Auxin-induced cell expansion of potato tuber and chicory root: Role of hydroxyproline synthesis

We have reinvestigated the role of protein-bound hydroxyproline (extensin) in auxininduced cell enlargement using discs excised from tubers of Solanum tuberosum L. cv. Pentland Crown and from roots of Cichorium intybus L. cv. Magdeburg. Extensin increases markedly in potato tuber discs treated with water and auxin, and the hydroxyproline is primarily in the cell wall. 2,2'-Dipyridyl totally inhibits both hydroxyproline synthesis and auxin-induced cell expansion in potato with the inhibitions being reversed in parallel by Fe²⁺. Free hydroxyproline also totally prevents induced cell enlargement. Pretreatment with gibberellic acid totally inhibits subsequent auxin-induced cell expansion but does not inhibit hydroxyproline synthesis. Therefore, the level of hydroxyproline does not control auxin-induced cell enlargement in potato tuber discs. Other interpretations are discussed but we conclude that extensin biosynthesis is necessary for auxin-dependent cell expansion as inhibition of the synthesis prevents the induced expansion. Dipyridyl and free hydroxyproline partially inhibit auxin-induced cell enlargement in chicory root discs. Thus a component of the auxin-dependent cell enlargement in chicory is also dependent on extensin synthesis.     

Extensin from suspension-cultured potato cells: a hydroxyproline-rich glycoprotein, devoid of agglutinin activity

Enhanced deposition and cross-linking of hydroxyproline-rich glycoproteins (HRGPs) in the plant cell wall is acknowledged to contribute to the formation of a resistant barrier against pathogen infection. We have isolated, from suspension-cultured potato (Solanum tuberosum L. cv. Desiree) cells, two forms of soluble HRGP, a cross-linked and a monomeric form; the latter can be converted to the cross-linked form by incubation with tomato extensin peroxidase and H₂O₂. The monomeric form was purified by Sephacryl S-200 gel-filtration, reverse-phase high-performance liquid chromatography and Mono-S cation-exchange chromatography into two isoforms (A, a minor form; B, a major form). The properties of the B isoform were further investigated. A quantitative enzyme-linked immuno-sorbent assay of the B isoform, using tomato extensin antiserum, showed a titration curve at a high antibody-dilution range comparable to that of purified tomato extensin monomer (M.D. Brownleader and P.M. Dey, 1993, Planta 191: 457–469). The amino acid and carbohydrate compositions were similar to those of tomato extensin, but did not match well with the other two HRGPs from potato, potato lectin and potato bacterial agglutinin. These observations demonstrate the similarities of the B isoform to extensin. The homogeneity of the B isoform was demonstrated by its ability to be fully cross-linked in vitro, leaving no residual protein, into a high-molecular-weight form by the action of extensin peroxidase. The trifluoroacetic acid-deglycosylated sample migrated as a single protein band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, SDS-PAGE and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry indicated a molecular weight of approximately 67 kDa. Circular-dichroism spectroscopy demonstrated that the molecule possesses an extended polyproline II helix conformation with no evidence of α- helix or β- sheet secondary structure. In conclusion, we refer to this HRGP as potato extensin. As proposed for other extensins, potato extensin is likely to play a role in cell wall architecture and plant disease resistance. Received: 25 November 1996 / Accepted 13 January 1997     

Change in phosphorylase isoenzymes during dedifferentiation of potato tuber cells

The phosphorylase isoenzyme composition of soluble preparations isolated from potato ( Solanum tuberosum L. cv. Spunta) tuber-derived callus has been studied by polyacrylamide gel electrophoresis and affinity electrophoresis. Native electrophoretic profiles indicate that dedifferentiated callus tissue contains a single form of phosphorylase that differs in primer requirement, charge and affinity towards branched α-1,4/1,6-glucans from the major phosphorylase form (phosphorylase II) in potato tuber. This latter molecular form is missing in dedifferentiated callus. However, callus phosphorylase appears to be closely related to tuber phosphorylase I, a minor form found in the original explant tissue.     

Oligoarabinosides of hydroxyproline isolated from potato lectin

The lectin from potato tubers is a glycoprotein containing 50% sugars and rich in hydroxyproline and arabinose moieties. The nature of the protein-sugar linkage has been compared to that of insoluble potato cell wall protein and the arabinose was shown to exist as short oligosaccharides of 3 or 4 residues attached to hydroxyproline. In the lectin there were no large oligosaccharides attached to hydroxyproline. Lectin activity with the same specificity as that of the tuber lectin was shown to be associated with particulate membrane fractions prepared from cultured potato roots.     

