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Dissecting the response to dehydration and salt (NaCl) in the resurrection plant Craterostigma plantagineum
Abstract
Although desiccation tolerant, the resurrection plant Craterostigma plantagineum is sensitive to relatively low levels of sodium chloride. Exposure to sodium chloride, but not dehydration, led to accumulation of sodium ions in leaves and roots and caused irreversible wilting. The effects of salt and dehydration on transcript accumulation patterns were studied by using selected cDNA clones that were related to water stress. Most of the clones represented genes that were up-regulated in response to both treatments. Among the transcripts specifically up-regulated by dehydration were RNAs encoding transcripts with homology to aquaporins. Expression analysis revealed dehydration-specific profiles of late embryogenesis abundant (LEA) genes, which differed from the patterns observed for the same genes under sodium chloride stress. The interconversion of octulose and sucrose, which is characteristic for the desiccation/rehydration cycle in C. plantagineum leaves, was not activated by sodium chloride. The present results suggest that dehydration-specific responses involve the synchronized expression of specific genes and the presence of a determined concentration of sucrose. These dehydration responses were not detected in response to sodium chloride treatment.
... In general, most LEA genes, such as rice Rab16A-D [13], barley HVA1 [14], soybean GmPM8 [15] and tomato Le25 [16], are expressed in both reproductive and vegetative organs. However, some, such as wheat Em [17], cotton D19 [18], carrot Dc8 [19], and B. napus Lea76 [20], are seed or embryo specific, and others, such as Arabidopsis Cor47 [21] and Lit29 [22], spinach Cap85 [23], and several Craterostigma plantagineum LEA II, III, IV, and V genes [24][25][26], are vegetative-tissue specific. Detailed analysis revealed that the expression of LEA genes is regulated significantly by temporal and spatial effects. ...
... Previous analysis showed several LEA V genes, such as cotton D-34 and D-95, carrot ECP31, or Arabidopsis PAP140 and AtRAB28, expressed in seeds or induced in young embryos by dehydration [18,49,63,64]. The expression of other LEA V genes, such as C. Plantagineum pcC27-45 and pcC16-81, maize RAB28, tomato ER5, and hot pepper CaLEA6, was induced or enhanced by salt or dehydration in vegetative tissues [24,26,[65][66][67][68]. Soybean GmPM17 and GmPM25 and Arabidopsis Atecp31 [64] are examples of LEA V genes expressed both in seeds and vegetative tissues. ...
... The transporter mobilized water and dissolved solutes from the vacuoles during drought (Willigen et al., 2004). Smith-Espinoza et al. (2003) reported the upregulation of some PIPs in Craterostigma plantagineum exposed to drought stress. Transgenic Arabidopsis plants overexpressing the endogenous gene, FaPIP2;1 from the perennial grass, Festuca arundinacea were tolerant to desiccation and leaf dehydration (Zhuang et al., 2015). ...
Aquaporins (AQPs) are crucial transporters in the plant cells, which mobilize the intercellular passage of water, solvent, and dissolved solutes from the source to the sink. Thus these transmembrane channel proteins are effective checkpoints of solvent transport across tissues. It is known that cellular water conservation is the major strategy for plant tolerance against multiple abiotic stresses, such as salinity, drought, desiccation, temperature, nutrient limitation, and heavy-metal toxicity. Hence, the roles of AQPs in abiotic stress tolerance are obvious. Transgenic plants overexpressing AQP genes have shown tolerance toward suboptimal environmental conditions. The basic mechanism behind such tolerance phenotype is efficient stomatal closure, low rate of water loss, and effective water circulation throughout the system. Among the different subclasses of AQPs, the plasma-membrane intrinsic proteins are involved in diverse stress responses. Thus these transporters have been highlighted as potential molecular targets, which can be expressed to generate multiple stress tolerance. This chapter exhaustively discusses the diverse roles of AQPs in promoting plant survival under challenging environmental situations.
... Transgenic plants were assayed for GUS activity at different developmental stages and upon salt stress. Salt stress was chosen instead of dehydration because this allowed more robust testing than dehydration and because it was previously shown that pcC13-62 accumulated to high levels following NaCl treatments (Smith-Espinoza et al., 2003). ...
Reproductive structures of plants (e.g. seeds) and vegetative tissues of resurrection plants can tolerate desiccation. Many genes encoding desiccation-related proteins (DRP) have been identified in the resurrection plant Craterostigma plantagineum, but the function of these genes remains mainly hypothetical. Here the importance of the DRP gene pcC13-62 for desiccation tolerance is evaluated by analysing the expression of this gene in C. plantagineum and in the closely related desiccation tolerant species Lindernia brevidens and desiccation sensitive species Lindernia subracemosa. Quantitative analysis revealed that pcC13-62 transcripts accumulate at a much lower level in desiccation sensitive species than in desiccation tolerant species. The study of pcC13-62 promoters from these species demonstrated a correlation between promoter activity and gene expression levels suggesting transcriptional regulation of gene expression. Comparison of promoter sequences identified a drought responsive element (DRE) motif in the promoters of tolerant species which is required for dehydration-induced GUS accumulation. We hypothesize that variations in the regulatory sequences of the pcC13-62 gene occurred to establish pcC13-62 expression in vegetative tissues which might be required for desiccation tolerance. The pcC13-62 promoters could also be activated by salt stress in Arabidopsis thaliana plants stably transformed with promoter::GUS constructs.
... The PIP family proteins are involved in plant response to environmental stresses, such as water deficiency, salt, heavy metal and cold stresses. 16 For instance, GmPIP1;6 confers salt tolerance to soybean plants. 17 In addition, PIPs are also important in controlling plant response to biotic stresses, such as plant-microbe symbiosis and plant immune response to pathogens. ...
Plasma membrane microdomains are plasma membrane sub-compartments enriched in sphingolipids and sterols, and composed by a specific set of proteins. They are involved in recognizing signal molecules, transducing these signals, and controlling endocytosis and exocytosis processes. In a recent study, applying biochemical and microscopic methods, we characterized the soybean GmFWL1 protein, a major regulator of soybean nodulation, as a new membrane microdomain-associated protein. Interestingly, upon rhizobia inoculation of the soybean root system, GmFWL1 and one of its interacting partners, GmFLOT2/4, both translocate to the root hair cell tip, the primary site of interaction and infection between soybean and Rhizobium. The role of GmFWL1 as a plasma membrane microdomain-associated protein is also supported by immunoprecipitation assays performed on soybean nodules, which revealed 178 GmFWL1 protein partners including a large number of microdomain-associated proteins such as GmFLOT2/4. In this addendum, we provide additional information about the identity of the soybean proteins repetitively identified as GmFWL1 protein partners. Their function is discussed especially in regard to plant-microbe interactions and microbial symbiosis. This addendum will provide new insights in the role of plasma membrane microdomains in regulating legume nodulation.
... Water deficit exposure to a drought tolerant rice cultivar revealed up-regulation of the OsPIP1;3 gene suggesting its role in drought avoidance [87]. Strong up-regulation of some PIPs in a dehydration tolerant succulent plant, Craterostigma plantagineum, was observed under water stress [88]. Characterization of a FaPIP2;1 gene isolated from a drought-tolerant perennial grass species Festuca arundinacea showed its involvement in leaf dehydration status during water stress by overexpressing this gene in Arabidopsis. ...
Aquaporins are membrane channel proteins ubiquitously present in all kingdoms of life. Although aquaporins were originally discovered as water channels, their roles in the transport of small neutral solutes, gasses, and metal ions are now well established. Plants contain the largest number and greatest diversity of aquaporin homologs with diverse subcellular localization patterns, gating properties, and solute specificity. The roles of aquaporins in physiological functions throughout plant growth and development are well known. As an integral regulator of plant–water relations, they are presumed to play an important role in plant defense responses against biotic and abiotic stressors. This review highlights involvement of various aquaporin homologs in plant stress responses against a variety of environmental stresses that disturb plant cell osmotic balance and nutrient homeostasis.
... The second protein that did not fall within a select functional group has sequence homology to plant desiccation-related proteins (pcc 13-62), which have been implicated in processes related to cell water availability [96]. Pcc 13-62 was first identified in the resurrection plant Craterostigma plantagineum [97], and is normally found in plants subjected to dehydration [98], or during late stages of seed development [99]. Despite the fact that both APs and pcc 13-62 were detected, and seem to have a role during seed development, the higher levels of these proteins during SE6 proliferation, apparently do not increase the responsiveness of this cell line to embryo formation. ...
... It seems that these protein channels can be facultatively expressed in response to the extracellular water potential. Smith-Espinoza et al. (75) demonstrated that a specific aquaporine gene was up-regulated by dehydration but down-regulated by salinity. The notion of water channels is relatively new in plant biology and presently is the subject of intense investigation focused to elucidate the ecological and physiological significance of these proteins in the water relations of plants. ...
Abstract On a world basis, water availability is considered the main constraint for crop production. Concerns about water accessibility have always accompanied crop production in dry areas, which are on the other hand the most extensive areas for agriculture on the earth. As a consequence, men had to develop agricultural strategies to cope with water shortage, growing the plants during the short climatic interval ofwater availability and selecting plants possessing a relatively superior tolerance to water ,deficiency. Although development of drought tolerant plants by conventional breeding methods has resultedin modest
... While some of these genes have a known function, others have roles that are not yet defined. Genes that are induced or upregulated by drying can be grouped into the following categories encoding: 1) enzymatic proteins related to metabolism of sugars or other compatible osmolytes or to cell antioxidant systems (Bartels and Salamini, 2001; Kirch et al., 2001; Mowla et al., 2002); 2) nonenzymatic proteins with cell stabilizing properties (Blomstedt et al., 1998; Ndima et al., 2001); 3) membrane proteins involved in transport processes (Mariaux et al., 1998; Smith-Espinoza et al., 2003; Marais et al., 2004); 4) regulatory proteins such as transcription factors or putative components of signaling pathways (Frank et al., 1998Frank et al., , 2000 Deng et al., 2002; Hilbricht et al., 2002); 5) proteins with unknown function (Furini et al., 1997; Garwe et al., 2003). On the contrary, genes related to photosynthesis are downregulated (Bochel et al., 1998; Collett et al., 2003). ...
