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  • Author or Editor: Ann Callahan x
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NADP-dependent Malic Enzyme (NADP-ME, EC 1.1.1.40) catalyzes the decarboxylation of malate, resulting in the release of CO2. In C3 plants the enzyme does not contribute CO2 directly to photosynthesis. Rather, it is associated with the supplemental synthesis of glycolytic and Krebs Cycle intermediates, although it may also be involved in regulating intracellular pH. NADP-ME activity increases during ripening of several fruits e.g. tomato and apple, usually in association with increased respiration of the developing fruit. We examined expression of NADP-ME during ripening in peach using a cDNA probe derived from F. trinervia (C4 dicot). The probe hybridized to a single RNA species of the predicted size and was low in abundance as expected for a C3 NADP-ME. As fruit matured, the RNA levels increased to a maximum around 133-140 days after bloom (fully ripe). NADP-ME RNA was not detectable from leaves isolated at the same time.

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A gene (Lhcb2*Pp1) encoding a type II chlorophyll a/b-binding protein (LHCB) was isolated from peach [Prunus persica (L.) Batsch]. The gene was sequenced and compared to a variety of other genes encoding LHCB polypeptides associated with photosystem II. Similarity at the nucleotide and amino acid level was highest between Lhcb2*Pp1 and other type II genes and was lowest with type I and III genes from other species. Expression of Lhcb2*Pp1 was followed by determining abundance of transcripts in developing leaves of field grown trees in the absence of flowering. Expression was monitored at three times during the first phase of the growing season and was shown to be highest in leaves which were at or near full expansion at each sampling time. These results are consistent with the function of this gene and indicate that it can serve as a marker of photosynthetic maturity under field conditions.

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Abstract

Peach [Prunus persica (L.) Batsch] RNA was extracted from fruit at six times during development, from leaves and from isolated ovules. These RNAs were translated in a wheat germ extract and the resulting polypeptides were compared by one-dimensional NaDodSO4/PoIyacrylamide gel electrophoresis (PAGE). RNAs encoding polypeptides of 100,85, 59,54,42, 41, and 25.5 kDa increased, while those encoding polypeptides of 46, 40, 25.5, and 25 kDa decreased during peach fruit development. These RNAs are present in fruit but not in ovules or leaves. This study suggests that fruit development in peach is at least partially regulated at the transcriptional level or at the level of mRNA stability.

Open Access

Cold acclimation in temperate, woody plants involves distinct changes in gene activity and protein expression. We have been identifying proteins and genes that are associated with seasonal changes in cold hardiness. Seasonal changes in a 60-kDa dehydrin and its corresponding transcript have been identified, as well as seasonal changes in 16- and 19-kDa storage proteins. Further screening of a cDNA library, constructed from cold-acclimated bark tissues collected in December, identified a 700–800-bp clone that was seasonally expressed in Northern blots. The transcript began to accumulate in October, reached a peak in November–December, and then began to decline. By April, the transcript was no longer present in bark tissues. The transcript size indicates that this gene my be related to either the 16- or 19-kDa storage proteins previously identified; however, an amino acid sequence of the protein for comparison has not yet been obtained. Interestingly, the transcript is also expressed during the early stages of peach fruit development. A similar pattern between seasonal expression and fruit development has been observed for a peach dehydrin transcript. Analysis of a partial sequence of the clone has indicated a similarity to genes encoding proteinase inhibitors and thionins (a class of biocidal proteins). More definitive characterization of the gene and identification of its corresponding protein are in progress.

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The plum (Prunus domestica) cultivar Stoneless was characterized to determine if the lack of stone was the result of reduced endocarp development or a decrease in lignification. Fruit were sampled at several times from 37 days before stone hardening (DBH) until the stone was too hard to cut with a knife and were compared with plum fruit that had normal stones. At all sampling times there was less endocarp tissue and reduced lignin staining in the ‘Stoneless’ plum fruit. The tissue that did stain appeared to be small endocarp remnants present in the ‘Stoneless’ plum, and was concentrated at the suture side and at the blossom end as well as the stem end. The lignin stain was detected in these regions beginning at 19 DBH, while the normal plums had a progression of staining beginning at the blossom end, suture side at 23 DBH and radiating up to the stem end and throughout the presumptive stone tissue at 8 DBH. Comparison of dry weight for dissected tissues agreed with the specific lack of endocarp tissue in the ‘Stoneless’ plum. Gene activity for the lignin pathway was analyzed using four known genes required for lignification. All four genes showed endocarp-specific expression in ‘Stoneless’ similar to that observed for the control. These results support the idea that the phenotype of ‘Stoneless’ plum fruit is due to a decrease in endocarp formation rather than a decrease in endocarp lignification.

