Plant dehydrins and stress tolerance: Versatile proteins for complex mechanisms

Dehydrins (DHNs), or group 2 LEA (Late Embryogenesis Abundant) proteins, play a fundamental role in plant response and adaptation to abiotic stresses. They accumulate typically in maturing seeds or are induced in vegetative tissues following salinity, dehydration, cold and freezing stress. The generally accepted classification of dehydrins is based on their structural features, such as the presence of conserved sequences, designated as Y, S and K segments. The K segment representing a highly conserved 15 amino acid motif forming amphiphilic a-helix is especially important since it has been found in all dehydrins. Since more than 20 y, they are thought to play an important protective role during cellular dehydration but their precise function remains unclear. This review outlines the current status of the progress made toward the structural, physico-chemical and functional characterization of plant dehydrins and how these features could be exploited in improving stress tolerance in plants.Key words: abiotic stress, dehydration stress, drought, cold acclimation, freezing tolerance, LEA proteins, dehydrins     

Overexpression of a <Emphasis Type="Italic">Panax ginseng</Emphasis> tonoplast aquaporin alters salt tolerance,drought tolerance and cold acclimation ability in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

Water movement across cellular membranes is regulated largely by a family of water channel proteins called aquaporins (AQPs). Since several abiotic stresses such as, drought, salinity and freezing, manifest themselves via altering water status of plant cells and are linked by the fact that they all result in cellular dehydration, we overexpressed an AQP (tonoplast intrinsic protein) from Panax ginseng, PgTIP1, in transgenic Arabidopsis thaliana plants to test its role in plant’s response to drought, salinity and cold acclimation (induced freezing tolerance). Under favorable conditions, PgTIP1 overexpression significantly increased plant growth as determined by the biomass production, and leaf and root morphology. PgTIP1 overexpression had beneficial effect on salt-stress tolerance as indicated by superior growth status and seed germination of transgenic plants under salt stress; shoots of salt-stressed transgenic plants also accumulated greater amounts of Na⁺ compared to wild-type plants. Whereas PgTIP1 overexpression diminished the water-deficit tolerance of plants grown in shallow (10 cm deep) pots, the transgenic plants were significantly more tolerant to water stress when grown in 45 cm deep pots. The rationale for this contrasting response, apparently, comes from the differences in the root morphology and leaf water channel activity (speed of dehydration/rehydration) between the transgenic and wild-type plants. Plants overexpressed with PgTIP1 exhibited lower (relative to wild-type control) cold acclimation ability; however, this response was independent of cold-regulated gene expression. Our results demonstrate a significant function of PgTIP1 in growth and development of plant cells, and suggest that the water movement across tonoplast (via AQP) represents a rate-limiting factor for plant vigor under favorable growth conditions and also significantly affect responses of plant to drought, salt and cold stresses.     

NMR assignments of the intrinsically disordered K2 and YSK2 dehydrins

Dehydrins are proteins expressed by plants during various dehydrative stresses (drought, cold, and high salinity) to reduce cellular damage. These intrinsically disordered proteins are thought to function by binding to proteins to prevent denaturation, and to membranes to prevent leakage. Here, we report the ¹H, ¹⁵N, and ¹³C chemical shift assignments of the K₂ and YSK₂ dehydrins from Vitis riparia (wild grape). Our results show that the segmental nature of dehydrins allows for the assignments of the shorter dehydrins to be used in that of the longer forms.     

Seasonal Changes in the Composition and Content of Dehydrins in Winter Wheat Plants

Seasonal changes in the pattern and content of dehydrins in winter wheat (Triticum aestivum) plants grown under field and laboratory conditions were studied by one-dimensional PAGE and immunochemical methods. During hardening, plants accumulated dehydrin-like polypeptides with mol wts of 209, 196, 66, 50, and 41 kD. In winter, low-molecular-weight dehydrins with mol wts of 24, 22, 17, 15, and 12 kD were synthesized and accumulated as well. Their content dropped sharply in spring when plants became unhardened. Accumulation/disappearance of these proteins corresponded to the fluctuations in wintering plant frost tolerance before winter and in spring. It is assumed that both high- and medium-molecular-weight dehydrins are involved in plant stress responses and adaptation, whereas low-molecular-weight dehydrins are evidently involved only in the process of low-temperature adaptation.     

