In the January 2002 issue of Trends in Plant Science (7:35-41), Alisdair R. Fernie and Lothar Willmitzer (Max-Planck-Institut, Golm, Germany) and Richard N. Trethewey (Metanomics GmbH, Berlin, Germany) review recent research projects in which molecular plant physiological techniques were used to study carbon metabolism in potato tubers. Many of these studies have employed transgenic plants in which the expression of genes encoding enzymes critical for sucrose-to-starch conversion has been altered. The authors also discuss some unexpected observations made during detailed study of these metabolic processes.
The authors state that nearly all genes encoding enzymes believed to participate directly in the sucrose-to-starch conversion have been cloned. Modulation of the expression of these genes, both individually and in combination, has been achieved in a variety of transgenic lines. Studies of these lines confirm most existing hypotheses regarding metabolic regulation that had previously been proposed based on indirect methods. In spite of this impressive progress some questions, such as identification of critical steps for determination of potato tuber yield or the source of pyrophosphate required for mobilization of sucrose, still remain unanswered.
Sucrose is the carbon source for starch synthesis in potato tubers, but the mechanism by which sucrose is transferred to the cytosol of tuber cells from phloem has been a contentious subject. Phloem unloading has been proposed to occur by either a process mediated by the cell wall (apoplastic) or a process mediated by plasmodesmata (symplastic). Evidence exists for both processes; for example, plasmodesmatal connections between phloem and parenchyma cells have been observed, and impairment of these connections by plasmolysis inhibits sucrose flux, providing support for a symplastic process. On the other hand, isolated tuber tissue can accumulate exogenously supplied sucrose, pointing to an apoplastic mode of transfer. A recent study of this process using confocal microscopy, autoradiography and biochemical analyses indicates that both apoplastic and symplastic processes are involved, but are developmentally regulated. Sucrose transfer from phloem to parenchyma cells appears to occur apoplastically during stolon elongation and symplastically during the initial phases of tuberization. The mechanism of hexose entry into the cell depends upon their source. Hexoses generated in the apoplast could enter the vacuole through an endocytosis-like mechanism, in which vesicles arising from invaginations of the plasma membrane are transported to the vacuole. Alternatively, a sucrose proton transporter named SUT1 has been identified and characterized, though additional questions remain about its role in planta.
Cytosolic carbon metabolism involves sucrose import, cleavage, and conversion of substrates into glucose-6-phosphate, which is shuttled to the chloroplast for starch synthesis. The genes encoding enzymes involved in cytosolic carbon metabolism such as hexokinases, cytosolic phosphoglucomutase and sucrose phosphate phosphatase have been cloned. This paves the way for understanding the regulatory points of the sucrose-to-starch conversion pathway.
Sucrose delivered to the tuber may be cleaved by three different mechanisms: by invertase in the apoplast or by either invertase or sucrose synthase in the cytosol. While invertase activity is high at the initiation of tuber formation, sucrose synthase predominates in the developing tuber, reflecting the developmental regulation of phloem unloading described above. The products of sucrose synthase cleavage are acted on by fructokinase and UDP-glucose pyrophosphorylase, while the products of invertase are acted on by fructokinase and hexokinases. Cytosolic phosphoglucose isomerase and phosphoglucomutase equilibrate levels of these hexose phosphates.
Sucrose is not only degraded in the tuber, as a substantial amount of sucrose re-synthesis also occurs. Re-synthesis can occur either as a reverse of the sucrose synthase degradative pathway or through action of sucrose phosphate synthase and sucrose phosphate phosphatase. Feeding experiments indicate that both pathways function in the tuber to fulfill demands for carbon and respond to fluxes in sucrose supply. The authors describe recent studies that examine the regulation of these sucrose synthesis pathways in tubers.
Once generated, sucrose-derived carbon must enter the amyloplast. Results of experiments with radiolabeled sucrose molecules have demonstrated that hexose monophosphate, rather than triose phosphates, enters amyloplasts. Though both glucose-1-phosphate and glucose-6-phosphate have been suggested as forms entering the plastid, an answer to this debate has recently been found. A gene encoding a potato hexose monophosphate transporter has been identified, and the homologous gene in cauliflower encodes a transporter highly specific for glucose 6-phosphate.
Once substrates arrive in the amyloplast, a number of enzymes are involved in synthesis of starch. These include plastidial isoforms of phosphoglucomutase, AGPase, branching enzymes and starch synthase. These enzymes work together to participate in a number of reactions leading to the formation of starch molecules. By producing transgenic plants with altered levels of one or more of these enzymes, their roles in the classical starch synthesis pathway have been confirmed. The authors describe studies on AGPase, which is thought to act as a major regulatory point for the rate of starch synthesis. AGPase is activated by 3-phosphoglycerate (3-PGA) and inactivated by inorganic phosphate. Recent studies have shown that there is a correlation between changes in 3-PGA concentration and the rate of starch synthesis in the tuber. Plants with a reduced amount of AGPase activity also show decreased starch content. Furthermore, manipulating the plastidial levels of ATP, a substrate of AGPase, also affects the rate of starch synthesis. These experiments provide support for a critical role of AGPase in the biosynthetic pathway. Later polymerization reactions don’t seem to control the rate of starch accumulation, but do affect starch structure.
In two sidebars, the authors discuss two aspects of starch metabolism in depth: the role of pyrophosphate as a co-factor in starch synthesis and the potential role of sugars as regulatory signals. Pyrophosphate is required for each sucrose molecule cleaved by the sucrose synthase-dependant pathway. Pyrophosphate is thought to be generated by recycling across the amyloplast membrane or by a cycling process involving pyrophosphate-fructose-6-phosphate 1-phosphotransferase or tonoplast pyrophosphatase. However, manipulation of pyrophosphatase levels in vivo has resulted in a variety of phenotypic effects depending on specific lines and conditions, indicating that more study is needed to determine the regulatory role of this substrate in the sucrose-to-starch transition. In the second sidebar, the authors describe research focused on determining whether sugars are capable of regulating their own metabolism in vivo. Work with yeast has indicated that glucose may serve as a regulatory signal, and the authors describe studies that provide evidence both for and against the presence of glucose-dependent signaling processes in the tuber. The authors also describe evidence for plasma membrane sugar carriers that may affect tuber metabolism. However, additional work in sugar sensing is also needed.
Finally, the authors discuss the prospects of using emerging technologies to produce a more efficient tuber. The advent of genomic analyses in potato and tomato, as well as metabolic profiling in which gas chromatography-mass spectrometry techniques have been used to identify hundreds of metabolites in a single sample, enable a broader analysis of metabolic events in the potato tuber than ever before. Other emerging approaches that the authors believe will continue to impact the field include the development of non-aqueous fractionation techniques for metabolite profiling of specific cellular compartments in the tuber cell, allowing assessment of subcellular substrate pool sizes. Single-cell analyses are beginning to allow all of these techniques to be applied to specific cell types. The authors believe that application of these approaches to transgenic lines will allow rational design of metabolic engineering strategies.
Studies of transgenic lines with altered expression of genes encoding starch synthesis enzymes have confirmed a number of hypotheses about individual steps that constitute the sucrose-to-starch biosynthetic pathway. The authors point out that, in spite of recent knowledge gained through molecular plant physiology, work remains before the complex regulatory mechanisms underlying starch biosynthesis become clear. The authors note the progress that has been made and advocate additional systems approaches that will provide a broad picture of metabolic events.