Physiological characterization of drought stress response and expression of two transcription factors and two LEA genes in three Prunus genotypes

Global warming has led to a progressive decrease in rainfall, which is reflected by a reduction of water resources in the soil and a negative effect on crop production in Mediterranean areas. Under drought stress, many plants react by inducing a different series of responses at both physiological and molecular levels, allowing them to survive for a variable period of time. Therefore, in order to understand the response of roots to drought conditions, the genotypes peach × almond ‘Garnem’ [P. amygdalus Batsch × P. persica (L.) Batsch] and their progeny, the hybrid ‘P.2175’ × ‘Garnem’-3 and OP-‘P.2175’ (P. cerasifera Ehrh.) were subjected to a period of water deficit. Drought conditions with a subsequent re-watering period were tested for potted plants for one month. Stomatal conductance and leaf water potential were measured to monitor the plant physiological responses. Significant differences among the drought stress and drought stress recovery treatments and among the genotypes were observed. In addition, four genes related to the ABA biosynthesis pathway were studied for their expression by RT-qPCR: an AN20/AN1 zinc finger protein (ppa012373m); a bZIP transcription factor (ppa013046m); a dehydrin (ppa005514m) and a LEA protein (ppa008651m). Their expression profiles correlated with our physiological results of drought response, being higher in roots than in phloem tissue. In general, the expression of the four studied genes was higher after 15 days under drought conditions. Under drought and recovery conditions, the zinc finger and bZIP transcription factors showed significant differences in their relative expression levels from LEA and dehydrin. These results suggest the role of LEA and dehydrin in the regulatory response to drought stress in Prunus genotypes. Therefore, the dehydrin and the protein LEA might be potential biomarkers to select rootstocks for tolerance to drought conditions.We wish to thank Michael Glenn for helpful comments and ideas on the ash content discussion. This work was supported by RTA2014-00062 from the Instituto Nacional de Investigación y Tecnología Agraria (INIA) and by the Research Group A12 of Aragon, Spain. We kindly appreciate the FPI-INIA 2012 grant for B. Bielsa

Cold treatment breaks dormancy but jeopardizes flower quality in Camellia japonica L.

Camellia japonica L. is an evergreen shrub whose cultivars are of great ornamental value. In autumn, after flower bud differentiation, dormancy is initiated. As in many other spring flowering woody ornamentals, winter low temperatures promote dormancy release of both flower and vegetative buds. However, warm spells during late autumn and winter can lead to unfulfilled chilling requirements leading to erratic and delayed flowering. We hypothesized that storing plants at no light and low temperature could favor dormancy breaking and lead to early and synchronized flowering in response to forcing conditions in C. japonica ‘Nuccio’s Pearl’. Plants with fully developed floral primordia were stored at dark, 7∘C, and RH > 90% for up to 8 weeks. To monitor endodormancy release during the storage, we evaluated the content of abscisic acid (ABA) in flower buds and the expression profiles of five putative genes related to dormancy and cold acclimation metabolism in leaves and flower buds. In addition, the expression of four anthocyanin biosynthesis pathway genes was profiled in flower buds to assess the effect of the treatment on flower pigment biosynthesis. At 0, 4, 6, and 8 weeks of cold treatment, 10 plants were transferred to the greenhouse and forced to flower. Forced plant flower qualities and growth were observed. The ABA content and the expression profiles of two dormancy-related genes (CjARP and CjDEH) suggested that dormancy breaking occurred after 6–8 weeks of cold treatment. Overall, plants treated for 6–8 weeks showed earlier vegetative sprouting, enhanced, and homogeneous flowering with reduced forcing time. Prolonged cold treatments also reduced flower size and longevity, anthocyanin content, and pigment biosynthesis-related gene transcripts. In conclusion, the cold treatment had a promotive effect on dormancy breaking but caused severe drawbacks on flower quality

Analysis of the variability of dehydrin codifying genes in ecotypes of Vigna unguiculata (L.) Walp from Mozambique

