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.     

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.     

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.     

Blue Light Inhibition of Tuberization in a Day-Neutral Potato

In tests on the effects of light quality on potato tuberization, continuous blue light was found to consistently inhibit tuberization of tissue-cultured plantlets of Solanum tuberosum ssp. tuberosum cv. ??Norland??. Other tested cultivars, including sports of ??Norland??, formed tubers under continuous blue light. Microarrays identified BL, GA7ox, and Nudix genes as exhibiting altered expression in response to blue light treatment. Quantitative RT-PCR (qRT-PCR) showed that GA7ox RNA increased in ??Norland?? but not in ??Sangre?? plantlets in blue light compared to darkness. RNA levels of genes identified in the literature as having roles in potato tuberization were also measured using qRT-PCR. Levels of GA20o1x, but not GA2ox, RNA increased in response to blue light in ??Norland?? plantlets. BEL5 RNA content was greater under blue light compared to darkness for both ??Norland?? and ??Sangre?? plants. Levels of FT were not significantly different in blue light compared to dark-treated ??Norland?? plants, but were low in blue light-treated compared to dark-treated ??Sangre?? plants. Addition of ancymidol to ??Norland?? plants exposed to blue light overcame blue light inhibition of tuberization. Ancymidol prevents the oxidation of ent-kaurene to ent-kaurenoic acid, thus inhibiting gibberellin biosynthesis. These data suggest that blue light may increase GA accumulation in ??Norland?? plants, as has been shown to occur in Arabidopsis plants. The novel effect of blue light in inhibiting tuberization of ??Norland?? plants suggests that this system could be a useful tool in further elucidating the mechanisms of day-neutral potato tuberization.     

Jasmonic Acid induces tuberization of potato stolons cultured in vitro

The aim of the study was to assess the potential in vitro effects of jasmonic acid and kinetin on tuberization of potato (Solanum tuberosum). Of the two, the former was by far the stronger in vitro promoter of stolon tuberization. Number of tubers induced per stolon, tuberization rate, and final tuber weight were higher by factors of 2.8, 2.3, and 6.4, respectively. Bioassay sensitivity of jasmonic acid, measured in terms of the point at which the concentration for inducing tuberization was saturating, was more than 20 times greater than that of kinetin. Tuberization in both cases was associated with a decrease in rooting ability. Jasmonic acid also triggered a general state of induction throughout the stolon.     

Phytochrome F plays critical roles in potato photoperiodic tuberization

The transition to tuberization contributes greatly to the adaptability of potato to a wide range of environments. Phytochromes are important light receptors for the growth and development of plants, but the detailed functions of phytochromes remain unclear in potato. In this study, we first confirmed that phytochrome F ( St PHYF ) played essential roles in photoperiodic tuberization in potato. By suppressing the St PHYF gene, the strict short‐day potato genotype exhibited normal tuber formation under long‐day ( LD ) conditions, together with the degradation of the CONSTANTS protein St COL 1 and modulation of two FLOWERING LOCUS  T ( FT ) paralogs, as demonstrated by the repression of St SP 5G and by the activation of St SP 6A during the light period. The function of St PHYF was further confirmed through grafting the scion of St PHYF ‐silenced lines, which induced the tuberization of untransformed stock under LD s, suggesting that St PHYF was involved in the production of mobile signals for tuberization in potato. We also identified that St PHYF exhibited substantial interaction with St PHYB both in vitro and in vivo . Therefore, our results indicate that St PHYF plays a role in potato photoperiodic tuberization, possibly by forming a heterodimer with St PHYB .     

Molecular cloning and expression analysis of a novel <Emphasis Type="Italic">CONSTANS</Emphasis>-like gene from potato

A full-length cDNA of a StCONSTANS-like (StCOL) gene was cloned from potato (Solanum tuberosum L.) by RTPCR and RACE. The predicted amino acid sequence of this cDNA has a high degree of identity with other homologous members of the CO or COL family. Analysis of mRNA levels for StCOL shows that it is highly expressed in leaves and becomes weaker during tuberization; moreover, is independent of gibberellin A₃ and sucrose. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 11, pp. 1525–1531.     

Interaction between day length and phytohormones in the control of potato tuberization in the in vitro culture

We studied the interaction of the day length, cytokinins, and gibberellins in the control of tuberization in potato (Solanum tuberosum L, cv. Desire) plants and derived transgenic plants with the inserted PHYB gene from Arabidopsis encoding the synthesis of phytochrome B apoprotein and put under the control of the 35S CaMV promoter. Plantlets were cultured in vitro on hormone-free MS medium containing 5% sucrose and kinetin (1 mg/l) or/and GA (0.5 and 1.0 mg/l), at long day (LD, a 16-h photoperiod), short day (SD, a 10-h photoperiod), or continuous darkness conditions. The content of cytokinins (Ck, zeatin, and zeatin riboside) in various plant organs was determined by the immunoenzyme method, and GA activity was measured in bioassay with dwarf pea. Potato plant transformation with the PHYB gene enhanced substantially tuber initiation inhibition by LD. Kinetin addition to culture medium enhanced tuberization and reduced Ck content in aboveground shoots and Ck redistribution in the favor of underground organs. GA addition to the culture medium suppressed tuberization and induced Ck accumulation in aboveground organs. We concluded that Ck role in tuberization depends on their predominant localization in above- or underground potato organs. The involvement of Ck and GA in the competitive relations between growing tubers and shoots is considered.     

