Luca Ferrarese

Differential ethylene-inducible expression of cellulase in pepper plants

Ethylene promotes the abscission of leaves and the ripening of fruits in pepper plants, and in both events an increase in cellulase activity is observed. However, two enzyme isoforms (pI 7.2 and 8.5, respectively) are differentially involved in the two physiological phenomena. The pI 8.5 form has been purified from ripe fruits. It is a glycoprotein with an apparent molecular mass of 54 kDa. Two short peptides were sequenced and a very high homology to a tomato cellulase was observed. Polyclonal antibodies, raised against the purified enzyme, have allowed us to demonstrate that the observed ethylene-induced increase in cellulase activity is paralleled by de novo synthesis of protein. Three cDNAs (CX1, CX2 and CX3), encoding different cellulases, were obtained and characterized and their expression investigated. Accumulation of all three mRNAs is induced by ethylene treatment, though to different levels. CX1 is mainly expressed in ripe fruits while CX2 is especially found in abscission zones. CX3 accumulates at very low levels in activated abscission zones. Comparisons with other known cellulases demonstrate clear heterogeneity within the higher plant cellulases. Differences in ethylene inducibility and molecular structure suggest different physiological roles for cellulase in pepper plants.

Characterization of ppEG1, a member of a multigene family which encodes endo-beta-1,4-glucanase in peach.

Three cDNA clones (pCel10, pCel20 and pCel30), each encoding different endo-β-1,4-glucanases in peach, were obtained by RT-PCR and their expression investigated by northern analysis during leaf and fruit abscission and during fruit development. This analysis allowed the detection of only the pCel10-related mRNA. A 2.2 kb transcript accumulated in ethylene activated abscission zones of leaves and fruits, and ppEG1 (Prunus persica endoglucanase 1) the gene coding for pCel10, was isolated and characterized. A cDNA (termed pCel1), containing the entire open reading frame of ppEG1, was obtained and its sequence used to define the structure of the gene and the exon/intron boundaries. ppEG1 consists of 7 exons and encodes a 497 amino acid polypeptide including a putative signal peptide at the N-terminus. The similarity of this peach endo-β-1,4-glucanase (EGase, EC 3.2.1.4) is high (76.3%) with the ripening avocado and low (47.3%) with the bean abscission EGase. A 1639 bp region at the 5′ of the transcription start site shows regulatory functions in transgenic tobacco plants, as judged by its ability to drive GUS expression in cell separation-related events.

Two Different Endo-β-1,4-Glucanases Contribute to the Softening of the Strawberry Fruits

Summary The expression of different endo-(β-l,4-glucanases (EGases) and the ultrastructural details of the cell wall dismantling were studied throughout the development of the strawberry fruits. Detectable enzyme activity is first observed in large green fruits, but a steep increase occurs in white fruits when the ripening process proper starts. This process is then accompanied by a further increase in EGase activity which appears to be doubled in red ripe fruits. Two isoforms with different isoelectric points (pI 7.9 and 9.0, respectively) contribute to the observed enzyme activity, and both isoenzymes increase in activity as fruits ripen. Two cDNA fragments, encoding two divergent endo-(β-l,4-glucanases (faEGlO and faEG30, respectively), were obtained by RT-PCR and the expression of their related mRNAs examined by northern analysis. faEG30-related transcripts start to be detected in large green fruits, while faEGlO expression is detected in white fruits. Thereafter, the amount of both EGase mRNAs increases throughout the ripening process up to the stage of red ripe fruits. The fine structure of the cell wall weakening which occurs during the ripening is well in accordance with the above described biochemical and molecular data.

Secretion, purification and activity of two recombinant pepper endo-β-1,4-glucanases expressed in the yeast Pichia pastoris

Two pepper endo‐β‐1,4‐glucanases, involved in fruit softening (cCel1) and leaf and flower abscission (cCel2), have been expressed in Pichia pastoris. Secretion was obtained by using either the mouse α‐factor signal (cCel1) or the native signal sequence (cCel2). Times for optimal expression of the two proteins were different and cCel2 appeared very sensitive to proteolytic degradation. A one‐step purification protocol yielded cCel2 in a pure form, while an additional chromatography step was necessary to purify cCel1. The two recombinant proteins are highly active and able to degrade carboxymethylcellulose in viscometric assays. Moreover, they have both a molecular mass (54 kDa) and an isoelectric point (7.2 for cCel2 and 8.5 for cCel1) equal to those of the native proteins, thus suggesting that post‐translational modifications have properly occurred.

Endo-β-1,4-glucanase activity is involved in the abscission of pepper flowers

Summary The expression of endo-β-1,4-glucanase (EGase) and ultrastructural details were examined in the abscission zone (AZ) of pepper flowers. Induction of abscission caused an increase in EGase activity which was due to two isoforms with different isoelectric points (pI 7.2 and 8.5, respectively). In particular, ethylene had a promotive effect on the 7.2 isoform while the basic isoenzyme did not show any significant increase. The observed change of EGase activity was due to de novo protein synthesis, as demonstrated by both RNA and protein blots. By means of a cDNA fragment encoding a pepper EGase and an antibody raised against a pepper fruit EGase, it was possible to show the appearance of a 2.2 kb transcript and a 54 kDa polypeptide, respectively, following activation of flower abscission. A cDNA library was constructed, and the cDNA fragment was used to isolate a 1.8 kb clone which was sequenced and characterised. Ultrastructural analyses of the cell separation events in the flower AZ showed clear consistency with the demonstrated contribution of the EGase activity to abscission. Massive hydrolysis phenomena were particularly observed at the level of primary wall of the separating cells. Immunolocalization of EGase confirmed both the involvement and the target of the enzyme.

Different Endo-β-1,4-Glucanases are Expressed During Abscission and Fruit Ripening in Pepper and Peach Plants

Senescence of leaves, flowers and fruits usually ends with the formation of an abscission zone at the base of the organ involved. The abscission zone consists of a few layers of cells whose walls undergo extensive digestion processes leading to loss of adhesion between cells [1]. Also ripening of fleshy fruits includes an aging requirement which involves similar cell wall changes during the softening of the tissues. Though the rate of senescence can be either delayed or increased by plant hormones, in many cases ethylene has been found to have promotive effects on the senescence phenomena. In particular, this hormone accelerates abscission and ripening, although, in term of regulation, different responses are shown by climacteric and nonclimacteric fruits. These ethylene-mediated physiological processes involve synthesis of different mRNAs which include those encoding cell wall hydrolases [2].

Cellulase and cell separation.

Abstract The involvement of cellulase (endo-b-l,4-glucanase, EC 3.2.1.4) in a number of different cell separation events which occur in higher plants has been well established. Besides their significance for the plant growth and differentiation, these events can be economically important since they also comprise softening of fleshy fruits and abscission of fruits, flowers and leaves. In higher plants cellulase is present in a number of different biochemical isoforms which are encoded by different genes. This finding is in accordance with the wide range of physiological events which require the intervention of cellulase activity, and whose peculiarities and amplitudes can be quite various.