Evolutionary Migration of the Disjunct Salt Cress Eutrema salsugineum (= Thellungiella salsuginea,Brassicaceae) between Asia and North America

Eutrema salsugineum (= Thellungiella salsuginea Brassicaceae), a species growing in highly saline habitats, is a good model for use in salt-stress research. However, its evolutionary migrations and genetic variations within and between disjunct regions from central Asia to northern China and North America remain largely unknown. We examined genetic variations and phylogeographic patterns of this species by sequencing ITS, 9 chloroplast (cp) DNA fragments (4379 bp) and 10 unlinked nuclear loci (6510 bp) of 24 populations across its distributional range. All markers suggested the high genetic poverty of this species and the limited number of genetic variations recovered was congruently partitioned between central Asia, northern China and North America. Further modelling of nuclear population-genetic data based on approximate bayesian computation (ABC) analyses indicated that the long-distance dispersals after the recent origin of E. salsugineum may have occurred from central Asia to the other two regions respectively within 20000 years. The fast demographic expansions should have occurred in northern China in a more recent past. Our study highlights the importance of using ABC analyses and nuclear population genetic data to trace evolutionary migrations of the disjunct distributions of the plants in the recent past.     

Lipid profiling and tolerance to low-temperature stress in Thellungiella salsuginea in comparison with Arabidopsis thaliana

Changes in membrane lipid composition is a fundamental strategy for plants to resist low-temperature stress. We compared members of 11 membrane glycerolipid classes in Thellungiella salsuginea and its close relative Arabidopsis thaliana at normal growth temperature, and during cold acclimation (CA), freezing (FR), and post-freezing recovery (PFR). The results showed several properties of T. salsuginea distinct from that in A. thaliana, which included: 1) low relative content of phosphatidic acid (PA) and a rapid increase and decrease of PA during FR and PFR respectively; 2) insensitivity of lyso-phospholipids to freezing; and 3) high ratio of phosphatidylcholine to phosphatidylethanolamine. All these properties were in favour of maintaining membrane integrity and stability and therefore enable T. salsuginea to be more tolerant to freezing than A. thaliana.     

Cold shock domain proteins in the extremophyte Thellungiella salsuginea (salt cress): gene structure and differential response to cold

Four genes encoding cold shock domain (CSD) proteins have been identified in salt cress [Thellungiella salsuginea (halophila), an extremophyte currently recognized as a promising model for studying stress tolerance]. The deduced proteins prove highly homologous to those of Arabidopsis thaliana (up to 95% identity) and are accordingly enumerated TsCSDP1-TsCSDP4; after the N-proximal conserved CSD, they have respectively 6, 2, 7, and 2 zinc finger motifs evenly spaced by Gly-rich stretches. Much lower similarity (approximately 45%) is observed in the regions upstream of TATA-box promoters of TsCSDP1 vs. AtCSP1, with numerous distinctions in the sets of identifiable cis-regulatory elements. Plasmid expression of sCSDP1 rescues a cold-sensitive cup-lacking mutant of Escherichia coli, confirming that the protein is functional. In leaves of salt cress plants under normal conditions, the mRNA levels for the four TsCSDPs relate as 10: 27: 1: 31. Chilling to 4 degrees C markedly alters the gene expression; the 4-day dynamics are different for all four genes and quite dissimilar from those reported for their Arabidopsis homologues under comparable conditions. Thus, the much greater cold hardiness of Thellungiella vs. Arabidopsis cannot be explained by structural distinctions of its CSDPs, but rather may be due to expedient regulation of their expression at low temperature.     

Phospholipases C and D modulate proline accumulation in Thellungiella halophila/salsuginea differently according to the severity of salt or hyperosmotic stress

Proline accumulation is one of the most common responses of plants to environmental constraints. Thellungiella halophila/salsuginea, a model halophyte, accumulates high levels of proline in response to abiotic stress and in the absence of stress. Recently, lipid signaling pathways have been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. Here we investigated the relationship between lipid signaling enzymes and the level of proline in T. salsuginea. Inhibition of phospholipase C (PLC) enzymes by the specific inhibitor U73122 demonstrated that proline accumulation is negatively controlled by PLCs in the absence of stress and under moderate salt stress (200 mM NaCl). The use of 1-butanol to divert some of the phospholipase D (PLD)-derived phosphatidic acid by transphosphatidylation revealed that PLDs exert a positive control on proline accumulation under severe stress (400 mM NaCl or 400 mM mannitol) but have no effect on its accumulation in non-stress conditions. This experimental evidence shows that positive and negative lipid regulatory components are involved in the fine regulation of proline metabolism. These signaling pathways in T. salsuginea are regulated in the opposite sense to those previously described in A. thaliana, revealing that common signaling components affect the physiology of closely related glycophyte and salt-tolerant plants differently.     

