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    1. 1. Lateral Root Development & Root Gravitropism in Arabidopsis thaliana<br />Simon Wright<br />
    2. 2. Lateral Root Development – An Overview<br />Many higher plants use lateral roots in the acquisition of water and nutrients, it is therefore of great agronomic importance to understand the mechanisms that control and regulate their formation(Johnstone, 2011).<br />Lateral root development (LRD) is initiated when pericycle cells which are adjacent xylem pole cells become primed to form founder cells, which have a different developmental fate from the parent cells. This process takes place behind the root apical meristem (RAM) in the basal meristem and is dependent on both temporal oscillations of auxin and a mechanical stimulus.(Johnstone, 2011.Péret, Rybel et al, 2009)<br />Lateral root primordia (LRP) develop through the endodermis cortex and then epidermis in 8 conventional stages. In stage Ievent of LRD, 2 pericycle founder cells undergo simultaneous polarized asymmetric divisions, daughter cells continue to divide accompanied by a series of mitotic divisions by flanking pericycle cell files. Following radial expansion, central daughter cells divide periclinally, creating a primordium consisting of an inner and outer layer, this stage of development is termed stage II. After stage II, the root divides periclinally and anticlinally until a dome shape is formed and the primordium emerges. (Johnstone, 2011.Péret, Rybel et al, 2009)<br />
    3. 3. Lateral Root Development – An Overview<br />Once pericycle cellls are primed, LRD is transcriptionally regulated by auxin in an extensive signal cascade, although data from DR5:GUS & DR5:Luciferase show that auxin functions in tandem with other signals. (Johnstone, 2011)<br />When auxin in present, it binds to a TIR motif signalling for the targeted degradation of AUX proteins via ubiquitination. AUX proteins function to repress a number of key transcription factors, such as ARF7&19, which activate further downstream genes such as LBD16&18. (Johnstone, 2011)<br />During LRD, a substantial amount of cellular remodelling occurs, this is facilitated by cell wall remodelling (CWR) enzymes, which are regulated by SHY2 in the endodermis, and by LAX3 in the cortex and epidermis. Once LRP have emerged, the lateral root meristem is activated, capable of producing its own auxin, independent of the RAM. (Johnstone, 2011)<br />
    4. 4. Project Description in brief<br />1.RNA was extracted and a microarray data set was generated at 3 hour intervals identifying some 27000 potential loci involved with LRD.<br />7.Plants were scored for a phenotype, by measuring root number, length and density<br />8.Seedlings were sampled at 18h and 42h for RNA extraction to determine primordia staging.<br />2.Target transcription factors that were differentially expressed were identified.<br />9.RNA was then extracted.<br />3.T-DNA SALK lines were ordered from NASC.<br />10.cDNA was reverse transcribed from the mRNA and a RT-qPCR assay was performed<br />4.DNA was extracted for genotyping.<br />5.A length based PCR assay was performed, and samples run on ethidiumbromide gels.<br />11.A time courses analysis was performed to determine relative expression levels at time intervals.<br />6.Homozygotes were selected and the F1 seeds were grown on 0.5MS.<br />
    5. 5. Mutant Lines <br />at5g65640<br />At5g23420<br />At5g65310<br />at2g46680<br />at1g73410<br />
    6. 6. Mutant Lines <br />at1g28470<br />At2g24430<br />AT1G75520 <br />AT2G18230<br />AT1G17840<br />AT4G36260<br />
    7. 7. Microarray Dataset – Data from Ute Voss/Ewan Johnstone<br />
    8. 8. Analysis<br />Steep decrease in expression in At1g73410after 42h, At1g73410 involved in regulating transcription and secondary cell wall biogenesis, steep decrease in expression could coincide with root emergence and lower levels of cell wall biogenesis?<br />Levels of At1g73410 remain constant until 12h, suggesting little involvement with initial stages of LRD.<br />Many genes decrease in expression after 42h, these genes are down-regulated once lateral roots fully emerged?<br />At5g65310 levels remain only slightly elevated throughout, At5g65310 involved in positively regulating ABA response, mediating ABA effect during seedling establishment, minoraffect on LRD?<br />Further Comments/thoughts<br />Volume of data was limited by the duration of time worked here, no visual phenotype could be observed reliably as plants with target mutations were not mature enough.<br />Progress was also halted by problems with PCR assays, as inneffective annealing temperatures and primers were used.<br />
