A new class of regulatory genes underlying the cause of pear-shaped tomato fruit

Abstract

A common, recurring theme in domesticated plants is the occurrence of pear-shaped fruit. A major quantitative trait locus (termed ovate) controlling the transition from round to pear-shaped fruit has been cloned from tomato. OVATE is expressed early in flower and fruit development and encodes a previously uncharacterized, hydrophilic protein with a putative bipartite nuclear localization signal, Von Willebrand factor type C domains, and an approximately equal 70-aa C-terminal domain conserved in tomato, Arabidopsis, and rice. A single mutation, leading to a premature stop codon, causes the transition of tomato fruit from round- to pear-shaped. Moreover, ectopic, transgenic expression of OVATE unevenly reduces the size of floral organs and leaflets, suggesting that OVATE represents a previously uncharacterized class of negative regulatory proteins important in plant development.

Figure 1

Molecular cloning of the OVATE QTL. (A) The location of the major QTLs for both fruit elongation (red) and fruit neck constriction (blue) on chromosome 2. (B) High-resolution mapping of the ovate region. Molecular marker TG645 was used to isolate a BAC clone, the BAC19, which covers the ovate region. The recombinant events in the original mapping population (3, 4), shown by “x”, delimit ovate to a 55-kb region. (C) Schematic diagram of the 105-kb BAC19 insert. Red horizontal arrows orient and size the 17 ORFs (11). Vertical arrows and a delta symbol (Δ) position the SNPs and the 2-bp indel between wild-type round-fruited (TA496) and pear-shaped (TA493) genotypes, respectively. (D) Structure of the ORF6 on BAC19. Two exons (black bar) are separated by an intron (thin line) where a crossover event in TA1792, a homozygous recombinant from the original mapping population (3, 4), is indicated as “x”. The start codon (ATG), stop codon (TAG), and polyA site are as marked. A G_TA496-to-T_TA493 polymorphism (in red) in the second exon causes an early stop codon in TA493.

Figure 2

Complementation of pear-shaped phenotypes by OVATE. Images represent ovaries (A) and mature fruit (C) as well as their corresponding longitudinal cross sections (B and D, respectively) from wild-type round-fruited TA496 (column 1) or a pear-shaped line (TA503) transformed with vector, native, or 35S∷OVATE (columns 2, 3, and 4, respectively). (E–G) Progressive rescue of pear-shaped phenotypes by native OVATE. Fruit at early, late, and mature developmental stages, respectively, from an inflorescence of a native transformant.

Figure 3

Previously uncharacterized phenotypes of OVATE-overexpression lines. The pear-shaped line, TA503, was transformed with the binary vector alone (column 1) or 35S∷OVATE (columns 2–4). Lines 1, 2, and 3 represent weaker, modest, and stronger overexpression lines, respectively. (A) Stigma exsertion phenotype of overexpression lines. (B) Measurements of the length of different floral organs (n = 10). (C) Plant growth inhibition by overexpression of OVATE. (D) Suppression of leaf size by overexpression of OVATE. Shown are the tenth leaves from shoot apices of each line. (E) The corresponding top leaflets in D. Stronger overexpression lines (columns 3 and 4) show changed leaflet shape.

Figure 4

Relative expression of OVATE in vegetative and floral organs of round-fruited (TA496, black box) and pear-shaped (TA503, white box) genotypes. DBA, day before anthesis; DAA, day after anthesis.

Figure 5

Structure and sequence alignments of OVATE. (A) Hydrophilicity plot of OVATE. (B) Schematic diagram of OVATE structure. Green box, bipartite nuclear localization signal (NLS); blue boxes, VWFC protein–protein interaction domains; orange box, conserved C-terminal domains from tomatoes, arabidopsis, and rice. (C) Sequence alignment of the conserved carboxyl termini of OVATE with matches from the GenBank EST and nucleotide databases. The consensus sequences are shaded in black boxes. *, location of the premature translation termination in ovate (TA503, TA493).