Avocado cellulase: nucleotide sequence of a putative full-length cDNA clone and evidence for a small gene family

A cDNA library was prepared from ripe avocado fruit (Persea americana Mill. cv. Hass) and screened for clones hybridizing to a 600 bp cDNA clone (pAV5) coding for avocado fruit cellulase. This screening led to the isolation of a clone (pAV363) containing a 2021 nucleotide transcribed sequence and an approximately 150 nucleotide poly(A) tail. Hybridization of pAV363 to a northern blot shows that the length of the homologous message is approximately 2.2 kb. The nucleotide sequence of this putative full-length mRNA clone contains an open reading frame of 1482 nucleotides which codes for a polypeptide of 54.1 kD. The deduced amino acid composition compares favorably with the amino acid composition of native avocado cellulase determined by amino acid analysis. Southern blot analysis of Hind III and Eco RI endonuclease digested genomic DNA indicates a small family of cellulase genes.     

Interrelationship of Gene Expression, Polysome Prevalence, and Respiration during Ripening of Ethylene and/or Cyanide-Treated Avocado Fruit

Upon initiation of ripening in avocado fruit (Persea americana Mill. cv Hass) with 10 microliters/liter ethylene, polysome prevalence and associated poly(A)⁺ mRNA increase approximately 3-fold early in the respiratory climacteric and drop off to preclimacteric levels at the peak of the respiratory climacteric. The increase in poly(A)⁺ mRNA on polysomes early in the respiratory climacteric constitutes a generic increase in constitutive mRNAs. New gene expression associated with ripening is minimal but evident after 10 hours of ethylene treatment and continues to increase relative to constitutive gene expression throughout the climacteric. The respiratory climacteric can be temporally separated into two phases. The first phase is associated with a general increase in protein synthesis, whereas the second phase reflects new gene expression and accumulation of corresponding proteins which may be responsible for softening and other ripening characteristics. A major new message on polysomes that arises concomitantly with the respiratory climacteric codes for an in vitro translation product of 53 kilodaltons which is immunoprecipitated by antiserum against avocado fruit cellulase.

Cyanide at 500 microliters/liter fails to affect the change in polysome prevalance or new gene expression associated with the ethylene-evoked climacteric in avocado fruit. Treatment of fruit with 500 microliters/liter cyanide alone initiates a respiratory increase within 4 hours, ethylene biosynthesis within 18 hours, and new gene expression akin to that educed by ethylene within 20 hours of exposure to cyanide.

     

Changes in NADP+-linked isocitrate dehydrogenase during tomato fruit ripening

The activity of NADP⁺-specific isocitrate dehydrogenase (NADP⁺-IDH, EC 1.1.1.42) was investigated during the ripening of tomato (Lycopersicon esculentum Mill.) fruit. In the breaker stage, NADP⁺-IDH activity declined but a substantial recovery was observed in the late ripening stages when most lycopene synthesis occurs. These changes resulted in higher NADP⁺-IDH activity and specific polypeptide abundance in ripe than in green fruit pericarp. Most of the enzyme corresponded to the predominant cytosolic isoform which was purified from both green and ripe fruits. Fruit NADP⁺-IDH seems to be a dimeric enzyme having a subunit size of 48 kDa. The K _m values of the enzymes from green and ripe pericarp for NADP⁺, isocitrate and Mg²⁺ were not significantly different. The similar molecular and kinetic properties and chromatographic behaviour of the enzymes from the two kinds of tissue strongly suggest that the ripening process is not accompanied by a change in isoenzyme complement. The increase in NADP⁺-IDH in the late stage of ripening also suggests that this enzyme is involved in the metabolism of C₆ organic acids and in glutamate accumulation in ripe tissues.     

