CdCl2 for 14 days compare to no-Cd control. Meanwhile, the photosynthetic oxygen evolution rate significantly decreased and the chlorophyll content slightly decreased. The MDA and hydrogen peroxide (H2O2) contents of leaves increased as the Cd concentrations increased, and the oxidative damage in Cd-treated seedlings was greater under Cd stress than under the no-Cd controls. Though the translocation factor (TF) was always under 1, T. halophila can tolerate and accumulate more than 100 mg kg-1 Cd in shoot dry biomass. T. halophila mitigated the oxidative damage by increasing enzymatic components, glutathione (GSH), and ascorbic acid (AsA). In Cd-treated seedlings, both the leaf GSH and AsA and the activities of both peroxidase (POD) and catalase (CAT) were significantly greater under Cd stress than under no-Cd control. No significant differences were found in the activity of ascorbate peroxidase (APX), glutathione reductase (GR) and superoxide dismutase (SOD). These results critically summarize the effects of Cd stress in T. halophila and these results above indicate that the antioxidants are responsible for Cd tolerance in T. halophila.

TsNAC1 Is a Key Transcription Factor in Abiotic Stress Resistance and Growth.

NAC proteins constitute one of the largest families of plant-specific transcription factors, and a number of these proteins participate in the regulation of plant development and responses to abiotic stress. T. HALOPHILA STRESS RELATED NAC1 (TsNAC1), cloned from the halophyte Thellungiella halophila, is a NAC transcription factor gene, and its overexpression can improve abiotic stress resistance, especially in salt stress tolerance, in both T. halophila and Arabidopsis (Arabidopsis thaliana) and retard the growth of these plants. In this study, the transcriptional activation activity of TsNAC1 and RD26 from Arabidopsis was compared with the target genes' promoter regions of TsNAC1 from T. halophila, and the results showed that the transcriptional activation activity of TsNAC1 was higher in tobacco (Nicotiana tabacum) and yeast. The target sequence of the promoter from the target genes also was identified, and TsNAC1 was shown to target the positive regulators of ion transportation, such as T. HALOPHILA H+-PPASE1, and the transcription factors MYB HYPOCOTYL ELONGATION-RELATED and HOMEOBOX12 In addition, TsNAC1 negatively regulates the expansion of cells, inhibits LIGHT-DEPENDENT SHORT HYPOCOTYLS1 and UDP-XYLOSYLTRANSFERASE2, and directly controls the expression of MULTICOPY SUPPRESSOR OF IRA14 Based on these results, we propose that TsNAC1 functions as an important upstream regulator of plant abiotic stress responses and vegetative growth.

Salt-induced photoinhibition of PSII is alleviated in halophyte Thellungiella halophila by increases of unsaturated fatty acids in membrane lipids

The effect of salinity on plant growth, chlorophyll content, photosynthetic parameters, photochemical efficiency of PSII, membrane lipid content and fatty-acid composition of halophyte Thellungiella halophila and glycophyte Arabidopsis thaliana was investigated to examine the possible role of unsaturated fatty acids in photosynthesis under saline conditions. The growth of Arabidopsis was significantly decreased by the 100 and 200 mM NaCl treatments; however, there was no significant difference in the fresh and dry weight of Thellungiella at different concentrations of NaCl. Exposure of Arabidopsis to salt resulted in a progressive decline in chlorophyll content, while there was no significant change in that of Thellungiella. The net photosynthetic rate, maximal photochemical efficiency of PSII (Fv/Fm) and actual PSII efficiency were significantly reduced in Arabidopsis but remained unaffected in Thellungiella. Arabidopsis under NaCl treatment showed decreased PG levels and decreased values for the unsaturated fatty acid content and the double bond index (DBI) of monogalactosyldiacylglycerols and phosphatidylglycerols; these values significantly increased in Thellungiella under NaCl treatment, as did the DBI values of digalactosyldiacylglycerols and sulphoquinovosyldiacylglycerols. These results suggest that Thellungiella under salt stress displays high resistance to photoinhibition and that increased concentrations of unsaturated fatty acids in membrane lipids enhances the tolerance of photosystem II to salt stress.

Over-expression of TsCBF1 gene confers improved drought tolerance in transgenic maize

The DREB/CBF transcription factors play important roles during low temperature, drought, and high-salt stress in higher plants. In this paper, we transferred TsCBF1 gene from a dicotyledonous halophyte Thellungiella halophila into the monocotyledonous crop maize (Zea mays L.). PCR, Southern blot, and RT-PCR analysis indicated that the TsCBF1 gene had been integrated into the genome of transgenic plants and was expressed in their progeny. After 14 days of drought stress treatments, transgenic plants showed improved drought tolerance with higher Relative Water Content (RWC), higher solute accumulation, and less cell damage compared with wild-type (WT) plants. Most importantly, they showed shorter anthesis-silking interval (ASI) and produced much higher grain yield than WT under drought stress. The results indicate that the TsCBF1 gene conferred enhanced drought tolerance to maize plants and may have utility for improving tolerance to other abiotic stresses as well.

