▪ Abstract Many plants increase in freezing tolerance upon exposure to low nonfreezing temperatures, a phenomenon known as cold acclimation. In this review, recent advances in determining the nature and function of genes with roles in freezing tolerance and the mechanisms involved in low temperature gene regulation and signal transduction are described. One of the important conclusions to emerge from these studies is that cold acclimation includes the expression of certain cold-induced genes that function to stabilize membranes against freeze-induced injury. In addition, a family of Arabidopsis transcription factors, the CBF/DREB1 proteins, have been identified that control the expression of a regulon of cold-induced genes that increase plant freezing tolerance. These results along with many of the others summarized here further our understanding of the basic mechanisms that plants have evolved to survive freezing temperatures. In addition, the findings have potential practical applications as freezing te...

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PLANT COLD ACCLIMATION: Freezing Tolerance Genes and Regulatory Mechanisms

Summary
The diets of over two-thirds of the world's population lack one or more essential mineral elements. This can be remedied through dietary diversification, mineral supplementation, food fortification, or increasing the concentrations and/or bioavailability of mineral elements in produce (biofortification). This article reviews aspects of soil science, plant physiology and genetics underpinning crop biofortification strategies, as well as agronomic and genetic approaches currently taken to biofortify food crops with the mineral elements most commonly lacking in human diets: iron (Fe), zinc (Zn), copper (Cu), calcium (Ca), magnesium (Mg), iodine (I) and selenium (Se). Two complementary approaches have been successfully adopted to increase the concentrations of bioavailable mineral elements in food crops. First, agronomic approaches optimizing the application of mineral fertilizers and/or improving the solubilization and mobilization of mineral elements in the soil have been implemented. Secondly, crops have been developed with: increased abilities to acquire mineral elements and accumulate them in edible tissues; increased concentrations of ‘promoter’ substances, such as ascorbate, β-carotene and cysteine-rich polypeptides which stimulate the absorption of essential mineral elements by the gut; and reduced concentrations of ‘antinutrients’, such as oxalate, polyphenolics or phytate, which interfere with their absorption. These approaches are addressing mineral malnutrition in humans globally.

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Biofortification of crops with seven mineral elements often lacking in human diets--iron, zinc, copper, calcium, magnesium, selenium and iodine.

Winter wheats require several weeks at low temperature to flower. This process, vernalization, is controlled mainly by the VRN1 gene. Using 6,190 gametes, we found VRN1 to be completely linked to MADS-box genes AP1 and AGLG1 in a 0.03-centimorgan interval flanked by genes Cysteine and Cytochrome B5. No additional genes were found between the last two genes in the 324-kb Triticum monococcum sequence or in the colinear regions in rice and sorghum. Wheat AP1 and AGLG1 genes were similar to Arabidopsis meristem identity genes AP1 and AGL2, respectively. AP1 transcription was regulated by vernalization in both apices and leaves, and the progressive increase of AP1 transcription was consistent with the progressive effect of vernalization on flowering time. Vernalization was required for AP1 transcription in apices and leaves in winter wheat but not in spring wheat. AGLG1 transcripts were detected during spike differentiation but not in vernalized apices or leaves, suggesting that AP1 acts upstream of AGLG1. No differences were detected between genotypes with different VRN1 alleles in the AP1 and AGLG1 coding regions, but three independent deletions were found in the promoter region of AP1. These results suggest that AP1 is a better candidate for VRN1 than AGLG1. The epistatic interactions between vernalization genes VRN1 and VRN2 suggested a model in which VRN2 would repress directly or indirectly the expression of AP1. A mutation in the promoter region of AP1 would result in the lack of recognition of the repressor and in a dominant spring growth habit.

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Positional cloning of the wheat vernalization gene VRN1

A number of proteins have been identified that typically accumulate in plants in response to any environmental stimulus that has a dehydrative component or is temporally associated with dehydration. This includes drought, low temperature, salinity and seed maturation. Among the induced proteins, dehydrins (late embryogenesis abundant [LEA] D-II family) have been the most commonly observed, yet we still have an incomplete knowledge of their fundamental biochemical role in the cell. Current research trends are changing this situation: immunolocalization and in vitro biochemical analyses are, through analogies to other more fully characterized proteins and molecules, shaping our understanding. In brief, dehydrins may be structure stabilizers with detergent and chaperone-like properties and an array of nuclear and cytoplasmic targets. Recent progress on the mapping of dhn genes and the inheritance of freezing tolerance in barley and other Triticeae species tentatively points to dehydrins as key components of dehydration tolerance.

