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Plant Cell Advance Publication. Published on September 28, 2016, doi:10.1105/tpc.16.00612

RESEARCH PAPER

Transcriptional Activation of Two Palmitoyl-ACP ∆9 Desaturase Genes by MYB115 and MYB118 is Critical for Biosynthesis of Omega-7 Monounsaturated Fatty Acid in the Endosperm of Arabidopsis Seeds Manuel Adrián Troncoso-Ponce1‡, Guillaume Barthole1,2‡, Geoffrey Tremblais1, Alexandra To1, Martine Miquel1, Loïc Lepiniec1, and Sébastien Baud1 1

Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, 78000 Versailles, France 2 Univ. Paris-Sud, Université Paris-Saclay, 91405 Orsay, France ‡ Both authors contributed equally to this work Corresponding author e-mail: [email protected] Short title Regulation of ω-7 fatty acid synthesis in seeds One-sentence summary The MYB115 and MYB118 transcription factors activate two palmitoyl-ACP desaturases, AAD2 and AAD3, which are responsible for omega-7 fatty acid biosynthesis in the endosperm of Arabidopsis seeds.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Sébastien Baud ([email protected]). Contact information: Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, INRA Centre de Versailles-Grignon, Route de Saint-Cyr (RD10), 78026 Versailles Cedex, France. Tel. +33 1 30 83 33 25 ABSTRACT In angiosperms, double fertilization of the embryo sac initiates the development of the embryo and the endosperm. In Arabidopsis thaliana, an exalbuminous species, the endosperm is reduced to one cell layer during seed maturation and reserves such as oil are massively deposited in the enlarging embryo. Here, we consider the strikingly different fatty acid (FA) compositions of the oils stored in the two zygotic tissues. Endosperm oil is enriched in ω-7 monounsaturated FAs, that represent more than 20 Mol% of total FAs, whereas these molecular species are ten-fold less abundant in the embryo. Two closely related transcription factors, MYB118 and MYB115, are transcriptionally induced at the onset of the maturation phase in the endosperm and share a set of transcriptional targets. Interestingly, the endosperm oil of myb115 myb118 double mutants lacks ω-7 FAs. The identification of two Δ9 palmitoyl-ACP desaturases responsible for ω-7 FA biosynthesis, which are activated by MYB115 and MYB118 in the endosperm, allows us to propose a model for the transcriptional control of oil FA composition in this tissue. In addition, an initial characterization of the structure-function relationship for these desaturases reveals that their particular substrate specificity is conferred by amino acid residues lining their substrate pocket that distinguish them from the archetype Δ9 stearoyl-ACP desaturase.

©2016 American Society of Plant Biologists. All Rights Reserved

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INTRODUCTION

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In spermatophyta, also known as seed plants, the double fertilization of the

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embryo sac initiates the development of zygotic tissues, namely the embryo and the

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endosperm. They are protected by the seed coat, which comprises several cell layers

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of maternal origin derived from the ovular integuments. Seed formation therefore

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requires the coordinated growth of tissues of distinct origins that undergo two

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successive developmental phases: morphogenesis and maturation (Vicente-

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Carbajosa and Carbonero, 2005). Maturing seeds accumulate reserve compounds

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that are remobilized to fuel post-germinative seedling establishment. Depending on

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the species considered, the nature, relative proportion, and tissue localization of

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these reserve components vary greatly. Exalbuminous seeds of Arabidopsis store

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approximately equivalent amounts of oil (triacylglycerols, TAGs) and storage proteins

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(2S albumins and 12S globulins), these compounds being mostly deposited in a large

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embryo structure acquired at the expense of the endosperm (Baud et al., 2002). In

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mature dry seeds, the residual endosperm consists of a thin peripheral cell layer that

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contains no more than 10% of total seed reserves (Li et al., 2006). The fine

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biochemical characterization of the endosperm has revealed a reserve composition

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clearly different from that of the embryo, with a strongly decreased abundance of

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globulins (Barthole et al., 2014) and a unique oil fatty acid (FA) composition (Penfield

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et al., 2004). If all the FAs detected in the endosperm are also present in the embryo,

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the former contains ten-fold higher proportions of ω-7 monounsaturated FAs, like

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vaccenic acid (cis-ω-7 C18:1) and paullinic acid (cis-ω-7 C20:1), that account for

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more than 50% of the total ω-7 FAs present in the whole seed.

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In plants, de novo synthesis of FAs occurs in plastids (Harwood, 1996).

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Production of 16- or 18-carbon saturated FAs is catalyzed by the type II fatty acid

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synthase. Stromal ∆9 acyl-ACP desaturases (AADs) can introduce a carbon-carbon

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double bond (also called unsaturation) within these saturated acyl chains to form cis-

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monoenes (Lindqvist et al., 1996). AAD isoforms with different substrate specificities

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catalyze the formation of distinct monoenes differing by the position of the

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unsaturation within their aliphatic chains (referred to as ω-x). For instance, ∆9

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stearoyl-ACP desaturases (SADs) efficiently desaturate C18:0 to form cis-ω-9 C18:1

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(oleic acid). SADs represent the predominant AAD isoforms in most seed plants.

