In Planta Evaluation of Gene Regulation by Soybean Auxin Response Factors

Nitrogen (N) is an important plant nutrient but its abundance in the soil is not sufficient for profitable crop production. N input in the form of chemical fertilizers helps fill this need; however, an alternative for fertilizers is an immediate need due to the environmental pollution resulting from excessive use of fertilizers. Leguminous plants such as soybean (Glycine max) that form root nodules through symbiotic association with N-fixing rhizobia and therefore need little or no chemical N fertilizers. The plant hormone auxin plays a crucial role in determining the number of nodules and their rate of maturity in soybean. Auxin action is mediated by a group of transcriptional factors named auxin response factors (ARFs) that bind conserved DNA elements named auxin response elements (AuxREs) and regulate gene expression. One of the soybean ARFs, GmARF16-2, is believed to regulate auxinresponsive gene expression during soybean nodule development; however, its mechanism of action is still unclear. This project seeks to resolve the mechanism of GmARF16-2 action using transient expression with agroinfiltration method in Nicotiana benthamiana. Previous studies have characterized GmARF16-2 as a repressor while GmARF8a as an activator ARF. We hypothesized that the balance between GmARF16-2 and GmARF8a plays a role in determining the nature and extent of auxin-responsive gene expression. In this study, GmARF16-2 transactivated direct repeat AuxRE (DR5:GUS) suggesting GmARF16-2 can potentially act as an activator ARF; however, it failed to transactivate evert repeat AuxREs (ER7GG and ER7TC). In contrast, GmARF8a, was able to transactivate evert repeat AuxREs but not with DR5:GUS. It appears that monomeric ARFs not capable of dimerization are more likely to bind and transactivate direct repeat or single-copy AuxREs and ARFs capable of dimerization are more likely to bind and transactivate evert repeat AuxREs. Therefore, the repertoire of genes regulated by each ARF is likely to be determined by the orientation and sequence of the AuxREs present in target genes. A better understanding of how auxin regulates gene expression during nodule development can help devise strategies to optimize the number of nodules and their rate of maturity to enhance nitrogen fixation

Plant lateral organs: development, growth and ufe span

RIASSUNTO Le piante sono fondamentali per il mantenimento del benessere dell\u2019umanit\ue0, in quanto forniscono numerosi servizi indispensabili per il mantenimento di un ecosistema correttamente funzionante (Whelan et al., 2005). Entro il 2050, la popolazione mondiale avr\ue0 raggiunto pi\uf9 o meno 9 miliardi di persone, quindi le richieste di cibo, di materie prime e di energia rinnovabile aumenteranno drasticamente (Grierson et al., 2011). Per soddisfare questa crescente domanda di beni, \ue8 necessaria una forte collaborazione interdisciplinare tra gli scienziati che lavorano per migliorare le colture e gli scienziati che si occupano dell\u2019ambiente, in modo da tradurre specifiche conoscenze di laboratorio in soluzioni pratiche sul campo. In questo contesto, la biologia vegetale svolge un ruolo importante perch\ue9 consente l'identificazione e la manipolazione di caratteri utili e interessanti che possono essere utilizzati nei programmi di breeding per selezionare nuove linee di colture con caratteristiche desiderabili come una minor necessit\ue0 di input lasciando invariata la resa, e una maggior adattabilit\ue0 all\u2019ambiente. Solo lavorando in questa direzione sar\ue0 possibile arginare i problemi dell'attuale pratica agronomica come la perdita di biodiversit\ue0, il degrado del suolo, l'inquinamento chimico e l'esaurimento delle risorse idriche (Khush, 2001). In particolare, i frutti rappresentano la parte pi\uf9 preziosa della produzione agricola. Infatti rappresentano la parte commestibile di molte colture, comprese quelle utilizzate come frutta da dessert (mele, fragole, uva), come verdure (cetrioli, fagioli, pomodori), come fonti di oli culinari (oliva, olio di palma) o per altri prodotti culinari (vaniglia). I frutti sono importanti anche per la produzione di semi (colza, cereali) e diverse sostanze non commestibili (cotone, oli industriali) e possono essere sfruttati per la produzione di molti altri prodotti, compresi quelli farmaceutici. Da un punto di vista botanico, il frutto \ue8 il risultato dello sviluppo dell'ovario dopo l'impollinazione e la fecondazione. Questa struttura rappresenta inoltre una delle principali innovazioni evolutive di Angiosperme (Ferrandiz, 2011). Infatti, i frutti sono essenziali per la