StGA2ox1 is induced prior to stolon swelling and controls GA levels during potato tuber development

The formation and growth of a potato ( Solanum tuberosum ) tuber is a complex process regulated by different environmental signals and plant hormones. In particular, the action of gibberellins (GAs) has been implicated in different aspects of potato tuber formation. Here we report on the isolation and functional analysis of a potato GA 2-oxidase gene ( StGA2ox1 ) and its role in tuber formation. StGA2ox1 is upregulated during the early stages of potato tuber development prior to visible swelling and is predominantly expressed in the subapical region of the stolon and growing tuber. 35S-over-expression transformants exhibit a dwarf phenotype, reduced stolon growth and earlier in vitro tuberization. Transgenic plants with reduced expression levels of StGA2ox1 showed normal plant growth, an altered stolon swelling phenotype and delayed in vitro tuberization. Tubers of the StGA2ox1 suppression clones contain increased levels of GA₂₀, indicating altered GA metabolism. We propose a role for StGA2ox1 in early tuber initiation by modifying GA levels in the subapical stolon region at the onset of tuberization, thereby facilitating normal tuber development and growth.     

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.     

Modification of gibberellin production and plant development in Arabidopsis by sense and antisense expression of gibberellin 20-oxidase genes

Gibberellin (GA) 20-oxidase catalyses consecutive steps late in GA biosynthesis in plants. In Arabidopsis, the enzyme is encoded by a gene family of at least three members (AtGA20ox1, AtGA20ox2 and AtGA20ox3) with differential patterns of expression. The genes are regulated by feedback from bioactive GAs, suggesting that the enzymes may be involved in regulating GA biosynthesis. To investigate this, we produced transgenic Arabidopsis expressing sense or antisense copies of each of the GA 20-oxidase cDNAs. Over-expression of any of the cDNAs gave rise to seedlings with elongated hypocotyls; the plants flowered earlier than controls in both long and short days and were 25% taller at maturity. GA analysis of the vegetative rosettes showed a two- to threefold increase in the level of GA4, indicating that GA 20-oxidase normally limits bioactive GA levels. Plants expressing antisense copies of AtGA20ox1 had short hypocotyls and reduced rates of stem elongation. This was reflected in reduced levels of GA4 in both rosettes and shoot tips. In short days, flowering was delayed and the reduction in the rate of stem elongation was greater. Antisense expression of AtGA20ox2 had no apparent effects in long days, but stem growth in one transgenic line grown in short days was reduced by 20%. Expression of antisense copies of AtGA20ox3 had no visible effect, except for one transgenic line that had short hypocotyls. These results demonstrate that GA levels and, hence, plant growth and development can be modified by manipulation of GA 20-oxidase expression in transgenic plants.     

Dissection of GA 20-oxidase members affecting tomato morphology by RNAi-mediated silencing

GA 20-oxidase is a key enzyme involved in gibberellin (GA) biosynthesis. In tomato, the GA 20-oxidase gene family consists of three members: GA20ox1, GA20ox2, and GA20ox3. To investigate the roles of these three genes in regulating plant growth and development, we used RNA interference technology to generate three kinds of transgenic tomato plants with suppressed expression of each three individual genes. Suppression of GA20ox1 or GA20ox2 resulted in shorter stems, a decreased length of internodes, and small dark green leaves while plants with decreased expression of GA20ox3 had no visible changes on stems and leaves. The plants of the three transgenic lines can flower and set fruits normally, but the seeds from these plants germinated slower than that from the normal plants. Decreased levels of endogenous GAs were detected in the apex of the three transgenic lines. These results demonstrate that the three GA 20-oxidase genes play different roles in the control of plan vegetative growth, but show no effects on flower and fruit development.Equal contribution authors: J. Xiao and H. Li.     

Daylength and spatial expression of a gibberellin 20-oxidase isolated from hybrid aspen (Populus tremula L. × P. tremuloides Michx.)