Tolerance to near complete desiccation of vegetative organs is a widespread capability in bryophytes and is also shared by a small group of vascular plants known as resurrection plants. To date more than 300 species, belonging to pteridophytes and angiosperms, have been identified that possess this kind of desiccation-tolerance. The vegetative desiccation-tolerance of resurrection plants is an inductive process displayed only under environmental stress with or without the involvement of abscisic acid as molecular signal. The different problems associated with desiccation encountered by resurrection plants render the employment of many interacting mechanisms necessary. Preservation of cell order and correct structure of membranes and macromolecules is underpinned by the synthesis of large amounts of sugars, amino acids, and small polypeptides such as late embryogenesis abundant (LEA) proteins and dehydrins. Some of these compatible solutes, such as sucrose and LEA proteins, are also involved in cytoplasm vitrification, which occurs during the last phase of desiccation. Mechanical damage due to vacuole shrinkage in dehydrating cells is avoided by cell wall folding or by replacing the water in vacuoles with nonaqueous substances. Oxidative stress, due to enhanced production of reactive oxygen species (ROS) especially by chloroplasts, is minimized through two different strategies. The homoiochlorophyllous resurrection plants, which conserve chloroplasts with chlorophylls and thylakoids upon drying, fold leaf blades and synthesize anthocyanins, as both sunscreens and free radical scavengers, and additionally increase the activity of antioxidant systems in cells. In contrast, the chloroplasts in poikilochlorophyllous species degrade chlorophylls and thylakoid membranes yielding desiccoplasts that are devoid of any internal structures. These adaptive mechanisms preserve cells from damage by desiccation and allow them to resume vital functions once rehydrated. Even if based mainly on cell protection during drying, the vegetative desiccation-tolerance of resurrection plants also relies on systems of cell recovery and repair upon rehydration. However, most of these systems are prepared during cell dehydration.
... A role for aquaporins in fine tuning of water availability is supported by a recent example from the resurrection plant C. plantagineum. It is shown that a specific aquaporin gene is upregulated by dehydration but downregulated by salt stress, which coincides with the tolerance behavior of this plant (Smith-Espinoza et al., 2003). Direct evidence for a role of aquaporins in maintaining the water status of plants has been derived from aquaporin antisense experiments in Arabidopsis or tobacco (Kaldenhoff et al., 1998;Siefritz et al., 2002). ...
Agricultural productivity worldwide is subject to increasing environmental constraints, particularly to drought and salinity due to their high magnitude of impact and wide distribution. Traditional breeding programs trying to improve abiotic stress tolerance have had some success, but are limited by the multigenic nature of the trait. Tolerant plants such as Craterostigma plantagenium, Mesembryanthemum crystallinum, Thellungiella halophila and other hardy plants could be valuable tools to dissect the extreme tolerance nature. In the last decade, Arabidopsis thaliana, a genetic model plant, has been extensively used for unravelling the molecular basis of stress tolerance. Arabidopsis also proved to be extremely important for assessing functions for individual stress-associated genes due to the availability of knock-out mutants and its amenability for genetic transformation. In this review, the responses of plants to salt and water stress are described, the regulatory circuits which allow plants to cope with stress are presented, and how the present knowledge can be applied to obtain tolerant plants is discussed.
... Desiccation tolerance in plants is a complex and multifaceted response (Alpert, 2006;Buitink and Leprince, 2004;Clegg, 1986). The response may include the structural or component alteration of the cell wall, organelles, or organs (Farrant, 2000;Farrant et al., 2003;Jones and McQueen-Mason, 2004;Peeva and Maslenkova, 2004;Tuba et al., 1996;Vicre et al., 2004a,b); induction of the repair system (Potts, 1996;Smith-Espinoza et al., 2003;Wilson et al., 2004); removal of free radicals (França et al., 2007;Leprince et al., 1996;Oliver et al., 2001;Sunkar et al., 2003); and accumulation of macromolecules (Buitink and Leprince, 2004;Collins and Clegg, 2004;Crowe et al., 1992Crowe et al., , 1998Crowe et al., , 2005Dure et al., 1989;Hoekstra, 2005;Hoekstra and Golovina, 2002;Murphy et al., 2001;Oliver et al., 2001;Quartacci et al., 2002;Xu et al., 1996). During drying, the accumulation of macromolecules such as oligosaccharides or proteins greatly increases cytoplasmic viscosity and usually causes the formation of bioglasses. ...
During the late maturation stage of seed development, water content decreases greatly. One of the most striking characteristics of mature orthodox seeds is their ability to withstand severe desiccation. Mechanisms of plant drought/desiccation tolerance have been studied by numerous groups, and a broad range of molecules have been identified to play some roles. Examples are proline, oligosaccharide, and late embryogenesis abundant (LEA) proteins, and so on. LEA proteins were first described from mature cotton seeds decades ago. Since then, many LEA proteins were identified from vascular and nonvascular plants, fungi, algae, and microbes, as well as anhydrobiotic animals such as protozoa, nematodes, insects, and crustaceans, and so on. The extensive distribution of LEA genes among diverse taxa implies that these genes might be primitive yet important and therefore maintained by these species. As a result of evolution, they may have a certain universal function—osmoprotection. Hydrophilic LEA proteins are members of natively unfolded proteins in solution. After the removal of bulk cytoplasmic water, the structures of LEA proteins undergo desiccation-induced folding. These biophysical features suggest that LEA proteins may carry out a bipartite function under different water states. During drought, LEA proteins may establish a water shell and decrease ion strength. After desiccation, they may enhance the bioglass strength and act as a water replacement to stabilize cellular components.
... Resurrection plants can undergo total dehydration and recover upon rehydration (Phillips et al., 2008). This requires a wide range of mechanisms of detoxification and protection (Smith-Espinoza et al., 2003), which can be transferred to more common plants (Sunkar et al., 2003). It is therefore tempting to consider that the genetic engineering of these mechanisms is a promising avenue for drought tolerance. ...
Most traits associated with drought tolerance have a dual effect, positive in very severe scenarios and negative in milder
scenarios, or the opposite trend. Their effects also depend on other climatic conditions such as evaporative demand or light,
and on management practices. This is the case for processes associated with cell protection and with avoidance, but also for
the maintenance of growth or photosynthesis, high water use efficiency, large root systems or reduced abortion rate under
water deficit. Therefore, spectacular results obtained in one drought scenario may have a limited interest for improving food
security in other geographical areas with water scarcity. The most relevant questions on drought tolerance are probably, ‘Does
a given allele confer a positive effect on yield in an appreciable proportion of years/scenarios in a given area or target
population of environment (TPE)?’; ‘In a given site or TPE, what is the trade-off between risk avoidance and maintained performance?’;
and ‘Will a given allele or trait have an increasingly positive effect with climate change?’ Considerable progress has already
occurred in drought tolerance. Nevertheless, explicitly associating traits for tolerance to drought scenarios may have profound
consequences on the genetic strategies, with a necessary involvement of modelling.
... Because DT-related transcripts are relatively abundant at all times, the DT response in this moss is not as sensitive to the rate of drying and is based more heavily on cellular repair mechanisms that become active upon rehydration ( Oliver et al., 1993( Oliver et al., , 2000aWood et al., 2000 ). Considerable attention has been directed at understanding the proteins involved in the vegetative DT of resurrection plants ( Piatkowski et al., 1990 ;Bartels et al., 1996 ;Ingram and Bartels, 1996 ;Bockel et al., 1998 ;Ditzer et al., 2001 ;Smith-Espinoza et al., 2003, 2005Collett et al., 2004 ;Bartels, 2005 ;Rascio and La Rocca, 2005 ). One the most signifi cant components of the DT response characterized in these studies is the group known as the late embryogenesis abundant (LEA) proteins. ...
Desiccation tolerance is a complex trait that is broadly but infrequently present throughout the evolutionary tree of life. Desiccation tolerance has played a significant role in land plant evolution, in both the vegetative and reproductive life history stages. In the land plants, the late embryogenesis abundant (LEA) gene families are involved in both abiotic stress tolerance and the development of reproductive propagules. They are also a major component of vegetative desiccation tolerance. Phylogenies were estimated for four families of LEA genes from Arabidopsis, Physcomitrella, and the desiccation tolerant plants Tortula ruralis, Craterostigma plantagineum, and Xerophyta humilis. Microarray expression data from Arabidopsis and a subset of the Physcomitrella LEAs were used to estimate ancestral expression patterns in the LEA families and to evaluate alternative hypotheses for the origins of vegetative desiccation tolerance in the flowering plants. The results contradict the idea that vegetative desiccation tolerance in the resurrection angiosperms Craterostigma and Xerophyta arose through the co-option of genes exclusively related to stress tolerance, and support the propagule-derived origin of vegetative desiccation tolerance in the resurrection plants.
... Desiccation tolerance in plants is a complex and multifaceted response (Alpert, 2006;Buitink and Leprince, 2004;Clegg, 1986). The response may include the structural or component alteration of the cell wall, organelles, or organs (Farrant, 2000;Farrant et al., 2003;Jones and McQueen-Mason, 2004;Peeva and Maslenkova, 2004;Tuba et al., 1996;Vicre et al., 2004a,b); induction of the repair system (Potts, 1996;Smith-Espinoza et al., 2003;Wilson et al., 2004); removal of free radicals (França et al., 2007;Leprince et al., 1996;Oliver et al., 2001;Sunkar et al., 2003); and accumulation of macromolecules (Buitink and Leprince, 2004;Collins and Clegg, 2004;Crowe et al., 1992Crowe et al., , 1998Crowe et al., , 2005Dure et al., 1989;Hoekstra, 2005;Hoekstra and Golovina, 2002;Murphy et al., 2001;Oliver et al., 2001;Quartacci et al., 2002;Xu et al., 1996). During drying, the accumulation of macromolecules such as oligosaccharides or proteins greatly increases cytoplasmic viscosity and usually causes the formation of bioglasses. ...
