Abstract
Sorbitol, the primary photosynthate and translocated carbohydrate in apple (Malus3domestica Borkh.), is converted to fructose by sorbitol dehydrogenase (SDH; EC 171.132.149.166) which is active in apple fruit throughout development. In the apple genome, nine SDH genes have been isolated and their sequences characterized, but their individual expression patterns during apple fruit set and development have not been determined. The objective of this work was to ascertain if SDH genes are differentially expressed and how their patterns of expression may relate to SDH activity in apple seed and cortex during early fruit development. Seed SDH activity was found to be much higher than cortex SDH activity per mg and g fresh weight (FW), and seed SDH activity contributed significantly to whole fruit SDH activity during weeks 2-5 after bloom. Five of the nine SDH genes present in the apple genome were expressed in apple fruit. Two SDH genes, SDH1 and SDH3, were expressed in both seed and cortex tissues. SDH2 expression was limited to cortex, while SDH6 and SDH9 were expressed in seed tissues only. SDH isomeric proteins of different pI values were detected in apple fruit. SDH isomers with pI values of 4.2, 4.8, 5.5, and 6.3 were found in seeds, and SDH isomers with pI values of 5. 5, 6.3, 7.3, and 8.3 were found in cortex. The present work is the first to show that SDH is highly active in apple seed and that SDH genes are differentially expressed in seed and cortex during early development.
Introduction
Apple (Malus3domestica Borkh.) fruit set and development depend on carbohydrate import and metabolism. The main translocated carbohydrate in apple is sorbitol, and oxidation of sorbitol to fructose by sorbitol dehydrogenase (SDH, EC 171.132.149.166), using NAD + as a cofactor, is the first step of sorbitol utilization. SDH has been identified as the primary enzyme that metabolizes sorbitol in apple fruit (Beruter, 1985; Yamaki and Ishiwaka, 1986; Yamaguchi et al., 1996) . A significant level of SDH activity per g fresh weight (FW) and per mg protein has been found immediately after fruit growth starts, 1 week after bloom (AB), and during the ensuing weeks (Nosarzewski et al., 2004) . In addition, SDH exhibits high levels of activity at the transition from cell division to cell expansion, and in some cultivars during ripening (Yamaguchi et al., 1996; Yamada et al., 1999; Park et al., 2002) . Analysis of SDH protein levels during the first 5 weeks of apple fruit development, often termed fruit set, showed that the amount of protein present in the tissue did not change significantly, while SDH activity per mg protein fluctuated depending on the cultivar during that time period (Nosarzewski et al., 2004) .
Variation in SDH activity may be due to limitations in sorbitol availability to the fruit. Apple fruit set and early development are very sensitive to carbohydrate availability, as shown by partial shading and chemical treatments that reduce photosynthetic activity (Bepete and Lakso, 1998; Byers et al., 1990) . This availability is also affected by competition among the many reproductive and vegetative sinks. Because fruit are weaker sinks than growing shoots (Corelli Grappadelli et al., 1994; Lakso et al., 1998) , the inability of many fruit to persist and grow, and/or the low growth rate of some fruit that do persist and that results in poor size and quality at harvest, may be due to less efficient utilization of uploaded carbohydrates compared with shoots and other vegetative sinks. Were fruit more competitive for carbohydrate resources during early development, more fruit could develop without compromising size and quality. Zhang et al. (2005) have recently shown that fruit growth in pear (Pyrus pyrifolia) is limited by sink strength of the fruit rather than the capacity of the transport pathway; sorbitol is the major photoassimilate in pear as it is in apple. This strongly suggests that the capacity for utilization of carbohydrate is critical for achieving sufficient yield and quality. It is likely that SDH plays a critical role in establishing young apple fruit as sinks during the fruit set phase.
