Salt Cress Research Articles

Structural analysis of 83-kb genomic DNA from Thellungiella halophila: Sequence features and microcolinearity between salt cress and Arabidopsis thaliana

Salt cress ( Thellungiella halophila) has become a desirable plant model for molecular analysis of the mechanisms of salt tolerance. Analysis of its physiological action and expressed EST has resulted in better understanding. However, less is known about its genomic features. Here we determined a continuous sequence approximately 83 kb from a salt cress BAC clone, providing the first insight into the genomic feature for this species. The gene density is approximately one gene per 3.6 kb in this sequence. Many types of repetitive sequences are present in this salt cress sequence, including LTR retroelements, DNA transposons and a number of simple sequence repeats. Comparison of sequence similarity indicated that salt cress shares a close relationship with Arabidopsis. Extensive conservation and high-level microcolinearity were uncovered for both genomes. Our study also indicated that genomic DNA alternations (involving chromosome inversion, sequence loss and gene translocation) contributed to the genomic discrepancies between salt cress and Arabidopsis.

Characterization of glucosinolate—myrosinase system in developing salt cress Thellungiella halophila

Glucosinolates are specialized plant metabolites derived from amino acids. They can be hydrolyzed by myrosinases into different degradation products, which have a variety of biological activities. In this study, the compositions and contents of glucosinolates in salt cress (Thellungiella halophila) at different developmental stages were analyzed by high performance liquid chromatography and mass spectrometry (HPLC-MS). Myrosinase activities were also measured. Seven glucosinolates were identified in T. halophila throughout its life cycle. The glucosinolate profiles varied significantly among different tissues. The roots at stage 4 contained the highest concentrations of total, aromatic and indole glucosinolates among all tissues. Whereas roots, flowers and siliques contained all seven glucosinolates, seeds contained only four aliphatic glucosinolates. During development, the concentrations also displayed significant changes. From seeds to cotyledons and from stage 4 roots to stage 5 roots, there were dramatic declines of glucosinolates, which correlated well with changes in myrosinase activities. In other tissues, myrosinase activity alone could not explain the glucosinolate concentration changes. Certain tissues of T. halophila contained Arabidopsis myrosinase TGG1 and TGG2 orthologs. The molecular basis and functional significance of our findings are discussed here.

Functional gene‐mining for salt‐tolerance genes with the power of Arabidopsis

Here we report on a functional gene-mining method developed to isolate stress tolerance genes without any prior knowledge of the genome or genetic mapping of the source germplasms. The feasibility of this approach was demonstrated by isolating novel salt stress tolerance genes from salt cress (Thellungiella halophila), an extremophile that is adapted to a harsh saline environment and a close relative of the model plant Arabidopsis thaliana. This gene-mining method is based on the expression of salt cress cDNA libraries in Arabidopsis. A cDNA expression library of the source germplasm, salt cress, was constructed and used to transform Arabidopsis via Agrobacterium-mediated gene transfer. A transgenic seed library consisting of >125,000 independent lines was generated and screened for salt-tolerant lines via a high-throughput genetic screen. A number of salt-tolerant lines were isolated, and the salt cress cDNAs were identified by PCR amplification and sequencing. Among the genes isolated, several novel small protein-encoding genes were discovered. The homologs of these genes in Arabidopsis have not been experimentally analyzed, and their functions remain unknown. The function of two genes isolated by this method, ST6-66 and ST225, and their Arabidopsis homologs, were investigated in Arabidopsis using gain- and loss-of-function analyses, and their importance in salt tolerance was demonstrated. Thus, our functional gene-mining method was validated by these results. Our method should be applicable for the functional mining of stress tolerance genes from various germplasms. Future improvements of the method are also discussed.

