Expression of ACC synthase is enhanced earlier than that of ACC oxidase during fruit ripening of mume (Prunus mume)

Mume (Japanese apricot: Prunus mume Sieb. et Zucc.) is a climacteric fruit that produces large amounts of ethylene as it ripens. Ripening is accompanied by marked increases in the activities of two ethylene-biosynthetic enzymes, namely, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. To study the molecular aspects of ripening of mume, we isolated cDNA clones for proteins that we considered likely to be involved in the biosynthesis and perception of ethylene during ripening, namely, ACC synthase, ACC oxidase and the ethylene receptor. Northern blotting analysis revealed the markedly increased expression of ACC synthase prior to that of ACC oxidase and the increase in ethylene production during ripening. Overall, the levels of the mRNAs for the genes corresponded closely to the levels of activity of the ethylene-biosynthetic enzymes. Exposure of mature green mume fruit to ethylene for 12 h induced strong expression of ACC synthase, as well as of ACC oxidase. Wounding of the pericarp of mume fruit induced the expression of ACC synthase but not of ACC oxidase. The rate of ethylene production increased only slightly after wounding. These results suggest that expression of the genes for ACC synthase and ACC oxidase must be activated sequentially for maximum production of ethylene during ripening of mume fruit and that several mechanisms regulate the expression of ethylene-biosynthetic genes during ripening.     

Coexpression of a defensin gene and a thionin-like gene via different signal transduction pathways in pepper and Colletotrichum gloeosporioides interactions

The anthracnose fungus, Colletotrichum gloeosporioides, interacts incompatibly with the ripe fruit of pepper (Capsicum annuum). It interacts compatibly with the unripe-mature fruit. We isolated a defensin gene, j1-1, and a thionin-like gene, PepThi, expressed in the incompatible interaction by using an mRNA differential display method. Both genes were developmentally regulated during fruit ripening, organ-specifically regulated, and differentially induced during the compatible and incompatible interactions. Expression of the PepThi gene was rapidly induced in the incompatible-ripe fruit upon fungal infection. The fungus-inducible PepThi gene is highly inducible only in the unripe fruit by salicylic acid. In both ripe and unripe fruit, it was induced by wounding, but not by jasmonic acid. Expression of the j1-1 gene is enhanced by jasmonic acid in the unripe fruit but suppressed in the ripe fruit. These results suggest that both small and cysteine-rich protein genes are induced via different signal transduction pathways during fruit ripening to protect the reproductive organs against biotic and abiotic stresses.     

Expression of multiple forms of polygalacturonase gene during ripening in banana fruit.

The activity of polygalacturonase (PG, E.C 3.2.1.15) during ripening in climacteric fruits has been positively correlated with softening of the fruit tissue and differential expression of its gene is suspected to be regulated by the plant hormone ethylene. We have cloned four partial cDNAs, MAPG1 (acc. no. AF311881), MAPG2 (acc. no. AF311882), MAPG3 (acc. no. AF542382) and MAPG4 (acc. no. AY603341) for PG genes and studied their differential expression during ripening in banana. MAPG3 and MAPG4 are believed to be ripening related and regulated by ethylene whereas MAPG2 is associated more with senescence. MAPG1 shows constitutive expression and is not significantly expressed in fruit tissue. The genomic clone MAGPG (acc. No. AY603340) includes the complete MAPG3 gene, which consists of four exons and three introns. The structure of the gene has more similarity to tomato abscission PG rather than tomato fruit PG. It is concluded that softening during ripening in banana fruit results from the concerted action of at least four PG genes, which are differentially expressed during ripening.     

Characterization of ripening-regulated cDNAs and their expression in ethylene-suppressed charentais melon fruit