riproduzione e l'adattamento delle piante e migliorano notevolmente l'efficienza della dispersione dei semi. La capacit\ue0 dei semi di germogliare e crescere lontano dalla pianta madre ha consentito alle Angiosperme di colonizzare nuove aree, riducendo il rischio di inbreeding e competizione tra sibling. Il presente lavoro pu\uf2 essere diviso in due diverse linee di ricerca. La prima (primo e secondo capitolo) riguarda la regolazione dell'architettura delle piante e dell'attivit\ue0 dei meristemi negli organismi modello Antirrhinum majus e Arabidopsis thaliana. La seconda invece (terzo e quarto capitolo) rappresenta il progetto principale di questa tesi di dottorato e mira a identificare un solido strumento per la delucidazione dei meccanismi molecolari che controllano la formazione dei frutti in Arabidopsis thaliana. Concentrandosi sulla seconda linea di ricerca, per esplorare i meccanismi che controllano la formazione e la maturazione dei frutti, abbiamo eseguito un'analisi trascrittomica sul tessuto delle valve della siliqua di Arabidopsis thaliana, utilizzando la strategia RNAseq. In tal modo, abbiamo generato un set di dati di geni differenzialmente regolati che aiuteranno a chiarire i meccanismi molecolari che sono alla base della fase iniziale della crescita del frutto, e successivamente della fase di maturazione. La robustezza del nostro set di dati \ue8 stata testata atraverso studi di genomica funzionale. Utilizzando un approccio di genetica inversa, abbiamo selezionato 10 geni differenzialmente espressi ed esplorato le conseguenze della loro distruzione sulla crescita e la senescenza delle silique. Abbiamo scoperto che i geni contenuti nel nostro set di dati (codificanti per fattori di trascrizione, proteine del citoscheletro ed enzimi che modulano l'omeostasi degli ormoni) svolgono ruoli essenziali in diversi stadi dello sviluppo e della maturazione della siliqua. Inoltre, dal nostro set di dati, tra i geni down-regolati, abbiamo trovato il gene AUXIN RESPONSE FACTOR 8 (ARF8), il cui trascritto diminuisce costantemente dal primo all'ultimo stadio di sviluppo della siliqua. ARF8 codifica per un fattore di trascrizione che pu\uf2 agire specificamente nel pistillo, in risposta al segnale dell\u2019auxina. L'ormone vegetale auxina regola i principali aspetti dello sviluppo della pianta principalmente attraverso la sua distribuzione differenziale all'interno dei tessuti vegetali. In particolare, ARF8 sembra essere il legame tra il segnale ormonale e il meccanismo molecolare di formazione del frutto (Goetz et al., 2006). In Arabidopsis, la formazione del frutto viene generalmente repressa fino a quando avviene la fecondazione. Tuttavia, nel mutante auxin response factor 8-4 (arf8-4), gi\ue0 precedentemente caratterizzato, sembra che la formazione del frutto sia disgiunta dalla fecondazione, risultando nella formazione di un frutto privo di semi (partenocarpico; Goetz et al., 2006). La struttura che si sviluppa dal pistillo non fecondato del mutante arf8-4, \ue8 stata considerata per anni una silique partenocarpica in quanto risulta pi\uf9 lunga del pistillo non fecondato wild-type e presenta un pattern di deiscenza, processo essenziale per l\u2019apertura del frutto maturo e il rilascio dei semi. Nonostante ci\uf2, nel 2010, Carbonell-Bejerano e collaboratori hanno riferito che esiste un processo di senescenza inerente allo sviluppo che include lo sviluppo della zona di deiscenza e che risulta essere indipendente alla fecondazione. Questo processo di senescenza quindi \ue8 comune tra pistilli fecondati e non fecondati. In linea con questo studio, i nostri risultati suggeriscono che il mutante arf8-4 non presenta un vero fenotipo partenocarpico ma piuttosto mostra un\u2019alterazione nella regolazione del cross-talk ormonale, probabilmente dovuta a una proteina tronca. Questa alterazione pu\uf2 a sua volta influenzare il coordinamento tra crescita e senescenza del pistillo, modificando quindi la corretta progressione dei processi di sviluppo. Per questo motivo, almeno in Arabidopsis, l'unica caratteristica strutturale che differenzia il frutto parthenocarpic di arf8-4 dal pistillo non fecondato di tipo wild-type \ue8 la dimensione aumentata. Ulteriori analisi saranno necessarie per continuare a studiare il fenotipo di arf8-4, incluse analisi molecolari ad alto rendimento (spettrometria di massa) sul contenuto di ormoni nel tessuto delle valve e analisi western-blot per confermare definitivamente la presenza della proteina tronca nelle piante mutanti arf8-4. Referenze Carbonell-Bejerano, P., Urbez, C., Carbonell, J., Granell, A. and Perez-Amador, M.A. (2010) A Fertilization-Independent Developmental Program Triggers