Separate signal pathways regulate the expression of a low-temperature-induced gene in Arabidopsis thaliana (L.) Heynh.

A cDNA clone corresponding to a novel low-temperature-induced Arabidopsis thaliana gene, named lti140, was employed for studies of the environmental signals and the signal pathways involved in cold-induced gene expression. The single-copy lti140 gene encodes a 140 kDa cold acclimation-related polypeptide. The lti140 mRNA accumulates rapidly in both leaves and roots when plants are subject to low temperature or water stress or are treated with the plant hormone abscisic acid (ABA), but not by heat-shock treatment. The low-temperature induction of lti140 is not mediated by ABA, as shown by normal induction of the lti140 mRNA in both ABA-deficient and ABA-insensitive mutants and after treatment with the ABA biosynthesis inhibitor fluridone. The effects of low temperature and exogenously added ABA are not cumulative suggesting that these two pathways converge. The induction by ABA is abolished in the ABA-insensitive mutant abi-1 indicating that the abi-1 mutation defines a component in the ABA response pathway. Accumulation of the lti140 mRNA in plants exposed to water stress was somewhat reduced by treatment with fluridone and in the ABA-insensitive mutant abi-1 suggesting that the water stress induction of lti140 could be partly mediated by ABA. It is concluded that three separate but converging signal pathways regulate the expression of the lti140 gene.     

The expression of a rab-related gene,rab 18, is induced by abscisic acid during the cold acclimation process of Arabidopsis thaliana (L.) Heynh

Erratum

The expression of a rab-related gene, rab 18, is induced by abscisic acid during the cold acclimation process of Arabidopsis thaliana (L.) Heynh     

Differential expression of two related,low-temperature-induced genes in Arabidopsis thaliana (L.) Heynh

Plant cold acclimation is correlated to expression of low-temperature-induced (lti) genes. By using a previously characterized lti cDNA clone as a probe we isolated a genomic fragment that carried two closely located lti genes of Arabidopsis thaliana. The genes were structurally related with the coding regions interrupted by three similarly located short introns and were transcribed in the same direction. The nucleotide sequences of the two genes, lti78 and lti65, predict novel hydrophilic polypeptides with molecular weights of 77856 and 64510, respectively, lti78 corresponding to the cDNA probe. Of the 710 amino acids of LTI78 and 600 amino acids of LTI65, 346 amino acids were identical between the polypeptides, which suggests that the genes may have a common origin.Both lti78 and lti65 were induced by low temperature, exogenous abscisic acid (ABA) and drought, but the responsiveness of the genes to these stimuli was markedly different. Both the levels and the temporal pattern of expression differed between the genes. Expression of lti78 was mainly responsive to low temperature, that of lti65 to drought and ABA. In contrast to the induction of lti78, which follows separate signal pathways during low-temperature, ABA and drought treatment, the drought induction of lti65 is ABA-dependent and the low-temperature induction appears to be coupled to the ABA biosynthetic pathway. This differential expression of two related genes may indicate that they have some-what different roles in the stress response.     

Low temperature acclimation and treatment with exogenous abscisic acid induce common polypeptides in Arabidopsis thaliana (L.) Heynh

Summary Exogenously applied abscisic acid (ABA) induced frost hardening of Arabidopsis thaliana (L.) Heynh. The freezing tolerance of A. thaliana plantlets treated with ABA (15 mg/l) at a non-acclimating temperature (20 °C) appeared to increase even more rapidly than following a low temperature (4 °C) acclimation. Analysis of in vivo-labelled soluble proteins by two-dimensional gel electrophoresis revealed several low temperature — or ABA — induced proteins, which where not produced in non-acclimated plants. A subset of these proteins was induced by both low temperature and ABA treatments, suggesting that they might be directly involved in the frost hardening process in A. thaliana.     