Dehydrins: Emergence of a biochemical role of a family of plant dehydration proteins

Changes in cytosolic free calcium concentration ([Ca2+]cyt) in response to mannitol (drought) and salt treatments were detected in vivo in intact whole Arabidopsis seedlings. Transient elevations of [Ca2+]cyt to around 1.5 microM were observed, and these were substantially inhibited by pretreatment with the calcium-channel blocker lanthanum and to a lesser extent, the calcium-chelator EGTA. The expression of three genes, p5cs, which encodes delta(1)-pyrroline-5-carboxylate synthetase (P5CS), the first enzyme of the proline biosynthesis pathway, rab18 and Iti78 which both encode proteins of unknown function, was induced by mannitol and salt treatments. The induction of all three genes by mannitol was inhibited by pretreatment with lanthanum. Salt-induced p5cs, but not rab18 and Iti78, expression was also inhibited by lanthanum. Induction of p5cs by mannitol was also inhibited by the calcium channel-blockers gadolinium and verapamil and the calcium chelator EGTA, further suggesting the involvement of calcium signalling in this response. Mannitol induced greater levels of p5cs gene expression than an isoosmolar concentration of salt, at both relatively high and low concentrations. However, calcium transients were of a similar magnitude and duration in response to both mannitol and isoosmolar concentrations of salt, suggesting that a factor other than calcium is involved in the discrimination between drought and salinity signals in Arabidopsis. In order to gauge the involvement of the vacuole as an intracellular calcium store in the response of Arabidopsis to mannitol, [Ca2+]cyt was measured at the microdomain adjacent to the vacuolar membrane. The results obtained were consistent with a significant calcium release from the vacuole contributing to the overall mannitol-induced [Ca2+]cyt response. Data obtained by using inhibitors of inositol signalling suggested that this release was occurring through IP3-dependent calcium channels.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.1997.12051067.x

Calcium signalling in Arabidopsis thaliana responding to drought and salinity

A population of single chromosome recombinant lines was developed from the cross between a frost-sensitive, vernalization-insensitive substitution line, 'Chinese Spring' (Triticum spelta 5A) and a frost-tolerant, vernalization-sensitive line, 'Chinese Spring' ('Cheyenne' 5A), and used to map the genes Vrn1 and Fr1 controlling vernalization requirement and frost tolerance, respectively, relative to RFLP markers located on this chromosome. The Vrn1 and Fr1 loci were located closely linked on the distal portion of the long arm of 5AL, but contrary to previous observations, recombination between them was found. Three RFLP markers, Xpsr426, Xcdo504 and Xwg644 were tightly linked to both. The location of Vrn1 suggests that it is homoeologous to other spring habit genes in related species, particularly the Sh2 locus on chromosome 7 (5H) of barley and the Sp1 locus on chromosome 5R of rye.

RFLP mapping of the vernalization (Vrn1) and frost resistance (Fr1) genes on chromosome 5A of wheat.

A population of single chromosome recombi- nant lines was developed from the cross between a frost- sensitive, vernalization-insensitive substitution line, 'Chi- nese Spring' (Triticum spelta 5A) and a frost-tolerant, ver- nalization-sensitive line, 'Chinese Spring' ('Cheyenne' 5A), and used to map the genes Vrnl and Frl controlling vernalization requirement and frost tolerance, respectively, relative to RFLP markers located on this chromosome. The Vrnl and Frl loci were located closely linked on the dis- tal portion of the long arm of 5AL, but contrary to previ- ous observations, recombination between them was found. Three RFLP markers, Xpsr426, Xcdo504 andXwg644 were tightly linked to both. The location of Vrnl suggests that it is homoeologous to other spring habit genes in related species, particularly the Sh2 locus on chromosome 7 (5H) of barley and the Spl locus on chromosome 5R of rye.