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Accordingly, the majority of the FAs found in embryo oil of Arabidopsis consists of 2

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oleic acid and of its derivatives. However, other AAD isoforms prefer C16:0 instead of

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C18:0 as a substrate. These ∆9 palmitoyl-ACP desaturases (PADs) catalyze the

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formation of cis-ω-7 C16:1 (palmitoleic acid), which can be further elongated to cis-ω-

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7 C18:1 (vaccenic acid), then to cis-ω-7 C20:1 (paullinic acid). Monoenes of the ω-7

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FA series occur infrequently in most seed plants, with the noticeable exception of a

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few plant species that produce unusual oils enriched in these ω-7 monounsaturated

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FAs, such as cat’s claw vine (Doxantha unguis-cati) or sea buckthorn (Hippophae

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rhamnoides) (Bondaruk et al., 2007; Fatima et al., 2012).

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The genome of the model plant Arabidopsis thaliana contains seven closely

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related genes coding for AADs (Kachroo et al., 2007). FAB2, the best-characterized

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member of the family, encodes a SAD (Lightner et al., 1994), whereas the other

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members of the family have been poorly characterized. The PAD(s) responsible for

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the production ω-7 monounsaturated FAs that accumulate at high levels in the

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endosperm oil of Arabidopsis seeds remain to be identified.

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Over the last decade, our knowledge on the regulation of storage compound

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metabolism in maturing seeds has increased tremendously. This knowledge has

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arisen mostly from genetic analyses carried out in Arabidopsis. Transcriptional

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regulators ensuring that maturation-related programs, such as oil biosynthesis, are

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correctly deployed during the transition phase between embryogenesis and seed

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maturation have been identified (Santos Mendoza et al., 2008). These transcription

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factors (TFs) participate in a complex network essential for completion of seed filling

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(Roscoe et al., 2015). Master regulators of the maturation program include members

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of the AFL (ABSCISIC ACID INSENSITIVE3/FUSCA3/LEAFY COTYLEDON2)

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network. These TFs belong to the B3 domain superfamily of DNA binding proteins

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and cooperate with LEAFY COTYLEDON1 (LEC1), a protein homologous to the

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HAP3 subunit of CCAAT-box binding proteins (Lotan et al., 1998; Suzuki and

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McCarty, 2008). Next to these master regulators, other TFs like basic leucine zippers

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bZIP53 or bZIP67 confer correct expression patterns to maturation genes (Mendes et

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al., 2013). Several genes encoding storage proteins or actors involved in TAG

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assembly and storage were shown to be direct targets of the above-mentioned TFs.

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By contrast, transcriptional activation of many glycolytic and FA biosynthetic genes,

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which is essential to support sustained rates of oil production, is indirectly mediated

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by WRINKLED1 (WRI1), a TF of the AP2-EREBP family (Cernac et al., 2004; Baud

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and Lepiniec, 2010). All together, these studies have led to a significant breakthrough

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in our understanding of the activation of reserve compound synthesis in the embryo,

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while the regulation of endosperm metabolism has scarcely been investigated in

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Arabidopsis. The recent characterization of MYB118, a TF transcriptionally induced in

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the maturing endosperm and repressing storage compound accumulation in this seed

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compartment, has shed some new light on the differential regulation of reserve

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partitioning between the embryo and endosperm (Barthole et al., 2014). However, the

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regulatory mechanisms explaining the contrasting compositions of these reserves

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remain completely unknown.

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To isolate new regulators of endosperm maturation and elucidate the peculiar

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composition of endosperm reserves, new screening procedures have been

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undertaken. Here, we report the functional characterization of MYB115 (At5g40360),

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a close homolog of MYB118 also induced in the endosperm at the onset of seed

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maturation. We provide evidence that the master regulator LEC2 positively regulates

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the two genes. This regulation and the negative feedback exerted by MYB118 on

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LEC2 expression suggest a partial compensation of the myb118 mutation by an

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overexpression of MYB115 in this genetic background. This hypothesis was

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confirmed by the thorough characterization of maturing myb115 myb118 mutant

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seeds demonstrating that the two MYBs are positive regulators of ω-7

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monounsaturated FA synthesis in the endosperm. We finally describe the

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identification of two targets of MYB115 and MYB118 belonging to the AAD multigene

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family. We show that these targets, namely AAD2 and AAD3, encode two ∆9 PADs

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responsible for the biosynthesis of ω-7 FA in the maturing endosperm. Taken

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together, these results allow us to establish a model for a transcriptional activation

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cascade participating in the control of oil FA composition within the maturing

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endosperm of Arabidopsis seeds.