Characterization and differential expression of dhn/lea/rab-like genes during cold acclimation and drought stress in Arabidopsis thaliana

We have characterized cDNAs for two new dhn/lea/rab (dehydrin, late embryogenesis-abundant, responsive to ABA)-related genes from Arabidopsis thaliana. The two genes were strongly induced in plants exposed to low temperature (4 °C) and were accordingly designated lti45 and lti30 (low temperature-induced). The lti45 gene product contains the conserved serine stretch and three lysine-rich repeats characteristic of DHN/LEA/RAB proteins and is very similar to another low temperature-responsive protein of A. thaliana, COR47 [17]. Both proteins have the same repeat structure and an overall amino acid identity of 64%. This structural similarity of the proteins and the tandem array of the genes suggest that this gene pair arose through a duplication. The other polypeptide, LTI30, consists of several lysine-rich repeats, a structure found in CAP85, a low temperature-and water stress-responsive protein in spinach [41] and similar proteins found in wheat [20].The expression pattern of the five dhn/lea/rab-related genes (cor47, dhnX, lti30, lti45 and rab18) identified so far in A. thaliana, was characterized in plants exposed to low temperature, drought and abscisic acid (ABA). Expression of both lti30 and lti45 was mainly responsive to low temperature similar to cor47. The lti45 and lti30 genes show only a weak response to ABA in contrast to cor47, which is moderately induced by this hormone. The three genes were also induced in severely water-stressed plants although the expression of lti30 and lti45 was rather low. In contrast to these mainly low temperature-induced genes, the expression of rab18 was strongly induced both in water-stressed and ABA-treated plants but was only slightly responsive to cold. The dhnX gene showed a very different expression pattern. It was not induced with any of the treatments tested but exhibited a significant constitutive expression. The low-temperature induction of the genes in the first group, lti30 and lti45, is ABA-independent, deduced from experiments with the ABA-deficient (aba-1) and ABA-insensitive (abi1) mutants of A. thaliana, whereas the induction of rab18 is ABA-mediated. The expression of dhnX was not significantly affected in the ABA mutants.     

Potato cold hardiness development and abscisic acid. I. Conjugated abscisic acid is not the source of the increase in free abscisic acid during potato (Solanum commersonii) cold acclimation

Free and conjugated abscisic acid (ABA) levels in stem-cultured plantlets of potato ( Solanum commersonii Dun, PI 458317) during cold acclimation were measured. The levels of free and conjugated ABA were measured by an enzyme immunoassay (EIA) with rabbit anti-ABA-serum. The use of immunoglobulin G fraction purified from rabbit antiserum and the methylated form of ABA resulted in an improved measuring range (0.01 to 10 pmol ABA) and precision (slope of logit-log plot, −1.35) of EIA, compared to the use of antiserum and free ABA. Estimates of the EIA were consistent with those resulting from a commercial EIA. Under a 4/2°C (day/night) temperature regime, the potato plantlets increased cold hardiness from −5°C (warm-grown control) to −10°C by the 7th day. During the same period, there were two transitory increases in free ABA, the first one three-fold from 1.5 to 5.3 nmol (g dry weight)⁻¹ on the 2nd day and the second one five-fold from 1.5 to 7.6 nmol (g dry weight)⁻¹ on the 6th day. Each increase in ABA concentration was followed by an increase in cold hardiness. There was no significant change in conjugated ABA content (4.2±0.6 nmol [g dry weight]⁻¹) throughout the cold acclimation period. The lack of an interrelationship between levels of free and conjugated ABA suggested that the transitory increase in free ABA during cold acclimation was not a result of the conversion of conjugated ABA. The increase in free ABA due to biosynthesis of ABA during potato cold acclimation is discussed.     

Differential remodeling of the lipidome during cold acclimation in natural accessions of Arabidopsis thaliana

Freezing injury is a major factor limiting the geographical distribution of plant species and the growth and yield of crop plants. Plants from temperate climates are able to increase their freezing tolerance during exposure to low but non‐freezing temperatures in a process termed cold acclimation. Damage to cellular membranes is the major cause of freezing injury in plants, and membrane lipid composition is strongly modified during cold acclimation. Forward and reverse genetic approaches have been used to probe the role of specific lipid‐modifying enzymes in the freezing tolerance of plants. In the present paper we describe an alternative ecological genomics approach that relies on the natural genetic variation within a species. Arabidopsis thaliana has a wide geographical range throughout the Northern Hemisphere with significant natural variation in freezing tolerance that was used for a comparative analysis of the lipidomes of 15 Arabidopsis accessions using ultra‐performance liquid chromatography coupled to Fourier‐transform mass spectrometry, allowing the detection of 180 lipid species. After 14 days of cold acclimation at 4°C the plants from most accessions had accumulated massive amounts of storage lipids, with most of the changes in long‐chain unsaturated triacylglycerides, while the total amount of membrane lipids was only slightly changed. Nevertheless, major changes in the relative amounts of different membrane lipids were also evident. The relative abundance of several lipid species was highly correlated with the freezing tolerance of the accessions, allowing the identification of possible marker lipids for plant freezing tolerance.     