Zinc induces disorder-to-order transitions in free and membrane-associated Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2: a solution CD and solid-state ATR-FTIR study

Dehydrins are intrinsically unstructured proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. Although their role is not completely understood, it has been suggested that they stabilize proteins and membrane structures during environmental stress and also sequester metals such as zinc. Here, we investigate two dehydrins (denoted as TsDHN-1 and TsDHN-2) from Thellungiella salsuginea. This plant is a crucifer that thrives in the Canadian sub-Arctic (Yukon Territory) where it grows on saline-rich soils and experiences periods of both extreme cold and drought. We show using circular dichroism and attenuated total reflection-Fourier transform infrared spectroscopy that ordered secondary structure is induced and stabilized in these proteins, both in free and vesicle-bound form, by association with zinc. In membrane-associated form, both proteins have an increased proportion of β-strand conformation induced by the cation, in addition to the amphipathic α-helices formed by their constituent K-segments. These results support the hypothesis that dehydrins stabilize plant plasma and organellar membranes in conditions of stress, and further that zinc may be an important co-factor in stabilization. Whereas dehydrins in the cytosol of a plant cell undergoing dehydration or temperature stress form bulk hydrogels and remain primarily disordered, dehydrins with specific membrane- or protein-associations will have induced ordered secondary structures.     

植物抗寒及其基因表达研究进展

植物经过逐渐降低的温度从而提高抗寒能力 ,这个过程被人们称为低温驯化。植物低温驯化过程是一个复杂的生理、生化和能量代谢变化过程 ,这些变化主要包括膜系统的稳定性、可溶性蛋白的积累和小分子渗透物质 ,比如脯氨酸、糖等 ,这些变化中的一些是植物抗寒必需的 ,而另外一些变化不是必需的。主要对冷害和低温生理生化变化、低温诱导表达基因的功能和作用、低温驯化的调节机制及其信号转导方面进行了综述。通过差别筛选 c DNA文库的方法已经鉴定了许多低温诱导表达、进而提高植物抗寒能力的基因 ,其中有脱水素、COR基因和 CBF1转录因子等。低温信号的感受、转导和调节表达是低温驯化的关键环节 ,低温信号的转导过程与干旱胁迫之间具有一定的交叉 ,这为利用 ABA等来提高植物抗寒能力成为可能 ,相信不久的将来人们可以通过提高植物抗寒能力从而增加经济产量成为现实。     

Plant resistance to cold stress: Mechanisms and environmental signals triggering frost hardening and dehardening

This introductory overview shows that cold, in particular frost, stresses a plant in manifold ways and that the plant’s response, being injurious or adaptive, must be considered a syndrome rather than a single reaction. In the course of the year perennial plants of the temperate climate zones undergo frost hardening in autumn and dehardening in spring. Using Scots pine (Pinus sylvestris L.) as a model plant the environmental signals inducing frost hardening and dehardening, respectively, were investigated. Over 2 years the changes in frost resistance of Scots pine needles were recorded together with the annual courses of day-length and ambient temperature. Both act as environmental signals for frost hardening and dehardening. Climate chamber experiments showed that short day-length as a signal triggering frost hardening could be replaced by irradiation with far red light, while red light inhibited hardening. The involvement of phytochrome as a signal receptor could be corroborated by respective night-break experiments. More rapid frost hardening than by short day or far red treatment was achieved by applying a short period (6 h) of mild frost which did not exceed the plant’s cold resistance. Both types of signals were independently effective but the rates of frost hardening were not additive. The maximal rate of hardening was − 0.93°C per day and frost tolerance of < − 72°C was achieved. For dehardening, temperature was an even more effective signal than day-length.     

Stress-induced accumulation and tissue-specific localization of dehydrins in Arabidopsis thaliana

Stress-induced accumulation of five (COR47, LTI29, ERD14, LTI30 and RAB18) and tissue localization of four (LTI29, ERD14, LTI30 and RAB18) dehydrins in Arabidopsis were characterized immunologically with protein-specific antibodies. The five dehydrins exhibited clear differences in their accumulation patterns in response to low temperature, ABA and salinity. ERD14 accumulated in unstressed plants, although the protein level was up-regulated by ABA, salinity and low temperature. LTI29 mainly accumulated in response to low temperature, but was also found in ABA- and salt-treated plants. LTI30 and COR47 accumulated primarily in response to low temperature, whereas RAB18 was only found in ABA-treated plants and was the only dehydrin in this study that accumulated in dry seeds.Immunohistochemical localization of LTI29, ERD14 and RAB18 demonstrated tissue and cell type specificity in unstressed plants. ERD14 was present in the vascular tissue and bordering parenchymal cells, LTI29 and ERD14 accumulated in the root tip, and RAB18 was localized to stomatal guard cells. LTI30 was not detected in unstressed plants. The localization of LTI29, ERD14 and RAB18 in stress-treated plants was not restricted to certain tissues or cell types. Instead these proteins accumulated in most cells, although cells within and surrounding the vascular tissue showed more intense staining. LTI30 accumulated primarily in vascular tissue and anthers of cold-treated plants.This study supports a physiological function for dehydrins in certain plant cells during optimal growth conditions and in most cell types during ABA or cold treatment. The differences in stress specificity and spatial distribution of dehydrins in Arabidopsis suggest a functional specialization for the members of this protein family.     

