Characterization of a gene for spinach CAP160 and expression of two spinach cold-acclimation proteins in tobacco

Abstract

The cDNA sequence for CAP160, an acidic protein previously linked with cold acclimation in spinach (Spinacia oleracea L.), was characterized and found to encode a novel acidic protein of 780 amino acids having very limited homology to a pair of Arabidopsis thaliana stress-regulated proteins, rd29A and rd29B. The lack of similarity in the structural organization of the spinach and Arabidopsis genes highlights the absence of a high degree of conservation of this cold-stress gene across taxonomic boundaries. The protein has several unique motifs that may relate to its function during cold stress. Expression of the CAP160 mRNA was increased by low-temperature exposure and water stress in a manner consistent with a probable function during stresses that involve dehydration. The coding sequences for CAP160 and CAP85, another spinach cold-stress protein, were introduced into tobacco (Nicotiana tabacum) under the control of the 35S promoter using Agrobacterium tumefaciens-based transformation. Tobacco plants expressing the proteins individually or coexpressing both proteins were evaluated for relative freezing-stress tolerance. The killing temperature for 50% of the cells of the transgenic plants was not different from that of the wild-type plants. As determined by a more sensitive time/temperature kinetic study, plants expressing the spinach proteins had slightly lower levels of electrolyte leakage than wild-type plants, indicative of a small reduction of freezing-stress injury. Clearly, the heterologous expression of two cold-stress proteins had no profound influence on stress tolerance, a result that is consistent with the quantitative nature of cold-stress-tolerance traits.

Figure 1

Constructs used in the tobacco transformation experiments (Malik and Wahab, 1993). The right (RB) and left (LB) borders are indicated by the open boxes at the ends of the constructs.

Figure 2

RNA-blot analyses of the influence of cold acclimation, deacclimation, water stress, and heat shock on the CAP160 steady-state mRNA abundance. A, Acclimation, deacclimation, and water-stress responses. B, Short-term response to low temperature. C, Response to heat shock. The cDNA hybridizes to a major 2.7-kb mRNA. Each lane was loaded with 5 μg of total RNA, and equal loading was verified by ethidium-bromide staining before blotting. Similar results were observed in several hybridization experiments. Details regarding the physiologic parameters for the water-stressed samples have been published elsewhere (Guy et al., 1992; Neven et al., 1993).

Figure 3

Nucleotide and deduced amino acid sequence of CAP160 clone IIa. Amino acids in boldface and underlined correspond to cyanogen bromide-generated peptide sequences from purified CAP160. The dark-shaded box denotes a motif rich in negatively charged residues, and the lighter-shaded box denotes a repeat sequence. An imperfectly tandem repeated sequence is double underlined, and a repeating four-residue motif of P(I/S)(T/K)(G/W) is underlined.

Figure 4

CAP160 and CAP85 protein expression in E. coli XL1-Blue cells. A, Lane 1, pBluescript without an insert; lane 2, CAP160 clone IIa; lane 3, clone IIb; lane 4, clone VIII; lane 5, clone VII; lane 6, clone IX; lane 7, spinach leaf CAP160; lane 8, soluble fraction of pBluescript without an insert minus IPTG; lane 9, insoluble fraction of pBluescript without an insert minus IPTG; lane 10, soluble fraction of pBluescript without an insert plus IPTG; lane 11, insoluble fraction of pBluescript without an insert plus IPTG; lane 12, soluble fraction of CAP160 IIa minus IPTG; lane 13, insoluble fraction of CAP160 IIa minus IPTG; lane 14, soluble fraction of CAP160 IIa plus IPTG; lane 15, insoluble fraction of CAP160 IIa plus IPTG; and lane 16, spinach leaf CAP160. B, Lane 1, Spinach leaf CAP85; lanes 2 and 3, CAP85 out-of-frame clone S42a plus IPTG; and lanes 4 and 5, CAP85 in-frame clone S42aE plus IPTG.

Figure 5

Alignment of CAP160 protein sequence with the Arabidopsis rd29A and rd29B proteins. Identical residues are indicated by dots.

Figure 6

Genomic DNA blot for CAP160. Results of three different experiments are shown. Clone IIa was used to make the hybridization probe.

Figure 7

Structure of the CAP160 gene. Flanking sequences are indicated by dark shading, noncoding and intron sequences are lightly shaded, and exons are shown as unshaded boxes. The hatched area in intron 1 shows the position of a large deletion present in some sequences.

Figure 8

Subcellular localization of CAP160. The presence of CAP160 was detected by protein blot using a monoclonal antibody, 2H8, which is specific for CAP160. For marker protein analysis and relative contamination for each subcellular fraction, see Neven et al. (1993).

Figure 9

Analysis of spinach CAP160 RNA and protein expression in transgenic lines of tobacco. Spinach RNA and protein are shown for reference. Nt10 was transformed with the vector only. Hybridizations were done with radiolabeled cDNA clone IIa, and protein detection was with monoclonal antibody 2H8. RNA blots were loaded with 5 μg of total RNA per lane. Protein blots were loaded with 20 μg of total buffer-soluble protein per lane.

Figure 10

Analysis of spinach CAP85 expression in tobacco (A) and coexpression of CAP85 and CAP160 in progeny from a cross of lines expressing individually either CAP85 or CAP160 (B). Cross-reacting proteins were detected with a mixture of monoclonal antibody 2H8 for CAP160 and monoclonal antibody 5A10 for CAP85.

Figure 11

Influence of CAP85 or CAP160 expression or coexpression of both proteins on the severity of injury after freezing. Error bars represent the sd of two to four experiments.

Figure 12

Differential sensitivity of tobacco expressing spinach cold-stress proteins in response to a time-course freeze/thaw regime. Error bars represent the sd of five experiments.