Leaf ureide degradation and N(2) fixation tolerance to water deficit in soybean

Accumulation of ureides in leaves is associated with the sensitivity of N(2) fixation in soybean to soil water deficit. Consequently, ureide degradation in leaves may be a key to increasing soybean tolerance to dry soils. Previous research indicated that allantoic acid degradation is catalysed by different enzymes in cultivars Maple Arrow and Williams. The enzyme found in Williams requires manganese as a cofactor. The first objective of this study was to determine if the two degradation pathways were associated with differences in N(2) sensitivity to soil water deficits. N(2) fixation of Williams grown on low-Mn soil was sensitive to stress, but it was relatively tolerant when grown on soil amended with Mn. N(2) fixation in Maple Arrow was relatively tolerant of soil drying regardless of the Mn treatment. The second objective of this study was to expand the study of the degradation pathway to nine additional genotypes. Based on ureide degradation in the presence and absence of Mn, these genotypes also segregated for the two degradation pathways. Those genotypes with the Mn-dependent pathway tended to have drought-sensitive N(2) fixation, but there was one exception. The genotypes not requiring Mn for ureide degradation were drought-tolerant except for one genotype. These results demonstrated the possibility for increasing N(2) fixation tolerance to soil water deficits in soybean by selection of lines with high ureide degradation rates, which were commonly associated with the Mn-independent pathway.     

The Functional and Regulatory Mechanisms of the Thellungiella salsuginea Ascorbate Peroxidase 6 (TsAPX6) in Response to Salinity and Water Deficit Stresses

Soil salinization is a resource and ecological problem in the world. Thellungiella salsuginea is becoming a new model plant because it resembles its relative species, Arabidopsis thaliana, in small genome and short life cycle. It is highly tolerant to salinity and drought stresses. Ascorbate peroxidase (APX) is an enzyme that clears H₂O₂ in plants. The function and molecular and regulation mechanisms of APX in T. salsuginea have rarely been reported. In this study, an APX gene, TsApx6, was cloned from T. salsuginea and its responses to abiotic stresses in transgenic Arabidopsis were studied. Under high salinity treatment, the expression of TsApx6 was significantly induced. Under drought treatment, overexpression of TsApx6 increased the survival rate and reduced leaf water loss rate in Arabidopsis. Compared to the wild type plants, high salinity treatment reduced the concentrations of MDA, H₂O₂ and proline but elevated the activities of APX, GPX, CAT and SOD in the TsApx6-overexpressing plants. Meanwhile, germination rate, cotyledon greening, and root length were improved in the transgenic plants compared to the wild type plants under salt and water deficit conditions. Based on these findings, TsApx6 has an important function in the resistance of plants to certain abiotic stresses. The TsApx6 promoter sequence was obtained using Genome Walking technology. Bioinformatics analysis indicated that it contains some cis-acting elements related to stress response. The treatments of salt, dehydration, and ABA induced the expression of Gus gene under the regulation of the TsApx6 promoter. Mutation analysis showed that the MBS motif present in the TsApx6 promoter might be a key negative regulatory element which has an important effect on the growth and developmental process of plants.     

盐芥miR396的表达分析、前体克隆及靶基因预测

利用实时定量PCR的方法分析了tsa-mi R396在盐芥不同组织的表达情况及盐、冷胁迫下的表达量变化,结果显示tsami R396在盐芥的不同组织均有表达,且在盐芥幼苗受盐、冷胁迫处理2 h后其表达量明显上升,随后呈波动性变化。利用在线软件ps RNATarget预测到25个tsa-mi R396的靶基因并进行功能分类。成功克隆tsa-mi R396前体,并利用Mfold在线软件分析该前体能够折叠形成茎环结构。     

Metabolic responses of Thellungiella halophila/salsuginea to biotic and abiotic stresses: Metabolite profiles and quantitative analyses

