Heterologous Expression of Wheat VERNALIZATION 2 (TaVRN2) Gene in Arabidopsis Delays Flowering and Enhances Freezing Tolerance

The vernalization gene 2 (VRN2), is a major flowering repressor in temperate cereals that is regulated by low temperature and photoperiod. Here we show that the gene from Triticum aestivum (TaVRN2) is also regulated by salt, heat shock, dehydration, wounding and abscissic acid. Promoter analysis indicates that TaVRN2 regulatory region possesses all the specific responsive elements to these stresses. This suggests pleiotropic effects of TaVRN2 in wheat development and adaptability to the environment. To test if TaVRN2 can act as a flowering repressor in species different from the temperate cereals, the gene was ectopically expressed in the model plant Arabidopsis. Transgenic plants showed no alteration in morphology, but their flowering time was significantly delayed compared to controls plants, indicating that TaVRN2, although having no ortholog in Brassicaceae, can act as a flowering repressor in these species. To identify the possible mechanism by which TaVRN2 gene delays flowering in Arabidopsis, the expression level of several genes involved in flowering time regulation was determined. The analysis indicates that the late flowering of the 35S::TaVRN2 plants was associated with a complex pattern of expression of the major flowering control genes, FCA, FLC, FT, FVE and SOC1. This suggests that heterologous expression of TaVRN2 in Arabidopsis can delay flowering by modulating several floral inductive pathways. Furthermore, transgenic plants showed higher freezing tolerance, likely due to the accumulation of CBF2, CBF3 and the COR genes. Overall, our data suggests that TaVRN2 gene could modulate a common regulator of the two interacting pathways that regulate flowering time and the induction of cold tolerance. The results also demonstrate that TaVRN2 could be used to manipulate flowering time and improve cold tolerance in other species.     

Changes in wheat leaf phenolome in response to cold acclimation

A study of wheat (Triticum aestivum L.) leaves phenolome was carried out during cold acclimation of the winter (Claire) and spring (Bounty) varieties using a combination of HPLC–ESI–MS techniques. A total of 40 phenolic and flavonoid compounds were identified, and consisted mainly of two coumarin derivatives, eight simple phenolic derivatives, 10 hydroxycinnamoyl amides and 20 flavonoid derivatives. Identification and quantification of individual compounds were performed using an HPLC system coupled with a photodiode array detector and two different ESI–MS systems, in combination with a multiple reaction monitoring (MRM) technique. The analyses indicated that, although there were no qualitative differences in their profiles, the winter variety exhibited a higher phenolic content compared to the spring variety when both were grown under non-acclimated (control) conditions. Cold acclimation, on the other hand, resulted in a significant differential accumulation of phenolic compounds in both varieties: mostly as luteolin C-glycosides and their O-methyl derivatives in the winter variety (Claire) and a derivative of hydroxycinnamoyl amide in the spring variety (Bounty). These compounds accumulated in relatively large amounts in the apoplastic compartment. The accumulation of the O-methylated derivatives was associated with a marked increase in O-methyltransferase (OMT) activity. In addition, the trimethylated flavone, 3′,4′,5′-trimethyltricetin was identified for the first time in the native extracts of both control and cold-acclimated wheat leaves. The accumulation of a mixture of beneficial flavonoids, such as iso-orientin, vitexin and tricin in cold acclimated wheat leaves, attests for its potential as an inexpensive source of a health-promoting supplement to the human diet.     

Energy state of spring and winter wheat during cold hardening. Soluble sugars and adenine nucleotides

Marked increases were found in the content of total soluble sugars, reducing sugars and ATP in winter wheat ( Triticum aestivum L. cv. Frederick) during cold hardening. The changes in soluble sugars and ATP of spring wheat ( T. aestivum L. cv. Glenlea) grown under similar conditions were less pronounced. The increase in ATP content during hardening of winter wheat was not associated with significant changes in the content of ADP or AMP. The adenylate energy charge did not change during hardening in either cultivar, but it was higher in the winter cultivar under both growth conditions. This difference could be related to the cold hardiness capacity of winter wheat.     

Purification, Characterization and cDNA Cloning of the 200 kDa Protein Induced by Cold Acclimation in Wheat

We have purified to homogeneity the 200 kDa protein inducedspecifically by low temperature in wheat (Triticum aestivumL.). The boiling solubility of the protein has been used asa main step in the purification procedure. Amino acid compositionindicates that the 200 kDa has a compositional bias for glycine(11.4%), threonine (13.3%), and alanine (22.0%). Using oligonucleotideprobes, we have isolated a clone (pWcs200) from a cold-acclimatedwinter wheat cDNA library. Northern analysis demonstrated thatthe expression of the corresponding gene was specifically upregulatedby low temperature. Southern analysis showed that the gene organizationand the relative copy number were identical in two cultivarsdiffering in their capacity to develop freezing tolerance. Proteinsequence and immunological analyses indicate that this proteinshares similar features with the 50 kDa protein induced duringcold acclimation of wheat. The two proteins are boiling-soluble,and possess similar repeated elements. These elements may beimportant for the development of freezing tolerance. We haveshown that the 200 kDa protein is the largest member of a familyof immunologically-related cold-induced proteins in wheat. Expressionof pWcs200 in E. coli yielded a product of around 200 kDa, indicatingthat the clone contains most of the coding region for this protein. (Received August 18, 1992; Accepted October 14, 1992)     

Daphnetin methylation stabilizes the activity of phosphoribulokinase in wheat during cold acclimation

The methylation of daphnetin (7,8-dihydroxycoumarin) to its 8-methyl derivative is catalyzed by a wheat (Triticum aestivum L.) O-methyltransferase (TaOMT1). This enzyme is regulated by cold and photosystem II excitation pressure (plastid redox state). Here, we investigated the biological significance of this methylation and its potential role in modulating the activity of kinases in wheat. To identify the potential kinases that may interact with daphnetin in wheat, the soluble protein extract from aerial parts of cold-acclimated wheat was purified by DEAE-cellulose separation and affinity chromatography on a daphnetin derivative (7,8-dihydroxy-4-coumarin acetic acid)-EAH sepharose column. Mass spectrometric analysis indicated that wheat phosphoribulokinase (TaPRK) is the major kinase that binds to daphnetin. This TaPRK plays an important role in regulating the flow of carbon through the Calvin cycle, by catalyzing the final step in the regeneration of ribulose 1,5-bisphosphate from ribulose-5-phosphate (Ru5P) and ATP. The activities of TaPRK, endogenous or recombinant, are inhibited by daphnetin in a specific and dose-dependent manner, but not by its monomethyl derivative (7-methyl, 8-hydroxycoumarin). Furthermore, HPLC-MS analysis of wheat extracts reveals that 7,8-dimethoxycoumarin is more abundant than its monomethyl derivative. The results also show that cold acclimation does not alter the level of TaPRK mRNA or its enzyme activity, and thus ensures the stable generation of ribulose 1,5-biphosphate.