Bean abscission cellulase : characterization of a cDNA clone and regulation of gene expression by ethylene and auxin

The physiology and anatomy of abscission has been studied in considerable detail; however, information on the regulation of gene expression in abscission has been limited because of a lack of probes for specific genes. We have identified and sequenced a 595 nucleotide bean (Phaseolus vulgaris cv Red Kidney) abscission cellulase cDNA clone (pBACl). The bean cellulase cDNA has extensive nucleic and amino acid sequence identity with the avocado cellulase cDNA pAV363. The 2.0 kilobase bean mRNA complementary to pBACl codes for a polypeptide of approximately 51 kilodalton (shown by hybrid-selection followed by in vitro translation). Bean cellulase antiserum is shown to immunoprecipitate a 51 kilodalton polypeptide from the in vitro translation products of abscission zone poly(A)⁺ RNA. Ethylene initiates bean leaf abscission and tissue-specific expression of cellulase mRNA. If ethylene treatment of bean explants was discontinued after 31 h and then 2,5-norbornadiene given to inhibit responses resulting from endogenously synthesized ethylene, polysomal cellulase mRNA hybridizing to pBACl decreased. Thus, ethylene is required not only to initiate abscission and cellulase gene expression but also to maintain continued accumulation of cellulase mRNA. Explants treated with auxin 4 hours prior to a 48 hour treatment with ethylene showed no substantial accumulation of RNA hybridizing to pBACl or expression of cellulase activity.     

Gene expression during fruit ripening in avocado

The poly(A) ⁺RNA populations from avocado fruit (Persea americana Mill cv. Hass) at four stages of ripening were isolated by two cycles of oligo-dT-cellulose chromatography and examined by invitro translation, using the rabbit reticulocyte lysate system, followed by two-dimensional gel electrophoresis (isoelectric focusing followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis) of the resulting translation products. Three mRNAs increased dramatically with the climacteric rise in respiration and ethylene production. The molecular weights of the corresponding translation products from the ripening-related mRNAs are 80,000, 36,000, and 16,500. These results indicate that ripening may be linked to the expression of specific genes.     

Proteinase inhibitors I and II in fruit of wild tomato species: Transient components of a mechanism for defense and seed dispersal

The juice of unripe fruit from a wild species of tomato, Lycopersicon peruvianum (L.) Mill., LA 107, contains over 50% of its soluble proteins as the sum of two proteinase inhibitors. These are the highest levels of proteinase inhibitors and highest percentage of soluble proteins as proteinase inhibitors of any plant or animal tissue found to date. Fruit of the modern tomato, L. esculentum Mill., contains only negligible quantities of the two inhibitors. The two proteinase inhibitors in the fruit of L. peruvianum are members of the Inhibitor I and II families previously found in potato tubers and in leaves of wounded potato and tomato plants. The levels of the two inhibitors in the unripe fruit decrease significantly during ripening. Unripe fruit from other wild Lycopersicon species such as L. parviflorum Rick, Kesicki, Fobes et Holle, L. hirsutum Humb. et Bonpe., L. pimpinellifolium Mill., and other lines of L. peruvianum contain moderate levels of the inhibitors that also decrease during ripening. Another wild tomato species, L. pennellii Corr., is similar to L. esculentum in not containing the two proteinase inhibitors in either unripe or ripe fruit. The transient levels of the inhibitors in fruit of wild species indicate that they are present in unripe fruit as defensive chemicals against insects, birds or small mammals and their disappearance during ripening may render them edible to facilitate seed dispersal. High levels of mRNAs coding for Inhibitors I and II in unripe fruit of L. peruvianum, LA 107, indicate that strong promoters may regulate the developmentally expressed proteinase-inhibitor genes in tomato fruit that may have a substantial potential for use in genetic-engineering experiments to enhance the production of large quantities of proteinase inhibitors or other proteins in field tomatoes.Abbreviations poly(A)⁺ mRNA polyadenylated mRNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide electrophoresis Project 1791, College of Agriculture and Home Economics Research Center, Washington, State University     

Characterization of fruit specific cDNAs from tomato

Summary Differential hybridization was used to screen a cDNA library made from ripe tomato fruit poly(A⁺)RNA. Clones were identified representing genes expressed predominantly at the unripe and/or ripe stage of the fruit development. Northern analysis was used for further characterization of the clones and in this report we describe four cDNA clones expressed at varying stages of fruit development. Three of these cDNAs were found to represent low-copy number genes and one was found to represent a gene family. Dot blot analysis revealed that the expression of these four genes was reduced between 2-fold and 100-fold in three ripening mutants of tomato.     