Abscisic acid deficiency prevents development of freezing tolerance in Arabidopsis thaliana (L.) Heynh.

Summary Abscisic acid (ABA) has been implicated as a regulatory factor in plant cold acclimation. In the present work, the cold-acclimation properties of an ABA-deficient mutant (aba) of Arabidopsis thaliana (L.) Heynh. were analyzed. The mutant had apparently lost its capability to cold acclimate: the freezing tolerance of the mutant was not increased by low temperature treatment but stayed at the level of the nonacclimated wild type. The mutational defect could be complemented by the addition of exogenous ABA to the growth medium, restoring freezing tolerance close to the wild-type level. This suggests that ABA might have a central regulatory function in the development of freezing tolerance in plants. Cold acclimation has been previously correlated to the induction of a specific set of proteins that have been suggested to have a role in freezing tolerance. However, these proteins were also induced in the aba mutant by low temperature treatment.     

Effects of abscisic acid and abscisic acid analogs on the induction of freezing tolerance of winter rye (Secale cereale L.) seedlings

The ability of abscisic acid (ABA) and abscisic acid analogs to induce freezing tolerance in fall rye (Secale cereale cv Puma) seedlings grown at nonhardening temperatures was investigated. Analogs were constructed with systematic alterations at C-1 (acid replaced with methyl ester, aldehyde or alcohol), at C-4, C-5 (trans double bond replaced with a triple bond), and at C-2, C-3 (double bond replaced with a single bond so that the side chain and C-2 methyl groups were cis). Freezing tolerance (LT₅₀) was determined 3, 4 and 6 days after the first of two consecutive applications of chemical (100 µM) to either the leaves or roots. All analogs were more effective when applied to the plant roots than when applied to the leaves. ABA, acetylenic ABA and 2,3-dihydroacetylenic ABA decreased the LT₅₀ from –3 °C (control) to –9 °C. Consistent structure-activity relationships were only detected following root application. No single functional group altered was absolutely required for activity. The effect of any given change to the molecule was modified by the presence of other functional groups. For example, substituting the double bond in the ring with a single bond decreased activity, but concomitant substitution of the trans double bond in the side chain with a triple bond restored activity. In general, analogs with a cis, trans side chain were more active initially but rapidly lost activity, whereas acetylenic analogs maintained or gained activity over the three sampling times. The application of gibberellin biosynthesis inhibitors (100 µM; tetcyclacis or mefluidide) did not increase freezing tolerance beyond that induced by ABA, either alone or in combination with ABA. It can be concluded that ABA and certain ABA analogs can induce limited freezing tolerance in whole rye seedlings, and partially substitute for low temperature acclimation.     

Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thaliana

We have examined the cold-induced enhancement of freezing tolerance and expression of cold-regulated (cor) genes in Arabidopsis thaliana (L.) Heynh (Landsberg erecta) and abscisic acid (ABA)-deficient (aba) and ABA-insensitive (abi) mutants derived from it. The results indicate that the abi mutations had no apparent effect on freezing tolerance, while the aba mutations did: cold-acclimated aba mutants were markedly impaired in freezing tolerance compared to wild-type plants. In addition, it was observed that non-frozen leaves from both control and cold-treated aba mutant plants were more ion-leaky than those from corresponding wild-type plants. These data are consistent with previous observations indicating that ABA levels can affect freezing tolerance. Whether ABA has a direct role in the enhancement of freezing tolerance that occurs during cold acclimation, however, is uncertain. Several studies have suggested that ABA might mediate certain changes in gene expression that occur during cold acclimation. Our data indicate that the ABA-induced expression of three ABA-regulated Arabidopsis cor genes was unaffected in the abi2, abi3, and aba-1 mutants, but was dramatically impaired in the abi1 mutant. Cold-regulated expression of all three cor genes, however, was nearly the same in wild-type and abi1 mutant plants. These data suggest that the cold-regulated and ABA-regulated expression of the three cor genes may be mediated through independent control mechanisms.     