Nine SDH genes were found in the apple genome, each encoding a polypeptide of an approximate molecular weight (deduced from mRNA sequences) of 39-40 kDa (Nosarzewski et al., 2004) . The presence of such an abundance of SDH genes in apple suggests tissue-specific regulation of SDH expression. Park et al. (2002) reported that expression of three apple SDH genes was restricted to sink tissues such as young leaves, stems, roots, and maturing fruit, while a fourth SDH gene was expressed in both immature and mature leaves. Similar results were obtained from the developing buds of Japanese pear (Pyrus serotina, another Rosaceae family species) where partial fragments of five SDH genes were isolated (Ito et al., 2005) . The derived amino acid sequence of one of the pear isomers (PpySDH5) has 94-98% homology with apple SDH1 and is distinct from the other four isomers of pear SDH (71% homology). The other four isomers are similar to each other with 88-95% homology. Due to the observed expression pattern in buds, the authors suggested that the pear SDH genes could be categorized into two groups: one expressed in the bud for growth and development (i.e. PpySDH1) and the others of unknown tissue specificity (such as PpySDH5). The expression patterns of all SDH genes during apple fruit set and development have not been determined, although the presence of SDH1 and SDH2 transcripts was detected in fruit starting at 90 d AB, and SDH3 and SDH4 transcripts were expressed at 120 d AB (Park et al., 2002) . Although none of these four SDH transcripts was detected at 30 d AB (Park et al., 2002) , SDH mRNA was present in apple fruit during the first 5 weeks AB, indicating that at least one SDH gene was expressed (Nosarzewski et al., 2004) .
Prior to the present work, studies of fruit SDH activity have focused on either whole fruit or cortex activity only. There have been no data for apple that compare SDH activity in seeds with that of cortex tissue. As the early fruit development period after fertilization is critical to eventual yield, an analysis of both tissues can provide a greater understanding of the dynamics of sorbitol metabolism during this important phase. One of the objectives of this study was to determine if SDH is expressed and is active in apple seed and cortex, and how analyses of whole fruit SDH expression and SDH activity patterns might be related to those in seed and cortex separately. Another objective of this study was to determine if differences in activity in seed and cortex are related to tissue-specific expression of SDH genes. For these purposes, seed and cortex tissues from apple collected during early fruit development were subjected to analysis of SDH activity, isolation of SDH isomeric protein by two-dimensional (2D) electrophoresis, and detection of individual SDH transcripts using reverse transcription-polymerase chain reaction (RT-PCR).
Materials and methods

Materials
The apple cultivars, 'Redchief Delicious' and 'Mutsu', were harvested from the University of Kentucky Horticulture Research Farm orchard, Lexington, KY, once a week for 5 weeks in 2003 , and 2005 ('Redchief Delicious' and 'Mutsu') starting immediately after bloom when fruit growth was first evident, defined as week 1. Whole fruit were immediately frozen in liquid N 2 upon harvest at week 1. Starting in week 2, when separation of seed from cortex was first possible, fruit were placed on ice and transported to the laboratory where seeds were separated from cortex, and both were frozen in liquid N 2 . All fruit tissues were stored at -80°C until further use.
SDH activity measurements
Cortex and seed tissue samples of apple were weighed, and SDH enzyme was extracted and assayed from these samples as in Nosarzewski et al. (2004) , with the exclusion of dithiothreitol (DTT) from the extraction solution since DTT has been reported to be an SDH inhibitor (El-Kabbani et al., 2004) . The Bradford assay (1976) 
Northern analysis
Total RNA was extracted from a composite sample comprised of weekly samples of seed or cortex tissue of 'Redchief Delicious' apple using a hot borate protocol (Wan and Wilkins, 1994) . Northern analyses were run as described in Nosarzewski et al. (2004) .
RNA blot results indicated the presence of SDH transcript in seed and cortex tissues of apple fruit (Fig. 9 ). Despite the slightly lower level of seed RNA loaded on the gel (Fig. 9, lower panel) , the amounts of SDH transcript per lg total RNA appeared to be much greater in seed than in cortex (Fig. 9, upper panel) .
Western analysis
Western blots were performed on weekly composite samples using the ImmunoPure ABC Phosphatase Staining Kit (Pierce) at room temperature as described in Nosarzewski et al. (2004) .
2D electrophoresis
Extracted proteins, as described above, from weekly composite samples were precipitated with a 43 volume of cold acetone overnight at -20°C. After centrifugation for 10 min at 10 000 g and 4°C, the pellet was air-dried and dissolved in sample
Each immobilized protein gradient (IPG) strip was passively rehydrated with 125 ll of prepared sample containing 200-500 lg of protein for 24 h. First dimension focusing was done using a Protean isoelectric focusing (IEF) cell (Bio-Rad) and using rapid ramp mode up to 400 V and 20 000 Vh. After a 10 min equilibration of the IPG strip in equilibration solution [6 M urea, 20% (w/v) SDS, 1.5 M TRIS (pH 8.8), 50% (v/v) glycerol] containing 2% (w/v) DTT and another 10 min equilibration of the IPG strip in the equilibration solution containing 2.5% (w/v) iodoacetamide, the second dimension resolution was performed using SDS containing 12.5% (w/v) acrylamide gels. The resulting gels were subjected to western analysis as described in Nosarzewski et al. (2004) .