Expression of SK3-type dehydrin in transporting organs is associated with cold acclimation in Solanum species

The expression of a gene, encoding a dehydrin protein designated as DHN24 was analyzed at the protein level in two groups of Solanum species differing in cold acclimation ability. The DHN24 protein displays consensus amino acid sequences of dehydrins, termed K- and S-segments. The S-segment precedes three K-segments, classifying the protein into SK₃-type dehydrins. A group of Solanum species able to cold acclimation constituted by S. sogarandinum and S. tuberosum, cv. Aster, and a second one composed of a S. sogarandinum line, that lost ability to cold acclimation, and of S. tuberosum, cv. Irga, displaying low ability to cold acclimation were studied. Under control conditions, noticeable levels of the DHN24 protein was observed in stems, tubers, and roots of Solanum species. No protein was detected in leaves. During low temperature treatment the DHN24 protein level substantially increased in tubers, in transporting organs and in apical parts, and only a small increase was observed in leaves. The increase in protein abundance was only observed in the plants able to cold acclimate and was found to parallel the acclimation capacity. Upon drought stress, the DHN24 level decreased in stems and in leaves, but increased in apical parts. These results suggest that Dhn24 expression is regulated by organ specific factors in the absence of stress and by factors related to cold acclimation processes during low temperature treatment in collaboration with organ-specific factors. A putative function of the SK₃-type dehydrin proteins during plant growth and in the tolerance to low temperature is discussed.     

Dehydrins associated with the development of frost resistance of Asian white birch

Seasonal changes in the content of dehydrins in Asian white birch (Betula platyphylla Sukacz.) growing under extreme cold conditions of Eastern Siberia (Central Yakutia) were studied for the first time by SDS-PAGE and immunoblotting. Several polypeptides, including putative storage proteins, which content was higher in winter than in other periods, were observed. Intraspecies polymorphism of dehydrins was detected during plant dormancy. The two groups of dehydrins were found: dehydrins with mol wts of 56-73 kD, which were present year-round, and dehydrins with mol wts of 15–21 kD, evidently related to the development of frost resistance because they were absent in summer but present in large amounts in winter. Under low winter temperatures, the highest level of dehydrins coincided with the lowest content of water in buds, which was accompanied by increased plant frost resistance to the highest values.     

A dehydrin cognate protein from pea (Pisum sativum L.) with an atypical pattern of expression

Dehydrins are a family of proteins characterised by conserved amino acid motifs, and induced in plants by dehydration or treatment with ABA. An antiserum was raised against a synthetic oligopeptide based on the most highly conserved dehydrin amino acid motif, the lysine-rich block (core sequence KIKEK-LPG). This antiserum detected a novel M _r 40 000 polypeptide and enabled isolation of a corresponding cDNA clone, pPsB61 (B61). The deduced amino acid sequence contained two lysine-rich blocks, however the remainder of the sequence differed markedly from other pea dehydrins. Surprisingly, the sequence contained a stretch of serine residues, a characteristic common to dehydrins from many plant species but which is missing in pea dehydrin.The expression patterns of B61 mRNA and polypeptide were distinctively different from those of the pea dehydrins during seed development, germination and in young seedlings exposed to dehydration stress or treated with ABA. In particular, dehydration stress led to slightly reduced levels of B61 RNA, and ABA application to young seedlings had no marked effect on its abundance.The M _r 40 000 polypeptide is thus related to pea dehydrin by the presence of the most highly conserved amino acid sequence motifs, but lacks the characteristic expression pattern of dehydrin. By analogy with heat shock cognate proteins we refer to this protein as a dehydrin cognate.     

Differential accumulation of dehydrin-like proteins by abiotic stresses in Deschampsia antarctica Desv.