Selective vital staining of companion cells of potato tuber and parsnip root with neutral red.

When staining the internal phloem region of a potato tuber with the vital stain neutral red, it was observed that files of elongated cells of narrow diameter were heavily stained and were easily distinguishable from the more isodiametric parenchyma cells, many of which did not stain with neutral red. The elongated cells were identified as companion cells by locating the adjacent sieve-tube members through counterstaining with aniline blue and reviewing under violet light. Of a number of other plants surveyed, only parsnip roots possessed companion cells exhibiting a similar slective staining. In other plants both the companion cells and the surrounding parenchyma cells usually stained. Sieve-tube members never accumulated neutral red. It was concluded that the vacuoles of the companion cells of the potato tuber were stained by the ion trap mechanism because of the color of the accumulated stain, the lack of staining when neutral red was applied in an acidic solution, and the complete destaining after soaking in dilute ammonium hydroxide.     

The effect of genetic transformations for pest resistance on foliar solanidine-based glycoalkaloids of potato (Solatium tuberosuni)

Foliage of potato cv. Desiree was harvested from glasshouse‐cultivated plants of five experimental transgenic lines expressing three different insecticidal proteins (snowdrop lectin, Galanthus nivalis agglutinin (GNA); jackbean lectin, Concanavalin A (Con A), cowpea trypsin inhibitor; (CpTi)), tissue‐cultured control plants and standard control (non‐tissue cultured) plants. The foliage was subdivided into stems, upper, middle and lower leaves and analysed separately by HPLC for the solanidine‐based glycoalkaloids a‐solanine and a‐chaconine. The results demonstrate that one or more stages in the plant transformation process (i.e. insecticidal‐ and marker‐gene insertions, gene expression and tissue culture) resulted in a lower level of leaf glycoalkaloids than that found in either the tissue‐cultured controls or standard controls, based on the selected potato lines transformed for insecticidal protein expression. However, the distribution of glycoalkaloids throughout the plant foliage was unaffected by genetic transformation and tissue culture, with the highest glycoalkaloid levels being observed in the top third of the plant. The importance of investigating unexpected effects of genetic engineering on plant secondary metabolism is discussed from an ecological viewpoint.     

Rhizobacteria that produce auxins and contain 1‐amino‐cyclopropane‐1‐carboxylic acid deaminase decrease amino acid concentrations in the rhizosphere and improve growth and yield of well‐watered and water‐limited potato (Solanum tuberosum)

Plant‐growth‐promoting rhizobacteria (PGPR) utilise amino acids exuded from plant root systems, but hitherto there have been no direct measurements of rhizosphere concentrations of the amino acid 1‐amino‐cyclopropane‐1‐carboxylic acid (ACC) following inoculation with PGPR containing the enzyme ACC deaminase. When introduced to the rhizosphere of two potato (Solanum tuberosum) cultivars (cv. Swift and cv. Nevsky), various ACC deaminase containing rhizobacteria (Achromobacter xylosoxidans Cm4, Pseudomonas oryzihabitans Ep4 and Variovorax paradoxus 5C‐2) not only decreased rhizosphere ACC concentrations but also decreased concentrations of several proteinogenic amino acids (glutamic acid, histidine, isoleucine, leucine, phenylalanine, serine, threonine, tryptophan, tyrosine, valine). These effects were not always correlated with the ability of the bacteria to metabolise these compounds in vitro, suggesting bacterial mediation of root amino acid exudation. All rhizobacteria showed similar root colonisation following inoculation of sand cultures, thus species differences in amino acid utilisation profiles apparently did not confer any selective advantage in the potato rhizosphere. Rhizobacterial inoculation increased root biomass (by up to 50%) and tuber yield (by up to 40%) in pot trials, and tuber yield (by up to 27%) in field experiments, especially when plants were grown under water‐limited conditions. Nevertheless, inoculated and control plants showed similar leaf water relations, indicating that alternative mechanisms (regulation of phytohormone balance) were responsible for growth promotion. Rhizobacteria generally increased tuber number more than individual tuber weight, suggesting that accelerated vegetative development was responsible for increased yield.     

