Photoperiodic control of growth, development and phytohormone balance in Solanum tuberosum

Some lines of Solanum tuberosum ssp. andigena are strictly photoperiodic, forming tubers only in short days and flowers only in long days. We used this advantageous phenomenon to study phytohormone involvement in the development of the plants, mainly that of tuber formation. Plants grown for 2 months under short days (SD) of 14 h darkness, night break (1 h white light in the middle of the 14‐h dark period) and continuous light (LD) were compared. Short day‐grown plants formed tubers, while plants in LD flowered. Night break prevented tuber formation, but caused flowering, although it was weaker than in LD. Plants grown under night break displayed many growth characteristics intermediate between SD and LD. Under LD and night break regimes ABA levels in all organs were about one‐fourth of those under SD. An opposite trend was found for gibberellin content: it was very low in SD‐grown plants and 4‐10 times higher under both other conditions with the exception of roots and stolons in night break. Thus, the ratio of ABA/GA, known to be important for tuber formation, was high in SD and low in night break and LD. The level of free indoleacetic acid (IAA) was high in LD‐ and night break‐grown plants and it was much lower in SD‐grown plants, including tubers. Cytokinin (CK) levels were also high in LD‐ and night break‐grown plants. In SD, leaves had slightly decreased and stems and roots + stolons had more pronouncedly decreased cytokinin levels. The CK/IAA ratio was thus higher in SD in stems and roots + stolons; it was also high in tubers and no difference was found in leaves. These results indicate that the effect of photoperiod on tuber formation and development may be mediated by photoperiod‐induced changes in hormone levels.     

Somatic hybrid potato plants after electrofusion of diploid Solanum tuberosum and Solanum phureja

Protoplasts from diploid S. tuberosum and diploid S. phureja were electrofused followed by selection of the heterokaryons with a micromanipulator. Visual identification of the heterokaryons was facilitated by fluorescein diacetate staining of the protoplasts from one of the parents, which was grown on herbicide containing medium to induce bleaching of the chlorophyll. In total, 840 heterokaryons showing red (chlorophyll) and yellow-green (fluorescein) fluorescence were selected and cultured at relatively low densities using various feeder systems. Finally, 18 putative hybrid plantlets were obtained and grown to maturity. DNA histograms indicated that the plants were hexaploid, octoploid or mixoploid. With Giemsa C-band pattern analysis of the chromosomes the hybrid character and the combinations of the chromosome sets of all plants investigated could be established.     

Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.)

Sink strength of growing potato tubers is believed to be limited by sucrose metabolism and/or starch synthesis. Sucrose synthase (Susy) is most likely responsible for the entire sucrose cleavage in sink tubers, rather than invertases. To investigate the unique role of sucrose synthase with respect to sucrose metabolism and sink strength in growing potato tubers, transgenic potato plants were created expressing Susy antisense RNA corresponding to the T-type sucrose synthase isoform. Although the constitutive 35S CaMV promotor was used to drive the expression of the antisense RNA the inhibition of Susy activity was tuber-specific, indicating that independent Susy isoforms are responsible for Susy activity in different potato organs. The inhibition of Susy leads to no change in sucrose content, a strong accumulation of reducing sugars and an inhibition of starch accumulation in developing potato tubers. The increase in hexoses is paralleled by a 40-fold increase in invertase activities but no considerable changes in hexokinase activities. The reduction in starch accumulation is not due to an inhibition of the major starch biosynthetic enzymes. The changes in carbohydrate accumulation are accompanied by a decrease in total tuber dry weight and a reduction of soluble tuber proteins. The reduced protein accumulation is mainly due to a decrease in the major storage proteins patatin, the 22 kDa proteins and the proteinase inhibitors. The lowered accumulation of storage proteins is not a consequence of the availability of the free amino acid pool in potato tubers. Altogether these data are in agreement with the assumption that sucrose synthase is the major determinant of potato tuber sink strength. Contradictory to the hypothesis that the sink strength of growing potato tubers is inversely correlated with the tuber number per plant, no increase in tuber number per plant was found in Susy antisense plants.     

