High K<Superscript>+</Superscript> does not affect potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) tuber induction,but represses its development <Emphasis Type="Italic">in vitro</Emphasis>

The role of K⁺ in potato (Solanum tuberosum L.) tuberization, based on the effects of K fertilizer and soil exchangeable K⁺, appears to be mostly contradictory. Here, we provide evidence that K⁺ at high concentrations is detrimental to tuber development in vitro once induction has taken place. An experimental system using in vitro-cultured single-node cuttings showed that K⁺ at ≥30.0 mM significantly reduced tuber fresh mass concomitant with a corresponding decline in starch content. However, high K⁺ did not affect tuber induction in terms of number of tubers developed per cutting. High K⁺-induced inhibitory effect on tuber development was attributed to a reduced rate of assimilate partitioning. ⁸⁶Rb(K) transport to stolons, and tubers that acted as strong sinks in vitro were proportional to exogenous K⁺ levels; however, ⁸⁶Rb accumulation and K⁺ deposition were markedly reduced in tubers as compared with that in stolons, especially at higher K⁺ levels. The results indicated a diminishing sink strength developed by tubers with increasing K nutrition. Highly significant negative correlations between ⁸⁶Rb accumulation/K⁺ deposition in both the sink organs and tuber fresh mass reinforced the inhibitory effect of high K⁺ on tuber development. The rate of tuber K removal in vitro was similar to that of crop K removal reported in vivo, suggesting highly conserved K uptake and transport mechanisms during tuberization process. The results have been discussed in the context of possible effects of high K⁺ on impairing sucrose uptake and metabolism.     

Patatin and four serine proteinase inhibitor genes are differentially expressed during potato tuber development

A highly efficient and synchronousin vitro tuberization system is described. One-node stem pieces from potato (Solanum tuberosum cv. Bintje) plants grown under short day-light conditions containing an axillary bud were cultured in the dark on a tuber-inducing medium. After 5 or 6 days all axillary buds started to develop tubers. To study gene expression during tuber development, RNA isolated from tuberizing axillary buds was used for bothin vitro translation and northern blot hybridizations. The genes encoding the proteinase inhibitors I and II (PI-I and PI-II), a Kunitz-and a Bowman-Birk-type proteinase inhibitor were already expressed in uninduced axillary buds. The length of the day-light conditions differently influenced the expression level of the individual genes. In addition, the expression of each of these genes changed specifically during the development of the axillary bud to tuber. In contrast to the expression of these proteinase inhibitor genes, patatin gene expression was only detectable from the day tuberization was manifested as a radial expansion of the axillary bud.These results are discussed with respect to the regulation of the expression of the genes studied in relation to the regulation of tuber development.     

Induction and accumulation of major tuber proteins of potato in stems and petioles

A family of immunologically identical glycoproteins with apparent molecular weights of approximately 40,000 are among the major tuber proteins of potato (Solanum tuberosum L.). These proteins, as purified by ion-exchange and affinity chromatography, have been given the trivial name `patatin.' To determine if patatin can be used as a biochemical marker to study the process of tuberization, its amount was measured in a variety of tissues by rocket immunoelectrophoresis and by enzyme-linked immunosorbent assay (ELISA).

Patatin comprises 40 to 45% of the soluble protein in tubers regardless of whether they are formed on underground stolons or from axillary buds of stem cuttings. Under normal conditions, patatin is present in only trace amounts, if at all, in leaves, stems, or roots of plants which are either actively forming tubers or which have been grown under long days to prevent tuberization. However, if tubers and axillary buds are removed, patatin can accumulate in stems and petioles. This accumulation occurred without any obvious tuber-like swelling and would occur even under long days. In all tissues containing large amounts of patatin, the other tuber proteins were also found as well as large amounts of starch.