Avocado fruit protoplasts: a cellular model system for ripening studies

Summary Mesocarp protoplasts were isolated from mature avocado fruits (Persea americana cv. Hass) at varying stages of propylene-induced ripening. Qualitative changes in the pattern of radiolabel incorporation into polypeptides were observed in cells derived from fruit at the different stages. Many of these differences correlate with those observed during radiolabeling of polypeptides from fresh tissue slices prepared from unripe and ripe fruit. Protoplasts isolated from fruit treated with propylene for one day or more were shown to synthesize cellulase (endo-ß-1,4-glucanase) antigen, similar to the intact propylene-treated fruit. These results suggest that the isolated protoplasts retain at least some biochemical characteristics of the parent tissue. The cells may also be used in transient gene expression assays. Protoplasts isolated from preclimacteric and climacteric fruit were equally competent in expressing a chimeric test gene, composed of the CaMV 35S RNA promoter fused to the bacterial chloramphenicol acetyltransferase gene, which was introduced by electroporation.Abbreviations PCM Murashige and Skoog salts and growth factors, supplemented with 3% sucrose, 0.3 % glucose, 0.3% enzymatic casein hydrolysate, 0.5 M mannitol, and 5 mM CaCl₂ - CAT chloramphenicol acetyltransferase     

Peroxisomal thiolase mRNA is induced during mango fruit ripening

Fruit ripening is a complex, developmentally regulated process. A series of genes have been isolated from various ripening fruits encoding enzymes mainly involved in ethylene and cell wall metabolism. In order to aid our understanding of the molecular basis of this process in a tropical fruit, a cDNA library was prepared from ripe mango (Mangifera indica L. cv. Manila). By differential screening with RNA poly(A)⁺ from unripe and ripe mesocarp a number of cDNAs expressing only in ripe fruit have been isolated. This paper reports the characterization of one such cDNA (pTHMF 1) from M. indica which codes for a protein highly homologous to cucumber, rat and human peroxisomal thiolase (EC 2.3.1.16), the catalyst for the last step in the -oxidation pathway.The cDNA for the peroxisomal mango thiolase is 1305 bp in length and codes for a protein of 432 amino acids with a predicted molecular mass of 45 532 Da. Mango thiolase is highly homologous to cucumber thiolase (80%), the only other plant thiolase whose cloning has been reported, and to rat and human thiolases (55% and 55% respectively).It is shown by northern analysis that during fruit ripening THMF 1 is up-regulated. A similar pattern of expression was detected in tomato fruit. Wounding and pathogen infection do not appear to affect THMF 1 expression. The possible involvement of thiolase in fatty acid metabolism during fruit ripening will be discussed. To our knowledge this is the first report cloning of a plant gene involved in fatty acid metabolism showing an induction during fruit ripening.     

Influence of avocado (Persea americana) Cx-cellulase on the structural features of avocado cellulose

Avocado (Persea americana Mill.) fruit produce copious quantities of the enzyme Cx-cellulase (EC 3.2.1.4) during ripening. The possibility that Cx-cellulase is able to disrupt cellulose microfibril oranization was investigated using molecular weight (M_r), x-ray diffraction, and ultrastructural analyses of cell walls from unripe avocado fruit incubated with the purified enzyme. Results indicate that Cx-cellulase causes a downshift in the M_r of unbranched cell-wall polymers in the M_r range of 10⁶–10⁷ Da. There is an increase in the proportion of crystalline cellulose, and cellulose fibrils appear to lose cohesiveness in response to enzyme activity. We propose that Cx-cellulase attacks avocado cellulose at accessible sites in the peripheral and integral noncrystalline regions of the microfibril, resulting in a loss of cohesiveness within the fibril structure and an alteration in the binding of associated cell-wall matrix polysaccharides. The initial loss of avocado mesocarp firmness during fruit ripening may be linked to the onset of Cx-cellulase activity.Abbreviations CMC carboxymethylcellulose - DMAC dimethylacetamide - DS developmental stage - M molecular weight - XG xyloglucan     

Cloning of a cDNA encoding 1-aminocyclopropane-1-carboxylate synthase and expression of its mRNA in ripening apple fruit