Partial Fruit Development and Senescence Processes in Pistils of Arabidopsis. Plant Physiol., 154, 163\u2013172. Ferrandiz, C. (2011) Fruit Structure and Diversity. Encycl. Life Sci. Goetz, M., Vivian-Smith, A., Johnson, S.D. and Koltunow, A.M. (2006) AUXIN RESPONSE FACTOR8 Is a Negative Regulator of Fruit Initiation in Arabidopsis. Plant Cell, 18, 1873\u20131886. Grierson, C.S., Barnes, S.R., Chase, M.W., et al. (2011) One hundred important questions facing plant science research. New Phytol., 192, 6\u201312. Khush, G.S. (2001) Green revolution: the way forward. Nat. Rev. Genet., 2, 815\u2013822. Whelan, C.J., Wenny, D.G. and Marquis, R.J. (2005) Ecosystems and Human Well-being: Synthesis. Isl. Press. Washington, DC, 1\u2013137.ABSTRACT Plants can be consider fundamental for maintaining human well-being, since they provide several benefits that humans freely gain from the natural environment and from properly functioning ecosystems (Whelan et al., 2005). By 2050, the world population will have reached more or less 9 billion people, therefore, the demands for energy-intensive food, shelter, clothes, fibre, and renewable energy will dramatically increase (Grierson et al., 2011). To satisfy such increasing goods demand it requires a strong interdisciplinary collaboration between plant scientists, working to improve crop, and environmental scientists, working on environmental stability to translate the specific knowledge into field-based solutions. In this contest, plant developmental biology has an important role because it allow the identification and manipulation of useful and interesting traits which then can be used for breading programs to select new crop cultivars that need less inputs and are adapted to live in their environment. So they can help to overcome the problems of current agronomic practice like loss of biodiversity, soil degradation, chemical pollution and depletion of water resources (Khush, 2001). Particularly, fruit represent the most valuable part of crop production. Actually, they are the edible part of many crops, including those used as dessert fruits (apples, strawberries, grapes), as vegetables (cucumbers, beans, tomatoes), as sources of culinary oils (olive, oil palm), or for other culinary products (vanilla). Fruits are also important for seed production (canola, cereals) and several non-edible substances (cotton, industrial oils), and can be adapted to the production of many other products, including pharmaceuticals. From a botanical point of view, fruit is the result of the development of ovary after pollination and fertilization and it represent a major evolutionary innovation of Angiosperms (Ferrandiz, 2011). Actually, fruits are essential for plant reproduction and adaptation, and greatly enhance the efficiency of seed dispersal. The ability of the seeds to germinate and grow far away from the parent plant allows Angiosperms to colonize new areas, reducing the risk of inbreeding and sibling competition. The present work can be divided in two different research lines. The first one (first and second chapters) concerns the regulation of plant architecture and meristem activity in the model organisms Antirrhinum majus and Arabidopsis thaliana. The second one (third and fourth chapters) represents the main project of this PhD thesis and it aims to identify a powerful tool for the elucidation of the molecular mechanisms controlling fruit formation in Arabidopsis thaliana. Focusing on the second research line, to explore the mechanisms controlling fruit formation and maturation, we performed a transcriptomic analysis on the valve tissue of the Arabidopsis thaliana silique, using the RNAseq strategy. In doing so, we have generated a dataset of differentially regulated genes that will help to elucidate the molecular mechanisms that underpin the initial phase of fruit growth, and subsequently trigger fruit maturation. The robustness of our dataset has been tested by functional genomic studies. Using a reverse genetics approach, we selected 10 differentially expressed genes and explored the consequences of their disruption for both silique growth and senescence. We found that genes contained in our dataset (encoding for transcription factors, cytoskeletal proteins, and enzymes that modulate hormone homeostasis) play essential roles in different stages of silique development and maturation. Moreover, from our dataset, among down-regulated genes, we found the AUXIN RESPONSE FACTOR 8 (ARF8) gene, whose transcript diminishes steadily from the first time-point to the last. ARF8 encodes for a transcription factor that can act specifically in the pistil, in response to auxin signal. The plant hormone