Cold-treated Plants Research Articles

UVH6, a Plant Homolog of the Human/Yeast TFIIH Transcription Factor Subunit XPD/RAD3, Regulates Cold-stress Genes in Arabidopsis thaliana

The UVH6 gene from Arabidopsis thaliana is predicted to function in transcription regulation, based on known activities of its human and yeast homologs. In this study, we show that uvh6-1 mutants are ultra-sensitive to cold and suggest that this defect results from reduced expression of cold-stress genes. Comparison of mRNA levels in cold-treated wild-type and uvh6-1 plants reveals that expression of two cold-stress genes (Cor6.6 and Cor15a) is impaired in the mutant. In contrast, the mutant shows normal cold induction of three transcription factor genes (CBF1, 2, 3), which regulate these Cor genes, and normal induction of several additional CBF-targeted genes. Thus, we propose that UVH6 promotes cold resistance by specifically regulating transcription of Cor6.6 and Cor15a genes. We further find features among the regulatory sites in the Cor6.6 and Cor15a promoters which suggest unique regulation of these genes.

Gene expression and genetic mapping analyses of a perennial ryegrass glycine-rich RNA-binding protein gene suggest a role in cold adaptation

A perennial ryegrass cDNA clone encoding a putative glycine-rich RNA binding protein (LpGRP1) was isolated from a cDNA library constructed from crown tissues of cold-treated plants. The deduced polypeptide sequence consists of 107 amino acids with a single N-terminal RNA recognition motif (RRM) and a single C-terminal glycine-rich domain. The sequence showed extensive homology to glycine-rich RNA binding proteins previously identified in other plant species. LpGRP1-specific genomic DNA sequence was isolated by an inverse PCR amplification. A single intron which shows conserved locations in plant genes was detected between the sequence motifs encoding RNP-1 and RNP-2 consensus protein domains. A significant increase in the mRNA level of LpGRP1 was detected in root, crown and leaf tissues during the treatment of plants at 4 degrees C, through which freezing tolerance is attained. The increase in the mRNA level was prominent at least 2 h after the commencement of the cold treatment, and persisted for at least 1 week. Changes in mRNA level induced by cold treatment were more obvious than those due to treatments with abscisic acid (ABA) and drought. The LpGRP1 protein was found to localise in the nucleus in onion epidermal cells, suggesting that it may be involved in pre-mRNA processing. The LpGRP1 gene locus was mapped to linkage group 2. Possible roles for the LpGRP1 protein in adaptation to cold environments are discussed.

Winter disruption of the circadian clock in chestnut

Circadian clock performance during winter dormancy has been investigated in chestnut by using as marker genes CsTOC1 and CsLHY, which are homologous to essential components of the central circadian oscillator in Arabidopsis. During vegetative growth, mRNA levels of these two genes in chestnut seedlings and adult plants cycled daily, as expected. However, during winter dormancy, CsTOC1 and CsLHY mRNA levels were high and did not oscillate, indicating that the circadian clock was altered. A similar disruption was induced by chilling chestnut seedlings (to 4 degrees C). Normal cycling resumed when endodormant or cold-treated plants were returned to 22 degrees C. The behavior of CsTOC1 and CsLHY during a cold response reveals a relevant aspect of clock regulation not yet encountered in Arabidopsis.

Chilling stress suppresses chloroplast development and nuclear gene expression in leaves of mung bean seedlings

Etiolated leaves of 28 degrees C-dark-grown mung bean (Vigna radiata L. cv. 2937) seedlings fail to turn green after being shifted to a light and cold environment. At the visible phenotypic level, incapability of leaf greening is the only failure event for the de-etiolation of mung bean seedlings at low temperature. Ultrastructural studies revealed that chloroplast development was completely suppressed by chilling treatment. A cDNA library originating from 28 degrees C-light-grown seedling leaves was constructed for screening cold-suppressed (cos) genes. Thirteen full-length cDNA clones were obtained, with 12 clones encoding chloroplast proteins, which, according to their known physiological functions, were important for chloroplast development and photosynthesis. Another cos cDNA encodes CYP90A2, which is a cytochrome P450 protein involved in the biosynthesis of brassinosteroid hormones. All cos genes are light-regulated at normal temperature. The influence of chilling stress on cos expression was examined in 10 degrees C-light- and 10 degrees C-dark-grown etiolated seedlings, and in 10 degrees C-light-grown green plants. The data show that cos expression in these three treatments is severely suppressed. This suppression is controlled at the transcriptional level, as demonstrated by nuclear runoff experiments, and is reversible because cos mRNAs accumulate again after the cold-treated plants have been transferred to 28 degrees C.