Cloning and characterization of a flowering time gene fromThellungiella halophila

Thellungiella halophila (T. halophila) (salt cress) is a close relative of Arabidopsis and a model plant for salt tolerance research. However, the nature of its later flowering causes some difficulties in genetic analysis. The FRIGIDA (FRI) gene plays a key role in the Arabidopsis vernalization flowering pathway, whose homolog in T. halophila may also be a key factor in controlling flowering time. In order to study the molecular mechanism of vernalization responses in T. halophila, a full length cDNA named ThFRI (Thellungiella halophila FRIGIDA) was isolated from the young seedlings of T. halophila by RT-PCR and RACE. The ThFRI cDNA was 2017 bp in length and contained an open reading frame encoding a putative protein of 605 amino acids. The ThFRI showed significant homology to AtFRI (74.5% at the nucleotide level and 63.9% at the amino acid level). To study its function, ThFRI cDNA was transformed into Arabidopsis thaliana, driven by CaMV 35S promoter. Transgenic plants expressing ThFRI exhibited late-flowering phenotype, which suggests that ThFRI is the funtional FRI homolog in T. halophila. The cloning and funtional characterization of the FRI homolog of T. halophila will faciliate further study of flowering time control in T. halophila.

Cloning and characterization of a flowering time gene from <italic>Thellungiella halophila</italic>

<title>Abstract</title> <italic>Thellungiella halophila (T. halophila)</italic> (salt cress) is a close relative of <italic>Arabidopsis</italic> and a model plant for salt tolerance research. However, the nature of its later flowering causes some difficulties in genetic analysis. The <italic>FRIGIDA (FRT)</italic> gene plays a key role in the <italic>Arabidopsis</italic> vernalization flowering pathway, whose homolog in <italic>T. halophila</italic> may also be a key factor in controlling flowering time. In order to study the molecular mechanism of vernalization responses in <italic>T. halophila</italic>, a full length cDNA named <italic>ThFRI (Thellungiella halophila FRIGIDA)</italic> was isolated from the young seedlings of <italic>T. halophila</italic> by RT-PCR and RACE. The <italic>ThFRI cDNA</italic> was 2017 bp in length and contained an open reading frame encoding a putative protein of 605 ami no acids. The <italic>ThFRI</italic> showed significant homology to <italic>AtFRI</italic> (74.5% at the nucleotide level and 63.9% at the ami no acid level). To study its function, <italic>ThFRI</italic> cDNA was transformed into <italic>Arabidopsis thaliana</italic>, driven by CaMV 35S promoter. Transgenic plants expressing <italic>ThFRI exhibited</italic> late-flowering phenotype, which suggests that <italic>ThFRI</italic> is the funtional FRI homolog in <italic>T. halophila</italic>. The cloning and funtional characterization of the <italic>FRI</italic> homolog of <italic>T. halophila</italic> will faciliate further study of flowering time control in <italic>T. halophila</italic>. <b>FullText for HTML: </b><a target="_blank" class="col_4A90E2" href="https://doi.org/10.1093/abbs/40.8.747">https://doi.org/10.1093/abbs/40.8.747</a>

Molecular Cloning and Functional Analysis of a Na+/H+ Antiporter Gene ThNHX1 from a Halophytic Plant Thellungiella halophila

Na+/H+ exchanger catalyzes the countertransport of Na+ and H+ across membranes. Using the rapid amplification of cDNA ends method, a Na+/H+ antiporter gene (ThNHX1) was isolated from a halophytic plant, salt cress (Thellungiella halophila). The deduced amino acid sequence contained 545 amino acid residues with a conserved amiloride-binding domain (87LFFIYLLPPI96) and shared more than 94% identity with that of AtNHX1 from Arabidopsis thaliana. The ThNHX1 mRNA level was upregulated by salt and other stresses (abscisic acid, polyethylene glycol, and high temperature). This gene partially complemented the Na+/Li+-sensitive phenotype of a yeast mutant that was deficient in the endosomal–vacuolar Na+/H+ antiporter ScNHX1. Overexpression of ThNHX1 in Arabidopsis increased salt tolerance of transgenic plants compared with the wild-type plants. In addition, the silencing of ThNHX1 gene in T. halophila caused the transgenic plants to be more salt and osmotic sensitive than wild-type plant. Together, these results suggest that ThNHX1 may function as a tonoplast Na+/H+ antiporter and play an important role in salt tolerance of T. halophila.

