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Detection of Expansin Proteins and Activity During Tomato Fruit Ontogeny1

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Detection of Expansin Proteins and Activity During Tomato Fruit Ontogeny1 Detection of Expansin Proteins and Activity during Tomato Fruit Ontogeny1 Jocelyn K.C. Rose, Daniel J. Cosgrove, Peter Albersheim, Alan G. Darvill, and Alan B. Bennett* Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, 220 Riverbend Road, Athens, Georgia 30602–4712 (J.K.C.R., P.A., A.G.D.); Department of Biology, 208 Mueller Laboratory, The Pennsylvania State University, University Park, Pennsylvania 16802 (D.J.C.); and Mann Laboratory, Department of Vegetable Crops, University of California, Davis, California 95616 (A.B.B.) Expansins are plant proteins that have the capacity to induce extension in isolated cell walls and are thought to mediate pH-dependent cell expansion. J.K.C. Rose, H.H. Lee, and A.B. Bennett ([1997] Proc Natl Acad Sci USA 94: 5955–5960) reported the identification of an expansin gene (LeExp1) that is specifically expressed in ripening tomato (Lycopersicon esculentum) fruit where cell wall disassembly, but not cell expansion, is prominent. Expansin expression during fruit ontogeny was examined using antibodies raised to recombinant LeExp1 or a cell elongation-related expansin from cucumber (CsExp1). The LeExp1 antiserum detected expansins in extracts from ripe, but not preripe tomato fruit, in agreement with the pattern of LeExp1 mRNA accumulation. In contrast, antibodies to CsExp1 cross-reacted with expansins in early fruit development and the onset of ripening, but not at a later ripening stage. These data suggest that ripening-related and expansion-related expansin proteins have distinct antigenic epitopes despite overall high sequence identity. Expansin proteins were detected in a range of fruit species and showed considerable variation in abundance; however, appreciable levels of expansin were not present in fruit of the rin or Nr tomato mutants that exhibit delayed and reduced softening. LeExp1 protein accumulation was ethylene-regulated and matched the previously described expression of mRNA, suggest- ing that expression is not regulated at the level of translation. We report the first detection of expansin activity in several stages of fruit development and while characteristic creep activity was detected in young and developing tomato fruit and in ripe pear, avocado, and pepper, creep activity in ripe tomato showed qualitative differences, suggesting both hydrolytic and expansin activities. Synthesis, assembly, and modification of the pri- driven cell expansion relies on the coordination of mary cell wall are features common to both cell ex- cell wall loosening with wall biosynthesis and assem- pansion and cell differentiation and represent funda- bly to maintain structural integrity and strength. Un- mental determinants of plant architecture. An der acidic conditions in vitro, plant tissues grow excellent model system in which to study cell wall more rapidly, giving rise to the “acid-growth” theory dynamics, including wall assembly, restructuring, of cell expansion (Rayle and Cleland, 1992). Simi- and disassembly, are the contiguous stages of fruit larly, when cell walls are clamped in a constant-load development. In most plant species fruit ontogeny extensometer and placed in low pH buffers, they may be divided into several distinct developmental exhibit prolonged elongation (Cosgrove, 1989), con- phases (Gillaspy et al., 1993). These comprise early sistent with a pH-dependent mechanism of wall ex- fruit growth accompanied by cell division, a period tension or wall creep. Heat-inactivated walls can be of cell expansion that generates more substantial and induced to elongate in a similar fashion by applying prolonged growth until maximal fruit size is reached, a protein extract from growing walls and purification and finally ripening, involving numerous metabolic of the growth-promoting activities from cucumber pathways including those leading to textural changes hypocotyls and oat coleoptiles identified a class of and softening. Similar changes in the structure and proteins that were termed expansins (McQueen- composition of the cell wall and in the complement of Mason et al., 1992; Li et al., 1993). A number of associated enzymes have been observed in expand- expansin genes have now been cloned and divided ing cells and ripening fruit (Rose and Bennett, 1999). into two broad classes based on sequence homology ␣ In growing tissues the cell wall acts to restrict (Cosgrove, 1998). One class, termed -expansins, in- increases in cell volume, and so controlled turgor- clude the original cucumber expansin gene (CsExp1) and homologs from numerous species, many of 1 which have been associated with rapidly growing This work was supported by the U.S. Department