Modifications of abscisic acid level in winter oilseed rape leaves during acclimation of plants to freezing temperatures

An almost twofold increase in abscisic acid (ABA) content was observed in the leaves of winter oilseed rape plants (Brassica napus L., var. oleifera L., cv. Jantar) grown in the cold (>0°C). This ABA increase took place during the first three days of cold treatment. After 6 days of plant growth in the cold, the level of ABA started to decline or remained constant, depending on the calculation basis: dry weight or disc area units, respectively. The exposure of cold-acclimated plants to night frost (–5°C for 18 h) induced a further increase (65%) in the ABA level, which begun during the first few hours after thawing. The comparison of time courses of frost resistance increments and ABA content changes showed that modifications of ABA level in the cold-treated leaves preceded those of frost resistance, whereas in the frost-pretreated tissues the ABA increase occurred later than that of frost tolerance. Possible interrelations between ABA content, frost tolerance and tissue water potential modifications in the low temperature-affected tissues are discussed.     

Regulation of proline accumulation in Arabidopsis thaliana (L.) Heynh during development and in response to desiccation

Significant differences were observed in the amount and proportion of free amino acids in different organs of Arabidopsis thaliana (L.) Heynh, ecotype Columbia. The most notable were found for proline, which formed 17–26% of the total free amino acid concentration in reproductive tissues (floret and seed), but only 1–3% of the total free amino acid concentration in vegetative tissues (rosette leaf and root). Proline accumulation was associated with tissues that had relatively low water contents. Tissues which displayed high water contents, such as rosette leaves, contained low levels of proline. A significant increase in the levels of proline accumulation occurred in plants subjected to experimentally induced low water potentials as compared to unstressed plants. For instance, an 8–10-fold increase in proline was observed in the presence of 120 mmol kg^?1 NaCl or KCl, and a 20-fold increase was stimulated by 60 mmol kg^?1 PEG. However, in addition to the accumulation of proline, massive accumulation of Na⁺, K⁺ and Cl^? ions occurred in tissues of plants stressed with salt. No significant differences were observed in mineral ions in plants stressed with PEG. Isotope tracer experiments with ¹⁴C compounds established that glutamate, ornithine and arginine are precursors of the proline biosynthesis induced by PEG in response to low water potentials in Arabidopsis thaliana. We conclude that the accumulation of proline in response to PEG occurs through increased biosynthesis.     

Monitoring expression profiles of Arabidopsis genes during cold acclimation and deacclimation using DNA microarrays

A comparative analysis of gene expression profiles during cold acclimation and deacclimation is necessary to elucidate the molecular mechanisms of cold stress responses in higher plants. We analyzed gene expression profiles in the process of cold acclimation and deacclimation (recovery from cold stress) using two microarray systems, the 7K RAFL cDNA microarray and the Agilent 22K oligonucleotide array. By both microarray analyses, we identified 292 genes up-regulated and 320 genes down-regulated during deacclimation, and 445 cold up-regulated genes and 341 cold down-regulated genes during cold acclimation. Many genes up-regulated during deacclimation were found to be down-regulated during cold acclimation, and vice versa. The genes up-regulated during deacclimation were classified into (1) regulatory proteins involved in further regulation of signal transduction and gene expression and (2) functional proteins involved in the recovery process from cold-stress-induced damages and plant growth. We also applied expression profiling studies to identify the key genes involved in the biosynthesis of carbohydrates and amino acids that are known to play important roles in cold acclimation. We compared genes that are regulated during deacclimation with those regulated during rehydration after dehydration to discuss the similarity and difference of each recovery process.Electronic Supplementary Material Supplementary materials are available for this article at     

Effect of abscisic acid and cold acclimation on the cytoskeletal and phosphorylated proteins in different cultivars of Triticum aestivum L

