β-Maltose Is the Metabolically Active Anomer of Maltose during Transitory Starch Degradation  

Abstract

Maltose is the major form of carbon exported from the chloroplast at night as a result of transitory starch breakdown. Maltose exists as an a-or b-anomer. We developed an enzymatic technique for distinguishing between the two anomers of maltose and tested the accuracy and specificity of this technique using b-maltose liberated from maltoheptose by b-amylase. This technique was used to investigate which form of maltose is present during transitory starch degradation in bean (Phaseolus vulgaris), wild-type Arabidopsis (Arabidopsis thaliana), two starch deficient Arabidopsis lines, and one starch-excess mutant of Arabidopsis. In Phaseolus and wild-type Arabidopsis, b-maltose levels were low during the day but were much higher at night. In Arabidopsis plants unable to metabolize maltose due to a T-DNA insertion in the gene for the cytosolic amylomaltase, (Y. Lu, T.D. Sharkey [2004] Planta 218: 466-473) levels of a-and b-maltose were high during both the day and night. In starchless mutants of Arabidopsis, total maltose levels were low and almost completely in the a-form. We also found that the subcellular concentration of b-maltose at night was greater in the chloroplast than in the cytosol by 278 mM. We conclude that b-maltose is the metabolically active anomer of maltose and that a sufficient gradient of b-maltose exists between the chloroplast and cytosol to allow for passive transport of maltose out of chloroplasts at night.In leaves, transitory starch is formed in the chloroplasts during the day and broken down at night. Transitory starch acts as an energy reserve, providing the plant with carbohydrate during the night when sugars cannot be made by photosynthesis, and as an overflow, allowing photosynthesis to go faster than Suc synthesis during the day (Ludewig et al., 1998).It has long been known that maltose is an intermediate in the breakdown of storage starch, for example during malting, but the importance of maltose in transitory starch degradation has only recently been proven. Early reports of significant maltose levels in leaves undergoing transitory starch breakdown (Levi and Gibbs, 1976;Peavey et al., 1977;Kruger and ap Rees, 1983;Neuhaus and Schulte, 1996;Servaites and Geiger, 2002) were counterbalanced by reports concluding that phosphorolytic starch breakdown was the predominant pathway in leaves (Heldt et al., 1977;Stitt et al., 1978; ap Rees, 1979, 1980;Stitt and Heldt, 1981).The form of carbon exported from the chloroplast at night was reexamined in light of accumulating genetic and biochemical evidence against phosphorolytic degradation for export. We found that at least two-thirds of the carbon exported from dark-adapted, isolated chloroplasts was in the form of maltose . The maltose is likely used by an amylomaltase enzyme (Chia et al., 2004;Lu and Sharkey, 2004) in metabolism that may be similar to that used by Escherichia coli (Boos and Shuman, 1998).Maltose transport across the chloroplast membrane has been known for some time (Rost et al., 1996) and recently the maltose transporter was ...

Mutants of Arabidopsis Lacking Starch Branching Enzyme II Substitute Plastidial Starch Synthesis by Cytoplasmic Maltose Accumulation

“…The absence of maltose in the starch-free pgm1 mutant (Caspar et al, 1985) and in the very-low-starch ss2 ss3 double mutant (M. James and A. Myers, personal communication) confirms this conclusion. The amount of maltose in the be2-1 be3-2 mutant is 300-to 600-fold higher than that assayed at midcourse of the light phase in the wild type but is more or less equivalent to that measured in dpe2 mutants defective for cytosolic transglucosidase (Chia et al, 2004;Lu and Sharkey, 2004;Weise et al, 2005). The stunted growth phenotype of the double mutant is equally reminiscent of that observed in the dpe2 mutants.…”

“…The lack of maltose assimilation in the dpe2 mutant can explain why such a high level of maltose accumulates in the cell. Spontaneous mutarotation of maltose could explain why equivalent amounts of both anomers are assayed in the mutant (Weise et al, 2005). In our study, >90% of maltose assayed in the be2-1 be3-2 double mutant was in the a-form.…”

“…This test was performed on fresh material immediately after extraction of the MOS to limit the impact of mutarotation in the samples and after immediate boiling to inactivate enzymes. Mutarotation occurs spontaneously when molecules are conserved in solution, although its rate is quite low according to Weise et al (2005) and based on our own observations. Mutarotation can be defined as the spontaneous conversion of either a-to b-maltose or b-to a-maltose.…”

“…Transglucosidase DPE2 Requires b-Maltose to Be Active Weise et al (2005) previously suggested that DPE2, a cytosolic transglucosidase required for the metabolization of maltose during leaf starch degradation at night (Chia et al, 2004;Lu and Sharkey, 2004), could be specific for b-maltose. The accumulation of a-maltose in the be2-1 be3-2 double mutant provided us with a reliable source that enables us to prove or rule out this hypothesis.…”

Overexpression of an extraplastidic β-amylase which accumulates in the radish taproot influences the starch content of Arabidopsis thaliana

“…Recent molecular genetic studies have provided convincing evidence that plastidic β-amylases are involved in endogenous starch degradation in Arabidopsis leaves (Smith et al 2005;Fettke et al 2009;Zeeman et al 2010). β-Maltose is a primary product of starch degradation in Arabidopsis leaves (Weise et al 2005). The Arabidopsis genome has nine β-amylase genes.…”