Antisense inhibition of cytosolic phosphorylase in potato plants (Solanum tuberosum L.) affects tuber sprouting and flower formation with only little impact on carbohydrate metabolism

To determine the function of cytosolic phosphorylase (Pho2; EC 2.4.1.1), transgenic potato plants were created in which the expression of the enzyme was inhibited by introducing a chimeric gene containing part of the coding region for cytosolic phosphorylase linked in antisense orientation to the 35S CaMV promotor. As revealed by Northern blot analysis and native polyacrylamide gel electrophoresis, the expression of cytosolic phosphorylase was strongly inhibited in both leaves and tubers of the transgenic plants. The transgenic plants propagated from stem cuttings were morphologically indiscernible from the wild-type. However, sprouting of the transgenic potato tubers was significantly altered: compared with the wild-type, transgenic tubers produced 2.4 to 8.1 times more sprouts. When cultivated in the greenhouse, transgenic seed tubers produced two to three times more shoots than the wild-type. Inflorescences appeared earlier in the resulting plants. Many of the transgenic plants flowered two or three times successively. Transgenic plants derived from seed tubers formed 1.6 to 2.4 times as many tubers per plant as untransformed controls. The size and dry matter content of the individual tubers was not noticeably altered. Tuber yield was significantly higher in the transgenic plants. As revealed by carbohydrate determination of freshly harvested and stored tubers, starch and sucrose pools were not noticeably affected by the antisense inhibition of cytosolic phosphorylase; however, glucose and fructose levels were markedly reduced after prolonged storage. These results favour the view that cytosolic phosphorylase does not participate in starch degradation. The possible links between the reduced levels of cytosolic phosphorylase and the observed changes with respect to sprouting and flowering are discussed.     

Altered plastidic ATP/ADP-transporter activity influences potato ( Solanum tuberosum L.) tuber morphology, yield and composition of tuber starch

The metabolic function of the plastidic ATP/ADP transporter (AATP) in heterotrophic plastids was examined in transgenic potato plants that exhibited increased or decreased amounts of the protein. Altered mRNA levels correlated with activities of the plastidic ATP/ADP transporter. Potato tubers with decreased plastidic ATP/ADP transporter activities exhibited reduced starch contents whereas sense lines accumulated increased amounts of tuber starch. Starch from wild-type tubers had an amylose content of 18.8%, starch from antisense plants contained 11.5–18.0% amylose, whereas starch from sense plants had levels of 22.7–27.0%. The differences in physiological parameters were accompanied with altered tuber morphology. These changes are discussed with respect to the stromal ATP supply during starch biosynthesis.     

RNAi-mediated silencing of endogenous <Emphasis Type="Italic">Vlnv</Emphasis> gene confers stable reduction of cold-induced sweetening in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L. cv. Désirée)

Potato tubers must be cold-stored to extend their shelf life and maintain an uninterrupted supply chain for food processors. However, a side-effect of low-temperature storage is manifested in terms of cold-induced sweetening (CIS) of potato tubers, which reduces the processing quality and the commercial value of the end-products. RNA interference (RNAi) technology, whereby transgene-derived small interfering RNAs can trigger the homology-based knockdown of cognate host genes and can initiate gene silencing, has been successfully applied in crop improvement through targeted gene knockout in host plants. In the current study, transgenic potato plants (Solanum tuberosum cv. Désirée) were generated, expressing a 300 bp hairpin loop nucleotide sequence targeting the potato vacuolar invertase gene (VInv), under the constitutive Cauliflower mosaic virus 35S promoter. Tubers collected from transgenic lines showed a significant reduction in reducing sugar content after 180 days of cold storage, without showing any measurable off-target effects on plant morphology and tuberization compared to non-transformed control plants. The cold-stored tubers were further assayed for chip color, which showed a fairly light colored quality in the samples originating from RNAi lines. Together with similar effects seen in previously published experiments involving other potato varieties, the Désirée results described here establish the efficacy of using RNAi for the successful reduction of CIS in potato tubers.     

