Photoperiod and temperature interactions regulate low-temperature-induced gene expression in barley.

Vernalization and photoperiod (PP) responses are developmental mechanisms that allow plants to synchronize their growth and reproductive cycles with the seasonal weather changes. Vernalization requirement has been shown to influence the length of time that low-temperature (LT)-induced genes are up-regulated when cereal species are exposed to acclimating temperatures. The objective of the present study was to determine whether expression of LT-induced Wcs and Wcor gene families is also developmentally regulated by PP response. The LT-tolerant, highly short-day (SD)-sensitive barley (Hordeum vulgare L. cv Dicktoo) was subjected to 8-h SD and 20-h long-day PPs at cold-acclimating temperatures over a period of 70 d. A delay in transition from the vegetative to the reproductive stage under SD resulted in an increased level and longer retention of LT tolerance. Similar WCS and WCOR protein homologs were expressed, but levels of expression were much higher in plants acclimated under SD, indicating that the poor LT tolerance of long-day plants was the result of an inability to maintain LT-induced genes in an up-regulated state. These observations indicate that the PP and vernalization genes influence the expression of LT-induced genes in cereals through separate pathways that eventually converge to activate genes controlling plant development. In both instances, the delay in the transition from the vegetative to the reproductive stage produces increased LT tolerance that is sustained for a longer period of time, indicating that the developmental genes determine the duration of expression of LT-induced structural genes.

Survey of gene expression in winter rye during changes in growth temperature, irradiance or excitation pressure.

Previous comparisons of winter rye plants (Secale cereale L. cv. Musketeer) grown in a combination of specific temperature (degrees C)/irradiance (micromol m(-2) s(-1)) regimes (20/50; 20/250; 20/800; 5/50; 5/250) revealed (1) that photosynthetic acclimation to low temperature mimics photosynthetic acclimation to high light because both conditions result in comparable reduction states of photosystem II (PSII), that is, comparable PSII excitation pressure; (2) that the relative redox state of PSII also appears to regulate a specific cold acclimation gene, Wcs19. In order to identify additional genes regulated differentially by either low temperature, irradiance or excitation pressure, we initiated a detailed analysis of gene expression. We identified and characterized 42 differentially expressed genes from wheat and rye. Based on their patterns of regulation under the five growth conditions employed, 37 of the cDNAs could be classified into four groups: genes regulated by PSII excitation pressure, low temperature, growth irradiance and interaction between growth temperature and irradiance. Partial sequence analyses revealed that several of these genes encode known chloroplastic proteins such as ELIPs, transketolase, carbonic anhydrase and Mg-chelatase. However, five of the genes could not be classified unambiguously into any one of these four categories. The implications of these results and the limitations of the experimental design are discussed in terms of larger-scale genomic studies designed to understand the interactions of multiple abiotic stresses to which a plant may be exposed when examining regulation of gene expression.

Regulation of a wheat actin-depolymerizing factor during cold acclimation.

We have previously shown that the wheat (Triticum aestivum) TaADF gene expression level is correlated with the plants capacity to tolerate freezing. Sequence analysis revealed that this gene encodes a protein homologous to members of the actin-depolymerizing factor (ADF)/cofilin family. We report here on the characterization of the recombinant TaADF protein. Assays for ADF activity showed that TaADF is capable of sequestering actin, preventing nucleotide exchange, and inducing actin depolymerization. In vitro phosphorylation studies showed that TaADF is a substrate for a wheat 52-kD kinase. The activity of this kinase is modulated by low temperature during the acclimation period. Western-blot analyses revealed that TaADF is expressed only in cold-acclimated Gramineae species and that the accumulation level is much higher in the freezing-tolerant wheat cultivars compared with the less tolerant ones. This accumulation was found to be regulated by a factor(s) encoded by a gene(s) located on chromosome 5A, the chromosome most often found to be associated with cold hardiness. The induction of an active ADF during cold acclimation and the correlation with an increased freezing tolerance suggest that the protein may be required for the cytoskeletal rearrangements that may occur upon low temperature exposure. These remodelings might be important for the enhancement of freezing tolerance.