Molecular characterization of ThIPK2, an inositol polyphosphate kinase gene homolog from Thellungiella halophila, and its heterologous expression to improve abiotic stress tolerance in Brassica napus

Inositol polyphosphate kinases play important roles in diverse cellular processes. In this study, the function of an inositol polyphosphate kinase gene homolog named ThIPK2 from a dicotyledonous halophyte Thellungiella halophila was investigated. The deduced translation product (ThIPK2) shares 85% identity with the Arabidopsis inositol polyphosphate kinase AtIPK2beta. Transient expression of ThIPK2-YFP fusion protein in tobacco (Nicotiana tabacum) protoplasts indicates that the protein is localized to the nucleus and plasma membrane, with a minor localization to the cytosol. Heterologous expression of ThIPK2 in ipk2Delta (also known as arg82Delta), a yeast mutant strain that lacks inositol polyphosphate multikinase (Ipk2) activity, rescued the mutant's salt-, osmotic- and temperature-sensitive growth defects. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed ubiquitous expression of ThIPK2 in various tissues, including roots, rosette leaves, cauline leaves, stem, flowers and siliques, and shoot ThIPK2 transcript was strongly induced by NaCl or mannitol in T. halophila as exhibited by real-time PCR analysis. Transgenic expression of ThIPK2 in Brassica napus led to significantly improved salt-, dehydration- and oxidative stress resistance. Furthermore, the transcripts of various stress responsive marker genes increased in ThIPK2 transgenic plants under salt stress condition. These results suggest that ThIPK2 is involved in plant stress responses, and for the first time demonstrate that ThIPK2 could be a useful candidate gene for improving drought and salt tolerance in important crop plants by genetic transformation.

Comparison Analysis of Transcripts from the Halophyte Thellungiella halophila

The Brassicaceae family halophyte Thellungiella halophila has a high salinity tolerance and serves as a valuable halophytic genetic model plant with experimental convenience similar to Arabidopsis thaliana. A cDNA library of Thellungiella was generated from salt-treated seedlings including rosettes and roots. More than 1 000 randomly selected clones were sequenced and 946 expressed sequence tags (ESTs) were generated. The accession numbers of our EST data are available online in the GenBank database from EC598928 to EC599965. In total 679 unique clusters were assembled, and 632 (93%) had BLASTX hits in the nr databases and 7% are Thellungiella unique. According to the Gene Ontology (GO) hierarchy, 385 of 679 unigenes were categorized. Compared with public Arabidopsis microarray data, our results provide more potential salt tolerance genes in Thellungiella. These results will provide a broader coverage into Thellungiella transcriptome and benefit the discovery of salt tolerance related genes.

Contrasting response of photosynthesis to salt stress in the glycophyte Arabidopsis thaliana and the halophyte Thellungiella halophila

Contrasting response of photosynthesis to salt stress in the glycophyte Arabidopsis thaliana and the halophyte Thellungiella halophila

Overexpression of aThellungiella halophila CBl9 homolog,ThCBL9, confers salt and osmotic tolerances in transgenicarabidopsis thaliana

The calcineurin B-like (CBL) proteins, comprising a large subfamily of calcium sensors in plant cells, play an important role in many stress responses. We cloned a gene from the halophyteThellungiella halophila that is homologous toAtCBL9 inArabidopsis thaliana. The 1008-bpThCBL9 contains an ORF of 639 bp and encodes 213 amino acids, with a 5“-untranslated region of 193 bp and a 3”-untranslated region of 176 bp. Its amino acid sequence shares high homology with AtCBLs.ThCBL9 is up-regulated by ABA, NaCI, and PEG inThellungiella leaves. Using molecular biological methods, we over-expressedThCBL9 inA. thaliana and found that this enhanced tolerances to both high salt and osmotic stress in transgenicArabidopsis.