Growth and tuberization of transgenic potato plants expressing sense and antisense sequences of Cu/Zn superoxide dismutase from lily chloroplasts

Overexpression of a chloroplast-localized Cu/Zn superoxide dismutase (chCu/ZnSOD) obtained from lily significantly affects the growth and shape of potato tubers from anin vitro culture system (Kim et al., 2007). Here, we further characterized the sense and antisense transgenic potatoes grown and pots and the greenhouse to investigate the potential for more practical field applications of such phenotypic manipulations. Underin vitro conditions, antisense transgenic plants showed increased shoot growth, delayed tuberization, and altered tuber shapes. When antisense plants were treated with paclobutrazol, an inhibitor of GA biosynthesis, tuberization efficiency and tuber shape were recovered to a status very similar to that ofin vitro- grown wild-type plants. Our results strongly support the idea that potato tuberization and shape is mediated by SOD-catalyzed reactive oxygen species, possibly via the GA biosynthesis pathway.     

Acceleration of potato tuber sprouting by the expression of a bacterial pyrophosphatase

Potato is a globally important crop. Unfortunately, potato farming is plagued with problems associated with the sprouting behavior of seed tubers. The data presented here demonstrate that using transgenic technology can influence this behavior. Transgenic tubers cytosolically expressing an inorganic pyrophosphatase gene derived from Escherichia coli under the control of the tuber-specific patatin promoter display significantly accelerated sprouting. The period of presprouting dormancy for transgenic tubers planted immediately after harvest is reduced by six to seven weeks when compared to wild-type tubers. This study demonstrates a method with which to regulate dormancy, an important aspect of potato crop management.     

QTL mapping of potato chip color and tuber traits within an autotetraploid family

Cultivated potato (Solanum tuberosum L.) is a highly heterozygous autotetraploid crop species, and this creates challenges for traditional line development and molecular breeding. Recent availability of a single-nucleotide polymorphism (SNP) array with 8303 features and software packages for linkage and association mapping in autotetraploid species present new opportunities for the identification of genomic regions that contribute to high-value traits in cultivated potato. A biparental tetraploid potato population was evaluated across three field seasons and storage trials in order to identify quantitative trait loci (QTL) for multiple tuber traits including fried chip color after 5.5–7.2 °C storage. Tetra-allelic dosage information was used to construct a genetic linkage map that covered 1041 cM and contained 2095 SNP markers with a median marker interval of 0.4 cM. A total of 41 QTL were identified for flower color, tuber yield, tuber number per plant, tuber weight, tuber size, and chip color after various storage regimes. Moderate effect QTL for chip color at 3 months were identified that co-localized with candidate genes vacuolar invertase (VInv), invertase inhibitor (INH2), and apoplastic invertase (Inv _ap -b). A separate QTL for chip color after 6 months of storage was identified in the short arm of chromosome 2, and this locus may contribute to variation in senescent sweetening resistance. QTL for tuber weight, length, and width co-localized with a known QTL for plant maturity on chromosome 5. Genome-wide association mapping using a polyploid model detected the tuber size QTL and identified a number of candidate SNPs, but was unable to detect markers significantly associated with chip color.     

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.     

Changes in GA 20-oxidase gene expression strongly affect stem length, tuber induction and tuber yield of potato plants

Gene StGA20ox1 encoding potato GA 20-oxidase is expressed to relatively high levels in leaves and regulated by daylength. To investigate whether this gene is involved in photoperiodic regulation of tuber formation, we have obtained transgenic potato plants expressing sense and antisense copies of the StGA20ox1 cDNA. Over-expression of this cDNA resulted in taller plants that required a longer duration of a short day photoperiod (SD) to tuberize. Tubers from these plants had a decreased time of dormancy and developed sprouts with elongated internodes. Plants expressing antisense copies of the StGA20ox1 cDNA had shorter stems, a decreased length of the internodes and tuberized earlier than control plants, showing increased tuber yields. Antisense inhibition of this gene had no visible effect on the time of dormancy of the tubers, although at the end of dormancy these formed sprouts with shortened internodes. Decreased levels of endogenous GA20 and GA1 were detected in the apex and first leaves of the antisense lines. These results demonstrate the involvement of the GA 20-oxidase activity encoded by StGA20ox1 in the control of stem elongation and in tuber induction but not in tuber dormancy, indicating that the latter may be regulated by another member of the gene family.     

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.     