During the late maturation stage of seed development, water content decreases greatly. One of the most striking characteristics of mature orthodox seeds is their ability to withstand severe desiccation. Mechanisms of plant drought/desiccation tolerance have been studied by numerous groups, and a broad range of molecules have been identified to play some roles. Examples are proline, oligosaccharide, and late embryogenesis abundant (LEA) proteins, and so on. LEA proteins were first-described from mature cotton seeds decades ago. Since then, many LEA proteins were identified from vascular and nonvascular plants, fungi, algae, and microbes, as well as anhydrobiotic animals such as protozoa, nematodes, insects, and crustaceans, and so on. The extensive distribution of LEA genes among diverse taxa implies that these genes might be primitive yet important and therefore maintained by these species. As a result of evolution, they may have a certain universal function-osmoprotection. Hydrophilic LEA proteins are members of natively unfolded proteins in solution. After the removal of bulk cytoplasmic water, the structures of LEA proteins undergo desiccation-induced folding. These biophysical features suggest that LEA proteins may carry out a bipartite function under different water states. During drought. LEA proteins may establish a water shell and decrease ion strength. After desiccation, they may enhance the bioglass strength and act as a water replacement to stabilize cellular components
... The molecular aspects of desiccation tolerance in resurrection plants such as Craterostigma plantagineum (Bartels et al. 1990; Bartels and Salamini 2001; Phillips et al. 2002; Bartels 2005 ), Xerophyta viscosa (Mundree et al. 2000; Mowla et al. 2002; Dahlia et al. 2003), Xerophyta humilis (Collett et al. 2003), Sporobolus stapWanus (Neale et al. 2000) and Selaginella lepidophylla (Iturriaga et al. 2000 ) reveal complex mechanisms of desiccation tolerance (Bernacchia and Furini 2004). Although some mechanisms of stress response are common to all cells, there exist major diVerences in strategies adopted by plants to cope with desiccation stress (Ramanjulu and Bartels 2002; Smith-Espinoza et al. 2003). Realizing this, it would be more rewarding to explore crop species with higher levels of stress tolerance at molecular level. ...
Peanut, found to be relatively drought tolerant crop, has been the choice of study to characterize the genes expressed under gradual water deficit stress. Nearly 700 genes were identified to be enriched in subtractive cDNA library from gradual process of drought stress adaptation. Further, expression of the drought inducible genes related to various signaling components and gene sets involved in protecting cellular function has been described based on dot blot experiments. Fifty genes (25 regulators and 25 functional related genes) selected based on dot blot experiments were tested for their stress responsiveness using northern blot analysis and confirmed their nature of differential regulation under different field capacity of drought stress treatments. ESTs generated from this subtracted cDNA library offered a rich source of stress-related genes including signaling components. Additional 50% uncharacterized sequences are noteworthy. Insights gained from this study would provide the foundation for further studies to understand the question of how peanut plants are able to adapt to naturally occurring harsh drought conditions. At present functional validation cannot be deemed in peanut, hence as a proof of concept seven orthologues of drought induced genes of peanut have been silenced in heterologous N. benthamiana system, using virus induced gene silencing method. These results point out the functional importance for HSP70 gene and key regulators such as Jumonji in drought stress response.
... The molecular aspects of desiccation tolerance in resurrection plants such as Craterostigma plantagineum (Bartels et al. 1990; Bartels and Salamini 2001; Phillips et al. 2002; Bartels 2005 ), Xerophyta viscosa (Mundree et al. 2000; Mowla et al. 2002; Dahlia et al. 2003), Xerophyta humilis (Collett et al. 2003), Sporobolus stapWanus (Neale et al. 2000) and Selaginella lepidophylla (Iturriaga et al. 2000 ) reveal complex mechanisms of desiccation tolerance (Bernacchia and Furini 2004). Although some mechanisms of stress response are common to all cells, there exist major diVerences in strategies adopted by plants to cope with desiccation stress (Ramanjulu and Bartels 2002; Smith-Espinoza et al. 2003). Realizing this, it would be more rewarding to explore crop species with higher levels of stress tolerance at molecular level. ...
Peanut, found to be relatively drought tolerant crop, has been the choice of study to characterize the genes expressed under gradual water deficit stress. Nearly 700 genes were identified to be enriched in subtractive cDNA library from gradual process of drought stress adaptation. Further, expression of the drought inducible genes related to various signaling components and gene sets involved in protecting cellular function has been described based on dot blot experiments. Fifty genes (25 regulators and 25 functional related genes) selected based on dot blot experiments were tested for their stress responsiveness using northern blot analysis and confirmed their nature of differential regulation under different field capacity of drought stress treatments. ESTs generated from this subtracted cDNA library offered a rich source of stress-related genes including signaling components. Additional 50% uncharacterized sequences are noteworthy. Insights gained from this study would provide the foundation for further studies to understand the question of how peanut plants are able to adapt to naturally occurring harsh drought conditions. At present functional validation cannot be deemed in peanut, hence as a proof of concept seven orthologues of drought induced genes of peanut have been silenced in heterologous N. benthamiana system, using virus induced gene silencing method. These results point out the functional importance for HSP70 gene and key regulators such as Jumonji in drought stress response.
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... In dehydration experiments, mature plants were removed from their pots and water was withheld. The water content of untreated plants was set to 100% of relative water content and the water loss was monitored during the dehydration process (Smith-Espinoza et al. 2003). Plants were treated for salt stress in hydroponic cultures in Hoagland solution supplemented with the indicated NaCl concentrations. ...
The resurrection plant Craterostigma plantagineum Hochst. is used as an experimental system to investigate desiccation tolerance in higher plants. A search for genes activated during early stages of dehydration identified the gene CpEdi-9, which is expressed in mature seeds and in response to dehydration in the phloem cells of vascular tissues of leaves. Elements for the tissue-specific expression pattern reside in the isolated promoter of the CpEdi-9 gene, as shown through the analysis of transgenic plants. The
CpEdi-9 promoter could be a suitable tool for expressing genes in the vascular system of dehydrated plants.
CpEdi-9 encodes a small (10 kDa) hydrophilic protein, which does not have significant sequence homologies to known genes. The predicted protein CpEDI-9 shares some physicochemical features with LEA proteins from plants and a nematode. Based on the unique expression pattern and on the nucleotide sequence we propose that CpEdi-9 defines a new class of hydrophilic proteins that are supposed to contribute to cellular protection during dehydration. This group of proteins may have evolved because desiccation tolerance requires the abundant expression of protective proteins during early stages of dehydration in all tissues.
... The lowest K leaf was found in W-leaves, where transcellular water flow likely crossed the largest number of mesophyll cell membranes, suggesting that membranes reduce K leaf . The strong positive correlation between leaf osmotic potential and K leaf may be causal, a result of the negative effect of rising osmotic concentration on the expression of aquaporins both in the plasmalemma (Yamada et al. 1995, Kawasaki et al. 2001, Martínez-Ballesta et al. 2003a, Smith-Espinoza et al. 2003 ) and in the tonoplast (Weig et al. 1997, Kirch et al. 2000). However, because of TREE PHYSIOLOGY ONLINE at http://heronpublishing.com POPULUS TREMULA LEAF HYDRAULIC CONDUCTANCE 1415Figure 5. Relationships between leaf hydraulic conductance and cell wall characteristics: leaf dry mass per area (A) and the leaf bulk modulus of elasticity (B) in 3 – 6-week-old P. tremula leaves. ...
Leaf hydraulic conductance (K(leaf)) and several characteristics of hydraulic architecture and physiology were measured during the first 10 weeks of leaf ontogeny in Populus tremula L. saplings growing under control, mild water deficit or elevated temperature conditions. During the initial 3 weeks of leaf ontogeny, most measured characteristics rapidly increased. Thereafter, a gradual decrease in K(leaf) was correlated with a decrease in leaf osmotic potential under all conditions, and with increases in leaf dry mass per area and bulk modulus of elasticity under mild water deficit and control conditions. From about Week 3 onward, K(leaf) was 33% lower in trees subjected to mild water deficit and 33% higher in trees held at an elevated temperature relative to control trees. Mild water deficit and elevated temperature treatment had significant and opposite effects on most of the other characteristics measured. The ontogenetic maximum in K(leaf) was correlated positively with the width of xylem conduits in the midrib, but negatively with the overall width of the midrib xylem, number of lateral ribs, leaf dry mass per area and bulk modulus of elasticity. The ontogenetic maximum in K(leaf) was also correlated positively with the proportion of intercellular spaces and leaf osmotic potential, but negatively with leaf thickness, volume of mesophyll cells and epidermis and number of cells per total mesophyll cell volume, the closest relationships being between leaf osmotic potential and number of cells per total mesophyll cell volume. It was concluded that differences in protoplast traits are more important than differences in xylem or parenchymal cell wall traits in determining the variability in K(leaf) among leaves growing under different environmental conditions.
... In plants, folding cell walls (Vander Willigen et al., 2004) In plants, replacement of water in vacuoles by non-aqueous compounds and fragmentation of vacuoles (Farrant, 2000; Vicre et al., 2004a) Physiological damage at low intermediate water contents Upregulation of proteins that increase membrane permeability (Smith-Espinoza et al., 2003; Vander Willigen et al., 2004) ...
Drying to equilibrium with the air kills nearly all animals and flowering plants, including livestock and crops. This makes drought a key ecological problem for terrestrial life and a major cause of human famine. However, the ability to tolerate complete desiccation is widespread in organisms that are either <5 mm long or found mainly where desiccation-sensitive organisms are scarce. This suggests that there is a trade-off between desiccation tolerance and growth. Recent molecular and biochemical research shows that organisms tolerate desiccation through a set of mechanisms, including sugars that replace water and form glasses, proteins that stabilize macromolecules and membranes, and anti-oxidants that counter damage by reactive oxygen species. These protections are often induced by drying, and some of the genes involved may be homologous in microbes, plants and animals. Understanding how mechanisms of desiccation tolerance may constrain growth might show how to undo the constraint in some economically important macroorganisms and elucidate the much-studied but elusive relationship between tolerance of stress and productivity.
... Several control sequences including actin and ubiquitin were also spotted. GUS, desmin and neblin ( Bellin et al. 2002) gene fragments were spotted in different amounts and their transcripts generated by in vitro transcription were included to prepare RNA probes, as spiking controls ( Bellin et al, 2002;Smith et al, 2003). For the wild-type (Col-0) and three CpHB-7 expressing lines, leaf tissue was harvested from 10 plants and pooled prior to total RNA extraction. ...
A subset of homeodomain leucine zipper proteins (HDZip) play a role in regulating adaptation responses including developmental adjustment to environmental cues in plants. Here we report the structural and functional characterisation of a dehydration responsive nuclear-targeted HDZip transcriptional regulator, CpHB-7. DNA-protein interaction studies suggest that CDeT6-19, a known ABA and dehydration responsive dehydrin gene, is a potential target gene of CpHB-7 in the desiccation-tolerant plant Craterostigma plantagineum. Transgenic plants that ectopically express CpHB-7 display reduced sensitivity towards ABA during seed germination and stomatal closure. Expression analysis reveals that genes with induced or repressed expression in CpHB-7 ectopic expression lines are either mostly repressed or induced by ABA, drought or salt treatment respectively, thus demonstrating that CpHB-7 modifies ABA-responsive gene expression as a negative regulator. CpHB-7 gene expression is also linked to early organ development, leading to the suggestion that CpHB-7 is functionally similar to the Arabidopsis transcription factor, ATHB-6.