Physiologically active gibberellins (GAs) are key regulators of shoot growth in trees. To investigate this mechanism of GA-controlled growth in hybrid aspen, we cloned cDNAs encoding gibberellin 20-oxidase (GA 20-oxidase), a key, highly regulated enzyme in the biosynthesis of GAs. Clones were isolated from leaf and cambium cDNA libraries using probes generated by polymerase chain reaction, based on conserved domains of GA 20-oxidases. Upon expression in Escherichia coli, the GST-fusion protein was shown to oxidise GA12 as well as oxidising the 13-hydroxylated substrate GA53, successively to GA9 and GA20, respectively. The gene PttGA20ox1 was expressed in meristematic cells and growing tissues such as expanding internodes, leaves and roots. The expression was negatively regulated by both GA4 and overexpression of phytochrome A. RNA analysis also showed that the expression was down-regulated in late-expanding leaf tissue in response to short days (SDs). Actively growing tissues such as early elongating internodes, petioles and leaf blades had the highest levels of C19-GAs. Upon transfer to SDs an accumulation of GA19 was observed in early elongating internodes and leaf blades. The levels of C19-GAs were also to some extent changed upon transfer to SDs. The levels of GA20 were down-regulated in internodes, and those of GA1 were significantly reduced in early expanding leaf blades. In roots the metabolites GA19 and GA8 decreased upon shifts to SDs, while GA20 accumulated slightly. The down-regulation of GA 20-oxidase activity in response to SDs was further indicated by studies of [14C]GA12 metabolism in shoots, demonstrating that the substrate for GA 20-oxidase, [14C]GA53, accumulates in SDs.     

Function and transcript analysis of gibberellin-biosynthetic enzymes in wheat

The enzymes gibberellin (GA) 20-oxidase and 3-oxidase are major sites of regulation in GA biosynthesis. We have characterised one member of each of the gene families encoding these enzymes that are highly expressed in elongating stems and in developing and germinating grains of wheat and are therefore likely to have prominent developmental roles in these tissues. We mapped the three homoeologues of the GA 20-oxidase gene TaGA20ox1 to chromosomes 5BL, 5DL and 4AL. TaGA20ox1 is expressed mainly in the nodes and ears of the elongating stem, and also in developing and germinating embryos. Expression in the nodes, ears and germinating embryos is predominantly from the A and D genomes. Each homoeologous cDNA encodes a functional enzyme that catalyses the multi-step conversions of GA12-GA9, and GA53-GA20. Time course and enzyme kinetic studies indicate that the initial oxidation steps from GA12 and GA53 to the free alcohol forms of GA15 and GA44, respectively, occur rapidly but that subsequent steps occur more slowly. The intermediate GA19 has an especially low affinity for the enzyme, consistent with its accumulation in wheat tissues. The three homoeologous cDNAs for the 3-oxidase gene TaGA3ox2 encode functional enzymes, one of which was shown to possess low levels of 2beta-hydroxylase, 2,3-desaturase, 2,3-epoxidase and even 13-hydroxylase activities in addition to 3beta-hydroxylase activity. In contrast to TaGA20ox1, TaGA3ox2 is expressed in internodes, as well as nodes and the ear of the elongating stem. It is also highly expressed in developing and germinated embryos.     

Expression of a gibberellin 2-oxidase gene around the shoot apex is related to phase transition in rice

A major catabolic pathway for gibberellin (GA) is initiated by 2beta-hydroxylation, a reaction catalyzed by GA 2-oxidase. We have isolated and characterized a cDNA, designated Oryza sativa GA 2-oxidase 1 (OsGA2ox1) from rice (Oryza sativa L. cv Nipponbare) that encodes a GA 2-oxidase. The encoded protein, produced by heterologous expression in Escherichia coli, converted GA(1), GA(4), GA(9), GA(20), and GA(44) to the corresponding 2beta-hydroxylated products GA(8), GA(34), GA(51), GA(29), and GA(98), respectively. Ectopic expression of the OsGA2ox1 cDNA in transgenic rice inhibited stem elongation and the development of reproductive organs. These transgenic plants were deficient in endogenous GA(1). These results indicate that OsGA2ox1 encodes a GA 2-oxidase, which is functional not only in vitro but also in vivo. OsGA2ox1 was expressed in shoot apex and roots but not in leaves and stems. In situ hybridization analysis revealed that OsGA2ox1 mRNA was localized in a ring at the basal region of leaf primordia and young leaves. This ring-shaped expression around the shoot apex was drastically decreased after the phase transition from vegetative to reproductive growth. It was absent in the floral meristem, but it was still present in the lateral meristem that remained in the vegetative phase. These observations suggest that OsGA2ox1 controls the level of bioactive GAs in the shoot apical meristem; therefore, reduction in its expression may contribute to the early development of the inflorescence meristem.     

