Point mutation of a plastidic invertase inhibits development of the photosynthetic apparatus and enhances nitrate assimilation in sugar-treated Arabidopsis seedlings

Abstract

Because the photosynthetic apparatus contains a massive amount of nitrogen in plants, the regulation of its development by sugar signals is important to the maintenance of the carbon-nitrogen balance. In this study we isolated an Arabidopsis mutant (sicy-192) whose cotyledon greening was inhibited by treatments with sugars such as sucrose, glucose, and fructose. In the mutant, the gene encoding plastidic alkaline/neutral invertase (INV-E) was point-mutated at codon 294, with Tyr substituted for Cys (C294Y). Interestingly, the greening of cotyledons in the knock-out INV-E lines was not inhibited by treatment with the sugars. In addition, the knock-out INV-E lines expressing an INV-E:C294Y or INV-E:C294A gene had the same phenotype as sicy-192 mutants, whereas the lines expressing a wild-type INV-E gene had the same phenotype as wild-type plants. A recombinant INV-E:C294Y protein had the same enzymatic activity as a recombinant INV-E protein, suggesting that the Cys-294 residue of INV-E is important for its functions in the chloroplasts. On treatment with sucrose, the expression of photosynthesis-related genes was weaker in seedlings of mutant plants than wild-type seedlings, whereas the activity of nitrate reductase was stronger in the mutant plants than wild-type plants. These findings suggest that Cys-294 of INV-E is associated with the development of the photosynthetic apparatus and the assimilation of nitrogen in Arabidopsis seedlings to control the ratio of sucrose content to hexose content.

FIGURE 1.

Phenotypes of the wild-type and sicy-192 mutant plants. A, shown are visual phenotypes of the wild-type and sicy-192 mutant grown for 7 days in MS medium with or without 100 mm sucrose. B, shown is the chlorophyll content of the wild-type (open bars) and sicy-192 mutant (solid bars) plants grown for 7 days in MS medium with or without 100 mm of various sugars. C, growth of wild-type (open bars) and sicy-192 mutant (solid bars) plants with various carbon-nitrogen balances is shown. Shown is the wet weight of shoots of the plants grown for 2 weeks in MS medium without sucrose (control), MS medium with sucrose (+Suc), 1/10 n (6 mm nitrogen) MS medium without sucrose (0.1 n), or 1/10 n (6 mm nitrogen) MS medium with sucrose (0.1 n+ Suc). Each value is the mean and S.E. for three replicates. Asterisks indicate that mean values were significantly different compared with those in wild-type plants when analyzed by Student's t test (*, p < 0.05; **, p < 0.01).

FIGURE 2.

Map-based cloning of the causative gene of sicy-192 mutant plants and alignment of A/N invertase proteins. A, the locus of the causative gene was mapped to between the markers KUCAPS5–742 and KUCAPS5–748 on chromosome 5. The start codon (ATG) and stop codon (TGA) are indicated. The G to A nucleotide substitution in exon 4 converted Cys to Tyr. B, alignments of A/N invertase proteins of Arabidopsis, rice, and Synechocystis sp. PCC 6803 are shown. An arrow indicates the mutated residue in sicy-192.

FIGURE 3.