auxin regulates the major aspects of plant development mainly through its differential distribution within plant tissues. Particularly, ARF8 seems to be the link between hormone and molecular mechanism in fruit initiation (Goetz et al., 2006). In Arabidopsis, fruit initiation is generally repressed until fertilization occurs. However, in the already characterized auxin response factor 8-4 (arf8-4) mutant, it seems that fruit initiation is uncoupled from fertilization, resulting in the formation of seedless fruit (parthenocarpic fruit), if fertilization is prevented before anthesis with the removal of anthers (Goetz et al., 2006). The structure that develops from arf8-4 unfertilized pistil, has been considered for years a parthenocarpic silique because it is longer than wild-type unfertilized pistil and it shows a dehiscence pattern. However, in 2010 Carbonell-Bejerano and collaborators reported that there is a developmental senescence program (that includes the development of the dehiscence zone) which is independent form fertilization and so it is in common between seeded and unfertilized Arabidopsis pistils. In line with this study, our findings suggest that arf8-4 mutant has not a real parthenocarpic phenotype but rather it shows a mis-regulation in the hormones crosstalk, likely due to a truncated protein. This alteration can affect the coordination between growth and senescence of the pistil, modifying the correct progression of the developmental processes. For this reason, at least in Arabidopsis, the only structural characteristic that differentiates arf8-4 parthenocarpic fruit from wild-type unfertilized pistil is the increased size. Further analyses will be necessary to continue investigating arf8-4 phenotype, including high-throughput molecular analyses (mass-spectrometry) about hormones content in the valve tissue and western-blot analysis to confirm definitely the presence of the truncated protein in arf8-4 plants. Overall, the main outcome of this work was that the transcriptome-based gene list on the valve tissue of the Arabidopsis thaliana silique represents a powerful tool for the elucidation of the molecular mechanisms controlling fruit formation. References Carbonell-Bejerano, P., Urbez, C., Carbonell, J., Granell, A. and Perez-Amador, M.A. (2010) A Fertilization-Independent Developmental Program Triggers Partial Fruit Development and Senescence Processes in Pistils of Arabidopsis. Plant Physiol., 154, 163\u2013172. Ferrandiz, C. (2011) Fruit Structure and Diversity. Encycl. Life Sci. Goetz, M., Vivian-Smith, A., Johnson, S.D. and Koltunow, A.M. (2006) AUXIN RESPONSE FACTOR8 Is a Negative Regulator of Fruit Initiation in Arabidopsis. Plant Cell, 18, 1873\u20131886. Grierson, C.S., Barnes, S.R., Chase, M.W., et al. (2011) One hundred important questions facing plant science research. New Phytol., 192, 6\u201312. Khush, G.S. (2001) Green revolution: the way forward. Nat. Rev. Genet., 2, 815\u2013822. Whelan, C.J., Wenny, D.G. and Marquis, R.J. (2005) Ecosystems and Human Well-being: Synthesis. Isl. Press. Washington, DC, 1\u2013137

Control de la proliferación y diferenciación celular en plantas por microARNs

El tamaño final de los órganos está determinado mayormente por la extensión de una fase inicial de proliferación celular, seguida de una fase de expansión y diferenciación celular. El control de estos procesos involucra la acción concertada de varias vías de señalización hormonal y de redes de factores de transcripción, los cuales pueden, a su vez, ser regulados por miARNs. Los microARNs (miARNs) son ARN pequeños de 20-21 nucleótidos y constituyen un sistema ampliamente distribuido para controlar la expresión génica.El miARN miR396 regula los factores de transcripción de la familia GROWTH REGULATING FACTORs (GRFs). Estos factores de transcripción se caracterizan por presentar dos dominios conservados llamados WRC y QLQ, que median la unión al ADN y la interacción con otras proteínas, respectivamente. En Arabidopsis thaliana, siete de los nueve GRFs son regulados por miR396. A su vez, los GRFs forman complejos con los GRF-INTERACTING FACTORS (GIFs). El sistema miR396/GRFs se encuentra conservado en angiospermas y gimnospermas. Los GRFs estimulan la proliferación celular, mientras que la represión de los mismos por el miARN miR396 detiene la división celular y dispara la diferenciación. Durante el desarrollo de la hoja este sistema regulatorio tiene un rol fundamental en la determinación el tamaño y forma del órgano, así como también participa en el control de la senescencia.En este trabajo de tesis, en primer lugar, se analizó el control del balance miR396/GRFs como potencial herramienta