Dehydrins are proteins that accumulate during environmental stresses leading to cell dehydration. Deschampsia antarctica is one of the two vascular plants that have colonized the Maritime Antarctic. This plant is usually exposed to cold, salt and desiccating winds in the field. We proposed that among the factors that allow D. antarctica to survive the harsh environmental conditions is the presence of dehydrins. We studied the accumulation of dehydrins by abscisic acid (ABA), dehydration, NaCl and low osmotic potential. Western blots using an anti-dehydrin antibody revealed a complex pattern of dehydrin-like proteins (DLPs) accumulation in the different treatments. DLPs with apparent molecular weight of 58, 57, 55, 53, 48, 42, 32, 30, 28 and 25 kDa were detected in the different treatments. DLPs accumulation was associated with a decrease in the relative water content (RWC) of the plants. These results suggest that DLPs accumulation could contribute to explain how D. antarctica can survive under adverse Antarctic conditions.     

CBL1, a calcium sensor that differentially regulates salt,drought, and cold responses in Arabidopsis

Although calcium is a critical component in the signal transduction pathways that lead to stress gene expression in higher plants, little is known about the molecular mechanism underlying calcium function. It is believed that cellular calcium changes are perceived by sensor molecules, including calcium binding proteins. The calcineurin B-like (CBL) protein family represents a unique group of calcium sensors in plants. A member of the family, CBL1, is highly inducible by multiple stress signals, implicating CBL1 in stress response pathways. When the CBL1 protein level was increased in transgenic Arabidopsis plants, it altered the stress response pathways in these plants. Although drought-induced gene expression was enhanced, gene induction by cold was inhibited. In addition, CBL1-overexpressing plants showed enhanced tolerance to salt and drought but reduced tolerance to freezing. By contrast, cbl1 null mutant plants showed enhanced cold induction and reduced drought induction of stress genes. The mutant plants displayed less tolerance to salt and drought but enhanced tolerance to freezing. These studies suggest that CBL1 functions as a positive regulator of salt and drought responses and a negative regulator of cold response in plants.     

Diverse accumulation of several dehydrin-like proteins in cauliflower (Brassica oleracea var. botrytis), Arabidopsis thaliana and yellow lupin (Lupinus luteus) mitochondria under cold and heat stress

Background  

Dehydrins represent hydrophilic proteins acting mainly during cell dehydration and stress response. Dehydrins are generally thermostable; however, the so-called dehydrin-like (dehydrin-related) proteins show variable thermolability. Both groups immunoreact with antibodies directed against the K-segment of dehydrins. Plant mitochondrial dehydrin-like proteins are poorly characterized. The purpose of this study was to extend previous reports on plant dehydrins by comparing the level of immunoprecipitated dehydrin-like proteins in cauliflower (Brassica oleracea var. botrytis), Arabidopsis thaliana and yellow lupin (Lupinus luteus) mitochondria under cold and heat stress.     

Interaction of Temperature and Light in the Development of Freezing Tolerance in Plants

Freezing tolerance is the result of a wide range of physical and biochemical processes, such as the induction of antifreeze proteins, changes in membrane composition, the accumulation of osmoprotectants, and changes in the redox status, which allow plants to function at low temperatures. Even in frost-tolerant species, a certain period of growth at low but nonfreezing temperatures, known as frost or cold hardening, is required for the development of a high level of frost hardiness. It has long been known that frost hardening at low temperature under low light intensity is much less effective than under normal light conditions; it has also been shown that elevated light intensity at normal temperatures may partly replace the cold-hardening period. Earlier results indicated that cold acclimation reflects a response to a chloroplastic redox signal while the effects of excitation pressure extend beyond photosynthetic acclimation, influencing plant morphology and the expression of certain nuclear genes involved in cold acclimation. Recent results have shown that not only are parameters closely linked to the photosynthetic electron transport processes affected by light during hardening at low temperature, but light may also have an influence on the expression level of several other cold-related genes; several cold-acclimation processes can function efficiently only in the presence of light. The present review provides an overview of mechanisms that may explain how light improves the freezing tolerance of plants during the cold-hardening period.     

蒙古沙冬青AmRD22基因的克隆及耐逆功能研究

脱水应答蛋白22(RD22)属于植物特有的BURP蛋白家族中的一个亚族,与耐逆性关系密切。该研究从中国西北荒漠区特有的强耐逆植物蒙古沙冬青克隆到一个RD22基因(AmRD22)的全长cDNA,并对其编码蛋白、表达模式和耐逆功能进行了研究。结果表明：(1)AmRD22蛋白(360 aa)的初级结构中含有RD22亚族共有的4个结构域,预测其定位于细胞壁;在功能已知的RD22蛋白中,AmRD22与大豆GmRD22的进化关系最近。(2)在室内培养的蒙古沙冬青幼苗中,AmRD22的表达受失水、高盐、低温和ABA胁迫的诱导显著上调,其中失水和低温胁迫诱导其上调幅度较大;在野外生长的蒙古沙冬青植株嫩叶中,其表达量从中秋至隆冬远高于其他季节。(3)转AmRD22基因拟南芥的耐盐性显著提高且Na⁺含量降低,其耐旱性也有较明显的改善且在种子萌发早期对外源ABA的敏感性降低,但耐冷性和耐冻性无明显变化。     