INV-E expression level and chlorophyll content in seedlings of KO-INV-E lines expressing mutated INV-E. A, transcript levels of INV-E are shown. These lines were grown in SUC⁺ medium for 5 days. The values were normalized to the eIF4A-1 expression of each sample and are shown in relation to the RNA level measured in wild-type plants. Each value is the mean and S.E. of three replicates. B, shown is A/N invertase activity in crude extracts of the plants grown in SUC⁺ medium for 5 days. Each value is the mean and S.E. for three replicates. Bars marked with a different letter were significantly different (p < 0.05) when analyzed by the Student-Newman-Keuls analysis of variance. C, the accumulated levels of INV-E in seedlings of the wild-type, sicy-192 mutants, and KO-INV-E lines are shown. Seedlings were grown in the SUC⁺ medium (Suc) or SUC⁻ medium (control) for 5 days, and their soluble proteins (10 μg lane⁻¹) were separated by 12% SDS-PAGE and analyzed by immunoblotting using antibody against INV-E. Molecular masses of marker proteins are indicated on the left. CBB, Coomassie Brilliant Blue. D, chlorophyll content of the plants grown in the SUC⁺ medium (solid bars) or SUC⁻ medium (open bars) is shown. Each value is the mean and S.E. for three replicates. Asterisks indicate that mean values were significantly different compared with those for the control treatment when analyzed by Student's t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). FW, fresh weight. E, shown is the phenotype of 5-day-old seedlings in 100 mm sucrose-containing medium.

FIGURE 4.

Enzymatic properties of recombinant INV-E and INV-E:C294Y proteins. A, purified His-tagged INV-E and His-tagged INV-E:C294Y proteins (0.5 μg of protein lane⁻¹) were separated on a12% SDS-PAGE gel and detected by silver staining. B, shown is the effect of thiol-modifying reagents on the invertase activity of recombinant INV-E and INV-E:C294Y. The specific activities of the recombinant INV-E and INV-E:C294Y proteins without the reagent (none) were 32.8 and 40.8 mmol of product min⁻¹ mg⁻¹ protein, respectively, and were normalized (= 100%). DTT, dithiothreitol; pCMB, p-chloromercuribenzoate.

FIGURE 5.

A, chlorophyll contents of single and double mutant lines grown in the SUC⁺ medium (Suc) or SUC⁻ medium (control) for 7 days are shown. Each value is the mean and S.E. for three replicates. Bars marked with a different letter were significantly different (p < 0.05) when analyzed by the Student-Newman-Keuls analysis of variance. B, shown is the phenotype of 5-day-old seedlings in 100 mm sucrose-containing medium. FW, fresh weight.

FIGURE 6.

The transcript levels of photosynthesis and nitrate assimilation-related genes in seedlings of the wild type and sicy-192 mutants and KO-INV-E lines. Seedlings were grown in the SUC⁺ medium (Suc) or SUC⁻ medium (Control) for 5 days, and their RNA was analyzed by real time reverse transcription-PCR. The values were normalized to the eIF4A-1 (At3g13920) expression of each sample and are shown in relation to the RNA level measured in sucrose-treated wild-type plants. Each value is the mean and S.E. for three or four replicates. Bars marked with a different letter were significantly different (p < 0.05) when analyzed by the Student-Newman-Keuls analysis of variance. CAB, chlorophyll a/b-binding protein; SBPase, sedoheptulose-1,7- bisphosphatase; FBPase, fructose-1,6-bisphosphatase.

FIGURE 7.

The accumulated levels of rbcL, sedoheptulose-1,7-bisphosphatase (SBPase), and GSs in seedlings of the wild type and sicy-192 mutants and KO-INV-E lines. Seedlings were grown in the SUC⁺ medium (Suc) or SUC⁻ medium (Control) for 5 days, and their soluble proteins (10 μg lane⁻¹) were separated by 12% SDS-PAGE and analyzed by immunoblotting with antibody against rbcL, sedoheptulose-1,7-bisphosphatase (SBPase), or GS. Molecular masses of marker proteins are indicated on the left. CBB, Coomassie Brilliant Blue.

FIGURE 8.

Hexose (A) and sucrose (B) levels in seedlings of the wild type and sicy-192 mutants and KO-INV-E lines. The seedlings were grown in the SUC⁺ medium (+Suc) or SUC⁻ medium (Control) for 5 days, and then hexose and sucrose levels in the seedlings were determined. Each value is the mean and S.E. for four replicates. Bars marked with a different letter were significantly different (p < 0.05) when analyzed by the Student-Newman-Keuls analysis of variance. FW, fresh weight.