biotecnológica. Obtuvimos plantas con hojas más grandes a través de la reducción de la actividad de miR396. Demostramos que versiones resistentes a la regulación de miR396 de los genes de Arabidopsis thaliana AtGRF2 (rGRF2) y AtGRF3 (rGRF3) aumentan el tamaño de las hojas, pero que rGRF3 resulta ser más eficiente. La introducción de At-rGRF3 en Brassica oleracea produce aumento de hojas, raíz y semillas. Además, cuando genes homólogos At-rGRF3 de soja y arroz son introducidos en Arabidopsis, también se incrementa el tamaño de la hoja. Esto sugiere que la regulación de miR396 sobre GRF3 es importante para el control del crecimiento de los órganos en un amplio rango de especies. Las plantas que expresan rGRF3 presentaron hojas más grandes que las silvestres, incluso en condiciones de estrés por sequía, estado en el cual se estimula la expresión de miR396. Por otro lado, se analizó la evolución de la regulación de miR396 sobre los GRFs y se encontró que la relación se encuentra ampliamente conservada evolutivamente. En la mayoría de las especies de dicotiledóneas todos los genes GRF están regulados por miR396. Sin embargo, observamos que dentro de las Brasicáceas existe un subgrupo de genes GRFs que han perdido dicha regulación. En Arabidopsis thaliana, estos genes son GRF5 y GRF6. Posiblemente, el subgrupo de GRFs no regulados por miR396 provenga de un único ancestro común que perdió originalmente la regulación. Estudios sobre AtGRF5 muestran que presenta funciones redundantes con los GRFs involucrados en el control del tamaño de la hoja y regulados por miR396. Análisis del promotor de GRF5 mostraron secuencias altamente conservadas en las Brasicáceas. En particular, la eliminación de uno de estos motivos conservados causa la expresión ectópica de GRF5. Estos resultado sugieren que la expresión de GRF5 se controla por represión transcripcional, en contraste con los otros GRFs reprimidos post-transcripcionalmente por miR396.A partir del análisis de distintos candidatos, identificamos que el represor transcripcional es el factor de transcripción AUXIN RESPONSE FACTOR2 (ARF2). Mutantes por perdida de función de arf2 presentan efectos fenotípicos pleiotrópicos, incluyendo hojas más grandes, que se asemejan a los observados en plantas que sobreexpresan GRF5. Un análisis de mutantes arf2 reveló que presentan incremento de los niveles de expresión de GRF5 y otros GRFs. Finalmente, a través de análisis genéticos y moleculares describimos como ARF2 controla la proliferación celular en hojas a través de GRF5. En forma más general, describimos una red regulatoria donde se vincula a ARF2 con el sistema de miR396/GRFs/GIFs en el control de la proliferación celular durante el desarrollo de las hojas.Fil: Beltramino, Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Structural basis for specific gene regulation by Auxin Response Factors

Auxin is a plant hormone that triggers a broad variety of responses during plant development. These responses range from correct cell division patterns during embryogenesis to formation and growth of different organs. Due to its importance for plant growth and development, many aspects of the biology of auxin have been studied. In Chapter 2, we use Arabidopsis embryogenesis as a stage to describe generalities about its biosynthesis, transport, components of its signaling pathway and transcriptional control of some know target genes. As most of the players involved in transcriptional regulation in response to auxin have been identified, the question of how the same signal can elicit so many different responses remains open. In this thesis we approach this issue by focusing on the ultimate effectors of the auxin signaling pathway: the ARF family of transcription factors. In Chapter 3 we present the crystal structure of the DNA binding Domain (DBD) of two divergent members of the family: ARF1 and ARF5. Careful observation of the structures, followed by in vitro and in vivo experiments led to the following conclusions: 1) ARF DBDs dimerize through a conserved alpha-helix, and bind cooperatively to an inverted repeat of the canonical TGTCTC AuxRE. Dimerization of this domain is important for high-affinity DNA binding and in vivo activity. 2) Monomeric ARFs have the same binding preference for the DNA sequence TGTCGG (determined by protein binding microarray). 3) DNA-contacting residues are almost completely conserved within the ARF family members. 