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. 2000 Jul;123(3):939-48.
doi: 10.1104/pp.123.3.939.

Glucose and disaccharide-sensing mechanisms modulate the expression of alpha-amylase in barley embryos

E Loreti  1 , A Alpi, P Perata
Affiliations

Affiliation

  • 1 Department of Crop Plant Biology, University of Pisa, Via Mariscoglio 34, 56124 Pisa, Italy.

Glucose and disaccharide-sensing mechanisms modulate the expression of alpha-amylase in barley embryos

E Loreti et al. Plant Physiol. 2000 Jul.
. 2000 Jul;123(3):939-48.
doi: 10.1104/pp.123.3.939.

Authors

E Loreti  1 , A Alpi, P Perata

Affiliation

  • 1 Department of Crop Plant Biology, University of Pisa, Via Mariscoglio 34, 56124 Pisa, Italy.

Abstract

The aim of this study was to investigate the sugar-sensing processes modulating the expression of alpha-amylase in barley (Hordeum vulgaris L. var Himalaya) embryos. The results highlight the existence of independent glucose (Glc) and disaccharides sensing. Glc treatment destabilizes the alpha-amylase mRNA. Non-metabolizable disaccharides repress alpha-amylase induction, but have no effects on transcript stability. Structure-function analysis indicates that a fructose (Fru) moiety is needed for disaccharide sensing. Lactulose (beta-galactose [Gal][1-->4]Fru), palatinose (Glc[1-->6]Fru), and turanose (Glc[1-->3]Fru) are not metabolized but repress alpha-amylase. Disrupting the fructosyl moiety of lactulose and palatinose, or replacing the Fru moiety of beta-Gal[1-->4]Fru with Glc or Gal results in molecules unable to repress alpha-amylase. Comparison of the molecular requirements for sucrose transport with those for disaccharide sensing suggests that these sugars are perceived possibly at the plasma membrane level independently from sucrose transport.

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Figures

Figure 1

Figure 1

Glc and Suc repression of…

Figure 1

Glc and Suc repression of the induction of α-amylase in barley embryos. A,…

Figure 1
Glc and Suc repression of the induction of α-amylase in barley embryos. A, GA induction of α-amylase in barley embryos and repression by Glc and Suc. Embryos were incubated in 5 mm CaCl2, 10 μm uniconazole, and, when used, 1 μm GA3, 50 mm Glc, or 50 mm Suc for 24 h. Blots were probed with the α-amylase probe. B, Effect of Glc and Suc on the α-amylase mRNA level in barley embryos. Embryos were incubated for 24 h in 1 μm GA3 and 5 mm CaCl2 in the presence of increasing concentrations of Glc or Suc. Embryos were extracted for RNA gel-blot analysis. Blots were probed with the α-amylase probe. Membranes were reprobed with an rRNA probe (not shown). RNA levels of both α-amylase and rRNA were quantified and the relative α-amylase transcript level normalized to rRNA (100 = transcript level in the embryos not treated with exogenous sugars) is reported in the histograms. Data are means ± se of three separate experiments. The mRNA band relative to 0 mm was duplicated in the two reproductions for the sake of clarity.
Figure 2

Figure 2

Screening of carbohydrates able to…

Figure 2

Screening of carbohydrates able to repress α-amylase induction in barley embryos. A, Effect…

Figure 2
Screening of carbohydrates able to repress α-amylase induction in barley embryos. A, Effect of various carbohydrates on the α-amylase mRNA level in barley embryos. Embryos were incubated for 24 h in 1 μm GA3 and 5 mm CaCl2 in the presence of 100 mm carbohydrates. Embryos were extracted for RNA gel-blot analysis. Blots were probed with the α-amylase probe. Membranes were reprobed with an rRNA probe (not shown). RNA levels of both α-amylase and rRNA were quantified and the relative α-amylase transcript level normalized to rRNA (100 = Control) is reported in the histograms. Data are means ± se of three separate experiments. The mRNA bands were rearranged in the reproduction for the sake of clarity. B, Glc, Fru, and Suc content in barley embryos fed with various carbohydrates. Embryos were incubated for 24 h in 1 μm GA3 and 5 mm CaCl2 in the presence of various carbohydrates (100 mm) and extracted for sugar assay. Data are means ± se of four separate experiments.
Figure 3

Figure 3

Effect of various carbohydrates on…

Figure 3

Effect of various carbohydrates on the germination of isolated barley embryos. A, Barley…

Figure 3
Effect of various carbohydrates on the germination of isolated barley embryos. A, Barley embryos were dissected from dry barley grains and incubated in 1 μm GA3 and 5 mm CaCl2 in the presence of 200 mm carbohydrates. After 48 h, the embryos were collected and photographed. A representative photograph is shown for each treatment. B, Dry weight of barley embryos (100 = Control) treated as described in A. Data are means ± se of three replicates.
Figure 4

Figure 4

The disaccharide signaling is independent…

Figure 4

The disaccharide signaling is independent of ABA and of effects on gibberellin biosynthesis.…

