The Maize (<i>Zea mays</i> L.) <i>AUXIN/INDOLE-3-ACETIC ACID</i> Gene Family: Phylogeny, Synteny, and Unique Root-Type and Tissue-Specific Expression Patterns during Development

<div><p>The plant hormone auxin plays a key role in the coordination of many aspects of growth and development. <i>AUXIN/INDOLE-3-ACETIC ACID</i> (<i>Aux/IAA</i>) genes encode instable primary auxin responsive regulators of plant development that display a protein structure with four characteristic domains. In the present study, a comprehensive analysis of the 34 members of the maize <i>Aux/IAA</i> gene family was performed. Phylogenetic reconstructions revealed two classes of Aux/IAA proteins that can be distinguished by alterations in their domain III. Seven pairs of paralogous maize Aux/IAA proteins were discovered. Comprehensive root-type and tissue-specific expression profiling revealed unique expression patterns of the diverse members of the gene family. Remarkably, five of seven pairs of paralogous genes displayed highly correlated expression patterns in roots. All but one (<i>ZmIAA23</i>) tested maize <i>Aux/IAA</i> genes were auxin inducible, displaying two types of auxin induction within three hours of treatment. Moreover, 51 of 55 (93%) differential <i>Aux</i>/<i>IAA</i> expression patterns between different root-types followed the expression tendency: crown roots > seminal roots > primary roots > lateral roots. This pattern might imply root-type-specific regulation of <i>Aux/IAA</i> transcript abundance. In summary, the detailed analysis of the maize <i>Aux/IAA</i> gene family provides novel insights in the evolution and developmental regulation and thus the function of these genes in different root-types and tissues.</p> </div

Correlation of gene expression of the seven paralogous <i>Aux/IAA</i> gene pairs in maize roots.

<p>Five of seven paralogous <i>Aux/IAA</i> pairs showed a significant correlation in their gene expression patterns in roots (coefficient of determination R² >0.5; <i>p</i> ≤0.01). Only <i>ZmIAA30</i>/<i>ZmIAA18</i> and <i>ZmIAA13</i>/<i>ZmIAA3</i> did not display significant expression correlation in the tested root-types and tissues.</p

Expression of <i>Aux/IAA</i> genes during development in different root-types and tissues.

<p>Relative expression of 30 <i>Aux/IAA</i> genes was surveyed via qRT-PCR relative to <i>myosin</i> during primary root development (A), in different primary root tissues (B), and in seminal, crown and lateral roots (C).</p

Repression of a <i>firefly</i> luciferase reporter gene by Aux/IAA proteins fused to a GAL4 DNA binding domain.

<p>Aux/IAA proteins fused to GAL4 DNA binding domains were co-transformed with a <i>firefly</i> luciferase reporter into <i>Arabidopsis</i> protoplasts. All luciferase activities were normalized relative to the empty effector vector (GAL4-DBD) and an internal luciferase control (<i>renilla</i> luciferase). Error bars: SD, n = 3, <i>p</i>≤0.001.</p

Diversity of Stability, Localization, Interaction and Control of Downstream Gene Activity in the Maize Aux/IAA Protein Family

<div><p>AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins are central regulators of auxin signal transduction. They control many aspects of plant development, share a conserved domain structure and are localized in the nucleus. In the present study, five maize Aux/IAA proteins (ZmIAA2, ZmIAA11, ZmIAA15, ZmIAA20 and ZmIAA33) representing the evolutionary, phylogenetic and expression diversity of this gene family were characterized. Subcellular localization studies revealed that ZmIAA2, ZmIAA11 and ZmIAA15 are confined to the nucleus while ZmIAA20 and ZmIAA33 are localized in both the nucleus and the cytoplasm. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107346#s1" target="_blank">Introduction</a> of specific point mutations in the degron sequence (VGWPPV) of domain II by substituting the first proline by serine or the second proline by leucine stabilized the Aux/IAA proteins. While protein half-life times between ∼11 min (ZmIAA2) to ∼120 min (ZmIAA15) were observed in wild-type proteins, the mutated forms of all five proteins were almost as stable as GFP control proteins. Moreover, all five maize Aux/IAA proteins repressed downstream gene expression in luciferase assays to different degrees. In addition, bimolecular fluorescence complementation (BiFC) analyses demonstrated interaction of all five Aux/IAA proteins with RUM1 (ROOTLESS WITH UNDETECTABLE MERISTEM 1, ZmIAA10) while only ZmIAA15 and ZmIAA33 interacted with the RUM1 paralog RUL1 (RUM-LIKE 1, ZmIAA29). Moreover, ZmIAA11, ZmIAA15 ZmIAA33 displayed homotypic interaction. Hence, despite their conserved domain structure, maize Aux/IAA proteins display a significant variability in their molecular characteristics which is likely associated with the wide spectrum of their developmental functions.</p></div

