Normal growth of Arabidopsis requires cytosolic invertase but not sucrose synthase

Abstract

The entry of carbon from sucrose into cellular metabolism in plants can potentially be catalyzed by either sucrose synthase (SUS) or invertase (INV). These 2 routes have different implications for cellular metabolism in general and for the production of key metabolites, including the cell-wall precursor UDPglucose. To examine the importance of these 2 routes of sucrose catabolism in Arabidopsis thaliana (L.), we generated mutant plants that lack 4 of the 6 isoforms of SUS. These mutants (sus1/sus2/sus3/sus4 mutants) lack SUS activity in all cell types except the phloem. Surprisingly, the mutant plants are normal with respect to starch and sugar content, seed weight and lipid content, cellulose content, and cell-wall structure. Plants lacking the remaining 2 isoforms of SUS (sus5/sus6 mutants), which are expressed specifically in the phloem, have reduced amounts of callose in the sieve plates of the sieve elements. To discover whether sucrose catabolism in Arabidopsis requires INVs rather than SUSs, we further generated plants deficient in 2 closely related isoforms of neutral INV predicted to be the main cytosolic forms in the root (cinv1/cinv2 mutants). The mutant plants have severely reduced growth rates. We discuss the implications of these findings for our understanding of carbon supply to the nonphotosynthetic cells of plants.

Fig. 1.

Location of SUS6 transcript and protein in WT and quadruple mutant plants. Pictures are representative of multiple plants for each genotype and treatment. (A) Blots of hypocotyl extracts (from 11-week-old plants grown in short days), stems (as in Table 1), whole root systems (from 5-week-old plants grown in an inert medium), and purified SUS proteins (28) (Std, SUS5 protein for SUS5 antiserum; SUS6 protein for SUS6 antiserum) were probed with SUS5 or SUS6 antiserum (28). In each panel, lanes are from the same gel and blot. Equal fractions of soluble and pellet material were loaded. Note that bands recognized by the SUS5 antiserum in root soluble fractions are not SUS5 protein. They migrate more slowly than authentic SUS5 (arrowed), and are present in the sus5/sus6 mutant. The SUS6 antiserum recognizes 2 bands in extracts (arrowed in WT). Both are missing in the sus5/sus6 mutant, so both are probably SUS6 protein. (B) Tissue prints of stem (Upper) and hypocotyl (Lower) sections probed with SUS6 antiserum. (Left to Right) WT, sus5/sus6 mutant, sus1/sus2/sus3/sus4 (Quad) mutant, and a stem section stained with toluidine blue. Black arrows show typical antiserum reactions; red arrows show the equivalent region on the section. (C) Location of SUS6 transcript in roots by whole-mount in situ RNA hybridization. Fixed and cleared roots of 4-day-old seedlings were treated with RNA probes for SUS6. (Left) WT with SUS6 antisense probe; (Center) WT with SUS6 sense probe; (Right) sus1/sus2/sus3/sus4 mutant with SUS6 antisense probe. The appearance of sus6 mutant roots with SUS6 antisense probe was the same as WT roots with SUS6 sense probe. Arrows show RNA hybridization. Root diameter is ≈130 μm. (D) Location of SUS transcript in root sections. Roots as in C were embedded and sectioned at the level at which RNA hybridization was observed. Arrows show hybridization. (Upper Left and Right) Sections of WT roots with SUS6 antisense probe and SUS6 sense probe respectively; (Lower Left) a sus1/sus2/sus3/sus4 mutant root with SUS6 antisense probe.

Fig. 2.

Anatomy and composition of WT and quadruple mutant plants. (A) Scanning electron micrographs of freeze-fractured leaves, showing cell walls in cross-section (Scale bars, 2 μm.) (Left); and face-on (Scale bars, 1 μm.) (Right). (Upper) WT leaves. (Lower) The sus1/sus2/sus3/sus4 mutant leaves. (B) Light micrographs of cross-sections of the basal 4 cm of flowering stems. (Left) WT; (Right) sus1/sus2/sus3/sus4 mutant. (C) FTIR spectra derived from the insoluble (cell wall) fraction of roots of 4-day-old seedlings. Spectra are averaged from analyses on 20 samples from a preparation of 40 primary roots. (Upper) WT roots; (Lower) sus1/sus2/sus3/sus4 mutant roots. Similar results were obtained with a different batch of seedlings. (D) Scanning FTIR array microscopy of stems (as in Table 1). (Upper) “Heatmap” images of 25-μm stem sections at a wavenumber dominated by cellulose (1,056 cm⁻¹). White is the highest and black the lowest cellulose content. (Lower) Averaged spectra derived from xylem regions (arrowed). (Left) WT plant; (Right) sus1/sus2/sus3/sus4 mutant plant.

Fig. 3.

Appearance of the cinv1/cinv2 mutant. (A) Mature WT (Left) and cinv1/cinv2 mutant (Right) plants of the same age, grown in the same conditions. (B) Seven-day-old seedlings grown vertically on solid medium. (Left) WT seedlings (at the left) and cinv1/cinv2 seedlings (at the right); (Right) a cinv1/cinv2 seedling. (C and D) Cross-sections of roots of WT (C) and cinv1/cinv2 (D) seedlings. Sections are close to the base of the root cap (outer layer of cells) for WT roots and at an equivalent position for mutant roots. Plants were from the same plate. Magnifications are the same. (E and F) Longitudinal sections of roots of WT (E) and cinv1/cinv2 (F) seedlings. Plants were from the same plate. Magnifications are the same. The partial collapse of the root was seen in all mutant roots, but no WT roots. (C–F) Results are typical of those for many seedlings.