Phytochrome B mediates the photoperiodic control of tuber formation in potato

To determine whether phytochrome B is involved in the response of potato plants to photoperiod, a potato PHYB cDNA fragment was inserted in the antisense orientation behind the 35S CaMV promoter in Bin19 and this construct was transformed into Solanum tuberosum ssp. andigena plants which normally require short days for tuberization. Two independent transformants were obtained that had much lower levels of PHYB mRNA and protein, and which exhibited phenotypes characteristic of phyB mutants, for example, elongated stems and decreased chlorophyll content. The level of phyA, and of several phytochrome A-controlled responses, was unaffected in these plants. The photoperiodic control of tuberization in these antisense PHYB plants was abolished, the plants tuberizing in short day, long day, or short day plus night break conditions. This result shows that phytochrome B is required for the photoperiodic control of tuberization in potato ( Solanum tuberosum ssp. andigena ) and that it regulates this developmental process by preventing tuber formation in non-inductive photoperiods rather than by promoting tuberization in inductive photoperiods.     

Key players associated with tuberization in potato: potential candidates for genetic engineering

Tuberization in potato (Solanum tuberosum L.) is a complex biological phenomenon which is affected by several environmental cues, genetic factors and plant nutrition. Understanding the regulation of tuber induction is essential to devise strategies to improve tuber yield and quality. It is well established that short-day photoperiods promote tuberization, whereas long days and high-temperatures inhibit or delay tuberization. Worldwide research on this complex biological process has yielded information on the important bio-molecules (proteins, RNAs, plant growth regulators) associated with the tuberization process in potato. Key proteins involved in the regulation of tuberization include StSP6A, POTH1, StBEL5, StPHYB, StCONSTANS, Sucrose transporter StSUT4, StSP5G, etc. Biomolecules that become transported from “source to sink” have also been suggested to be important signaling candidates regulating the tuberization process in potatos. Four molecules, namely StSP6A protein, StBEL5 RNA, miR172 and GAs, have been found to be the main candidates acting as mobile signals for tuberization. These biomolecules can be manipulated (overexpressed/inhibited) for improving the tuberization in commercial varieties/cultivars of potato. In this review, information about the genes/proteins and their mechanism of action associated with the tuberization process is discussed.     

AGO1 controls arabidopsis inflorescence architecture possibly by regulating TFL1 expression

Background and Aims

The TERMINAL FLOWER 1 (TFL1) gene is pivotal in the control of inflorescence architecture in arabidopsis. Thus, tfl1 mutants flower early and have a very short inflorescence phase, while TFL1-overexpressing plants have extended vegetative and inflorescence phases, producing many coflorescences. TFL1 is expressed in the shoot meristems, never in the flowers. In the inflorescence apex, TFL1 keeps the floral genes LEAFY (LFY) and APETALA1 (AP1) restricted to the flower, while LFY and AP1 restrict TFL1 to the inflorescence meristem. In spite of the central role of TFL1 in inflorescence architecture, regulation of its expression is poorly understood. This study aims to expand the understanding of inflorescence development by identifying and studying novel TFL1 regulators.

Methods

Mutagenesis of an Arabidopsis thaliana line carrying a TFL1::GUS (β-glucuronidase) reporter construct was used to isolate a mutant with altered TFL1 expression. The mutated gene was identified by positional cloning. Expression of TFL1 and TFL1::GUS was analysed by real-time PCR and histochemical GUS detection. Double-mutant analysis was used to assess the contribution of TFL1 to the inflorescence mutant phenotype.

Key Results

A mutant with both an increased number of coflorescences and high and ectopic TFL1 expression was isolated. Cloning of the mutated gene showed that both phenotypes were caused by a mutation in the ARGONAUTE1 (AGO1) gene, which encodes a key component of the RNA silencing machinery. Analysis of another ago1 allele indicated that the proliferation of coflorescences and ectopic TFL1 expression phenotypes are not allele specific. The increased number of coflorescences is suppressed in ago1 tfl1 double mutants.

Conclusions

The results identify AGO1 as a repressor of TFL1 expression. Moreover, they reveal a novel role for AGO1 in inflorescence development, controlling the production of coflorescences. AGO1 seems to play this role through regulating TFL1 expression.