Potato (Solanum tuberosum L.) can reproduce sexually through flowering and asexually through tuberization. While tuberization has been thoroughly studied, little research has been done on potato flowering. Flower bud development in the strictly short-day tuberizing S. tuberosum group Andigena is impaired under short-day conditions. This impaired development may indicate that tuberization negatively influences flowering. Here, we determine how tuberization affects flower bud development. To find out whether the absence of tubers improves flowering, we prevented tuberization by: (i) grafting potato scions onto wild potato rootstocks, which were unable to form tubers; (ii) removing stolons, the underground structures on which tubers form; and (iii) using plants that were silenced in the tuberization signal StSP6A. Additionally, transgenic plants with increased StSP6A expression were used to determine if flower bud development was impaired. The absence of a tuber sink alone did not accelerate flower bud development, nor did it allow more plants to reach anthesis (open flowering stage) or have more open flowers. Interestingly, reducing StSP6A expression improved flower bud development, and increasing expression impaired it. Our results show that flower bud development in potato is repressed by the tuberization signal StSP6A, and not by competition with the underground tuber sink.

Sucrose is the principal transport form of assimilates in most plants. In many species, translocation of assimilates from the mesophyll into the phloem for long distance transport is assumed to be carrier mediated. A putative sucrose proton cotransporter cDNA has been isolated from potato and shown to be expressed mainly in the phloem of mature exporting leaves. To study the in vivo role and function of the protein, potato plants were transformed with an antisense construct of the sucrose transporter cDNA under control of the CaMV 35S promoter. Upon maturation of the leaves, five transformants that expressed reduced levels of sucrose transporter mRNA developed local bleaching and curling of leaves. These leaves contained > 20-fold higher concentrations of soluble carbohydrates and showed a 5-fold increase in starch content as compared with wild type plants, as expected from a block in export. Transgenic plants with a reduced amount of sucrose carrier mRNA show a dramatic reduction in root development and tuber yield. Maximal photosynthetic activity was reduced at least in the strongly affected transformants. The effects observed in the antisense plants strongly support an apoplastic model for phloem loading, in which the sucrose transporter located at the phloem plasma membrane represents the primary route for sugar uptake into the long distance distribution network.

Sucrose (Suc) transporters belong to a large gene family. The physiological role of SUT1 proteins has been intensively investigated in higher plants, whereas that of SUT4 proteins is so far unknown. All three known Suc transporters from potato (Solanum tuberosum), SUT1, SUT2, and SUT4, are colocalized and their RNA levels not only follow a diurnal rhythm, but also oscillate in constant light. Here, we examined the physiological effects of transgenic potato plants on RNA interference (RNAi)-inactivated StSUT4 expression. The phenotype of StSUT4-RNAi plants includes early flowering, higher tuber production, and reduced sensitivity toward light enriched in far-red wavelength (i.e. in canopy shade). Inhibition of StSUT4 led to tuber production of the strict photoperiodic potato subsp. andigena even under noninductive long-day conditions. Accumulation of soluble sugars and Suc efflux from leaves of transgenic plants are modified in StSUT4-RNAi plants, leading to modified Suc levels in sink organs. StSUT4 expression of wild-type plants is induced by gibberellins and ethephon, and external supply of gibberellic acid leads to even more pronounced differences between wild-type and StSUT4-RNAi plants regarding tuber yield and internode elongation, indicating a reciprocal regulation of StSUT4 and gibberellins.

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.

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 promoter 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.

The concept of florigen, postulated in the early 1930s, has taken form after the identification of the FLOWERING LOCUS T (FT) protein as the flowering-inducing signal. Besides their role in flowering, FT genes were subsequently reported to play additional functions in other biological processes. This is particularly relevant in the nightshades, where the FT genes appear to have undergone considerable expansion at the functional level and gained a new role in the control of storage organ formation in potato (Solanum tuberosum). Neofunctionalization of FT homologs in the nightshades identifies these proteins as a new class of primary signaling components that modulate development and organogenesis in these agronomic relevant species. Copyright © 2013 Elsevier Ltd. All rights reserved.

The BEL1-like family of transcription factors is ubiquitous in plants and plays important roles in regulating development. They function in tandem with KNOTTED1 types to bind to a double TTGAC motif in the upstream sequence of target genes. StBEL5 of potato (Solanum tuberosum) functions as a mobile RNA signal that is transcribed in leaves, moves down into stolons in response to short days, and induces tuber formation. Despite their importance, however, very little is known about the targets of BEL1-like transcription factors. To better understand this network, we made use of a phloem-mobile BEL5 induction model, an ethanol-inducible system coupled with RNA sequencing analysis, and a screen for tandem TTGAC cis-elements in the upstream sequence to catalog StBEL5 target genes. Induction of StBEL5 activated several genes that are also induced by StSP6A (S. tuberosum SELF-PRUNING 6A), a FLOWERING LOCUS T coregulator that functions as a signal for tuberization. Both enhancement and suppression of StBEL5 expression were also closely linked to StSP6A transcriptional activity. Site mutagenesis in tandem TTGAC motifs located in the upstream sequence of StSP6A suppressed the short day-induced activity of its promoter in both young tubers and leaves. The expression profile of StBEL5 induced in stolons from plants grown under long-day conditions revealed almost 10,000 differentially expressed genes, including important tuber marker genes and genes involved in cell growth, transcription, floral development, and hormone metabolism. In a random screen of 200 differentially expressed targets of StBEL5, 92% contained tandem TTGAC motifs in the upstream sequence within 3 kb of the transcription start site. © 2016 American Society of Plant Biologists. All Rights Reserved.

