Allelic variation of a dehydrin gene cosegregates with chilling tolerance during seedling emergence

Abstract

Dehydrins (DHNs, LEA D-11) are plant proteins present during environmental stresses associated with dehydration or low temperatures and during seed maturation. Functions of DHNs have not yet been defined. Earlier, we hypothesized that a approximately 35-kDa DHN and membrane properties that reduce electrolyte leakage from seeds confer chilling tolerance during seedling emergence of cowpea (Vigna unguiculata L. Walp.) in an additive and independent manner. Evidence for this hypothesis was not rigorous because it was based on correlations of presence/absence of the DHN and slow electrolyte leakage with chilling tolerance in closely related cowpea lines that have some other genetic differences. Here, we provide more compelling genetic evidence for involvement of the DHN in chilling tolerance of cowpea. We developed near-isogenic lines by backcrossing. We isolated and determined the sequence of a cDNA corresponding to the approximately 35-kDa DHN and used gene-specific oligonucleotides derived from it to test the genetic linkage between the DHN presence/absence trait and the DHN structural gene. We tested for association between the DHN presence/absence trait and both low-temperature seed emergence and electrolyte leakage. We show that allelic differences in the Dhn structural gene map to the same position as the DHN protein presence/absence trait and that the presence of the approximately 35-kDa DHN is indeed associated with chilling tolerance during seedling emergence, independent of electrolyte leakage effects. Two types of allelic variation in the Dhn gene were identified in the protein-coding region, deletion of one Phi-segment from the DHN-negative lines and two single amino acid substitutions.

Figure 1

Percentage emergence of cowpea lines with (open symbols) or without (closed symbols) the ≈35-kDa DHN at 14°C using seeds with 6% moisture content. Parental lines are indicated by dotted lines and the vertical bars are the standard errors.

Figure 2

(a) Deduced amino acid sequence of the ≈35-kDa cowpea dehydrin cDNA with the amino acid sequence of the putative-coding region presented in a single-letter code. The Φ-segment landmarks are shown in boldface. (b) Schematic representation showing the Y₂K structure with eight repeats (Φ-segments), six of them with 14-aa residues and two with 10 residues each. Arrows indicate positions of the PCR primers. (c) PCR products from each line; note that Φ₇ is deleted in the alleles represented by smaller PCR products.

Figure 3

PCR products (Upper) and Western blots (Lower) from parental cowpea lines and a subset of the recombinant inbred (RI) lines (a) and cowpea near-isogenic lines and parents (b). Total DNA was isolated from etiolated young shoots for PCR analysis. Total protein was extracted from dry, mature seeds for Western blot analysis.