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Jeanine M. Davis and Wayne H. Loescher

Diurnal fluctuations in soluble carbohydrates and starch were monitored in young (expanding), mature (first fully expanded), and old (nearing senescence) celery (Apium graveolens L.) leaves. In all tissues, mannitol and sucrose were the carbohydrates present in the highest concentrations. In old and young leaflets and their petioles, there was little change in levels of mannitol and sucrose in 26 hours. In mature leaflets, sucrose accumulated in the light and decreased in the dark; mannitol increased slightly in late afternoon. Starch concentration, although quite low, showed definite diurnal fluctuations in mature leaflets, but only small changes in young and old leaflets. Both sucrose and mannitol were present in mature petiole phloem tissues. Mannitol concentrations were high in the adjacent storage parenchyma tissue, but sucrose was almost undetectable. These data support earlier findings that sucrose is produced, translocated, and metabolized throughout the celery plant. Mannitol is also translocated, but also serves as a major storage carbohydrate in leaf tissues, especially petiole parenchyma. Starch serves as a minor short-term storage compound in leaflets.

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Wayne Loescher, Zhulong Chan, and Rebecca Grumet

Soil salinization is an increasing problem worldwide and is often intensified by irrigation. Unfortunately, few new crop cultivars have been developed resistant to saline soils, a consequence, in part, of the complexity of plant responses to salt stress. There are now, however, several non-traditional options to improving salt tolerance as a result of recent progress in better understanding the mechanisms involved. These mechanisms include 1) exclusion of Na+ and Cl from plant tissues; 2) inclusion of these ions in inert compartments or tissues; and/or 3) some means of osmotic adjustment with solutes that are compatible with the metabolic machinery of the cell. Although there are very few horticultural examples, several lines of evidence indicate that reductions in salt sensitivity through exclusion or inclusion can be achieved by single gene modifications of the ion transport system. Similarly, single genes resulting in osmotic adjustment with solutes compatible with the metabolic machinery of the cell have resulted in significant increases in salt tolerance. Recent advances in sequencing, use of quantitative trait loci, and marker-assisted selection promise to provide other options for improving salt tolerance.

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John Everard, Rebecca Grumet, and Wayne Loescher

In celery, photosynthetic carbon partitioning between mannitol and sucrose is highly dependent on developmental (leaf age) and environmental (salt stress) factors. Mannose 6-phosphate reductase (M6PR) mediates a key step in mannitol biosynthesis and may regulate partitioning between sucrose and mannitol. We have constructed a cDNA library and have isolated M6PR-specific clones. Before library construction, poly(A)+ RNA, extracted from newly fully expanded leaves, was translated in vitro. A single polypeptide (35.1 kD), immunoprecipitated with M6PR-specific antisera, accounted for ≈5% of the total 35S incorporated into TCA-precipitated products. Parity between the molecular masses of the immunoprecipitated product and authentic M6PR indicated minimal posttranslational modification. The unidirectional primary library, constructed in UniZap XR vector (Stratagene), consisted of 1.53 million plaque forming-units (pfus) of which <0.4% were nonrecombinant, as estimated by “blue/white”' screening. After a single amplification, ≈0.14% of the 200,000 pfus screened with M6PR-specific antisera were identified as putative M6PR clones. Following two further rounds of screening and in vivo excision of the pBluescript phagemids their identity as full length M6PR clones was confirmed as follows: 1) IPTG-induced expression of M6PR activity in crude extracts; 2) IPTG-induced expression of a polypeptide that specifically interacted with M6PR antisera and with identical mobility (on SDS gels) to authentic M6PR; 3) 100% sequence homology to an internal peptide from a tryptic digest of purified M6PR. Based on these criteria, we conclude that we successfully cloned M6PR. The sequence is similar to several reductases from both plants and animals including an aldose 6-phosphate reductase from apple. Supported by USDA-NRI grant 940-1439.

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Yufei Xu*, Eric Hanson, James Flore, and Wayne Loescher

In Michigan boron (B) deficiencies in sour cherry have resulted in routine use of B sprays to enhance fruit set and increase fruit yield. However, field observations indicate that high B levels are associated with premature softening, making fruit unacceptable for processing. Our fertilization studies show that fruit B levels are higher, but B generally has little or no effect on fruit size, maturity, color, or pull force. However, at some locations, B applications increase the number of soft fruit, especially when harvest is delayed well after the optimum maturity date (as indicated by pull force). B-induced yield increases can be achieved without inducing excessive fruit softening by careful monitoring of fruit maturation and prompt harvest. Leaf and fruit B levels will be presented.

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Guo-qing Song, Aaron E. Walworth, and Wayne H. Loescher

Grafting is a well-established agricultural practice, and it now has implications for the commercialization of transgenic plants. In transgrafted plants, only one part (scion or rootstock) is transgenic with the other part untransformed. However, transgenes may affect both mobile and immobile endogenous metabolites (e.g., RNAs, proteins, and phytohormones) and mobility has implications for transgrafting. In the phloem, long-distance transport of mobile metabolites can play important roles in plant development and signaling. In a transgrafted plant, an immobile transgene product (ITP) is not likely to be translocated across the graft union. In contrast, mobile transgene products (MTP) may be translocated across the graft. Regardless of the mobility of transgene products (TP), interaction of transgenic and nontransgenic parts in transgrafted plants through either the MTP or ITP has been demonstrated to be effective in facilitating changes in nontransgenic portions of the plant. Consequently, and particularly in fruit crops, transgrafting provides the potential for improving products from their nontransgenic parts with the possibility of minimizing the controversy over transgenic crops. This review focuses mainly on the mobility of TP and effects on the whole transgrafted plant.