Chitinase genes responsive to cold encode antifreeze proteins in winter cereals.

Antifreeze proteins similar to two different chitinases accumulate during cold acclimation in winter rye (Secale cereale). To determine whether these cold-responsive chitinases require post-translational modification to bind to ice, cDNAs coding for two different full-length chitinases were isolated from a cDNA library produced from cold-acclimated winter rye leaves. CHT9 is a 1,193-bp clone that encodes a 31.7-kD class I chitinase and CHT46 is a 998-bp clone that codes for a 24.8-kD class II chitinase. Chitinase-antifreeze proteins purified from the plant were similar in mass to the predicted mature products of CHT9 and CHT46, thus indicating that there was little chemical modification of the amino acid sequences in planta. To confirm these results, the mature sequences of CHT9 and CHT46 were expressed in Escherichia coli and the products of both cDNAs modified the growth of ice. Transcripts of both genes accumulated late in cold acclimation in winter rye. Southern analysis of winter rye genomic DNA indicated the presence of a small gene family homologous to CHT46. In hexaploid wheat, CHT46 homologs mapped to the homeologous group 1 chromosomes and were expressed in response to cold and drought. We conclude that two novel cold-responsive genes encoding chitinases with ice-binding activity may have arisen in winter rye and other cereals through gene duplication.

Two novel intrinsic annexins accumulate in wheat membranes in response to low temperature.

Four immunologically related proteins that belong to the annexin family were identified in cold acclimated wheat (Triticum aestivum). Two soluble forms with molecular masses of 34 and 36 kDa were found to bind phospholipid membranes in a calcium-dependent manner. These two forms are similar to the previously reported doublet in several plant species. The other two forms, with molecular masses of 39 and 22.5 kDa, were found associated with the microsomal fraction. Biochemical analysis showed that both forms are intrinsic membrane proteins and their association with the membrane is calcium independent. This is, to our knowledge, the first report of the presence of these annexin forms in plants. Membrane purification by two phase partitioning demonstrated that the p39 form is localized to the plasma membrane. Immunoblot analysis showed that the protein level of both p39 and p22.5 increases gradually reaching a maximum level after one day of low temperature exposure. The protein accumulation was similar in both hardy and less hardy cultivars, suggesting that the accumulation is not correlated with freezing tolerance. The results are discussed with respect to the possible role of these new intrinsic membrane annexins in low temperature signal transduction pathway.

Biotechnological applications of plant freezing associated proteins.

Plants use a wide array of proteins to protect themselves against low temperature and freezing conditions. The identification of these freezing tolerance associated proteins and the elucidation of their cryoprotective functions will have important applications in several fields. Genes encoding structural proteins, osmolyte producing enzymes, oxidative stress scavenging enzymes, lipid desaturases and gene regulators have been used to produce transgenic plants. These studies have revealed the potential capacity of different genes to protect against temperature related stresses. In some cases, transgenic plants with significant cold tolerance have been produced. Furthermore, the biochemical characterization of the cold induced antifreeze proteins and dehydrins reveals many applications in the food and the medical industries. These proteins are being considered as food additives to improve the quality and shelf-life of frozen foods, as cryoprotective agents for organ and cell cryopreservation, and as chemical adjuvant in cancer cryosurgery.

Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation of wheat

Expression of the acidic dehydrin gene wcor410 was found to be associated with the development of freezing tolerance in several Gramineae species. This gene is part of a family of three homologous members, wcor410, wcor410b, and wcor410c, that have been mapped to the long arms of the homologous group 6 chromosomes of hexaploid wheat. To gain insight into the function of this gene family, antibodies were raised against the WCOR410 protein and affinity purified to eliminate cross-reactivity with the WCS120 dehydrin-like protein of wheat. Protein gel blot analyses showed that the accumulation of WCOR410 proteins correlates well with the capacity of each cultivar to cold acclimate and develop freezing tolerance. Immunoelectron microscope analyses revealed that these proteins accumulate in the vicinity of the plasma membrane of cells in the sensitive vascular transition area where freeze-induced dehydration is likely to be more severe. Biochemical fractionation experiments indicated that WCOR410 is a peripheral protein and not an integral membrane protein. These results provide direct evidence that a subtype of the dehydrin family accumulates near the plasma membrane. The properties, abundance, and localization of these proteins suggest that they are involved in the cryoprotection of the plasma membrane against freezing or dehydration stress. We propose that WCOR410 plays a role in preventing the destabilization of the plasma membrane that occurs during dehydrative conditions.

The wheat wcs120 promoter is cold-inducible in both monocotyledonous and dicotyledonous species.

The wcs120 gene is specifically induced by low temperature (LT) and encodes a protein that is thought to play an important role in the cold acclimation process in wheat. To identify the regulatory elements involved in its LT responsiveness, the transient expression activity of different promoter regions was determined using the luciferase reporter gene. The data indicate the involvement of putative enhancer elements, negative and positive regulatory regions in the transcriptional regulation of this gene. The promoter was found to be cold-inducible in different freezing-tolerant and -sensitive monocot and dicot species, suggesting that universal transcription factors responsive to LT may be present in all plants. This promoter could be used to drive the genes needed for LT tolerance in sensitive species.

Low temperature-stimulated phosphorylation regulates the binding of nuclear factors to the promoter of Wcs120, a cold-specific gene in wheat.

The Wcs120 gene encodes a highly abundant protein which appears to play an important role during cold acclimation of wheat. To understand the regulatory mechanism controlling its expression at low temperature, the promoter region has been characterized. Electrophoretic mobility shift assays using short promoter fragments revealed the presence in nuclear extracts from non-acclimated (NA) plants of multiple DNA-binding proteins which interact with several elements. In contrast, no DNA-binding activity was observed in the nuclear extracts from cold-acclimated (CA) plants. In vitro dephosphorylation of these CA nuclear extracts with alkaline phosphatase restored the binding activity. Moreover, okadaic acid (a potent phosphatase inhibitor) markedly stimulated the in vivo accumulation of the WCS120 family of proteins. This suggests that protein phosphatases PP1 and/or PP2A negatively regulate the expression of the Wcs120 gene. In addition, both Ca(2+)-dependent and Ca(2+)-independent kinase activities were found to be significantly higher in the CA nuclear extracts. Western analysis using antibodies directed against protein kinase C (PKC) isoforms showed that a PKCgamma homolog (84 kDa) is selectively translocated into the nucleus in response to low temperature. Taken together, our results suggest that, in vivo, the expression of the Wcs120 gene may be regulated by nuclear factors whose binding activity is modulated by a phosphorylation/dephosphorylation mechanism.

Gene expression during cold acclimation in strawberry.

To elucidate the molecular basis of cold acclimation in strawberry (Fragaria x anannassa), we have begun studies to identify genes associated with low temperature (LT) acclimation. Differential screening of a cDNA library prepared from cold-acclimated strawberry plants allowed us to isolate several cDNAs showing differential expression at LT. Northern analysis showed that the transcript level of Fcor1 (Fragaria Cold-Regulated) peaked after 2 days of LT exposure while that of Fcor2 peaked after 2 weeks. On the other hand, the level of Fcor3 transcript decreased within 24 hours of LT exposure and remained low during the 8 weeks acclimation period. Fcor1 and Fcor2 are expressed in all tissues while Fcor3 is specific to leaves. The Fcor1-encoded protein has a compositional bias for leucine, isoleucine, glycine, proline and serine. This protein shares homology with the proteins encoded by blt101, a LT-responsive gene from barley, and ESI3, a gene induced by salt stress in Lophopyrum. The FCOR2 protein is rich in lysine, leucine, valine, alanine and arginine, and shows no homology with any known gene products. The partial Fcor3 cDNA clone encodes a polypeptide that shows a very high identity with the spinach PSI subunit V and with the PSI PsaG polypeptide from barley. The level of Fcor1 transcript accumulation is correlated with the freezing tolerance of the strawberry cultivars used in our study. This suggests that Fcor1 may be useful as a molecular marker to select for this trait in resulted species of the Rosaceae family.

