Tuber physiology and properties of starch from tubers of transgenic potato plants with altered plastidic adenylate transporter activity

Abstract

We showed recently that antisense plants with decreased activity of the plastidic ATP/ADP-transporter protein exhibit drastically reduced levels of starch and a decreased amylose/amylopectin ratio, whereas sense plants with increased activity of the transporter possessed more starch than wild-type plants and an increased amylose/amylopectin ratio. In this paper we investigate the effect of altered plastidic ATP/ADP-transporter protein expression on primary metabolism and granule morphology in more detail. Tuber tissues from antisense and sense plants exhibited substantially increased respiratory activity compared with the wild type. Tubers from antisense plants contained markedly increased levels of free sugars, UDP-Glc, and hexose phosphates, whereas phosphoenolpyruvate, isocitrate, ATP, ADP, AMP, UTP, UDP, and inorganic pyrophosphate levels were slightly decreased. In contrast, tubers from sense plants revealed a slight increase in adenine and uridine nucleotides and in the levels of inorganic pyrophosphate, whereas no significant changes in the levels of soluble sugars and metabolites were observed. Antisense tubers contained 50% reduced levels of ADP-Glc, whereas sense tubers contained up to 2-fold increased levels of this sole precursor for starch biosynthesis. Microscopic examination of starch grain morphology revealed that the size of starch grains from antisense tubers was substantially smaller (50%) compared with the wild type. The large starch grains from sense tubers appeared of a more angular morphology, which differed to the more ellipsoid shape of wild type grains. The results suggest a close interaction between plastidial adenylate transport and starch biosynthesis, indicating that ADP-Glc pyrophosphorylase is ATP-limited in vivo and that changes in ADP-Glc concentration determine starch yield, as well as granule morphology. Possible factors linking starch synthesis and respiration are discussed.

Figure 1

Respiration rates in slices from tubers with altered expression of the AATP. Slices were incubated in 100 mm Suc at a temperature of 25°C. Data points are means ± sd of three independent samples. Each sample represents four discs taken from four individual tubers of a line.

Figure 2

Sugars and metabolite levels in tubers with altered expression of the AATP. Tuber material was frozen in liquid nitrogen and then extracted in trichloroacetic acid to analyze the levels of Suc (A), Glc (B), Fru (C), Glc1P (D), Glc6P (E), Fru6P (F), triose-P (G), 3PGA (H), PEP (I), pyruvate (J), isocitrate (K), α-ketoglutarate (L), malate (M), and PPi (N). Results are means ± se (n = 5).

Figure 3

Uridine nucleotide levels in tubers with altered expression of the AATP. The same extracts as in Figure 2 were used to analyze total uridine nucleotide levels (A), which is the sum of UTP (B), UDP (C), and UDP-Glc (D). The ratio of UTP/UDP-Glc (E) is also shown. Results are means ± se (n = 5).

Figure 4

Adenine nucleotide levels in tubers with altered expression of the plastidic ATP/ADP translocator. The same extracts as in Figure 2 were used to analyze total adenine nucleotide levels (A), which is the sum of ATP (B), ADP (C), AMP (D), and ADP-Glc (E). The ratio of ATP/ADP (F) is also shown. Results are means ± se (n = 5).

Figure 5

Microscopic examination of starch grains using polarized light. Hand sections of potato tubers (top panel), accumulation of isolated starch grains (middle panel), and single isolated starch grains (bottom panel) are depicted from wild type (A), antisense JT 654 (B), and sense JT 62 (C) transformants.