     

Characterization of gene expression during potato tuber development in individuals and populations using the luciferase reporter system

Analysis of gene expression and enzyme activity in pooled tuber samples has previously indicated different developmental events occurring in a fixed sequential order during tuber development, starting with the up-regulation of starch synthesis then induction of protein storage followed by cell division and cell enlargement. In this report we analysed in vivo promoter activity of genes related to cell division and storage of reserves during tuber development in individual in vitro tubers, using the non invasive firefly luciferase reporter system. The average activity of the storage related promoters (AGPaseS and Pat21) was up-regulated prior to visible swelling, while the average activity of both cell cycle genes (cycB1;1 and CDC2a) showed an up-regulation after the onset of swelling. However, this novel system allowed expression analysis in individual tubers, which showed a variable up-regulation of both storage genes in relation to the moment of swelling, from 4 days before to 10 days after the onset of swelling. We conclude that during the first stages of tuber development, the moment of storage gene induction is independent from swelling. These results indicate that the developmental program of potato tubers does not consist of a fixed sequential order of events, but consists of independent developmental programs (storage and swelling), together resulting in the formation of a potato tuber. It is concluded that analysis of developmental programs by studying individuals may result in new insights, possibly obscured when using pooled samples.     

Tuberization in Potato at High Temperatures: Promotion by Disbudding

The role of the terminal and axillary buds, as presumptive organsof gibberellin synthesis, in the control of tuberization inpotato (Solanum tuberosum L., cv. Sebago) at high temperatureswas studied. Decapitation alone strongly promoted the outgrowthof axillary buds, but did not promote tubenzation. When growthof the axillary buds was suppressed by the use of chemical pruningagents (MH, TIBA or 1-decanol), tuberization was promoted. Manualremoval of the buds promoted tuberization to a similar extent.The results are consistent with the hypothesis that the budsare major sites of gibberellin synthesis in the potato, andthat high temperatures stimulate the synthesis of gibberellinsand their export to the stolons, where they inhibit tuber formation. Solarium tuberosum L., potato, tuberization, temperature, disbudding, chemical pruning, gibberellins     

Tuberization in potato involves a switch from apoplastic to symplastic phloem unloading

Phloem unloading was studied in potato plants in real time during the early stages of tuberization using carboxyfluorescein (CF) as a phloem-mobile tracer, and the unloading pattern was compared with autoradiography of tubers that had transported (14)C assimilates. In stolons undergoing extension growth, apoplastic phloem unloading predominated. However, during the first visible signs of tuberization, a transition occurred from apoplastic to symplastic transport, and both CF and (14)C assimilates subsequently followed identical patterns of phloem unloading. It is suggested that the switch to symplastic sucrose unloading may be responsible for the upregulation of several genes involved in sucrose metabolism. A detailed analysis of sugar levels and (14)C sugar partitioning in tuberizing stolons revealed a distinct difference between the apical region of the tuber and the subapical region. Analysis of invertase activity in nontuberizing and tuberizing stolons revealed a marked decline in soluble invertase in the subapical region of swelling stolons, consistent with the switch from apoplastic to symplastic unloading. However, cell wall-bound invertase activity remained high in the apical 1 to 2 mm of tuberizing stolons. Histochemical analysis of potato lines transformed with the promoter of an apoplastic invertase gene (invGE) linked to a reporter gene also revealed discrete gene expression in the apical bud region. Evidence is presented that the apical and lateral tuber buds function as isolated domains with respect to sucrose unloading and metabolism.     

Tuberization in Potato at High Temperatures: Gibberellin Content and Transport from Buds

Warm temperatures (35°C day/30°C night) which inhibittuberization in potato (Solanum tuberosum L., cv. Sebago) increasedgibberellin activity in crude extracts from buds, but not frommature leaves, as determined by the lettuce hypocotyl bioassay.Changes in the growth of tubers and stolons indicate the occurrenceof basipetal movement of GA₃ applied to the terminal bud ora mature leaf. ¹⁴C labelling from GA₃ or mevalonic acid injectedjust below the terminal bud was recovered in the lower shoot,stolons and tubers, but the amount transported was greater atcool temperatures (20/15°C). It is concluded that high temperaturespromote the synthesis of gibberellin in the buds rather thantransport to the stolons. Solanum tuberosum L., potato, tuberization, gibberellin     