    9. 9. Supplementary Gene Information<br />At5g65640 - sequence-specific DNA binding transcription factor , no evidence at present of involvement in LRD<br />At5g65310 - positive regulator of ABA-responsiveness, mediating the inhibitory effect of ABA on growth during seedlings establishment, expressed in root and shoot systems, potential involvement with ABA in LRD?<br />At5g23420 - Binds to supercoiled DNA in vitro, expressed in roots and is involved in the regulation of transciption. HMGB6 is phosphorylated by protein kinase CK2alpha within its acidic C-terminal domain.<br />At2g46680 - encodes a putative transcription factor that contains a homeodomain closely linked to a leucine zipper motif. Transcript is detected in all tissues examined. Is transcriptionally regulated in an ABA-dependent manner and may act in a signal transduction pathway which mediates a drought response.<br />At1g73410 -  transcription factor  of the R2R3-MYB family. Regulation of transciption, secondary cell wall biogenesis? Expressed in roots<br />At1g28470 - NAC domain containing protein, regulation of transciption and biogenesis of secondary cell wall, expressed in roots<br />At2g24430 - NAC domain containing protein, multicellular organismal development, regulation of transcription, expressed in roots<br />AT1G75520 -  SHI gene family, one of ten members, function and synergistically promote gynoecium, stamen and leaf development, no indication of functions related to LRD<br />AT2G18230 GATA23 - Encodes a protein that might have inorganic pyrophosphatase activity. Phosphate metabolism? Expressed in roots.<br />AT1G17840 GATA23 - Encodes a plasma membrane-localized ATP-binding cassette transporter ATP catabolic process, cutin transport, fatty acid transport, response to abscisic acid stimulus, response to salt stress, response to wounding, transmembrane transport. Expressed in roots<br />AT4G36260 GATA23 - SHI gene family, one of ten members, function and synergistically promote gynoecium, stamen and leaf development,  Encodes protein with a single zinc finger motif and a members of a small gene family of putative transcription factors, expressed in roots.<br />
    10. 10. Gravitropism Overview<br />The primary root of plants can correct for changes in orientation allowing maximum possible acquisition of water and nutrients via a process called gravitropism. (Swarup, Bennett. 2010)<br />Gravitropism can be split into 3 phases;1.Gravity Perception-<br /><ul><li>Occurs in S1 & S2 columella cells in the root cap.
    11. 11. Perceived by sedimenting starch filled amyloplasts termed statoliths of columella cells.
    12. 12. Auxin efflux carrier PIN3 is rapidly redistributed creating an auxin response gradient following gravistimulus.
    13. 13. Cytoplasmic pH of columella cells is also increased from 7.2 to 7.6 following gravistimulus.</li></ul>2.Gravitropic Signal Transduction<br /><ul><li>Auxin represents the main candidate for a gravitropic signal shown by gravitropic defects in auxin mutants.
    14. 14. The signal is channelled from columella cells to the elongation zone by auxin influx and efflux carriers AUX1 and PIN2.
    15. 15. Asymmetric localisation of PIN2 to the apical plasma membrane of lateral root cap (LRC) cells directs the auxin gradient to the elongation zone, whilst AUX1is essential for efficient uptake of auxin in LRC cells, elevating levels 15x higher than in passive uptake. (Swarup, Bennett. 2010)</li></li></ul><li>Gravitropism Overview<br />3.Gravitropic Response<br /><ul><li>High levels of cellular auxin in epidermal cells cause differential cell elongation.
    16. 16. Late phase Gravitropic reponse is dependent on auxin regulated transcription.
    17. 17. Auxin binds to AUX proteins degrading them, derepressingauxin response factors (ARF) 7 and 19, initiating the transciption of many target genes, some of which are believed to be cell wall remodelling enzymes involved in cell expansion.
