High-throughput transient transformation of Arabidopsis roots enables systematic colocalization analysis of GFP-tagged proteins

Determination of the subcellular localization of an unknown protein is a major step towards the elucidation of its function. Lately, the expression of proteins fused to fluorescent markers has been very popular and many approaches have been proposed to express these proteins. Stable transformation using Agrobacterium tumefaciens generates stable lines for downstream experiments, but is time-consuming. If only colocalization is required, transient techniques save time and effort. Several methods for transient assays have been described including protoplast transfection, biolistic bombardment, Agrobacterium tumefaciens cocultivation and infiltration. In general colocalizations are preferentially performed in intact tissues of the same species, resembling the native situation. High transformation rates were described for cotyledons of Arabidopsis, but never for roots. Here we report that it is possible to transform Arabidopsis root epidermal cells with an efficiency that is sufficient for colocalization purposes

Combination of the ALCR/alcA ethanol switch and GAL4/VP16-UAS enhancer trap system enables spatial and temporal control of transgene expression in **Arabidopsis**

Dermience Alice. A. Esser, K. Von Kellenbach, A. Mehlhorn, et al. (eds.), Feminist Approaches to Interreligious Dialogue – Perspectivas feministas acerca del diálogo interreligioso – Feministische Zugänge zum interreligiösen Dialog (coll. Journal of the European Society of Women in Theological Research, 17), 2009. In: Revue théologique de Louvain, 42ᵉ année, fasc. 1, 2011. pp. 130-131

Over-expression of AtEXLA2 alters etiolated arabidopsis hypocotyl growth

Background and Aims Plant stature and shape are largely determined by cell elongation, a process that is strongly controlled at the level of the cell wall. This is associated with the presence of many cell wall proteins implicated in the elongation process. Several proteins and enzyme families have been suggested to be involved in the controlled weakening of the cell wall, and these include xyloglucan endotransglucosylases/hydrolases (XTHs), yieldins, lipid transfer proteins and expansins. Although expansins have been the subject of much research, the role and involvement of expansin-like genes/proteins remain mostly unclear. This study investigates the expression and function of AtEXLA2 (At4g38400), a member of the expansin-like A (EXLA) family in arabidposis, and considers its possible role in cell wall metabolism and growth. Methods Transgenic plants of Arabidopsis thaliana were grown, and lines over-expressing AtEXLA2 were identified. Plants were grown in the dark, on media containing growth hormones or precursors, or were gravistimulated. Hypocotyls were studied using transmission electron microscopy and extensiometry. Histochemical GUS (β-glucuronidase) stainings were performed. Key Results AtEXLA2 is one of the three EXLA members in arabidopsis. The protein lacks the typical domain responsible for expansin activity, but contains a presumed cellulose-interacting domain. Using promoter::GUS lines, the expression of AtEXLA2 was seen in germinating seedlings, hypocotyls, lateral root cap cells, columella cells and the central cylinder basally to the elongation zone of the root, and during different stages of lateral root development. Furthermore, promoter activity was detected in petioles, veins of leaves and filaments, and also in the peduncle of the flowers and in a zone just beneath the papillae. Over-expression of AtEXLA2 resulted in an increase of >10 % in the length of dark-grown hypocotyls and in slightly thicker walls in non-rapidly elongating etiolated hypocotyl cells. Biomechanical analysis by creep tests showed that AtEXLA2 over-expression may decrease the wall strength in arabidopsis hypocotyls. Conclusions It is concluded that AtEXLA2 may function as a positive regulator of cell elongation in the dark-grown hypocotyl of arabidopsis by possible interference with cellulose metabolism, deposition or its organization

Characterization of a Small Auxin-Up RNA (SAUR)-Like Gene Involved in Arabidopsis thaliana Development

<div><p>The root of Arabidopsis thaliana is used as a model system to unravel the molecular nature of cell elongation and its arrest. From a micro-array performed on roots that were treated with aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, a Small auxin-up RNA (SAUR)-like gene was found to be up regulated. As it appeared as the 76th gene in the family, it was named SAUR76. Root and leaf growth of overexpression lines ectopically expressing SAUR76 indicated the possible involvement of the gene in the division process. Using promoter::GUS and GFP lines strong expression was seen in endodermal and pericycle cells at the end of the elongation zone and during several stages of lateral root primordia development. ACC and IAA/NAA were able to induce a strong up regulation of the gene and changed the expression towards cortical and even epidermal cells at the beginning of the elongation zone. Confirmation of this up regulation of expression was delivered using qPCR, which also indicated that the expression quickly returned to normal levels when the inducing IAA-stimulus was removed, a behaviour also seen in other SAUR genes. Furthermore, confocal analysis of protein-GFP fusions localized the protein in the nucleus, cytoplasm and plasma membrane. SAUR76 expression was quantified in several mutants in ethylene and auxin-related pathways, which led to the conclusion that the expression of SAUR76 is mainly regulated by the increase in auxin that results from the addition of ACC, rather than by ACC itself.</p> </div

Expression analysis of SAUR76.

<p><b>A</b>) In silico expression analysis of SAUR76 using Genevestigator expressed as signal intensities on the 22k array. Expression of SAUR76 seen as GUS activity in promoter-reporter lines, in seedlings (<b>B</b>-<b>C</b>), flowers (<b>D</b>), stamens (<b>E</b>-<b>F</b>), filaments (<b>G</b>), leaves (<b>H</b>) and during several phases of lateral root development (<b>I</b>-<b>L</b>). Scale bar is 100 μm in B and C, 25 μm in I-L.</p

Expression analysis of SAUR76 in control roots and roots treated with ACC and IAA.

<p><b>A</b>) Expression of SAUR76 seen in promoter::GUS (left) and promoter::GFP lines (right) in control conditions. <b>B</b>) Expression of SAUR76 in roots after 3 h treatment with 5μM ACC or C) 5μM IAA. <b>D</b>) Confocal mid-plane longitudinal and <b>E</b>) transverse section in the differentiation zone of untreated promotor::GFP roots and <b>F</b>) after 3, 6 and 24 hrs treatment with 5μM IAA. Scale bars are 100μm in A-C, 30μm in D-E and 20 μm in F.</p

Effect of SAUR76-overexpression on leaf development.

<p><b>A</b>) Area of leaf series of the wild type and two independent overexpression lines. <b>B</b>) Leaf area of WT and 2 overexpressor lines. <b>C</b>) Cell area, <b>D</b>) number of cells and <b>E</b>) stomatatal index in leaf 4 of WT and 2 overexpressor lines. Asterisks represent statistical differences towards the wild type and results are presented as means ± SE.</p