Abstract Cowpea (Vigna unguiculata (L.) Walp) is a tropical grain legume, which plays an important nutritional role in developing countries of the tropics and subtropics, especially in sub-Saharian Africa, Asia, Central and South America. Its production is limited by a lot of environmental stresses and drought seems to be one of the most important. It has been reported that cowpea has in its genome genes encoding proteins associated with environmental stresses. Among the stress induced proteins there is a group called dehydrins. They are produced on the last stages of embryogenesis or in response to drought, salinity, low temperature or to ABA application. It has been hypothesized that dehydrins function by stabilizing large-scale hydrophobic interactions, such as membrane structures or hydrophobic patches of proteins. We hypothesized that, as dehydrins have been proposed to protect cellular macromolecules and their expression increase in response to dehydration, it should be used as molecular marker for drought stress tolerance. To test this hypothesis, a fragment of dehydrin was amplified and used for analysis of variability dehydrin gene and cowpea. We used also semi-quantitative RT-PCR (Reverse transcription PCR) to evaluate dehydrin gene expression in plants subjected to water stress. An anti-dehydrin antibody was used to study dehydrin protein accumulation under water stress. Analysis of the dehydrin gene variability revealed a high diversity of this protein family and a high identity to Vigna unguiculata dehydrin database associated to chilling tolerance. Expression of dehydrins was high on the wild ecotypes evidencing more adaptability of this group to water deficit. Western blot analysis revealed an apparent absence of dehydrins in leaves and a presence of a constitutive dehydrin in seeds of unstressed plants

Comparison of drought stress response and gene expression between a GM maize variety and a near-isogenic non-GM variety

Maize MON810, grown and commercialised worldwide, is the only cultivated GM event in the EU. Maize MON810, variety DKC6575, and the corresponding near-isogenic line Tietar were studied in different growth conditions, to compare their behaviour in response to drought. Main photosynthetic parameters were significantly affected by water stress in both GM and non –GM varieties to a similar extents. Though DKC6575 (GM) had a greater sensitivity in the early phase of stress response as compared with Tietar (non GM), after six days of stress they behaved similarly, and both varieties recovered from stress damage. Profiling gene expression in water deficit regimes and in a generalised water stress condition showed an up-regulation of many stress- responsive genes, but a greater number of differentially expressed genes was observed in Tietar, with genes belonging to transcription factor families and genes encoding HSPs, LEAs and detoxification enzymes. Since induction of these genes have been indicated from the literature as typical of stress responses, their activation in Tietar rather than in DKC6575 may be reminiscent of a more efficient response to drought. DKC6575 was also analysed for the expression of the transgene CryIAb (encoding the delta-endotoxin insecticidal protein) in water deficit conditions. In all the experiments, the CryIAb transcript was not influenced by water stress, but was expressed at a constant level.. This suggests that though possessing a different pattern of sensitivity to stress, the GM variety maintains the same expression level for the transgene

Dehydrins in maritime pine (Pinus pinaster) and their expression related to drought stress response

Maritime pine (Pinus pinaster) is an important commercial species throughout its Atlantic distribution. With the anticipated increase in desiccation of its habitat as a result of climate change, the selection of genotypes with increased survival and growth capability under these conditions for breeding programs is of great interest for this species. We aimed to study the response to a realistic drought stress under controlled conditions, looked for a method to measure dehydration resistance, and analyzed dehydrin expression in drought-resistant and drought-sensitive clones from different ecotypes. We report here the sequence characteristics and the expression patterns of five dehydrins from P. pinaster, along with the physiological characterization of drought stress responses in different genotypes (clonally replicated plants), originating from a broad geographical distribution across France and Spain (provenances). In total, we distinguished five different dehydrin genes in silico, grouped into two types—K2 and SKn. Three of the dehydrin genes had several sequence variants, differing by multiple or single amino acid substitutions. Only two of the dehydrins (PpinDhn3 and PpinDhn4) showed an increase in transcription with increased drought stress which was dependent on provenance and genotype, suggesting their involvement in drought resistance. The other dehydrins showed decreased expression trends with increased severity of the drought stress. The lack of close association between the drought stress and expression patterns of these dehydrin genes suggest that they could have other functions and not be involved in drought resistance. Our results suggest large differences in function between different dehydrin genes

Profiling dehydrin gene sequence and physiological parameters in drought tolerant and susceptible spring wheat cultivars.

