Comparative transcriptome analysis coupled to X-ray CT reveals sucrose supply and growth velocity as major determinants of potato tuber starch biosynthesis

<p>Abstract</p> <p>Background</p> <p>Even though the process of potato tuber starch biosynthesis is well understood, mechanisms regulating biosynthesis are still unclear. Transcriptome analysis provides valuable information as to how genes are regulated. Therefore, this work aimed at investigating transcriptional regulation of starch biosynthetic genes in leaves and tubers of potato plants under various conditions. More specifically we looked at gene expression diurnally in leaves and tubers, during tuber induction and in tubers growing at different velocities. To determine velocity of potato tuber growth a new method based on X-ray Computed Tomography (X-ray CT) was established.</p> <p>Results</p> <p>Comparative transcriptome analysis between leaves and tubers revealed striking similarities with the same genes being differentially expressed in both tissues. In tubers, oscillation of granule bound starch synthase (GBSS) expression) was observed which could be linked to sucrose supply from source leaves. X-ray CT was used to determine time-dependent changes in tuber volume and the growth velocity was calculated. Although there is not a linear correlation between growth velocity and expression of starch biosynthetic genes, there are significant differences between growing and non-growing tubers. Co-expression analysis was used to identify transcription factors positively correlating with starch biosynthetic genes possibly regulating starch biosynthesis.</p> <p>Conclusion</p> <p>Most starch biosynthetic enzymes are encoded by gene families. Co-expression analysis revealed that the same members of these gene families are co-regulated in leaves and tubers. This suggests that regulation of transitory and storage starch biosynthesis in leaves and tubers, respectively, is surprisingly similar. X-ray CT can be used to monitor growth and development of belowground organs and allows to link tuber growth to changes in gene expression. Comparative transcriptome analysis provides a useful tool to identify transcription factors possibly involved in the regulation of starch biosynthesis.</p