Heterologous expression of the TsVP gene improves the drought resistance of maize

In this study, it was shown that the TsVP gene [vacuolar H+-pyrophosphatase (V-H+-PPase) gene from a dicotyledonous halophyte Thellungiella halophila] could be transferred into the monocotyledonous crop maize (Zea mays L.), and that the heterologous expression of the transgene improved the drought resistance of transgenic plants. Polymerase chain reaction amplification and Southern blotting confirmed the existence of the foreign gene in transformed plants and their progeny. Expression differences of the TsVP gene in different transgenic lines were monitored by reverse transcriptase-polymerase chain reaction. The measurement of isolated vacuolar membrane vesicles from the TsVP transgenic and wild-type (WT) plants demonstrated that the transgenic plants had higher V-H+-PPase activity, and the performance of maize-expressed TsVP in response to osmotic/drought stress was better in lines with higher V-H+-PPase activity. Transgenic plants showed a higher percentage of seed germination, better developed root systems and greater biomass, greater solute accumulation and less cell membrane damage relative to WT plants under osmotic stress. After drought stress treatment, transgenic plants showed less growth retardation and shorter anthesis-silking interval, and produced much larger grain yields, than WT plants. It was concluded that the high V-H+-PPase activity of transgenic maize improved the drought resistance of plants. This report provides a feasible approach to increase monocotyledonous crop yields under conditions of soil water deficit by the heterologous expression of TsVP.

Comparative salt tolerance analysis between Arabidopsis thaliana and Thellungiella halophila, with special emphasis on K +/Na + selectivity and proline accumulation

The eco-physiology of salt tolerance, with an emphasis on K(+) nutrition and proline accumulation, was investigated in the halophyte Thellungiella halophila and in both wild type and eskimo-1 mutant of the glycophyte Arabidopsis thaliana, which differ in their proline accumulation capacity. Plants cultivated in inert sand were challenged for 3 weeks with up to 500mM NaCl. Low salinity significantly decreased A. thaliana growth, whereas growth restriction was significant only at salt concentrations equal to or exceeding 300mM NaCl in T. halophila. Na(+) content generally increased with the amount of salt added in the culture medium in both species, but T. halophila showed an ability to control Na(+) accumulation in shoots. The analysis of the relationship between water and Na(+) contents suggested an apoplastic sodium accumulation in both species; this trait was more pronounced in A. thaliana than in T. halophila. The better NaCl tolerance in the latter was associated with a better K(+) supply, resulting in higher K(+)/Na(+) ratios. It was also noteworthy that, despite highly accumulating proline, the A. thaliana eskimo-1 mutant was the most salt-sensitive species. Taken together, our findings indicate that salt tolerance may be partly linked to the plants' ability to control Na(+) influx and to ensure appropriate K(+) nutrition, but is not linked to proline accumulation.

Ectopic expression of ThCYP1, a stress-responsive cyclophilin gene from Thellungiella halophila, confers salt tolerance in fission yeast and tobacco cells

The halophyte Thellungiella halophila (salt cress) is an ideal model system for studying the molecular mechanisms of salinity tolerance in plants. Herein, we report the identification of a stress-responsive cyclophilin gene (ThCYP1) from T. halophila, using fission yeast as a functional system. The expression of ThCYP1 is highly inducible by salt, abscisic acid (ABA), H(2)O(2) and heat shock. Ectopic overexpression of the ThCYP1 gene enhance the salt tolerance capacity of fission yeast and tobacco (Nicotiana tabacum L.) cv. Bright Yellow 2 (BY-2) cells significantly. ThCYP1 is expressed constitutively in roots, stems, leaves and flowers, with higher expression occurring in the roots and flowers. The ThCYP1 proteins are distributed widely within the cell, but are enriched significantly in the nucleus. The present results suggest that ThCYP1 may participate in response to stresses in the salt cress, perhaps by regulating appropriate folding of certain stress-related proteins, or in the signal transduction processes.

Cloning, characterization and genetic engineering of FLC homolog in Thellungiella halophila

The halophyte Thellungiella halophila is used as a model for the study of salt tolerance in plants. However, it flowers late and requires vernalization to promote flowering. In Arabidopsis thaliana, a close relative of T. halophila, FLC is the key gene controling the vernalization response pathway. Here, we report the cloning and functional characterization of T. halophila FLC gene (ThFLC). ThFLC showed significant homology to AtFLC and was negatively regulated upon vernalization treatment. Ectopic expression of ThFLC in Arabidopsis functionally complemented FLC-Ler and caused a late-flowering phenotype. An RNAi construct, developed from a 309bp fragment of ThFLC cDNA, was used for gene specific silencing of endogenous ThFLC in T. halophila. In transgenic T. halophila plants the endogenous ThFLC transcript was eliminated, producing an early flowering phenotype while retaining the same salt tolerance as wild-type. This engineered T. halophila provides a better research model for plant salt tolerance studies than wild-type T. halophila.

Molecular and genetic aspects of plant responses to osmotic stress.

Drought, high salinity and freezing impose osmotic stress on plants. Plants respond to the stress in part by modulating gene expression, which eventually leads to the restoration of cellular homeostasis, detoxification of toxins and recovery of growth. The signal transduction pathways mediating these adaptations can be dissected by combining forward and reverse genetic approaches with molecular, biochemical and physiological studies. Arabidopsis is a useful genetic model system for this purpose and its relatives including the halophyte Thellungiella halophila, can serve as valuable complementary genetic model systems.