Lipolytic and peroxidative enzyme expressions in cultured potato tuber cells

Potato cells (cv. Norchip) were cultured from tuber parenchymal tissue and subcultured to dissociate and habituate the despecialized cells. After several subculturings on a minimal nutrient media, this line of cells demonstrated repeatable physical growth profiles for dry weight (DW), fresh weight (FW) and protein. Two enzymes of plant lipid metabolism were investigated, lipolytic acyl hydrolase (LAH) and lipoxygenase (LOX), which respectively liberate and peroxidize fatty acids from lipid in cellular membranes. LAH, measured as p-nitrophenyl palmitate hydrolase, was present in this line of cells in easily detectable amounts (317 units g^-1 DW) albeit much lower than that found in mother tuber (9878 units g^-1 DW). The presence of LAH in this line is significant because LAH isozymes are often described as storage proteins, yet activity per gram fresh weight in these unorganized cells is reasonably constant until culture growth exits the linear phase. However, LOX, the most active free fatty acid metabolizing enzyme in potato tubers (89,800 units g^-1 DW), was not detectable in this line of callus or suspension cultured cells. The absence of LOX activity in this line of cells was verified by a number of assay approaches and was confirmed by activity staining of extracted enzymes separated in polyacrylamide gels. The absence of LOX in these cultured cells is especially important in determining the functions of this lipid peroxidation system and how it may be genetically regulated.Mention of company or trade name does not imply endorsement by the United States Department of Agriculture over others not named.A laboratory cooperatively operated by the Midwest Area, Agricultural Research Service, U.S. Department of Agriculture, The Minnesota Agricultural Experiment Station, the North Dakota Agrcultural Experiment Station, and the Red River Valley Potato Grower's Association.     

Tuber induction and initiation during production and early field growth of transplants from in vitro-derived potato plants

In transplants from in vitro‐derived plantlets from very early potato (Solanum tuberosum) cultivars, a lower degree of tuber induction at the time of field planting is thought to increase tuber production. Leaf‐bud cuttings were used to assess the progress to tuber induction in in vitro‐derived potato plantlets during the transplant production phase and after subsequent transplanting into the field. Induction and initiation of tubers on the same plants were assessed to study the effects of the duration of transplant production and conditions during transplant production for cv. Gloria (very early) and cv. Bintje (mid‐early). In vitro‐produced plantlets were not induced by the time of planting but rapidly progressed to the induced state thereafter. The progress in induction with time and the change in percentage of plants showing tubers fitted typical sigmoid curves. Plantlets achieved 50% induction ca 15 days after planting into in vivo conditions, and 50% tuber initiation usually occurred 10 days later. Shorter transplant production periods reduced the degree of induction of the transplants at field planting. Transplant production for more than 2 weeks was required to allow conditions during that period to affect induction or initiation. Long‐term non‐inducing conditions delayed the progress to tuber induction in cv. Gloria and delayed tuber initiation in both cultivars. Cv. Gloria showed no faster progress to induction than cv. Bintje but initiated tubers earlier. The results suggest that the relation between progress to induction and tuber initiation is cultivar dependent and that leaf‐bud cuttings can be used successfully in very young in vitro‐derived plants for assessing the progress to tuber induction.     

Production of dextran in transgenic potato plants

The production of dextran in potato tubers and its effect on starch biosynthesis were investigated. The mature dextransucrase (DsrS) gene from Leuconostoc mesenteroides was fused to the chloroplastic ferredoxin signal peptide (FD) enabling amyloplast entry, which was driven by the highly tuber-expressed patatin promoter. After transformation of two potato genotypes (cv. Kardal and the amylose-free (amf) mutant), dextrans were detected by enzyme-linked immunosorbent assay (ELISA) in tuber juices of Kardal and amf transformants. The dextran concentration appeared two times higher in the Kardal (about 1.7 mg/g FW) than in the amf transformants. No dextran was detected by ELISA inside the starch granule. Interestingly, starch granule morphology was affected, which might be explained by the accumulation of dextran in tuber juices. In spite of that, no significant changes of the physicochemical properties of the starches were detected. Furthermore, we have observed no clear changes in chain length distributions, despite the known high acceptor efficiency of DSRS.     

Growth pattern, tuber formation and hormonal balance in in vitro potato plants carrying ipt gene