Analysis of the regulation of potato tuber dormancy

Da Kartoffelknollen vor allem frisch verarbeitet bzw. verzehrt werden besteht das ganze Jahr über ein hoher Bedarf an frischem Erntegut. Aufgrund der klimatischen Bedingungen können Kartoffelknollen jedoch nicht das ganze Jahr über angebaut werden, so dass eine Langzeitlagerung der Knollen erforderlich ist. Nachdem das Auskeimen von Kartoffelknollen mit einer Remobilisierung von Speicherstoffen und Wasserverlust einhergeht, ist dies ein Hauptfaktor für eine Verringerung der Erntequalität. Daher ist es von großem Interesse die molekulare Regulation der Dormanz von Kartoffelknollen besser zu verstehen. Durch die Identifizierung von Kandidatengenen für potentielle Regulatoren der Knollendormanz bzw. Knollenmeristem-spezifischer Promotoren könnte es möglich werden, die Länge der Keimruhe von Kartoffelknollen gezielt zu beeinflussen. Wie zuvor bekannt kann die Keimruhe von Kartoffelknollen durch Zugabe von Gibberellin (GA) gebrochen werden. Daher sollte die Rolle dieses Phytohormons bei der Knollenkeimung näher untersucht werden. Mithilfe transgener Pflanzen mit erhöhtem bzw. verringertem endogenen GA-Gehalt konnte gezeigt werden, dass die Erhöhung des endogenen GA-Gehalts unter anderem zu einer leicht verfrühten Keimung und stark verlängerten Keimen führt. Pflanzen mit verringertem GA-Gehalt wiesen dagegen eine verlängerte Dormanzphase und stark verkürzte Keime auf. GA ist demnach sowohl an der Brechung der Keimruhe als auch am Elongationswachstum des Knollenkeimes beteiligt. Da über die molekularen Mechanismen der Knollenkeimung noch wenig bekannt ist, sollten Mikroarrayexperimente zu einem besseren Verständnis der Keimung von Kartoffelknollen beitragen. Zudem sollten Kandidatengene für potentielle Regulatoren der Knollendormanz ausgewählt und mithilfe transgener Pflanzen näher charakterisiert werden. Die Analyse globaler transkriptioneller Unterschiede in dormanten und keimenden Knollenaugen führte unter anderem zur Identifizierung von GARP („GA-regulated protein“) als potentiellem Regulator der Knollenkeimung. Knollen transgener Pflanzen mit erhöhter bzw. verringerter GARP-Genexpression zeigten jedoch kein verändertes Keimverhalten, was zu dem Schluss führte, dass GARP keinen Hauptregulator der Knollenkeimung darstellt. Zusätzlich führte die Transkriptomanalyse dormanter und keimender Knollenaugen zur Identifizierung der dUTPase als molekularem Marker für die Reaktivierung des Knollenmeristems. Die Analyse transkriptioneller Veränderungen während eines „sprout release assay“-Experiments konnte zu einem besseren Verständnis der molekularen Veränderungen während der Knollenkeimung beitragen. Es zeigte sich, dass die Zellwandbiosynthese bzw. modifikation zu den sehr frühen Prozessen der Knollenkeimung zählt. Wie erwartet war auch der Wiedereintritt in den Zellzyklus entsprechend der Mikroarraydaten ein sehr früher Prozess der Initiation der Knollenkeimung. Eine frühe Induktion von Zellzyklusregulatoren des G1/ S-Phasenübergangs wurde mittels „Real-Time“ PCR-Analysen verifiziert. Durch den Vergleich von Mikroarraydaten aus GA3- bzw. BA-induzierter Knollenkeimung und dormanten bzw. keimenden Knollenaugen konnten früh, generell bzw. spät während der Keimung regulierte Gene identifiziert werden. Aus diesen Ergebnissen wurde ein Modell über die molekularen Veränderungen während der Knollenkeimung abgeleitet. Zusätzlich wurde das KNOX-Gen StKn2 als möglicher Regulator der Knollenkeimung identifiziert und mithilfe transgener Pflanzen weiter charakterisiert. StKn2 ist sehr wahrscheinlich an der Reaktivierung des Knollenmeristems bei der Initiation der Keimung beteiligt. Durch vergleichende Analyse von Genexpressionsprofilen induzierter Stolone und aktiver Knollenmeristeme konnte die Hypothese bestätigt werden, dass die Knolleninduktion und die Knollenkeimung auf transkriptioneller Ebene eher antagonistisch reguliert werden. Mittels vergleichender Analysen von Transkriptionsprofilen aktiver Knollenmeristeme mit Mikroarraydaten aus verschiedenen Meristem-haltigen Kartoffelgeweben sollten gemeinsam und vor allem auch spezifisch exprimierte Gene identifiziert werden. Zuletzt wurden durch einen Vergleich von Mikroarraydaten von verschiedenen Zeitpunkten der Knollenkeimung und einer Reihe verschiedenster Kartoffelgewebe eine sehr wahrscheinlich spezifisch bei der Knollenkeimung exprimierte putative Pektinesterase (pPE) identifiziert.Because potato tubers are mainly processed and consumed in the fresh formed, there is a strong year-round demand for fresh tubers. Due to the climatic conditions in the main growing areas it is impossible to grow potato throughout the year. Therefore, lengthy storage is necessary and maintenance of postharvest market quality is of prime importance. Tuber sprouting is accompanied by physiological changes such as remobilization of storage reserves, water loss and tuber quality is thus impaired. For this reason it is of high interest to get insights in the molecular regulation of potato tuber dormancy. A better understanding of the molecular changes during this developmental process might allow the manipulation of the length of the dormancy period. As was known before, application of GA leads to dormancy breakage of potato tubers which ends up with visible sprouting. Therefore, transgenic lines with increased and decreased endogenous gibberellin content were analyzed. It could be shown that high endogenous GA content led to a shortened dormancy period and strongly elongated sprouts whereas a decreased GA content resulted in a prolonged dormancy period and reduced sprout length. Therefore GA is involved in dormancy breakage and strongly affects sprout outgrowth. Little is known about the molecular changes involved in the transition from dormant to sprouting potato tubers, so transcriptome profiling of different timepoints during tuber sprouting was performed. Additionally, candidate genes putatively regulating tuber dormancy were identified and further characterized using transgenic lines. The analysis of global transcriptional changes in dormant and sprouting tuber buds resulted in the identification of GARP („GA-regulated protein“) as a potential regulator of tuber sprouting. Tubers with either reduced or increased GARP gene expression did not show any changes in their sprouting behaviour. It was therefore concluded that GARP is not a main regulator of tuber sprouting. Analysis of microarray data from dormant and sprouting tuber buds also led to the identification of the dUTPase as a molecular marker for reactivation of potato tuber meristems. Further investigations also revealed an early up-regulation of dUTPase gene expression before visible sprouting occurs. Anlysis of transcriptional changes during a “sprout release assay” experiment led to a better understanding of the molecular changes during tuber sprouting. Interestingly, cell wall biosynthesis and modification seem to be one of the earliest steps in the transition from dormant to sprouting potato tubers. Re-entry of meristematic cells into cell cycle was also an early event at the beginning of tuber sprouting. Using real-time PCR, an early induction of genes encoding for cell cycle regulators of the G1/ S-phase transition could be verified. This confirmed the re-entry of tuber meristem cells of the at the end of tuber dormancy via G1-phase. A comparative analysis of microarray data derived from GA3- and BA induced tuber sprouting, as well as dormant and sprouting tuber buds resulted in the identification of early, general and late regulated genes during tuber sprouting. From this the KNOX-family gene StKn2 was identified as a potential regulator of tuber sprouting and a model of molecular changes during tuber sprouting was deduced. Transgenic lines with an increased StKn2 expression showed strong phenotypic alterations. Interestingly, tuber buds from StKn2 overexpressing tubers showed earlier sprouting during a sprout release assay experiment. Thus, StKn2 is most likely involved in the reactivation of the tuber meristem at the end of the dormancy period. Comparison of transcriptional profiles from induced stolons and active tuber meristems confirmed the hypothesis that tuber induction and sprouting are on the transcriptional level mainly antagonistically regulated. Comparative analysis of microarray data from tuber meristems and apical and axillary shoot meristems revealed a similar similar regulation on the transcriptional level. More detailed analysis revealed that at the transcriptional level, active tuber meristems are more similar to axillary than to apical shoot meristems. Moreover, comparative analysis of potato meristems led to the identification of specifically expressed genes. Most interesting of these was a TCP-transcription factor which might be a good candidate to regulate potato tuber dormancy. To identify candidate genes for the isolation of tuber meristem-specific genes, an analysis of transcriptome data from active tuber meristems and different meristematic and non-meristematic potato tissues was performed. Through this analysis it was revealed that pPE is expressed only in tuber meristems, making it an attractive candidate for the isolation of a tuber meristem-specific promoter

