Trehalose-6-phosphate synthase/phosphatase regulates cell shape and plant architecture in Arabidopsis

Abstract

The vacuole occupies most of the volume of plant cells; thus, the tonoplast marker delta-tonoplast intrinsic protein-green fluorescent protein delineates cell shape, for example, in epidermis. This permits rapid identification of mutants. Using this strategy, we identified the cell shape phenotype-1 (csp-1) mutant in Arabidopsis thaliana. Beyond an absence of lobes in pavement cells, phenotypes included reduced trichome branching, altered leaf serration and stem branching, and increased stomatal density. This result from a point mutation in AtTPS6 encoding a conserved amino-terminal domain, thought to catalyze trehalose-6-phosphate synthesis and a carboxy-terminal phosphatase domain, is catalyzing a two-step conversion to trehalose. Expression of AtTPS6 in the Saccharomyces cerevisiae mutants tps1 (encoding a synthase domain) and tps2 (encoding synthase and phosphatase domains) indicates that AtTPS6 is an active trehalose synthase. AtTPS6 fully complemented defects in csp-1. Mutations in class I genes (AtTPS1-AtTPS4) indicate a role in regulating starch storage, resistance to drought, and inflorescence architecture. Class II genes (AtTPS5-AtTPS11) encode multifunctional enzymes having synthase and phosphatase activity. We show that class II AtTPS6 regulates plant architecture, shape of epidermal pavement cells, and branching of trichomes. Thus, beyond a role in development, we demonstrate that the class II gene AtTPS6 is important for controlling cellular morphogenesis.

Figure 1.

The csp-1 mutant displays cell shape, size, and trichome aberrations. A to F, Confocal microscopy images of cells of 7-d-old seedlings expressing the tonoplast marker GFP∷δ-TIP. A and B, Leaf epidermal pavement cells of parental (A) and csp-1 mutant lines (B). C and D, Hypocotyl cells of the parent (C) and mutant (D). E and F, Root cells of the parent (E) and mutant (F). G to K, Scanning electron microscopy images of similar 14-d-old seedlings. G and H, Leaf epidermal pavement cells of the parental line (G) and mutant (H). I, Leaves of the csp-1 mutant (left) and the parental line (right) display obvious differences in trichome biogenesis. J and K, Several individual trichomes are shown from csp-1 (J) and the parental line (K), highlighting the differences in branch number. Arrows indicate positions of several epidermal cell lobes in the parent (A and G) and mutant (B and H). Bars = 50 μm (A and B); 150 μm (C–F); 100 μm (G and H); 500 μm (I); and 200 μm (J and K).

Figure 2.

The csp-1 mutant is affected in growth from seedling to adult stages. A, Nine-day-old seedlings of the parental line (left two seedlings) and csp-1 (right two seedlings) show obvious retardation in growth of the mutant when grown and viewed on agar medium. B and C, Sixteen weeks after sowing, rosettes of the parent (B) and mutant (C) are different morphologically, with the leaves of csp-1 displaying prominent serration and strong epinasty. D, Differences in leaf morphology are apparent when leaves of the parent (left leaf) are viewed in more detail relative to a mutant leaf (right leaf).

Figure 3.

The csp-1 mutant is altered in a gene encoding TPS (AtTPS6). A, The physical position of the csp-1 locus is indicated with the positions of nearby insertion-deletion markers (top bar). The location of AtTPS6 on BAC T23K23 is indicated with other BACs in the region. B, Schematic of the AtTPS6 gene indicating start and stop codons. The positions of an A-to-C substitution in csp-1 at nucleotide position 456 and a T-DNA allele (csp-2) are shown. Numbers indicate nucleotide positions. C, Amino acid residues in single-letter code are shown for the parental line (top sequence) and csp-1 (bottom sequence) in the region of an Arg-to-Ser substitution (indicated by line). Numbers indicate amino acid positions.

Figure 4.

AtTPS6 has functional synthase and phosphatase domains. A, Schematic showing protein domains of TPS1 and TPS2 from yeast ScTPS1 and ScTPS2 and their homologs in Arabidopsis AtTPS1 and AtTPS6, respectively. B, Complementation of yeast with corresponding Arabidopsis genes. Top, As a control, wild-type yeast (wild type), a ScTPS1 deletion mutant (Δtps1), and the deletion mutant expressing AtTSP6 (Δtps1 + AtTPS6) grew on Gal as a carbon source. When grown on Glc, wild-type yeast grew normally, whereas the deletion mutant (Δtsp1) could not utilize Glc as a carbon source. When AtTPS6 was expressed in Δtps1 (AtTPS6 + Δtps1), yeast grew vigorously, indicating that AtTPS6 complemented Δtsp1 functionally. Bottom, When grown at the permissive temperature (28°C), wild-type yeast, a deletion mutant (Δtps2), and AtTPS6 expressed in Δtps2 (Δtps2 + AtTPS6) all grew normally. However, at the nonpermissive temperature, Δtps2 could not grow, whereas the strain expressing AtTPS6 (Δtps2 + AtTPS6) displayed growth similar to that of the control (wild type), indicating functional complementation by AtTSP6.

Figure 5.

AtTPS6 fully complements the cell shape and developmental defects in csp-1. A, Cell morphology of leaf pavement cells of 7-d-old Arabidopsis seedlings coexpressing GPF∷δ-TIP and a construct encoding wild-type AtTPS6 (TPS6Pro∷TPS6; compare to Fig. 1, A and B). Normal cell morphology indicated complementation. B and C, Adult plants of the complemented mutant (C) displayed overall morphology similar to that of the control (wild type; B), including rosettes with leaves showing typical morphology without pronounced serration as in csp-1 (compare to Fig. 2C). D, Trichome of the complemented line displaying three branches.

Figure 6.

A second allele (csp-2) displays cell shape defects and overexpression lines of AtTPS6 showing dramatic alterations in development. A, A T-DNA loss-of-function allele csp-2 when stained with FM4-64 also shows cell shape defects similar to those of csp-1. Bar = 50 μm. B, However, csp-2 does not display leaf serration or epinasty as observed for csp-1. C, Representative category I overexpression mutant displaying increased rosette and leaf size. D, Scanning electron micrograph showing a representative trichome from a line overexpressing AtTPS6 with four branches compared to the normal three found in the parental line (compare to Fig. 1K). Bar = 300 μm. E, Category I AtTPS6 overexpression lines displaying increased rosette and leaf size and decreased bolts (increased apical dominance). Inset, Representative quantity of transcript by RT-PCR (top band, AtTPS6; bottom band, ribosomal control; left lane, wild-type Col ecotype; right lane, representative overexpression line). F, Category II AtTPS6 overexpression lines displaying delayed growth and increased bolts (reduced apical dominance) but not enlarged rosettes. Inset, Representative quantity of transcript by RT-PCR (top band, AtTPS6; bottom band, ribosomal control; left lane, wild-type Columbia ecotype; right lane, category II overexpression line).