Down regulation of StGA3ox genes in potato results in altered GA content and affect plant and tuber growth characteristics

GA biosynthesis and catabolism has been shown to play an important role in regulating tuberization in potato. Active GAs are inactivated in the stolon tips shortly after induction to tuberization. Overexpression of a GA inactivation gene results in an earlier tuberization phenotype, while reducing expression of the same gene results in delayed tuberization. In addition, overexpression of genes involved in GA biosynthesis results in delayed tuberization, while decreased expression of those genes results in earlied tuberization. The final step in GA biosynthesis is catalysed by StGA3ox1 and StGA3ox2 activity, that convert inactive forms of GA into active GA₁ and GA₄. In this study we cloned StGA3ox2 gene in an RNAi construct and used this construct to transform potato plants. The StGA3ox2 silenced plants were smaller and had shorter internodes. In addition, we assayed the concentrations of various GAs in the transgenic plants and showed an altered GA content. No difference was observed on the time point of tuber initiation. However, the transgenic clones had increased number of tubers with the same yield, resulting in smaller average tuber weight. In addition, we cloned the promoter of StGA3ox2 to direct expression of the GUS reporter gene to visualize the sites of GA biosynthesis in the potato plant. Finally, we discuss how changes of several GA levels can have an impact on shoot, stolon and tuber development, as well as the possible mechanisms that mediate feed-forward and feed-back regulation loops in the GA biosynthetic pathway in potato.     

Gibberellin A1 metabolism contributes to the control of photoperiod-mediated tuberization in potato

Some potato species require a short-day (SD) photoperiod for tuberization, a process that is negatively affected by gibberellins (GAs). Here we report the isolation of StGA3ox2, a gene encoding a GA 3-oxidase, whose expression is increased in the aerial parts and is repressed in the stolons after transfer of photoperiod-dependent potato plants to SD conditions. Over-expression of StGA3ox2 under control of constitutive or leaf-specific promoters results in taller plants which, in contrast to StGA20ox1 over-expressers previously reported, tuberize earlier under SD conditions than the controls. By contrast, StGA3ox2 tuber-specific over-expression results in non-elongated plants with slightly delayed tuber induction. Together, our experiments support that StGA3ox2 expression and gibberellin metabolism significantly contribute to the tuberization time in strictly photoperiod-dependent potato plants.     

The Role of Gibberellin, Abscisic Acid, and Sucrose in the Regulation of Potato Tuber Formation in Vitro

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 GA_4/7 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.     

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.     

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.     

A crosstalk of auxin and GA during tuber development

Several hormones have been studied for their effect on tuber initiation and development. Until recently, the hormone with the most prominent role in tuber initiation was attributed to GA. Genes involved in GA degradation do exhibit an upregulated profile during early stages of tuber development, leading to a rapid decrease of active GA content, thereby facilitating stolon-tip swelling. While GA is known to be involved in shoot and stolon elongation, the development of the new tuberorgan requires changes in meristem identity and the reorientation ofthe plane of cell division. In other developmental processes, such as embryo patterning, flower development and lateral root initiation auxin plays a key role. Recent evidence on the involvement of auxin in tuber formation was providedby the measurement of auxin content in swelling stolons. Auxin content in the stolon tips increased several fold prior to tuber swelling. In vitro tuberisation experiments with auxin applications support the role of auxin during tuber initiation. Taken together, it is becoming clear that the initiation and induction of tubers in potato is a developmental process that appears to be regulated by a crosstalk between GA and auxin.     

Temporal dynamics of tuber formation and related processes in a crossing population of potato (Solanum tuberosum)

The chronological relationships between stolon formation, stolon tip swelling, tuber initiation, flowering, senescence, growth and resorption of tubers were studied under field conditions in a diploid population of potato with 238 genotypes, the parental clones and seven tetraploid cultivars. Timing of tuber initiation was not closely related to the timing of stolon formation, flowering and duration of the plant cycle. Tuber initiation very often preceded stolon branching. The number and size distribution of tubers were largely influenced by the degree of stolon branching, the length of the stolon swelling period and tuber resorption. The peak production of stolons and swollen stolon tips largely took place within the flowering period, although in most genotypes, some stolon tip swelling took place until the end of the plant cycle. More information on the general temporal relationships between events related to tuber formation and plant development will contribute to a better understanding of the physiological and genetic basis of the processes leading to the production of harvestable tubers.     