Isolation of a novel barley cDNA encoding a nuclear protein involved in stress response and leaf senescence.

In order to isolate genes involved in the early acclimation of winter barley (Hordeum vulgare L. cv. Trixi) to a combined cold and light stress of 2 degrees C and 600 micromol m(-2) s(-1) restriction fragment differential display-polymerase chain reaction was performed. Impact of the cold-treatment on the leaves was characterized by measuring chlorophyll content and photosystem II efficiency. By this approach several cDNAs of genes that quickly and transiently up-regulated during early stages of the stress were identified. One of these genes (HvFP1) includes sequence motifs representing a heavy metal associated domain (HMA), nuclear localization signals (NLS) and a farnesylation motif. This gene is also induced at drought stress, during leaf senescence and after exposure to abscisic acid. Analysis of its spatial expression patterns in barley plants either grown at 21 or 2 degrees C showed that in contrast to the situation in leaves transcript level of this gene is high not only in cold-treated plants but also in controls kept at 21 degrees C in plant compartments enriched in meristematic tissues. The nuclear localization of the protein was confirmed by confocal laser scanning microscopy of epidermal onion cells after particle bombardment with chimeric HVFP1-GFP constructs. Using a construct with a modified farnesylation motif yielded a different pattern of nuclear distribution of the chimeric protein.

Regulation of phosphatidylcholine biosynthesis under salt stress involves choline kinases in Arabidopsis thaliana

Increasing evidence suggests a major role for phosphatidylcholine (PC) in plant stress adaptation. The present work investigated the regulation of choline, PC and interconnected phosphatidylethanolamine biosynthesis in Arabidopsis thaliana L. as a function of cold- and salt- or mannitol-mediated hyperosmotic stresses. While PC synthesis is accelerated in both salt- and cold-treated plants, the choline kinase (CK) and phosphocholine cytidylyltransferase genes are oppositely regulated with respect to these abiotic treatments. Salt stress also stimulates CK activity in vitro. A possible regulatory role of CK in stimulating PC biosynthesis rate in salt-stressed plants is discussed.

Chilling tolerance of potato plants transformed with a yeast-derived invertase gene under the control of the B33 patatin promoter

Tolerance to chilling was compared under in vitro conditions in potato plants ( Solanum tuberosum L., cv. Desiree) transformed with a yeast-derived invertase gene under the control of the B33 class 1 tuber-spe- cific promoter (the B33-inv plants) and potato plants transformed only with a reporter gene (the control plants). The expression of the inserted yeast invertase gene was proved by following the acid and alkaline invertase activities and sugar contents in the leaves under the optimum temperature (22 ° C). The total activities of acid and alkaline invertases in the B33-inv plants exceeded those in the control plants by the factors of 2-3 and 1.3, respectively. In the B33-inv plants, the activity of acid invertase twice exceeded that of the alkaline invertase, whereas the difference equaled 12% in the control plants. The contents of sucrose and glucose increased in the B33-inv plants by 21 and 13%, respectively, as compared to the control. Chilling at +3 and -1 ° C for 1, 3, and 6 h did not affect the rate of lipid peroxidation, as measured by the content of malonic dialdehyde (MDA) in the leaves of the genotypes under study. Only the longer exposures (24 h at +3 and -1 ° C and 7 days at +5 ° C) produced a significant decline in the MDA content in the B33-inv plants, as compared to the control. Following short freezing (20 min at -9 ° C), the content of MDA increased by 50% in the leaves of the control plants, while in the B33-inv plants, cold-treated and control plants did not differ in the MDA content. The authors presume that the potato plants transformed with the yeast invertase gene acquire a higher tolerance to low temperatures as compared to the control plants, apparently due to the changes in sugar ratio produced by the foreign invertase.

Cold stress decreases the capacity for respiratory NADH oxidation in potato leaves

Cold stress effects on the expression of genes for respiratory chain enzymes were investigated in potato ( Solanum tuberosum L., cv. Desiree) leaves. The nda1 and ndb1 genes, homologues to genes encoding the non-proton-pumping respiratory chain NADH dehydrogenases of Escherichia coli and yeast, were compared to genes encoding catalytic subunits of the proton-pumping NADH dehydrogenase (complex I). Using a real-time PCR system, we demonstrate a specific and gradual decrease of the NDA1 transcript after exposing the plants to 5°C. After 6 days of cold treatment the NDA1 transcript abundance is 10% of the original level. This decrease is accompanied by specific decreases of immunodetected NDA protein and internal rotenone-insensitive NADH oxidation in mitochondria isolated from cold-treated plants. The alternative oxidase is not cold-induced neither at the protein nor at the activity level. The results are discussed in relation to the recent finding that the nda1 gene expression is completely light-dependent.