In winter wheat, the tubulin and 60 kDa-phosphorylated proteins/actin ratio is considerably higher in the roots than in the leaves. Differences in the content of the main cytoskeletal proteins were also found in the leaves of the different cultivars. It is suggested that the lower amount of the tubulin and 60 kDa-phosphorylated proteins and higher content of actin determine the greater tubulin cytoskeletal stability in the leaves and their higher frost resistance, as compared with the roots. Also, it is possible that the higher content of the tubulin and 60 kDa-phosphorylated proteins defines the lower microtubule (MT) stability in the leaves of the low frost resistant cultivar than in the leaves of the more frost resistant ones. In the roots and leaves of the low frost resistant cultivar, the low stability of the numerous tubulin structures is apparently one reason for the abscisic acid (ABA)-induced reduction of the cytoskeletal and 60 kDa-phosphorylated proteins in the cells. The cold acclimation compensated the ABA effect in the roots of the very frost resistant cultivar in the most extent. This suggests the existence of the different pathways in the increased plant cell frost resistance through the action of ABA and low temperature.     

Potato cold hardiness development and abscisic acid. II. De novo synthesis of proteins is required for the increase in free abscisic acid during potato (Solanum commersonii) cold acclimation

During cold acclimation of potato plantlets ( Solanum commersonii Dun, PI 458317), there are two transitory increases in free ABA content corresponding to a three-fold increase on the 2nd day and a five-fold increase on the 6th day (Ryu and Li 1993). During this period, plantlets increased in cold hardiness from −5°C (killing temperature, control grown at 22/18°C, day/night) to −10°C by the 7th day of exposure to 4/2°C (day/night). This increase in free ABA was not found when cycloheximide (CHI), an inhibitor of cytoplasmic protein synthesis, was added to the culture medium 6 h before exposure to low temperatures. Plantlets treated with CHI did not acclimate to cold, maintaining a hardiness level (−5°C) similar to that of the 22/18°C-grown plantlets. When the CHI-treated plantlets were exposed to low temperatures for 3 days, transferred to CHI-free culture medium and grown at low temperatures, the plantlets showed a transitory increase in free ABA 2 days later. This increase was followed by the development of cold hardiness (−8°C). Application of CHI to the culture medium after 3 days of cold acclimation, when the first ABA peak and a partial development of cold hardiness (−8°C) had occurred, blocked the second transitory increase in free ABA and resulted in no further development of cold hardiness. These results suggest that de novo synthesis of proteins is required for these transitory increases in free ABA during cold acclimation of potato plantlets.     

Characterization of a cDNA from Arabidopsis thaliana encoding a potential thiol protease whose expression is induced independently by wilting and abscisic acid

The sequence and expression characteristics are described of a wilt-inducible gene in Arabidopsis thaliana. A 1494 encodes a potential thiol protease whose mRNA accumulates rapidly in shoot tissue upon the loss of turgor. A1494 mRNA levels peaked after ca. 4 h and declined thereafter. Dehydration also induced rapid biosynthesis of the phytohormone abscisic acid (ABA), which continued for at least 9 h. Exogenous ABA induced the accumulation of A1494 mRNA, with kinetics similar to those after wilting. Rehydration of wilted shoots led to a rapid decline in the content of both ABA and A1494 mRNA. Wilting and ABA independently induced A1494 expression as evidenced by the effects of ABA and wilting on the ABA-deficient aba-1 and ABA-insensitive abi-1 and abi-3 genotypes. A1494 mRNA was not detectable in aba-1 shoots but accumulated rapidly after either wilting or ABA treatment, whereas the shoot ABA content was increased only by ABA treatment. ABA had no effect on A1494 mRNA levels in the abi-1 and abi-3 mutants but wilting did result in enhanced A1494 expression. Heat shock had only a minor effect on A1494 mRNA levels, whereas exposure to low temperature resulted in substantial accumulation of A1494 mRNA in wild-type shoots. However, this latter response, unlike that to drought, was mediated exclusively via ABA synthesis as demonstrated by the lack of A1494 mRNA accumulation in cold-treated aba-1 shoots.