beta-Galactosidases with a lectin-like domain are expressed in strawberry.

Strawberry fruits (Fragaria x ananassa Duch.) undergo a marked softening during their ripening, and the process is accompanied by a release of free sugars with galactose among them. In this work total beta-galactosidase activity was measured in cell wall proteins from strawberry fruits at different developmental stages. Three full-length cDNAs (Fa beta gal1, Fa beta gal2 and Fa beta gal3, respectively) encoding different beta-galactosidases (EC 3.2.1.23) were isolated from a library representing red fruit transcripts. All of them could be detected both in fruits and in vegetative tissues. However, only Fa beta gal1 showed an increasing expression during the ripening stages up to a maximum in the red fruits, while the other two (Fa beta gal2 and Fa beta gal3) were mostly found in green fruits and became barely detectable during ripening proper. The three beta-galactosidase-encoding cDNAs were expressed in the yeast Pichia pastoris, and it was thus possible to demonstrate that each of them encode a beta-galactosidase. The expression of the three beta-galactosidase genes appears to be down-regulated by auxin, as already observed for other ripening-related genes of the non-climacteric strawberry. An unusual characteristic of two strawberry beta-galactosidases (Fa beta gal1 and Fa beta gal2) is that at the C-terminus of the enzymes a domain is found which is structurally related to known animal peptides with a sugar-binding ability.

A simple protocol for transient gene expression in ripe fleshy fruit mediated by Agrobacterium.

Fleshy fruits represent a very important economic resource and, therefore, they are an ideal target for biotechnological ameliorations. However, because of their physiological and anatomical characteristics, ripe fleshy fruits represent an extremely difficult material for transient gene expression assays aimed at the study of gene promoters in a short time. To this purpose, a fast and efficient Agrobacterium-mediated transient gene expression system was developed for ripe fleshy fruits. A beta-glucuronidase reporter gene interrupted by an intron was used in order to prevent the possible expression of GUS activity by the Agrobacterium cells. The contemporary use of another reporter gene was used to check the transformation efficiency. This method is based on the injection of an Agrobacterium suspension into the fruits, and allows both qualitative and quantitative assays in a wide range of fruits to be carried out.

A novel E-type endo-beta-1,4-glucanase with a putative cellulose-binding domain is highly expressed in ripening strawberry fruits.

Two full-length cDNA clones (faEG1 and faEG3, respectively) have been isolated by screening a cDNA library representing transcripts from red strawberry fruits. Southern blot analysis of genomic DNA suggests that the strawberry endo-beta-1,4-glucanases (EGases) are encoded by a multigene family. The cognate genes are predominantly expressed during the ripening process proper, although, in the case of faEG3, some expression has also been observed in large green fruits and, at low amounts, in young vegetative green tissues. In agreement with other ripening-related genes in strawberry, also the expression of faEG1 and faEG3 is down-regulated by treatment with an auxin analogue (1-naphthaleneacetic acid, NAA). Differences in temporal expression of the two EGase genes in fruits are not accompanied by differences in spatial expression. The pattern of expression and the sequence characteristics of the two polypeptides suggest that the two strawberry EGases operate in a synergistic and coordinate manner. The protein encoded by faEG1 looks like one of the usual higher-plant EGases (average molecular mass of 54 kDa), while the protein encoded by faEG3 has a greater deduced molecular mass (about 68 kDa) due to the presence of an extra peptide of about 130 amino acids at the C-terminus. Such unusual peptide shows some features also found in microbial cellulases and contains a putative cellulose-binding domain. We propose that the faEG3-encoded EGase might especially hydrolyse the xyloglucans coating the cellulose microfibrils, thus rendering the cell wall more susceptible to the subsequent hydrolytic activity of the faEG1-encoded EGase.

Characterization of cCel3, a member of the pepper endo-beta-1,4-glucanase multigene family.

In pepper plants the enzyme endo-beta-1,4-glucanase (EGase) is encoded by a multigene family. Here is described the characterization of cCel3, a cDNA which codes for the third EGase known so far in this plant. The known members of this family are present as single copy genes, as demonstrated by a Southern analysis of the genomic DNA. Analysis of the expression of cCel3 demonstrates that the highest levels of the cCel3 mRNA are found in abscission zones of leaves and flowers activated by treatments with the plant hormone ethylene. However, the amounts of the cCel3 transcripts (detectable only by RT-PCR) are always much lower than those of cCel2 so that the latter can be regarded as the "abscission" EGase while cCel3 is likely to perform a role ancillary to that of cCel2.

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

Two pepper endo-beta-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 alpha-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.

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

Three cDNA clones (pCel 10, pCel 20 and pCel 30), each encoding different endo-beta-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 pCel 10-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 pCel 10, was isolated and characterized. A cDNA (termed pCel 1), containing the entire open reading frame of ppEGC1, 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-beta-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.

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 abscission zones in the flowers and fruits of peach [Prunus persica (L.) Batsch].

Cell wall hydrolases, their mRNAs, and ultrastructural details of cell wall digestion have been studied in peach abscission zones (AZ) located at the base of flower bud (AZ1) and the base of flower receptacle (AZ2), respectively. Induction of abscission was obtained by treatment of explants with exogenous ethylene. Cell separation patterns of the two examined abscission zones have been compared with those of other already known AZs of peach, i.e. the AZs located between fruit and peduncle and the leaf AZ. Analyses have shown similarities in response to ethylene treatment between AZ1 and leaf AZ and between AZ2 and AZs, respectively. Results have been discussed considering the precise position of AZ1 and AZ2 on the flower bud. The timing of functional differentiation, evaluated as the cells'ability to respond to induction by ethylene treatments, showed that AZ1 and AZ2 became functional after bud breaking and bud scale shedding. Later on, they lost their functionality at about 6-7 wk from anthesis. AZ3 became functional very precociously and could be activated 1 wk after anthesis in the fertilized flowers. In the latter zone the cells could also undergo a morphological predifferentiation, even though it occurred a long time after the acquisition of the ethylene responsiveness. This finding shows that morphological differentiation is not necessarily a prerequisite for those cells to become competent to respond to the abscission inducing stimuli.

