Transverse sectioning of Arabidopsis thaliana leaves using resin embedding

The leaf is the major functional part of the shoot performing the bulk of photosynthetic activity. Its development is relatively plastic allowing the plant to adapt to environmental changes by modifying leaf size and anatomy. Moreover, a leaf is made up of various distinct cell layers, each having specialized functions. To understand functional adaptation and the development of the leaf it is essential to obtain cross sections throughout leaf development and at maturity (Kalve et al., 2014). Here, we describe a protocol for transverse sectioning of Arabidopsis thaliana leaves using resin embedding. This protocol provides a reliable platform to yield high quality images of cross sections allowing study of development of various tissue layers across the transversal axis of the leaf. As this method is an adaptation of the protocol developed for the Arabidopsis root tip by Beeckman and Viane (1999) and De Smet et al. (2004), it can easily be modified to accommodate other organs and species

Alteration in Auxin Homeostasis and Signaling by Overexpression Of PINOID Kinase Causes Leaf Growth Defects in Arabidopsis thaliana

In plants many developmental processes are regulated by auxin and its directional transport. PINOID (PID) kinase helps to regulate this transport by influencing polar recruitment of PIN efflux proteins on the cellular membranes. We investigated how altered auxin levels affect leaf growth in Arabidopsis thaliana. Arabidopsis mutants and transgenic plants with altered PID expression levels were used to study the effect on auxin distribution and leaf development. Single knockouts showed small pleiotropic growth defects. Contrastingly, several leaf phenotypes related to changes in auxin concentrations and transcriptional activity were observed in PID overexpression (PIDOE) lines. Unlike in the knockout lines, the leaves of PIDOE lines showed an elevation in total indole-3-acetic acid (IAA). Accordingly, enhanced DR5-visualized auxin responses were detected, especially along the leaf margins. Kinematic analysis revealed that ectopic expression of PID negatively affects cell proliferation and expansion rates, yielding reduced cell numbers and small-sized cells in the PIDOE leaves. We used PIDOE lines as a tool to study auxin dose effects on leaf development and demonstrate that auxin, above a certain threshold, has a negative affect on leaf growth. RNA sequencing further showed how subtle PIDOE-related changes in auxin levels lead to transcriptional reprogramming of cellular processes

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

Perturbation of Auxin Homeostasis and Signaling by PINOID Overexpression Induces Stress Responses in Arabidopsis

Under normal and stress conditions plant growth require a complex interplay between phytohormones and reactive oxygen species (ROS). However, details of the nature of this crosstalk remain elusive. Here, we demonstrate that PINOID (PID), a serine threonine kinase of the AGC kinase family, perturbs auxin homeostasis, which in turn modulates rosette growth and induces stress responses in Arabidopsis plants. Arabidopsis mutants and transgenic plants with altered PID expression were used to study the effect on auxin levels and stress-related responses. In the leaves of plants with ectopic PID expression an accumulation of auxin, oxidative burst and disruption of hormonal balance was apparent. Furthermore, PID overexpression led to the accumulation of antioxidant metabolites, while pid knockout mutants showed only moderate changes in stress-related metabolites. These physiological changes in the plants overexpressing PID modulated their response toward external drought and osmotic stress treatments when compared to the wild type. Based on the morphological, transcriptome, and metabolite results, we propose that perturbations in the auxin hormone levels caused by PID overexpression, along with other hormones and ROS downstream, cause antioxidant accumulation and modify growth and stress responses in Arabidopsis. Our data provide further proof for a strong correlation between auxin and stress biology

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