Expression of bacterial cellulase genes in transgenic alfalfa (Medicago sativa L.), potato (Solanum tuberosum L.) and tobacco (Nicotiana tabacum L.)

The genes encoding thermostable cellulases E2 and E3 of Thermomonospora fusca were expressed in plants under the control of the constitutive, hybrid Mac promoter. For both E2 and E3, the genes were modified so as to remove the sequence encoding the bacterial leader peptide. Western blot analysis indicated that expression levels of recombinant cellulase in tobacco lines ranged up to about 0.1% (E2) and 0.02% of soluble protein (E3). No phenotypic effect of cellulase expression was noted. Recombinant E2 expressed in either tobacco or alfalfa was active and retained heat stability. These findings are an important first step in the development of crop plants as a production system for cellulases.     

Identification and quantification of catecholamines in potato plants (Solanum tuberosum) by GC-MS

Dopamine, norepinephrine, and normetanephrine were identified by GC-MS in potato (Solanum tuberosum L.) plants, the latter was new for plants. The highest amount of catecholamines was found in leaves. A developmental stage dependent variation in potato leaf catecholamines accumulation was also observed with highest level in third leaves. Catecholamine contents decrease during cold storage of tubers to undetectable levels. Mechanical wounding of leaves led to a small increase in the level of catecholamines investigated.     

Stacking of antimicrobial genes in potato transgenic plants confers increased resistance to bacterial and fungal pathogens

Solanum tuberosum plants were transformed with three genetic constructions expressing the Nicotiana tabacum AP24 osmotine, Phyllomedusa sauvagii dermaseptin and Gallus gallus lysozyme, and with a double-transgene construction expressing the AP24 and lysozyme sequences. Re-transformation of dermaseptin-transformed plants with the AP24/lysozyme construction allowed selection of plants simultaneously expressing the three transgenes. Potato lines expressing individual transgenes or double- and triple-transgene combinations were assayed for resistance to Erwinia carotovora using whole-plant and tuber infection assays. Resistance levels for both infection tests compared consistently for most potato lines and allowed selection of highly resistant phenotypes. Higher resistance levels were found in lines carrying the dermaseptin and lysozyme sequences, indicating that theses proteins are the major contributors to antibacterial activity. Similar results were obtained in tuber infection tests conducted with Streptomyces scabies. Plant lines showing the higher resistance to bacterial infections were challenged with Phytophthora infestans, Rhizoctonia solani and Fusarium solani. Considerable levels of resistance to each of these pathogens were evidenced employing semi-quantitative tests based in detached-leaf inoculation, fungal growth inhibition and in vitro plant inoculation. On the basis of these results, we propose that stacking of these transgenes is a promising approach to achieve resistance to both bacterial and fungal pathogens.     

Changes in jasmonate and gibberellin levels during development of potato plants (Solanum tuberosum)

Among the multiple environmental signals and hormonal factors regulatingpotato plant morphogenesis and controlling tuber induction, jasmonates (JAs)andgibberellins (GAs) are important components of the signalling pathways in theseprocesses. In the present study, with Solanum tuberosum L.cv. Spunta, we followed the endogenous changes of JAs and GAs during thedevelopmental stages of soil-grown potato plants. Foliage at initial growthshowed the highest jasmonic acid (JA) concentration, while in roots the highestcontent was observed in the stage of tuber set. In stolons at the developmentalstage of tuber set an important increase of JA was found; however, in tubersthere was no change in this compound during tuber set and subsequent growth.Methyl jasmonate (Me-JA) in foliage did not show the same pattern as JA; Me-JAdecreased during the developmental stages in which it was monitored, meanwhileJA increased during those stages. The highest total amount of JAs expressed asJA+Me-JA was found at tuber set. A very important peak ofJA in roots was coincident with that observed in stolons at tuber set. Also, aprogressive increase of this compound in roots was shown during the transitionof stolons to tubers. Of the two GAs monitored, gibberellic acid(GA₃) was the most abundant in all the organs. While GA₁and GA₃ were also found in stolons at the time of tuber set, noothermeasurements of GAs were obtained for stolons at previous stages of plantdevelopment. Our results indicate that high levels of JA and GAs are found indifferent tissues, especially during stolon growth and tuber set.     

