Purification and characterization of an antifungal chitinase in jelly fig (Ficus awkeotsang) achenes

A method was developed to purify a 30-kDa protein from jelly fig (Ficus awkeotsang) pericarp, including preparation of jelly curd from achenes, extraction of proteins from the curd, and isolation of the 30-kDa protein by anion-exchanger and gel filtration. Chitinase activity was detected in the purified 30-kDa protein by activity staining in both non-denaturing gel electrophoresis and SDS-PAGE. Isoelectrofocusing showed that the isoelectric point of the 30-kDa protein was lower than pH 3.5. The K(m), k(cat), optimal pH and temperature of this putative chitinase were determined to be 0.076 mM, 0.089 s(-1), pH 4, and 60 degrees C, respectively. The purified 30-kDa protein was thermostable (retaining activity up to 65 degrees C for several hours) and could be stored at 4 degrees C for a year without apparent loss of chitinase activity. Antifungal activity of this putative chitinase was measured in terms of inhibition of Colletotrichum gloeosporioides spore germination.     

Characterization of oil bodies in jelly fig achenes.

Thin-layer chromatography analysis revealed that the contents stored in oil bodies isolated from jelly fig (Ficus awkeotsang Makino) achenes were mainly neutral lipids (>90% triacylglycerols and approximately 5% diacylglycerols). Fatty acids released from the neutral lipids of achene oil bodies were highly unsaturated (62.65% alpha-linolenic acid, 18.24% linoleic acid, and 10.62% oleic acid). The integrity of isolated oil bodies was presumably maintained via electronegative repulsion and steric hindrance provided by their surface proteins. Immunological cross-recognition using antibodies against sesame oil-body proteins indicated that two oleosin isoforms and one caleosin were present in these oil bodies. MALDI-MS analyses confirmed that the three full-length cDNA fragments obtained by PCR cloning from maturing achenes encoded the two jelly fig oleosin isoforms and one caleosin identified by immunological screening.     

Hormonal regulation of ripening in the strawberry,a non-climacteric fruit

Anthocyanin accumulation is one measure of ripening in the strawberry (Fragaria ananassa Duch.), a non-climacteric fruit. Neither aminoethoxyvinylglycine, an inhibitor of 1-aminocyclopropane carboxylic acid synthase, nor inhibitors of ethylene action (silver, norbornadiene) affected anthocyanin accumulation in ripening fruit. When the achenes were removed from one half of an unripe fruit there was an accelerated accumulation of anthocyanin and induction of phenylalanine ammonia lyase on the de-achened portion of the ripening fruit. These effects of achene removal could be prevented by the application of the synthetic auxins 1-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid to the de-achened surface. The introduction of 1-naphthalene acetic acid into intact unripe strawberry fruit through the peduncle delayed their subsequent ripening, as measured by the accumulation of anthocyanin, loss of chlorophyll and decrease in firmness. These findings suggest that the decline in the concentration of auxin in the achenes as strawberry fruit mature modulates the rate of fruit ripening.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - NAA 1-naphthaleneacetic acid - PA1 phenylalanine ammonia-lyase - POA phenoxyacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Gene Families Encoding 11S Globulin and 2S Albumin Isoforms of Jelly Fig (Ficus awkeotsang) Achenes

Seed storage proteins of plants commonly comprise several groups of multiple isoforms encoded by gene families. From about 300 expressed sequence tag (EST) clones in maturing jelly fig (Ficus awkeotsang Makino) achenes, gene families encoding precursor polypeptides of two storage protein classes, including six 11S globulin isoforms and two 2S albumin isoforms, were identified. Complete sequences encoding the precursor polypeptides of these eight storage proteins were obtained by sequencing the pertinent EST clones that contained full-length cDNA fragments. Matrix-assisted laser desorption/ionization mass spectrometry analysis confirmed the presence of these storage protein isoforms in the extract of jelly fig achenes resolved in SDS–PAGE. The amino acid compositions of the deduced storage proteins indicated that achene proteins in jelly fig are nutritive, for both isoforms of 2S albumin are sulfur-rich, and one of them is also rich in tryptophan.     

