Fathey Sarhan

Energy balance and acclimation to light and cold

Changes in environmental conditions such as light intensity or temperature result in an imbalance between the light energy absorbed through photochemistry versus the energy utilized through metabolism. Such an energy imbalance is sensed through alterations in photosystem II excitation pressure, which reflects the relative reduction state of the photosystem. Modulation of this novel, chloroplastic redox signal either by excess light or by low temperature initiates a signal transduction pathway. This appears to coordinate photosynthesis-related gene expression and to influence the nuclear expression of a specific cold-acclimation gene, plant morphology and differentiation in cyanobacteria. Thus, in addition to its traditional role in energy transduction, the photosynthetic apparatus might also be an environmental sensor.

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.

TaVRT-1, a Putative Transcription Factor Associated with Vegetative to Reproductive Transition in Cereals

The molecular genetics of vernalization, defined as the promotion of flowering by cold treatment, is still poorly understood in cereals. To better understand this mechanism, we cloned and characterized a gene that we named TaVRT-1 (wheat [Triticum aestivum] vegetative to reproductive transition-1). Molecular and sequence analyses indicated that this gene encodes a protein homologous to the MADS-box family of transcription factors that comprises certain flowering control proteins in Arabidopsis. Mapping studies have localized this gene to the Vrn-1 regions on the long arms of homeologous group 5 chromosomes, regions that are associated with vernalization and freezing tolerance (FT) in wheat. The level of expression of TaVRT-1 is positively associated with the vernalization response and transition from vegetative to reproductive phase and is negatively associated with the accumulation of COR genes and degree of FT. Comparisons among different wheat genotypes, near-isogenic lines, and cereal species, which differ in their vernalization response and FT, indicated that the gene is inducible only in those species that require vernalization, whereas it is constitutively expressed in spring habit genotypes. In addition, experiments using both the photoperiod-sensitive barley (Hordeum vulgare cv Dicktoo) and short or long day de-acclimated wheat revealed that the expression of TaVRT-1 is also regulated by photoperiod. These expression studies indicate that photoperiod and vernalization may regulate this gene through separate pathways. We suggest that TaVRT-1 is a key developmental gene in the regulatory pathway that controls the transition from the vegetative to reproductive phase in cereals.

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.

A new cold-induced alfalfa gene is associated with enhanced hardening at subzero temperature.

When alfalfa (Medicago sativa L. cv Apica) plants grown at room temperature are transferred to 2[deg]C, the temperature at which 50% of the plants fail to survive (LT50) decreases from -6 to -14[deg]C during the first 2 weeks but then increases to -9[deg]C during the subsequent 2 weeks. However, when plants are kept for 2 weeks at 2[deg]C and then transferred to -2[deg]C for another two weeks, the LT50 declines to -16[deg]C. These changes in freezing tolerance are paralleled by changes in transcript levels of cas15 (cold acclimation-specific gene encoding a 14.5-kD protein), a cold-induced gene. Cold-activation of cas15 occurs even when protein synthesis is inhibited by more than 90%, suggesting that cold-initiated events up to and including the accumulation of cas15 transcripts depend on preexisting gene products, cas15 shows little homology to any known gene at the nucleotide or amino acid level. The deduced polypeptide (CAS15) of 14.5 kD contains four repeats of a decapeptide motif and possesses a bipartite sequence domain at the carboxy terminus with homology to the reported nuclear-targeting signal sequences. Although the relative amount of cas15 DNA as a fraction of the total genomic DNA is similar in cultivars with different degrees of freezing tolerance, its organization in the genome is different. The possible role of cas15 in the development of cold-induced freezing tolerance is discussed.

Cold-regulated cereal chloroplast late embryogenesis abundant-like proteins. Molecular characterization and functional analyses.

Cold acclimation and freezing tolerance are the result of complex interaction between low temperature, light, and photosystem II (PSII) excitation pressure. Previous results have shown that expression of the Wcs19 gene is correlated with PSII excitation pressure measured in vivo as the relative reduction state of PSII. Using cDNA library screening and data mining, we have identified three different groups of proteins, late embryogenesis abundant (LEA) 3-L1, LEA3-L2, and LEA3-L3, sharing identities with WCS19. These groups represent a new class of proteins in cereals related to group 3 LEA proteins. They share important characteristics such as a sorting signal that is predicted to target them to either the chloroplast or mitochondria and a C-terminal sequence that may be involved in oligomerization. The results of subcellular fractionation, immunolocalization by electron microscopy and the analyses of target sequences within the Wcs19 gene are consistent with the localization of WCS19 within the chloroplast stroma of wheat (Triticum aestivum) and rye (Secale cereale). Western analysis showed that the accumulation of chloroplastic LEA3-L2 proteins is correlated with the capacity of different wheat and rye cultivars to develop freezing tolerance. Arabidopsis was transformed with the Wcs19 gene and the transgenic plants showed a significant increase in their freezing tolerance. This increase was only evident in cold-acclimated plants. The putative function of this protein in the enhancement of freezing tolerance is discussed.

The CBF gene family in hexaploid wheat and its relationship to the phylogenetic complexity of cereal CBFs.

Most temperate plants tolerate both chilling and freezing temperatures whereas many species from tropical regions suffer chilling injury when exposed to temperatures slightly above freezing. Cold acclimation induces the expression of cold-regulated genes needed to protect plants against freezing stress. This induction is mediated, in part, by the CBF transcription factor family. To understand the evolution and function of this family in cereals, we identified and characterized 15 different CBF genes from hexaploid wheat. Our analyses reveal that wheat species, T. aestivum and T. monococcum, may contain up to 25 different CBF genes, and that Poaceae CBFs can be classified into 10 groups that share a common phylogenetic origin and similar structural characteristics. Six of these groups (IIIc, IIId, IVa, IVb, IVc and IVd) are found only in the Pooideae suggesting they represent the CBF response machinery that evolved recently during colonization of temperate habitats. Expression studies reveal that five of the Pooideae-specific groups display higher constitutive and low temperature inducible expression in the winter cultivar, and a diurnal regulation pattern during growth at warm temperature. The higher constitutive and inducible expression within these CBF groups is an inherited trait that may play a predominant role in the superior low temperature tolerance capacity of winter cultivars and possibly be a basis of genetic variability in freezing tolerance within the Pooideae subfamily.

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 molecular marker to select for freezing tolerance in Gramineae

SummaryWe isolated, and expressed in Escherichia coli, a gene (Wcs120) that is strongly induced during cold acclimation of wheat. The gene product was purified and used to produce antibodies. Immunoblotting experiments with the anti-WCS120 antibody identified several cold-induced proteins named FTMs for Freezing Tolerance Markers since they are associated with the development of freezing tolerance. This protein family was found to be coordinately regulated specifically by low temperature, highly hydrophilic, stable to boiling, and to have a pI above 6.5. The accumulation kinetics during the acclimation period indicated a positive correlation with the capacity of each genotype to develop freezing tolerance. Accumulation of the proteins was higher in the freezing-tolerant genotype than in the less tolerant one. In addition, their accumulation was more pronounced in the crown and leaf tissues compared with roots, confirming a relationship to the capacity of the different tissues to develop freezing tolerance. Analysis of different species (eight monocots and four dicots) indicated that this protein family is specific for freezing-tolerant cereals. The antibody did not cross-react with any of the non-cereal species examined. The anti-FTMs antibody represents a potential tool for breeders to select for freezing tolerance traits in the Gramineae.

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.