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R. López-Gómez and M.A. Gómez-Lim

A rapid and simple procedure is described for efficiently extracting intact RNA from mango (Mangifera indica L. cv. Manila) mesocarp, a tissue rich in polysaccharides. The RNA can be used for in vitro translation, northern blots, and cDNA synthesis. This method is applicable to other fleshy fruits rich in polysaccharides.

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Shahid N. Chohan and Terence A. Brown

The RNA content of tomato seeds was shown to increase when the seeds were imbibed in water. This increase was due mainly to an increase in nuclear RNA, the polysomal content declining and the ribonucleoprotein fraction remaining constant. The poly(A)+RNA population also showed a gradual increase, again due to a high de novo synthesis rate in the nucleus. In the presence of 200 μM abscisic acid (ABA), the total nuclear DNA failed to increase in the manner seen with water imbibition, leading to an overall decline in RNA during the first 1.5 h. The polysomal and ribonucleoprotein fractions were unaffected by ABA. The decline in total nuclear RNA was due primarily to a major decrease in the nuclear poly(A)+ content of seeds imbibing with ABA. This reduction in de novo transcription may be a factor responsible for the inhibitory effect that ABA has on germination of tomato seeds.

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Ann M. Callahan and Carole L. Bassett

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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P.B. McGarvey, M.S. Montasser, and J.M. Kaper

Transgenic tomato plants (Lycopersicon esculentum Mill.) expressing cucumber mosaic virus (CMV) satellite RNA fused to a gene for β-glucuronidase were produced using Agrobacterium-mediated transformation. The R1 progeny of self-crossed R0 plants were challenge-inoculated with virion or RNA preparations of CMV or tomato aspermy virus (TAV). The transgenic plants challenged with CMV-1 showed mild disease symptoms in the first 2 weeks postchallenge followed by a decrease in symptoms, resulting in little difference between the transgenic and uninfected control group by the fourth week. Enzyme-linked immunosorbent assay results showed about a 10-fold decrease in virus accumulation in the transgenic plants compared to controls. Tolerance was evident only in plants that contained the recombinant insert and produced mature unit-length satellite RNA after CMV infection. Plants challenged with TAV showed no significant tolerance to virus-induced symptoms.

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Carole L. Bassett and Ann M. Callahan

Leaf expansion in peach (Prunus persica) cv Loring was monitored by measuring the increase in blade length during the spring and early summer of 1994, a season in which no flowers were observed on the sampled trees. Expansion was correlated with time after vegetative budbreak and with leaf position on growing apical shoots. In preliminary studies, information from these measurements was used to identify the relative maturity of leaves during the growing season in order to define sampling times that would represent “old,” “mature,” and “young” leaves. Leaves in these categories were sampled and pooled on two different dates, and total RNA was isolated from each sample. The RNAs were examined by Northern blot analysis using a 32P-labeled cDNA clone encoding a peach cab (chlorophyll a/b binding protein) gene. Estimates of abundance based on the intensities of RNA bands hybridizing to the probe indicated that RNAs representing the cab gene family were most abundant in “mature” leaves. Further examination of abundance in pooled, individual leaves representing positions 1 through 19 (numbered acropetally) revealed a substantial decline in abundance in leaves from positions 1 through 5, which were already showing signs of senescence. These results are consistent with enhanced expression of the cab gene in the most photosynthetically active leaves.

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Faye M. Rosin and David Hannapel

MADS-box genes are an important family of highly conserved regulatory genes in plants, animals, and yeast. Genetic analyses have shown that plant MADS-box genes are homeotic and control both the spatial and temporal location of specific organs. While MADS-box genes have been extensively studied and characterized in floral organ development, their involvement in other developmental processes, such as fruit development, is not well understood. From a strawberry fruit cDNA library, we have identified a strawberry AGAMOUS-like MADS-box gene (SAG1) that is expressed in developing fruit, but not in leaves. This is the first MADS-box gene to be isolated from strawberry. The hypothesis guiding this research is that SAG1 plays an important role in the development of the fruit. Nucleotide sequence analysis showed that this cDNA had the highest sequence match to genes from the AGAMOUS family. Comparison of amino acid sequence similarity between SAG1 and members of this family ranged from 70 to 75% overall, and between 98% to100% within the MADS-box. Involvement in stamen and carpel identity is one function of this family of MADS-box genes. Northern hybridizations were performed in order to analyze the expression of this gene at the RNA level. RNA was extracted from various organs of Fragaria ×ananassa, c.v. Calypso. SAG1 RNA expression was specific to stamens, carpels and all stages of fruit and seed development. No expression was detected in roots, leaves, or sepals. Thus, we conclude that SAG1 RNA is involved in reproductive organ and fruit development.

