An ABA and GA modulated gene expressed in the barley embryo encodes an aldose reductase related protein.

In most higher plants a period of desiccation is the terminal event in embryogenesis. Excised barley embryos acquire desiccation tolerance at a precise developmental stage and cDNA clones have been isolated which are temporally linked with desiccation tolerance. One such clone (pG22-69) with a putative gene product of 34 kd displays high structural homology to mammalian genes encoding an NADPH dependent aldose reductase involved in the synthesis of sorbitol. This first aldose reductase gene of plants is expressed constitutively during embryo maturation and is modulated by the plant hormones abscisic acid (ABA) and gibberellic acid (GA). Immunohistochemistry showed that the protein is preferentially expressed in tissues formed at early stages in embryogenesis. Measurements of enzymatic activity indicate that pG22-69 encodes an active aldose reductase. The finding of this reductase activity and the cloning of the corresponding gene supports the existence of a metabolic pathway in plants playing a role in the synthesis of osmolytes like sorbitol. The significance of this work is that genes of related structure and functions are being used in diverse organisms to fulfil stress related biological requirements.     

Molecular characterization of two alanine-rich Lea genes abundantly expressed in the resurrection plant C. plantagineum in response to osmotic stress and ABA

Expression of many genes is induced during dehydration in vegetative tissues of the desiccation tolerant resurrection plantCraterostigma plantagineum. The most abundant group of desiccation-related gene products belong to the LEA (= Late Embryogenesis Abundant) proteins. Here we describe structures and expression patterns of members of group 3 and group 4Lea genes fromC. plantagineum. The most intriguing observation is the strong conservation of repeat motifs inLea genes found across divers plant species includingC. plantagineum and non-desiccation tolerant plants. This conservation of structural elements leads to speculations about evolution of desiccation tolerance in the resurrection plant.     

The lysine-rich motif of intrinsically disordered stress protein CDeT11-24 from Craterostigma plantagineum is responsible for phosphatidic acid binding and protection of enzymes from damaging effects caused by desiccation

The late embryogenesis abundant (LEA)-like protein CDeT11-24 is one of the major desiccation-related phosphoproteins of the resurrection plant Craterostigma plantagineum. In this study, it was shown that CDeT11-24 is mostly intrinsically disordered and protects two different enzymes, citrate synthase and lactate dehydrogenase, against damaging effects caused by desiccation. Lipid-binding assays revealed that CDeT11-24 is able to interact with phosphatidic acid, although electrostatic repulsion was expected due to the overall negative net charge of the protein under the tested physiological conditions. CDeT11-24 carries an N-terminal lysine-rich sequence, which is predicted to form an amphipathic α-helix. Analysis of the truncated CDeT11-24 protein identified this region to be responsible for both activities: enzyme protection and phosphatidic acid interaction. Possible functions of the CDeT11-24 protein are discussed in the context of desiccation tolerance.     

MS-desi, a desiccation-related protein in the floral nectar of the evergreen velvet bean (Mucuna sempervirens Hemsl): molecular identification and characterization

Plant desiccation-related proteins (DRPs) were first identified as pcC13-62 from the resurrection plant Craterostigma plantagineum and it has been suggested they are involved in plant desiccation tolerance. We identified and characterized a plant DRP, which we called MS-desi, in the floral nectar of a subtropical bean species, Mucuna sempervirens (MS). MS-desi is a major nectar protein (nectarin) of the bean plant and expresses exclusively in the stylopodium, where the nectary is located. The full-length MS-desi gene encodes for a protein of 306 amino acids with a molecular mass of 33,248 Da, and possesses a ferritin-like domain and a signal peptide of 30 amino acids. Structural and phylogenetic analysis demonstrated MS-desi has high similarity to members of the plant DRPs, including pcC 13-62 protein. MS-desi has a similar hydropathy profile to that of pcC13-62 with a grand average of hydropathy index of 0.130 for MS-desi and 0.106 for pcC13-62 protein, which is very different from those of dehydrins and late embryogenesis abundant proteins. The protein’s secondary structures, both predicted from the amino acid sequence and directly analysed by far UV circular dichroism, showed that MS-desi is mainly composed of alpha helices and is relatively temperature dependent. The structure change is reversible within a wide range of temperatures. Purified MS-desi and raw MS floral nectar showed dose-dependent citrate synthase inhibition activity, but insensitivity to lactate dehydrogenase, suggesting that, unlike dehydrins, it does not act as a chaperone. The overall results constitute, to our knowledge, the first study on a desiccation-related protein in plant floral nectar.     

