Transcriptomic analysis of potato tuber development and tuber quality traits using microarray technology

The potato crop is one of the most important food crops in the world and in order to understand how in potato plants the formation of potato tubers and the determination of different tuber quality traits are regulated, we need to identify and characterize the genes that are involved in regulating these processes. The aim of the research described in this PhD thesis was to implement a relatively new genomic tool (i.e. microarray technology) in an ongoing study to understand the process of potato tuber development and the mechanisms underlying differences in tuber quality traits through the identification and analysis of candidate genes. The induction and formation of a potato tuber from underground stem-like structures, the stolons, follows a developmental program that requires the coordinated regulation of many metabolic pathways. We have developed a dedicated potato cDNA-microarray specifically designed to study the different aspects of the potato tuber life cycle. Genes selected for representation on the dedicated array were retrieved from EST databases using a text mining approach based on functional homology with genes thought to be linked to the process of tuber formation or determining tuber quality traits.

In a large scale in vivo tuberization experiment we have analyzed the expression profiles of genes during the transition of a stolon into a tuber and subsequent tuber growth stages using the dedicated potato cDNA-microarray. Expression analysis of eight tuber developmental stages revealed both unique as well as more common gene expression profiles including, up-regulation, down-regulation and transiently up- or down regulation at tuber organogenesis. Based on the observed expression profiles during tuber development, tissue specificity studies and functional homology, promising candidate genes were identified and selected for further analysis. Reverse genetic approaches were implemented to identify functional roles for candidate genes in relation to the process of tuber development or quality traits. One of the most intriguing candidate genes that we identified showed homology to a GA2-oxidase gene involved in the breakdown of bioactive gibberellins (GA) and was named StGA2ox1 . GA is a plant hormone that has been shown to be an important regulator of tuber formation. When potato plants are induced to tuberize, stolon growth ceases and a reduction in the levels of GA is thought to result in the longitudinal reorientation of the cell micro tubules and micro fibrils, allowing lateral cell expansion and division. StGA2ox1 is up-regulated during the early stages of potato tuber development prior to visible swelling. Transgenic clones over-expressing StGA2ox1 exhibit a dwarfed phenotype, reduced stolon length and earlier in vitro tuberization. Transgenic plants with reduced expression levels of StGA2ox1 showed normal plant growth, an altered stolon swelling phenotype and delayed in vitro tuberization. Furthermore, tubers of silencing clones contain increased levels of GA ₂₀ , a precursor of bioactive GAs, indicating altered gibberellin metabolism. Based on these results we propose a role for StGA2ox1 in tuber development through the action of regulating GA levels in the subapical stolon region during early tuberization events.

Another candidate gene that was selected for gene function assessment showed homology to an Aux/IAA protein and was named StIAA2 . Aux/IAA proteins are short-lived transcription factors that can promote or repress auxin induced gene expression under the control of the plant hormone auxin. Auxins have long been implicated to play a regulatory role in potato tuber development. However, a precise function for this plant hormone in tuber development has never been established. During the early stages of potato tuber development StIAA2 transcript levels are dramatically reduced indicating strong transcriptional control. Targeted down-regulation of StIAA2 transcript levels, through post-transcriptional-gene-silencing (PTGS), results in distinctive phenotypes that include increased plant height, petiole hyponasty and extreme curvature of growing leaf primordia in the shoot apex. Due to lack of a visible potato tuber phenotype in the StIAA2 transgenic clones, no direct role for StIAA2 in controlling potato tuber development could be assigned. However, additional gene expression analysis of transgenic plants with reduced StIAA2 transcript levels resulted in the identification of a number of genes with altered expression profiles including another member of the Aux/IAA gene family ( StIAA ), providing new leads in auxin regulated gene expression in potato.

Besides the identification and analysis of candidate genes for a role in potato tuber development, we have used the dedicated cDNA microarray in a novel screening method for the identification of candidate genes related to tuber quality in a segregating diploid potato population (C x E). Isolated mRNA from potato tubers of individuals were pooled based on the observed difference in potato tuber cooking type. Transcript profiling of the contrasting bulks, consisting of individuals with either a mealy or non-mealy texture character, led to the identification of a candidate gene involved in determining potato tuber texture after cooking. The candidate gene exhibits strong homology to a t yrosine and l ysine r ich p rotein family and was named StTLRP . TLRP proteins are extra cellular cell wall proteins thought to be involved in cross-linking other proteins to the cell wall making them insoluble, thereby potentially modifying cell wall characteristics. It has been well documented that potato tuber cooking type is largely dependent on cell wall characteristics such as rigidity, permeability and level of cell adhesion. Variation in the level of gene expression of the candidate gene was confirmed by quantitative RT-PCR for a subset of individuals of the C x E population. Through the identification of an allelic variant of StTLRP and treating the expression levels of StTLRP as a quantitative trait, the identified potato StTLRP gene was mapped, both as a PCR marker and expression QTL, to the same chromosomal location as the QTL identified for tuber cooking type, strengthening its status as a candidate gene. The combination of transcript profiling with population genetics is often referred to as genetical genomics and is likely to greatly enhance candidate gene identification and subsequent cloning steps. Furthermore, we show that the use of a pooling strategy in a segregating population is a powerful and more cost-effective alternative to hybridizing individuals, and can be easily copied for other potato tuber quality traits or transcript profiling techniques.

Identification of the key regulatory genes for potato tuber development or tuber quality traits will greatly enhance the capabilities for potato tuber crop improvement through marker assisted breeding methods or genetic engineering. The work described in this thesis shows the successful application of a dedicated potato cDNA-microarray for the study of potato tuber development and quality traits and has resulted in the identification of candidate genes that have provided new insights into the complex biology of the potato crop.