1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) purified from apple (Malus sylvestris Mill.) fruit was subjected to trypsin digestion. Following separation by reversed-phase high-pressure liquid chromatography, ten tryptic peptides were sequenced. Based on the sequences of three tryptic peptides, three sets of mixed oligonucleotide probes were synthesized and used to screen a plasmid cDNA library prepared from poly(A)⁺ RNA of ripe apple fruit. A 1.5-kb (kilobase) cDNA clone which hybridized to all three probes were isolated. The clone contained an open reading frame of 1214 base pairs (bp) encoding a sequence of 404 amino acids. While the polyadenine tail at the 3-end was intact, it lacked a portion of sequence at the 5-end. Using the RNA-based polymerase chain reaction, an additional sequence of 148 bp was obtained at the 5-end. Thus, 1362 bp were sequenced and they encode 454 amino acids. The deduced amino-acid sequence contained peptide sequences corresponding to all ten tryptic fragments, confirming the identity of the cDNA clone. Comparison of the deduced amino-acid sequence between ACC synthase from apple fruit and those from tomato (Lycopersicon esculentum Mill.) and winter squash (Cucurbita maxima Duch.) fruits demonstrated the presence of seven highly conserved regions, including the previously identified region for the active site. The size of the translation product of ACC-synthase mRNA was similar to that of the mature protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that apple ACC-synthase undergoes only minor, if any, post-translational proteolytic processing. Analysis of ACC-synthase mRNA by in-vitro translation-immunoprecipitation, and by Northern blotting indicates that the ACC-synthase mRNA was undetectable in unripe fruit, but was accumulated massively during the ripening proccess. These data demonstrate that the expression of the ACC-synthase gene is developmentally regulated.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AdoMet S-adenosyl-l-methionine - HPLC high-pressure liquid chromatography - kDa kilodalton - kb kilobase - mAb monoclonal antibody - Met methionine - PCR polymerase chain reaction - poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported by grants DCB-9004129 and INT-8915155 from the National Science Foundation.     

Ripening and in vitro retention of respiratory control by avocado and pear mitochondria

The retention of respiratory control (“survival”) by mitochondria held at 25 C was studied in relation to the ripening of two varieties of avocado (Persea americana Mill. var. `Fuerte' and `Hass') and one variety of pear (Pyrus communis. L. var. `Bartlett') fruit. The survival of avocado mitochondria increased from 8 to 10 hours when isolated from unripe, preclimacteric fruit, to 48 hours when isolated from fully ripe, postclimacteric fruits. Although rates of α-ketoglutarate oxidation, respiratory control, and ADP/O decreased somewhat in the postclimacteric phase, survival per se was not affected. Pear mitochondria survived for more than 30 hours regardless of the physiological age of the source.     

An ethylene-related cDNA from ripening apples

We report the isolation of a ripening-related apple cDNA which is complementary to a mRNA which may be involved in ethylene production. Poly(A)⁺ RNA was extracted from cortical tissue of ripe apple fruit (Malus domestica Borkh cv. Golden Delicious) and a cDNA library constructed in the plasmid vector pSPORT. The library was screened with pTOM13, a tomato cDNA clone thought to code for ACC oxidase in that fruit. An apple cDNA clone (pAP4) was isolated and sequenced. The 1182 bp cDNA insert includes an open reading frame of 942 bp, and shows strong homology with reported tomato and avocado sequences, both at the nucleic acid and amino acid levels. The polypeptide has a calculated molecular mass of 35.4 kDa and a calculated pI of 5.15. In apple cortical tissue, expression of pAP4-complementary RNA increased with ethylene production by the fruit during ripening. Expression was also enhanced in both ethylene-treated and wounded fruit.     

