Plastidial alpha-glucan phosphorylase is not required for starch degradation in Arabidopsis leaves but has a role in the tolerance of abiotic stress

Abstract

To study the role of the plastidial alpha-glucan phosphorylase in starch metabolism in the leaves of Arabidopsis, two independent mutant lines containing T-DNA insertions within the phosphorylase gene were identified. Both insertions eliminate the activity of the plastidial alpha-glucan phosphorylase. Measurement of other enzymes of starch metabolism reveals only minor changes compared with the wild type. The loss of plastidial alpha-glucan phosphorylase does not cause a significant change in the total accumulation of starch during the day or its remobilization at night. Starch structure and composition are unaltered. However, mutant plants display lesions on their leaves that are not seen on wild-type plants, and mesophyll cells bordering the lesions accumulate high levels of starch. Lesion formation is abolished by growing plants under 100% humidity in still air, but subsequent transfer to circulating air with lower humidity causes extensive wilting in the mutant leaves. Wilted sectors die, causing large lesions that are bordered by starch-accumulating cells. Similar lesions are caused by the application of acute salt stress to mature plants. We conclude that plastidial phosphorylase is not required for the degradation of starch, but that it plays a role in the capacity of the leaf lamina to endure a transient water deficit.

Figure 1.

T-DNA insertion mutations at the AtPHS1 locus. Structure of the AtPHS1 gene; exons are depicted as closed boxes.T-DNA left border sequence is on a hatched background. Underlined sequence is unidentified DNA lacking similarity to the AtPHS1 locus or the T-DNA sequence. The T-DNA insertion in Atphs1-1 starts 12 bp into intron 6, whereas the insertion site in Atphs1-2 is 99 bp upstream of the ATG translational start codon.

Figure 2.

Loss of plastidial phosphorylase activity in Atphs1-1 and Atphs1-2. Soluble proteins from crude extracts of leaves were subjected to native PAGE and stained to reveal phosphorylase isoforms. The cytosolic form (H-form) is present in all extracts. The chloroplast form (L-form) is missing in extracts of Atphs1-1 and Atphs1-2.

Figure 3.

Starch and sugar contents of wild-type and Atphs1-1plants during the diurnal cycle. A, Samples comprising all the leaves of individual wild-type (white symbols, solid line) or Atphs1-1plants (black symbols, dashed line) plants were harvested and immediately frozen in liquid N₂. Leaf material was ground to a powder and extracted in ice-cold perchloric acid. Starch in the insoluble material and sugars in the soluble fraction were measured. Each point is the mean ± se from four replicate samples. B, Starch samples (1 mg) from leaves of the wild type (gray bars) and Atphs1-1 (black bars) were debranched with isoamylase, derivatized with the fluorophore APTS, and chains of different lengths separated by PAGE in an Applied Biosystems DNA sequencer (Foster City, CA). Peak areas of chains between 6- and 41-Glc units in length were summed and the areas of individual peaks expressed as a percentage of the total. Representative data from one of three independent experiments are shown. C, Starch samples (0.5 mg) of leaves from wild type (gray circles, solid line) and Atphs1-1 (black circles, dashed line) were separated by Sepharose CL2B chromatography. The absorbance of each fraction at 595 nm was determined after the addition of an iodine solution. To normalize each data set, the absorbance of each fraction was divided by the summed absorbencies of all the fractions. Representative data from one of three independent experiments are shown.

Figure 4.

The formation of lesions bordered by starch-accumulating cells in Atphs1 mutants. A, A typical 5-week-old Atphs1-1 plant grown in a growth chamber with a 12-h light/12-h dark regime (175 μmol photons m⁻² s⁻¹), 20°C, 75% relative humidity. Plants were sown onto germination medium and transplanted after 2 to 3 weeks into individual pots. The arrowhead marks a visible lesion. Bar represents 1 cm. B, The same plant as in A, decolorized with 80% ethanol and stained with iodine at the end of the dark period. The arrowhead marks the local accumulation of starch around the lesion site. Bar represents 1 cm. C, Light micrograph of leaf lamina bordering the lesion site shown in B revealing cell-specific accumulation of starch. Bar represents 0.25 mm.

Figure 5.

Lesion formation in Atphs1 can be induced by transient water stress. A, Wild-type (top two rows) and Atphs1-1 plants (bottom two rows) grown as described in Figure 4A, except that still air with a humidity near 100% was maintained with a clear plastic cover. No lesions were visible on mutant plants at this stage of growth. The plastic cover was then removed and the plants exposed to circulating air with 60% ± 5% relative humidity. B, The same plants as in A 3 d after the plastic cover was removed. Wild-type plants exhibit only infrequent wilt symptoms at leaf margins and continue to grow. Mutant plants exhibit severe wilt symptoms, with the death of entire leaves, and an overall suppression of growth. C, Enlargement of a single mature leaf of an Atphs1-1 plant 1, 2, 3, and 6 d after the switch from high to low humidity described in A. D, Wild-type and Atphs1-2 plants grown as described in A were irrigated with 0.5 m NaCl as described in “Materials and Methods.” Wild-type and mutant plants exhibited a loss of turgor in existing leaves and the growth of new, pale green, wrinkled leaves. Plants were photographed 6 d after the beginning of the salt treatment. Identical results were obtained with Atphs1-1.

Figure 6.

Quantification of lesion formation on leaves of wild type and Atphs1-1. A, Five-week-old wild-type plants (pictured in Fig. 5, A and B) were scored for lesions (day 0) then shifted from high to low humidity as described. Leaves present on day 0 were scored for lesions on days 1, 2, 3, and 6. The percentage of leaves bearing no lesion (white bars), lesions covering less that 30% of the lamina (light gray bars), lesions covering more than 30% of the lamina (dark gray bars), and dead leaves (black bars) is presented. Leaves that developed after day 0 were not scored for lesion formation. se were lower than 5% (n = 8). B, Five-week-old Atphs1-1 plants (pictured in Fig. 5, A and B) were scored as described in A.

Figure 7.

Stomatal function in wild type (white symbols), Atphs1-1 (gray symbols), and Atphs1-2 (black symbols). A, Rate of water loss from detached 5-week-old rosettes of wild type, Atphs1-1, and Atphs1-2 (black symbols). Plants were detached and weighed at intervals over the course of 60 min. Each point is the mean ± se of measurements on four replicate plants. B, Stomatal conductance of single leaves from wild type, Atphs1-1, and Atphs1-2. Gas exchange was measured over the course of 60 min following the detachment of the leaf and used to calculate stomatal conductance. Each point is the mean ± se of measurements from four replicate leaves.