... Similar to freezing, desiccation places an organism under osmotic stress. In desiccated plants, Smith-Espinoza et al. (Smith-Espinoza et al., 2003) and Barrieu et al. (Barrieu et al., 1999) found an increase in aquaporin transcription and protein concentration. Likewise, mammals increase aquaporin expression in response to dehydration (Ishibashi et al., 1997). ...
Survival of freezing not only requires organisms to tolerate ice formation within their body, but also depends on the rapid redistribution of water and cryoprotective compounds between intra- and extracellular compartments. Aquaporins are transmembrane proteins that serve as the major pathway through which water and small uncharged solutes (e.g. glycerol) enter and leave the cell. Consequently, we examined freeze-tolerant larvae of the goldenrod gall fly, Eurosta solidaginis, to determine whether aquaporins are present and if their presence promotes freeze tolerance of specific tissues. Immunoblotting with mammalian anti-AQP2, -AQP3 and -AQP4 revealed corresponding aquaporin homologues in E. solidaginis, whose patterns of expression varied depending on acclimation temperature and desiccation treatment. To examine the role of aquaporins in freeze tolerance, we froze fat body, midgut and salivary gland tissues in the presence and absence of mercuric chloride, an aquaporin inhibitor. Survival of fat body and midgut cells was significantly reduced when mercuric chloride was present. In contrast, survival of the salivary gland did not decrease when it was frozen with mercuric chloride. Overall, this study supports our hypothesis that naturally occurring aquaporins in E. solidaginis are regulated during desiccation and promote cell survival during freezing.
Cellular transport of water, i.e., essential for cell survival, takes place through a specialized water channel, referred to as an aquaporin (AQP). It falls under the category of the membrane intrinsic protein (MIP) superfamily and possesses several isoforms. AQP functions are directly related to an osmotic adjustment of plants facing different types of stresses. PIPs are the conserved ones among plant AQP subfamilies as found in most of the plant groups. About 30 kDa-sized monomers constitute the tetrameric structure of AQPs. Six transmembrane α-helices constitute the water transport channel where NPA, AEFL, and HW[V/I][F/Y]WXGP are the conserved sequences. Rapid water movement through this water channel occurs as a single-file configuration of water by means of channel selectivity with respective pore size. Specialized alignment of water molecules along with dipole charges plays a significant role in maintaining this water conduction. Basically, water stress is one of the substantial reasons for creating osmotic imbalances. Plasma membrane intrinsic proteins (PIPs) and tonoplast intrinsic proteins (TIPs) are expressed in most of the plants with deficiency of optimum water content for normal physiological activities. Each type of biotic and abiotic stress condition eventually causes cell-damaging osmotic stress. There are different factors like protons (H +), divalent cations, pH status, reactive oxygen species (ROS), hormones, phosphorylation, cellular trafficking, etc., which regulate the expression of AQPs in response to osmotic imbalance by different mechanisms. Presently, study of the role of AQPs in osmoregulation is very important in advanced research. Moreover, a multidirectional investigation is essential to answer questions regarding unrevealed issues in this area of plant physiology.
The aim of this research is to investigate on the osmotic adjustment and pigment preservation by soluble sugar accumulation in salt stressed rice using exogenous glucose and abscisic acid (ABA) application, leading to plant growth and development. Soluble sugars including sucrose, glucose and fructose in the salt-stressed root tissues were continuously increased in the conditions of 111-222 mM glucose and 20-60 μM ABA treatments and then drop in the extreme 333-444 mM glucose and 80 μM ABA treatments. Osmolarity in the salt-stressed root tissues showed the similar pattern to the sugar responses and was negatively related to soluble sugar concentration (r = 0.91). Chlorophyll a, chlorophyll b and total carotenoid concentrations in the salt-stressed seedlings were significantly maintained by endogenous sugar osmotic adjustment. In addition to, the high osmolarity in salt-stressed seedlings was negatively related to total chlorophyll stabilization (r = 0.83). The total chlorophyll degradation in the salt-stressed leaf tissues was positively correlated with plant growth defined by shoot height (r = 0.81). Root length, root number and root cortex thickness of salt-stressed rice seedlings showed the highest at 222 mM glucose and 60 μM ABA treatments for 146.1 cm, 17.3 and 1.3 μm, respectively. An exogenous application of glucose and ABA in this investigation is an alternative way to acclimatize the rice crop before exposed to soil salinity and further applied for rice cultivation in salinity filed trial.
Under natural conditions, the plants are exposed to changing environmental conditions that determine complex respuests that influence in the growth, development and productivity of the crops. The conditions of drought and salinity in the soils are the greater causes of stress in the crops and cause lost economic in the world agriculture. The drought as the salinity are osmotic stress that inhibits the growth and cause interferences at metabolic level. The recognition of the biochemical and physiologic mechanisms involved in the osmoregulation involved in the osmotic stress allows to implement new strategies for the handling and improvement of the cultivations under conditions of stress hidric and saline. Different mechanisms are recognized through which the plants manage to adapt and to tolerate the changes in the water potential, between which stand: the water transport by acuaporins, the estomatic closing, the synthesis of compatible osmolites, the ión transport through selective systems of sodium and potassium and those not selective located in the biological membranes and the etrution and compartimentalization of sodium. The osmoregulation confers to the plants the capacity to tolerate conditions of drought and high salinity, with the expression of adaptative mechanisms that avoid the reduction of the photosynthesis, alterations in the allocation photoasimilates and losses in yield, facts that have significant transcendency in the normal physiology of the plant and in the productivity of the crops. The present review has as central objective, to report the discoveries more recent envelope the smorregulation process in exposed cultivated plants to stress by water deficit and by salinity in the soils.
Introduction Quantification of Desiccation Tolerance and Damage Kinetics Define Desiccation Damage Moisture “rafts” Reactions When Cells Dry Conclusions
Introduction Whole Plant Effects: Growth and Metabolism Control of Water Permeability: The Role of Aquaporins Sensing of Water Deficit Signal Transduction: Second Messengers and Signaling Molecules Molecular Responses: Gene Expression in Response to Dehydration and Desiccation Craterostigma Desiccation Tolerant (CDT) Genes Integration of Genetic Pathways: MAP Kinase Cascades Oxidative Damage and Reactive Oxygen Species Conservation of Pathways Conclusions
Biographical SketchScientific AchievementsThe ManAcknowledgmentsLiterature CitedPublications of Francesco Salamini
Metabolic depression is relatively uncommon among the fishes with the greatest number of species exhibiting dormancy as embryos.
Dormancy in fish embryos is largely associated with deposition of embryos into terrestrial habitats to avoid embryo predation
or to survive intermittent drying of aquatic habitats. Killifish embryos in general, and especially the embryos of annual
killifish, are highly adapted for life at the interface between land and water and thus have evolved a suite of characters
that allows them to survive in an aerial environment. Here we review the available literature on embryonic dormancy and dehydration
tolerance in killifish embryos.
The accumulation of cryoprotectants and the redistribution of water between body compartments play central roles in the capacity of insects to survive freezing. Aquaporins (AQPs) allow for rapid redistribution of water and small solutes (e.g. glycerol) across the cell membrane and were recently implicated in promoting freeze tolerance. Here, we examined whether aquaporin-like protein abundance correlated with the seasonal acquisition of freezing tolerance in the goldenrod gall fly, Eurosta solidaginis (Diptera: Tephritidae). Through the autumn, larvae became tolerant of freezing at progressively lower temperatures and accumulated the cryoprotectant glycerol. Furthermore, larvae significantly increased the abundance of membrane-bound aquaporin and aquaglyceroporin-like proteins from July through January. Acute exposure of larvae to cold and desiccation resulted in upregulation of the AQP3-like proteins in October, suggesting that their abundance is regulated by environmental cues. The seasonal increase in abundance of both putative aquaporins and aquaglyceroporins supports the hypothesis that these proteins are closely tied to the seasonal acquisition of freeze tolerance, functioning to permit cells to quickly lose water and take-up glycerol during extracellular ice formation, as well as reestablish water and glycerol concentrations upon thawing.
The ubiquitous cell membrane proteins called aquaporins are now firmly established as channel proteins that control the specific transport of water molecules across cell membranes in all living organisms. The aquaporins are thus likely to be of fundamental significance to all facets of plant growth and development affected by plant-water relations. A majority of plant aquaporins have been found to share essential structural features with the human aquaporin and exhibit water-transporting ability in various functional assays, and some have been shown experimentally to be of critical importance to plant survival. Furthermore, substantial evidence is now available from a number of plant species that shows differential gene expression of aquaporins in response to abiotic stresses such as salinity, drought, or cold and clearly establishes the aquaporins as major players in the response of plants to conditions that affect water availability. This review summarizes the function and regulation of these genes to develop a greater understanding of the response of plants to water insufficiency, and particularly, to identify tolerant genotypes of major crop species including wheat and rice and plants that are important in agroforestry.
Anhydrobiosis (‘life without water’) is the remarkable ability of certain organisms to survive almost total dehydration. It requires a coordinated series of events during dehydration that are associated with preventing oxidative damage and maintaining the native structure of macromolecules and membranes. The preferential hydration of macromolecules is essential when there is still bulk water present, but replacement by sugars becomes important upon further drying. Recent advances in our understanding of the mechanism of anhydrobiosis include the downregulation of metabolism, dehydration-induced partitioning of amphiphilic compounds into membranes and immobilization of the cytoplasm in a stable multicomponent glassy matrix.
A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
Water profoundly affects the stability of biological membranes, and its removal leads to destructive events including fusion and liquid crystalline to gel phase transitions. In heterogeneous mixtures such as those found in biological membranes the phase transitions can lead to increases in permeability and lateral phase separations that often are irreparable. Certain sugars are capable of preventing these deleterious events by inhibiting fusion during drying and by maintaining the lipid in a fluid state in the absence of water. As a result, the increased permeability and lateral phase separations that accompany dehydration are absent. The weight of the evidence suggests strongly that there is a direct interaction between the sugars and lipids in the dry state. Although the evidence is less clear about whether these sugars can interact directly with hydrated bilayers, there are strong suggestions in the literature that sugars free in solution or covalently linked to membrane constituents can also affect the physical properties and presumably the stability of bilayers. Finally, we have far less evidence concerning the mechanism by which they do so, but the same sugars are also capable of preserving the structure and function of both membrane-bound and soluble proteins in the absence of water. We believe these effects may be important in the survival of intact cells and organisms such as seeds in the absence of water. Furthermore, in view of the practical importance of preserving biological structures we suspect that the results described here will ultimately have important applications in biology and medicine.
