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.     

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.     

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. These authors contributed equally to this work.     

盐胁迫对盐芥（Thellungiella halophila）生长和抗氧化酶活性的影响

在盐芥抽苔期用不同浓度NaCl进行处理,测定单株生长量、苔茎叶和根系的质膜透性、MDA含量、苔茎叶的超氧阴离子（O^-₂）含量,苔茎叶的超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）等的活性。结果表明：低浓度NaCl处理盐芥单株干重增加,高浓度NaCl处理则降低盐芥单株的干重,鲜重有抑制作用;盐处理后盐芥地上部质膜透性逐渐增加,地下部质膜透性、叶片中的丙二醛（MDA）和超氧阴离子（O^-₂）含量先降低后升高。抗氧化酶系统中的超氧化物歧化酶（SOD）活性先升高后降低,过氧化物酶（POD）、过氧化氢酶（CAT）的活性呈上升趋势。表明低浓度的盐处理对盐芥生长有利,活性氧及丙二醛（MDA）含量减少,而高浓度的盐处理后,抗氧化酶不能及时将活性氧类清除,从而导致活性氧及MDA积累,引起质膜伤害,盐芥生长量降低。     

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.     

Effects of NaCl salinity on growth, morphology, photosynthesis and proline accumulation of Salvinia natans

The effects of NaCl salinity on growth, morphology and photosynthesis of Salvinia natans (L.) All. were investigated by growing plants in a growth chamber at NaCl concentrations of 0, 50, 100 and 150 mM. The relative growth rates were high (ca. 0.3 d⁻¹) at salinities up to 50 mM and decreased to less than 0.2 d⁻¹ at higher salinities, but plants produced smaller and thicker leaves and had shorter stems and roots, probably imposed by the osmotic stress and lowered turgor pressure restricting cell expansion. Na⁺ concentrations in the plant tissue only increased three-fold, but uptake of K⁺ was reduced, resulting in very high Na⁺/K⁺ ratios at high salinities, indicating that S. natans lacks mechanisms to maintain ionic homeostasis in the cells. The contents of proline in the plant tissue increased at high salinity, but concentrations were very low (<0.1 μmol g⁻¹ FW), indicating a limited capacity of S. natans to synthesize proline as a compatible compound. The potential photochemical efficiency of PSII (F_v/F_m) of S. natans remained unchanged at 50 mM NaCl but was reduced at higher salinities, and the photosynthetic capacity (ETR_max) was significantly reduced at 50 mM NaCl and higher. It is concluded that S. natans is a salt-sensitive species lacking physiological measures to cope with exposure to high NaCl salinity. At low salinities salts are taken up and accumulate in old leaves, and high growth rates are maintained because new leaves are produced at a higher rate than for plants not exposed to salt.     

Establishment of an efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated leaf disc transformation of <Emphasis Type="Italic">Thellungiella halophila</Emphasis>

Thellungiella halophila is a salt-tolerant close relative of Arabidopsis, which is adopted as a halophytic model for stress tolerance research. We established an Agrobacterium tumefaciens-mediated transformation procedure for T. halophila. Leaf explants of T. halophila were incubated with A. tumefaciens strain EHA105 containing a binary vector pCAMBIA1301 with the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene as a reporter gene. Following co-cultivation, leaf explants were cultured on selective medium containing 10 mg l⁻¹ hygromycin and 500 mg l⁻¹ cefotaxime. Hygromycin-resistant calluses were induced from the leaf explants after 3 weeks. Shoot regeneration was achieved after transferring the calluses onto fresh medium of the same composition. Finally, the shoots were rooted on half strength MS basal medium supplemented with 10 mg l⁻¹ hygromycin. Incorporation and expression of the transgenes were confirmed by PCR, Southern blot analysis and GUS histochemical assay. Using this protocol, transgenic T. halophila plants can be obtained in approximately 2 months with a high transformation frequency of 26%.     