    18. 18. Other signals such as Nitric Oxide and Reactive Oxygen Species (ROS) may alsoplay a role a role downstream of auxin. (Swarup, Bennett. 2010)</li></ul>Recent evidence also suggests the presence of a 4th phase in Gravitropism termed gravitropic signal attenuation which serves to reduce the gravitropic signal preventing further bending. (Swarup, Bennett. 2010)<br />Despite these recent advances, many of the genes regulating Gravitropism are yet to be identified<br />
    19. 19. Project Description – as described by Hussein Hijazi<br />1.A transciptomics data set was generated from control plants and plants given a 90° stimulus.<br />2.RNA was extracted and a microarray was generated, it was found that ~500 genes were differentially expressed.<br />4.Over 24h and using only the naked eye, an initial phenotype was scored to detect any defects in root tip bending.<br />3.Knock-out T-DNA lines of these genes were ordered from NASC. (of the 500 identified, 130-140 were available)<br />6.Phenotypes of homozygotes were rescored in light using the robot and tip angles measured using ImageJsoftware.<br />5.The plants suspected of showing a gravitropic defect were then genotyped and homozygotes for the KO were selected.<br />My Input<br />7.~15 mutant lines that showed significant differences from the wild type (wt) were scored n light using an IR camera.<br />8.Genes of interest were then further analysed using molecular techniques, such as protein fusion constructs, promoter fusions and auxin reporters such as DII:Venus or DR5:Venus for later analysis using confocal microscopy.<br />
    20. 20. Personal Input<br />Methods:<br />1. Mutant and wt plants that showed no initial bend at time 0h were chosen for analysis and recorded for future<br />2.The tip angles were then recorded every 1 hour for a total of 8 hours.<br />3. Tip angles were then averaged for that plate, and a graph was generated comparing the bending in mutant and wt plants. <br />4. Standard deviation calculations and T tests were performed to assess quality and significance of the data respectively.<br />The images were initially analysed using the software Roottrace, however the project demanded a level of precision too high Roottrace to deliver, and despite Roottrace being reprogrammed specifically for the project, Roottrace seemed prone to errors as shown to the left.<br />Several genes were shown to differ significantly from the wt in plants of the same size and age.<br />
    21. 21. Some Data – showing variation in root tip angle with time after a 90 degrees Gravitropism signal<br />
    22. 22. Analysis (Aramennon. 2010) <br />Results forAT4G30290 showing high expression levels in root cap.  Intensive expression at the dividing and elongating regions. Expression ceases after LRP begins to grow.<br />Results showAT4G32460 isprimarily expressed in stele, endodermis, stem epidermis and stigma tissues. Protein of unknown function, gravitropic defect suggests involvement in gravitropism.<br />Arr3,4 double mutant (arr3 =AT1G59940, arr4 = AT1G10470). Arr3 acts redundantly with arr4 in the control of circadian period in a cytokinin-independent manner. Induced rapidly by cytokinin and involved in red light signalling.<br />Arr1 = AT3G16857 , Arr2 = AT4G16110, Arr10 = AT4G31920, Arr12 = AT2G25180. Genes involved in cytokinin signalling pathways. Together with auxin and other phytohormes, cytokinin regulates root gravitropism, supported by findings on previous slide. (Aloni, Langhans et al. 2006)<br />For further evidence, protein fusion constructs need to be made such as DII:Venus for confocal analysis <br />
    23. 23. Further Comments<br />Data only shown if results were significant at a p = 0.05 level shown by t-test, and results only accepted as significant if wt and mutant plants were the same size.<br />Quality of data could be improved by removing human error component associated with ImageJ.<br />Roottrace eliminates human error aspect, however it is not ideal if the project demands absolute precision. It could however be used if the volume of data was large enough for errors to not dramatically affect the averages.<br />Care needs to be taken when plating seeds as a large number of plants grew into the agar making them difficult to clearly visualise.<br />
    24. 24. Acknowledgements<br />Ute Voss and Malcolm Bennett for giving me the invaluable opportunity to gain lab experience, and Ute in particular for her time invested in teaching me some scientific theory and many tasks around the lab.<br />Hussein Hijazi for the time invested teaching me how to use ImageJ and for his general assistance and support during the previous 3 weeks.<br />
    25. 25. References<br />1.Johnstone. E, 2011. Analysis Of Transcription Factors For Lateral Root Development In Arabidopsis thaliana. BSc. University of Nottingham<br />2. Péret, B., B. De Rybel, et al. (2009). "Arabidopsis lateral root development: an emerging story." Trends in Plant Science 14(7): 399-408.<br />3. RanjanSwarup & Malcolm J. Bennett, 2010, Root Gravitropism, Tom Beeckman, 2010, Annual Plant Reviews, Volume 37, RootDevelopment, BlackwellPublishing Ltd, ch. 2. pp. 157 – 192.<br />4. Aramennon, 2010. University of Cologne URL :<br />5. R, Aloni. E, Aloni. M, Langhans. C, I, Ullrich. (2006). Role of Cytokinin and Auxin in Shaping Root Architecture: Regulating Vascular Differentiation, Lateral Root Initiation, Root Apical Dominance and Root Gravitropism . Animals of Botany. 97(5):883-893<br />Google images:<br /> 2031%20Plant%20Structures/Roots/root.gif<br /><br /><br /><br /><br /><br /> <br /> <br /> <br /> <br /><br />