A rice membrane-bound calcium-dependent protein kinase is activated in response to low temperature.

Calcium-dependent protein kinases (CDPKs) are found in various subcellular localizations, which suggests that this family of serine/threonine kinases may be involved in multiple signal transduction pathways. CDPKs are believed to be involved in the response of plants to low temperatures, but the precise role in the signal transduction pathway is largely unknown. Previous reports described changes in CDPKs' mRNA levels in response to cold treatment, but whether these changes are accompanied by increases in protein level and/or kinase activities is unknown. In the present study, we identify in rice (Oryza sativa L. cv Don Juan) plants a 56-kD membrane-bound CDPK that is activated in response to cold treatment. Immunoblot analysis of the enzyme preparations from control and cold-treated plants showed that the kinase level was similar in both preparations. However, both kinase and autophosphorylating activities of the enzyme prepared from cold-treated plants were significantly higher than that obtained from control plants. The activation of the CDPK is detected after 12 to 18 h of cold treatment, which indicates that the kinase does not participate in the initial response to low temperature but in the adaptative process to adverse conditions. To our knowledge, this is the first demonstration of a CDPK that is posttranscriptionally activated in response to low temperature.

Low temperature treatment advances flowering in Clivia miniata (Lindley) Regel

Clivia miniata plants were exposed to a cold treatment of 7.5–10 °C for 14 days in the dark from 24 April 2000. Compared to the non-cold treated control plants, the cold-treated plants started flowering four weeks earlier resulting in 80% of the cold treated plants being ready for sale before or during the first week in August as compared to 76% of the control plants that were marketable by the last week of August.

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.

Effect of Low Temperature on the Protein Metabolism of Wheat Leaves

The effect of low temperature on the protein metabolism of wheat primary leaves was examined. In seedlings transferred from 25 to 5 °C, total soluble protein accumulation, in vivo protein synthesis and breakdown, in vitro protein breakdown, and SDS-PAGE profiles of proteinases in gelatine-containing gels were analysed. Leaf protein content increased within a 7-d period (70 % over the initial value) in plants exposed to 5 °C. The fast protein accumulation observed on days 0 – 2 was mainly attributed to a decreased breakdown. In further days, parallelly to a slowdown in the rate of protein accumulation, the leaf proteolytic activity increased. The incubation temperature also had an influence on the proteolytic activity: Q 10 values for the 15 – 5 °C range were 80 – 200 % higher than those observed for the 25 – 15 °C range. On the other hand, the in vivo protein synthesis capacity, at either 25 or 55 °C, was not significantly modified in cold-treated plants. In addition to the enhanced activities of two serine-proteinases (previously found in control plants by SDS-PAGE analysis), cold-treated plants displayed a new proteinase, which had not been detected so far.

Paraquat Resistance of Horseweed (Erigeron canadensis L.) Is Not Caused by Polyamines

Abstract The correlation between polyamine content and paraquat resistance in horseweed ( Erigeron canadensis L.) was investigated. Untreated paraquat-resistant and paraquat/atrazine-coresistant plants contained about two to three times higher amounts of polyamines, especially putrescine, compared to sensitive ones. A marked increase of polyamine content was detected after paraquat treatment in each biotype which decreased to the initial level in 6 h. Functional activity (characterized by variable fluorescence) of sensitive plants continuously decreased after paraquat treatment, while resistant biotypes recovered their activities after a transitory inhibition. Changes in polyamine content did not correlate with the changes in functional activity. Exogenously added 10 −4 mol L −1 putrescine combined with 10 −5 mol L −1 paraquat in detached sensitive leaves showed a slight protective effect, while paraquat-resistant and paraquat/atrazine-coresistant leaves remained unaffected. The polyamine level of the cycloheximide-treated or long-term cold-treated plants also changed independently from their functional activity. It can be concluded that the higher polyamine level of the treated resistant leaves and the increase of the polyamine content after paraquat treatment seem to be a general stress response rather than the specific reason for paraquat resistance.

