ABSTRACTpropose that they should be more correctly termed sucrolysis and sucroneogenesis. Before recent work it was customary to assume that sucrose synthase action resulted in the formation of UDP-glucose and then other nucleotide sugars leading into sugar polymer synthesis, such as plant cell walls. However, a substrate level pool of PPi was measured in plants (2,7,27), and we successfully tested the pyrophosphorolysis of UDPglucose feeding glucose 1-P directly into plant metabolism (32). These steps are an integral part of the recently proposed sucrose synthase pathway (1,15,30). Then, of course, from glycolysis carbon can be directed into essentially every metabolic activity of a cell. Therefore, the ability of a tissue or organ to metabolize sucrose must be one determinant of sink strength. Here we have tested the feasibility of biochemically measuring sucrose sink strength by assaying sucrose cleavage activities, i.e. sucrolysis via either the invertases or by the sucrose synthase pathway. The reasons we developed this UDP and PPi-dependent assay for sucrose synthase activity were (a) to measure activity in the sucrose breakdown direction whereas many other workers measured the opposite, namely sucrose synthesis (3,4,22,26), and (b) others who measured sucrose breakdown often assayed for UDP-glucose accumulation as a precursor of the synthesis of cell walls or other nucleotide sugars (6, 22). But in our assay we couple through the PPi-dependent UDP-glucopyrophosphorylase, which is very active in plants, to the formation of glucose 1-P which feeds carbon directly into glycolysis and possibly on to starch formation (31,32
Plant cells have two cytoplasmic pathways of glycolysis and gluconeogenesis for the reversible interconversion of fructose 6‐phosphate (F‐6‐P) and fructose 1,6‐bisphosphate (F‐1,6‐P2). One pathway is described as a maintenance pathway that is catalyzed by a nucleotide triphosphate‐dependent phosphofructokinase (EC 2.7.1.11; ATP‐PFK) glycolytically and a F‐1,6 bisphosphatase (EC 3.1.3.11) gluconeogenically. These are non‐equilibrium reactions that are energy consuming. The second pathway, described as an adaptive pathway, is catalyzed by a readily reversible pyrophosphate‐dependent phosphofructokinase (EC 2.7.1.90; PP‐PFK) in an equilibrium reaction that conserves energy through the utilization and the synthesis of pyrophosphate. A constitutive regulator cycle is also present for the synthesis and hydrolysis of fructose 2,6‐bisphosphate (F‐2,6‐P2) via a 2‐kinase and a 2‐phosphatase, respectively. The pathway catalyzed by the ATP‐PFK and F‐1,6‐bisphosphatase, the maintenance pathway, is fairly constant in maximum activity in various plant tissues and shows less regulation by F‐2,6‐P2. Plants use F‐2,6‐P2 initially to regulate the adaptive pathway at the reversible PPi‐PFK step. The adaptive pathway, catalyzed by PPi‐PFK, varies in maximum activity with a variety of phenomena such as plant development or changing biological and physical environments. Plants can change F‐2,6‐P2 levels rapidly, in less than 1 min when subjected to rapid environmental change, or change levels slowly over periods of hours and days as tissues develop. Both types of change enable plants to cope with the environmental and developmental changes that occur during their lifetimes. The two pathways of sugar metabolism can be efficiently linked by the cycling of uridylates and pyrophosphate required for sucrose breakdown via a proposed sucrose synthase pathway. The breakdown of sucrose via the sucrose synthase pathway requires half the net energy of breakdown via the invertase pathway. Pyrophosphate occurs in plant tissues as a substrate pool for biosynthetic reactions such as the PPi‐PFK or uridine diphosphate glucose pyrophosphorylase (EC 2.7.7.9; UDPG pyrophosphorylase) that function in the breakdown of imported sucrose. Also, pyrophosphate links the two glycolytic/gluco‐neogenic pathways; and in a reciprocal manner pyrophosphate is produced as an energy source during gluconeogenic carbon flow from F‐1,6‐P2 toward sucrose synthesis.
1988. A reassessment of glycolysis and gluconeogenesis in higher plants. -Physiol. Plant. 72: 650-654.Sucrose is the starting point of glycolysis and end point of gluconeogenesis in higher plants. During both glycolysis and gluconeogenesis alternative enzymes are present at various steps to carry out parallel pathways; alternatives are available for utilizing nucleotide triphosphates and pyrophosphate; fructose 2.6-bisphosphate serves as a strong internal regulator; and plants use these cytoplasmic alternatives as they develop and as their environments change.
The breakdown of sucrose to feed both hexoses into glycolytic carbon flow can occur by the sucrose synthase pathway. This uridine diphosphate (UDP) and pyrophosphate (PPi)-dqpendent pathway was biochemically characterized using soluble extracts from several plants.
Developing and germinating lima bean (Phaseolus lunatus var Cangreen) seeds were used for testing the sucrose synthase pathway, to examine the competition for uridine diphosphate (UDP) and pyrophosphate (PPi), and to identify adaptive and maintenance-type enzymes in glycolysis and gluconeogenesis. In developing seeds, sucrose breakdown was dominated by the sucrose synthase pathway; but in the seedling embryos, both the sucrose synthase pathway and acid invertase were active. UDPase activity was low and seemingly insufficient to compete for UDP during sucrose metabolism in seed development or germination. In contrast, both an acid and alkaline pyrophosphatase were active in seed development and germination. The set of adaptive enzymes identified in developing seeds were sucrose synthase, PPi-dependent phosphofructokinase, plus acid and alkaline pyrophosphatase; and, the adaptive enzymes identified in germinating seeds included the same set of enzymes plus acid invertase. The set of maintenance enzymes identified during development, in the dry seed, and during germination were UDPglucopyrophosphorylase, neutral invertase, ATP and UTP-dependent fructokinase, glucokinase, phosphoglucomutase, ATP and UTP-dependent phosphofructokinase and sucrose-P synthase.Sucrose is a primary nutrient for essentially all higher plant cells. Recently, a new pathway of sucrose breakdown was proposed which is dependent upon UDP and PPi and that involves a cyclic series of reactions to produce the required UDP and PPi (4,13,23,31). When sucrose is broken down by this pathway, which is called the sucrose synthase pathway, both hexoses from sucrose can feed into glycolysis. In addition, we have proposed that in the plant cell cytoplasm, alternative enzymes are present at various steps in glycolysis and gluconeogenesis for the interconversion of sucrose and pyruvate such that they form a network of reactions rather than the classical textbook-type pathways. (3,19,24). In view of these and other recent discoveries such as a substrate level pool of PPi and fructose 2,6-P2 regulation (3, 4, 10, 21, 30), we have reassessed all ofplant glycolysis and gluconeogenesis, beginning with sucrose (24).Because the glycolytic and the gluconeogenic metabolism of sucrose are at a central junction in plant metabolism (7,12,28) MATERIALS AND METHODS All studies were conducted with lima beans, Phaseolus lunatus var Cangreen, purchased locally and grown with good cultural practices either in field plots or in the greenhouse. The studies were conducted over the last 3 years with both field and greenhouse grown plants. Each study was repeated in at least three similar tests and the data presented are illustrative of developmental patterns, recognizing that variations occurred, e.g. seedling emergence in a given study might occur on d 5 or 6, etc. Lima bean has an indeterminate flower production; hence, seed development work based on freshly harvested weights was highly facilitated. And seedling development work also was facilitated because the lima bean...
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