Involvement of endogenous gibberellins in potato tuber dormancy and early sprout growth: a critical assessment

The role of endogenous gibberellins (GAs) in the regulation of potato (Solanum tuberosum) tuber dormancy was examined by determining: 1. changes in endogenous GA levels during natural dormancy progression, 2. the effects of GA biosynthesis inhibitors on tuber dormancy duration and 3. the dormancy status and tuber GA levels in a dwarf mutant of potato. The tubers (cv. Russet Burbank) used in these studies were still completely dormant after 98 days of storage. Between 98 and 134 days of storage, dormancy began to end and tubers exhibited limited (< 2 mm) sprout growth. Tuber dormancy weakened with further storage and tubers exhibited greater rates of sprout growth after 187 days of storage. Tubers stored for 212 days or longer were completely non-dormant and exhibited vigorous sprout growth. Immediately after harvest, the endogenous contents of GA19, GA20, and GA1 were relatively high (0.48-0.62 ng g fresh weight(-1)). The content of these GAs declined between 33 and 93 days of storage. Internal levels of GA19, GA20, and GA, rose slightly between 93 and 135 days of storage reaching levels comparable to those found in highly dormant tubers immediately after harvest. Levels of GA19, GA20, and GA1 continued to increase as sprout growth became more vigorous. Neither GA4 nor GA8 was detected in any tuber sample regardless of dormancy status. Dormant tubers exhibited a time-dependent increase in apparent GA sensitivity. Freshly harvested tubers were completely insensitive to exogenous GAs. As postharvest storage continued, exogenous GAs promoted premature dormancy release with GA1 and GA20 eliciting the greatest response. Injection of up to 5 microg tuber(-1) of kaurene, GA12, GA19 or GA8 had no effect on dormancy release. Sprout growth from non-dormant tubers was also promoted by exogenous GA in the following sequence of activity: GA1 = GA20 > GA19. Kaurene, GA12, and GA8 were inactive. Continuous exposure of developing tubers to inhibitors of GA biosynthesis (AMO-1618, ancymidol, or tetcyclasis) did not extend tuber dormancy but rather hastened dormancy release. Comparison of tuber dormancy and GA1 content in tubers of a wild-type and dwarf mutant of S. tuberosum ssp. andigena revealed a near-identical pattern of dormancy progression in spite of the absence of detectable levels of GA1 in tubers of the dwarf sibling at any time during dormancy progression. Collectively, these results do not support a role for endogenous GA in potato tuber dormancy release but are consistent with a role for GAs in the regulation of subsequent sprout growth.     

Flowering in tobacco needs gibberellins but is not promoted by the levels of active GA1 and GA4 in the apical shoot

Flowering of Nicotiana tabacum cv Xhanti depends on gibberellins because gibberellin-deficient plants, due to overexpression of a gibberellin 2-oxidase gene (35S:NoGA2ox3) or to treatment with the gibberellin biosynthesis inhibitor paclobutrazol, flowered later than wild type. These plants also showed inhibition of the expression of molecular markers related to floral transition (NtMADS-4 and NtMADS-11). To investigate further the role of gibberellin in flowering, we quantified its content in tobacco plants during development. We found a progressive reduction in the levels of GA1 and GA4 in the apical shoot during vegetative growth, reaching very low levels at floral transition and beyond. This excludes these two gibberellins as flowering-promoting factors in the apex. The evolution of active gibberellin content in apical shoots agrees with the expression patterns of gibberellin metabolism genes: two encoding gibberellin 20-oxidases (NtGA20ox1 = Ntc12, NtGA20ox2 = Ntc16), one encoding a gibberellin 3-oxidase (NtGA3ox1 = Nty) and one encoding a gibberellin 2-oxidase (NtGA2ox1), suggesting that active gibberellins are locally synthesized. In young apical leaves, GA1 and GA4 content and the expression of gibberellin metabolism genes were rather constant. Our results support that floral transition in tobacco, in contrast to that in Arabidopsis, is not regulated by the levels of GA1 and GA4 in apical shoots, although reaching a threshold in gibberellin levels may be necessary to allow meristem competence for flowering.