Nodal cuttings of in vitro grown potato plants (Solanum tuberosum, cv. Miranda) were transformed by a vector plasmid carrying ipt gene of Agrobacterium tumefaciens. From the initial teratoma stage 5 clones of transgenic plants (1, 2, 11, 13 and 15) were obtained, which displayed in varying degree shortening of the internodes, decrease of the leaf size, decrease of apical dominance and poor rooting. In addition, two of the clones (11 and 13) showed increased stolon and tuber formation. In all these clones the endogenous level of free cytokinins (CKs) was increased: from 40% in clone 11 to almost 300% in clone 1. Also free indole-3-acetic acid (IAA) level was increased, but to a lower degree; the maximal increase was 160% (clone 13). Applied kinetin or IAA (1 mg·l^-1) strongly suppressed root and tuber formation in clones 11 and 13, although they did not affect or even stimulated these processes in control plants. For control plants the minimal medium sucrose concentration necessary for tuber initiation was 6% whereas in clone 11 plants 2% was sufficient. Different distribution of endogenous CKs and IAA was observed in clone 11 and control plants. The highest CK content was found in transgenic plants in stems and in controls in leaves. In clone 11 plants abscisic acid (ABA) level was significantly increased in comparison to the control throughout the cultivation period. Ethylene formation was strongly increased the first week after the subcultivation and later on the difference between transgenic and control plants rapidly diminished. Reactions of clone 11 plants to red (RL) and blue light (BL) were similar to reactions of control plants. In RL clone 11 plants were tall and thin with stunted leaves; in BL they had a teratoma-like appearance and formed a very high number of tubers. The role of hormones in these changes in growth and tuber formation is discussed.     

Induction mechanism of 3-hydroxy-3-methylglutaryl-CoA reductase in potato tuber and sweet potato root tissues

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC1.1.1.34), the key enzyme in isoprenoid biosynthesis, was purified from microsomes of potato tuber tissue, and a polyclonal antibody and two monoclonal antibodies against the purified enzyme were prepared. HMGR protein content was measured by immunotitration and radioimmunoassay using these antibodies. HMGR activity was very low in the fresh tissues of both potato tuber and sweet potato root. The activity in potato tuber was increased by cutting and further by additional fungal infection of the cut tissues. In sweet potato root tissue, the activity was scarcely increased after cutting alone, but was markedly increased by additional fungal infection or chemical treatment. The HMGR protein contents in both fresh potato tuber and sweet potato root tissues were also very low, and increased markedly in response to cutting and fungal infection. From these results, we proposed a hypothesis on the induction mechanism of HMGR after cutting and fungal infection in potato tuber and sweet potato root tissues.     

The restricted distribution of potato leafroll luteovirus antigen in potato plants with transgenic resistance resembles that in clones with one type of host genemediated resistance

The distribution of virus-infected cells was examined, by fluorescence microscopy, within plants of a range of potato clones infected with potato leafroll luteovirus (PLRV). This range included nine PLRV-resistant clones, of which four were transgenic lines carrying the PLRV coat protein gene and five were conventionally bred. Plants of these clones were resistant to PLRV multiplication and accumulated less virus antigen in leaf tissue than did susceptible clones. Indirect fluorescent antibody staining of thin sections from carbodiimide-fixed petiole tissue revealed that in plants of PLRV-susceptible clones, virus-infected cells were abundant within both external (abaxial) and internal (adaxial) phloem bundles. In plants of the PLRV-resistant conventionally bred clones and in resistant transgenic lines of cv. Pentland Squire, virus-infected cells were much fewer in number and largely restricted to internal phloem bundles. In resistant transgenic lines of cv. Désirée, this restricted distribution of PLRV antigen was only detected in petioles of young leaves. The results suggest that the transgenic and a host-mediated type of resistance that restricts virtis multiplication have underlying similarities.     

Cysteine proteinase forms in sprouting potato tuber

Transformation of plants with exogenous proteinase inhibitor genes represents an attractive strategy for the biological control of insect pests. However, such a strategy necessitates a thorough characterization of endogenous proteinases. which represent potential target enzymes for the exogenous inhibitors produced. In the present study. changes in general endoproteolytic activity were monitored during sprouting of potato ( Solanum tuberosum L. cv. Kennebec) tuber. Quantitative data obtained using standard procedures showed that an increase in cysteine proteinase (EC 3.4.22) activity occurs during sprouting. This increased activity results from the gradual appearance of new cysteine proteinase forms, as demonstrated by the use of class-specific proteinase activity gels. While only one cysteine proteinase form was present during early sprouting, at least six new active forms of the same class were shown to appear gradually after the mature tuber was sown, suggesting the involvement of a complex cysteine proteolytic system in the last stages of tuber protein breakdown. Interestingly, oryzacystatins I and II. two cysteine proleinase inhibitors potentially useful for insect control, had no effect on any tuber proteinase delected. Similar results were obtained with leaf, stem and stolon proteinases. This apparent absence of direct interference supports the potential of oryzacystatin genes for production of insect-tolerant transgenie potato plants.     