Reactivation of Meristem Activity and Sprout Growth in Potato Tubers Require Both Cytokinin and Gibberellin1[C][W][OA]

Reactivation of dormant meristems is of central importance for plant fitness and survival. Due to their large meristem size, potato (Solanum tuberosum) tubers serve as a model system to study the underlying molecular processes. The phytohormones cytokinins (CK) and gibberellins (GA) play important roles in releasing potato tuber dormancy and promoting sprouting, but their mode of action in these processes is still obscure. Here, we established an in vitro assay using excised tuber buds to study the dormancy-releasing capacity of GA and CK and show that application of gibberellic acid (GA3) is sufficient to induce sprouting. In contrast, treatment with 6-benzylaminopurine induced bud break but did not support further sprout growth unless GA3 was administered additionally. Transgenic potato plants expressing Arabidopsis (Arabidopsis thaliana) GA 20-oxidase or GA 2-oxidase to modify endogenous GA levels showed the expected phenotypical changes as well as slight effects on tuber sprouting. The isopentenyltransferase (IPT) from Agrobacterium tumefaciens and the Arabidopsis cytokinin oxidase/dehydrogenase1 (CKX) were exploited to modify the amounts of CK in transgenic potato plants. IPT expression promoted earlier sprouting in vitro. Strikingly, CKX-expressing tubers exhibited a prolonged dormancy period and did not respond to GA3. This supports an essential role of CK in terminating tuber dormancy and indicates that GA is not sufficient to break dormancy in the absence of CK. GA3-treated wild-type and CKX-expressing tuber buds were subjected to a transcriptome analysis that revealed transcriptional changes in several functional groups, including cell wall metabolism, cell cycle, and auxin and ethylene signaling, denoting events associated with the reactivation of dormant meristems

Genome-wide analysis of starch metabolism genes in potato (Solanum tuberosum L.)

