Differential expression of expansin gene family members during growth and ripening of tomato fruit

cDNA clones encoding homologues of expansins, a class of cell wall proteins involved in cell wall modification, were isolated from various stages of growing and ripening fruit of tomato (Lycopersicon esculentum). cDNAs derived from five unique expansin genes were obtained, termed tomato Exp3 to Exp7, in addition to the previously described ripening-specific tomato Exp1 (Rose et al. (1997) Proc Natl Acad Sci USA 94: 5955–5960). Deduced amino acid sequences of tomato Exp1, Exp4 and Exp6 were highly related, whereas Exp3, Exp5 and Exp7 were more divergent. Each of the five expansin genes showed a different and characteristic pattern of mRNA expression. mRNA of Exp3 was present throughout fruit growth and ripening, with highest accumulation in green expanding and maturing fruit, and lower, declining levels during ripening. Exp4 mRNA was present only in green expanding fruit, whereas Exp5 mRNA was present in expanding fruit but had highest levels in full-size maturing green fruit and declined during the early stages of ripening. mRNAs from each of these genes were also detected in leaves, stems and flowers but not in roots. Exp6 and Exp7 mRNAs were present at much lower levels than mRNAs of the other expansin genes, and were detected only in expanding or mature green fruit. The results indicate the presence of a large and complex expansin gene family in tomato, and suggest that while the expression of several expansin genes may contribute to green fruit development, only Exp1 mRNA is present at high levels during fruit ripening.     

Ethylene receptor expression is regulated during fruit ripening, flower senescence and abscission

Using theArabidopsis ethylene receptorETR1 as a probe, we have isolated a tomato homologue (tETR) from a ripening cDNA library. The predicted amino acid sequence is 70% identical toETR1 and homologous to a variety of bacterial two component response regulators over the histidine kinase domain. Sequencing of four separate cDNAs indicates that tETR lacks the carboxyl terminal response domain and is identical to that encoded by the tomatoNever ripe gene. Ribonuclease protection showed tETR mRNA was undetectable in unripe fruit or pre-senescent flowers, increased in abundance during the early stages of ripening, flower senescence, and in abscission zones, and was greatly reduced in fruit of ripening mutants deficient in ethylene synthesis or response. These results suggest that changes in ethylene sensitivity are mediated by modulation of receptor levels during development.     

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.     

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     

Cellulase gene expression in ripening avocado fruit: The accumulation of cellulase mRNA and protein as demonstrated by cDNA hybridization and immunodetection

Summary A cDNA library was constructed from poly(A)⁺RNA of ripe avocado fruit. Colony hybridization identified a number of ripening specific clones of which one, pAV5, was shown to be specific for cellulase. Hybrid selection with pAV5 provided a message from ripe fruit that on in vitro translation yielded a polypeptide of 53kD, comigrating with purified avocado cellulase on SDS polyacrylamide gel electrophoresis. The translation product was selectively immunoprecipitated by antiserum to purified avocado cellulase. Immunoblotting of unripe and ripe avocado fruit extracts following SDS-PAGE showed a plentiful immunoreactive polypeptide in ripe fruit, and essentially none in unripe fruit. Hybridization of pAV5 to poly(A)⁺-RNA from unripe and ripe avocado fruit demonstrated that there is at least a 50-fold increase in the cellulase message concentration during ripening. Thus, the expression of cellulase enzyme activity during ripening is regulated by the appearance of mRNA coding for cellulase rather than by either translational or post-translational control mechanisms.Abbreviations poly(A)⁺ polyadenylated - DS sodium dodecyl sulfate - D kilodalton - bp base pairs Supported by Research Grant GM 19807 from the United States Public Health Service and by additional funds from the University of California Research Council.     

Control and manipulation of gene expression during tomato fruit ripening

Ripening is a complex developmental process involving changes in the biochemistry, physiology and gene expression of the fruit. It is an active process characterised by changes in all cellular compartments. cDNA cloning has been used as an approach to analyse changes in gene expression during fruit ripening. This has revealed that several genes are switched on specifically during fruit ripening, including one encoding polygalacturonase (PG), a major cell wall protein. These cDNA clones have been used to study the expression of the genes in normal and ripening mutant fruits, and under environmental stress conditions.The PG gene has been isolated and it has been demonstrated that 1450 bases 5 of the coding region are sufficient for the tissue- and development-specific expression of a bacterial marker gene in transgenic tomatoes. Antisense RNA techniques have been developed to generate novel mutant tomatoes in which the biochemical function of this enzyme and its involvement in fruit softening has been tested.     

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 from 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.     

果实成熟过程相关调控基因研究进展

果实成熟过程中,多聚半乳糖醛酸酶（ＰＧ）参与果胶的分解,从而在果实软化中起作用,新近发现,果实软化过程中,协同展蛋白具有一定的作用：ＡＣＣ合成酶（ＡＣＳ）、ＡＣＣ氧化酶（ＡＣＯ）和ＡＣＣ脱氨酶与乙烯合成直接有关,ＡＣＳ是乙烯形成的关键酶,由多基因家族编码,各个基因协同表达,每一基因都有自己的转录特性,新近不断发现果实中ＡＣＳ基因家族中的新成员;ＡＣＯ是一种与膜结合的酶,这种酶具有结构上的立体专一性     

Modification of matrix polysaccharides during avocado (Persea americana) fruit ripening: an assessment of the role of Cx-cellulase