4) The distance between the AuxREs may play a role for binding of specific ARF dimers as for example, ARF5 can accommodate and bind to different spacing (6-9 bp) compared to ARF1 which is more rigid (7-8 bp). In Chapter 4 we follow up on the observations made. First we again used structural biology to determine the reason of the high binding affinity to the TGTCGG sequence compared to the previously identified canonical TGTCTC element. We found that in complex with TGTCGG, His137 (ARF1) could rotate and make hydrogen bonds with either G5 or G6, as well as a hydrogen bond with the C opposing to G6. This rotation is not possible when in complex with TGTCTC and there the same histidine can make only one hydrogen bond with the G opposing to C6. We conclude then that this histidine plays a role in determining the strength of binding to TGTCNN elements and that this also reflects in its specific transcriptional activity as mutating the corresponding histidine in ARF5 renders a semi-functional protein in vivo (Chapter 3). The next observation we followed up in Chapter 4 is the biological meaning of ARF DBDcooperative binding to DNA. We identified AuxRE inverted repeats (IR) in the promoter of the TMO5 gene and mutated them. This brought the expression of the gene to very low levels despite the presence of other multiple single AuxREs. Thus, the single inverted AuxRE repeat in the TMO5 promoter is essential for ARF5 binding and gene regulation. Importantly, mutating only a single AuxRE element within the inverted repeat led to very pronounced loss of activity, consistent with requirement of both AuxRE sites for high-affinity ARF5 binding. We then concluded that IR AuxREs have a significant effect in gene regulation by ARFs. Next we search the genome for bipartite AuxREs that correlated to auxin response and found two main elements: inverted repeat with 8 bases of spacing (IR8) and direct repeat with 5 bases of spacing (DR5). As this kind of bipartite AuxREs are rarer to find than single AuxREs, we tested their presence in promoters as predictors of auxin responsiveness by qPCR. We found that about 75% of the selected genes containing either IR8 or DR5 responded to auxin. The expression study also show that genes containing the DR5 sequence were only up-regulated when regulated. Interestingly, Surface Plasmon Resonance study showed that only class A (activator) ARFs can bind the DR5 sequence cooperatively. As the structural differences of ARFs DBDs are subtle, we then asked if specific gene targeting is determined by this domain alone. In Chapter 5 we used a DBD swap experiment and conclude that the DBD is necessary for specific gene targeting but not sufficient and the other domains of an ARF also contribute in its specific activity. In Chapter 5 we expand our focus from the DBD to the other ARF domains, Middle Region (MR) and C-terminal (CT). As ARFs have protein-protein interaction interfaces in all three domains, we expressed the isolated domains of ARF5 and perform immuno-precipitation followed by tandem mass-spectrometry. Although the procedure needs optimization, some interactions expected for each domain could be identified. The DBD showed to interact with the general transcription machinery and the CT could interact with another ARF and 3 Aux/IAA. These interactions seem to be specific as the Aux/IAA recovered are not the most abundant in the sampled tissue. Finally, in Chapter 6 all the obtained results are put in a broader context and new questions derived from our results are proposed.</p

Computational Methods for the Analysis of Genomic Data and Biological Processes

In recent decades, new technologies have made remarkable progress in helping to understand biological systems. Rapid advances in genomic profiling techniques such as microarrays or high-performance sequencing have brought new opportunities and challenges in the fields of computational biology and bioinformatics. Such genetic sequencing techniques allow large amounts of data to be produced, whose analysis and cross-integration could provide a complete view of organisms. As a result, it is necessary to develop new techniques and algorithms that carry out an analysis of these data with reliability and efficiency. This Special Issue collected the latest advances in the field of computational methods for the analysis of gene expression data, and, in particular, the modeling of biological processes. Here we present eleven works selected to be published in this Special Issue due to their interest, quality, and originality

Unravelling the regulatory network controlling lateral root hydropatterning

