Novel insight into vascular, stress, and auxin-dependent and -independent gene expression programs in strawberry, a non-climacteric fruit

Abstract

Using cDNA microarrays, a comprehensive investigation of gene expression was carried out in strawberry (Fragaria x ananassa) fruit to understand the flow of events associated with its maturation and non-climacteric ripening. We detected key processes and novel genes not previously associated with fruit development and ripening, related to vascular development, oxidative stress, and auxin response. Microarray analysis during fruit development and in receptacle and seed (achene) tissues established an interesting parallelism in gene expression between the transdifferentiation of tracheary elements in Zinnia elegans and strawberry. One of the genes, CAD, common to both systems and encoding the lignin-related protein cinnamyl alcohol dehydrogenase, was immunolocalized to immature xylem cells of the vascular bundles in the strawberry receptacle. To examine the importance of oxidative stress in ripening, gene expression was compared between fruit treated on-vine with a free radical generator and non-treated fruit. Of 46 genes induced, 20 were also ripening regulated. This might suggest that active gene expression is induced to cope with oxidative stress conditions during ripening or that the strawberry ripening transcriptional program is an oxidative stress-induced process. To gain insight into the hormonal control of non-climacteric fruit ripening, an additional microarray experiment was conducted comparing gene expression in fruit treated exogenously with auxin and control fruit. Novel auxin-dependent genes and processes were identified in addition to transcriptional programs acting independent of auxin mainly related to cell wall metabolism and stress response.

Figure 1

Using cDNA microarrays to follow gene expression patterns in strawberry fruit during development, in different tissues, under oxidative stress conditions and hormonal treatment. A, Strawberry fruit developmental stages. 1, Small flower bud; 2, large flower bud; 3, anthesis; 4, small green; 5, medium green; 6, large green; 7, small white; 8, large white; 9, turning; 10, red. Stages 5 and 8–10 were used for microarray experiments (referred to as G, W, T, and R, respectively). B, Experimental schematic diagram of first (I–IV) and second (V and VI) generation microarray experiments.

Figure 2

Differential gene expression in strawberry fruit. The diagrams show the numbers of overlapping and nonoverlapping genes differentially expressed during fruit development (Dev), receptacle associated (RecA), and achene associated (AchA) as detected in experiments I through IV (A). Those genes are auxin repressed (AuxRep), ripening regulated (RipR), receptacle associated (RecA), and stress induced (StrInd), detected in experiments V and VI and based on experiments I through IV (B).

Figure 3

The vascular system and lignin-associated gene expression and protein localization in strawberry fruit. A, RNA gel-blot analysis of strawberry CCR and CAD expression in various strawberry tissues and during fruit development. 1, Petiole; 2, leaf; 3, flower; 4, green fruit; 5, white fruit; 6, turning fruit; 7, red fruit; 8, red fruit without achenes; 9, achenes; 10, overripe fruit. The entire strawberry CCR and CAD cDNAs were used as probes for hybridizations. B, Section of a green and red ripe strawberry fruit showing fibrovascular strands (vb, vascular bundles) connecting the achenes (a) to the interior of the receptacle (p, pith). C and D, Presence of lignin in the vascular system (xylem vessels) in the receptacle, visualized after staining with phloroglucinol, E and F, Cross sections of the receptacle stained by immunolocalization of CAD in the lignified vascular tissue (immature xylem) with the strawberry anti-CAD (F193) antiserum. G and H, Receptacle stained by immunolocalization with pre-immune antiserum (negative controls). Sections C, E, and G and D, F, and H are green and red stage strawberry receptacle, respectively. In C and F, bar = 12 μm; in D and E, bar = 6 μm; in H and G, bar = 7 μm.

Figure 4

Identification of ripening-regulated genes induced by oxidative stress. A, Ferritin gene expression in strawberry fruit after treatment with the free radical generator AAPH. Two RNA gel-blot experiments were performed: I, comparing expression in non-treated fruit (Ntr), fruit treated with buffer only (buffer), and treated with buffer containing 100 mm AAPH (100 mm); II, comparing expression in fruit treated with 10 and 100 mm AAPH (depicted as 10 and 100 mm, respectively). Gene expression in fruit treated with 100 mm AAPH and control fruit treated with buffer only was compared using the second generation microarray (experiment V). B, Graph representing cDNAs shown to be ripening regulated from microarray experiments (I–III) and oxidative stress induced from microarray experiment V. Gray bars represent genes showing differential and elevated expression in receptacle tissue compared with achenes, whereas black bars represent genes showing no differential expression between the two tissues as deduced from experiment IV. The number in parentheses after the putative definitions represent the number of the sequence contig in the case when more than one sequence showed a similar BLAST result but did not align in the sequence alignment. Genes marked with an asterisk did not show any change in expression upon application of exogenous auxin (as detected by experiment VI).