Glycolytic gene expression in amphibious Acorus calamus L. under natural conditions

Acorus calamus L is an amphibious plant, which is exposed to periods of flooding and consequently hypoxic conditions as a part of its natural life cycle. Previous experiments under laboratory conditions have shown that the plant can survive for two months in the complete absence of oxygen, and that during this period the expression of genes encoding the glycolytic enzymes fructose-1,6-bisphosphate aldolase (ALD), pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) is induced in leaves and rhizomes (Bucher and Kuhlemeier, 1993). Here we studied the expression of ALD and ADH through two years in the natural habitat of A. calamus. Under natural conditions roots and rhizomes were always submerged but newly grown leaves emerged in spring; in autumn the leaves senesced and the whole plant was submerged again. High Ald and Adh mRNA levels in leaf and rhizome were found only in winter when the leaves were entirely submerged. Upon leaf emergence in spring the mRNA levels rapidly declined. Under controlled experimental conditions expression of Ald and Adh was not induced by low temperature. The combination of laboratory and field experiments supports the hypothesis that oxygen deprivation rather than low temperature is a major regulator of glycolytic gene expression in A. calamus. The possible role of other environmental factors is also discussed.Abbreviations ADH alcohol dehydrogenase - Adh gene encoding ADH - ALD cytoplasmic fructose-1,6-bisphosphate aldolase - Ald gene encoding ALD - PDC pyruvate decarboxylase - Pdc gene encoding PDC     

Flight muscle function in Drosophila requires colocalization of glycolytic enzymes.

Structural relationships between the myofibrillar contractile apparatus and the enzymes that generate ATP for muscle contraction are not well understood. We explored whether glycolytic enzymes are localized in Drosophila flight muscle and whether localization is required for function. We find that glycerol-3-phosphate dehydrogenase (GPDH) is localized at Z-discs and M-lines. The glycolytic enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are also localized along the sarcomere with a periodic pattern that is indistinguishable from that of GPDH localization. Furthermore, localization of aldolase and GAPDH requires simultaneous localization of GPDH, because aldolase and GAPDH are not localized along the sarcomere in muscles of strains that carry Gpdh null alleles. In an attempt to understand the process of glycolytic enzyme colocalization, we have explored in more detail the mechanism of GPDH localization. In flight muscle, there is only one GPDH isoform, GPDH-1, which is distinguished from isoforms found in other tissues by having three C-terminal amino acids: glutamine, asparagine, and leucine. Transgenic flies that can produce only GPDH-1 display enzyme colocalization similar to wild-type flies. However, transgenic flies that synthesize only GPDH-3, lacking the C-terminal tripeptide, do not show the periodic banding pattern of localization at Z-discs and M-lines for GPDH. In addition, neither GAPDH nor aldolase colocalize at Z-discs and M-lines in the sarcomeres of muscles from GPDH-3 transgenic flies. Failure of the glycolytic enzymes to colocalize in the sarcomere results in the inability to fly, even though the full complement of active glycolytic enzymes is present in flight muscles. Therefore, the presence of active enzymes in the cell is not sufficient for muscle function; colocalization of the enzymes is required. These results indicate that the mechanisms by which ATP is supplied to the myosin ATPase, for muscle contraction, requires a highly organized cellular system.     

Triglyceride pools, flight and activity variation at the Gpdh locus in Drosophila melanogaster

We have created a set of P-element excision-derived Gpdh alleles that generate a range of GPDH activity phenotypes ranging from zero to full activity. By placing these synthetic alleles in isogenic backgrounds, we characterize the effects of minor and major activity variation on two different aspects of Gpdh function: the standing triglyceride pool and glycerol-3-phosphate shuttle-assisted flight. We observe small but statistically significant reductions in triglyceride content for adult Gpdh genotypes possessing 33-80% reductions from normal activity. These small differences scale to a notable proportion of the observed genetic variation in triglyceride content in natural populations. Using a tethered fly assay to assess flight metabolism, we observed that genotypes with 100 and 66% activity exhibited no significant difference in wing-beat frequency (WBF), while activity reductions from 60 to 10% showed statistically significant reductions of approximately 7% in WBF. These studies show that the molecular polymorphism associated with GPDH activity could be maintained in natural populations by selection in the triglyceride pool.