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The theme running through many of Luther Burbank’s breeding programs was to make plants more tailored to human uses. Mr. Burbank thought that the stone in plum fruits was unessential to a tree that was propagated vegetatively, so he chose stoneless plums as a breeding goal. He made two releases, ‘Miracle’ in 1903 and his final and almost perfect ‘Conquest’ in 1916, which he considered one of his best accomplishments in plum breeding. ‘Conquest’ had only a grain of stone and flavor and size comparable to the best French types of the time but was not commercially successful. In view of the current desire for convenience food such as seedless fruit (citrus, grapes, watermelon) and advanced knowledge of genetics and breeding technologies, we have taken up where Mr. Burbank left off in the production of a better than “almost perfect” stoneless plum. We began by locating what were most likely remnants from Mr. Burbank’s breeding program and we are now using 21st century technology to achieve a completely stoneless, high-quality plum fruit. These technologies include molecular markers, genetic engineering, and accelerated breeding cycles (FasTrack). Initial experiments had characterized the stoneless trait as a decrease in the number of endocarp cells that form the stone. We defined the time critical to the formation of endocarp by analyzing gene expression of a number of transcription factors involved with determining endocarp cells. We identified genes that were expressed differently during this period between normal stone cultivars and one of the stoneless cultivars. In addition, we targeted genes for genetic engineering to reduce the lignification in endocarp and to reduce or convert endocarp cells to non-lignifying cells. A system, FasTrack, using a flowering gene from poplar, has been incorporated to reduce the juvenility period and eliminate the seasonal aspect of fruiting to see the results of the breeding as well as the genetic engineering approach much faster. The combination of these approaches is now in place to attempt to improve on Mr. Burbank’s stoneless plum.

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Peach [Prunus persica (L.) Batsch] cDNA libraries have been constructed from RNA isolated from immature (30 days after bloom) and ripe fruit. cDNA clones of interest have been identified by differential hybridization among the cDNAs of various peach cultivars or from several stages in fruit development. In addition, several clones were isolated by low stringency hybridizations with oligonucleotides derived from a tomato polygalacturonase cDNA sequence and a cucumber peroxidase amino acid sequence. The pattern of accumulation of the corresponding mRNAs during fruit development was examined by RNA gel-blot analyses in the commercial cultivar Suncrest. Three cDNA clones, pch201, pch307, and pch313, were related to mRNAs that accumulate during the softening stage of fruit development. cDNA clones pchl03, pch205, and pch306 were related to an mRNAs that increase in abundance throughout development, with maximum levels in ripe fruit. cDNA clones pch104 and pch202 were related to mRNAs that exhibit maximum abundance in midfruit development, and clone pch108 was related to mRNA that decreases as the fruit matures. Southern analyses indicated that seven of the cDNAs are represented by only a few genes, while pch104 detects a repetitive family, and pch307 detects a small family of genes. These clones will provide the initial source of genes to manipulate and affect fruit development.

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We are interested in identifying and isolating genes which affect the rate of softening in peach fruit. It may be possible through the engineering of these genes to delay or extend the softening. This could ultimately allow for the harvest and transport of more mature, higher quality fruit. The clone, pch313, was isolated from a ripe peach fruit cDNA library. RNA homologous to this clone is detected at a low abundance in fruit until softening when a >100 fold increase in abundance of the RNA occurs. Pch313 RNA is also detected 30 min after wounding leaf or fruit tissue and peaks in accumulation within 2-8 hours. Wound ethylene was measured from the same tissue and its rate of evolution paralleled the accumulation of the RNA. The cDNA was sequenced and found to have 78% sequence identity with pTom13, a tomato gene that is expressed during fruit ripening and wounding (Holdsworth et al., NAR 15:731-739, 1987). To determine the universality of pch313 related gene expression, RNA accumulation was measured in other fruits during softening, and in leaf tissue upon wounding.

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We are interested in identifying and isolating genes which affect the rate of softening in peach fruit. It may be possible through the engineering of these genes to delay or extend the softening. This could ultimately allow for the harvest and transport of more mature, higher quality fruit. The clone, pch313, was isolated from a ripe peach fruit cDNA library. RNA homologous to this clone is detected at a low abundance in fruit until softening when a >100 fold increase in abundance of the RNA occurs. Pch313 RNA is also detected 30 min after wounding leaf or fruit tissue and peaks in accumulation within 2-8 hours. Wound ethylene was measured from the same tissue and its rate of evolution paralleled the accumulation of the RNA. The cDNA was sequenced and found to have 78% sequence identity with pTom13, a tomato gene that is expressed during fruit ripening and wounding (Holdsworth et al., NAR 15:731-739, 1987). To determine the universality of pch313 related gene expression, RNA accumulation was measured in other fruits during softening, and in leaf tissue upon wounding.

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