Analysis of desiccation-induced candidate phosphoproteins from Craterostigma plantagineum isolated with a modified metal oxide affinity chromatography procedure

Reversible protein phosphorylation/dephosphorylation is crucial for regulation of many cellular events, and increasing evidence indicates that this post-translational modification is also involved in the complex process of acquisition of desiccation tolerance. To analyze the phosphoproteome of the desiccation tolerant resurrection plant Craterostigma plantagineum, MOAC-enriched proteins from leaves at different stages of a de-/rehydration cycle were separated by 2-D PAGE and detected by phosphoprotein-specific staining. Using this strategy 20 putative phosphoproteins were identified by MALDI-TOF MS and MS/MS, which were not detected when total proteins were analyzed. The characterized desiccation-related phosphoproteins CDeT11-24 and CDeT6-19 were used as internal markers to validate the specificity of the analyses. For 16 of the identified proteins published evidence suggests that they are phosphoproteins. Comparative analysis of the 2-D gels showed that spot intensities of most identified putative phosphoproteins change during the de-/rehydration cycle. This suggests an involvement of these proteins in desiccation tolerance. Nearly all changes in the phosphoproteome of C. plantagineum, which are triggered by dehydration, are reversed within 4 days of rehydration, which is in agreement with physiological observations. Possible functions of selected proteins are discussed in the context of the de-/rehydration cycle.     

The isolation of genes from the resurrection grass Sporobolus stapfianus which are induced during severe drought stress

A modification of the ‘cold plaque’ screening technique (Hodge et al., Plant Journal1992, 2, 257–260) was used to screen a cDNA library constructed from drought‐stressed leaf tissue of the desiccation tolerant (‘resurrection’) grass Sporobolus stapfianus. This technique allowed a large number of clones representing genes expressed at low abundance to be isolated. An examination of expression profiles revealed that several of these genes are induced in desiccation‐tolerant tissue experiencing severe drought stress. Further characterization indicated that the gene products encoded include an eIF1 protein translation initiation factor and a glycine‐ and proline‐rich protein which have not previously been associated with drought stress. In addition, genes encoding a serine/threonine phosphatase type 2C, a tonoplast‐intrinsic protein (TIP) and an early light‐inducible protein (ELIP) were isolated. A number of these genes are expressed differentially in desiccation‐tolerant and desiccation‐sensitive tissues, suggesting that they may be associated with the desiccation tolerance response of S. stapfianus. The results indicate that there may be unique gene regulation processes occurring during induction of desiccation tolerance in resurrection plants which allow different drought‐responsive genes to be selectively expressed at successive levels of water loss.     

Proteins in development and germination of a desiccation sensitive (recalcitrant) seed species

In the recalcitrant seeds of Avicennia marina, protein content and the rates of protein synthesis increase during histodifferentiation. This is similar to the situation in desiccation tolerant seeds. During the stage of reserve accumulation the protein content and rates of synthesis remain constant and there is no de novo synthesis of proteins which might qualify as storage proteins. There is also no change in the nature of proteins present in either axis or cotyledonary tissues during development or germination. Similarly, fluorographs of axis proteins show only very limited changes in the patterns of protein synthesis during development and germination, at least until the onset of root growth. Heat-stable proteins are present from an early developmental stage. However, no late embryogenic abundant (LEA) proteins are synthesised during the late stages of development, indicating that seedling establishment is independent of such maturation proteins. It is suggested that the lack of desiccation tolerance of A. marina seeds might be related to the absence of desiccation-related LEAs. Although the rate of protein synthesis increases during germination, protein metabolism appears to remain qualitatively the same as that occurring during development. The present results suggest that in these desiccation sensitive seeds, protein metabolism characterising development changes imperceptibly into that of germination.     

砂藓抗旱相关基因RcSOD的克隆及表达分析

超氧化物歧化酶(superoxide dismutase, SOD)是植物抗氧化系统的关键酶,在植物应对各种环境胁迫的过程中发挥重要的作用。本研究在砂藓(Racomitrium canescens)干旱胁迫转录组数据中发现一个表达量上调的SOD基因,将其命名为RcSOD。通过RT-PCR技术对RcSOD的开放阅读框进行了克隆及相关的生物信息学预测分析。通过实时荧光定量PCR (qPCR)对砂藓复水和脱水过程中RcSOD基因的表达量变化进行了检测分析。结果显示,该基因c DNA全长1 230 bp,开放阅读框长555 bp。RcSOD编码蛋白质的氨基酸数为184,相对分子质量为18.9 kD,理论等电点为5.94,不稳定指数为18.35,总平均亲水性为-0.172,预测该蛋白为疏水性蛋白,属于非跨膜、非分泌型蛋白,含有Cu/Zn SOD结构域。系统进化分析表明,RcSOD与藓类Cu/Zn SOD蛋白属于同一分枝。实时荧光定量PCR结果显示,RcSOD基因在复水和干旱的条件下均有差异性表达,在脱水过程中显著上调,初步推测RcSOD可能参与砂藓干旱胁迫应答反应。本研究对进一步研究砂藓中Cu/Zn SOD基因的生物学功能具有推动作用。     