The Plant Dehydrins: Structure and Putative Functions

This review deals with recent data on the structure and biochemical properties of dehydrins, proteins that are normally synthesized in maturating seeds during their desiccation, and also in vegetative tissues of plants treated with abscisic acid or exposed to environmental stress factors that result in cellular dehydration. The dehydrins are considered as stress proteins involved in formation of plant protective reactions against dehydration. The generally accepted classification of dehydrins is based on their structural features, such as the presence of conserved sequences, designated as Y-, S-, and K-segments. The K-segment representing a highly conserved 15 amino acid motif (EKKGIMDKIKEKLPG) forming amphiphilic -helix has been found in all dehydrins. The pathways of regulation of dehydrin gene expression, putative functions of dehydrins, and molecular mechanisms of their actions are discussed.     

Drought survival,summer dormancy and dehydrin accumulation in contrasting cultivars of Dactylis glomerata

To study survival under prolonged and severe drought in the perennial grass Dactylis glomerata we compared dormant, resistant and sensitive cultivars (cvs.) in both field and glasshouse experiments. Water status, membrane stability and expression of dehydrins were assessed in the immature leaf bases, which are the last surviving organs. Analysis of leaf elongation and senescence of aerial tissues showed that dormancy was exhibited by the potentially dormant cultivar (cv.) only in the field. This cultivar exhibited a high survival rate, similar levels of dehydration and expression of a low-molecular weight (22–24 kDa) dehydrin in both drought and irrigated plants, whether fully dormant or not. At the same level of soil water deficit, there were no differences between the non-dormant drought resistant and drought sensitive cultivars in plant water status and membrane stability. However, the accumulation of dehydrins as drought progressed was markedly different between these cultivars and was associated with their contrasting survival. The possible role of the major low-molecular dehydrins in maintenance of cell integrity under dehydration is discussed with reference to both summer dormancy and survival under severe drought.     

Dormancy development during cold hardening of in vitro cultured Malus domestica Borkh. plants in relation to their frost resistance and cryotolerance

The development of dormancy, frost resistance and cryotolerance of in vitro apple plants (Malus domestica Borkh.), cv. Greensleeves during their exposure to cold hardening was studied. In vitro cultures were cold hardened at 4°C under a short photoperiod up to 25?weeks. The dormancy status, non-structural saccharides, proline, water content and frost resistance were evaluated for optimization of cryopreservation. According to regrowth tests, in vitro cultures exhibited endogenous dormancy after the maximal frost resistance was reached. The highest regeneration ability of shoot tips after cryopreservation by encapsulation–dehydration method coincided with the period of the plant’s dormant state and maximum of frost resistance. All studied saccharides and proline exhibited the maximal values at the beginning of cold hardening and/or the dormancy phase. Contrary to the accumulation of saccharides and proline, water content showed the inverse time behaviour. According to these results, the cold hardening-induced endodormancy, high frost resistance and accumulation of saccharides and proline are the important prerequisites for the successful cryopreservation of shoot tips of in vitro grown apple plants.     

Cryoprotective mechanism of a small intrinsically disordered dehydrin protein

Dehydration proteins (Dehydrins) are expressed during dehydration stress in plants and are thought to protect plant proteins and membranes from the loss of water during drought and at cold temperatures. Several different dehydrins have been shown to protect lactate dehydrogenase (LDH) from damage from being frozen and thawed. We show here that a 48 residue K₂ dehydrin from Vitis riparia protects LDH more effectively than bovine serum albumin, a protein with known cryoprotective function. Light scattering and 8‐anilino‐1‐naphthalene sulfonate fluorescence experiments show that dehydrins prevent aggregation and unfolding of the enzyme. The cryoprotective effects of LDH are reduced by the addition of salt, suggesting that the positively charged K‐segments are attracted to a negatively charged surface but this does not result in binding. Overall K₂ is an intrinsically disordered protein; nuclear magnetic resonance relaxation experiments indicate that the two‐terminal, Lys‐rich K‐segments show a weak propensity for α‐helicity and are flexible, and that the central, polar rich phi‐segment has no secondary structure preference and is highly flexible. We propose that the phi‐segments in dehydrins are important for maintaining the disordered structure so that the protein can act as a molecular shield to prevent partially denatured proteins from interacting with one another, whereas the K‐segments may help to localize the dehydrin near the enzyme surface.