|Title||Cell cycle and storage related gene expression in potato tubers|
|Source||Wageningen University. Promotor(en): L.H.W. van der Plas; A.R. van der Krol; D. Vreugdenhil. - S.l. : S.n. - ISBN 9789058086587 - 133|
Laboratory of Plant Physiology
|Publication type||Dissertation, internally prepared|
|Keyword(s)||solanum tuberosum - aardappelen - knollen - genexpressie - celcyclus - methodologie - genetische transformatie - luciferasen - plantenfysiologie - in vivo experimenten - solanum tuberosum - potatoes - tubers - gene expression - cell cycle - methodology - genetic transformation - luciferases - plant physiology - in vivo experimentation|
|Categories||Plant Cell Biology / Plant Physiology / Plant Molecular Biology|
Potato tubers are vegetative structures aimed at survival of the period when conditions are not optimal for the growth of the potato plant. Understanding the process of tuber formation in potatoes ( Solanum tuberosum ) is important, both from a scientific view, and for practical applications. During the initial stages of tuber formation an enhanced synthesis of starch and storage proteins is observed at the site of the stolon that develops into a tuber. Besides an enhanced synthesis of storage products, longitudinal cell division and cell enlargement is observed resulting in a radial expansion of the stolon. Because cell division and the synthesis of storage products are two important events during tuber development we analyzed the expression of two sets of genes: two cell cycle genes (cycB1;1 and CDC2a) and two storage related genes (AGPaseS andlPat21). To analyze cell cycle and storage related gene expression we fused the promoter sequences of these genes to the firefly luciferase reporter gene from North American firefly ( Photinus pyralis ). Luciferase (LUC) activity can be observed non- destructively in individual tubers and correlates with the luciferase mRNA level in both in vitro grown as well as in soil grown tubers, indicating that in planta LUC activity reflects the ongoing expression level of the reporter gene. More important, LUC activity correlates with the mRNA level of the endogenous genes.
In chapter 2 we describe the expression of cell cycle and storage related gene expression in in vitro grown explants in relation to the onset of swelling marking the morphological onset of tuber formation. The average activity quantified in multiple tubers indicated that the storage related genes were upregulated well before visible swelling, viz., four days before swelling for AGPaseS, and three days before swelling forlPat21, while the average activities of both cell cycle genes showed an upregulation after the onset of swelling. However with the ability to observe changes in gene expression in individual tubers a variable upregulation for both storage related genes could be shown in relation to the moment of visible swelling, varying between four days before till ten days after the onset of swelling. The results indicate that tuber development does not occur via a fixed sequential order of events, but consists of independent individual programs that occur in the same region of the stolon resulting in the formation of a potato tuber.
When after tuber initiation, environmental conditions become adverse for tuber induction, second growth may occur. Adverse tuber-inducing conditions may result in the outgrowth of the apical bud (second growth) and a reduced growth of the tuber (primary growth). We mimicked second growth inducing conditions by transferring developing in vitro tubers to medium with gibberellin (chapter 3) or to medium with a low level of ethanol (chapter 4). GA-induced second growth resulted in the outgrowth of the apical bud followed by the formation of a secondary stolon. Transfer of tubers to medium supplemented with ethanol resulted in the formation of a secondary tuber when combined with 8% sucrose, whereas ethanol combined with 1% sucrose resulted in the formation of a secondary stolon. Preceding bud growth cycB1;1, AGPaseS andlPat21 expression declined in the complete tuber both in young (1-5 days after visible swelling) and in mature tubers (25-30 days after visible swelling) after transfer to medium with GA 4+7 or medium with ethanol, whereas CDC2a activity only declined when tubers were transferred to medium with ethanol. Second growth inducing conditions did not affect the activity of the CaMV35S promoter, indicating that the decline in the cell cycle and storage related promoter activity in the tubers during second growth is not related to a general effect on gene expression. Although a decline in cell cycle and storage related gene expression is observed before second growth, there is no causal relation between this decline and the outgrowth of the bud, as concluded from analysis of tubers transferred to medium with GA or ethanol combined with ABA which prevents outgrowth of the bud. Under these conditions cell cycle and storage related gene expression also declined. Transferring tubers to medium with different alcohols and combining ethanol with an inhibitor of alcohol dehydrogenase (4-methyl pyrazole) showed that ADH activity is a key factor during ethanol-induced second growth and the changes observed in gene expression in bud and tuber region.
Preceding second growth, a higher cycB1;1 and AGPaseS expression is observed in the bud region as compared to the activity in the tuber region. CycB1;1 expression in the bud region indicates a specific induction of the cell cycle genes during second growth. However, a relatively higher cycB1;1 expression in the bud is not sufficient for bud growth both during GA or ethanol-induced second growth. These results indicate that bud growth is not controlled by cycB1;1. AGPaseS expression in the bud region during second growth possibly indicates the enhanced import of sugars into the bud region during second growth. Preventing bud outgrowth by combining GA with ABA counteracts the difference in AGPaseS expression between bud and tuber in mature tubers, indicating that tuber and bud region respond differentially upon GA and ABA. During ethanol-induced second growth, relatively higher AGPaseS expression was observed in the bud region in young tubers when transferred to medium with ethanol and 8% sucrose whereas tubers transferred to medium with ethanol and 1% sucrose showed a decline in AGPaseS expression both in bud and tuber region during second growth. These results indicate that the type of second growth and the enhanced AGPaseS expression in the bud region during ethanol-induced second growth are related to the amount of sugars available for the bud. Mature tubers transferred to medium with ethanol showed higher AGPaseS expression in the bud region, irrespectively of the sucrose concentration in the medium. Combining ethanol with ABA could not prevent AGPaseS expression in the bud region during ethanol-induced second growth. These results indicate that ABA affects sugar import in the bud region during GA-induced second growth whereas ABA does not affect sugar import during ethanol-induced second growth.
In chapter 5 we described the relation between the expression of the cell cycle and storage related genes in the tubers and the differences in tuber growth rate of tubers growing on complete plants, to determine if the expression of these genes can be used as a measure for differences in sink strength and sink activity between tubers. Tuber growth rate was determined during the complete period of tuber development as an indication for sink strength and the correlation was determined with the level of gene expression quantified per tuber and per unit tuber area. By using hydroponics pot assemblies we were able to observe tuber growth and to quantify gene expression non-destructively during the complete period of tuber development. Three different stages of tuber growth were distinguished viz., initiation, linear tuber growth and maturation. During initiation, a low correlation was observed between tuber growth rate and the expression level of the cell cycle and storage related genes. During the period of linear tuber growth a good correlation was observed forlPat21 while during maturation the expression of cycB1;1, AGPaseS andlPat21 correlated well with the sink strength, as reflected by the growth rate. The results presented in chapter 5 indicate that patatin gene expression is a good predictive marker for differences in sink strength between tubers initiated on the same potato plant.
It has become clear that non-invasive methods to study gene expression are useful tools for developmenta l studies as described in this thesis on potato tuber development. Changes in gene expression can be observed in individual tuber samples, showing the exact relation between changes in gene expression and developmental changes.