Auxin-regulated genes encoding cell wall-modifying proteins are expressed during early tomato fruit growth

Abstract

An expansin gene, LeExp2, was isolated from auxin-treated, etiolated tomato (Lycopersicon esculentum cv T5) hypocotyls. LeExp2 mRNA expression was restricted to the growing regions of the tomato hypocotyl and was up-regulated during incubation of hypocotyl segments with auxin. The pattern of expression of LeExp2 was also studied during tomato fruit growth, a developmental process involving rapid cell enlargement. The expression of genes encoding a xyloglucan endotransglycosylase (LeEXT1) and an endo-1, 4-beta-glucanase (Cel7), which, like LeExp2, are auxin-regulated in etiolated hypocotyls (C. Catalá, J.K.C. Rose, A.B. Bennett [1997] Plant J 12: 417-426), was also studied to examine the potential for synergistic action with expansins. LeExp2 and LeEXT1 genes were coordinately regulated, with their mRNA accumulation peaking during the stages of highest growth, while Cel7 mRNA abundance increased and remained constant during later stages of fruit growth. The expression of LeExp2, LeEXT1, and Cel7 was undetectable or negligible at the onset of and during fruit ripening, which is consistent with a specific role of these genes in regulating cell wall loosening during fruit growth, not in ripening-associated cell wall disassembly.

Figure 1

Phylogenetic tree of the alignment of LeExp2 deduced amino acid sequence with other α-expansins. Protein sequences were aligned using CLUSTAL, and a phylogenetic tree was constructed using PHYLIP software with the PROTPARS program. Numbers above the branches indicate the bootstrap values. A Phleum pollen allergen (Phlp1), identified as a β-expansin, was used as an outgroup. Full details and accession numbers are given in “Materials and Methods.”

Figure 2

Genomic DNA analysis of LeExp2. Genomic DNA (10 μg per lane) was digested with the indicated restriction enzymes, gel blot hybridized with the LeExp2 cDNA probe, and washed with 0.2× SSC, 0.5% (w/v) SDS at 65°C (8°C below the Tm).

Figure 3

Analysis of LeExp2 mRNA abundance in tomato vegetative tissues. a, Poly(A⁺) RNA-blot analysis of LeExp2 expression in vegetative tissues. Each lane contained 1 μg of poly(A⁺) RNA from leaf, stem, hypocotyl, or root tissues. b, Total RNA gel-blot analysis of LeExp2 expression along the tomato etiolated hypocotyl. Each lane contained 15 μg of total RNA isolated from consecutive 1-cm regions of the hypocotyl. Ethidium bromide staining of the gel is shown below the blot as a loading control. The growth rates of regions A through D were measured in 15 seedlings over a 12-h period, as described in “Materials and Methods.” RNA gel blots were hybridized and washed at the same stringency as in Figure 2.

Figure 4

Auxin regulation of LeExp2 mRNA levels in etiolated hypocotyl segments. a, Effect of auxin concentration on LeExp2 accumulation. Apical segments were pre-incubated for 2 h in buffer and then transferred to fresh buffer plus the indicated auxin concentration and incubated for 24 h. NI, Non-incubated; BR, brassinolide. b, Time course of LeExp2 mRNA accumulation. Apical segments were pre-incubated for 2 h in buffer (0-h time point) and then for the indicated times in buffer alone (control) or buffer plus 5 μm 2,4-D. c, Effect of other auxins on LeExp2 mRNA accumulation. Apical segments were pre-incubated for 2 h in buffer (0-h time point) and then for 24 h in buffer alone (control) or buffer plus the indicated concentration of 2,4-D, αNAA, or IAA. After incubation, RNA was isolated and total RNA-blot analysis (15 μg per lane) was performed. Ethidium bromide staining of the gel is shown below the blots as a loading control. RNA gel blots were hybridized and washed at the same stringency as in Figure 2.

Figure 5

Expression of expansin (LeExp2), XET (LeEXT1), and EGase (Cel7) genes during tomato fruit development. Flowers were tagged at anthesis and fruit diameter measured over a period of 50 d. A regression curve was established from data obtained with 15 fruit. Poly(A⁺) RNA-blot analysis of LeExp2, LeEXT1, and Cel7 expression was done during tomato fruit development. Northern blots (1 μg per lane) were hybridized successively with the LeExp2, LeEXT1, Cel7, and actin cDNA probes. (I, 0.5- to 1.0-cm diameter fruit; II, 2- to 3-cm diameter fruit; III, 4- to 6-cm diameter fruit; IG, immature green; MG, mature green; Br, breaker; Tu, turning; Pi, pink; LR, light red; RR, red-ripe). RNA gel blots were hybridized and washed at the same stringency as in Figure 2.