Charentais melons (Cucumis melo cv Reticulatus) are climacteric and undergo extremely rapid ripening. Sixteen cDNAs corresponding to mRNAs whose abundance is ripening regulated were isolated to characterize the changes in gene expression that accompany this very rapid ripening process. Sequence comparisons indicated that eight of these cDNA clones encoded proteins that have been previously characterized, with one corresponding to ACC (1-aminocyclopropane-1-carboxylic acid) oxidase, three to proteins associated with pathogen responses, two to proteins involved in sulfur amino acid biosynthesis, and two having significant homology to a seed storage protein or a yeast secretory protein. The remaining eight cDNA sequences did not reveal significant sequence similarities to previously characterized proteins. The majority of the 16 ripening-regulated cDNAs corresponded to mRNAs that were fruit specific, although three were expressed at low levels in vegetative tissues. When examined in transgenic antisense ACC oxidase melon fruit, three distinct patterns of mRNA accumulation were observed. One group of cDNAs corresponded to mRNAs whose abundance was reduced in transgenic fruit but inducible by ethylene treatment, indicating that these genes are directly regulated by ethylene. A second group of mRNAs was not significantly altered in the transgenic fruit and was unaffected by treatment with ethylene, indicating that these genes are regulated by ethylene-independent developmental cues. The third and largest group of cDNAs showed an unexpected pattern of expression, with levels of mRNA reduced in transgenic fruit and remaining low after exposure to ethylene. Regulation of this third group of genes thus appears to ethylene independent, but may be regulated by developmental cues that require ethylene at a certain stage in fruit development. The results confirm that both ethylene-dependent and ethylene-independent pathways of gene regulation coexist in climacteric fruit.     

Antisense inhibition of the Nr gene restores normal ripening to the tomato Never-ripe mutant, consistent with the ethylene receptor-inhibition model

The hormone ethylene regulates many aspects of plant growth and development, including fruit ripening. In transgenic tomato (Lycopersicon esculentum) plants, antisense inhibition of ethylene biosynthetic genes results in inhibited or delayed ripening. The dominant tomato mutant, Never-ripe (Nr), is insensitive to ethylene and fruit fail to ripen. The Nr phenotype results from mutation of the ethylene receptor encoded by the NR gene, such that it can no longer bind the hormone. NR has homology to the Arabidopsis ethylene receptors. Studies on ethylene perception in Arabidopsis have demonstrated that receptors operate by a "receptor inhibition" mode of action, in which they actively repress ethylene responses in the absence of the hormone, and are inactive when bound to ethylene. In ripening tomato fruit, expression of NR is highly regulated, increasing in expression at the onset of ripening, coincident with increased ethylene production. This expression suggests a requirement for the NR gene product during the ripening process, and implies that ethylene signaling via the tomato NR receptor might not operate by receptor inhibition. We used antisense inhibition to investigate the role of NR in ripening tomato fruit and determine its mode of action. We demonstrate restoration of normal ripening in Nr fruit by inhibition of the mutant Nr gene, indicating that this receptor is not required for normal ripening, and confirming receptor inhibition as the mode of action of the NR protein.     

Ethylene and Wound-Induced Gene Expression in the Preclimacteric Phase of Ripening Avocado Fruit and Mesocarp Discs

Whereas intact postharvest avocado (Persea americana Mill.) fruit may take 1 or more weeks to ripen, ripening is hastened by pulsing fruit for 24 h with ethylene or propylene and is initiated promptly by cutting slices, or discs, of mesocarp tissue. Because the preclimacteric lag period constitutes the extended and variable component of the ripening syndrome, we postulated that selective gene expression during the lag period leads to the triggering of the climacteric. Accordingly, we sought to identify genes that are expressed gradually in the course of the lag period in intact fruit, are turned on sooner in response to a pulse, and are induced promptly in response to wounding (i.e. slicing). To this end, a mixed cDNA library was constructed from mRNA from untreated fruit, pulsed fruit, and aged slices, and the library was screened for genes induced by wounding or by pulsing and/or wounding. The time course of induction of genes encoding selected clones was established by probing northern blots of mRNA from tissues variously treated over a period of time. Four previously identified ripening-associated genes encoding cellulase, polygalacturonase (PG), cytochrome P-450 oxidase (P-450), and ethylene-forming enzyme (EFE, or 1-aminocyclopropane-1-carboxylic acid synthase), respectively, were studied in the same way. Whereas cellulase, PG, and EFE were ruled out as having a role in the initiation of the climacteric, the time course of P-450 induction, as well as the response of same to pulsing and wounding met the criteria[mdash]together with several clones from the mixed library[mdash]for a gene potentially involved in preclimacteric events leading to the onset of the climacteric. Further, it was established that the continuous presence of ethylene is required for persisting induction, and it is suggested that in selected cases wounding may exert a synergistic effect on ethylene action.