Figure 4
The disaccharide signaling is independent of ABA and of effects on gibberellin biosynthesis. A, Effect of ABA on α-amylase induction. Embryos were incubated for 24 h in 1 μm GA3 and 5 mm CaCl2 together with increasing ABA concentrations. Embryos were extracted for RNA gel-blot analysis. Blots were probed with the α-amylase probe. Membranes were reprobed with an rRNA probe as well as with an ubiquitin probe. B, Effect of disaccharides on the expression of the ABA-inducible Rab16A gene. Embryos were incubated for 24 h in 1 μm GA3 and 5 mm CaCl2. When present as a control, ABA was used at a concentration of 10 μm. Disaccharides were used at a concentration of 40 mm. Embryos were extracted for RNA gel-blot analysis. Blots were probed with the Rab16A probe. Membranes were reprobed with an rRNA probe as well as with an ubiquitin probe. C, Effect of Glc and palatinose on α-amylase induction at the mRNA level in embryos from the constitutive GA-response slender mutant. Both wild-type and slender embryos were treated with uniconazole, a GA-biosynthesis inhibitor (Izumi et al., 1984). Embryos were incubated in 5 mm CaCl2, 10 μm uniconazole, and, when used, 1 μm GA3, 40 mm Glc, or 40 mm palatinose for 24 h. Blots were probed with the α-amylase probe. Membranes were reprobed with an rRNA probe as well as with an ubiquitin probe. Amy, α-amylase; Ubi, ubiquitin; Rab, Rab16A; sln, slender.
Figure 5

Figure 5

Glc triggers destabilization of the …

Figure 5

Glc triggers destabilization of the α-amylase mRNA through a transcription and protein synthesis-dependent…

Figure 5
Glc triggers destabilization of the α-amylase mRNA through a transcription and protein synthesis-dependent pathway. A, Effect of ActD and Glc on α-amylase mRNA stability. ActD (10 μg/mL) and Glc (40 mm) were added to embryos pretreated for 12 h (time = 0) in 1 μm GA3 and 5 mm CaCl2; samples were collected every 2 h. Embryos were extracted for RNA gel-blot analysis. Blots were probed with the α-amylase probe. Membranes were reprobed with an rRNA probe (not shown) as well as with an ubiquitin probe. RNA levels of both α-amylase and rRNA were quantified and the relative α-amylase transcript level normalized to rRNA (100 = transcript level in the embryos pretreated with GA3 for 12 h) is reported in the histograms. Data are means ± se of three separate experiments. The mRNA band relative to 0 h was duplicated in the four reproductions for the sake of clarity. B, Effect of CHX and Glc, on the α-amylase mRNA stability. Embryos were pretreated for 12 h in 1 μm GA3 and 5 mm CaCl2; CHX (200 μm) and Glc (40 mm) were added to the embryos for an additional 8 h, up to a total time of 20 h. Presence of chemicals is as indicated by the + sign. Embryos were extracted for RNA gel-blot analysis. Blots were probed with the α-amylase probe. Membranes were reprobed with an rRNA probe (not shown) as well as with an ubiquitin probe. RNA levels of both α-amylase and rRNA were quantified and the relative α-amylase transcript level normalized to rRNA (100 = transcript level in the embryos pretreated with GA3 for 12 h) is reported in the histograms. Data are means ± se of three separate experiments. Amy, α-amylase; Ubi, ubiquitin.
Figure 6

Figure 6

Suc but not turanose triggers…

Figure 6

Suc but not turanose triggers destabilization of the α-amylase mRNA through a transcription…

Figure 6
Suc but not turanose triggers destabilization of the α-amylase mRNA through a transcription dependent pathway. A, ActD (10 μg/mL) and Suc (40 mm) were added to embryos pretreated for 12 h (time = 0) in 1 μm GA3 and 5 mm CaCl2; samples were collected every 3 h. Embryos were extracted for RNA gel-blot analysis, performed as described in Figure 5. Data are means ± se of three separate experiments. B, ActD (10 μg/mL) and turanose (40 mm) were added to embryos pretreated for 12 h (time = 0) in 1 μm GA3 and 5 mm CaCl2; samples were collected every 3 h. Embryos were extracted for RNA gel-blot analysis, performed as described in Figure 5. Data are means ± se of three separate experiments. Amy, α-amylase; Ubi, ubiquitin.
Figure 7

Figure 7

Effects of increasing concentrations of…

Figure 7

Effects of increasing concentrations of disaccharides on α-amylase induction in barley embryos. A…

Figure 7
Effects of increasing concentrations of disaccharides on α-amylase induction in barley embryos. A and B, Effect of increasing concentration of lactulose, palatinose, turanose, lactitol, and palatinitol on the α-amylase mRNA level in barley embryos. Embryos were incubated for 24 h in 1 μm GA3 and 5 mm CaCl2 in the presence of 0 to 80 mm disaccharides. Embryos were extracted for RNA gel-blot analysis. Blots were probed with the α-amylase probe. Membranes were reprobed with an rRNA probe (not shown) as well as with an ubiquitin probe. RNA levels of both α-amylase and rRNA were quantified and the relative α-amylase transcript level normalized to rRNA (100 = transcript level in the embryos not treated with exogenous carbohydrates) is reported in the histograms. Data are means ± se of three separate experiments. The mRNA band relative to 0 mm (lactulose, palatinose, and turanose) was duplicated in the three reproductions for the sake of clarity. C, Effect of lactose, galactobiose (β-Gal[1→4]Gal, and Gal[1→3]Gal) on the α-amylase mRNA level in barley embryos. Embryos treated as described in A and B with 80 mm carbohydrates. RNA blots were obtained as described in A and B. Data are means ± se of three separate experiments. D, Effect of Fru epimers on the α-amylase mRNA level in barley embryos. Embryos treated as described in A and B with 80 mm tagatose or psicose. RNA blots were obtained as described in A and B. Amy, α-amylase; Ubi, ubiquitin.
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