Improvement of biomass accumulation of potato plants by transformation of cyanobacterial photorespiratory glycolate catabolism pathway genes

Transgenic potato (Solanum tuberosum L. cv. Desiree) plants expressing components of a novel cyanobacterial photorespiratory glycolate catabolism pathway were developed. Transgenic plant expressing glcD1 (glycolate dehydrogenase I) gene was referred to as synGDH and transgenic plants expressing gcl (glyoxylate carboligase) and tsr (tartronic semialdehyde reductase) genes simultaneously were designated as synGT. Both synGDH and synGT plants showed stable gene transformation, integration and expression. Enhanced glyoxylate contents in synGDH plants were detected as compared to synGT and non-transgenic (NT) plants. Phenotypic evaluation revealed that synGDH plants accumulated 11 % higher dry weight, while, tuber weight was 38 and 16 % higher than NT and synGT, respectively. Upon challenging the plants in high temperature and high light conditions synGDH plants maintained higher Fv/Fm and showed less bleaching of chlorophyll as compared to synGT and NT plants. These results indicate that genetic transformation of complete pathway in one plant holds promising outcomes in terms of biomass accumulation to meet future needs for food and energy.     

Expression of a Petunia inflata pectin methyl esterase in Solanum tuberosum L. enhances stem elongation and modifies cation distribution

Transgenic potato (Solanum tuberosum L.) plants were constructed with a Petunia inflata-derived cDNA encoding a pectin methyl esterase (PME; EC 3.1.1.11) in sense orientation under the control of the cauliflower mosaic virus 35S promoter. The PME activity was elevated in leaves and tubers of the transgenic lines but slightly reduced in apical segments of stems from mature plants. Stem segments from the base of juvenile PME-overexpressing plants did not differ in PME activity from the control, whereas in apical parts PME was less active than in the wild-type. During the early stages of development stems of these trangenic plants elongated more rapidly than those of the wild-type. Further evidence that overexpression of a plant-derived PME has an impact on plant development is based on modifications of tuber yield, which was reduced in the transgenic lines. Cell walls from transgenic tubers showed significant differences in their cation-binding properties in comparison with the wild-type. In particular, cell walls displayed increased affinity for sodium and calcium, while potassium binding was constant. Furthermore, the total ion content of transgenic potatoes was modified. Indications of PME-mediated differences in the distribution of ions in transgenic plants were also obtained by monitoring relaxations of the membrane potential of roots subsequent to changes in the ionic composition of the bathing solution. However, no effects on the chemical structure of pectin from tuber cell walls could be detected. Received: 24 March 1999 / Accepted: 20 August 1999     

Overexpression of a bacterial 1-deoxy-D-xylulose 5-phosphate synthase gene in potato tubers perturbs the isoprenoid metabolic network: implications for the control of the tuber life cycle

Potato tubers were engineered to express a bacterial gene encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS) in order to investigate the effects of perturbation of isoprenoid biosynthesis. Twenty-four independent transgenic lines out of 38 generated produced tubers with significantly elongated shape that also exhibited an early tuber sprouting phenotype. Expression analysis of nine transgenic lines (four exhibiting the phenotype and five showing a wild-type phenotype) demonstrated that the phenotype was strongly associated with dxs expression. At harvest, apical bud growth had already commenced in dxs-expressing tubers whereas in control lines no bud growth was evident until dormancy was released after 56-70 d of storage. The initial phase of bud growth in dxs tubers was followed by a lag period of approximately 56 d, before further elongation of the developing sprouts could be detected. Thus dxs expression results in the separation of distinct phases in the dormancy and sprouting processes. In order to account for the sprouting phenotype, the levels of plastid-derived isoprenoid growth regulators were measured in transgenic and control tubers. The major difference measured was an increase in the level of trans-zeatin riboside in tubers at harvest expressing dxs. Additionally, compared with controls, in some dxs-expressing lines, tuber carotenoid content increased approximately 2-fold, with most of the increase accounted for by a 6-7-fold increase in phytoene.     

Genotypic influence on in vitro induction, dormancy length, advancing age and agronomical performance of potato microtubers (Solanum tuberosum L.)

