Coordination of Tissue Cell Polarity by Auxin Transport and Signaling

Plants coordinate the polarity of hundreds of cells during vein formation, but how they do so is unclear. The prevailing hypothesis proposes that GNOM, a regulator of membrane trafficking, positions PIN-FORMED auxin transporters to the correct side of the plasma membrane; the resulting cell-to-cell, polar transport of auxin would coordinate tissue cell polarity and induce vein formation. Contrary to predictions of the hypothesis, we find that vein formation occurs in the absence of PIN-FORMED or any other intercellular auxin-transporter; that the residual auxin-transport-independent vein-patterning activity relies on auxin signaling; and that a GNOM-dependent signal acts upstream of both auxin transport and signaling to coordinate tissue cell polarity and induce vein formation. Our results reveal synergism between auxin transport and signaling, and their unsuspected control by GNOM in the coordination of tissue cell polarity during vein patterning, one of the most informative expressions of tissue cell polarization in plants

A coherent feed-forward loop drives vascular regeneration in damaged aerial organs of plants growing in a normal developmental context

Aerial organs of plants, being highly prone to local injuries, require tissue restoration to ensure their survival. However, knowledge of the underlying mechanism is sparse. In this study, we mimicked natural injuries in growing leaves and stems to study the reunion between mechanically disconnected tissues. We show that PLETHORA (PLT) and AINTEGUMENTA (ANT) genes, which encode stem cell-promoting factors, are activated and contribute to vascular regeneration in response to these injuries. PLT proteins bind to and activate the CUC2 promoter. PLT proteins and CUC2 regulate the transcription of the local auxin biosynthesis gene YUC4 in a coherent feed-forward loop, and this process is necessary to drive vascular regeneration. In the absence of this PLT-mediated regeneration response, leaf ground tissue cells can neither acquire the early vascular identity marker ATHB8, nor properly polarise auxin transporters to specify new venation paths. The PLT-CUC2 module is required for vascular regeneration, but is dispensable for midvein formation in leaves. We reveal the mechanisms of vascular regeneration in plants and distinguish between the wound-repair ability of the tissue and its formation during normal development.Peer reviewe

Patterning of Leaf Vein Networks by Convergent Auxin Transport Pathways

<div><p>The formation of leaf vein patterns has fascinated biologists for centuries. Transport of the plant signal auxin has long been implicated in vein patterning, but molecular details have remained unclear. Varied evidence suggests a central role for the plasma-membrane (PM)-localized PIN-FORMED1 (PIN1) intercellular auxin transporter of <i>Arabidopsis thaliana</i> in auxin-transport-dependent vein patterning. However, in contrast to the severe vein-pattern defects induced by auxin transport inhibitors, <i>pin1</i> mutant leaves have only mild vein-pattern defects. These defects have been interpreted as evidence of redundancy between PIN1 and the other four PM-localized PIN proteins in vein patterning, redundancy that underlies many developmental processes. By contrast, we show here that vein patterning in the Arabidopsis leaf is controlled by two distinct and convergent auxin-transport pathways: intercellular auxin transport mediated by PM-localized PIN1 and intracellular auxin transport mediated by the evolutionarily older, endoplasmic-reticulum-localized PIN6, PIN8, and PIN5. PIN6 and PIN8 are expressed, as PIN1 and PIN5, at sites of vein formation. <i>pin6</i> synthetically enhances <i>pin1</i> vein-pattern defects, and <i>pin8</i> quantitatively enhances <i>pin1pin6</i> vein-pattern defects. Function of <i>PIN6</i> is necessary, redundantly with that of <i>PIN8</i>, and sufficient to control auxin response levels, PIN1 expression, and vein network formation; and the vein pattern defects induced by ectopic <i>PIN6</i> expression are mimicked by ectopic <i>PIN8</i> expression. Finally, vein patterning functions of <i>PIN6</i> and <i>PIN8</i> are antagonized by <i>PIN5</i> function. Our data define a new level of control of vein patterning, one with repercussions on other patterning processes in the plant, and suggest a mechanism to select cell files specialized for vascular function that predates evolution of PM-localized PIN proteins.</p> </div

PIN8 expression in leaf development.

<p>(A–F,H–K) Top right: genotype, leaf age in days after germination (DAG). Bottom left: reproducibility index. Confocal laser scanning microscopy without (A–F) or with (H–K) transmitted light; first leaves. (A–E) Green: PIN8::PIN8:GFP expression; magenta: chlorophyll. (F) Expression at 3.25 DAG of PIN8::PIN8:GFP (left), staining by ER-Tracker Red (centre) and their overlay displayed with a dual-channel LUT (defined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003294#pgen-1003294-g002" target="_blank">Figure 2</a>) (right). (G) 5-DAG first leaf illustrating positions of close-ups in (D) and (K). (H–K) PIN6::YFPnuc expression. Bars: (A,H) 10 µm; (B–E,I–K) 50 µm; (F) 2 µm.</p

Control of <i>PIN1</i>-dependent vein patterning by intracellular auxin levels.