Gene families encoding 11S globulin and 2S albumin isoforms of jelly fig (Ficus awkeotsang) Achenes

Seed storage proteins of plants commonly comprise several groups of multiple isoforms encoded by gene families. From about 300 expressed sequence tag (EST) clones in maturing jelly fig (Ficus awkeotsang Makino) achenes, gene families encoding precursor polypeptides of two storage protein classes, including six 11S globulin isoforms and two 2S albumin isoforms, were identified. Complete sequences encoding the precursor polypeptides of these eight storage proteins were obtained by sequencing the pertinent EST clones that contained full-length cDNA fragments. Matrix-assisted laser desorption/ionization mass spectrometry analysis confirmed the presence of these storage protein isoforms in the extract of jelly fig achenes resolved in SDS-PAGE. The amino acid compositions of the deduced storage proteins indicated that achene proteins in jelly fig are nutritive, for both isoforms of 2S albumin are sulfur-rich, and one of them is also rich in tryptophan.     

Changes in gene expression during strawberry fruit ripening and their regulation by auxin

Changes in messenger RNA during the development of the strawberry (Fragaria ananassa Duch.), a non-climacteric fruit, were analysed by extracting total RNA and separating the in-vitro translated products by two-dimensional polyacrylamide gel electrophoresis. Alterations in numerous messenger RNAs accompanied fruit development between the immature green stage and the overripe stage, with prominent changes detected at or before the onset of ripening. A number of messenger RNAs undetectable in immature green fruit increased as the fruit matured and ripened. Others showed a marked decrease in advance of the ripening phase. A further group of messenger RNAs was prominent in immature and ripe fruit but absent just prior to the turning stage. Removing the achenes from a segment of the fruit accelerated anthocyanin accumulation in the de-achened portion and produced a pattern of translated polypeptides similar to normal ripe fruit. Application of the synthetic auxin 1-naphthaleneacetic acid to the de-achened receptacle produced a translation pattern similar to that in mature green fruit. These findings indicate that ripening in strawberry is associated with the expression of specific genes.     

Purification and Characterization of Pectinmethylesterase from Ficus awkeotsang Makino Achenes

Pectinmethylesterase from the pericarp of jelly fig (Ficus awkeotsang) achenes was extracted and purified to a specific activity of 289 micromole proton produced per minute per milligram protein. Pectinmethylesterase, a major protein with high specific activity in the crude extract, was monomeric with a molecular weight of 38,000. The enzyme preparation was stable in distilled water at 4°C for at least 6 months, and at 60°C for at least 10 minutes. This enzyme functioned optimally at pH 6.5 to 7.5 when the assay mixture contained no NaCl or at low NaCl concentration. The pH optimum shifted to lower pH as the NaCl concentration was increased. The K_m value for pectin was 0.75 milligram per milliliter pectin, corresponding to a V_max value of 310 micromoles per minute per milligram protein. Inhibition studies with antibodies indicated that jelly fig achene pectinmethylesterase and the two other pectinmethylesterases from orange and tomato were similar in their active site conformation; however, the surface determinants may be very different because no precipitation between anti-jelly fig pectinmethylesterase immune serum and the pectin methylesterase from orange and tomato could be observed in the double immunodiffusion analysis. Specific antisera raised against jelly fig achene pectinmethylesterase in a Western blot experiment also showed low similarity between jelly fig pectinmethylesterase with that from orange and tomato. This observation was also supported by the very low isoelectric point (pH 3.5) of jelly fig pectinmethylesterase, compared with high isoelectric points reported for most of the pectinmethylesterases. Amino acid composition and N-terminal sequence have been obtained. High homology of the N-terminal amino acid residues between jelly fig and tomato pectinmethylesterase (O Markovic, H Jornvall [1986] Eur J Biochem 158: 455-462) was observed. Pectinmethylesterase activity causes the release of protons from the deesterification of pectin such that a low pH environment is created, and this may be related to the cell growth. Pectinmethylesterase is not needed for jelly fig seed germination, however the gel formed from pectin and pectinmethylesterase may insure a water source for the germinating jelly fig seeds.     

Cloning and molecular characterization of a strawberry fruit ripening-related cDNA corresponding a mRNA for a low-molecular-weight heat-shock protein

We have isolated and characterized a cDNA from a strawberry fruit subtractive library that shows homology to class-I low-molecular-weight (LMW) heat-shock protein genes from other higher plants. The strawberry cDNA (clone njjs4) was a 779 bp full-length cDNA with a single open reading frame of 468 bp that is expected to encode a protein of ca. 17.4 kDa with a pI of 6.57. Southern analysis with genomic DNA showed several high-molecular-weight hybridization bands, indicating that the corresponding njjs4 gene is not present as a single copy in the genome. This strawberry gene was not expressed in roots, leaves, flowers and stolons but in fruits at specific stages of elongation and ripening. However, a differential pattern of mRNA expression was detected in the fruit tissues achenes and receptacle. The njjs4 gene expression increased in achenes accompanying the process of seed maturation whereas in the receptacle, a high mRNA expression was detected in the W2 stage, during which most of the metabolic changes leading to the fruit ripening are occurring. Our results clearly show a specific relationship of this njjs4 strawberry gene with the processes of seed maturation and fruit ripening, and strongly support that at least some of the class-I LMW heat-shock protein-like genes have a heat-stress-independent role in plant development, including fruit ripening.     

