Plant Physiology Preview. Published on May 20, 2016, as DOI:10.1104/pp.15.01625
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Running head: Roles for ANT and AIL6 in development and defense
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Beth A. Krizek
Corresponding Authors:
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Department of Biological Sciences
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University of South Carolina
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Columbia, SC 29205
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United States
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Tel: 1 803-777-1876
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Email:
[email protected]
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Ann E. Loraine
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Department of Bioinformatics and Genomics
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University of North Carolina at Charlotte
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Charlotte, NC 28223
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United States
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Tel: 1 704-250-5750
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Email:
[email protected]
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Research area: Genes, Development and Evolution
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Copyright 2016 by the American Society of Plant Biologists
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RNA-Seq links AINTEGUMENTA and AINTEGUMENTA-LIKE6 to cell wall remodeling
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and plant defense pathways in Arabidopsis
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Beth A. Krizek*, Carlton J. Bequette, Kaimei Xu, Ivory C. Blakley, Zheng Qing Fu,
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Johannes W. Stratmann, Ann E. Loraine*
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Department of Biological Sciences, University of South Carolina, Columbia, SC (B.A.K.,
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C.J.B., K.X., Z.Q.F., J.W.S.)
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Department of Bioinformatics and Genomics, University of North Carolina at Charlotte,
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Charlotte, NC (A.E.L., I.C.B.)
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*
Address correspondence to
[email protected] or
[email protected]
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Author contributions: B.A.K. and A.E.L. designed the research; B.A.K. prepared the
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samples for RNA-Seq and performed RT-qPCR, immunolocalization, and confocal
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microscopy; C.J.B. performed the SA and JA measurements; K.X. performed the
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bacterial growth assay; I.C.B. and A.E.L. performed bioinformatic analyses; Z.Q.F.
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supervised the bacterial growth assay; B.A.K., Z.Q.F., J.W.S., and A.E.L. interpreted the
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data; B.A.K. and A.E.L. wrote the article; all authors revised and approved the final
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article.
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Summary: Two related Arabidopsis transcription factors may regulate flower
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development through effects on the cell wall polysaccharide pectin and act in plant
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defense pathways.
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Financial source: This work was supported by a NIH INBRE Bioinformatics Pilot Project
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(BK) and National Science Foundation (NSF) grant IOS 1354452 (BK and AL).
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*
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Beth A. Krizek
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Email:
[email protected]
Corresponding authors:
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Ann E. Loraine
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Email:
[email protected]
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ABSTRACT
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AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) are two related
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transcription factors in Arabidopsis thaliana that have partially overlapping roles in
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several aspects of flower development including floral organ initiation, identity
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specification, growth and patterning. To better understand the biological processes
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regulated by these two transcription factors, we performed RNA-Seq on ant ail6 double
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mutants. We identified thousands of genes that are differentially expressed in the double
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mutant compared to wild type. Analyses of these genes suggest that ANT and AIL6
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regulate floral organ initiation and growth through modifications to the cell wall
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polysaccharide pectin. We find reduced levels of de-methyl-esterified homogalacturonan
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and altered patterns of auxin accumulation in early stages of ant ail6 flower development.
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The RNA-Seq experiment also reveals cross-regulation of AIL gene expression at the
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transcriptional level. The presence of a number of overrepresented GO terms related to
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plant defense in the set of genes differentially expressed in ant ail6 suggest that ANT and
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AIL6 also regulate plant defense pathways. Furthermore, we find that ant ail6 plants have
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elevated levels of two defense hormones: salicylic acid (SA) and jasmonic acid (JA) and
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show increased resistance to the bacterial pathogen Pseudomonas syringae. These results
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suggest that ANT and AIL6 regulate biological pathways that are critical for both
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development and defense.