Protein isoelectric point prediction
The Protein Modification Screening Tool (http://proteomics. mcw.edu/promost) was used to predict SDH isomer pI values.
RT-PCR analysis
Total RNA was isolated from seeds and cortex of 'Mutsu' apple (composite across weekly samples) using a hot borate technique (Wan and Wilkins, 1994) . Total RNA isolated from the above tissues was pretreated with DNase I using a kit (DNA-free, Ambion, Austin, TX, USA) according to the manufacturer's protocol. The first-strand cDNA for RT-PCR analysis was synthesized with oligo(dT) 18 primers using 1 lg of total RNA and SUPERSCRIPT III (Invitrogen) at 50°C for 1 h. The reaction was terminated (75°C, 15 min) and treated with RNase cocktail (Ambion; 37°C, 20 min). PCR was performed on 2 ll of first-strand cDNA, using gene-specific primers (Table 1) for all nine SDH mRNA species at 40 PCR cycles (annealing temperatures in Table 1 ). Amplicons were isolated on 1% (w/v) agarose gels and subjected to ethidium bromide staining.
Every gene-specific primer was tested by PCR to ensure its gene specificity. PCR using each of the nine pairs of gene-specific primers was performed for each SDH cDNA at the defined annealing temperature (Table 1) . DNA templates representing each of the nine SDH genes were obtained from available 'Mutsu' SDH cDNAs for SDH1, SDH2, and SDH9, and genomic clones for SDH3, SDH4, SDH5, SDH6, SDH7, and SDH8. PCR cycles were performed on ;5 pg of DNA template.
Results

SDH activity
Seed SDH activity per mg extractable protein during weeks 3-5 AB was up to 8-fold greater than cortex and whole fruit SDH activity (Fig. 1) . Seed SDH activity per mg protein increased 3-fold from the second to the third week AB, remained at that level throughout the fourth week AB, and rose again in the fifth week AB. In contrast, cortex and whole fruit SDH activity per mg protein were similar to one another, with slightly higher activity levels for whole fruit (Fig. 1) . Both cortex and whole fruit activity per mg protein appeared to decline slightly from the third to the fifth week AB.
Average seed and cortex SDH activity for 'Redchief Delicious' during 4 weeks of development (starting at the second week AB) reached values of 56.665.2 and 14.865.9 nmol NADH min À1 mg À1 protein, respectively. This is consistent (within one standard deviation) with the values found during a similar period for 'Mutsu', 53.463.8 and 13.363.4 nmol NADH min À1 mg À1 protein for seed and cortex, respectively.
Seed had much greater protein content per g FW than cortex or whole fruit ( Fig. 2A) . Due to changes in seedextractable protein content per g FW ( Fig. 2A ) and in seed SDH activity per mg protein (Fig. 1) , seed SDH activity pattern per g FW (Fig. 2B) varied. At the third week AB, seed SDH activity reached its peak, a 4-fold increase over the second week, due to a 3-fold increase in SDH activity 
SDH expression in apple fruit 1865 at Pennsylvania State University on February 20, 2013 http://jxb.oxfordjournals.org/ per mg protein and an increase in protein content per g FW. The seed SDH activity per mg protein did not change much by the fourth week AB but, due to a significant decrease in protein content, seed SDH activity per g FW dropped to half its activity in the third week AB. Another increase in seed SDH activity per g FW in the fifth week AB was not accompanied by an increase in seed-extractable protein but was related to elevated seed SDH activity per mg protein. Since the cortex and whole fruit protein content and SDH activities per mg protein changed only slightly over the 4 weeks, cortex and whole fruit SDH activities per g FW were fairly constant and considerably lower than seed SDH activity (Figs 1, 2A, B) .
Total SDH activity per fruit fluctuated (Fig. 3A) . After a 4-fold increase from the second to the third week AB and a small decline from the third to the fourth week AB, SDH activity nearly doubled from the fourth to the fifth week AB, reaching values nearly 8-fold greater than in the second week AB. This pattern was similar to the cortex SDH activity per fruit, and may be explained by the high contribution of cortex biomass to whole fruit biomass (Figs 4, 5) . Seed fresh weight accumulation and extractable protein content per fruit increased gradually during the second to fifth weeks AB ( Figs 4A, B, 5A, B) . Cortex fresh weight accumulation and extractable protein content gradually increased from the second to the fourth weeks AB and increased nearly 5-fold during the fifth week AB. In contrast to the relatively small contribution of seeds to whole fruit biomass and protein content (Figs 4C, 5C), seed SDH activity contributed significantly to total SDH activity per fruit (Fig. 3A, B) , reaching its highest proportion (30%) of total SDH activity per fruit at the fourth and fifth weeks AB.