Sensing Iron—A Whole Plant Approach

Regulatory mechanisms leading to cellular Fe homeostasis were investigated inPlantago (Plantago lanceolata L.) plants grown hydroponically at different temperature regimes either in the presence or absence of iron. During the experimental period of 6d, growth was not affected by Fe availability, but was decreased by lowering the root zone temperature (RZT) from 24 to 12°C. Cultivating plants at low RZT decreased the reduction activity for ferric chelates in Fe-deficient plants. In the presence of iron, the temperature regime did not affect Fe accumulation by root cells, but decreased translocation of Fe to the shoot, and chlorosis of young leaves was observed at suboptimal RZT. Under these conditions root-mediated reduction of ferric chelates was increased. In cold-treated plants this effect was specific to Fe and could not be evoked by Mn2+and Zn+2additions. Supplementing the medium with the ferrous scavenger ferrozine caused a further enhancement in reduction rates, probably due to mobilization of apoplastic Fe. These results can be explained plausibly if different sites of Fe sensing are postulated and if it is assumed that both the absence and presence of iron could be a signal increasing root reduction activity.

Accumulation of Dehydrin-like-proteins in the Mitochondria of Cold-treated Plants

Summary A number of proteins have been found to accumulate in plants during cold tolerance. One class of cold proteins that is associated with cold tolerance acclimation is the glycine-rich, hydrophilic, D-II LEA (late embryogenesis abundant) proteins known as dehydrins. Several members of the dehydrin-class of proteins are known to associate with the nucleus, in the cytosol, and with the plasma membrane. Dehydrins that localise with the mitochondria have not been found. We provide evidence that two dehydrin-like proteins (dlps) accumulate in the mitochondria only after low temperature treatment. We also provide evidence for a positive correlation between the relative accumulation of these proteins in the mitochondria in response to cold stress and relative cold tolerance of several species of plants.

Wild and cultivated barleys show differences in the expression pattern of a cold-regulated gene family under different light and temperature conditions.

Cold acclimation in plants involves the expression of many genes and gene families. The present study reports the expression analysis of three members of the blt14 gene family in barley. Gene-specific antisense oligonucleotides were used as probes in northern experiments so as to follow independently the expression of individual members of the gene family. Each clone revealed different accumulation kinetics when a spring and a winter cultivar were compared, suggesting that the different regulatory mechanisms leading to mRNA accumulation of an individual member of the blt14 gene family are genotype-dependent. In a collection of Hordeum spontaneum genotypes both qualitative and quantitative polymorphisms were found for the accumulation of blt14-related mRNAs, although no clear relationships were found between blt14 expression and frost resistance. The accumulation of the blt14-related mRNAs was also modulated by light and by the albino mutation a(n). The effects of light on the accumulation of the transcripts corresponding to the blt14 gene family were evaluated by comparing etiolated and green plants. Etiolated plants accumulate the blt14-related mRNAs at a detectable level already at 22 degrees C. When the same plants are exposed to cold in absence of light an increased mRNA accumulation above the level present in green cold-treated plants can be detected. On the contrary, etiolated plants showed a reduced blt14 accumulation when exposed to cold in the presence of light. Cold-induced expression of the blt14 gene family was strongly reduced in plants carrying the albino mutation a(n). This mutant showed a defective molecular response to cold even when probed with a cDNA coding for LEA type protein (paf93). The albino mutant a(n) was not able to harden when exposed to low temperature providing a direct evidence of the relationship between expression of cold-regulated (COR) genes and the development of cold hardening. Failure of cold acclimation in the mutant cannot be merely ascribed to the absence of photosynthetic activity, since etiolated wild-type plants accumulated COR mRNAs and improved frost resistance when exposed to cold.

Stress signaling in plants: a mitogen-activated protein kinase pathway is activated by cold and drought.

Yeast and animals use mitogen-activated protein (MAP) kinase cascades to mediate stress and extracellular signals. We have tested whether MAP kinases are involved in mediating environmental stress responses in plants. Using specific peptide antibodies that were raised against different alfalfa MAP kinases, we found exclusive activation of p44MMK4 kinase in drought- and cold-treated plants. p44MMK4 kinase was transiently activated by these treatments and was correlated with a shift in the electrophoretic mobility of the p44MMK4 protein. Although transcript levels of the MMK4 gene accumulated after drought and cold treatment, no changes in p44MMK4 steady state protein levels were observed, indicating a posttranslational activation mechanism. Extreme temperatures, drought, and salt stress are considered to be different forms of osmotic stress. However, high salt concentrations or heat shock did not induce activation of p44MMK4, indicating the existence of distinct mechanisms to mediate different stresses in alfalfa. Stress adaptation in plants is mediated by abscisic acid (ABA)-dependent and ABA-independent processes. Although ABA rapidly induced the transcription of an ABA-inducible marker gene, MMK4 transcript levels did not increase and p44MMK4 kinase was not activated. These data indicate that the MMK4 kinase pathway mediates drought and cold signaling independently of ABA.