Use of the growth retardant tetcyclacis for potato tuber formation in vitro

The effects of the plant growth retardant tetcyclacis on in vitro tuber formation in potatoes was studied, using two different approaches: 1. tuber formation in various lines that did not or hardly form tubers under control conditions, and 2. tuber formation by the variety Bintje, which readily forms tubers. The ABA-deficient (droopy) lines of S. phureja hardly formed tubers without the addition of tetcyclacis. In the presence of this growth retardant tuberization was nearly 100%, within three weeks of in vitro culture, even in the absence of cytokinin. A series of somatic hybrids between S. tuberosum and S. brevidens, that did not form tubers in field and pot experiments, were tested. They all formed tubers in vitro in the presence of tetcyclacis. Stoloniferous shoots formed on single-node cuttings from in vitro grown Solanum tuberosum var Bintje plantlets were transferred to media containing a high level of sucrose. In the presence of tetcyclacis, tuber formation started after 4 days, reaching a maximum level of 80% at day 7. Tubers formed in the presence of tetcyclacis, accumulated starch and expressed several tuber-specific genes. These effects were fully antagonized by gibberellic acid. It is concluded that the growth retardant tetcyclacis is a potent tool in the study of tuber formation in potatoes.Abbreviations ABA abscisic acid - BAP benzylaminopurine - GA₃ gibberellic acid - STS silver thiosulphate - TET tetcyclacis     

Shoot, Stolon, and Tuber Formation on Potato (Solanum tuberosum L.) Cuttings in Response to Photoperiod

The intensity of “tuberization stimulus” in potato shoots (Solanum tuberosum L.) can be assessed from cuttings containing one or more leaves. Cuttings maintained in a mist chamber under long days will form tubers from underground buds if prior to taking the cutting the leaves received sufficient exposure to photoperiods less than the critical photoperiod. The greatest tendency to tuberize was found in cuttings that consisted of a single, fully expanded leaf and its subtended bud. Grafts showed that genetical differences in critical photoperiod resided in properties of the leaf. Short days before cutting tended to shift growth from above ground buds of two-node cuttings to below ground buds, even if the number of short days was insufficient for tuber induction. As few as 6 short days reduced growth of shoots at the upper bud and increased underground growth of shoots and stolons.     

Genomic and functional characterization of StCDPK1

StCDPK1 is a calcium dependent protein kinase expressed in tuberizing potato stolons and in sprouting tubers. StCDPK1 genomic sequence contains eight exons and seven introns, the gene structure is similar to Arabidopsis, rice and wheat CDPKs belonging to subgroup IIa. There is one copy of the gene per genome and it is located in the distal portion of chromosome 12. Western blot and immunolocalization assays (using confocal and transmission electron microscopy) performed with a specific antibody against StCDPK1 indicate that this kinase is mainly located in the plasma membrane of swelling stolons and sprouting tubers. Sucrose (4–8%) increased StCDPK1 protein content in non-induced stolons, however the amount detected in swelling stolons was higher. Transgenic lines with reduced expression of StCDPK1 (β7) did not differ from controls when cultured under multiplication conditions, but when grown under tuber inducing conditions some significant differences were observed: the β7 line tuberized earlier than controls without the addition of CCC (GA inhibitor), developed more tubers than wild type plants in the presence of hormones that promote tuberization in potato (ABA and BAP) and was more insensitive to GA action (stolons were significantly shorter than those of control plants). StCDPK1 expression was induced by GA, ABA and BAP. Our results suggest that StCDPK1 plays a role in GA-signalling and that this kinase could be a converging point for the inhibitory and promoting signals that influence the onset of potato tuberization.     

Effects of High Air and Soil Temperature Stress on Growth and Tuberization in Solanum tuberosum

Potato plants (Solanum tuberosum L.) were grown at differentair and soil temperatures to determine the effects of high-temperaturestress on root, tuber, and shoot growth. Cooling the soil (17–27C) at high air temperatures (30–40 C) relieved noneof the visible symptoms of heat stress on shoot growth; norwas the degree of induction to tuberize in leaves increased,as reflected in tuberization of leaf-bud cuttings. Heating thesoil (27–35 C) at cool (17–27 C) air temperatureshad no apparent detrimental effect on shoot growth or inductionof leaves to tuberize. However, in each case hot soil largelyeliminated tuber development. In one experiment stolons grewup out of the hot soil and formed aerial tubers upon reachingthe cool air. When leaf-bud cuttings from induced plants wereused as a model system, high soil temperatures inhibited tuberdevelopment from the buried leaf buds, in the absence of anyroot growth. Apparently the induction of leaves to tuberizeis affected principally by air rather than soil temperature,but expression of the signal to tuberize can be blocked by highsoil temperature. Solanum tuberosum L., potato, temperature stress, soil temperature, tuberization     

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.     