Biochemical and molecular determinance of resistance and susceptibility in Solanum tuberosum (potato) plants challenged with Phytopthora infestans

Determinance of resistance was studied in four different varieties of Solanum tuberosum using biochemical and molecular parameters. It was clearly evident that due to infection of P. infestans, the total protein, total phenol and lignin were induced in all four verities; however, the induction was found more in resistant varieties compared to susceptible varieties. Induction of proteins was also determined by SDS-PAGE analysis. Deposition of lignin was showed by histological comparison using Phloroglucinol HCl staining. Higher deposition of lignin in resistant varieties could be considered as reliable characters related to disease resistance and could be used as biochemical markers for late blight resistance. The RAPD profile generated using eight different decameric primers showed both, polymorphic as well as monomorphic bands. There were many unique bands found only in resistant varieties. These polymorphic bands could also be served as molecular markers for screening of LB resistance potato varieties.     

Analysis of cytosolic heteroglycans from leaves of transgenic potato (Solanum tuberosum L.) plants that under- or overexpress the Pho 2 phosphorylase isozyme

During starch degradation, chloroplasts export neutral sugars into the cytosol where they appear to enter a complex glycan metabolism. Interactions between glycans and glucosyl transferases residing in the cytosol were studied by analyzing transgenic potato (Solanum tuberosum L.) plants that possess either decreased or elevated levels of the cytosolic (Pho 2) phosphorylase isoform. Water-soluble heteroglycans (SHGs) were isolated from these plants and were characterized. SHG contains, as major constituents, arabinose, rhamnose, galactose and glucose. Non-aqueous fractionation combined with other separation techniques revealed a distinct pool of the SHG that is located in the cytosol. Under in vitro conditions, the cytosolic heteroglycans act as glucosyl acceptor selectively for Pho 2. Acceptor sites were characterized by a specific hydrolytic degradation following the Pho 2-catalyzed glucosyl transfer. The size distribution of the cytosolic SHG increased during the dark period, indicating a distinct metabolic activity related to net starch degradation. Antisense inhibition of Pho 2 resulted in increased glucosyl and rhamnosyl contents of the glycans. Overexpression of Pho 2 decreased the content of both residues. Compared with the wild type, in both types of transgenic plants the size of the cytosolic glycans was increased.     

Cotransformation and differential expression of introduced genes into potato (Solanum tuberosum L.) cv Bintje

Summary The Dutch potato cultivar Bintje has been transformed by Agrobacterium strain LBA1060KG, which contains two plasmids carrying three different DNAs (TL- and TR-DNA on the Agrobacterium rhizogenes plasmid and TKG-DNA on the pBI121 plasmid). Several transformed root clones were obtained after transformation of leaf, stem, and tuber segments, and plants were then regenerated from these root clones. The expression of the various marker genes [rol, opine, -glucuronidase (GUS), and neomycin phosphotransferase (NPTII)] was determined in several root clones and in regenerated plants. The selection of vigorously growing root clones was as efficient as selection for kanamycin resistance. In spite of the location of NPTII and GUS genes on the same T-DNA, 17% of the root clones did not show GUS activity. Nevertheless, Southern blot analysis showed that these root clones contained at least three copies of the GUS gene. Sixty-four per cent of the root clones contained opines. The expression of these genes, however, was negatively correlated with plant regeneration capacity and normal plant development. The differential expression of the marker genes in the transgenic potato tissues is discussed.     