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Jean-Michel Hily, Ralph Scorza*, and Michel Ravelonandro

We have shown that high-level resistance to plum pox virus (PPV) in transgenic plum clone C5 is based on post-transcriptional gene silencing (PTGS), otherwise termed RNA silencing (Scorza et al. Transgenic Res. 10:201-209, 2001). In order to more fully characterize RNA silencing in woody perennial crops, we investigated the production of short interfering RNA (siRNA) in transgenic plum clones C3 and C5, both of which harbor the capsid protein (CP) gene of PPV. We used as a control, plum PT-23, a clone only transformed with the two marker genes, NPTII and GUS. We show in the current report that C5 constitutively produces two classes of siRNA, the short (21-22 nucleotides) and long (≈27 nucleotides) species in the absence of PPV inoculation. Transgenic susceptible clone C3 and the control clone PT-23, when healthy, produce no siRNA. Upon infection, these clones produce only the short siRNA (21-22 nt). This siRNA production suggests that plum trees naturally respond to virus infection by initiating PTGS or PTGS-like mechanisms. This study also suggests that high-level virus resistance in woody perennials may require the production of both the short and long size classes of siRNA, as are produced by the resistant C5 plum clone.

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C.A. Powell, A. Hadidi, and J.M. Halbrendt

The ability of 32P-labeled transcribed cRNA probes to detect tomato ringspot virus (TmRSV) RNA in nucleic acid extracts from roots, bark, and leaves of nectarine (Prunus persica [L.] Batsch) trees with the Prunus stem-pitting disease was assessed and compared with detection of TmRSV antigen by enzyme-linked immunosorbent assay (ELISA) in the same tissues. Neither TmRSV-specific nucleic acid nor antigen was detected in nectarine leaf tissue. ELISA detected TmRSV antigen in root extracts from 71% of the diseased trees, while dot hybridization detected virus-specific nucleic acid in 18% of the same samples. However, ELISA detected TmRSV antigen in only 47% of bark extracts; whereas TmRSV-specific nucleic acid was detected in 100% of the bark extracts from samples collected at or near the soil line. When nucleic acid extracts from bark were prepared from various locations on diseased trees and tested for TmRSV-specific nucleic acid by dot hybridization, there was an almost perfect correlation between the presence of stem-pitting symptoms and the detection of TmRSV nucleic acid. Detection of TmRSV RNA from the bark tissue of rootstock suckers from TmRSV-infected `Delicious'/MM.lO6 apple (Malus × domestica Borkh.) trees was unsuccessful using dot hybridization. The viral RNA, however, was usually detected in either leaf or root tissue of these same trees.

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Stacie L. Aragon, Keng-Chang Chuang, and Adelheid R. Kuehnle

Isolation of high quality nucleic acids from aroids can be difficult due to the presence of carbohydrates, phenolics, and other compounds that bind to and/or co-precipitate with the DNA or RNA. Methods previously used for marine algae, mango, and papaya were modified and successfully used for the simultaneous isolation of high quality genomic DNA and RNA from Anthurium, Colocasia, and Spathiphyllum leaves. Genomic DNA yields averaged 477 μg·g-1 fresh weight for Anthurium and 322 and 177 μg·g-1 fresh weight, respectively, for Colocasia and Spathiphyllum. Total RNA yields averaged 129 μg·g-1 fresh weight for Anthurium and 61 and 50 μg·g-1 fresh weight, respectively, for Colocasia Spathiphyllum. This method may be useful in co-isolating high quality nucleic acids from additional aroids and other plants.

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William R. Woodson

The target of many genetic engineering experiments is to inhibit the expression of an endogenous gene. For example, research in my laboratory attempts to suppress the expression of ethylene biosynthetic pathway genes to inhibit the production of ethylene and delay flower senescence. The silencing of endogenous genes is generally accomplished by engineering plants to express either antisense or sense RNAs homologous to the target sequence. The mechanism by which gene silencing occurs is not clearly understood. Genetic and molecular analyses of transgene-induced silencing has revealed both meiotically reversible and fully stable phenotypes resulting from the expression of the transgene. In several cases, the mechanisms potentially involved in the silencing of the transgene and concomitant reversion of phenotype have been studied. These include transgene copy number, configuration of the integrated DNA, level of transgene RNA, and environmental factors. In many cases the silencing of transgenes was correlated with DNA methylation. These phenomena and the implications for engineering horticultural crops to express transgenes will be discussed in this workshop.