Molecular cloning and expression of a Cu/Zn-Containing superoxide dismutase from <Emphasis Type="Italic">Thellungiella halophila</Emphasis>

Superoxide dismutases (SODs) constitute the first line of cellular defense against oxidative stress in plants. SODs generally occur in three different forms with Cu/Zn, Fe, or Mn as prosthetic metals. We cloned the full-length cDNA of the Thellungiella halophila Cu/Zn-SOD gene ThCSD using degenerate RT-PCR and rapid amplification of cDNA ends (RACE). Sequence analysis indicated that the ThCSD gene (GenBank accession number EF405867) had an open reading frame of 456 bp. The deduced 152-amino acid polypeptide had a predicted molecular weight of 15.1 kDa, an estimated pI of 5.4, and a putative Cu/Zn-binding site. Recombinant ThCSD protein was expressed in Escherichia coli and assayed for SOD enzymatic activity in a native polyacrylamide gel. The SOD activity of ThCSD was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide, confirming that ThCSD is a Cu/Zn-SOD. Northern blotting demonstrated that ThCSD is expressed in roots, stems, and leaves. ThCSD mRNA levels increased by about 30-fold when plants were treated with sodium chloride (NaCl), abscisic acid (ABA), and indole-acetic acid (IAA) and by about 50-fold when treated with UVB light. These results indicate that ThCSD is involved in physiological pathways activated by a variety of environmental conditions. These authors contributed equally to this work.     

Gender, season and habitat: Patterns of variation in photosynthetic activity, growth and fecundity in Thymelaea velutina

Changes in the ecophysiological performance of a plant species due to different environmental conditions generally reflect adaptations to the habitat where the plant grows and are often related to its survival capacity in a particular place. We examined this with the dioecious shrub Thymelaea velutina, in two contrasting populations representing the extremes of the altitudinal gradient where the species lives (coastal dunes and mountain habitats over 1000 m). We measured net photosynthetic rates and stomatal conductance, estimated the level of plant stress by chlorophyll fluorescence, and assessed their correlations with growth rate, plant size, flower production and fruit set. We hypothesized that plants at high altitude were more photosynthetically stressed than at sea level and expected a gender × habitat interaction in performance as females need more resources than males. Plants in the mountain experienced chronic photoinhibition during winter and a reduced photosynthetic performance both in winter and spring compared to plants in coastal dunes. However, there was no association between any of the fluorescence variables and either plant growth or fecundity, suggesting that other factors are involved determining performance. Mountain plants showed also an apparent lower capacity of heat dissipation to excessive radiation than dune plants. In the dunes, the greater leaf area and mass can lead to a higher photosynthetic carbon gain by whole individuals compared to plants in the mountain. No effect of gender was detected on the ecophysiological performance of this species, which we partly attribute to the small size of fruits of the female plants.     

盐芥叶片响应干旱胁迫的蛋白质组学初步分析

盐芥是新兴起的植物非生物逆境研究模式植物,研究盐芥叶片蛋白质组对于干旱胁迫的响应,以推进对植物干旱耐受机制的认识。该研究应用双向电泳技术分析了干旱胁迫对于盐芥叶片蛋白质组的影响,结果共鉴定了63个干旱胁迫差异表达蛋白,包括丰度上调的31个,新出现的蛋白点14个,丰度下调的15个,消失的蛋白点3个。应用生物质谱分析技术确定了包括硫氧还蛋白,铁蛋白-1和凝集素在内的9个干旱胁迫响应蛋白的身份,对这些干旱胁迫响应蛋白的功能分类分析表明,盐芥的耐旱机制可能涉及自由基清除能力的增强、能量代谢的调整以及光合作用的维持。     

Microsatellite DNA loci from the typical halophyte <Emphasis Type="Italic">Thellungiella salsuginea</Emphasis> (Brassicaceae)

Thellungiella salsuginea (Brassiaceae) is a typical halophyte which can tolerate extreme cold, drought, and salinity. In order to understand the adaptive evolution of this species in the arid habitats, it is important to know its genetic structure. In this study, 17 polymorphic microsatellite loci were isolated and characterized from an enrichment genomic library of this species. We further assessed the polymorphisms of each locus in 18 individuals from nine geographically distant populations. The number of alleles per locus ranged from six to fourteen. The observed and expected heterozygosity ranged from 0.17 to 0.28 and 0.32 to 0.45, respectively. These markers have been crossly checked in another congeneric species, T. halophila. These microsatellite markers will be useful for investigating population genetics and adaptive evolution of this species and morphological divergence between and it and the closely related species.     