Understanding tuberization in the major crop plant potato (Solanum tuberosum L.) is of importance to secure yield even under changing environmental conditions. Tuber formation is controlled by a homolog of the floral inductor FLOWERING LOCUS T, referred to as SP6A. To gain deeper insights into its function, we created transgenic potato plants overexpressing a codon-optimized version of SP6A, SP6A, to avoid silencing effects. These plants exhibited extremely early tuberization at the juvenile stage, hindering green biomass development and indicating a tremendous shift in the source sink balance. The meristem identity was altered in dormant buds of transgenic tubers. This strong phenotype, not being reported so far for plants overexpressing an unmodified SP6A, could be due to post-transcriptional regulation. In fact, a putative SP6A-specific small regulatory RNA was identified in potato. It was effectively repressing SP6A mRNA accumulation in transient assays as well as in leaves of young potato plants prior to tuber formation. SP6A expression is downregulated under heat, preventing tuberization. The molecular mechanism has not been elucidated yet. We showed that this small RNA is strongly upregulated under heat. The importance of the small RNA was demonstrated by overexpression of a target mimicry construct, which led to an increased SP6A expression, enabling tuberization even under continuous heat conditions, which abolished tuber formation in the wild-type. Thus, our study describes an additional regulatory mechanism for SP6A besides the well-known pathway that integrates both developmental and environmental signals to control tuberization and is therefore a promising target for breeding of heat-tolerant potato.Copyright © 2019 Elsevier Ltd. All rights reserved.

Potato plants form tuberous storage organs on underground modified stems called stolons. Tubers are rich in starch, proteins, and other important nutrients, making potato one of the most important staple food crops. The timing of tuber development in wild potato is regulated by day length through a mechanism that is closely related to floral transition [1, 2]. Tuberization is also known to be regulated by the availability of assimilates, in particular sucrose, the transported form of sugar, required for starch synthesis. During the onset of tuber development, the mode of sucrose unloading switches from apoplastic to symplastic [3]. Here, we show that this switch may be mediated by the interaction between the tuberization-specific FT homolog StSP6A and the sucrose efflux transporter StSWEET11 [4]. The binding of StSP6A to StSWEET11 blocked the leakage of sucrose to the apoplast, and is therefore likely to promote symplastic sucrose transport. The direct physical interaction between StSWEET11 and StSP6A proteins represents a link between the sugar and photoperiodic pathways for the regulation of potato tuber formation. Our data suggest that a previously undiscovered function for the FT family of proteins extends their role as mobile signals to mediators of source-sink partitioning, opening the possibility for modifying source-sink interactions.Copyright © 2019 Elsevier Ltd. All rights reserved.

Using the yeast (Saccharomyces cerevisiae) two-hybrid system and a potato (Solanum tuberosum) KNOX protein, designated POTH1, as bait, we have identified seven distinct interacting proteins from a stolon library of potato. All seven cDNAs are members of the BEL1-like family of transcription factors. Among these proteins, there are at least four regions of high sequence conservation including the homeodomain, the proline-tyrosine-proline three-amino acid loop extension, the SKY box, and a 120-amino acid region upstream from the homeodomain. Through deletion analysis, we identified a protein-binding domain present in the carboxy end of the KNOX domain of POTH1. The protein-binding domain in the BEL1 protein is located in the amino-terminal one-half of the 120-residue conserved region of the BELs. RNA-blot analysis showed differential patterns of RNA accumulation for the BELs in various potato organs. The level of StBEL5 mRNA increased in response to a short-day photoperiod in both leaves and stolons. Similar to sense mutants of POTH1, transgenic lines that overexpressed StBEL5 exhibited enhanced tuber formation even under noninductive conditions. Unlike POTH1 sense lines, however, these BEL lines did not exhibit the extreme leaf and stem morphology characteristic of KNOX overexpressers and displayed a more rapid rate of growth than control plants. Both StBEL5 and POTH1 sense lines exhibited an increase in cytokinin levels in shoot tips. StBEL5 lines also exhibited a decrease in the levels of GA 20-oxidase1 mRNA in stolon tips from long-day plants. Our results demonstrate an interaction between KNOX and BEL1-like transcription factors of potato that may potentially regulate processes of development.