Seawater-strength salt stress of the ice plant (Mesembryanthemum crystallinum) initially results in wilting, but full turgor is restored within approximately 2 days. We are interested in a mechanistic explanation for this behavior and, as a requisite for in-depth biochemical studies, have begun to analyze gene expression changes in roots coincident with the onset of stress. cDNAs that suggested changes in mRNA amount under stress were found; their deduced amino acid sequences share homologies with proteins of the Mip (major intrinsic protein) gene family and potentially encode aquaporins. One transcript, MipB, was found only in root RNA, whereas two other transcripts, MipA and MipC, were detected in roots and leaves. Transcript levels of MipB were of low abundance. All transcripts declined initially during salt stress but later recovered to at least prestress level. The most drastic decline was in MipA and MipC transcripts. MipA mRNA distribution in roots detected by in situ hybridization indicated that the transcript was present in all cells in the root tip. In the expansion zone of the root where vascular bundles differentiate, MipA transcript amounts were most abundant in the endodermis. In older roots, which had undergone secondary growth, MipA was highly expressed in cell layers surrounding individual xylem strands. MipA was also localized in leaf vascular tissue and, in lower amounts, in mesophyll cells. Transcripts for MipB seemed to be present exclusively in the tip of the root, in a zone before and possibly coincident with the development of a vascular system. MipA- and MipB-encoded proteins expressed in Xenopus oocytes led to increased water permeability. mRNA fluctuations of the most highly expressed MipA and MipC coincided with turgor changes in leaves under stress. As the leaves regained turgor, transcript levels of these water channel proteins increased.
Potassium (K+) nutrition and salt tolerance are key factors controlling plant productivity. However, the mechanisms by which plants regulate K+ nutrition and salt tolerance are poorly understood. We report here the identification of an Arabidopsis thaliana mutant, sos3 (salt-overly-sensitive 3), which is hypersensitive to Na+ and Li+ stresses. The mutation is recessive and is in a nuclear gene that maps to chromosome V. The sos3 mutation also renders the plant unable to grow on low K+. Surprisingly, increased extracellular Ca2+ suppresses the growth defect of sos3 plants on low K+ or 50 mM NaCl. In contrast, high concentrations of external Ca2+ do not rescue the growth of the salt-hypersensitive sos1 mutant on low K+ or 50 mM NaCl. Under NaCl stress, sos3 seedlings accumulated more Na+ and less K+ than the wild type. Increased external Ca2+ improved K+/Na+ selectivity of both sos3 and wild-type plants. However, this Ca2+ effect in sos3 is more than twice as much as that in the wild type. In addition to defining the first plant mutant with an altered calcium response, these results demonstrate that the SOS3 locus is essential for K+ nutrition, K+/Na+ selectivity, and salt tolerance in higher plants.
The resurrection plant Craterostigma plantagineum (Hochst) is able to survive almost complete tissue dehydration when water is withheld from it, and then can rehydrate rapidly
on rewatering. This ability is believed to be the result of the accumulation of sucrose in aerial tissues as a result of metabolism
of 2‐octulose. In this work the metabolic activity of well‐watered Craterostigma plantagineum plants has been investigated. It is shown that Craterostigma makes raffinose series oligosaccharides as a product of photosynthesis and translocates them in the phloem. Evidence is also
provided that 2‐octulose is a product of photosynthesis and accumulates in the leaves over the light period and is mobilized
at night. Thus 2‐octulose acts as a temporary storage carbohydrate in leaves during photosynthesis in a similar fashion to
starch in most C3 plants. Other potential roles of 2‐octulose are discussed. Other than these observations Craterostigma plants are very similar to other C3 plants under these conditions.
Full-length cDNAs are essential for functional analysis of plant genes. Using the biotinylated CAP trapper method, we constructed full-length Arabidopsis cDNA libraries from plants in different conditions, such as drought-treated, cold-treated, or unstressed plants, and at various developmental stages from germination to mature seed. We prepared a cDNA microarray using approximately 1300 full-length Arabidopsis cDNAs to identify drought- and cold-inducible genes and target genes of DREB1A/CBF3, a transcription factor that controls stress-inducible gene expression. In total, 44 and 19 cDNAs for drought- and cold-inducible genes, respectively, were isolated, 30 and 10 of which were novel stress-inducible genes that have not been reported as drought- or cold-inducible genes previously. Twelve stress-inducible genes were identified as target stress-inducible genes of DREB1A, and six of them were novel. On the basis of RNA gel blot and microarray analyses, the six genes were identified as novel drought- and cold-inducible genes that are controlled by DREB1A. Eleven DREB1A target genes whose genomic sequences have been registered in the GenBank database contained the dehydration-responsive element (DRE) or DRE-related CCGAC core motif in their promoter regions. These results show that our full-length cDNA microarray is a useful material with which to analyze the expression pattern of Arabidopsis genes under drought and cold stresses, to identify target genes of stress-related transcription factors, and to identify potential cis-acting DNA elements by combining the expression data with the genomic sequence data.
Transcript regulation in response to high salinity was investigated for salt-tolerant rice (var Pokkali) with microarrays including 1728 cDNAs from libraries of salt-stressed roots. NaCl at 150 mM reduced photosynthesis to one tenth of the prestress value within minutes. Hybridizations of RNA to microarray slides probed for changes in transcripts from 15 min to 1 week after salt shock. Beginning 15 min after the shock, Pokkali showed upregulation of transcripts. Approximately 10% of the transcripts in Pokkali were significantly upregulated or downregulated within 1 hr of salt stress. The initial differences between control and stressed plants continued for hours but became less pronounced as the plants adapted over time. The interpretation of an adaptive process was supported by the similar analysis of salinity-sensitive rice (var IR29), in which the immediate response exhibited by Pokkali was delayed and later resulted in downregulation of transcription and death. The upregulated functions observed with Pokkali at different time points during stress adaptation changed over time. Increased protein synthesis and protein turnover were observed at early time points, followed by the induction of known stress-responsive transcripts within hours, and the induction of transcripts for defense-related functions later. After 1 week, the nature of upregulated transcripts (e.g., aquaporins) indicated recovery.
Plant responses to salinity stress are reviewed with emphasis on molecular mechanisms of signal transduction and on the physiological consequences of altered gene expression that affect biochemical reactions downstream of stress sensing. We make extensive use of comparisons with model organisms, halophytic plants, and yeast, which provide a paradigm for many responses to salinity exhibited by stress-sensitive plants. Among biochemical responses, we emphasize osmolyte biosynthesis and function, water flux control, and membrane transport of ions for maintenance and re-establishment of homeostasis. The advances in understanding the effectiveness of stress responses, and distinctions between pathology and adaptive advantage, are increasingly based on transgenic plant and mutant analyses, in particular the analysis of Arabidopsis mutants defective in elements of stress signal transduction pathways. We summarize evidence for plant stress signaling systems, some of which have components analogous to those that regulate osmotic stress responses of yeast. There is evidence also of signaling cascades that are not known to exist in the unicellular eukaryote, some that presumably function in intercellular coordination or regulation of effector genes in a cell-/tissue-specific context required for tolerance of plants. A complex set of stress-responsive transcription factors is emerging. The imminent availability of genomic DNA sequences and global and cell-specific transcript expression data, combined with determinant identification based on gain- and loss-of-function molecular genetics, will provide the infrastructure for functional physiological dissection of salt tolerance determinants in an organismal context. Furthermore, protein interaction analysis and evaluation of allelism, additivity, and epistasis allow determination of ordered relationships between stress signaling components. Finally, genetic activation and suppression screens will lead inevitably to an understanding of the interrelationships of the multiple signaling systems that control stress-adaptive responses in plants.
This multi-author book gives a comprehensive account of desiccation and plant survival, and of how plant cells deal with extreme water stress. There is a general introduction on desiccation, and then four sections dealing with: The technical background to desiccation studies; the frequency and levels of dehydration stress tolerance in biological systems; mechanisms of damage and tolerance; and a brief retrospect and prospect. Orthodox and recalcitrant seeds, pollen and spores, vegetative parts, and other plant tissues are covered in detail.
Embryogenesis in plants can be thought of as a series of stages leading to the formation of a mature, differentiated, miniature plant within the seed. Embryogenesis is initiated by fertilisation, in which the fusion of a sperm nucleus and an egg nucleus gives rise to the diploid zygote. The fusion of a second sperm nucleus with the two polar nuclei within the embryo sac produces the lineage that ultimately becomes the triploid endosperm.
Plant growth is greatly affected by drought and low temperature. Expression of a number of genes is induced by both drought and low temperature, although these stresses are quite different. Previous experiments have established that a cis-acting element named DRE (for dehydration-responsive element) plays an important role in both dehydration- and low-temperature-induced gene expression in Arabidopsis. Two cDNA clones that encode DRE binding proteins, DREB1A and DREB2A, were isolated by using the yeast one-hybrid screening technique. The two cDNA libraries were prepared from dehydrated and cold-treated rosette plants, respectively. The deduced amino acid sequences of DREB1A and DREB2A showed no significant sequence similarity, except in the conserved DNA binding domains found in the EREBP and APETALA2 proteins that function in ethylene-responsive expression and floral morphogenesis, respectively. Both the DREB1A and DREB2A proteins specifically bound to the DRE sequence in vitro and activated the transcription of the b-glucuronidase reporter gene driven by the DRE sequence in Arabidopsis leaf protoplasts. Expression of the DREB1A gene and its two homologs was induced by low-temperature stress, whereas expression of the DREB2A gene and its single homolog was induced by dehydration. Overexpression of the DREB1A cDNA in transgenic Arabidopsis plants not only induced strong expression of the target genes under unstressed conditions but also caused dwarfed phenotypes in the transgenic plants. These transgenic plants also revealed freezing and dehydration tolerance. In contrast, overexpression of the DREB2A cDNA induced weak expression of the target genes under unstressed conditions and caused growth retardation of the transgenic plants. These results indicate that two independent families of DREB proteins, DREB1 and DREB2, function as trans-acting factors in two separate signal transduction pathways under low-temperature and dehydration conditions, respectively.