Statistical evaluation of BiFC data using a lineal model for logarithmized data and simulation-based multiple comparisons by Edwards and Berry [37].

a<p>value after using lineal model for logarithmized data.</p>b<p>t-test of value (log scale).</p>c<p><i>p</i>-value adjusted for simulation-based multiple comparisons by Edwards and Berry <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107346#pone.0107346-Edwards1" target="_blank">[37]</a>.</p>d<p>ns: not significant; *: significant (adjusted p-value <0.01).</p><p>Statistical evaluation of BiFC data using a lineal model for logarithmized data and simulation-based multiple comparisons by Edwards and Berry <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107346#pone.0107346-Edwards1" target="_blank">[37]</a>.</p

Homo- and heterointeraction of Aux/IAA proteins in BiFC experiments visualized by confocal microscopy.

<p><b>(A)</b> Homotypic protein interactions of Aux/IAA proteins were identified for ZmIAA11, ZmIAA15 and ZmIAA33 but not for ZmIAA20 and ZmIAA2. <b>(B)</b> Heterotypic interactions of RUM1 were detected for all five tested Aux/IAA proteins. RUL1 specifically interacted with ZmIAA15 and ZmIAA33. <b>(C)</b> Summary of the quantitative BiFC data obtained by flow cytometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107346#pone-0107346-t002" target="_blank">Table 2</a>) and the verification of these results by qualitative BiFC experiments surveying protoplasts under a confocal microscope (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107346#pone-0107346-g004" target="_blank">Figure 4A and B</a>). The arrowhead types are indicating the interacting partners. A full arrowhead describes an interaction of RUM1/RUL1-YFPN with Aux/IAA-YFPC, while a double arrowhead denotes an interaction of RUM1/RUL1-YFPC with Aux/IAA-YFPN. Homotypic interactions of ZmIAA11, ZmIAA15 and ZmIAA33 are highlighted with a double circle.</p

Subcellular localization of Aux/IAA-GFP fusion proteins in maize protoplasts by GFP-fluorescence.

<p>ZmIAA2, ZmIAA11 and ZmIAA15 were confined to the nucleus. Cytosolic and nuclear localization was identified for ZmIAA20 and ZmIAA33. Subcellular localization of mutated versions of the five Aux/IAA proteins and N-terminal GFP fusions that confirmed the subcellular localization of the C-terminal fusions are displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107346#pone.0107346.s003" target="_blank">Figure S3</a>.</p

Characteristics of maize <i>Aux/IAA</i> genes encoding proteins analyzed in this study.

a<p>according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107346#pone.0107346-Ludwig1" target="_blank">[5]</a>.</p><p>Characteristics of maize <i>Aux/IAA</i> genes encoding proteins analyzed in this study.</p

Degradation assay of maize Aux/IAA-GFP wild-type fusion proteins and Aux/IAA-GFP proteins containing mutations in their degron sequence.

<p>Detection of relative GFP-fluorescence of the GFP control, wild-type and two mutated forms of Aux/IAA proteins after 1-NAA and cycloheximide treatment prior to recording a two hour time course in <i>Arabidopsis</i> protoplasts. Red: wild-type, green: substitution of first proline by serine, blue: substitution of second proline by leucine, black: GFP control.</p