Carbohydrate Metabolism in Tuberizing Stolon Tips of the Strictly Short Day-Dependent Potato Species, Solanum Demissum Lindl.

Abstract: Tuber formation in the strictly short day-dependent potato species Solanum demissum Lindi. was studied. In order to accurately determine the developmental stage of individual stolon tips, a reliable parameter describing tuber formation is defined. Dry matter percentage of stolon tips was highly correlated with starch concentration and with swelling of the stoIon tips, independent of plant age and stolon diameter. Based on this parameter, changes in carbohydrate metabolism during the initial stages of tuber formation were analyzed. Glucose and fructose levels decreased significantly upon tuber formation. This decrease could be explained by a change in sucrose hydrolysis, dominated by acid invertases before tuber formation, and by sucrose synthase afterwards. A model of the temporal and spatial changes in sucrose unloading and hydrolysis in swelling stolon tips is discussed. The observed changes in carbohydrate metabolism are similar to changes observed in a simplified in vitro system, indicating that such model systems adequately reflect tuber development in intact plants.     

马铃薯块茎发育机理及其基因表达

马铃薯(Solanum tuberosum L.)块茎是有块茎马铃薯植物的地下变态器官,它由匍匐茎顶端膨大形成。对于马铃薯块茎形成的生理机制已有许多研究,这些研究表明,块茎发生受许多因素的影响,总体来讲短日照、较低的温度以及离体条件下培养基较高的蔗糖浓度等有利于块茎形成,同时,块茎形成过程中内源激素亦发生一系列变化。然而,对于块茎形成中相关基因表达,进而调控块茎形成的系统研究目前还较滞后。已有研究显示,块茎形成与膨大涉及到一系列基因的表达与关闭,同时它也与淀粉合成和块茎储藏蛋白基因的表达有关。综述了这一领域现有的研究进展。     

马铃薯块茎发育机理及其基因表达

马铃薯（Solanum tuberosum L.)块茎是有块茎马铃薯植物的地下变态器官,它由匍匐茎顶端膨大形成,对于马铃薯块茎形成的生理机制已有许多研究,这些研究表明,块茎发生受许多因素的影响,总体来讲短日 照,较低的温度以及离体条件下培养基较高的蔗糖浓度等有利于块茎形成,同时,块茎形成过程中内源激素亦发生一系列变化,然而,对于块茎形成中相关基因表达,进而调控块茎形成的系统研究目前还较滞后,已有研究显示,块茎形成与膨大涉及到一系列基因的表达与关闭,同时它也与淀粉合成和块茎储藏蛋白基因的表达有关,综述了这一领域现有的研究进展。     

Genome-wide identification,classification, evolutionary analysis and gene expression patterns of the protein kinase gene family in wheat and <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

Key message

This study provides the first genetic evidence for the role of PP2A in tuberization, demonstrating that the catalytic subunit StPP2Ac2b positively modulates tuber induction, and that its function is related to the regulation of gibberellic acid metabolism. The results contribute to a better understanding of the molecular mechanism controlling tuberization induction, which remains largely unknown.

Abstract

The serine/threonine protein phosphatases type 2A (PP2A) are implicated in several physiological processes in plants, playing important roles in hormone responses. In cultivated potato (Solanum tuberosum), six PP2A catalytic subunits (StPP2Ac) were identified. The PP2Ac of the subfamily I (StPP2Ac1, 2a and 2b) were suggested to be involved in the tuberization signaling in leaves, where the environmental and hormonal signals are perceived and integrated. The aim of this study was to investigate the role of PP2A in the tuberization induction in stolons. We selected one of the catalytic subunits of the subfamily I, StPP2Ac2b, to develop transgenic plants overexpressing this gene (StPP2Ac2b-OE). Stolons from StPP2Ac2b-OE plants show higher tuber induction rates in vitro, as compared to wild type stolons, with no differences in the number of tubers obtained at the end of the process. This effect is accompanied by higher expression levels of the gibberellic acid (GA) catabolic enzyme StGA2ox1. GA up-regulates StPP2Ac2b expression in stolons, possibly as part of the feedback system by which the hormone regulates its own level. Sucrose, a tuber-promoting factor in vitro, increases StPP2Ac2b expression. We conclude that StPP2Ac2b acts in stolons as a positive regulator tuber induction, integrating different tuberization-related signals mainly though the modulation of GA metabolism.