Full-length cDNAs are essential for functional analysis of plant genes. Using the biotinylated CAP trapper method, we constructed full-length Arabidopsis cDNA libraries from plants in different conditions, such as drought-treated, cold-treated, or unstressed plants, and at various developmental stages from germination to mature seed. We prepared a cDNA microarray using ∼1300 full-length Arabidopsis cDNAs to identify drought- and cold-inducible genes and target genes of DREB1A/CBF3, a transcription factor that controls stress-inducible gene expression. In total, 44 and 19 cDNAs for drought- and cold-inducible genes, respectively, were isolated, 30 and 10 of which were novel stress-inducible genes that have not been reported as drought- or cold-inducible genes previously. Twelve stress-inducible genes were identified as target stress-inducible genes of DREB1A, and six of them were novel. On the basis of RNA gel blot and microarray analyses, the six genes were identified as novel drought- and cold-inducible genes that are controlled by DREB1A. Eleven DREB1A target genes whose genomic sequences have been registered in the GenBank database contained the dehydration-responsive element (DRE) or DRE-related CCGAC core motif in their promoter regions. These results show that our full-length cDNA microarray is a useful material with which to analyze the expression pattern of Arabidopsis genes under drought and cold stresses, to identify target genes of stress-related transcription factors, and to identify potential cis-acting DNA elements by combining the expression data with the genomic sequence data.
Transcript regulation in response to high salinity was investigated for salt-tolerant rice (var Pokkali) with microarrays including 1728 cDNAs from libraries of salt-stressed roots. NaCl at 150 mM reduced photosynthesis to one tenth of the prestress value within minutes. Hybridizations of RNA to microarray slides probed for changes in transcripts from 15 min to 1 week after salt shock. Beginning 15 min after the shock, Pokkali showed upregulation of transcripts. Approximately 10% of the transcripts in Pokkali were significantly upregulated or downregulated within 1 hr of salt stress. The initial differences between control and stressed plants continued for hours but became less pronounced as the plants adapted over time. The interpretation of an adaptive process was supported by the similar analysis of salinity-sensitive rice (var IR29), in which the immediate response exhibited by Pokkali was delayed and later resulted in downregulation of transcription and death. The upregulated functions observed with Pokkali at different time points during stress adaptation changed over time. Increased protein synthesis and protein turnover were observed at early time points, followed by the induction of known stress-responsive transcripts within hours, and the induction of transcripts for defenserelated functions later. After 1 week, the nature of upregulated transcripts (e.g., aquaporins) indicated recovery.
Recently, a major transcription system that controls abscisic-acid-independent gene expression in response to dehydration and low temperature has been identified. The system includes the DRE/CRT (dehydration-responsive element/C-repeat) cis-acting element and its DNA-binding protein, DREB/CBF (DRE-binding protein/C-repeat binding factor), which has an AP2 domain. DREB/CBF contains two subclasses, DREB1/CBF and DREB2, which are induced by cold and dehydration, respectively, and control the expression of various genes involved in stress tolerance. Recent studies are providing evidence of differences between dehydration-signaling and cold-stress-signaling cascades, and of cross-talk between them.
Desiccation is the extreme form of dehydration. Tolerance of desiccation is acquired by seeds and in resurrection plants, a small group of angiosperms. Desiccation tolerance is the result of a complex cascade of molecular events, which can be divided into signal perception, signal trans-duction, gene activation and biochemical alterations leading to acquisition of tolerance. Many of these molecular processes are also observed during the dehydration of non-tolerant plants. Here we try to give an overview of the gene expression programmes that are triggered by dehydration, with particular reference to protective molecules and the regulation of their expression. Potential transgenic approaches to manipulating stress tolerance are discussed.
Lea genes code for mRNAs and proteins that are late embryogenesis abundant in higher plant seed embryos. They appear to be ubiquitous in higher plants and may be induced to high levels of expression in other tissues and at other times of ontogeny by ABA and/or desiccation. Presented here are the genomic and cDNA sequences for 6 of these genes from cotton seed embryos and the derived amino acid sequences of the corresponding proteins.
The Lea genes contain the standard sequence features of eucaryotic genes (TATA box and poly (A) addition sequences) and have 1 or more introns. Sequences differences between cDNA and genomic DNA confirm the existence of small multigene families for several Lea genes. The amino acid composition and sequence for the Lea proteins are unusual. Five are extremely hydrophilic, four contain no cys or trp and 4 have sequence domains that suggest amphiphilic helical structures. Hypothetical functions in desiccation survival, based on amino acid sequence, are discussed.
Leaves of the resurrection plant Craterostigma plantagineum Hochst, can be desiccated up to 1% relative water content and are still viable after rehydration. To clone genes related to this extreme desiccation tolerance, an in-vitro system was first developed which allows the induction of the same resurrection response in callus tissue upon treatment with abscisic acid (ABA). Several proteins and in-vitro-synthesized polypeptides were then identified which can be induced both in desiccation-tolerant, naturally dried leaves and in ABA-treated calli surviving after rehydration. Complementary-DNA clones corresponding to mRNAs expressed only in desiccation-tolerant tissues were obtained and classified into several gene families. In hybrid-selected translation experiments, representative cDNA clones were associated with water stress and ABA-inducible polypeptides abundantly expressed in dried leaves and ABA-treated calli. The expression pattern of several of these abundant transcripts was analyzed in RNA-hybridization experiments. Upon stress or ABA treatment the transcription levels increased rapidly, but they declined after relief from the stress state. This, together with data on genomic copy numbers indicated that a set of abundantly expressed genes are involved in the desiccation process of resurrection plants. Data on endogenous ABA contents before and after stress applications and on the physiological effects of exogenous ABA treatments indicate that in Craterostigma plantagineum the induction of an extreme desiccation tolerance is mediated by this plant hormone.
The resurrection plant Craterostigma plantagineum Hochst. (Fam. Scrophulariaceae) is unique in its ability to survive severe desiccation. To analyze the primary response of this plant to drought, genes were identified that are either transiently expressed or down-regulated during early phases of dehydration. A cDNA library was prepared using as template mRNA isolated from 1 h dried leaves. The library was differentially screened employing three experimental strategies. Partial sequencing of the isolated cDNAs and comparison of the cDNA sequences with those from the public data bases revealed that 49% of the cDNA clones displayed significant sequence similarity to previously identified genes. Northern and reverse Northern analysis confirmed that all genes so far tested showed differential expression upon drought stress: 58 % were transiently expressed, 35.8% were down-regulated and 6.2 % were found to be up-regulated during dehydration. The identification of a large spectrum of differentially expressed genes with diverse cellular functions points to a complex molecular response to drought stress in the resurrection plant C. plantagineum.
Improving salt tolerance in crop plants remains an urgent issue in plant molecular biology. The adaptation of plants to NaCl involves metabolic reactions (synthesis of organic solutes) and transport phenomena (ion extrusion at the plasma membrane and vacuolar compartmentation). In addition, a plethora of salt-induced genes with a bewildering variety of suggested functions have been described. The uncertainties about the physiological roles and/or molecular bases of many of these phenomena make it difficult to select genes that could improve salt tolerance (halotolerance) in transgenic plants. We suggest that the field of salt tolerance can benefit by complementing the present phenomenological or descriptive approaches with a functional strategy directed toward isolating genes that, by overexpression of the corresponding protein, could improve salt tolerance. These halotolerance genes not only could illuminate the critical steps for salt tolerance, but also could provide the tools for improvement. Microbial genetics facilitates the implementation of this genetic approach. Studies using the prokaryotic organism Escherichia coli suggest that the synthesis of organic solutes may be the crucial step for salt tolerance because the first described bacterial halotolerance gene (proB-74) determines the overaccumulation of proline. In the eukaryotic microorganism Saccharomyces cerevisiae, however, potassium homeostasis seems to be the most critical response to salt stress. The first halotolerance gene isolated from this organism (HAL1) seems to modulate potassium transport, increasing the intracellular level of this cation in NaCl-containing media. The existence of plant homologues to HAL1 indicates that yeast may be a useful model for the genetics of salt tolerance in plants.
Extreme tolerance to protoplastic desiccation is wide-spread among lower eukaryotes but is restricted to a small group of resurrection plants among higher plants. In many water-stressed organisms organic solutes accumulate and aid in retaining cell water while remaining compatible with macromolecular functions. Based on this observation, carbohydrates were analysed in the resurrection plant Craterostigma plantagineum. We found that the unusual carbohydrate 2-octulose is the dominant sugar in fully hydrated leaves but upon dehydration it is converted to sucrose. The possible metabolic pathways involved in this particular carbohydrate metabolism are discussed.
When plants do not receive sufficient water they are subjected to a stress called water deficit. Water deficit in the plant disrupts many cellular and whole plant functions, having a negative impact on plant growth and reproduction. Plants have evolved many different mechanisms to deal with the occurrence of this stress as it occurs in their environments. Availability of water is the most important factor in the environment that reduces the production of our crops.
Keywords:
water-deficit stress;
water relations;
gene regulation;
biotechnology
In order to understand the molecular mechanisms which are responsible for desiccation tolerance in the resurrection plant
Craterostigma plantagineum Hochst. a thorough analysis of the CDeT11-24 gene family was performed. CDeT11-24 comprises a small gene family whose genes
are expressed in response to dehydration, salt stress and abscisic acid (ABA) treatment in leaves. The gene products are constitutively
expressed in roots and disappear only when the plants are transferred to water. It is therefore suggested that the proteins
are involved in sensing water status. The predicted proteins are very hydrophilic; they share some features with late-embryogenesis-abundant
proteins, and sequence similarities were found with two ABA- and drought-regulated Arabidopsis genes. The analysis of β-glucuronidase reporter genes driven by the CDeT11-24 promoter showed high activity in mature seeds
in both transgenic Arabidopsis and tobacco. In vegetative tissues the promoter activity in response to ABA was restricted to young Arabidosis seedlings. The responsiveness to ABA during later developmental stages was regained in the presence of the Arabidopsis gene product ABI3. Dehydration-induced promoter activity was only observed in Arabidopsis leaves at a particular developmental stage. This analysis indicates that some components in the signal transduction pathway
of the resurrection plant are not active in tobacco or Arabidopsis.