Effects of Chilling on Chloroplast Development in Barley Primary Foliage Leaves

Summary Effects of chilling on chloroplast development were investigated in apical sections of barley primary foliage leaves. During chloroplast development at 23 °C photosynthetic capacity per unit chlorophyll decreases while PSII efficiency and chlorophyll content increase until day 8 after sowing. Thereafter, constant values are reached. Cold treatment at 5 °C retards the decrease in photosynthetic capacity per unit chlorophyll and the increase in total chlorophyll content occurring in the first phase of chloroplast development. PSII efficiency is affected even more drastically: it slightly decreases during the first day after transfer to 5 °C and then stays constant for at least 7 further days. Due to these impacts on chloroplast development, cold-treated chloroplasts exhibit a higher photosynthetic capacity per unit chlorophyll, a smaller chlorophyll content per fresh weight and a decreased efficiency of PSII centers. Besides the retardation of developmental processes, two specific impacts of cold treatment on the photosynthetic apparatus have been observed. On the one hand, immunological analysis of PSI and PSII content revealed a decrease in the level of PSI centers after exposure to 5 °C. Spectrophotometric analysis confirmed this result, showing that the PSIIIPSI ratio is clearly increased after cold treatment. On the other hand, immunological analysis also revealed a distinct reduction in the amount of CP 29, which plays an important role in energy transfer to PSII centers. This reduction correlates with the low efficiency of PSII centers in cold-treated plants. The results demonstrate that besides retarding chloroplast development chilling has a specific impact on the organization of the photosynthetic apparatus.

The 5'-region of Arabidopsis thaliana cor15a has cis-acting elements that confer cold-, drought- and ABA-regulated gene expression.

Previous nuclear run-on experiments indicated that the cor15a (cold-regulated) gene of Arabidopsis thaliana L. (Heyn) has a cold-inducible promoter (Hajela et al., Plant Physiol 93: 1246-1252, 1990). The data presented here indicate that the 5' region of cor15a between nucleotides -305 and +78 (relative to the start of transcription) contains a cis-acting element(s) that can impart cold-regulated gene expression. Histochemical staining experiments indicated that the cor15a promoter is inactive, or very weakly active, in most of the tissues and organs of plants grown at normal temperature and that it becomes activated throughout most of the plant in response to low temperature. Notable exceptions to this general pattern include constitutive activity of the promoter in anthers of control grown plants and apparent inactivity of the promoter in the roots and ovaries of cold-treated plants. Histochemical staining experiments also indicated that low temperature regulation of cor15a does not involve the synthesis of a regulatory molecule that can spread throughout the plant and induce cor gene expression at normal growth temperature. Finally, gene fusion experiments indicated that the 5' region of cor15a between nucleotides -305 and +78, in addition to imparting cold-regulated gene expression, can impart ABA- and drought-regulated gene expression.

Induction ofent-kaurene biosynthesis by low temperature in dwarf peas

Germinating pea (Pisum sativum L.) seeds of two dwarf cultivars, “Progress No. 9” and “Green Arrow”, and two tall cultivars, “Alaska” and “Alderman”, were treated with low temperature (3–5°C) for 14 days and then transferred to normal growing conditions (19–21°C for 16 h/14.5–16.5°C for 8 h) for an additional 10 days. Biosynthesis of [14C]ent-kaurene from [14C]2-mevalonic acid (2-MVA) was assayed in cell-free enzyme extracts prepared from shoot tips 10 days after cold treatment and was compared with activity in enzyme extracts prepared from noncold-treated, 10-day-old control plants. Shoot lengths of cold-treated plants were measured throughout a 35-day period and compared with shoot lengths of plants grown without cold treatment for 25–35 days. Low temperature induced a five-to 10-fold enhancement ofent-kaurene, hence potentially gibberellin (GA), biosynthesis in seedlings of the two dwarf cultivars but not in the tall cultivars. However, the lack of an increase in growth rate in the cold-treated dwarfs indicated that endogenous GA biosynthesis remained blocked at some point beyondent-kaurene in the biosynthetic pathway. Since the late-flowering “Alderman” cultivar did not exhibit enhanced biosynthesis ofent-kaurene, it appears that if vernalization in late-flowering cultivars of peas is correlated with enhanced GA biosynthesis, it is not the early part of the biosynthetic pathway which is affected.