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INTRODUCTION
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Flowers initiate on the flanks of the inflorescence meristem, a dome-shaped structure at
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the apex of a plant. After outgrowth of a flower primordium from the meristem, floral
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organ primordia appear in characteristic positions within the flower and undergo
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coordinated cellular behaviors and morphogenesis to develop into one of four organ
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types. A large number of proteins regulating flower development have been identified in
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the model plant Arabidopsis thaliana [reviewed in (Ó'Maoiléidigh et al., 2014)]. While
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certain aspects of flower development, such as the specification of floral organ identity,
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are well characterized, other aspects remain less well understood. For example, we know
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little about the molecular mechanisms regulating floral organ initiation, those controlling
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floral organ size, or those coordinating growth, differentiation and patterning during
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floral organogenesis. Four members of the AINTEGUMENTA-LIKE (AIL) transcription
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factor family: AINTEGUMENTA (ANT), AINTEGUMENTA-LIKE5 (AIL5),
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AINTEGUMENTA-LIKE6 (AIL6) and AINTEGUMENTA-LIKE7 (AIL7) have
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partially overlapping roles in some of these less well-understood processes as well as
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being required for the establishment of floral organ identity (Krizek, 2009, 2015) Within
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the AIL family, ANT and AIL6 make the most significant contributions to flower
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development, acting early to promote flower primordium initiation and later to direct
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several aspects of floral organ development (Krizek, 2009; Yamaguchi et al., 2013).
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Auxin maxima in the periphery of the inflorescence meristem are created through local
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auxin biosynthesis as well as polar transport of the hormone through the auxin efflux
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carrier, PIN-FORMED1 (PIN1) (Gälweiler et al., 1998; Benkova et al., 2003; Reinhardt
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et al., 2003; Heisler et al., 2005; Cheng et al., 2006). Flower primordium initiation is
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mediated by the transcription factor MONOPTEROS/AUXIN RESPONSE FACTOR5
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(MP/ARF5) (Przemeck et al., 1996). MP activates the floral meristem identity factor
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LEAFY (LFY) as well as ANT and AIL6 to promote flower primordium initiation
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(Yamaguchi et al., 2013). Within the flower primordium, sites of floral organ initiation
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are also correlated with sites of auxin accumulation (Chandler et al., 2011). It is not clear
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whether auxin specifies the cells that will give rise to floral organ primordia or 5
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accumulates in these cells after their specification. Furthermore, it is not known how
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auxin mediates floral organ outgrowth. After initiation of floral organ primordia, these
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primordia adopt fates as sepals, petals, stamens or carpels in response to distinct
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combinations of floral organ identity gene activities as described in the ABCE model
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[reviewed in (Krizek and Fletcher, 2005)]. The floral organ identity genes encode
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transcription factors that regulate target genes throughout floral organ development to
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bring about the elaboration of characteristic floral organ forms (Ito et al., 2004; Ito et al.,
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2007; Kaufmann et al., 2010; Wuest et al., 2012; Ó'Maoléidigh et al., 2013).
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ANT and AIL6 are key regulators of several aspects of floral organogenesis including
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floral organ initiation, identity specification, growth and patterning (Krizek, 2009).
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Flowers that form in ant ail6 double mutants are composed of fewer and smaller floral
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organs that arise in relatively random positions within the flower primordia. In addition,
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these flowers lack petals and normal stamens and carpels; they consist primarily of
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sepals, some stamenoid organs, unfused carpel valves and organs not present in normal
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flowers. Despite the importance of ANT and AIL6 in flower development, few regulatory
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targets of these transcription factors have been identified and little is known about the
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biological and cellular means by which they promote growth and development. Previous
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work has suggested that ANT may regulate organ growth by controlling the length of a
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cell-division competent state within developing lateral organs (Mizukami and Fischer,
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2000). The larger leaves of 35S:ANT plants are associated with prolonged expression of
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CycD3 and a longer period of growth, suggesting that ANT might regulate the expression
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of cell cycle genes. However, ANT does not appear to regulate CycD3 expression in
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petals (Randall et al., 2015).
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Thus, to gain insight into the biological roles of ANT and AIL6 in flower development,
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we used a transcriptomic approach to identify genes that are differentially expressed in
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ant ail6 inflorescences. Our results link ANT and AIL6 function in floral organ initiation
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to changes in the cell wall polysaccharide pectin. Unexpectedly, we also identify roles for
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ANT and AIL6 in plant defense pathways. 6
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RESULTS
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RNA-Seq of wild-type and ant ail6 inflorescences
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RNA-Seq libraries were constructed from four biological replicates of wild-type (Ler)
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and ant-4 ail6-2 inflorescences (Figure 1A, B). Reads that mapped uniquely to the
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genome were used to identify genes differentially expressed in ant ail6 inflorescences
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compared to Ler. Using a false discovery rate (FDR)