Physiological and yield traits such as stomatal conductance (mmol m-2s-1), Leaf relative water content (RWC %) and grain yield per plant were studied in a separate experiment. Results revealed that five out of sixteen cultivars viz. Anmol, Moomal, Sarsabz, Bhitai and Pavan, appeared to be relatively more drought tolerant. Based on morphophysiological results, studies were continued to look at these cultivars for drought tolerance at molecular level. Initially, four well recognized primers for dehydrin genes (DHNs) responsible for drought induction in T. durum L., T. aestivum L. and O. sativa L. were used for profiling gene sequence of sixteen wheat cultivars. The primers amplified the DHN genes variably like Primer WDHN13 (T. aestivum L.) amplified the DHN gene in only seven cultivars whereas primer TdDHN15 (T. durum L.) amplified all the sixteen cultivars with even different DNA banding patterns some showing second weaker DNA bands. Third primer TdDHN16 (T. durum L.) has shown entirely different PCR amplification prototype, specially showing two strong DNA bands while fourth primer RAB16C (O. sativa L.) failed to amplify DHN gene in any of the cultivars. Examination of DNA sequences revealed several interesting features. First, it identified the two exon/one intron structure of this gene (complete sequences were not shown), a feature not previously described in the two database cDNA sequences available from T. aestivum L. (gi|21850). Secondly, the analysis identified several single nucleotide polymorphisms (SNPs), positions in gene sequence. Although complete gene sequence was not obtained for all the cultivars, yet there were a total of 38 variable positions in exonic (coding region) sequence, from a total gene length of 453 nucleotides. Matrix of SNP shows these 37 positions with individual sequence at positions given for each of the 14 cultivars (sequence of two cultivars was not obtained) included in this analysis. It demonstrated a considerable diversity for this gene with only three cultivars i.e. TJ-83, Marvi and TD-1 being similar to the consensus sequence. All other cultivars showed a unique combination of SNPs. In order to prove a functional link between these polymorphisms and drought tolerance in wheat, it would be necessary to conduct a more detailed study involving directed mutation of this gene and DHN gene expression

High genetic diversity at the extreme range edge: nucleotide variation at nuclear loci in Scots pine (Pinus sylvestris L.) in Scotland

Nucleotide polymorphism at 12 nuclear loci was studied in Scots pine populations across an environmental gradient in Scotland, to evaluate the impacts of demographic history and selection on genetic diversity. At eight loci, diversity patterns were compared between Scottish and continental European populations. At these loci, a similar level of diversity (θsil=~0.01) was found in Scottish vs mainland European populations, contrary to expectations for recent colonization, however, less rapid decay of linkage disequilibrium was observed in the former (ρ=0.0086±0.0009, ρ=0.0245±0.0022, respectively). Scottish populations also showed a deficit of rare nucleotide variants (multi-locus Tajima's D=0.316 vs D=−0.379) and differed significantly from mainland populations in allelic frequency and/or haplotype structure at several loci. Within Scotland, western populations showed slightly reduced nucleotide diversity (πtot=0.0068) compared with those from the south and east (0.0079 and 0.0083, respectively) and about three times higher recombination to diversity ratio (ρ/θ=0.71 vs 0.15 and 0.18, respectively). By comparison with results from coalescent simulations, the observed allelic frequency spectrum in the western populations was compatible with a relatively recent bottleneck (0.00175 × 4Ne generations) that reduced the population to about 2% of the present size. However, heterogeneity in the allelic frequency distribution among geographical regions in Scotland suggests that subsequent admixture of populations with different demographic histories may also have played a role

Dehydrin Association with Supercomplexes of Pea Seedlings Mitochondria Under Hypothermia

The reaction of plant cell on many stressful conditions is accompanied by accumulation of protective proteins. Dehydrins are widespread in a plant kingdom and accumulated in reply to a drought, freezing, salt stress, and also high temperature. Earlier we found the accumulation of dehydrins in mitochondria of some plants under various stresses. This work aims to study the quantitative changes and localization of dehydrins in the mitochondria of pea seedlings under low temperature impact of varying intensity and duration. It has been found that the dehydrins content in mitochondria of pea seedlings subjected to the action of low temperatures increases. The maximum dehydrins content was founds after cold hardening which is accompanied by cryotolerance increasing. For the first time it was established that the part of dehydrins of plant mitochondria is localized in the several organellar supercomplexes

Production technologies for low-chill temperate fruits; Reports from the Second International Workshop, 19–23 April 2004, Chiang Mai, Thailand

Crop Production/Industries,