The metabolite profiles of the model crucifer Thellungiella salsuginea (salt cress) ecotype Shandong subjected to various biotic and abiotic stresses were analyzed using HPLC-DAD-ESI-MS. Two different cruciferous microbial pathogens, Albugo candida, a biotrophic oomycete, and Leptosphaeria maculans, a necrotrophic fungus, elicited formation of the phytoalexins wasalexins A and B without causing visual damage on inoculated leaves. Analyses of non-polar and polar metabolites led to elucidation of the chemical structures of five metabolites: 4′-O-(E)-sinapoyl-7-methoxyisovitexin-2″-O-β-d-glucopyranoside, 4′-O-(E)-sinapoylisovitexin-2″-O-β-d-glucopyranoside, 4-O-β-d-glucopyranosyl-7-hydroxymatairesinol, 5′-O-β-d-glucopyranosyldihydroneoascorbigen and 3-O-β-d-glucopyranosylthiane. 3-O-β-d-glucopyranosylthiane, an unique metabolite for which we suggest the name glucosalsuginin, is proposed to derive from the glucosinolate glucoberteroin. In addition, the identification of a broad range of polar metabolites identical to those of other crucifers was carried out. Quantification of several metabolites over a period of eight days showed that concentrations of the polar phytoanticipin 4-methoxyglucobrassicin increased substantially in leaves irradiated with UV light (λ_max 254 nm) relative to control leaves, but not in leaves subjected to other stresses.     

Effects of season-dependent irradiance levels and nitrogen-deficiency on photosynthesis and photoinhibition in field-grown rice (Oryza sativa)

Photoinhibition and acclimation of photosynthesis in rice plants grown under N-sufficient (NS) and N-deficient (ND) field conditions were investigated during the tropical wet (WS) and dry (DS) seasons in the Philippines. Diurnal patterns of CO₂ assimilation were examined. There was a transient peak in CO₂ assimilation in the leaves of the NS plants in the early morning during the DS and the WS, which was not seen in the ND plants in either season. ND leaves had lower Ribulose bisphosphate carboxylase/oxygenase (Rubisco) contents and lower chlorophyll contents. A lowered quantum yield of photosystem II (φPSII) was observed in the ND plants at an intermediate irradiance though no differences between N treatments were seen at high irradiance. Analysis of carotenoids indicated a small increase in the de-epoxidation state of the xanthophyll cycle (DES) at mid-day in the ND leaves compared to NS. Photoinhibition was greater in ND leaves when incident mid-day irradiance was increased by altering the leaf angle. Although Rubisco contents were lower in ND plants, photosynthesis in situ did not decline proportionally. For NS plants, Chlorophyll content, but not Rubisco content, was season-dependent and results are discussed in terms of the interaction between irradiance use and N content of rice leaves.     

Susceptibility to low-temperature photoinhibition and the acquisition of freezing tolerance in winter and spring wheat: The role of growth temperature and irradiance

Five winter and five spring wheat ( Triticum aestivum L.) cultivars were grown under either control conditions (20°C/250 photosynthetic photon flux density (PPFD) [μmol m⁻² s⁻¹]), high irradiance (20°C/800 PPFD) or at low temperature (either 5°C/250 PPFD or 5°C/50 PPFD). To eliminate any potential bias, the wheat cultivars were arbitrarily chosen without any previous knowledge of their freezing tolerance or photosynthetic competence. We show that the differential susceptibilities to photoinhibition exhibited between spring and winter wheat cultivars, as assessed by chlorophyll fluorescence cannot be explained on the basis of either growth irradiance or low growth temperature per se. The role of excitation pressure is discussed. We assessed the correlation between susceptibility to low-temperature photoinhibition, maximum ribulose 1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39) and NADP-dependent malate dehydrogenase (EC 1.1.1.82) activities, chlorophyll and protein concentrations and freezing tolerance determined by electrolyte leakage. Susceptibility to photoinhibition is the only parameter examined that is strongly and negatively correlated with freezing tolerance. We suggest that the assessment of susceptibility to photoinhibition may be a useful predictor of freezing tolerance and field survival of cereals.     

Cloning and characterization of TsMT3, 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.     