Plant responses to water deficit are dependent on the amount of water lost, the rate of loss and the duration of the stressed condition. The characterization of a large number of genes induced by stresses involving water deficit has significantly improved understanding of the response. There appear to be several pathways for gene induction involved, and these are being elucidated by the analysis of DNA elements, mutants and gene expression patterns. However, it has been difficult to resolve the functions of many drought-induced genes against the background of other stress-induced changes, and thus it is now important to integrate information about cellular and whole plant responses.
We isolated nine cDNAs for genes of Arabidopsis thaliana that are induced by the effects of water deficit by using the technique of differential hybridization and named them RD (RD for Responsive to Desiccation). The timing of induction of their mRNAs varies between these RD genes. Abscisic acid induces mRNAs that correspond to clones designated RD22 and RD29 but not to those designated RD19, RD21 and RD28, indicating that there are several signal-transduction pathways from water stress to expression of RD genes. Sequence analysis of the cDNA clone RD28 revealed that RD28 encodes a membrane protein with sequence homology to the majorintrinsic protein of bovine lens fiber gap junction, soybean nodulin-26 and the glycerolfacilitator from Escherichia coli, indicating that the putative RD28 protein is a member of the family of transmembrane channel proteins.
Wheat and barley DNA enriched for ribosomal RNA genes was isolated from actinomycin D-CsCl gradients and used to clone the ribosomal repeating units in the plasmid pAC184. All five chimeric plasmids isolated which contained wheat rDNA and eleven of the thirteen which had barley rDNA were stable and included full length ribosomal repeating units. Physical maps of all length variants cloned have been constructed using the restriction endonucleases Eco Rl, Bam Hl, Bgl II, Hind III and Sal I. Length variation in the repeat units was attributed to differences in the spacer regions. Comparison of Hae III and Hpa II digestion of cereal rDNAs and the cloned repeats suggests that most methylated cytosines in natural rDNA are in -CpG-. Incomplete methylation occurs at specific Bam Hl sites in barley DNA. Detectable quantities of ribosomal spacer sequences are not present at any genomic locations other than those of the ribosomal RNA gene repeats.
We believe we have established the major principles governing the stabilization of living cells in the unique condition known as anhydrobiosis. These findings have permitted us to design ways to stabilize membrane vesicles, liposomes, and proteins, and perhaps eventually even intact cells that do not normally survive dehydration. In a complex phenomenon as ancient as anhydrobiosis, one would expect a myriad of adaptations to be required for survival of drying. But the arguments presented here suggest that a single perturbation--synthesis of a disaccharide such as trehalose or sucrose--is sufficient to achieve survival. We hasten to add, however, that it is now certain that additional adaptations are required; for instance, cells containing highly unsaturated lipids may survive drying for a short time, but they are so susceptible to degradation that they survive for a short time only. Thus the interpretation placed on the finding that trehalose can stabilize dry membranes must be regarded from this perspective as well. Nevertheless, we believe that the underlying physical principles governing stability of dry biological materials are universal.
In Arabidopsis thaliana L., accumulation of abscisic acid (ABA) began to increase 2 h after plants had been subjected to dehydration stress and reached maximum levels after 10 h. Differential hybridization was used to isolate 26 Arabidopsis cDNAs with gene expression induced by a 1 h dehydration treatment. The cDNA clones were classified into 16 groups based on Southern blot hybridization, and named ERD (early-responsive to dehydration) clones. Partial sequencing of the cDNA clones revealed that three ERDs were identical to those of HSP cognates (Athsp70-1, Athsp81-2, and ubiquitin extension protein). Dehydration stress strongly induced the expression of genes for the three ERDs, while application of ABA, which is known to act as a signal transmitter in dehydration-stressed plants, did not significantly affect the ERD gene expression. This result suggests that these HSP cognates are preferentially responsive to dehydration stress in A. thaliana, and that signaling pathways for the expression of these genes under conditions of dehydration stress are not mainly mediated by ABA. We also discuss the possible functions of these three ERD gene products against dehydration stress.
Gridded on high density filters, a P1 genomic library of 17-fold coverage and a cosmid library of 8 genome equivalents, both made from S. pombe strain 972h-, were ordered by hybridizing genetic markers and individual clones from the two libraries. Yeast artificial chromosome (YAC) clones covering the entire genome were used to subdivide the libraries, and hybridization of short oligonucleotides and DNA pools made from randomly selected cosmids provided further mapping information. Restriction digests were generated as an independent confirmation of the clone order. The high resolution clone map was aligned to the genetic map and the physical Notl and YAC maps. The usefulness of the various mapping techniques and cloning procedures could be assessed upon the different data sets.
Recent efforts have defined a cis-acting DNA regulatory element in plants, the C-repeat/dehydration responsive element (DRE), that stimulates transcription in response to low temperature and water deficit. Here we report the isolation of an Arabidopsis thaliana cDNA that encodes a C-repeat/DRE binding factor, CBF1 (C-repeat/DRE Binding Factor 1). Analysis of the deduced CBF1 amino acid sequence indicates that the protein has a molecular mass of 24 kDa, a potential nuclear localization sequence, and a possible acidic activation domain. CBF1 also has an AP2 domain, which is a DNA-binding motif of about 60 aa present in the Arabidopsis proteins APETALA2, AINTEGUMENTA, and TINY; the tobacco ethylene response element binding proteins; and numerous other plant proteins of unknown function. The transcript levels for CBF1, which appears to be a single or low copy number gene, did not change appreciably in plants exposed to low temperature or in detached leaves subjected to water deficit. Binding of CBF1 to the C-repeat/DRE was demonstrated in gel shift assays using recombinant CBF1 protein expressed in Escherichia coli. Moreover, expression of CBF1 in yeast was found to activate transcription of reporter genes containing the C-repeat/DRE as an upstream activator sequence but not mutant versions of the DNA element. We conclude that CBF1 can function as a transcriptional activator that binds to the C-repeat/DRE DNA regulatory element and, thus, is likely to have a role in cold- and dehydration-regulated gene expression in Arabidopsis.
Plant growth is greatly affected by drought and low temperature. Expression of a number of genes is induced by both drought and low temperature, although these stresses are quite different. Previous experiments have established that a cis-acting element named DRE (for dehydration-responsive element) plays an important role in both dehydration- and low-temperature-induced gene expression in Arabidopsis. Two cDNA clones that encode DRE binding proteins, DREB1A and DREB2A, were isolated by using the yeast one-hybrid screening technique. The two cDNA libraries were prepared from dehydrated and cold-treated rosette plants, respectively. The deduced amino acid sequences of DREB1A and DREB2A showed no significant sequence similarity, except in the conserved DNA binding domains found in the EREBP and APETALA2 proteins that function in ethylene-responsive expression and floral morphogenesis, respectively. Both the DREB1A and DREB2A proteins specifically bound to the DRE sequence in vitro and activated the transcription of the b-glucuronidase reporter gene driven by the DRE sequence in Arabidopsis leaf protoplasts. Expression of the DREB1A gene and its two homologs was induced by low-temperature stress, whereas expression of the DREB2A gene and its single homolog was induced by dehydration. Overexpression of the DREB1A cDNA in transgenic Arabidopsis plants not only induced strong expression of the target genes under unstressed conditions but also caused dwarfed phenotypes in the transgenic plants. These transgenic plants also revealed freezing and dehydration tolerance. In contrast, overexpression of the DREB2A cDNA induced weak expression of the target genes under unstressed conditions and caused growth retardation of the transgenic plants. These results indicate that two independent families of DREB proteins, DREB1 and DREB2, function as trans-acting factors in two separate signal transduction pathways under low-temperature and dehydration conditions, respectively.
The molecular dissection of desiccation tolerance in the resurrection plant Craterostigma plantagineum led to the isolation of two dehydration-stress inducible homeo-domain-leucine zipper genes (CPHB-1 and -2). When the coding region of CPHB-1 was used as bait in the yeast two-hybrid system, the ability of CPHB-1 to form homodimers was demonstrated. The two-hybrid system was also used to isolate CPHB-2, which heterodimerises with CPHB-1. Both transcripts are inducible by dehydration in leaves and roots, but steady state levels vary in response to exogenously applied ABA. Although expression of CPHB-1 is not inducible by ABA, the transcript level of CPHB-2 increases during ABA-treatment. Both genes are expressed at very early stages of dehydration and thus may be involved in the regulation of gene expression during dehydration. CPHB-1 and -2 differential expression in response to ABA suggests that they act in different branches of the dehydration-induced signalling network. In vitro binding studies revealed that CPHB-1 specifically binds to the pseudopalindromic sequence CAAT(C/G)ATTG. Using this element for in vitro binding studies with nuclear proteins from dehydrated leaves, an inducible DNA-protein complex was identified.
Major intrinsic proteins (MIPs) are a family of channel proteins that are mainly represented by aquaporins in plants. These are divided into TIPs (tonoplast intrinsic proteins) and PIPs (plasma membrane intrinsic proteins) according to their subcellular localization. Homologues to PIPs and TIPs were isolated from the desiccation-tolerant resurrection plant Craterostigma plantagineum by two approaches: firstly, a cDNA library constructed from RNA of dehydrated C. plantagineum leaves was screened with an Arabidopsis thaliana Ath-PIP1b cDNA probe and, secondly, a cDNA library was screened differentially to isolate early drought-induced transcripts. According to sequence homologies the isolated cDNA clones were grouped as follows: Cp-PIPa, Cp-PIPb, Cp-PIPc and Cp-TIP. Cp-PIPa, Cp-PIPc and Cp-TIP transcript accumulation was regulated by dehydration and abscisic acid (ABA). Within the Cp-PIPa group transcripts were regulated either by drought only or by drought and ABA, indicating that ABA-dependent and -independent signal transduction pathways lead to Cp-PIPa expression. Comparison of Cp-PIPa expression in detached leaves and in whole plants suggested the involvement of a signal transmitted in the whole plant in response to drought. Cp-PIPb transcript levels were constitutive in all organs tested. Antibodies raised against a Cp-PIPA protein recognized a polypeptide with an apparent molecular mass of 28 kDa. Using these antibodies it was shown that both Cp-PIPA and Cp-PIPB proteins were localized to the plasma membrane. The role of different members of the MIP group in the dehydration response is discussed.