Cold Acclimation and Freezing Tolerance (A Complex Interaction of Light and Temperature).

By comparing growth under five different temperature and irradiance regimes (20[deg]C and 800, 250, and 50[mu]mol m-2 s-1 and 5[deg]C and 250 and 50 [mu]mul m-2 s-1), we have examined the effects of light, temperature, and the relative reduction state of photosystem II on plant morphology, freezing tolerance (lethal temperature at which freezing injury occurs [LT50]), transcript levels of Lhcb and two cold-stimulated genes (Wcs19 and Wcs120), and photosynthetic adjustment in winter rye (Secale cereale L. cv Musketeer). We show, for the first time to our knowledge, that in addition to adjustments in photosynthetic capacity, nonphotochemical quenching capacity and tolerance to photoinhibition, the accumulation of the cold-induced transcript Wcs19, and the compact plant morphology usually associated with cold-hardening are correlated with the relative reduction state of photosystem II rather than with growth temperature or growth irradiance per se. In contrast, the acquisition of maximal LT50, as well as Lhcb and Wcs120 mRNA accumulation, appears to be dependent on both growth temperature and growth irradiance but in an independent, additive manner. The results are discussed with respect to the possible role of the modulation of chloroplastic redox poise in photosynthetic acclimation to cold-hardening temperatures and the attainment of maximal LT50.

Chromosome mapping of low-temperature induced Wcs120 family genes and regulation of cold-tolerance expression in wheat.

Low-temperature (LT) induced genes of the Wcs120 family in wheat (Triticum aestivum) were mapped to specific chromosome arms using Western and Southern blot analysis on the ditelocentric series in the cultivar Chinese Spring (CS). Identified genes were located on the long arms of the homoeologous group 6 chromosomes of all 3 genomes (A, B, and D) of hexaploid wheat. Related species carrying either the A, D, or AB genomes were also examined using Southern and Western analysis with the Wcs120 probe and the WCS120 antibody. All closely related species carrying one or more of the genomes of hexaploid wheat produced a 50 kDa protein that was identified by the antibody, and a Wcs120 homoeologue was detected by Southern analysis in all species. In the absence of chromosome arm 6DL in hexaploid CS wheat no 50 kDa protein was produced and the high-intensity Wcs120 band was missing, indicating 6DL as the location of Wcs120 but suggesting silencing of the Wcs120 homoeologue in the A genome. Levels of proteins that cross-reacted with the Wcs120 antibody and degrees of cold tolerance were also investigated in the Chinese Spring/Cheyenne (CS/CNN) chromosome substitution series. CNN chromosome 5A increased the cold tolerance of CS wheat. Densitometry scanning of Western blots to determine protein levels showed that the group 5 chromosome 5A had a regulatory effect on the expression of the Wcs120 gene family located on the group 6 chromosomes of all three hexaploid wheat genomes.

Identification and characterization of a low temperature regulated gene encoding an actin-binding protein from wheat.