Potato Homeobox1 (POTH1) is a Knotted1-like transcription factor from the Three Amino Acid Loop Extension (TALE) superfamily that is involved in numerous aspects of development in potato (Solanum tuberosum L). POTH1 interacts with its protein partner, StBEL5, to facilitate binding to specific target genes to modulate hormone levels, mediate leaf architecture, and enhance tuber formation. In this study, promoter analyses show that the upstream sequence of POTH1 drives β-glucuronidase activity in response to light and in association with phloem cells in both petioles and stems. Because POTH1 transcripts have previously been detected in phloem cells, long-distance movement of its mRNA was tested. Using RT-PCR and transgenic potato lines over-expressing POTH1, in vitro micrografts demonstrated unilateral movement of POTH1 RNA in a rootward direction. Movement across a graft union into leaves from newly arising axillary shoots and roots of wild type stocks was verified using soil-grown tobacco heterografts. Leaves from the wild type stock containing the mobile POTH1 RNA exhibited a reduction in leaf size relative to leaves from wild type grafts. Both untranslated regions of POTH1 when fused to an expression marker β-glucuronidase, repressed its translation in tobacco protoplasts. RNA/protein binding assays demonstrated that the UTRs of POTH1 bind to two RNA-binding proteins, a polypyrimidine tract-binding protein and an alba-domain type. Conserved glycerol-responsive elements (GRE), specific to alba-domain interaction, are duplicated in both the 5' and 3' untranslated regions of POTH1. These results suggest that POTH1 functions as a mobile signal in regulating development.

MicroRNA156 (miR156) functions in maintaining the juvenile phase in plants. However, the mobility of this microRNA has not been demonstrated. So far, only three microRNAs, miR399, miR395, and miR172, have been shown to be mobile. We demonstrate here that miR156 is a potential graft-transmissible signal that affects plant architecture and tuberization in potato (Solanum tuberosum). Under tuber-noninductive (long-day) conditions, miR156 shows higher abundance in leaves and stems, whereas an increase in abundance of miR156 has been observed in stolons under tuber-inductive (short-day) conditions, indicative of a photoperiodic control. Detection of miR156 in phloem cells of wild-type plants and mobility assays in heterografts suggest that miR156 is a graft-transmissible signal. This movement was correlated with changes in leaf morphology and longer trichomes in leaves. Overexpression of miR156 in potato caused a drastic phenotype resulting in altered plant architecture and reduced tuber yield. miR156 overexpression plants also exhibited altered levels of cytokinin and strigolactone along with increased levels of LONELY GUY1 and StCyclin D3.1 transcripts as compared with wild-type plants. RNA ligase-mediated rapid amplification of complementary DNA ends analysis validated SQUAMOSA PROMOTER BINDING-LIKE3 (StSPL3), StSPL6, StSPL9, StSPL13, and StLIGULELESS1 as targets of miR156. Gel-shift assays indicate the regulation of miR172 by miR156 through StSPL9. miR156-resistant SPL9 overexpression lines exhibited increased miR172 levels under a short-day photoperiod, supporting miR172 regulation via the miR156-SPL9 module. Overall, our results strongly suggest that miR156 is a phloem-mobile signal regulating potato development.

The effects of plant hormones and sucrose (Suc) on potato (Solanum tuberosum L.) tuberization were studied using in vitro cultured single-node cuttings. Tuber-inducing (high Suc) and -noninducing (low Suc or high Suc plus gibberellin [GA]) media were tested. Tuberization frequencies, tuber widths, and stolon lengths were measured during successive stages of development. Endogenous GAs and abscisic acid (ABA) were identified and quantified by high-performance liquid chromatography and gas chromatography-mass spectrometry. Exogenous GA4/7 promoted stolon elongation and inhibited tuber formation, whereas exogenous ABA stimulated tuberization and reduced stolon length. Indoleacetic acid-containing media severely inhibited elongation of stolons and smaller sessile tubers were formed. Exogenous cytokinins did not affect stolon elongation and tuber formation. Endogenous GA1 level was high during stolon elongation and decreased when stolon tips started to swell under inducing conditions, whereas it remained high under noninducing conditions. GA1 levels were negatively correlated with Suc concentration in the medium. We conclude that GA1 is likely to be the active GA during tuber formation. Endogenous ABA levels decreased during stolon and tuber development, and ABA levels were similar under inducing and noninducing conditions. Our results indicate that GA is a dominant regulator in tuber formation: ABA stimulates tuberization by counteracting GA, and Suc regulates tuber formation by influencing GA levels.

Various transcriptional networks and plant hormones have been implicated in controlling different aspects of potato tuber formation. Due to its broad impact on many plant developmental processes, a role for auxin in tuber initiation has been suggested but never fully resolved. Here, auxin concentrations were measured throughout the plant prior to and during the process of tuber formation. Auxin levels increase dramatically in the stolon prior to tuberization and remain relatively high during subsequent tuber growth, suggesting a promoting role for auxin in tuber formation. Furthermore, in vitro tuberization experiments showed higher levels of tuber formation from axillary buds of explants where the auxin source (stolon tip) had been removed. This phenotype could be rescued by application of auxin on the ablated stolon tips. In addition, a synthetic strigolactone analogue applied on the basal part of the stolon resulted in fewer tubers. The experiments indicate that a system for the production and directional transport of auxin exists in stolons and acts synergistically with strigolactones to control the outgrowth of the axillary stolon buds, similar to the control of above-ground shoot branching.