Background Starch is the principle constituent of potato tubers and is of considerable importance for food and non-food applications. Its metabolism has been subject of extensive research over the past decades. Despite its importance, a description of the complete inventory of genes involved in starch metabolism and their genome organization in potato plants is still missing. Moreover, mechanisms regulating the expression of starch genes in leaves and tubers remain elusive with regard to differences between transitory and storage starch metabolism, respectively. This study aimed at identifying and mapping the complete set of potato starch genes, and to study their expression pattern in leaves and tubers using different sets of transcriptome data. Moreover, we wanted to uncover transcription factors co-regulated with starch accumulation in tubers in order to get insight into the regulation of starch metabolism. Results We identified 77 genomic loci encoding enzymes involved in starch metabolism. Novel isoforms of many enzymes were found. Their analysis will help to elucidate mechanisms of starch biosynthesis and degradation. Expression analysis of starch genes led to the identification of tissue-specific isoenzymes suggesting differences in the transcriptional regulation of starch metabolism between potato leaf and tuber tissues. Selection of genes predominantly expressed in developing potato tubers and exhibiting an expression pattern indicative for a role in starch biosynthesis enabled the identification of possible transcriptional regulators of tuber starch biosynthesis by co-expression analysis. Conclusions This study provides the annotation of the complete set of starch metabolic genes in potato plants and their genomic localizations. Novel, so far undescribed, enzyme isoforms were revealed. Comparative transcriptome analysis enabled the identification of tuber- and leaf-specific isoforms of starch genes. This finding suggests distinct regulatory mechanisms in transitory and storage starch metabolism. Putative regulatory proteins of starch biosynthesis in potato tubers have been identified by co-expression and their expression was verified by quantitative RT-PCR

What led Tel Aviv to become a leading entrepreneurial ecosystem?

“How has Tel Aviv become a leading entrepreneurial ecosystem? ” This thesis investigates the factors behind the growing success of Tel Aviv as an entrepreneurial scene. The economic capital of Israel has become one of the most important clusters of innovation in the World (Engel & del-Palacio, 2011). By analysing these factors with key entrepreneurial actors, the hopes were to find new qualitative evidence to back up the statistics. The aim of the thesis was to use qualitative interviews with chosen entrepreneurs and investors to give insights in Tel Aviv and how it has become a flourishing ecosystem. This has in turn enhanced the previous research on Knowledge-Intensive entrepreneurship by adding a singular case study. Analysing the Tel Aviv ecosystem allowed me to find ten different factors of its success. These can potentially be used as inspiration points for stimulating clusters of innovation around the World.MSc in Knowledge-based Entrepreneurshi

Additional file 2: of Genome-wide analysis of starch metabolism genes in potato (Solanum tuberosum L.)

Valid microarray identifiers for starch genes in the POCI 4x44k and 8x60k platforms. (XLS 46 kb

Additional file 1: of Genome-wide analysis of starch metabolism genes in potato (Solanum tuberosum L.)

Phylogenetic analysis of gene families involved in starch metabolism. Tree calculation was based on a global alignment with free end gaps, BLOSUM62 cost matrix and Jukes-Cantor genetic distance model. The tree was built by the Geneious 5.5.6 Tree Builder module employing a neighbour-joining method. a) alpha-amylases, b) beta-amylases, c) phosphoglucomutases, d) starch synthases, e) sucrose synthases, f) glucose-6-phosphate-phosphate translocators, g) starch branching enzymes, h) ADP-glucose pyrophosphorylases, i) isoamylases. The scale bar at the bottom represents the average substitutions per amino acid site. (PDF 110 kb