Western blot and 2D gel analyses
The presence of SDH during early development of apple seed and cortex tissues was confirmed by immunoblotting (Fig. 6) . A 37 kDa band representing SDH protein was clearly visible from both tissues every week. Furthermore, after 2D electrophoresis followed by immunoblotting detection of SDH protein, six SDH isomers with different pI values were detected in whole fruit tissue ( Table 2) . Four of the SDH isomers were found in seeds (Fig. 7) and four in cortex tissue (Fig. 8) . Two of the SDH isomers, with pIs of 5.5 and 6.3, were shared by the seed and cortex tissues, while the other isomers were tissue specific.
RT-PCR analyses
RT-PCR analyses (Fig. 10) were performed using total RNA extracted from seed and cortex tissues of 'Mutsu' collected during weeks 2-5 AB. Of the nine SDH genes present in the apple genome, five were expressed in fruit during this period (Table 3) . Two SDH genes, SDH1 and SDH3, were expressed in both seed and cortex tissues. SDH2 expression was limited to the cortex, while SDH6 and SDH9 were expressed in seed tissue only.
The gene specificity of the primers generating amplicons was confirmed by PCR analyses on SDH cDNAs and genomic DNA previously obtained from 'Mutsu' (Nosarzewski et al., 2004) (Fig. 11) .
The SDH1-specific primers recognized SDH1 cDNA only from nine SDH templates by amplifying a 281 bp amplicon (40 PCR cycles at 50°C annealing temperature) (Fig. 11A) .
The SDH2-specific primers recognized SDH2 cDNA from nine SDH templates by amplifying a 650 bp amplicon (40 PCR cycles at 55°C annealing temperature) (Fig. 11B) .
The SDH3-specific primers recognized SDH3 and SDH5 templates (40 PCR cycles at 55°C annealing temperature) (Fig. 11C) . Forty PCR cycles at 55°C were performed on the SDH5 and SDH3 templates using SDH5-specific primers (Fig. 11D) . SDH5-specific primers were able to recognize SDH5 template but not SDH3, and amplified the expected 611 bp fragment. Since SDH5-specific primers were not capable of recognizing SDH3 template, and since SDH5 expression was not evident in seed or cortex tissue, SDH3-specific primers were a valid gene-specific primer combination under the conditions of the study.
The SDH6-specific primers should have recognized SDH6 cDNA during 40 PCR cycles (at a 65°C annealing temperature) and a 283 bp fragment should have been amplified, but SDH6 templates obtained from genomic DNA should have amplified a 367 bp fragment. The difference in amplicon sizes is a consequence of the presence of a second intron in the SDH6 gene. SDH6 is the only apple SDH gene which possesses a second intron. The SDH6-specific primers recognized SDH6 and SDH5 templates (40 PCR cycles at a 55°C annealing temperature) amplifying the expected 367 bp and a 283 bp fragment of close to expected size (Fig. 11E) . The SDH6-specific primers in the presence of SDH2 and SDH7 templates also amplified an ;367 bp amplicon, a greater http://jxb.oxfordjournals.org/ than expected size. Thus, the SDH6-specific primers were a valid gene-specific primer combination under the following experimental conditions: (i) SDH5 expression was not found in seed, and SDH5-specific primers were not capable of recognizing SDH6 template, but the expected 611 bp fragment was amplified only in the presence of SDH5 template (Fig. 11D) ; (ii) SDH7 expression was not found in the seed, and SDH7 primers failed to amplify a band in the presence of SDH6 template despite being capable of a strong amplification when SDH7 template was present (Fig. 11H) ; and (iii) SDH2 expression was not found in seed, and SDH2-specific primers did not recognize SDH6 template despite being capable of a strong amplification when SDH2 template was present (Fig. 11B) .