Molecular cloning of a ripening-specific lipoxygenase and its expression during wild-type and mutant tomato fruit development.

A 94-kD protein that accumulates predominately in tomato (Ly-copersicon esculentum) fruit during ripening was purified, and antibodies specific for the purified protein were used to isolate cDNA clones from a red-ripe fruit cDNA library. A sequence analysis of these cDNAs and cross-reactivity of the 94-kD-specific antibodies to the soybean lipoxygenase (LOX) L-1, L-2, and L-3 proteins and soybean LOX L-1-specific antibodies to the 94-kD protein identified it as a member of the LOX gene family. Maximum levels of the 94-kD LOX mRNA and protein are present in breaker to ripe and red-ripe stages, respectively. Expression of 94-kD LOX in different tissues from mature green and red-ripe tomato fruits was found to be greatest in the radial walls of ripe fruit, but immunocytolocalization using tissue printing suggests that the highest accumulation of its protein occurs in locular jelly. None of 94-kD LOX is expressed in nonripening mutant fruits of any age. Never-ripe mutant fruit accumulate the 94-kD LOX mRNA to levels similar to those obtained in wild-type fruit, but fail to accumulate the 94-kD LOX protein. Collectively, the results show that expression of 94-kD LOX is regulated by the ripening process, and ethylene may play a role in its protein accumulation.     

Endogenous cytokinin in developing kiwifruit is implicated in maintaining fruit flesh chlorophyll levels

Background and Aims

Green kiwifruit (Actinidia deliciosa) retain high concentrations of chlorophyll in the fruit flesh, whereas in gold-fleshed kiwifruit (A. chinensis) chlorophyll is degraded to colourless catabolites during fruit development, leaving yellow carotenoids visible. The plant hormone group the cytokinins has been implicated in the delay of senescence, and so the aim of this work was to investigate the link between cytokinin levels in ripening fruit and chlorophyll de-greening.

Methods

The expression of genes related to cytokinin metabolism and signal transduction and the concentration of cytokinin metabolites were measured. The regulation of gene expression was assayed using transient activation of the promoter of STAY-GREEN2 (SGR2) by cytokinin response regulators.

Key Results

While the total amount of cytokinin increased in fruit of both species during maturation and ripening, a high level of expression of two cytokinin biosynthetic gene family members, adenylate isopentenyltransferases, was only detected in green kiwifruit fruit during ripening. Additionally, high levels of O-glucosylated cytokinins were detected only in green kiwifruit, as was the expression of the gene for zeatin O-glucosyltransferase, the enzyme responsible for glucosylating cytokinin into a storage form. Season to season variation in gene expression was seen, and some de-greening of the green kiwifruit fruit occurred in the second season, suggesting environmental effects on the chlorophyll degradation pathway. Two cytokinin-related response regulators, RRA17 and RRB120, showed activity against the promoter of kiwifruit SGR2.

Conclusions

The results show that in kiwifruit, levels of cytokinin increase markedly during fruit ripening, and that cytokinin metabolism is differentially regulated in the fruit of the green and gold species. However, the causal factor(s) associated with the maintenance or loss of chlorophyll in kiwifruit during ripening remains obscure.     

Mining secreted proteins that function in pepper fruit development and ripening using a yeast secretion trap (YST)

Plant cells secrete diverse sets of constitutively- and conditionally-expressed proteins under various environmental and developmental states. Secreted protein populations, or secretomes have multiple functions, including defense responses, signaling, metabolic processes, and developmental regulation. To identify genes encoding secreted proteins that function in fruit development and ripening, a yeast secretion trap (YST) screen was employed using pepper (Capsicum annuum) fruit cDNAs. The YST screen revealed 80 pepper fruit-related genes (CaPFRs) encoding secreted proteins including cell wall proteins, several of which have not been previously described. Transient GFP-fusion assay and an in planta secretion trap were used to validate the secretion of proteins encoded by selected YST clones. In addition, RNA gel blot analyses provided further insights into their expression and regulation during fruit development and ripening. Integrating our data, we conclude that the YST provides a valuable functional genomics tool for the identification of substantial numbers of novel secreted plant proteins that are associated with biological processes, including fruit development and ripening.     