A cDNA corresponding to a putative actin-binding protein was cloned from a cold-acclimated wheat cDNA library. The cDNA, designated Wcor719, encodes a polypeptide of 142 amino acids with a calculated molecular mass of 15.8 kDa and a pI of 4.27. The protein has the two conserved domains identified as actin and phosphatidylinositol 4,5-biphosphate (PIP2) binding sites found in members of the cofilin family. Northern analyses revealed that Wcor719 transcript accumulation is rapid and strongly up-regulated by low temperature. This accumulation was greater in the tolerant winter wheat and rye species compared to the less tolerant ones. The rapidity of transcript induction and the significant homology with actin-binding proteins (ABP) from different organisms suggest that the product of this gene might be involved in the dynamic reorganization of the cytoskeleton during low temperature acclimation. It may also serve as a key factor in the signal transduction pathway during cold acclimation.

The regulatory role of vernalization in the expression of low-temperature-induced genes in wheat and rye.

Low temperature is one of the primary stresses limiting the growth and productivity of wheat (Triticum aestivum L.) and rye (Secale cereale L.). Winter cereals low-temperature-acclimate when exposed to temperatures colder than 10°C. However, they gradually lose their ability to tolerate below-freezing temperatures when they are maintained for long periods of time in the optimum range for low-temperature acclimation. The overwinter decline in low-temperature response has been attributed to an inability of cereals to maintain low-temperature-tolerance genes in an up-regulated state once vernalization saturation has been achieved. In the present study, the low-temperature-induced Wcs120 gene family was used to investigate the relationship between low-temperature gene expression and vernalization response at the molecular level in wheat and rye. The level and duration of gene expression determined the degree of low-temperature tolerance, and the vernalization genes were identified as the key factor responsible for the duration of expression of low-temperature-induced genes. Spring-habit cultivars that did not have a vernalization response were unable to maintain low-temperature-induced genes in an up-regulated condition when exposed to 4°C. Consequently, they were unable to achieve the same levels of low-temperature tolerance as winter-habit cultivars. A close association between the point of vernalization saturation and the start of a decline in the Wcs120 gene-family mRNA level and protein accumulation in plants maintained at 4°C indicated that vernalization genes have a regulatory influence over low-temperature gene expression in winter cereals.

Immunolocalization of freezing-tolerance-associated proteins in the cytoplasm and nucleoplasm of wheat crown tissues.

A protein family associated with the development of freezing tolerance in wheat has been identified. This protein family is Gramineae-specific and coordinately regulated by low temperature. Antibodies directed against the 50 kDa (WCS120) protein recognize at least 5 members of this family. Using these antibodies, the cellular content and location of this protein family was determined in cold-acclimated wheat seedlings. Western analyses of subcellular fractions indicated the presence of all members of the family in the cytosolic and purified nuclear fractions. These proteins accumulated to 0.9% of soluble proteins after 21 days of cold acclimation in winter wheat. This represents a cellular concentration of 1.34 microM. Immunohistochemical localization showed that these proteins are highly expressed in the vascular transition zone. No detectable expression was found in mature xylem, in the shoot apical meristem or lateral root primordia. This differential tissue expression suggests that the sensitive cells near the regions where water tends to freeze first require a higher amount of these proteins. This observation is consistent with the fact that regrowth after freezing stress is highly dependent on the viability of this region of the crown. Electron microscopy analysis using immunogold labelling showed that these proteins are present in the cytoplasm and in the nucleoplasm. They are not found in cell walls or other organelles. In vitro cryoprotective assays indicated that the WCS120 protein (PD50 of 10 micrograms ml-1 or 0.2 microM) are as effective as BSA and sucrose (at 250 mM) against freezing denaturation of lactate dehydrogenase. These results suggest that this protein family may be involved in a general mechanism of protection in the soluble fraction of the cell. Their presence in the nucleoplasm may also suggest a possible protective function of the transcriptional machinery. The high hydrophilicity, the abundance and stability of these proteins to boiling suggest that they may provide a particular micro-environment needed for cell survival in the sensitive vascular transition zone during freezing stress.

Expression of the cold-induced wheat gene Wcs120 and its homologs in related species and interspecific combinations.