RFLP mapping of the vernalization (Vrnl) and frost resistance (Frl) genes on chromosome 5A of wheat

Two populations of single chromosome recombinant lines were used to map genes controlling flowering time on chromosome 5B of wheat, and one of the populations was also used to map a new frost resistance gene. Genetic maps were developed, mainly using microsatellite markers, and QTL analysis was applied to phenotypic data on the performance of each population collected from growth-room tests of flowering time and frost tolerance. Using a recombinant substitution-line mapping population derived from a cross between the substitution-line 'Chinese Spring' ('Cheyenne' 5B) and 'Chinese Spring' (CS), the gene Vrn-B1, affecting vernalization response, an earliness per se locus, Eps-5BL1, and a gene, Fr-B1, affecting frost resistance, were mapped. Using a 'Hobbit Sib' ('Chinese Spring' 5BL) × 'Hobbit Sib' recombinant substitution line mapping population, an earliness per se locus, Eps-5BL2 was mapped. The Vrn-B1 locus was mapped on the distal portion of the long arm of chromosome 5B, to a region syntenous with the segments of chromosomes 5A and 5D containing Vrn-A1 and Vrn-D1 loci, respectively. The two Eps-5BL loci were mapped close to the centromere with a 16-cM distance from each other, one in agreement with the position of a homoeologous locus previously mapped on chromosome 5H of barley, and suggested by the response of 'Chinese Spring' deletion lines. The Fr-B1 gene was mapped on the long arm of chromosome 5B, 40 cM from the centromeric marker. Previous comparative mapping data with rice chromosome 9 would suggest that this gene could be orthologous to the other Fr genes mapped previously by us on chromosomes 5A or 5D of wheat, although in a more proximal position. This study completes the mapping of these homoeoallelic series of vernalization requirement genes and frost resistance genes on the chromosomes of the homoeologous group 5 in wheat.

Mapping genes affecting flowering time and frost resistance on chromosome 5B of wheat

Genetic studies of frost resistance were performed on various wheat varieties using diallel, F2 monosomic and substitution analysis. A six-parental cross including reciprocals was carried out, and F1 hybrids and their parents were used for the freezing tests under controlled conditions. Both the general combining ability (GCA) and the specific combining ability (SCA) were significant, indicating additive and non-additive gene action in the inheritance of frost resistance. The high GCA∶SCA ratio revealed a preponderance of additive genetic variance. No significant reciprocal differences were found between the reciprocal crosses. The variance/covariance graphical analysis indicated the partial dominance of frost sensitivity. Frost sensitive varieties had the largest number of dominant genes, while frost resistant varieties had the highest proportion of recessive genes. The magnitude of the additive component of variation was higher than that of the dominance component, and the overall measure of the degree of dominance was smaller than one, so average dominance is incomplete. The increasing and decreasing alleles are not equally frequent at all loci. In this set of wheat varieties the values of narrow and broad heritability are relatively high. F2 monosomic analysis of the winter wheat variety ‘Arthur’ crossed with the monosomics of ‘Chinese Spring’ revealed that the average frost resistance of all the 21 monosomics was lower than that of the disomic. F2 monosomic hybrids 5A, 2B, 4B and 5D proved to be relatively frost resistant, while monosomics 3A, 3B and 6D were the most sensitive. The control of frost resistance in the set of chromosome substitution lines of the variety ‘Cheyenne’ into ‘Chinese Spring’ (with the exception of 2B) indicated that the genes responsible for the frost resistance of ‘Cheyenne’ are localised in chromosomes 5A, 7A, 4B, 5B, 4D and 5D. The genetic basis of frost resistance and problems of analysis are discussed.

Genetic studies of frost resistance in wheat

A gene for frost resistance on chromosome 5A of wheat was located using single chromosome recombinant lines from the cross between the substitution line Hobbit (Triticum spelta 5A) and Hobbit. In this sample of recombinant lines the locus for frost resistance, designated Fr1, is completely linked to the locus Vrn1 controlling vernalisation requirement. The results can be explained by a pleiotropic action of the Vrn1 locus or close genetic linkage between Vrn1 and Fr1. Further detailed study is necessary to resolve these alternative hypotheses.

József Sutka

Papers

RFLP mapping of the vernalization (Vrn1) and frost resistance (Fr1) genes on chromosome 5A of wheat.

RFLP mapping of the vernalization (Vrnl) and frost resistance (Frl) genes on chromosome 5A of wheat

Mapping genes affecting flowering time and frost resistance on chromosome 5B of wheat

Genetic studies of frost resistance in wheat

Location of a gene for frost resistance on chromosome 5A of wheat