The SDH9-specific primers recognized SDH9 and SDH5 templates (40 PCR cycles at a 60°C annealing temperature) by amplification of the expected 235 bp fragment (Fig. 11F) . Forty PCR cycles at 55°C were performed on SDH5 and SDH9 templates using SDH5-specific primers (Fig. 11D) . SDH5-specific primers recognized SDH5 template only, not SDH9 template. Since SDH5-specific primers were not capable of recognizing SDH9 template, and since SDH5 expression was not found in seed or cortex tissue, the SDH9-specific primer pair is a valid gene-specific primer under the study's conditions. The other four primers, designed to recognize SDH4, SDH5, SDH7, and SDH8 templates, were capable of recognizing the targeted SDH templates (Fig. 11G, D , H, and I, respectively). Since SDH4, SDH5, SDH7, and SDH8 were not expressed in the apple fruit tissues, any possible interactions between those primers and SDH templates other than the targeted ones are inconsequential in this study. The SDH4-specific primers recognized the SDH4 template during 40 PCR cycles (at a 60°C annealing temperature), and the expected 375 bp fragment was amplified (Fig. 11H) . The SDH5-specific primers recognized SDH5 template by amplifying a 611 bp amplicon during 40 PCR cycles (at a 55°C annealing temperature) (Fig. 11D) . The SDH7-specific primer recognized SDH7 template during 40 PCR cycles (at a 65°C annealing temperature), and the expected 283 bp fragment was amplified (Fig. 11H) . The SDH8-specific primer recognized SDH8 template during 40 PCR cycles (at a 65°C annealing temperature), and the expected 323 bp fragment was amplified (Fig. 11I ).
Discussion
SDH expression in both the seed and cortex of apple fruit during early fruit development was revealed in this study.
The increasing FW of apple fruit during weeks 2-5 of fruit development was correlated with increasing SDH activity per whole fruit ( Figs 3A, 4A, B) . The small decline in SDH activity per fruit around the fourth week AB was a result of a slight decrease in extractable protein content per fruit (Fig. 5A-C ) and was coincident with slower FW accumulation (Fig. 4A-C) . SDH activity in the whole fruit represented a composite of SDH activities extracted from both seed and cortex tissues. Despite the small amount of seed-extractable protein (Fig. 5A-C ) and seed FW (Fig. 4A-C) relative to total fruit protein and biomass, seed SDH activity contributed up to 30% to total SDH activity per fruit, peaking during the fourth and fifth weeks AB (Fig. 3A, B) . SDH expression in apple fruit 1869
Northern blot (Fig. 9 ) results indicated the presence of greater amounts of SDH transcript in seed than in cortex per lg total RNA, which supports the finding that seed SDH activity per mg protein was several fold greater than in the cortex. This high level of SDH transcript in seed might be due to more SDH genes being expressed, four versus three, respectively, in seed than in cortex (Fig. 10 ). This could also be related to a low transcriptional rate and/or low stability of SDH transcript in cortex.
High SDH activity in seed versus cortex could be related to catalytic differences among SDH isomers since two, SDH6 and SDH9, were seed specific (Table 2) . Posttranslational modification of seed SDH protein may also be a factor influencing SDH activities. Western analysis detected SDH protein in seed to a greater extent, i.e. greater signal strength, than in the cortex, possibly signifying that the SDH transcripts in seeds produced more or longer lasting SDH protein. Since the northern analysis (Fig. 9 ) and RT-PCR data (Fig. 10) did not distinguish between weekly samples but were a composite representing all 4 weeks together, it is not known if the high level of seed SDH activity per mg protein versus that in the cortex was associated with the high expression level of specific SDH genes at any particular time.