Cold-induced photoinhibition affects establishment of Eucalyptus nitens (Deane and Maiden) Maiden and Eucalyptus globulus Labill

The effects of cold-induced photoinhibition on Eucalyptus globulus and Eucalyptus nitens seedlings were assessed between planting and age 23 weeks. The seedlings were subjected to four treatments before planting: non-hardened (NH) E. globulus and E. nitens, cold-hardened (CH) E. nitens, and nutrient-starved (NS) E. nitens. Seedlings were planted alongside established 1-year-old E. nitens saplings. The experimental site was at 350 m above sea level, which is considered marginal for the establishment of E. globulus plantations due to low mean minimum temperatures. Cold and sunny conditions after frost increased photoinhibition in the order: NH E. globulus > NH and CH E. nitens > NS and established E. nitens. As a result there was 20% mortality of NH E. globulus seedlings. NS E. nitens seedlings were severely photoinhibited and had high anthocyanin levels at planting; levels of photoinhibition decreased after planting, anthocyanin levels remained high and there was no mortality. Carotenoid levels were low in E. globulus compared to E. nitens treatments. It was concluded that cold-induced photoinhibition is a factor determining the range of environments where E. globulus can be successfully planted, and not frost tolerance alone. Anthocyanin was synthesised in response to increased photoinhibition. Anthocyanin levels were correlated to the severity of the photoinhibition. Thus, E. nitens seedlings nutrient starved in the nursery are pre-conditioned to photoinhibitory conditions experienced soon after planting. This treatment is a useful risk-management tool where cold-induced photoinhibition is likely to occur during seedling establishment.     

Low irradiance stress tolerance in rice (Oryza sativa L.)

The effect of low irradiance on three rice cultivars (shade tolerant cvs. Swarnaprabha and CO 43 and shade susceptible cv. IR 20) was studied. The large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase with molecular mass of 55 kDa was reduced in cv. IR 20 grown under low irradiance (LI). Native protein profile studied showed, under LI, reduction in the contents of proteins with RF values 0.03, 0.11 and 0.37. Analysis of chloroplast polypeptides revealed an induction of light-harvesting chlorphyll-protein 2 (LHCP2) under shade. The induction was more expressed in cv. CO 43 than in cv. IR 20. Under LI, in vivo labelled protein bands in the molecular range of 26 - 27 kDa were induced. These proteins were highly turned over in the LI-grown plants of cv. CO 43 than in cv. IR 20. A signal for rbcL gene sequences in EcoRI digested lanes was also found. Isozyme analysis of peroxidase showed an induction of a new band with RF 0.43 in cv. IR 20 subjected to LI. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparison of inhibition of N2 fixation and ureide accumulation under water deficit in four common bean genotypes of contrasting drought tolerance

Background and Aims

Drought is the principal constraint on world production of legume crops. There is considerable variability among genotypes in sensitivity of nitrogen fixation to drought, which has been related to accumulation of ureides in soybean. The aim of this study was to search for genotypic differences in drought sensitivity and ureide accumulation in common bean (Phaseolus vulgaris) germplasm that may be useful in the improvement of tolerance to water deficit in common bean.

Methods

Changes in response to water deficit of nitrogen fixation rates, ureide content and the expression and activity of key enzymes for ureide metabolism were measured in four P. vulgaris genotypes differing in drought tolerance.

Key Results

A variable degree of drought-induced nitrogen fixation inhibition was found among the bean genotypes. In addition to inhibition of nitrogen fixation, there was accumulation of ureides in stems and leaves of sensitive and tolerant genotypes, although this was higher in the leaves of the most sensitive ones. In contrast, there was no accumulation of ureides in the nodules or roots of stressed plants. In addition, the level of ureides in the most sensitive genotype increased after inhibition of nitrogen fixation, suggesting that ureides originate in vegetative tissues as a response to water stress, probably mediated by the induction of allantoinase.

Conclusions

Variability of drought-induced inhibition of nitrogen fixation among the P. vulgaris genotypes was accompanied by subsequent accumulation of ureides in stems and leaves, but not in nodules. The results indicate that shoot ureide accumulation after prolonged exposure to drought could not be the cause of inhibition of nitrogen fixation, as has been suggested in soybean. Instead, ureides seem to be produced as part of a general response to stress, and therefore higher accumulation might correspond to higher sensitivity to the stressful conditions.     

Temperature acclimation affects thermal preference and tolerance in three Eremias lizards(Lacertidae)

We acclimated adult males of three Eremias lizards from different latitudes to 28℃,33℃ or 38℃ to examine whether temperature acclimation affects their thermal preference and tolerance and whether thermal preference and tolerance of these lizards correspond with their latitudinal distributions.Overall,selected body temperature(Tsel)and viable temperature range(VTR)were both highest in E.brenchleyi and lowest in E.multiocellata,with E.argus in between;critical thermal minimum(CTMin)was highest in E.multiocell...