High-Temperature Perturbation of Starch Synthesis Is Attributable to Inhibition of ADP-Glucose Pyrophosphorylase by Decreased Levels of Glycerate-3-Phosphate in Growing Potato Tubers

A cDNA (Cel1) encoding an endo-1,4-β-glucanase (EGase) was isolated from ripe fruit of strawberry (Fragaria × ananassa). The deduced protein of 496 amino acids contains a presumptive signal sequence, a common feature of cell wall-localized EGases, and one potential N-glycosylation site. Southern- blot analysis of genomic DNA from F. × ananassa, an octoploid species, and that from the diploid species Fragaria vesca indicated that the Cel1 gene is a member of a divergent multigene family. In fruit, Cel1 mRNA was first detected at the white stage of development, and at the onset of ripening, coincident with anthocyanin accumulation, Cel1 mRNA abundance increased dramatically and remained high throughout ripening and subsequent fruit deterioration. In all other tissues examined, Cel1 expression was invariably absent. Antibodies raised to Cel1 protein detected a protein of 62 kD only in ripening fruit. Upon deachenation of young white fruit to remove the source of endogenous auxins, ripening, as visualized by anthocyanin accumulation, and Cel1 mRNA accumulation were both accelerated. Conversely, auxin treatment of white fruit repressed accumulation of both Cel1 mRNA and ripening. These results indicate that strawberry Cel1 is a ripening-specific and auxin-repressed EGase, which is regulated during ripening by a decline in auxin levels originating from the achenes.     

Pectin methylesterase activity in vivo differs from activity in vitro and enhances polygalacturonase-mediated pectin degradation in tabasco pepper

Polygalacturonase (PG) and pectin methylesterase (PME) activities were analyzed in ripening fruits of two tabasco pepper (Capsicum frutescens) lines that differ in the extent of pectin degradation (depolymerization and dissolution). Ripe 'Easy Pick' fruit is characterized by pectin ultra-degradation and easy fruit detachment from the calyx (deciduous trait), while pectin depolymerization and dissolution in ripe 'Hard Pick' fruit is limited. PG activity in protein extracts increased similarly in both lines during fruit ripening. PME activity in vivo assessed by methanol production, however, was detected only in fruit of the 'Easy Pick' line and was associated with decreased pectin methyl-esterification. In contrast, methanol production in vivo was not detected in fruits of the 'Hard Pick' line and the degree of pectin esterification remained the same throughout ripening. Consequently, a ripening specific PME that is active in vivo appears to enhance PG-mediated pectin ultra-degradation resulting in cell wall dissolution and the deciduous fruit trait. PME activity in vitro, however, was detected in protein extracts from both lines at all ripening stages. This indicates that some PME isozymes are apparently inactive in vivo, particularly in green fruit and throughout ripening in the 'Hard Pick' line, limiting PG-mediated pectin depolymerization which results in moderately difficult fruit separation from the calyx.     

A constitutively expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1

A 36 kDa chitinase was purified by ion exchange and gel filtration chromatography from the culture supernatant of Bacillus thuringiensis HD-1. The chitinase production was independent of the presence of chitin in the growth medium and was produced even in the presence of glucose. The purified chitinase was active at acidic pH, had an optimal activity at pH 6.5, and showed maximum activity at 65 degrees C. Of the various substrates, the enzyme catalyzed the hydrolysis of the disaccharide 4-MU(GlnAc)(2) most efficiently and was therefore classified as an exochitinase. The sequence of the tryptic peptides showed extensive homology with Bacillus cereus 36 kDa exochitinase. The 1083 bp open reading frame encoding 36 kDa chitinase was amplified with primers based on the gene sequence of B. cereus 36 kDa exochitinase. The deduced amino-acid sequence showed that the protein contained an N-terminal signal peptide and consisted of a single catalytic domain. The two conserved signature sequences characteristic of family 18 chitinases were mapped at positions 105-109 and 138-145 of Chi36. The recombinant chitinase was expressed in a catalytically active form in Escherichia coli in the vector pQE-32. The expressed 36 kDa chitinase potentiated the insecticidal effect of the vegetative insecticidal protein (Vip) when used against neonate larvae of Spodoptera litura.