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。     

Effects of NaCl on growth, ion accumulation, protein, proline contents and antioxidant enzymes activity in callus cultures of Jatropha curcas

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. The effect of NaCl stress on growth, ion accumulation, contents of protein, proline, and antioxidant enzymes activity in callus cultures of J. curcas was investigated. Exposure of callus to NaCl decreased growth in a concentration dependent manner. NaCl treated callus accumulated Na and declined in K, Ca and Mg contents. Na/K ratio increased steadily as a function of external NaCl treatment. NaCl induced significant differences in quality and quantity of proteins, whereas, proline accumulation remained more or less constant with treatment. NaCl stress enhanced the activity of superoxide dismutase (SOD; E.C. 1.15.1.1) and peroxidase (POX; E.C. 1.11.1.7). Further in the isoenzyme studies, four SOD isoenzymes (SOD 1, 2, 3, and 4) and two POX isoenzymes (POX 1 and 2) were detected with the treatment. NaCl strongly induced activity of SOD 4 isoenzyme in 40, 60, 80 mM and POX 2 isoenzyme in 40 and 80 mM NaCl concentrations. Increase in antioxidant enzymes activity could be a response to cellular damage induced by NaCl. This increase could not stop the deleterious effects of NaCl, but it reduced stress severity and thus allowed cell growth to occur.     

Ectopic expression of <Emphasis Type="Italic">ThCYP1</Emphasis>, a stress-responsive cyclophilin gene from <Emphasis Type="Italic">Thellungiella halophila</Emphasis>, 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), H₂O₂ 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.     

Effects of Na2CO3 and NaCl stresses on the antioxidant enzymes of chloroplasts and chlorophyll fluorescence parameters of leaves of Puccinellia tenuiflora (Turcz.) scribn.et Merr.

Antioxidant enzymes in chloroplasts and chlorophyll fluorescence parameters of leaves of Puccinellia tenuiflora (Turcz.) scribn.et Merr. under isotonic Na₂CO₃ and NaCl stresses were studied. Ascorbate peroxidase (APX) and superoxide dismutase (SOD) activities showed a similar increasing trend and then decreased with the decreasing osmotic potential of culture solution, peaking at −4.74 × 10⁵ Pa under NaCl stress and at −3.40 × 10⁵ Pa under Na₂CO₃ stress. APX, glutathione transferase and SOD activities were higher under NaCl stress than those under Na₂CO₃ stress, and higher activities of antioxidant enzymes in chloroplasts were accompanied by lower MDA content under NaCl stress. F _v/F _m, F _v/F _o and F _v′/F _m′ all initially increased and then decreased with the decreasing osmotic potential of culture solution, while Φ _PSII, qNP and HDR showed a constant increase. F _v/F _m, F _v/F _o, Φ _PSII and qNP under NaCl stress were also shown to be higher than those under Na₂CO₃ stress. The present study suggested that acidity played an important role in the hurt toPuccinellia tenuiflora seedlings, which was due to higher activities of antioxidant enzymes, qNP and Φ _PSII, and the Na₂CO₃ resistance to Puccinellia tenuiflora was also supposed to be less than NaCl resistance in present work.     

缓释肥对杉木容器苗生长、光合生理和养分积累的影响

为探索杉木容器苗生长、光合特性及养分积累对不同缓释肥用量的响应特征,该文通过设置6种不同缓释肥处理(0、200、400、800、1 000、1 200 g·m^-3),研究不同缓释肥用量对杉木幼苗生长、光合色素含量、叶绿素荧光特性和养分含量的影响,并结合隶属函数法对各生长和生理指标进行综合评价,以期筛选出适合杉木容器苗生长的施肥水平,为杉木优质苗木的高效培育提供参考。结果表明：(1)与对照相比,缓释肥处理可不同程度促进杉木幼苗苗高、地径生长及植株总生物量的积累。(2)与对照相比,缓释肥处理可显著增加杉木叶片叶绿素和类胡萝卜素含量,提高叶片最大荧光(F_m)、可变荧光(F_v)、PSⅡ最大光化学效率(F_v/F_m)、PSⅡ潜在光化学效率(F_v/F_o)和实际量子产量(QY)值。(3)缓释肥处理可不同程度促进杉木幼苗养分的积累,其中锰、铁和锌积累量变化最显著。(4)隶属函数法分析结果表明,当缓释肥用量为1 000 g·m^-3时...