Low unidirectional sodium influx into root cells restricts net sodium accumulation in Thellungiella halophila, a salt-tolerant relative of Arabidopsis thaliana

Thellungiella halophila is a useful model species for research into plant salt tolerance. It is closely related to Arabidopsis thaliana, but shows considerably higher salt tolerance. Comparative analysis of ion homeostasis in the two species allows the identification of ion transport pathways that are critical for salt tolerance and provides the basis for future studies into their molecular features. Previous studies indicated that salt tolerance in T. halophila is accompanied by low accumulation of Na in the leaves. Kinetic analysis of net ion uptake over three days confirmed lower Na uptake and K loss in T. halophila compared with A. thaliana. Differential net Na uptake rates were still apparent after 6 weeks of salt treatment. To assess the contribution of unidirectional Na fluxes to net Na uptake, kinetic studies of (22)Na fluxes were carried out in both species. The results show that unidirectional root Na influx is significantly lower in salt-grown T. halophila plants than in A. thaliana exposed to the same level of salinity (100 mM). Quantitative comparison of unidirectional influx and net Na accumulation suggests that both species operate efficient Na efflux, which partly compensates for Na influx. Kinetic analysis of (22)Na efflux indicated higher root Na efflux in A. thaliana than in T. halophila. Thus A. thaliana appears to spend more energy on Na export while nevertheless accumulating more Na than T. halophila. It is proposed that limitation of Na influx is the main mechanism by which T. halophila secures low net Na accumulation in saline conditions. This strategy provides the basis for a positive balance between growth and net Na uptake rates, which is essential for survival in high salt.     

Thellungiella halophila, a salt-tolerant relative of Arabidopsis thaliana, has specific root ion-channel features supporting K+/Na+ homeostasis under salinity stress

Thellungiella halophila is a salt-tolerant relative of Arabidopsis thaliana with high genetic and morphological similarity. In a saline environment, T. halophila accumulates less sodium and retains more potassium than A. thaliana. Detailed electrophysiological comparison of ion currents in roots of both species showed that, unlike A. thaliana, T. halophila exhibits high potassium/sodium selectivity of the instantaneous current. This current differs in its pharmacological profile from the current through inward- and outward-rectifying K(+) channels insofar as it is insensitive to Cs(+) and TEA(+), but resembles voltage-independent channels of glycophytes as it is inhibited by external Ca(2+). Addition of Cs(+) and TEA(+) to the growth medium confirmed the key role of the instantaneous current in whole-plant sodium accumulation. A negative shift in the reversal potential of the instantaneous current under high-salt conditions was essential for decreasing sodium influx to twofold lower than the corresponding value in A. thaliana. The lower overall sodium permeability of the T. halophila root plasma membrane resulted in a smaller membrane depolarization during salt exposure, thus allowing the cells to maintain their driving force for potassium uptake. Our data provide quantitative evidence that specific features of ion channels lead to superior sodium/potassium homeostasis in a halophyte compared with a closely related glycophyte.     

Efficiency of biochemical protection against toxic effects of accumulated salt differentiates Thellungiella halophila from Arabidopsis thaliana

Thellungiella halophila and Arabidopsis thaliana were irrigated with medium containing NaCl at various concentrations. The salt treatment resulted in a restriction of rosette biomass deposition in both species. In A. thaliana leaves, this inhibition was stronger than for T. halophila and was associated with strong inhibition of both leaf initiation and leaf expansion. At highest medium salinity, A. thaliana accumulated Na(+) and Cl(-) at higher levels than T. halophila, but similar leaf dehydration was observed in the two species. Proline accumulation, which increased with NaCl concentration, did not differentiate the two species. The magnitude of the electrolyte leakage and the level of lipid peroxidation (assessed through hydroxy fatty acid content) were modest in T. halophila and quite marked in A. thaliana. The detrimental effects of the salt on photosynthetic activity and stomatal conductance of A. thaliana leaves were much more important than in T. halophila leaves. The abundance of the CDSP32 thioredoxin, a critical component of the defence system against oxidative damage and lipid peroxidation, was found to be higher in T. halophila than in A. thaliana under control conditions and salt treatment. These results suggest that the rosette leaves of T. halophila exhibit more efficient protective mechanisms against Na(+) metabolic toxicity than those of A. thaliana.     

盐芥热激同源蛋白70基因(Thhsc70)的分子克隆及鉴定

热激蛋白70(hsp70s)具有分子伴侣的功能,其中在非胁迫条件下表达的hsp70s称为热激同源蛋白70(hsc70).为更好地了解hsc70基因的特性,鉴定了盐芥(Thellungiella halophila(C.A.Mey.)O.E. Schulz)中编码胞质hsc70蛋白的基因Thhsc70.实验结果表明:在非胁迫条件下,Thhsc70基因具有组织特异性表达;Thhsc70基因受热胁迫和冷胁迫的诱导表达,但几乎不受盐诱导和干旱诱导.Thhsc70基因在拟南芥中过量表达后提高了转基因拟南芥的热耐受性和冷耐受性.     