<p>(A) Percentages of leaves in phenotype classes (defined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003294#pgen-1003294-g001" target="_blank">Figure 1</a>). Difference between <i>pin1</i> and WT, between <i>pin1</i>;<i>6</i> and <i>pin1</i>, and between PIN6::iaaL;<i>pin1</i> and <i>pin1</i> was significant at <i>P</i><0.001 (***) by Kruskal-Wallis and Mann-Whitney test with Bonferroni correction. Sample population sizes: WT, 50; PIN6::iaaL-15, 50; PIN6::iaaL-12, 50; <i>pin1</i>, 61; <i>pin1</i>;<i>6</i>, 61; PIN6::iaaL-15;<i>pin1</i>, 60; PIN6::iaaL-12;<i>pin1</i>, 62. (B,C) Dark-field illumination of cleared mature first leaves. Top right: genotype. Bottom left: phenotype class. Bars: (B,C) 2 mm.</p

Sufficiency of <i>PIN5</i>, <i>PIN6</i>, and <i>PIN8</i> for vein network formation.

<p>(A–G,J–P) Top right: genotype, markers and leaf age in days after germination (DAG). Bottom left: reproducibility index. (A–G) Confocal laser scanning microscopy; first leaves. LUT (in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003294#pgen-1003294-g002" target="_blank">Figure 2K</a>) visualizes expression levels. (J–P) Dark-field (J–L,N,P) or differential-interference-contrast (M,O) illumination of cleared mature first leaves. (A,B) DR5rev::YFPnuc expression. (C,D) ATHB8::CFPnuc expression. Magenta line connects nuclei in the first loop (l1). Dotted line: leaf outline. (E–G) PIN1::PIN1:YFP expression. (H) 4-DAG first leaf illustrating positions of close-ups in (A–D), (E–G) and (M,O). (I) Percentage of first leaves with 0, 1, 2 or ≥3 open loops. Difference between MP::PIN6 and WT, and between MP::PIN8 and WT was significant at <i>P</i><0.05 (*) or <i>P</i><0.001 (***) by Kruskal-Wallis and Mann-Whitney test with Bonferroni correction. Sample population sizes: WT, 43; MP::PIN6-38, 40; MP::PIN6-26, 42; MP::PIN8-10, 40; MP::PIN8-4, 40. (Q) Vein network complexity as mean ± SE number of vein branching points per first-leaf area unit in mm²<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003294#pgen.1003294-Candela1" target="_blank">[28]</a>. Difference between MP::PIN5 and WT was significant at <i>P</i><0.001 (***) by unpaired, two-tailed <i>t</i>-test. Sample population sizes: WT, 28; MP::PIN5, 28. l1, first loop; l2, second loop; l3, third loop. Bars: (A,B) 25 µm; (C–G) 50 µm; (J–L,N,P) 1.5 mm; (M,O) 0.2 mm.</p

Genetic interaction between <i>PIN1</i>, <i>PIN5</i>, <i>PIN6</i>, and <i>PIN8</i> in vein patterning.

<p>Percentages of leaves in phenotype classes (defined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003294#pgen-1003294-g001" target="_blank">Figure 1</a>). Difference between <i>pin1</i>;<i>6</i>;<i>8</i> and <i>pin1</i>;<i>6</i>, and between <i>pin1</i>;<i>5</i>;<i>6</i>;<i>8</i> and <i>pin1</i>;<i>6</i>;<i>8</i> was significant at <i>P</i><0.05 (*) or <i>P</i><0.001 (***) by Kruskal-Wallis and Mann-Whitney test with Bonferroni correction. Sample population sizes: <i>pin1</i>;<i>6</i>, 114; <i>pin1</i>;<i>5</i>;<i>6</i>, 92; <i>pin1</i>;<i>6</i>;<i>8</i>, 95; <i>pin1</i>;<i>5</i>;<i>6</i>;<i>8</i>, 114.</p

Vein patterning functions of Arabidopsis <i>PIN</i> genes.

<p>(A,B) Vein pattern of WT mature first leaf. In (A), dark grey, midvein; grey, loops; light grey, minor veins. (B–H) Dark-field illumination of cleared mature first leaves illustrating phenotype classes: unbranched, narrow midvein and scalloped vein-network outline (B); bifurcated midvein and scalloped vein-network outline (C); fused leaves with scalloped vein-network outline (D); conspicuous marginal vein (E); fused leaves with conspicuous marginal vein (F); wide midvein (G); fused leaves with wide midvein (H). (I) Percentages of leaves in phenotype classes. Difference between <i>pin1</i> and WT, between <i>pin1</i>;<i>6</i> and <i>pin1</i>, and between UBQ10::amiPIN6;<i>pin1</i> and <i>pin1</i> was significant at <i>P</i><0.001 (***) by Kruskal-Wallis and Mann-Whitney test with Bonferroni correction. Sample population sizes: WT, 65; <i>pin2</i>, 68; <i>pin3</i>, 68; <i>pin4</i>, 68; <i>pin5</i>, 68; <i>pin6</i>, 68; UBQ10::amiPIN6-5, 65; UBQ10::amiPIN6-10, 65; <i>pin7</i>, 68; <i>pin8</i>, 68; <i>pin1</i>, 71; <i>pin1</i>;<i>2</i>, 71; <i>pin1</i>;<i>3</i>, 77; <i>pin1</i>;<i>4</i>, 69; <i>pin1</i>;<i>5</i>, 72; <i>pin1</i>;<i>6</i>, 65; UBQ10::amiPIN6-5;<i>pin1</i>, 65; UBQ10::amiPIN6-10;<i>pin1</i>, 67; <i>pin1</i>;<i>7</i>, 77; <i>pin1</i>;<i>8</i>, 68. Bars: (B–F) 1.5 mm; (G,H) 0.75 mm.</p