Microtubers of 13 cultivars, largely grown in Italy and other European countries, were induced. They were stored in the dark at 3°C for different periods (28, 56, 84 and 105 days), prior to being transferred to 20°C for between 4 and 17 weeks. Following removal to room temperature, sprouting was recorded and dormancy duration quantified. Dormancy decreased from 28.1 to 19.9, 11.1 and 7.8 days with reduced time of storage. Cvs Arsy, Nicola and Jaerla took consistently more time for dormancy release. The dormancy duration was linearly and inversely correlated with the length of storage. After sprouting, tubers were held at 20°C for various intervals and a range of physiological ages (0, 368, 720 and 1008 degree days) were accumulated. The field comparison of microtubers evidenced a plant growth response and tuber yield/plant affected by the cultivar and physiological age. In early cultivars (Jaerla), a better performance was shown by younger tubers; the opposite trend was noted in Alpha (a later cultivar) with an increase in stems/plant, tubers/plant and tuber yield/plant for tubers with greater physiological age. Like conventional seed tubers, microtubers showed differences in optimum physiological age associated with cultivar earliness. This study has provided some indications on how to enhance emergence and haulm development of plants from microtubers.     

Antisense repression of StubGAL83 affects root and tuber development in potato

StubGAL83 is a potato gene that encodes the beta-subunit of a protein kinase complex similar to the yeast SNF1, and the mammalian AMPK complexes that are modulated by changes in the cellular AMP/ATP ratio and are important regulators of metabolic and stress responses. Here we show that the expression of StubGAL83 in potato foliage is much higher in the dark than in the light and can be repressed by metabolisable sugars in the dark. The amounts of StubGAL83 mRNA are higher in sink than in source leaves. To unravel the role of StubGAL83, transgenic potato plants expressing a part of the StubGAL83 cDNA in antisense orientation under the control of the constitutive CaMV35S promoter were generated. Northern analysis revealed a reduction up to 90-95% in StubGAL83 mRNA accumulation in leaves of seven lines. Five out of these seven lines exhibited a reduction of StubGAL83 mRNA levels also in root and tuber tissues. Independent on the type of repression, the transgenic lines showed a delay in rooting and an increased sensitivity to salt stress. The roots were stunted and possessed less pronounced tap roots than the controls albeit with different severity in the different transgenic lines. The root cells were smaller and some of them had irregular shape. Tuberisation of the antisense-StubGAL83 lines was delayed, the size of the tubers was reduced while the number of tubers per plant was increased. These results together suggest that StubGAL83 affects root and tuber development probably by altering the metabolic status of the leaves.     

Carbon partitioning in leaves and tubers of transgenic potato plants with reduced activity of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase

The role of fructose-2,6-bisphosphate (Fru-2,6-P₂) in regulation of carbon metabolism was investigated in transgenic potato plants ( Solanum tuberosum L. cv Dianella) transformed with a vector containing a cDNA-sequence encoding fructose-6-phosphate,2-kinase (F6P,2-K, EC 2.7.1.105)/fructose-2,6-bisphosphatase (F26BPase, EC 3.1.3.46) in sense or antisense direction behind a CaMV 35S promoter. The activity of F6P,2-K in leaves was reduced to 5% of wild-type (WT) activity, and the level of Fru-2,6-P₂ was reduced both in leaves (10% of the WT level) and in tubers (40% of the WT level). Analysis of photosynthetic ¹⁴CO₂ metabolism, showed that in plant lines with reduced Fru-2,6-P₂ level the carbon partitioning in the leaves was changed in favour of sucrose biosynthesis, and the soluble sugars-to-starch labelling ratio was doubled. The levels of soluble sugars and hexose phosphates also increased in leaves of the transgenic plants. Most notably, the levels of hexoses were four- to six-fold increased in the transgenic plants. In tubers with reduced levels of Fru-2,6-P₂ only minor effects on carbohydrate levels were observed. Furthermore, carbon assimilation in tuber discs supplied with [U-¹⁴C]-sucrose showed only a moderate increase in labelling of hexoses and a decreased labelling of starch. Similar results were obtained using [U-¹⁴C]-glucose. No differences in growth of the transgenic lines and the WT were observed. Our data provide evidences that Fru-2,6-P₂ is an important factor in the regulation of photosynthetic carbon metabolism in potato leaves, whereas the direct influence of Fru-2,6-P₂ on tuber metabolism was limited.