Low-temperature response was measured at the whole plant and at the molecular level in wheat-rye amphiploids and in other interspecific combinations. Cold tolerance of interspecifics whose parents diverged widely in hardiness levels resembled the less hardy higher ploidy level wheat parent. Expression of the low-temperature induced Wcs120 gene of wheat (Triticum aestivum L. em. Thell.) has been associated with freezing tolerance and was used here to study mRNA and protein accumulation in interspecific and parental lines during cold acclimation. Northern and Western analyses showed that homologous mRNAs and proteins were present in all the related species used in the experiments. Cold-tolerant rye (Secale cereale L.) produced a strong mRNA signal that was sustained throughout the entire 49-day cold-acclimation period. The wheats produced a mRNA signal that had diminished after 49 days of low-temperature exposure. The wheat-rye triticales did not exhibit the independent accumulation kinetics of the cold-tolerant rye parent but, rather, more closely resembled the wheat parent in that the mRNA signal was greatly diminished after 49 days of low-temperature exposure. The influence of the rye genome was manifest in slightly greater mRNA and protein accumulation in earlier stages of acclimation. Protein accumulations in the triticales were also maintained to a somewhat greater extent than found in the wheats at the end of the 49-day acclimation period. Protein accumulations in the wheat-crested wheatgrass (Agropyron cristatum L. Gaertner) interspecific resembled that of the wheat parent. The influence of the higher ploidy level wheats of the expression of homologous gene families from wheat-related hardy diploids in interspecific combinations may in part explain the poor cold tolerance observed.

Differential expression of a gene encoding an acidic dehydrin in chilling sensitive and freezing tolerant gramineae species.

We have characterized a new wheat cold-regulated cDNA clone, Wcor410, that accumulates to equivalent levels in root, crown and leaf tissues during cold acclimation. The Wcor410 cDNA contains an ORF encoding a dehydrin-like glutamate-rich protein of 28 kDa with a pI of 5.1. However, the acidic nature, the absence of the glycine-rich repeat and of the conserved N-terminal region, DEYGNP, suggest that Wcor410 belongs to a different subgroup of the D11 protein family. Northern analysis showed that this gene is expressed only in freezing tolerant gramineae, whereas Southern analysis showed that the Wcor410 gene is present in all monocot species tested. The presence of freezing tolerance-associated genes in sensitive species such as rice and corn is interesting. Characterization of the regulatory factors controlling these genes may help to establish an appropriate strategy to improve freezing tolerance.

A leaf-specific gene stimulated by light during wheat acclimation to low temperature.

We report here the identification and characterization of a new leaf-specific light-stimulated gene induced during cold acclimation of wheat. Sequence analysis revealed that the gene encodes a protein of 19 kDa with a pI of 8.8. This is a novel protein with a particular charge distribution. The C-terminal half has a high propensity to form an alpha-helix and contains all the acidic amino acids with a net negative charge of -7. On the other hand, the N-terminal half is rich in proline, lysine and arginine with a net positive charge of +10. These properties are commonly found in several transcription factors. The protein is also rich in alanine (21%), is hydrophilic but not boiling soluble in contrast to other alanine-rich proteins. During low temperature exposure, the corresponding mRNA accumulates rapidly in the leaf and remains at a constant level in two tolerant cultivars used. However, in a less tolerant cultivar, the mRNA level declines despite maintaining the plants at 4 degrees C. Southern blot analysis indicates that the differential expression in the less tolerant genotype is not due to a different genomic organization or gene copy number. The mRNA was specifically localized in leaf tissues and increased several-fold during the greening at 4 degrees C. Furthermore, this gene is not induced in callus cultures acclimated in the absence or presence of light. This suggests that the full expression of this gene is dependent on organized leaf tissue. The expression of this gene was not affected by ABA, drought, heat shock, salinity, wounding or anaerobiosis, demonstrating that it is specifically induced by low temperature. The Wcs19 mRNA is preferentially expressed in tolerant Gramineae species.