The metabolic and developmental roles of carotenoid cleavage dioxygenase4 from potato

Abstract

The factors that regulate storage organ carotenoid content remain to be fully elucidated, despite the nutritional and economic importance of this class of compound. Recent findings suggest that carotenoid pool size is determined, at least in part, by the activity of carotenoid cleavage dioxygenases. The aim of this study was to investigate whether Carotenoid Cleavage Dioxygenase4 (CCD4) activity affects potato (Solanum tuberosum) tuber carotenoid content. Microarray analysis revealed elevated expression of the potato CCD4 gene in mature tubers from white-fleshed cultivars compared with higher carotenoid yellow-fleshed tubers. The expression level of the potato CCD4 gene was down-regulated using an RNA interference (RNAi) approach in stable transgenic lines. Down-regulation in tubers resulted in an increased carotenoid content, 2- to 5-fold higher than in control plants. The increase in carotenoid content was mainly due to elevated violaxanthin content, implying that this carotenoid may act as the in vivo substrate. Although transcript level was also reduced in plant organs other than tubers, such as leaves, stems, and roots , there was no change in carotenoid content in these organs. However, carotenoid levels were elevated in flower petals from RNAi lines. As well as changes in tuber carotenoid content, tubers from RNAi lines exhibited phenotypes such as heat sprouting, formation of chain tubers, and an elongated shape. These results suggest that the product of the CCD4 reaction may be an important factor in tuber heat responses.

Figure 1.

CCD4 (MICRO.13362.C1) gene expression profile in tubers from Phureja (333-16, Mayan Gold) and Tuberosum (Desiree, Maris Piper) cultivars at two stages of tuber development (H1, developing tubers of 10–30 g fresh weight; H4, mature tubers at harvest) as determined by microarray analysis. Values are means of two biological replicates, and error bars represent se.

Figure 2.

CCD4 relative expression in different organs of the potato plant (cv Desiree). Values are means of triplicate assays ± se for tissues sampled from three replicate plants. DT, Developing tuber; SS, swelling stolon; S, stolon; St, stem; R, root; L, leaf; F, flower. Values are means of triplicate assays ± se for tissues sampled from three replicate plants.

Figure 3.

Expression levels (intensity values) of the annotated CCDs present on the POCI 44-K element Agilent microarray in CCD4 RNAi transgenic lines compared with Desiree controls as determined by microarray analysis. Sequences are named according to their unigene identifiers available at the POCI Web site (http://pgrc-35.ipk-gatersleben.de/pls/htmldb_pgrc/f?p=194:1:1942898893016362). For control lines, microarray data are mean values from two biological replicates from two wild-type and two empty vector plants. For the RNAi lines, data are mean values from one plant from each of the four RNAi lines that show the tuber color phenotype (lines 2, 4, 15, and 38). Error bars represent se. Gene names of the best BLAST hits have been included in Supplemental Table S1.

Figure 4.

CCD4 expression level in flower (A), stem (B), leaf (C), and root (D) in CCD4 RNAi lines, empty vector (EV) controls, and wild-type (WT) Desiree. Values are means of triplicate assays ± se for tissues sampled from three replicate plants and are expressed relative to the value in wild-type samples.

Figure 5.

Phenotypes of CCD4 RNAi tubers. A, Control tubers from wild-type Desiree grown under identical conditions to RNAi lines. B, Tubers from RNAi line 38 exhibiting dumbbell shape or chain tubers. C, Developing tubers from RNAi line 4 exhibiting a sprouting phenotype evident at harvest. D, Microtubers from RNAi line 15 exhibiting dumbbell shape, chain tubers, and sprouting during tuber development. E, Control microtubers grown under identical conditions to the RNAi microtubers.

Figure 6.

Tuber length-to-width ratios of CCD4 RNAi tubers compared with controls. Values shown are for mature tubers. Values are means of nine replicates pooled from three independent plants (se is shown for tuber length-to-width ratios). WT, Wild-type Desiree; EV, empty vector.