The 3-dimensional shape of a root system is of crucial importance to its ability to take up nutrients and water. As these resources are heterogeneously distributed in the soil, plants need to adapt their root growth to aid foraging. One such adaptive response is termed lateral root (LR) hydropatterning where roots branch towards areas with higher water availability. The main aim of this thesis is to investigate how plant roots sense water distribution and which regulatory pathways control lateral root branching towards available water using the model plant Arabidopsis thaliana. We observed that the choice of lateral root founder cells (LRFC) in the pericycle cell layer they originate from is influenced by external water availability. This allows lateral roots to angle towards water from the very first round of formative cell divisions. Additionally, the emerging lateral root primordium grows towards water availability through asymmetric rounds of cell division in its primordium flanks. LR hydropatterning is genetically regulated through a major regulator of LR initiation AUXIN RESPONSE FACTOR7 (ARF7). Knock-out mutants lose the ability to branch towards water and do not asymmetrically express the key transcription factor LBD16-GFP, a direct target for ARF7. This mechanism is regulated through post-translational regulation of ARF7. The ARF7 sequence contains four sites that can be SUMOylated. Transgenic lines expressing ARF7 with mutations in each of these four SUMO sites cannot rescue arf7-1 LR hydropatterning, revealing a key role for SUMOylation controlling water sensing by roots. ARF7 SUMO status appears to be controlled by SUMO protease OVERLY TOLERANT TO SALT1 (OTS1). Double knock-out mutants in OTS1 and its close homolog OTS2 have severely delayed root development and a LR hydropatterning defect. Additionally, ots1 ots2 mutants display reduced LR initiation and emergence defects that can be restored to Wild-Type levels by expressing an OTS1-Venus transgene. OTS1-Venus can be detected from the late elongation zone onwards in root pericycle cells and is stably expressed in the primordia. However, no asymmetrical localisation of OTS1-Venus is observed after a hydropatterning cue suggesting that SUMO protease activity, rather than stability, controls LR hydropatterning. This thesis highlights the early response of lateral roots to asymmetrical water distribution and role the deSUMOylation machinery plays in its molecular regulation

Unravelling the regulatory network controlling lateral root hydropatterning

The 3-dimensional shape of a root system is of crucial importance to its ability to take up nutrients and water. As these resources are heterogeneously distributed in the soil, plants need to adapt their root growth to aid foraging. One such adaptive response is termed lateral root (LR) hydropatterning where roots branch towards areas with higher water availability. The main aim of this thesis is to investigate how plant roots sense water distribution and which regulatory pathways control lateral root branching towards available water using the model plant Arabidopsis thaliana. We observed that the choice of lateral root founder cells (LRFC) in the pericycle cell layer they originate from is influenced by external water availability. This allows lateral roots to angle towards water from the very first round of formative cell divisions. Additionally, the emerging lateral root primordium grows towards water availability through asymmetric rounds of cell division in its primordium flanks. LR hydropatterning is genetically regulated through a major regulator of LR initiation AUXIN RESPONSE FACTOR7 (ARF7). Knock-out mutants lose the ability to branch towards water and do not asymmetrically express the key transcription factor LBD16-GFP, a direct target for ARF7. This mechanism is regulated through post-translational regulation of ARF7. The ARF7 sequence contains four sites that can be SUMOylated. Transgenic lines expressing ARF7 with mutations in each of these four SUMO sites cannot rescue arf7-1 LR hydropatterning, revealing a key role for SUMOylation controlling water sensing by roots. ARF7 SUMO status appears to be controlled by SUMO protease OVERLY TOLERANT TO SALT1 (OTS1). Double knock-out mutants in OTS1 and its close homolog OTS2 have severely delayed root development and a LR hydropatterning defect. Additionally, ots1 ots2 mutants display reduced LR initiation and emergence defects that can be restored to Wild-Type levels by expressing an OTS1-Venus transgene. OTS1-Venus can be detected from the late elongation zone onwards in root pericycle cells and is stably expressed in the primordia. However, no asymmetrical localisation of OTS1-Venus is observed after a hydropatterning cue suggesting that SUMO protease activity, rather than stability, controls LR hydropatterning. This thesis highlights the early response of lateral roots to asymmetrical water distribution and role the deSUMOylation machinery plays in its molecular regulation