Organ-specific distribution and subcellular localisation of ascorbate peroxidase isoenzymes in potato (Solanum tuberosum L.) plants

Summary. Ascorbate peroxidase (EC 1.11.1.11), a heme-containing homodimeric protein, is a hydrogen peroxide-scavenging enzyme, playing an important role in plants in order to protect them from oxidative stress, thus adverting cellular damage. Several ascorbate peroxidase isoenzymes have been reported but the understanding of their physiological role still depends on a better knowledge of their precise localisation within plant organs. Immunocytochemistry techniques were performed in order to elucidate the peroxisomal and cytosolic ascorbate peroxidase distribution within tissues of leaves and sprouts of potato plants. The peroxisomal isoenzyme was found to have a broad distribution in sprouts, but a differential one in leaves, being restricted to the spongy parenchyma. This differential expression may be associated to the mesophyll asymmetry and the diverse physiological processes that occur in it. The cytosolic isoenzyme was not detected in leaves under the used conditions, probably because it is present in low amounts in these tissues. The results obtained in sprouts were at least curious: cytosolic ascorbate was found to be adjacent to the amyloplasts. Given these results, it is possible to state that apart from their similarity, these two isoenzymes reside in different organelles and seem to take part in different physiological processes as suggested by their organ- and tissue-specific distribution. Correspondence and reprints: Plant Functional Biology Department, Institute for Cell and Molecular Biology, University of Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.     

Effects of plant genotype and growth stage on the structure of bacterial communities associated with potato (Solanum tuberosum L.)

The effects of genotype, plant growth and experimental factors (soil and year) on potato-associated bacterial communities were studied. Cultivars Achirana Inta, Désirée, Merkur and transgenic Désirée line DL12 (containing T4 lysozyme gene) were assessed in two field experiments. Cross-comparisons between both experiments were made using Désirée plants. Culture-dependent and -independent approaches were used to demonstrate effects on total bacterial, actinobacterial and Pseudomonas communities in bulk and rhizosphere soils and endospheres. PCR-denaturing gradient gel electrophoresis fingerprints prepared with group-specific primers were analyzed using multivariate analyses and revealed that bacterial communities in Achirana Inta plants differed most from those of Désirée and Merkur. No significant effects were found between Désirée and DL12 lines. Plant growth stage strongly affected different plant-associated communities in both experiments. To investigate the effect of plant-associated communities on plant health, 800 isolates from rhizospheres and endospheres at the flowering stage were tested for suppression of Ralstonia solanacearum biovar 2 and/or Rhizoctonia solani AG3. A group of isolates closely resembling Lysobacter sp. dominated in young plants. Its prevalence was affected by plant growth stage and experiment rather than by plant genotype. It was concluded that plant growth stage overwhelmed any effect of plant genotype on the bacterial communities associated with potato.     

The lack of enzymic browning in the wild potato species Solanum hjertingii compared with commercial Solanum tuberosum varieties

Enzymic browning of tuber tissue was evaluated quantitatively for 50 genotypes, representing seven accessions, of Solanum hjertingii, a wild potato species from north-east Mexico, together with five commercial varieties of Solanum tuberosum with a known range of enzymic browning. Ninety-four percent of S. hjertingii clones examined showed less browning than the commercial varieties, with 66% exhibiting half the ‘potential browning’ (a measure of the total enzymic browning resulting from disruption of cellular compartmentalisation in that system) of cv. Maris Piper, a low enzymic browning cultivar. Of these clones, 18 showed no visual discoloration when sliced. Results clearly indicate that the lack of enzymic browning in S. hjertingii is a ‘true’ character. The efficiency of one subjective and two objective methods for obtaining different measures of enzymic browning was assessed. Potential browning of a rehydrated freeze-dried powder was adopted as the most efficient technique for screening enzymic browning.     

Differential expression of invertase genes in internal and external phloem tissues of potato (Solanum tuberosum L.)

The cloning of promoter sequences of two invertase genes from potato (Solanum tuberosum L.) is described. Histochemical analysis of series of reporter transgenic lines reveals phloem-specific expression from both promoters, with one expressed preferentially in internal phloem and the other in external phloem of stem vascular bundles.