Cloning and characterization ofTsMT3, a type 3 metallothionein gene from salt cress (Thellungiella salsuginea)

A full-length type 3 plant metallothionein cDNA was isolated from 200 mM NaCl stressed shoots of the salt cress (Thellungiella salsuginea). The 447 bp TsMT3 cDNA sequence has a 207 bp open reading frame (ORF) and encodes a deduced 69 residue peptide of molecular weight 7.52 kDa. Southern blot analysis indicates that, there is only one copy of TsMT3 in the T. salsuginea genome. The accumulation of TsMT3 mRNA is enhanced by the stress imposed by PEG6000, 200 mM NaCl, 50 microM ABA, 4 degrees C, 40 microM CuSO(4) or 25 microM CdCl2. The expression vector pET28-TsMT3 was heterologously expressed in Escherichia coli to define the contribution of TsMT3 to heavy metal tolerance. In the presence of 2 mM CuSO4, 0.3 mM Pb(NO3)2 or 0.4 mM CdCl2, TsMT3 expressing cells exhibited enhanced metal tolerance and accumulated more metal than the controls. We believe that TsMT3 is probably involved in the processes of metal homeostasis, tolerance, and reactive oxygen species (ROS) scavenging.

Construction of a Plant Transformation‐ready Expression cDNA Library for Thellungiella halophila Using Recombination Cloning

Abstract Salt cress (Thellungiella halophila), a close relative of the model plant Arabidopsis thaliana L., is an extremophile that is adapted to harsh saline environments. To mine salt‐tolerance genes from this species, we constructed an entry cDNA library from the salt cress plant treated with salt‐stress by using a modified cDNA synthesis and an improved recombination‐assisted cDNA library construction method that is completely free of manipulations involving restriction enzymes and DNA ligase. This cDNA library construction procedure is significantly simplified and the quality of the cDNA library is improved. This entry cDNA library was subsequently shuttled into the destination binary vector pCB406 designed for plant transformation and expression via recombination‐assisted cloning. The library is plant transformation ready and is used to transform Arabidopsis on a large scale in order to create a large collection of transgenic lines for functional gene mining.

Functional screening of salt stress‐related genes fromThellungiella halophilausing fission yeast system

Salinity stress is one of the most serious factors limiting plant growth and threatening global food security. Identification of salt tolerance relevant genes from halophytes can provide more insights into the determinants of salt stress tolerance in plants. In this study, functional screening of genes for the salinity tolerance inThellungiella halophila(salt cress) was carried out using fission yeast as a host organism. A cDNA library of the salt cress, constructed in the yeast vector under the control of the inducible promoter nmt1, was transformed into the fission yeastSchizosaccharomyces pombe.Transgenic yeast cells that showed enhanced salt tolerance were selected. A total of 69 such clones were identified from 106of yeast transformants by one‐time screening. Functional assignment indicates that a large percentage (81%) of these cDNAs encode for homologues of proteins that are known to have roles in plant salt tolerance, while the other portion (19%) codes for unknown proteins. Among the proteins encoded by the known functional genes, the regulatory proteins account for a relatively higher proportion. Northern blot analysis showed that the expression of some isolated genes was salt stress inducible, whereas that of the others was constitutive. These results indicate that fission yeast is a simple and efficient system for functional and large‐scale identification of salt tolerance‐related genes from other organisms.