Aspects of resistance to sweet potato virus disease in sweet potato

In field trials during the first and the second rainy season of 1996 in Uganda, whiteflies were similarly abundant and aphids were absent on three clones of sweet potato (NIS-93–63, cv. Tanzania and cv. New Kawogo) although the three clones differed considerably in their resistance to sweet potato virus disease (SPVD), a complex disease resulting from infection by both the aphid-borne sweet potato feathery mottle virus (SPFMV) and the whitefly-borne sweet potato chlorotic stunt virus (SPCSV). This suggests that vector resistance does not determine the relative SPVD resistance of these genotypes. SPFMV alone had only a low virus titre in sweet potato cvs Tanzania and New Kawogo, became increasingly difficult to detect in plants of these cultivars and was seldom acquired by aphids. However, this resistance to SPFMV was not apparent in plants which were also infected with SPCSV. Plants then had a high SPFMV titre, appeared unable to eliminate SPFMV and provided good sources for aphids to acquire it.     

Involvement of 3-hydroxy-3-methylglutaryl coenzyme a reductase in the regulation of sesquiterpenoid phytoalexin synthesis in potato

The importance of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in the regulation of sesquiterpenoid phytoalexin accumulation in potato (Solanum tuberosum L. cv Kennebec) was examined. Wounding of potato tubers produced a large temporary increase in HMG-CoA reductase activity of the microsomal and organelle fractions. Treatment of wounded tuber tissue with the sesquiterpenoid phytoalexin elicitor arachidonic acid further increased and prolonged the HMG-CoA reductase activity in the microsomal but not the organelle fraction. Incubation of elicitor-treated tuber tissue in white light reduced organelle and microsomal HMG-CoA reductase activity to 50% and 10%, respectively, of the activity of tissues held in darkness. Constant light also reduced overall phytoalexin accumulation 58% by greatly reducing levels of lubimin. Rishitin accumulation was not significantly altered by light. Application of nanomolar amounts of mevinolin, a highly specific inhibitor of HMG-CoA reductase, to elicitor-treated tuber tissue produced a large decline in lubimin accumulation and did not markedly alter rishitin accumulation. These results indicate that HMG-CoA reductase has a role in the complex regulation of sesquiterpenoid phytoalexin accumulation in potato.     

Influence of changes in daylength and carbon dioxide on the growth of potato

Potatoes (Solanum tuberosum L.) are highly productive in mid- to high-latitude areas where photoperiods change significantly throughout the growing season. To study the effects of changes in photoperiod on growth and tuber development of potato cv. Denali, plants were grown for 112 d with 400 micromol m-2 s-1 photosynthetic photon flux (PPF) under a 12-h photoperiod (short days, SD), a 24-h photoperiod (long days, LD), and combinations where plants were moved between the two photoperiods 28, 56, or 84 d after planting. Plants given LD throughout growth received the greatest total daily PPF and produced the greatest tuber yields. At similar levels of total PPF, plants given SD followed by LD yielded greater tuber dry mass (DM) than plants given LD followed by SD. Stem DM per plant, leaf DM, and total plant DM all increased with an increasing proportion of LD and increasing daily PPF, regardless of the daylength sequence. When studies were repeated, but at an enriched (1000 micromol mol-1) CO2 concentration, overall growth trends were similar, with high CO2 resulting in greater stem length, stem DM, leaf DM, and total plant DM; but high CO2 did not increase tuber DM.     

A transgenic study on affecting potato tuber yield by expressing the rice sucrose transporter genes OsSUT5Z and OsSUT2M

In many plants, sucrose transporters are essential for both sucrose exports from sources and imports into sinks, indicating a function in assimilate partitioning. To investigate whether sucrose transporters can improve the yield of starch plant, potato plants (Solanum tuberosum L. cv. Désirée) were transformed with cDNAs of the rice sucrose transporter genes OsSUT5Z and OsSUT2M under the control of a tuber-specific, class-I patatin promoter. Compared to the controls, the average fructose content of OsSUT5Z transgenic tubers significantly increased. However, the content of the sugars and starch in the OsSUT2M transgenic potato tubers showed no obvious difference. Correspondingly, the average tuber yield, average number of tubers per plant and average weight of single tuber showed no significant difference in OsSUT2M transgenic tubers with controls. In the OsSUT5Z transgenic lines, the average tuber yield per plant was 1.9-fold higher than the controls, and the average number of tubers per plant increased by more than 10 tubers on average, whereas the average weight of a single tuber did not increase significantly. These results suggested that the average number of tubers per plant showed more contribution than the average weight of a single tuber to the tuber yield per plant.