Eco-genetics of desiccation resistance in Drosophila

Climate change globally perturbs water circulation thereby influencing ecosystems including cultivated land. Both harmful and beneficial species of insects are likely to be vulnerable to such changes in climate. As small animals with a disadvantageous surface area to body mass ratio, they face a risk of desiccation. A number of behavioural, physiological and genetic strategies are deployed to solve these problems during adaptation in various Drosophila species. Over 100 desiccation-related genes have been identified in laboratory and wild populations of the cosmopolitan fruit fly Drosophila melanogaster and its sister species in large-scale and single-gene approaches. These genes are involved in water sensing and homeostasis, and barrier formation and function via the production and composition of surface lipids and via pigmentation. Interestingly, the genetic strategy implemented in a given population appears to be unpredictable. In part, this may be due to different experimental approaches in different studies. The observed variability may also reflect a rich standing genetic variation in Drosophila allowing a quasi-random choice of response strategies through soft-sweep events, although further studies are needed to unravel any underlying principles. These findings underline that D. melanogaster is a robust species well adapted to resist climate change-related desiccation. The rich data obtained in Drosophila research provide a framework to address and understand desiccation resistance in other insects. Through the application of powerful genetic tools in the model organism D. melanogaster, the functions of desiccation-related genes revealed by correlative studies can be tested and the underlying molecular mechanisms of desiccation tolerance understood. The combination of the wealth of available data and its genetic accessibility makes Drosophila an ideal bioindicator. Accumulation of data on desiccation resistance in Drosophila may allow us to create a world map of genetic evolution in response to climate change in an insect genome. Ultimately these efforts may provide guidelines for dealing with the effects of climate-related perturbations on insect population dynamics in the future.     

Drought-Responsive Gene Expression in Sun and Shade Plants of <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis> Under Controlled Environment

Haberlea rhodopensis is a homoiochlorophyllous desiccation-tolerant plant growing mostly in shaded rock rifts below the trees at very low light intensity. These shade plants are very sensitive to photoinhibition and do not survive desiccation at irradiance of 350 μmol m^?2 s^?1, whereas plants growing on the top of rocks exposed to full sunlight (sun plants) can survive at even higher light intensities regularly. The aim of the present study was to establish how acclimation to different light intensities influences the expression of selected drought-responsive genes and the physiological activity during desiccation of shade and sun plants under controlled culture conditions. The photosynthetic activity was higher in sun plants not only when fully hydrated but also during dehydration. Thus, the higher photosynthetic capacity, reflected in PSII but especially in PSI activity, is accompanied by a reduced susceptibility to photodamage. For most of the genes examined, drought was the main factor in regulation; in addition, some were light modulated like genes coding for putative superoxide dismutase (SOD), ascorbate peroxidase (APX) and thioredoxin (TRX), whereby the former was almost purely light regulated. Differences between sun and shade plants concerned mainly on the time course. Whereas some genes reacted already at moderate desiccation only in sun plants (genes for monodehydroascorbate reductase (MDAR), plastidic translocase (PTL) similar to OEP16 and one of the genes, newly annotated ELIP-like, specific for H. rhodopensis), especially a gene for a putative UDP-glucuronic acid decarboxylase (UDP) retained its enhanced expression longer during recovery. Thus, these genes are probably especially important for survival and recovery in sun plants.     

Gene Expression in the Cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120 under Desiccation

The N₂-fixing cyanobacterium Anabaena sp. PCC7120 showed an inherent capacity for desiccation tolerance. A DNA microarray covering almost the entire genome of Anabaena was used to determine the genome-wide gene expression under desiccation. RNA was extracted from cells at intervals starting from early to late desiccation. The pattern of gene expression in DNA fragments was categorized into seven types, which include four types of up-regulated and three types of down-regulated fragments. Validation of the data was carried out by RT-PCR on selected up-regulated DNA fragments and was consistent with the changes in mRNA levels. Our conclusions regarding desiccation tolerance for Anabaena sp. PCC7120 are as follows: (i) Genes for osmoprotectant metabolisms and the K⁺ transporting system are up-regulated from early to mid-desiccation; (ii) genes induced by osmotic, salt, and low-temperature stress are up-regulated under desiccation; (iii) genes for heat shock proteins are up-regulated after mid-desiccation; (iv) genes for photosynthesis and the nitrogen-transporting system are down-regulated during early desiccation; and (v) genes for RNA polymerase and ribosomal protein are down-regulated between the early and the middle phase of desiccation. Profiles of gene expression are discussed in relation to desiccation acclimation.