Analysis of Microtubule-Associated-Proteins during IBA-Mediated Adventitious Root Induction Reveals KATANIN Dependent and Independent Alterations of Expression Patterns.

Adventitious roots (AR) are post embryonic lateral organs that differentiate from non-root tissues. The understanding of the molecular mechanism which underlies their differentiation is important because of their central role in vegetative plant propagation. Here it was studied how the expression of different microtubule (MT)-associated proteins (MAPs) is affected during AR induction, and whether expression differences are dependent on MT organization itself. To examine AR formation when MTs are disturbed we used two mutants in the MT severing protein KATANIN. It was found that rate and number of AR primordium formed following IBA induction for three days was reduced in bot1-1 and bot1-7 plants. The reduced capacity to form ARs in bot1-1 was associated with altered expression of MAP-encoding genes along AR induction. While the expression of MAP65-4, MAP65-3, AURORA1, AURORA2 and TANGLED, increased in wild-type but not in bot1-1 plants, the expression of MAP65-8 and MDP25 decreased in wild type plants but not in the bot1-1 plant after two days of IBA-treatment. The expression of MOR1 was increased two days after AR induction in wild type and bot1-1 plants. To examine its expression specifically in AR primordium, MOR1 upstream regulatory sequence was isolated and cloned to regulate GFP. Expression of GFP was induced in the primary root tips and lateral roots, in the pericycle of the hypocotyls and in all stages of AR primordium formation. It is concluded that the expression of MAPs is regulated along AR induction and that reduction in KATANIN expression inhibits AR formation and indirectly influences the specific expression of some MAPs

GFP expressed under the <i>MOR1</i> promoter is upregulated in response to IBA in the root and accumulates in LR and AR primordium.

<p>(A-D) <i>MOR1</i>_<i>pro</i>:<i>GFP</i> seedlings were grown on MS medium for 6 days. Half were then transferred to plates containing MS and 10 μ M IBA for 16h, after which a comparative examination by confocal microscopy was performed. (E-F) In another experiment, etiolated hypocotyls were excised (E) or excised and incubated in MS and 10 μ M IBA for 2d (F). (G) Fluorescence was measured in 3 plants from three independent transgenic lines (total of 9), asterisks show statistically significant differences as measured by Scheffe analysis p<0.05. Scale bars in A-F are 200 μ m.</p

AR induction is hampered in <i>KATANIN</i> mutant plants.

<p>Hypocotyls (5-6mm in length) were excised from etiolated seedlings and incubated in MS with 1% sucrose and 10 μ M IBA for 3 days or 6 days. A-H representative images, scale bar = 500 μ m. I. Quantitative analysis of hypocotyls without roots (no), with emerged roots (root) or with AR primordium (prim). Asterisks show significant difference from control wild-type plants as determined by Scheffe analysis p<0.05. Scale Bar = 500 μ m.</p

The kinetics of expression of various MAP-encoding genes in wild-type and <i>bot1-1</i> plants during AR induction.

<p>RNA was extracted from 3–4 biological replicas at the indicated time points and expression analysis was performed by the nanostring method. Scaling of gene expression signals was performed by subtracting the mean of the log₂ signals. An agglomerative hierarchical algorithm was applied with the “ward” method parameter on both the genes and the samples and a “heatmap” was illustrated by “Heatplus” R-package.</p

Expression of GFP under the <i>MOR1</i>_prom during AR induction.

<p>Etiolated hypocotyls were cut and induced to form AR by 10 μ M IBA. GFP fluorescence was followed before induction (A), and 1, 2, and 3 days post induction (B—E). (G). A hypocotyl that was incubated in MS without IBA for 3 days. The different primordium stages as in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143828#pone.0143828.ref008" target="_blank">8</a>] are shown by arrows. Scale bars in A, E, F are 50 μ M and in B -D are 20 μ M.</p

Statistical analysis of the expression of several MAPs during AR induction in wild-type and <i>bot1-1</i> plants.

<p>Hypocotyls (5-6mm in length) were excised from etiolated seedlings and incubated in MS with 1% sucrose and 10 μ M IBA. RNA was extracted at the indicated time periods and expression of the various transcripts was analyzed in 3–4 biological replicates by the nanostring method. Light and dark green asterisks show statistically significant differences of expression in Col+IBA in relation to <i>bot1-1</i>+IBA or Col respectively, as determined by Scheffe analysis p<0.05. Col = wild type <i>A</i>. <i>thaliana</i> Columbia.</p