Zinc finger protein 1 (ThZF1) from salt cress (Thellungiella halophila) is a Cys-2/His-2-type transcription factor involved in drought and salt stress

Zinc finger proteins (ZFPs) play important roles in growth and development in both animals and plants. Recently, some Arabidopsis genes encoding distinct ZFPs have been identified. However, the physiological role of their homologues with putative zinc finger motif remains unclear. In the present study, a novel gene, ThZF1, was characterized from salt stressed cress (Thellungiella halophila, Shan Dong), encoding a functional transcription factor. ThZF1 contains two conserved C(2)H(2) regions and shares conserved domains, including DNA-binding motif, with Arabidopsis thaliana ZFP family members. The transcript of the ThZF1 gene was induced by salinity and drought. Transient expression analysis of ThZF1-GFP fusion protein revealed that ThZF1 was localized preferentially in nucleus. A gel-shift assay showed that ThZF1 specially bind to the wild-type (WT) EP2 element, a cis-element present in the promoter regions of several target genes regulated by ZFPs. Furthermore, a functional analysis demonstrated that ThZF1 was able to activate HIS marker gene in yeast. Finally, ectopic expression of ThZF1 in Arabidopsis mutant azf2 suggested that ThZF1 may have similar roles as Arabidopsis AZF2 in plant development as well as regulation of downstream gene.

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.

Salinity stress adaptation competence in the extremophile Thellungiella halophila in comparison with its relative Arabidopsis thaliana

In stark contrast to Arabidopsis, a related species, Thellungiella halophila (Thellungiella salsuginea; salt cress), displays extreme tolerance to high salinity, low humidity and freezing. High nucleotide sequence identity permits the use of tools developed for Arabidopsis for Thellungiella transcript profiling, for which a microarray platform with >25,000 DNA elements (70-mer oligonucleotides) was used. Microarray transcript profiling and intensity analysis, quantitative RT-PCR, and metabolite profiles define genes and pathways that showed shared and divergent responses to salinity stress in the two species. Shared responses are exemplified by 40% of the regulated genes functioning in confining ribosomal functions, photosynthesis and cell growth, as well as activating osmolyte production, transport activities and abscisic acid-dependent pathways. An additional 60% of regulated genes distinguished Thellungiella from Arabidopsis. Analysis of the differences showed that Arabidopsis exhibited a global defense strategy that required bulk protein synthesis, while Thellungiella induced genes functioning in protein folding, post-translational modification and protein redistribution. At 150 mm NaCl, Thellungiella maintained unimpeded growth. Transcript intensity analyses and metabolite profiles supported the microarray results, pointing towards a stress-anticipatory preparedness in Thellungiella.

Salt stress in Thellungiella halophila activates Na+ transport mechanisms required for salinity tolerance.

Salinity is considered one of the major limiting factors for plant growth and agricultural productivity. We are using salt cress (Thellungiella halophila) to identify biochemical mechanisms that enable plants to grow in saline conditions. Under salt stress, the major site of Na+ accumulation occurred in old leaves, followed by young leaves and taproots, with the least accumulation occurring in lateral roots. Salt treatment increased both the H+ transport and hydrolytic activity of salt cress tonoplast (TP) and plasma membrane (PM) H(+)-ATPases from leaves and roots. TP Na(+)/H+ exchange was greatly stimulated by growth of the plants in NaCl, both in leaves and roots. Expression of the PM H(+)-ATPase isoform AHA3, the Na+ transporter HKT1, and the Na(+)/H+ exchanger SOS1 were examined in PMs isolated from control and salt-treated salt cress roots and leaves. An increased expression of SOS1, but no changes in levels of AHA3 and HKT1, was observed. NHX1 was only detected in PM fractions of roots, and a salt-induced increase in protein expression was observed. Analysis of the levels of expression of vacuolar H(+)-translocating ATPase subunits showed no major changes in protein expression of subunits VHA-A or VHA-B with salt treatment; however, VHA-E showed an increased expression in leaf tissue, but not in roots, when the plants were treated with NaCl. Salt cress plants were able to distribute and store Na+ by a very strict control of ion movement across both the TP and PM.