The role of C_x-cellulase (EC 3.2.1.4) in fruit ripening and softening is unknown. In the present study, avocado ( Persea americana ) fruit, a rich source of C_x-cellulase, were examined to determine if the enzyme plays a role in ripening-related hemicellulose metabolism. Hemicelluloses (4 M alkali-soluble) from avocado fruit exhibited a very broad distribution of polymer sizes and an overall decrease in M_r during ripening. Polymers affected were primarily those of large M_r (relative molecular mass). The characteristic total hemicellulose M_r distribution and changes with ripening were also evident for xyloglucan (XG), a putative substrate for avocado C_x-cellulase. Hydrolytic activity toward hemicelluloses from preripe fruit was detected in crude buffer-soluble protein extracts derived from ripe avocado mesocarp tissue. XG was also degraded, and in a pattern similar to that observed during ripening. Purified C_x-cellulase also exhibited activity against specific components of isolated hemicelluloses; however, in contrast to the crude protein. C_x-cellulase alone was without influence on the M_r distribution of avocado XG. Protein depleted of C_x-cellulase was capable of moderate XG depolymerization. We conclude from the present studies that the enzyme C_x-cellulase is not involved in the ripening-related depolymerization of XG in avocado fruit.     

果实成熟乙烯相关基因工程研究进展(综述)

果实成熟是一个复杂的生理生化过程,而乙烯是引发果实成熟的主要因素.本文简述乙烯合成过程中S-腺苷甲硫氨酸水解酶、ACC合成酶与ACC氧化酶、ACC脱氨酶基因和乙烯受体突变体的特性及克隆;同时,评述利用基因工程技术控制果实成熟的应用前景.     

The dual role of oxygen in avocado fruit respiration: Kinetic analysis and computer modelling of diffusion-affected respiratory oxygen isotherms

Abstract. Oxygen plays a dual role in affecting the rate of respiration of avocado fruit ( Persea americana Mill, cv. Hass). The respiration rate v. oxygen concentration curve for steady state avocado fruit respiration is biphasic. The curve becomes monophasic, however, when measured under conditions of rapidly changing oxygen concentration in a closed circulating system. The results are interpreted as indicating that oxygen at relatively high concentrations modulates respiration independent of its interaction with the terminal oxidase—presumably cytochrome oxidase.
A computer model is presented which takes into account the effect of diffusion barriers on the kinetics of oxygen utilization as a function of concentration in avocado fruit. The model is used to make predictions concerning the apparent K _m of the terminal oxidase or oxidases in avocado fruit. It is concluded that the apparent K _m of the terminal oxidase of uninhibited avocado fruit is that of cytochrome oxidase, and that the alternative, cyanide-resistant oxidase of avocado fruit does not contribute appreciably to the uninhibited respiration of preclimacteric or climacteric avocado fruit.     

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.     

The role of insect pollinators in avocado production: A global review

Insect pollination increases the yield and quality of many crops and therefore, understanding the role of insect pollinators in crop production is necessary to sustainably increase yields. Avocado Persea americana benefits from insect pollination, however, a better understanding of the role of pollinators and their contribution to the production of this globally important crop is needed. In this study, we carried out a systematic literature review and meta-analysis of studies investigating the pollination ecology of avocado to answer the following questions: (a) Are there any research gaps in terms of geographic location or scientific focus? (b) What is the effect of insect pollinators on avocado pollination and production? (c) Which pollinators are the most abundant and effective and how does this vary across location? (d) How can insect pollination be improved for higher yields? (e) What are the current evidence gaps and what should be the focus of future research? Research from many regions of the globe has been published, however, results showed that there is limited information from key avocado producing countries such as Mexico and the Dominican Republic. In most studies, insects were shown to contribute greatly to pollination, fruit set and yield. Honeybees Apis mellifera were important pollinators in many regions due to their efficiency and high abundance, however, many wild pollinators also visited avocado flowers and were the most frequent visitors in over 50% of studies. This study also highlighted the effectiveness of stingless bees (Meliponini) and blow flies (Calliphoridae) as avocado pollinators although, for the majority of flower visitors, there is a lack of data on pollinator efficiency. For optimal yields, growers should ensure a sufficient abundance of pollinators in their orchards either through increasing honeybee hive density or, for a more sustainable approach, by managing wild pollinators through practices that protect or promote natural habitat.     

Ethylene regulation of fruit ripening: Molecular aspects

Progress in ethylene regulating fruit ripening concerning itsperception and signal transduction and expression of ACC synthaseand ACC oxidase genes is reviewed. ACC synthase and ACC oxidasehave been characterized and their genes cloned from various fruittissues. Both ACC synthase and ACC oxidase are encoded bymultigene families, and their activities are associated withfruit ripening. In climacteric fruit, the transition toautocatalytic ethylene production appears to be due to a seriesof events in which ACC sythase and ACC oxidase genes have beenexpressed developmentally. Differential expression of ACCsynthase and ACC oxidase gene family members is probably involvedin such a transition that ultimately controls the onset of fruitripening.In comparison to ACC synthase and ACC oxidase, less is knownabout ethylene perception and signal transduction because of thedifficulties in isolating and purifying ethylene receptors orethylene-binding proteins using biochemical methods. However, theidentification of the Nr tomato ripening mutant as anethylene receptor, the applications of new potent anti-ethylenecompounds and the generation of transgenic fruits with reducedethylene production have provided evidence that ethylenereceptors regulate a defined set of genes which are expressedduring fruit ripening. The properties and functions of ethylenereceptors, such as ETR1, are being elucidated.Application of molecular genetics, in combination withbiochemical approaches, will enable us to better understand theindividual steps leading from ethylene perception and signaltransduction and expression of ACC synthase and ACC oxidase genefamily member to the physiological responses.