Functional characterization of the parthenocarpic fruit mutation in tomato (Solanum lycopersicum L.)
Author(s)
Ruiu, Fabrizio
Date Issued
June 11, 2013
Type
Doctoral Thesis
Abstract
Parthenocarpy is the production of seedless fruits in the absence of pollination and/or
fertilization. This process has been extensively studied in tomato (Solanum lycopersicum
L.) because it offers a method to overcome unfavourable environmental conditions that
reduce pollen production, anther dehiscence and, as a consequence, fruit set.
Among the different sources of genetic parthenocarpy described in tomato, the
parthenocarpic fruit (pat) mutation, object of the thesis, is of particular interest because of
its strong expressivity, high fruit set and enhanced fruit quality.
Previous studies have thoroughly characterized the pat mutation from the genetic,
biochemical and qualitative standpoint. More recent research described the complexity of
the pat syndrome, which associates a strong competence for parthenocarpy with a complex
floral phenotype, involving stamen (reduced length and carpelloid features) and ovule
(arrested integument growth and loss of viability) development.
By a positional cloning approach, the Pat locus was mapped on the long arm of
chromosome 3 and recently, by a candidate gene approach, nine candidates have been
addressed as potentially responsible for the mutant phenotype. Among them, four genes,
known to be involved in reproductive processes, were sequenced in the wild type (WT) and
near-isogenic pat line. After sequencing, a single point mutation was found only in the
tomato ortholog of ATHB15, also known as CORONA (CNA) or INCURVATA4 (ICU4), in
Arabidopsis. ATHB15/CNA/ICU4 is a transcription factor (TF) belonging to the class III HDZip
subfamily protein. Accordingly to this finding, in tomato, we named this gene Solanum
lycopersicum HB15 (SlHB15).
Starting from this background, the thesis aimed to confirm that SlHB15 is involved in
biological processes that, if deregulated, could lead to parthenocarpy and to highlight
differentially expressed genes in the WT and pat ovary during fruit set.
Using different bioinformatic tools, the amino acid substitution found in the SlHB15 protein
encoded by the pat allele was predicted to be not tolerated for the protein function. In
addition, following an in silico comparative approach between ATHB15/CNA/ICU4 in
Arabidopsis and SlHB15 in tomato, interesting highlights about its involvement in the
regulation of auxin homeostasis in plant tissues and biological processes (e.g. flower
development and fruit set) were found (Chapter 2).
So far, RNA interference and complementation experiments performed for the genetic
confirmation of the mutation were not definitive, but promising observations of putatively
SlHB15-silenced plants reinforced the hypothesis that SlHB15 underlies the pat mutation.
Furthermore, in order to find phenotypic similarities between pat and single mutants for its
ortholog ATHB15/CNA/ICU4 in Arabidopsis, a characterization of the cna-1 (loss-ofii
function) and icu4-1 (gain-of-function) mutants for this gene, was performed. Interestingly,
pleiotropic effects showed by pat (e.g. deviation of the number of cotyledons and aberrant
ovules) and parthenocarpy, were also observed in these mutants, indicating again that
SlHB15 could represent the locus of the pat mutation (Chapter 3).
A microarray experiment was performed in order to identify differentially expressed genes
(DEGs) in the WT and pat ovary during fruit set. Based on their expression pattern, the
DEGs were categorized into five groups of clusters representing different biological trends.
One of the groups deserving more attention was named ‘Controlling complex’ and
contained putative negative or positive regulators of fruit set (genes up- or down-regulated
at pre-athesis in the WT ovary and deregulated in the pat mutant). Interesting genes
belonging to this group encoded tomato orthologs of Arabidopsis TFs regulating the
meristem differentiation and development of floral organs, such as
SHOOTMERISTEMLESS (STM), BIG PETALp (BPEp), AINTEGUMENTA (ANT) and
CRABS CLAW (CRC). These findings represented new insights, because so far these
genes belonging respectively to the KNOX, bHLH, AP2/ERF and YABBY families of TFs
had never been so directly associated to parthenocarpy. Finally, these TFs and other
selected genes were studied also in other pathenocarpic systems (pat-2, pat-3/pat-4, EMSiaa9
and RNAi-ARF7) in order to increase the understanding of parthenocarpy in tomato
and to find homologies between these different sources for such an agronomically
important trait (Chapter 4).
Finally, a genetic interaction study between pat and Curl (Cu), a mutation responsible for
the overexpression of the gene LeT6/TKn2 (tomato ortholog of STM in Arabidopsis) in
vegetative and floral organs, was performed. Overall, the phenotypic and molecular
characterization of the pat Cu double mutant confirmed findings previously reported and
indicated that KNOX family members, such as LeT6/TKn2, may act as negative regulators
of the fruit set (Chapter 5).
Taken together, results obtained from this thesis increased the understanding of the genetic
and molecular bases of fruit set and parthenocarpy in tomato.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
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