of Energy ␤ (grant nos. DE–FG02–96ER20220 to J.K.C.R., P.A., and A.G.D. and vegetative tissues. Another class, -expansins, share DE–FG02–84ER13179 to D.J.C.). approximately 25% amino acid identity with * Corresponding author; e-mail [email protected]; fax ␣-expansins and include genes encoding a class of 530–752–4554. grass pollen allergens and closely related sequences Plant Physiology, August 2000, Vol. 123, pp. 1583–1592, www.plantphysiol.org © 2000 American Society of Plant Physiologists 1583 Rose et al. that are expressed in vegetative tissues (Cosgrove et al., 1997). The mechanism of action of expansins is still obscure; however, several pieces of evidence have suggested specific cell wall components that may be targeted (Cosgrove, 1998; Rose and Bennett, 1999). A recent, and perhaps surprising, finding was that an ␣-expansin gene from tomato (LeExp1) is specifi- cally and abundantly expressed at the level of mRNA accumulation in ripening fruit where cell expansion does not occur (Rose et al., 1997). Phylogenetic anal- ysis suggested that LeExp1 sequence aligned within an evolutionarily divergent clade of the super-family of expansin genes. Expansins were proposed to con- tribute to cell wall disassembly in ripening fruit, although this hypothesis was based solely on the expression patterns of LeExp1 mRNA. The mRNA expression patterns of other tomato expansins sug- gest roles for divergent expansins at different stages of fruit ontogeny (Brummell et al., 1999). In this paper we describe the expression of expan- sin proteins during fruit development in wild-type and ripening-impaired mutant tomato fruit, using antibodies raised to either a recombinant LeExp1 protein or to an expansin purified from cucumber hypocotyls (CsExp1). The results support the idea that distinct expansin isoforms are expressed in elon- gating cells and in ripening fruit. We also report the Figure 1. Phylogenetic analysis of expansin genes. The dendrogram first detection of expansin-like activity in protein ex- was generated based on an alignment of the deduced amino acid tracts from developing and ripening fruit and con- sequences of 27 expansin genes together with a pollen allergen trast the activities in ripening tomato fruit with those (Phlp1), using CLUSTAL. Tomato expansin genes are boxed and the from several other species. sequences corresponding to the polypeptides used to generate the antisera described in this paper (CsExp1 and LeExp1) are indicated with asterisks. Subgroups A through D are highlighted with vertical RESULTS lines, and values corresponding to matching percentages of branch- ing orders are indicated at each branch point. Additional details and Identification of Expansin Subgroups accession numbers are listed in “Materials and Methods.” Figure 1 shows a dendrogram generated from de- duced amino acid sequences corresponding to ing fruit and flowers (Brummell et al., 1999), and ␣-expansins from several species, together with the LeExp18, which is expressed in cells exhibiting high sequence of a related pollen allergen (Phlp1) that is meristematic activity (Reinhardt et al., 1998). Sub- more similar to ␤-expansins (Cosgrove, 1998). The group C includes LeExp5, which is expressed in late sequences of the deduced polypeptides, without the fruit growth and mature stems (Brummell et al., predicted signal sequences, were aligned using 1999). Subgroup B contains several sequences from CLUSTAL IV. Values corresponding to matching genes whose expression has been associated with percentages of branching orders are indicated at each rapid cell expansion, including CsExp1 from cucum- branch point. The sequences align within four prin- ber (McQueen-Mason et al., 1992), GhExp1 expressed cipal groups (labeled A–D, after Link and Cosgrove, during cotton fiber elongation (Orford and Timmis, 1998) in addition to two more distantly related se- 1998), and a rice expansin OsExp4 (Cho and Kende, quences from Arabidopsis (AtExp5) and tomato (Le- 1997a). In addition two newly submitted sequences Exp3). The sequences of six full-length tomato ex- from apricot (PaExp1 and PaExp2) are described in pansins that are currently in the databases align their database entries as being identified from ripen- within three of the groups and exhibit 50% to 90% ing fruit. amino acid identity to each other. Two partial length The polypeptides used to generate expansin anti- sequences, LeExp6 (accession no. AF059490) and Le- bodies for the western-blot analyses described in this Exp7 (accession no. AF059491), which are most sim- paper correspond to genes that align within sub- ilar to LeExp1 and LeExp3, respectively (Brummell et group A (LeExp1) and subgroup B (CsExp1) and are al., 1999), are not shown. Subgroup A includes Le- indicated in Figure 1 with an asterisk. LeExp1 and Exp1, the fruit ripening-related expansin
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