Tuber formation in the wild potato species Solanum demissum Lindl.

1. How does a potato plant form tubers?Potato plants produce sexual multiplication and survival structures, true seeds, and asexual multiplication and survival bodies, tubers. Berries of the potato plant contain a large number of minute seeds. Relatively large tubers are formed in the soil in the subapical part of the stolons. The genetically heterogeneous seeds of a potato plant will spread in a larger area than its tubers, whose radius of spread is restricted by the length of the stolons. Potato tubers from one plant are genetically identical (to each other and to the mother plant) and reside in the soil, a relatively sheltered environment. Contrary to germinating seeds, the sprouts on a potato tuber have access to a relatively large quantity of storage food. A potato plant invests a lot of dry matter into the tubers. Apparently, tubers are, apart from being a valuable agricultural product, important structures for the potato plant itself. In this thesis 1 focused on tuber induction (Chapters 2 and 3), and changes in sucrose metabolism in stolon tips as a result of tuber induction (Chapters 4, 5 and 6).A series of long nights and a relatively low night temperature favour tuber induction in tuber- forming Solanum species. As a result of exposure to these conditions a potato plant synthesizes a (set of) compound(s) in the leaves, which is (are) transported basipetally to the stolon tips. During the last thirty years, it was surmised that this (set of) compound(s) consisted of a (mix of) classical plant hormones. However, no (mix of) plant hormone(s) could be identified as the tuber inducing principle yet.The starting point for this research project was the finding of Struik et al. (1987) that tuber inducing activity is present in extracellular extracts from leaflets of tuber-bearing Solanum tuberosum plants. An important indication that this activity is related to tuber induction would be its absence in non-tuber-bearing plants from the same age. However, tuber formation in commercial West-European S. tuberosum cultivars cannot be prevented by a simple shortening of the nightlength. Therefore, it was decided to switch to a wild Solanum species that only forms tubers under short-day conditions. S.demissum, a Mexican Solanum species which habits resemble S.tuberosum, meets this prerequisite. Tubers are formed when plants are exposed to 10 h daylength, whereas no tuber formation is observed when the daylength is extended to 16 h.2. The role of (hydroxylated) jasmonic acids in tuber inductionYoshihara, et al. (1989) isolated and characterized a substance from leaves of tuber-bearing potato plants (Solanum tuberosum, cv. Irish Cobbler) which they called 'tuberonic acid'. 'Tuberonic acid' is a glucoside of 12-hydroxy-jasmonic acid (12-OH-JA). This finding prompted us to investigate whether the tuber-inducing activity found in extracts from potato leaflets could be attributed to the presence of 'tuberonic acid'. Assuming 'tuberonic acid' to be involved in tuber induction, it is to be expected that this substance is absent in leaflets from non- tuber-bearing S.demissum plants.Extracts of leaflets from S.demissum plants grown under long- (LD) and short-day (SD) conditions were analyzed. The aglycon of 'tuberonic acid', not its glucoside, was detected in leaflets from SD plants. Moreover, a second hydroxylated jasmonic acid was detected: 11-OH-JA. It was the first time that this compound was detected in higher plants (Chapter 2). As a native substance it was detected before in a fungus, Botryodiplodia theobromae, by Miersch et al. (1991). Because no deuterated hydroxylated JAs were available, we could not determine the absolute concentrations of 11- and (12-OH-JA). in SD leaflets. However, the level of 11-OH-JA of SD leaflets was higher than the (12-OH-JA). level. No hydroxylated JAs were detected in LD leaflets.Hydroxylated JAs are metabolization products of JA. The CA concentrations in LD and SD leaflets did not differ significantly. Hence, tuber induction in S.demissum is correlated with the hydroxylation of CA A number of diurnal cycles of long nights and relatively cool temperatures will finally result in either the formation or activation of CA hydroxylating enzymes or the neutralization of the spatial separation between enzyme and substrate.Whether hydroxylated JAs are causally related to tuber induction remains to be proven. CA was found to induce the formation of tubers on S.demissum explants in vitro . Hydroxylated JAs could not be tested because only minute amounts of these substances were available. The reason why CA itself is apparently not involved in tuber induction inplanta could be its apolarity. Apolarity prevents basipetal transport of a compound via the phloem. Hydroxylated JAs can be easily glycosylated, and become transportable. This hypothesis is only apparently conflicting with