Latency of Infection in Unripe Avocados by Colletotrichum gloeosporioides is Not Due to Inadequate Enzyme Potential

Colletotrichum gloeosporioides produced exo-pectin lyase and protease in a) liquid cultures with incorporated washed cell wall material from unripe or ripe avocado and b) autoclaved immature fruit. The activity of exo-pectin lyase and protease produced in liquid cultures incorporating washed cell walls from immature fruits was almost the same as when washed cell walls from ripe fruits were incorporated. Ripe fruit tissue rotted by the fungus contained exo-pectin lyase, endo-polygalacturonase (endo-PG) and protease. The endo-PG was found to be endogenous to avocado fruit, and had a pH optimum of 5.5. The pH optima of exo-pectin lyase and protease were 8.5 and 7.5 respectively in all three enzyme preparations. All these enzyme preparations completely macerated avocado fruit tissue discs in vitro in less than 3 h of incubation but not potato tuber discs. Neither immature nor ripe fruit contained substances, proteinaceous or otherwise, which could inhibit the exo-pectin lyase or protease activity of these preparations. The results indicated that C. gloeosporioides possesses sufficient enzyme potential to invade cell walls of unripe fruit and that the fruit tissue does not have a mechanism to inactivate such enzymes.     

The Effect of Ethylene and Propylene Pulses on Respiration, Ripening Advancement, Ethylene-Forming Enzyme, and 1-Aminocyclopropane-1-carboxylic Acid Synthase Activity in Avocado Fruit

When early-season avocado fruit (Persea americana Mill. cv Hass) were treated with ethylene or propylene for 24 hours immediately on picking, the time to the onset of the respiratory climacteric, i.e. the lag period, remained unchanged compared with that in untreated fruit. When fruit were pulsed 24 hours after picking, on the other hand, the lag period was shortened. In both cases, however, a 24 hour ethylene or propylene pulse induced a transient increase in respiration, called the pulse-peak, unaccompanied by ethylene production (IL Eaks [1980] Am Soc Hortic Sci 105: 744-747). The pulse also caused a sharp rise in ethylene-forming enzyme activity in both cases, without any increase in the low level of 1-aminocyclopropane-1-carboxylic acid synthase activity. Thus, the shortening of the lag period by an ethylene pulse is not due to an effect of ethylene on either of the two key enzymes in ethylene biosynthesis. A comparison of two-dimensional polyacrylamide gel electrophoresis polypeptide profiles of in vitro translation products of poly(A⁺) mRNA from control and ethylene-pulsed fruit showed both up- and down-regulation in response to ethylene pulsing of a number of genes expressed during the ripening syndrome. It is proposed that the pulse-peak or its underlying events reflect an intrinsic element in the ripening process that in late-season or continuously ethylene-treated fruit may be subsumed in the overall climacteric response. A computerized system that allows continuous readout of multiple samples has established that the continued presentation of exogeneous ethylene or propylene to preclimacteric fruit elicits a dual respiration response comprising the merged pulse-peak and climacteric peak in series. The sequential removal of cores from a single fruit has proven an unsatisfactory sampling procedure inasmuch as coring induces wound ethylene, evokes a positive respiration response, and advances ripening.     

Cloning and characterization of avocado fruit mRNAs and their expression during ripening and low-temperature storage

Differential sereening of a cDNA library made from RNA extracted from avocado (Persea americana Mill cv. Hass) fruit stored at low temperature (7°C) gave 23 cDNA clones grouped into 10 families, 6 of which showed increased expression during cold storage and normal ripening. Partial DNA sequencing was carried out for representative clones. Database searches found homologies with a polygalacturonase (PG), endochitinase, cysteine proteinase inhibitor and several stress-related proteins. No homologies were detected for clones from six families and their biological role remains to be elucidated. A full-length cDNA sequence for avocado PG was obtained and the predicted amino acid sequence compared with those from other PGs. mRNA encoding PG increased markedly during normal ripening, slightly later than mRNAs for cellulase and ethylene-forming enzyme (EFE). Low-temperature storage delayed ripening and retarded the appearance of mRNAs for enzymes known to be involved in cell wall metabolism and ethylene synthesis, such as cellulase, PG and EFE, and also other mRNAs of unknown function. The removal of ethylene from the atmosphere surrounding stored fruit delayed the appearance of the mRNAs encoding cellulase and PG more than the cold storage itself, although it hardly affected the expression of the EFE mRNA or the accumulation of mRNAs homologous to some other unidentified clones.AFRC Research Group in Plant Gene Regulation