盐芥热激同源蛋白70基因(Thhsc70)的分子克隆及鉴定(英文)

热激蛋白70(hsp70s)具有分子伴侣的功能,其中在非胁迫条件下表达的hsp70s称为热激同源蛋白70(hsc70)。为更好地了解hsc70基因的特性,鉴定了盐芥(Thellungiella halophila(C. A. Mey. )O. E. Schulz)中编码胞质hsc70蛋白的基因Thhsc70。实验结果表明:在非胁迫条件下,Thhsc70基因具有组织特异性表达;Thhsc70基因受热胁迫和冷胁迫的诱导表达,但几乎不受盐诱导和干旱诱导。Thhsc70基因在拟南芥中过量表达后提高了转基因拟南芥的热耐受性和冷耐受性。     

Phytoalexins and phytoanticipins from the wild crucifers Thellungiella halophila and Arabidopsis thaliana: rapalexin A, wasalexins and camalexin

Investigation of phytoalexin production using abiotic elicitation showed that the phytoalexin rapalexin A was produced by both Thellungiella halophila and Arabidopsis thaliana, but while A. thaliana produced camalexin, T. halophila produced wasalexins A and B and methoxybrassenin B. Considering that the genome of T. halophila is being sequenced currently and that the wasalexin pathway present in T. halophila is expected to involve a number of genes also present in Brassica species, our discovery should facilitate the isolation of genes involved in biosynthetic pathways of phytoalexins of the most economically important crucifer species.     

盐胁迫对盐芥生长及硝酸还原酶活性的影响

盐胁迫处理导致盐芥植株鲜重、干重、含水量、肉质化程度和根冠比都下降;根中有机物含量上升,而无机物含量下降,叶的变化与根的相反;渗透调节能力、Na 含量和根系活力上升;硝酸还原酶活性显著增加;超氧阴离子(O2-)含量先降低后升高.表面扫描电镜图像显示:盐芥叶片表面没有盐腺或盐囊泡,所以它不是泌盐盐生植物.盐芥生长状况、Na 含量和Na X-ray微区分析结果表明:盐芥也不是拒盐盐生植物,而很可能是稀盐盐生植物.     

Cloning and characterization of a flowering time gene from Thellungiella 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 (FRT) 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 ami no acids. The ThFRI showed significant homology to AtFRI (74.5% at the nucleotide level and 63.9% at the ami no 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 .     

Effects of NaCl on the growth, ion accumulation and photosynthetic parameters of Thellungiella halophila

Thellungiella halophila seedlings grown on a solid substrate for 25 days on standard medium were challenged with NaCl. Growth, tissue hydration, ion accumulation, photosynthesis, lipid peroxidation and antioxidant enzymatic activities were studied on rosette leaves. Three accessions of Arabidopsis thaliana were cultivated under the same conditions. During the first two weeks of salt treatment, the growth of T. halophila leaves was restricted by NaCl. No significant difference appeared between T. halophila and A. thaliana concerning biomass deposition, or hydric and ionic parameters. However, all A. thaliana plants displayed foliar damage, and died during the third week of salt (50mM NaCl) treatment. Almost all (94%) T. halophila plants remained alive, but did not display any sign of altered physiological condition. Tissue hydration, chlorophyll content, stomatal conductance, photosynthetic quantum yield, and photosynthetic rate were very similar to those of control plants. Lipid peroxidation, estimated from thermoluminescence, was very low and insensitive to salt treatment. Only slight changes occurred in antioxidant enzymatic activities (SOD, several peroxidases, and catalase). From the absence of physiological disorder symptoms, we infer that salt was efficiently compartmentalized in leaf vacuoles. In salt-treated A. thaliana, the photosynthetic quantum yield was diminished, and lipid peroxidation was augmented. These observations reinforce the conclusion that T. halophila could accumulate salt in its leaves without damage, in contrast to A. thaliana.     

盐胁迫对拟南芥和盐芥莲座叶芥子油苷含量的影响

芥子油苷是十字花科植物中一类含氮、含硫的次生代谢产物,与其水解产物在植物防御功能中有重要意义且与环境因子关系密切。以模式植物拟南芥（Arabidopsis thaliana）和盐生模式植物盐芥（Thellungiella halophila）为研究对象,系统地分析了盐胁迫下二者芥子油苷组成和含量的变化规律。拟南芥（生长4周）和盐芥（生长6周）叶片的芥子油苷组成在盐胁迫后没有改变。拟南芥的芥子油苷总量、脂肪族芥子油苷总量、吲哚族芥子油苷总量受盐胁迫的影响均不显著,而盐芥的则随盐胁迫增强先减少、后增加并高于对照水平。拟南芥脂肪族的3MSOP、5MSOP和吲哚族的4OHI3M、4MOI3M随盐胁迫增强而含量降低,而脂肪族的6MSOH、吲哚族的I3M以及盐芥脂肪族的3MSOP则随盐胁迫增强有含量增加的趋势。拟南芥脂肪族的8MSOO和吲哚族的1MOI3M,盐芥脂肪族的3MTP、Allyl、10MSD和吲哚族的4MOI3M,在盐胁迫下的含量变化与盐芥芥子油苷总量的变化趋势一致。     

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 500 mM NaCl. Low salinity significantly decreased A. thaliana growth, whereas growth restriction was significant only at salt concentrations equal to or exceeding 300 mM 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.     

Functional screening of salt stress-related genes from Thellungiella halophila using 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 in Thellungiella 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 yeast Schizosaccharomyces pombe. Transgenic yeast cells that showed enhanced salt tolerance were selected. A total of 69 such clones were identified from 10⁶ of 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.     

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.