The patterns of cortex SDH and whole fruit SDH activity per mg protein (Fig. 1 ) are similar, with slightly higher values for whole fruit. This similarity can be explained by the overwhelmingly high cortex biomass contribution to the whole fruit biomass, around 90%, during weeks 2-5 AB (Fig. 4A-C) . The overall fluctuations of cortex and whole fruit SDH activity per mg protein (Fig. 1) were small. These variations of whole fruit SDH activity per mg protein agree with the previous finding (Nosarzewski et al., 2004) where enzyme-linked immunosorbent assay (ELISA) analysis indicated that SDH protein levels per mg protein were constant. Although the pattern of whole fruit SDH activity per mg protein agrees with the previously reported whole fruit SDH activity pattern (Nosarzewski et al., 2004) , the previous data on SDH activity were lower than the present data due to the use of DTT in the extraction buffer in the previous work. DTT has an inhibitory effect on SDH activity (El-Kabbani et al., 2004) . The overall low and constant level of cortex SDH activity per mg protein (Fig. 1) during the first weeks of development is curious given the relatively high need for carbohydrate during this period. Perhaps this low level of cortex SDH activity per g FW (Fig. 2B) is sufficient for supporting cortex cell division and early cell expansion during these first weeks of fruit development since, even without considering seed SDH activity, total cortex SDH activity per fruit increased. It is also possible that at this Fig. 10 . RT-PCR analysis of RNA transcripts extracted from seed and cortex tissue of 'Mutsu' apple fruit between 2 and 5 weeks after bloom during early fruit development. The RT-PCR products were obtained at 40 reaction cycles using gene-specific primers for SDH cDNAs. b-Tubulin transcripts were amplified at 25 and 40 reaction cycles to show similar transcript abundance in seed and cortex RT-PCRs used for SDH cDNA detection. Table 3 . SDH genes expressed in 'Mutsu' apple fruit between 2 and 5 weeks after bloom
Gene
Tissue where gene was expressed
time the other sorbitol-metabolizing enzyme, sorbitol oxidase, which has not been studied in this early period for apple, assumes an important role. Sucrose or other sugars may also be important for sustaining fruit growth in this period. Acid invertase utilizes sucrose as a substrate, and it was active during all developmental stages in apple fruit starting from 10 d AB (Zhang et al., 2001) . In peach fruit, another Rosaceae species with sorbitol as the main translocated carbohydrate, acid invertase was found around the third week AB, reached a peak in the fourth week, rapidly dropped to negligible levels, and was again detected in the ninth week AB (Lo Bianco and Reiger, 1999) . Different SDH transcripts found by RT-PCR (Fig. 10 ) may be associated with specific SDH isomers detected by 2D gel analysis. SDH1 and SDH3 were expressed in both seed and cortex tissues. Similarly, two of the SDH isomers, one with a pI value of 5.5 and another with a pI value of 6.3, were also found by 2D gel analysis in both seed and cortex tissues (Table 1) . It is possible that those two proteins could be a product of the SDH1 and SDH3 genes, as the predicted pI value for SDH1 is 6.3 according to the Protein Modification Screening Tool. Since the SDH3 sequence is partially known, it can only be speculated that, if the isomer with a pI of 6.3 is in fact a product of SDH1, then the isomer with a 5.5 pI value would be a product of SDH3.
The other two SDH isomers found in the cortex have higher pI values, 7.3 and 8.3, than the two remaining isomers found in the seed with pIs of 4.2 and 4.8. Possibly, the cortex-specific isomer with the pI of 7.3 could be a product of SDH2 since the predicted pI value for SDH2 is 7.0. The two seed-specific SDH isomers with pI values of 4.2 and 4.8 could be products of SDH6 and SDH9. However, the correspondence between the individual isomers and individual genes is undetermined at this time. In the cortex, more SDH isomers than expressed SDH genes were found, four versus three, respectively. This may be the result of post-translational modifications altering the predicted pIs, an artefact of 2D gel analysis, or an indication that not all SDH genes have yet been discovered.
The northern analysis, RT-PCR analyses, and 2D gel results of this study contradict the findings of Park et al. (2002) , where none of the four SDH genes (SDH1, SDH2, SDH3, or SDH4) was expressed in apple fruit tissue during early development from 4 until 8 weeks AB. Expression of these four SDH genes was observed during the later period of fruit development, from ;12 to ;20 weeks AB. Additionally, they found that SDH2, SDH3, and SDH4 transcripts were limited to young leaves, while the SDH1 transcript was found in both young and old leaves.
In conclusion, the present work is the first to show that SDH was expressed in a tissue-specific manner and that SDH was active in both seed and cortex tissues during early fruit development. Five of nine SDH genes were expressed in apple fruit, two in seed, one in cortex, and Fig. 11 . Determination of the specificity of RT-PCR primers. A 5 pg aliquot of each of nine different SDH templates for every 40 PCR cycles was used in the presence of the following: (A) SDH1-specific primers, (B) SDH2-specific primers, (C) SDH3-specific primers, (D) SDH5-specific primers, (E) SDH6-specific primers, (F) SDH9-specific primers, (G) SDH4-specific primers, (H) SDH7-specific primers, and (I) SDH8-specific primers. See Table 1 for annealing temperatures used for each reaction. A DNA plus ladder was used as molecular size marker.
SDH expression in apple fruit 1871 two in both tissues during this period. The significantly higher level of seed SDH activity, combined with greater seed SDH transcript levels and more SDH genes being expressed, in seeds than in cortex tissue of apple suggest that SDH plays an important role in seed development. Because sorbitol is the primary photoassimilate in apple, it is probable that SDH is a critical enzyme responsible for carbohydrate metabolism during early apple seed and cortex development.

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