Recently, a major transcription system that controls abscisic-acid-independent gene expression in response to dehydration and low temperature has been identified. The system includes the DRE/CRT (dehydration-responsive element/C-repeat) cis-acting element and its DNA-binding protein, DREB/CBF (DRE-binding protein/C-repeat binding factor), which has an AP2 domain. DREB/CBF contains two subclasses, DREB1/CBF and DREB2, which are induced by cold and dehydration, respectively, and control the expression of various genes involved in stress tolerance. Recent studies are providing evidence of differences between dehydration-signaling and cold-stress-signaling cascades, and of cross-talk between them.
The ability of plant cells to maintain low cytosolic sodium concentrations is an essential process associated with the ability of plants to grow in high salt concentrations. Recent results have identified pathways for Na(+) entry, and the cloning of vacuolar Na(+)/H(+) antiporters has demonstrated the role of intracellular Na(+) compartmentation in plant salt tolerance.
Adverse environmental conditions restrict the pro- ductivity and the range of habitats available to plants. This represents a severe economic constraint on agricultural production. Plants as sessile organ- isms have evolved a wide spectrum of adaptations to cope with the challenges of environmental stress. Quite often, however, adaptation mechanisms them- selves adversely affect yield parameters, and a com- promise between biomass production and environ- mental fitness has to be accepted. One major factor that limits the productive potential of higher plants is the availability of water. The International Water Management Institute predicts that by the year 2025,
There is strong evidence that aquaporins are central components in plant water relations. Plant species possess more aquaporin genes than species from other kingdoms. According to sequence similarities, four major groups have been identified, which can be further divided into subgroups that may correspond to localization and transport selectivity. They may be involved in compatible solute distribution, gas-transfer (CO2, NH3) as well as in micronutrient uptake (boric acid). Recent advances in determining the structure of some aquaporins gives further details on the mechanism of selectivity. Gating behaviour of aquaporins is poorly understood but evidence is mounting that phosphorylation, pH, pCa and osmotic gradients can affect water channel activity. Aquaporins are enriched in zones of fast cell division and expansion, or in areas where water flow or solute flux density would be expected to be high. This includes biotrophic interfaces between plants and parasites, between plants and symbiotic bacteria or fungi, and between germinating pollen and stigma. On a cellular level aquaporin clusters have been identified in some membranes. There is also a possibility that aquaporins in the endoplasmic reticulum may function in symplasmic transport if water can flow from cell to cell via the desmotubules in plasmodesmata. Functional characterization of aquaporins in the native membrane has raised doubt about the conclusiveness of expression patterns alone and need to be conducted in parallel. The challenge will be to elucidate gating on a molecular level and cellular level and to tie those findings into plant water relations on a macroscopic scale where various flow pathways need to be considered.
CDeT27-45 is a lea-like gene from the resurrection plant Craterostigma plantagineum (Scrophulariaceae) which is strongly expressed in vegetative tissues in response to dehydration or treatment with abscisic acid (ABA). Expression of the gene is correlated with the acquisition of desiccation tolerance. Nuclear proteins bind to a 29-bp cis-regulatory region of the promoter which is essential for transcriptional activation of the CDeT27-45 gene by ABA. Using a yeast one-hybrid screen, the cDNA clone CpR18 was isolated, which encodes a protein with specific binding activity for the cis-regulatory element in the CDeT27-45 promoter. The protein contains an acidic region, a SAP-domain, a zinc finger of the C3H-type, and two motifs which are conserved in proteins from several plant species. One of the conserved regions is rich in basic residues and is predicted to form a helix-loop-helix structure. The R18 gene shows high similarities to genomic sequences and ESTs from other plant species. The tissue-specific expression pattern of the rare R18 mRNA and the distribution of nuclear protein binding activity for the CDeT27-45 promoter fragment are compared. The R18 protein is indeed localized in the nucleus, and activates transcription of CDeT27-45 promoter-GUS fusion constructs in tobacco protoplasts. DNA blot analysis and isolation of genomic clones reveal that two copies of R18 are present in the C. plantagineum genome.
Water deficit elicits a complex of responses beginning with stress perception, which initiates a signal transduction path- way(s) and is manifested in changes at the cellular, physio- logical, and developmental levels. The set of responses ob- served depends upon severity and duration of the stress, plant genotype, developmental stage, and environmental fac- tors providing the stress. Cellular water deficit may result from stresses such as drought, salt, and low temperature. This complexity makes it difficult to uncover the responses to water deficit that enhance stress tolerance. In recent years efforts have tumed toward isolation of genes that are induced during water deficit in order to study the function of drought- induced gene products and the pathways that lead to gene induction. Changes in gene expression are fundamental to the responses that occur during water deficit, and they control many of the short- and long-tem responses. Studies on the molecular responses to water deficit have identified multiple changes in gene expression using two- dimensional PAGE, and many genes that are water-deficit- induced have been isolated by differential screening of cDNA libraries. Functions for many of these genè products have been predicted from the deduced amino acid sequence of the genes. Genes expressed during stress are anticipated to pro- mote cellular tolerance of dehydration through protective functions in the cytoplasm, alteration of cellular water poten- tia1 to promote water uptake, control of ion accumulation, and further regulation of gene expression. Although these studies are promising, it continues to be difficult to ascertain the actual function of drought-induced gene products. Expression of a gene during stress does not guarantee that a gene product promotes the ability of the plant to survive stress. The expression of some genes may result from injury or damage that occurred during stress. Other genes may be induced, but their expression does not alter stress tolerance. Yet others are required for stress tolerance and the accumu- lation of these gene products is an adaptive response. Complex regulatory and signaling processes, most of which are not understood, control the expression of genes during water deficit. Multiple stresses may connect into the same or a similar transduction pathway, which is evidenced by the involvement of ABA in the induction of genes induced by a number of different stresses. In addition to induction by stress, the expression of water-deficit-associated genes is controlled with respect to tissue, organ, and developmental stage and may be expressed independently of the stress conditions. For example, some genes expressed during drought stress are also expressed during the maturation and desiccation phases of seed development. The regulation of specific processes will also depend upon the experimental conditions of stress application. Stress conditions that are applied in the laboratory may not accurately represent those that occur in the field. Frequently, laboratory stresses are rapid and severe, whereas stress in the field often develops over an extended period of time (Radin, 1993). These differ- ences must also be evaluated when studying the adaptive value of certain responses. The function of the gene products and the mechanisms of gene expression are intertwined, and both must be understood to fully comprehend the molecular response to water deficit.
Membrane vesicles were isolated from the roots of the halophyte Atriplex nummularia Lindl. H(+)-translocating Mg(2+)-ATPase activity was manifested by the establishment of a positive membrane potential (measured as SCN(-) accumulation); and also by the establishment of a transmembrane pH gradient (measured by quinacrine fluorescence quenching). H(+)-translocation was highly specific to ATP and was stable to oligomycin. Growing the plants in the presence of 400 millimolar NaCl doubled the proton-translocating activity per milligram of membrane protein and otherwise modulated it in the following ways. First, the flat pH profile observed in non-salt-grown plants was transformed to one showing a peak at about pH 6.2. Second, the lag effect observed at low ATP concentration in curves relating SCN(-) accumulation to ATP concentration was abolished; the concave curvature shown in the double reciprocal plot was diminished. Third, sensitivity to K-2 (N-morpholino)ethanesulfonic acid stimulation was shown in salt-grown plants (about 40% stimulation) but was absent in non-salt-grown plants. Fourth, the KCl concentration bringing about 50% dissipation of ATP-dependent SCN(-) accumulation was 20 millimolar for salt-grown plants and 50 millimolar for non-salt-grown plants. Vanadate sensitivity was shown in both cases. No clear NO(3) (-) inhibition was observed.
Na(+) and Cl(-) are the principal solutes utilized for osmotic adjustment in cells of Nicotiana tabacum L. var Wisconsin 38 (tobacco) adapted to NaCl, accumulating to levels of 472 and 386 millimolar, respectively, in cells adapted to 428 millimolar NaCl. X-ray microanalysis of unetched frozen-hydrated cells adapted to salt indicated that Na(+) and Cl(-) were compartmentalized in the vacuole, at concentrations of 780 and 624 millimolar, respectively, while cytoplasmic concentrations of the ions were maintained at 96 millimolar. The morphometric differences which existed between unadapted and salt adapted cells, (cytoplasmic volume of 22 and 45% of the cell, respectively), facilitated containment of the excited volume of the x-ray signal in the cytoplasm of the adapted cells. Confirmation of ion compartmentation in salt adapted cells was obtained based on kinetic analyses of (22)Na(+) and (36)Cl(-) efflux from cells in steady state. These data provide evidence that ion compartmentation is a component of salt adaptation of glycophyte cells.
Leaves of resurrection plants tolerate desiccation as do embryos of many higher plants. From the resurrection plant Craterostigma plantagineum a number of desiccation-related transcripts have recently been cloned; they are abundantly expressed in dried leaves and abscisic acid-treated dried callus (D Bartels, K Schneider, G Terstappen, D Piatkowski, F Salamini [1990] Planta 18: 27-34). Five distinct cDNA clones representing low copy number genes were selected for further characterization. Their nucleotide sequences were determined and proteins were predicted with a molecular mass between 16 and 34 kilodaltons. Three of these proteins have unusual amino acid compositions and extreme hydrophilic characters. Two of them contain a cluster of contiguous serine residues and lysine-rich repeats. These sequence motifs display homologies to desiccation-related genes expressed in embryos or dehydrated seedlings of several plants. A third cDNA clone contains tracts of sequences which are related to a cotton Lea (late embryogenesis abundant) gene (JC Baker, C Steele, L Dure III [1988] Plant Mol Biol II: 277-291). Secondary structure predictions are discussed and suggest that the deduced proteins could play a role in protecting core cell structures in a dehydrated cell. It is concluded that at least in part the gene products involved in the desiccation-induced pathways are common to leaves of resurrection plants and embryos. Two cDNA clones appear to code for Craterostigma-specific mRNAs. The expression patterns of all five transcripts were studied in comparison to desiccated leaves in dehydrated roots, in wound-stressed leaves and in salt-stressed callus. The data obtained point to the possibility that not only specificity of induction but also the expression level of specific gene products may be of importance for osmoprotection.
The South African flora contains a unique abundance of higher plants which withstand virtually complete desiccation. Water potentials of fielddry leaves corresponded to 30 to 40 percent relative humidity. Mature leaves survived from 15 to approximately 0 percent relative humidity. Known examples were increased from 4 to 15 species, which for the first time included grasses.