In vitro tuberization of potato clones from different maturity groups

Summary In vitro tuberization on shoot cultures of early, mid-season, late and very late potatoes was compared. Shoots were grown at 12, 16, or 20 h photoperiods; tuberization was then induced at 0, 8 or 16 h light. In the dark, shoots from early plants initially grown at 16 h consistently set tubers earlier than the other types, whereas the very late line tuberized later and produced significantly fewer tubers. Tuber setting of mid-season plants could not be distinguished from the late type. Tuberization of the very late line was significantly hastened by shortening the photoperiod from 20 h to 12 h during the shoot growth period. Light during tuber induction delayed tuberization. This system may be useful to screen callus-derived plants for maturity, and may also be suitable for in vitro study of the photoperiodic control of tuberization.     

Cell Cycling and the Fate of Potato Buds

The cell cycling characteristics of the regions of the apicalmeristems of underground shoots and buds of Solanum tuberosumL. were investigated by stathmokinesis and labelling. The apicaldomes of orthotropic shoots produce cells at twice the elementalrate of those of stolons, and their youngest leaf primordiaat twelve times the rate. Changing the fate of stolons so thatthey will become orthotropic by decapitating the tuber sproutsthat bear them results within 24 h in a general increase incell production especially in the leaf primordia. Axillary buds on tuber sprouts induced to become precursorsof orthotropic shoots instead of stolons undergo a spectacularincrease in cell division within 24 h in all regions, especiallyin the primordia and bud anlagen where the rate increases 20-foldor more. The summit is slower to react than other regions, but,by 24 h, its rate of cell division increases 11-fold and itis contributing 14 cells per day to the flanks from its 80 cells. In all the axillary buds the rate of mitosis in the summit ishalf that of the flanks of the apical dome, but, in both stolonsand orthotropic underground shoots, the rate is higher in thesummit than in the flanks or rib meristem. The results are discussed in relation to what is known of cellcycling changes after floral evocation. Solanum tuberosum L., potato, fate, cell division, apical meristem, stathmokinesis     

Occurrence of jasmonic acid in organs of Solanum tuberosum L. and its effect on tuberization

The aims of this study were to demonstrate the endogenous presence of jasmonic acid (JA) in roots, stolons and periderm of new formed tubers, by means of bioassays, ELISA and GC-MS, and to test a microdrop bioassay using the leaflets of potato cuttings cultured in vitro. Our results confirm the existence of JA by bioassays and GC-MS in foliage, stolons, roots and tuber periderm.Abbreviations DW dry weight - GC-MS gas chromatography-mass spectrometry - JA jasmonic acid - MeOH methanol - SD short day     

Regulation of potato tuber protein accumulation by gibberellic Acid

Many studies have shown that gibberellic acid (GA₃) inhibits tuberization in potato (Solanum tuberosum L.). In this study, we have utilized the 40 kilodalton glycoprotein, patatin, as a marker for biochemical events associated with the process of tuberization. To determine the effects of exogenous applications of GA₃ on the induction of the accumulation of this major tuber protein, we measured patatin levels in tubers from treated whole plants, petioles from a single-node cutting system with GA₃ applied in a lanolin paste, and stolon tips cultured in vitro on an inductive medium supplemented with GA₃. In all three systems, GA₃ inhibited the accumulation of patatin and the major 15 and 22 kilodalton tuber proteins. This effect appeared to be selective since most of the other proteins were not affected and, in tubers, at least one protein was stimulated by GA₃. These results suggest that GA₃ can reverse biochemical events of tuberization in tubers as well as prevent the accumulation of the major tuber proteins in other inducible tissues.     

Effect of ethylene on the endogenous cytokinin and gibberellin levels in tuberizing potatoes

High concentrations of 2-chloroethylphosphonic acid inhibited tuberization on aged potato tubers (Solanum tuberosum) that had been predisposed to the little tuber disorder. As a result of this treatment sprouts developed which contained relatively high levels of endogenous gibberellins and which elongated normally. The endogenous cytokinin levels in the different treatments did not change appreciably. It is suggested that tuberization is prevented by ethylene either as a direct inhibition of cell division or that it prevents the endogenous cytokinins from functioning. Irrespective of the mode of action of ethylene, cell division apparently is the primary process affected, the result being that storage tissue required for the accumulation of starch is not formed.