Functional characterization of a tomato COBRA-like gene functioning in fruit development and ripening
Ying Cao
1
Xiaofeng Tang
1
Jim Giovannoni
Fangming Xiao
Yongsheng Liu
0
1
0
School of Biotechnology and food Engineering, Hefei University of Technology
,
Hefei 230009
,
China
1
Ministry of Education Key Laboratory for Bio-resource and Eco-environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University
,
Chengdu 610064
,
China
Background: Extensive studies have demonstrated that the COBRA gene is critical for biosynthesis of cell wall constituents comprising structural tissues of roots, stalks, leaves and other vegetative organs, however, its role in fruit development and ripening remains largely unknown. Results: We identified a tomato gene (SlCOBRA-like) homologous to Arabidopsis COBRA, and determined its role in fleshy fruit biology. The SlCOBRA-like gene is highly expressed in vegetative organs and in early fruit development, but its expression in fruit declines dramatically during ripening stages, implying a primary role in early fruit development. Fruit-specific suppression of SlCOBRA-like resulted in impaired cell wall integrity and up-regulation of genes encoding proteins involved in cell wall degradation during early fruit development. In contrast, fruit-specific overexpression of SlCOBRA-like resulted in increased wall thickness of fruit epidermal cells, more collenchymatous cells beneath the epidermis, elevated levels of cellulose and reduced pectin solubilization in the pericarp cells of red ripe fruits. Moreover, transgenic tomato fruits overexpressing SlCOBRA-like exhibited desirable early development phenotypes including enhanced firmness and a prolonged shelf life. Conclusions: Our results suggest that SlCOBRA-like plays an important role in fruit cell wall architecture and provides a potential genetic tool for extending the shelf life of tomato and potentially additional fruits.
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Background
The ripening of fleshy fruits involves a number of
physiological processes including the production of
aromatic compounds, nutrients, pigmentation, and
softening of flesh to an edible texture [1,2]. These processes
have direct impacts not only on fruit firmness, color,
flavor and nutritional content, but also on shelf life,
consumer acceptability, processing qualities, in addition to
pre- and postharvest disease resistance [1,2]. Excessive
fruit softening is the main factor contributing to damage
during shipping, storage and post-harvest handling [3].
Fruit firmness and texture also affect the integrity of
chopped and diced fruit used for canning and fruit
products [4]. Because postharvest losses of fresh fruits
due to excessive softening can account for as much as
30~40% of total production, considerable research had
focused on mechanisms of fruit softening, often using
tomato (Solanum lycopersicum) as a model system [3].
Fruit softening during the ripening process results in
part from disassembly of the cell walls, leading to a
reduction in intercellular adhesion, depolymerization and
solubilization of pectins, depolymerization of
hemicelluloses, and loss of pectic galactose side chains [3].
Generally, the decline in fruit firmness due to softening is
accompanied by elevated expression of numerous cell
metabolism enzymes, including polygalacturonase (PG)
[5,6], pectin methylesterase (PME) [7], -galactosidase
[8], as well as cell wall loosening proteins such as
expansin [9,10]. Suppression of single genes encoding fruit PG
[3,11] or PME [7,12] in transgenic tomato plants had
limited impact on fruit softening during ripening, but
conferred longer shelf life resulting from reduced
susceptibility to postharvest pathogens. These results
suggest that suppression of certain enzymes acting on
cellulose, hemicellulose or pectin alone are not sufficient
to prevent softening, likely due to functional redundancy
of enzymes involved in what is likely a complex
metabolic process [1]. Nevertheless, a recent study has
shown that down-regulation of genes encoding the
N-glycan processing enzymes -mannosidase and
-D-N-acetylhexosaminidase significantly increased fruit
shelf life, which was attributed to decreased softening
during ripening [2]. These enzymes have been shown to
break glycosidic bonds between carbohydrates, or
between carbohydrates and noncarbohydrate structural
molecules [13].
Expansins are cell wall-localized proteins faciliating
wall loosening. They are involved in many aspects of cell
wall modification during development through
disruption of non-covalent bonds between matrix glycans and
cellulose microfibrils [9,10,14,15]. Transgenic silencing
of the tomato expansin gene LeExp1 resulted in
increased fruit firmness throughout ripening and improved
fruit integrity during storage [16].
Molecular and genetic investigations have identified
additional regulators of cell wall biosynthesis and
regulation of cell expansion. One such activity is encoded
by the COBRA gene previously reported in Arabidopsis,
rice and maize [17-20]. The COBRA gene (...truncated)