the observation that CA can induce tuber formation in vitro. In the bioassay, stem pieces with axillary buds were placed in the JA-containing, solidified nutrient medium, so no transport of CA via the phloem was needed.3. Reception of a tuber-inducing substance in a stolon tipTuber induction in potato plants is irregular: only a subset of the stolon tips available will start to swell subapically after exposure to tuber-inducing conditions. The vasculature of potato plants excludes the possibility that a subset of stolon tips would be solely connected with a certain subset of leaves that does or does not synthesize a tuber-inducing signal. Nevertheless, this misconception persists in the literature.We monitored external characteristics of 841 stolon tips from 6 plants exposed to SD conditions. It was investigated whether a correlation could be found between subapical swelling and branching order, stolon and stolon-branch age, longitudinal growth rates of stolons and stolon branches, and attachment of the stolon to the main stem. No correlation was found between tuber formation and one of these external characteristics (Chapter 3). It is concluded that tuber initiation in S.demissum depends on metabolic or hormonal conditions in stolons or stolon branches, which are insufficiently reflected in external characteristics of these stolons or stolon branches to indicate the change of longitudinal growth into radial growth.4. Tuber induction and concomitant changes in sucrose metabolism in stolon tipsHigh concentrations of sucrose induces tuber formation in S. tuberosum explants in vitro (e.g. Hussey and Stacey 1984) as well as the expression of genes coding two semi-tuberspecific proteins, patatin (Wenzler et al. 1989) and proteinase inhibitor II (Johnson and Ryan 1984). We decided to investigate whether changes in the sucrose metabolism could be observed in the stolon tip before or during tuber initiation.The mono- and disaccharide contents of individual stolon tips of different developmental stages were determined qualitatively and quantitatively. No (transient) increase of the sucrose concentration could be detected concomitant with tuber initiation. The sucrose concentration was constantly low in developing stolon tips, and only tended to increase in relatively large tubers. However, we could not be conclusive about this since sucrose measurements were done at stolon tips as a whole. Very local (transient) sucrose accumulations could not be excluded. Recently, Müller-Röber et al. (1992) showed that a constitutively high sucrose concentration in potato tuber does not lead to an enhanced expression of patatin or proteinase inhibitor II. It illustrates that phenomena observed in in vitro systems do not necessarily occur in planta. Apart from sucrose, glucose and fructose were the main sugars in stolon tips. The glucose concentration decreased gradually during the process of subapical swelling whereas the fructose concentration dropped (Chapter 4).Invertases catalyze the irreversible hydrolysis of sucrose into glucose and fructose. Acid invertase activity is high in non-swollen stolon tips and decreases during subapical swelling. A specific neutral invertase could not be detected in stolon tips (Chapter 5).Sucrose synthase catalyzes a reversible reaction in which UDP and sucrose are converted into UDP-glucose and fructose. Sucrose synthase activity is barely detectable in non-swollen stolon tips. From the onset of subapical swelling onwards sucrose synthase activity increases rapidly (Chapter 5).The decreasing glucose concentration could be attributed to an increase of the sucrose synthase activity relative to the acid invertase activity in the stolon tip. On the other hand, sucrose splitting results in the formation of fructose irrespective of the nature of the enzyme involved. The steep decrease of the fructose concentration could be explained by a spatial shift: acid invertase activity is restricted to the cell wall or the vacuole, an environment where hexose kinases are relatively inactive, whereas sucrose synthase activity occurs in the cytosol. The fructose level in stolon tips drops in developing tubers, because sucrose splitting takes place in the cytosol. Here, fructose is exposed to a high activity of hexose kinases. The rate of fructose phosphorylation in extracts from stolon tips of S.demissum is manifold higher than the rate of glucose phosphorylation (Chapter 4). In Chapter 6, the consequences of the above mentioned enzyme and spatial shift on levels of several phosphorylated sugars were determined.Potato tubers; everybody can tell you what they look like and how they taste. Most people will recognize a potato crop in the field. At the same time, nobody can tell you how a potato plant starts to form tubers. It is a fascinating subject to do research on, and maybe this thesis comprises a few little steps towards a better understanding of this phenomenon.