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Zhifang Gao, Sastry Jayanty, Randolph Beaudry, and Wayne Loescher

In apple (Malus ×domestica Borkh.), where sorbitol is a primary photosynthetic product that is translocated throughout the plant, accumulation of sorbitol in sink cells appears to require an active carrier-mediated membrane transport step. Recent progress in isolation and characterization of genes for sorbitol transporters in sour cherry (Prunus cerasus L.) and mannitol transporters in celery (Apium graveolens L.) suggested that similar transporters may be present in apple tissues. A defect in these transporters could also explain the occurrence of the fruit disorder watercore, characterized by the accumulation of fluids and sorbitol in the apoplasmic free space. Our objectives therefore included isolation and characterization of genes for sorbitol transporters in apple tissues and comparisons of expression of transporter genes, especially in various sink tissues including watercored and non-watercored fruit tissues. We have isolated and characterized two sorbitol transporter genes, MdSOT1 and MdSOT2. Sequence analyses indicated that these are members of the major facilitator transporter superfamily that gives rise to highly hydrophobic integral membrane proteins. Heterologous expression and measurement of sorbitol uptake in yeast indicated that these are specific and with high affinities for sorbitol, with Kms for sorbitol of 1.0 and 7.8 mm for MdSOT1 and MdSOT2, respectively. Sorbitol transporter expression was evident in all sink tissues tested with the exception of watercore-affected fruit tissues. Sorbitol accumulation in apple sink tissues thus involves an apoplasmic active membrane transport step and watercore results from a defect in that process.

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Riccardo Gucci, John Everard, James Flore, and Wayne Loescher

Photosynthetic rates (A) in celery-(Apium graveolens L.) and other polyol-synthesizers are sometimes high for C, species. In celery such rates have been related to a low CO2 compensation point typical of C4 and C3-C4 intermediate spp, although other data show celery photosynthesis as typically C3 Therefore, celery gas exchange was here reanalyzed, and while A was high (CO2 assimilation rates were 21.2 and 27.6 μ mol m-2s-1, average and maximum, photosynthesis was otherwise C,: CO, comp pt of 3.5-5.0 Pa, carboxylation efficiency of 0.99 μmol CO2m-2s-1Pa-1, light comp pt of 8-36 μ mol photon m-1s-1, optimum temp of 22-27°C for Amax. High A may relate to a capacity to synthesize both mannitol and sucrose. 14C pulse-chase studies, with different A obtained by imposing light gradients across opposite leaflets, showed 1-10% increases in mannitoll sucrose labelling. Higher A may reflect carbon partitioning into mannitol, agreeing with a hypothesis that polyol synthesis effectively recycles reductant in the cytosol.

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Wayne H. Loescher, Thaddeus McCamant, and John D. Keller

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Wayne Loescher, Tad Johnson, Randolph Beaudry, and Sastry Jayanty

Sorbitol is the major carbohydrate translocated into apple fruit where it is normally metabolized to fructose. In watercored apple fruit tissues, however, the intercellular spaces become flooded and sorbitol content is consistently higher than in nonwatercored apples, suggesting a defect in sugar alcohol metabolism or transport. Our previous results have identified and characterized two sorbitol transporters, MsSOT1 and MsSOT2, in apple fruit tissues. Sorbitol transporter gene expression has been implicated in development of watercore with MsSOT expression diminished or absent in certain watercored fruit tissues. To explore this further, we have investigated the relationships between watercore, fruit maturation, fruit composition, and MsSOT expression in a number of apple cultivars that differ in watercore susceptibility. We also compared transporter expression between affected (watercored) and healthy parts of the same fruit and between watercored and nonwatercored fruits throughout the maturation and ripening processes. The MsSOT expression was often dramatically reduced in fruit tissues exhibiting watercore. Thus, in susceptible cultivars, maturing (ripening) fruit parenchyma cells lose the ability to transport sorbitol, and this in turn leads to sorbitol accumulation in the apoplastic free space and subsequent flooding of these spaces. These results are consistent with a relationship between watercore and sorbitol transport and also with a genetic susceptibility to the disorder.

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Wayne H. Loescher, Paolo Sabbatini, Guo-Qing Song, Kenneth Sink, and James Flore

Mannitol, a sugar alcohol that appears to serve as an osmoprotectant/compatible solute to cope with salt stress, is synthesized in celery (Apium graveolens L.) via the action of a NADPH dependent mannose-6-phosphate reductase (M6PR). To evaluate the abiotic stress effects of mannitol biosynthesis, we transformed celery with an antisense construct of the celery leaf M6PR gene under control of the CaMV 35S promoter. Unlike wild type (WT) celery, independent antisense M6PR transformants did not accumulate significant amounts of mannitol in any tissue, with or without salt stress. In the absence of NaCl, and despite the lack of any significant accumulation of mannitol that is normally the major photosynthetic product, antisense transformants were mostly phenotypically similar to the WT celery. However, in the presence of NaCl, mature antisense transgenic plants were significantly less salt-tolerant, with reduced growth and photosynthetic rates, and some transformant lines were killed at 200 mM NaCl, a concentration that WT celery can normally withstand. Although mannitol biosynthesis is normally enhanced in salt-treated WT celery, no such increase was observed in the antisense transformants. Like our previous gain of function results showing enhanced salt tolerance in Arabidopsis plants transgenic for a sense M6PR construct, these loss of function results, using an antisense construct in celery, demonstrate a major role for mannitol biosynthesis in developing salt-tolerant plants.