Abscission in leaf and fruit explants of Prunus persica (L.) Batsch.

Structural and enzymatic aspects of leaf and fruit abscission in explants treated with exogenous ethylene were studied and compared with those previously observed under field conditions. Light and electron microscopy observations reconfirm that differences in abscission occur in leaf and fruit, and in explants cell separation is accelerated, with a more dramatic degradation of the cell walls. In fruit, digestion starts from the middle lamella, and subsequently extends to the entire parietal mass. In the leaf the process involves the primary cell wall, leaving, at the end, undigested materials. In fruit the lytic activity is sustained by endocellulase, and by exo- as well as endopolygalacturonase. In the leaf only endocellulase activity has been found. Cell enlargement phenomena occur subsequent to abscission zone activation in both leaf and fruit explants, involving the cells of either the separation layer or the adjacent region.

Cellulase and polygalacturonase involvement in the abscission of leaf and fruit explants of peach.

Ethylene-induced abscission in leaf and fruit explants of peach involves different enzymes. In leaves abscission is accompanied by increased occurrence of cellulase forms differing in isoelectric point (pI 6.5 and 9.5). A polypeptide with a molecular mass of 51 kDa gives in a western blot a strong cross-reaction with an antibody raised against a maturation cellulase from avocado fruit. Cellulase activity is also found in abscising fruit explants but the amount is very low compared to that of the leaf explants. A northern analysis with a cellulase clone from avocado reveals the presence of two hybridizing mRNAs with a size of 2.2 kb and 1.8 kb, respectively. The steady-state level of the 2.2 kb mRNA is significantly increased by treatment with ethylene. Polygalacturonases are not detected in abscising leaves, but are strongly induced by ethylene in fruit explants. Of the three forms found, two are exopolygalacturonases while the third is an endoenzyme. Ethylene activates preferentially the endoenzyme and the basic exoenzyme but depresses the acid exopolygalacturonases. A northern analysis carried out with a cDNA coding for tomato endopolygalacturonase shows hybridization only with one endopolygalacturonase mRNA form in the fruit abscission zone. Treatment with ethylene causes an increase in the steady-state level of this mRNA. The differences in the enzyme patterns observed in fruit and leaf abscission zones and a differential enzyme induction suggest the feasibility to regulate fruit abscission in peach with the aid of antisense RNA genes.

Characterization of a cDNA clone for barley leaf glutamine synthetase.

A barley cDNA clone (1182 bp) encoding chloroplastic glutamine synthetase was isolated with a heterologous cDNA probe of the gene specifying the enzyme from alfalfa. The clone, named pGS8, was found in a lambda gtII cDNA library prepared from dark grown barley leaves even though the chloroplastic glutamine synthetase is absent from such leaves. In agreement therewith the clone hybridized in Northern blot analyses with a 1.7 kb mRNA species present the in poly A+ mRNA fraction of both dark grown and greened primary leaves of barley. The nucleotide sequence of the barley clone reveals 75% identity to the Phaseolus vulgaris and Pisum sativum clones encoding chloroplastic glutamine synthetase, while only 69% identity is observed in comparisons with the clones specifying the cytosolic isozymes. At the amino acid level 85% identity is found between the deduced barley glutamine synthetase sequence and that of the corresponding chloroplastic isoenzymes from bean and pea. The chloroplastic glutamine synthetases contain cysteins in the putative ATP and and substrate binding sites. In the cytosolic forms these positions are occupied by alanine residues.

Cotyledonal chloroplasts in the hypogeal seeds of clementine.

Clementine (Citrus nobilisxCitrus aurantium amara pumila) is a chloroembryophyte with green quiescent embryos and hypogeal germination. The cotyledonal chloroplasts have been studied during germination in the dark and under two different irradiances 120 and 240 µmol·m(-2)·s(-1) throughout a period of three weeks. The plastids of the outer adaxial and inner regions develop differently. In the light, the former differentiate a photosynthetically active thylakoid system with an ultrastructural organization and a polypeptide composition resembling that of leaf chloroplasts. The "inner" chloroplasts maintain an organization reminiscent of chloroplasts of the quiescent embryo and never get beyond the photosynthesis/respiration compensation point; their differentiation pattern appears essentially the same under the two different irradiances. These observations and the germination in the dark indicate that the above differentiation is not strictly photodependent. The greening ability of the cotyledons provides, on occasion, an additional photosynthetic supply to this plant.

Structural and biochemical aspects of peach fruit abscission (Prunus persica L. Batsch).

The physiological drop of immature fruits was studied in relation to the activation of the abscission zone located between the fruit and the receptacle. Light- and electron-microscopy observations demonstrated that this zone consisted of two types of parenchymatous cells: in the distal region, closer to the fruit, were groups of small thick-walled cells with few intercellular spaces; in the proximal region, closer to the pedicel, the stillgrouped cells were larger, polyphenolic-rich, and thick-walled but with many wide intercellular spaces. Separation of the fruit occurred by dissolution of the middle lamella of the cells of this zone followed by an increase in the size of the intercellular spaces. Lysis of the middle lamella began at the corners of the cells and spread from there across the entire wall surface. Structural changes were paralleled by an increase in soluble proteins, endo-cellulase and exo-polygalacturonase activity. Isoelectric focusing indicated that both enzymes were present as isoenzymes whose patterns were affected by embryoctomy and 2-chloroethylphosphonic acid treatments.