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RESULTS

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MYB115 is induced in the endosperm of maturing seeds

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Functional redundancy between related TFs has been previously documented in

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Arabidopsis and recent studies suggest that MYB118 and MYB115, two close

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relatives of the MYB family (Wang et al., 2009; Zhang et al., 2009; Dubos et al.,

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2010), share transcriptional targets involved in glucosinolate biosynthesis (Zhang et 4

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al., 2015). To determine whether overlaps in function between MYB118 and close

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homologs occur in the maturing endosperm, we first examined the expression

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patterns of the three closest paralogs of MYB118, namely MYB115, MYB22, and

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MYB100 (Dubos et al., 2010) by reverse transcription quantitative PCR (RT-qPCR)

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on a set of cDNAs prepared from a range of plant organs of the wild-type accession

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Columbia-0 (Col-0). In all analyzed tissues, the accumulation of MYB22 and MYB100

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transcripts was below detectable levels; this was consistent with previously published

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transcriptomic analyses (Schmid et al., 2005; Le et al., 2010). By contrast, MYB115

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appeared to be expressed at very low levels in vegetative organs and induced in

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reproductive organs (flowers and developing siliques) (Figure 1A). To further

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characterize the expression pattern of MYB115, a time course analysis of MYB115

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mRNA abundance was carried out in developing seeds excised from siliques, which

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revealed a peak of transcript accumulation at the onset of seed maturation (Figure

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1B). Maturing seeds were then dissected and the two fractions obtained, namely

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embryo and endosperm fraction (comprising the endosperm and the seed coat; see

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methods), were independently analyzed. MYB115 mRNA abundance was high in the

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endosperm fraction during early maturation and hardly detected in the embryo

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(Figure 1C).

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To gain complementary information about the expression pattern of MYB115, the

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spatiotemporal activity of the MYB115 promoter was investigated. A 1-kb promoter

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fragment was transcriptionally fused to the uidA reporter gene. The corresponding

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construct was assayed for the resulting uidA expression pattern in transgenic

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Arabidopsis lines (Figure 1D-N). β-Glucuronidase (GUS) activity was observed in

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pollen grains (Figure 1D-E) and in seeds. A closer examination of developing seeds

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showed that the endosperm was stained (Figure 1G-J), whereas the seed coat and

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the embryo (Figure 1K-N) were not. The intense staining observed in the chalazal

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endosperm 12 days after anthesis (DAA; Figure 1J) was consistent with previous

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results of laser-capture microdissection of maturing seeds followed by mRNA

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quantification using stringent analyses of Affymetrix ATH1 GeneChip hybridization

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data (Le et al., 2010). MYB115 therefore appears to be co-expressed with MYB118 in

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the endosperm of early-maturing seeds (Barthole et al., 2014).

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Transcriptional activation of MYB115 by LEC2

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The master regulator LEC2 plays a key role in the transcriptional activation of

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MYB118 (Barthole et al., 2014). To test whether LEC2 also influences the

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transcriptional activation of MYB115, analysis of MYB115 mRNA abundance was first

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carried out in lec2 mutants. MYB115 transcripts were analyzed by RT-qPCR on

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cDNA prepared from 10-DAA-old seeds. MYB115 transcript steady-state levels were

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significantly reduced in lec2 alleles, suggesting the down-regulation of MYB115

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expression in the absence of LEC2 (Figure 1O). To test the transcriptional activation

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of MYB115 by LEC2, we used a dexamethasone (DEX; a synthetic glucocorticoid

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that activates the rat glucocorticoid receptor GR) inducible system (Santos Mendoza

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et al., 2005). The relative expression level of MYB115 was quantified by RT-qPCR in

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leaves of Pro35S:LEC2:GR plants. In transgenic plants treated for two weeks with

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DEX, a specific and significant accumulation of MYB115 mRNA was observed

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(Figure 1P). A time course analysis of MYB115 mRNA accumulation in rosette leaves

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treated with DEX revealed a marked increase of MYB115 mRNA levels from four

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days after induction onwards (Figure 1Q). Finally, the ProMYB115:uidA construct

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was introduced into transgenic Pro35S:LEC2:GR lines. The seedlings obtained were

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grown for 14 days on DEX-containing medium and were then assayed for the

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resulting uidA expression pattern (Figure 1R). GUS staining was detected in rosette

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leaves of these seedlings, confirming the ability of the LEC2:GR fusion protein to

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trigger MYB115 transcription.

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Transcriptional repression of MYB115 by MYB118 is LEC2-dependent

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MYB115 transcripts were analyzed by RT-qPCR on cDNA prepared from 10-

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DAA-old myb118 mutant seeds. MYB115 transcript steady-state levels were

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significantly increased both in myb118-1 seeds and in the OE3 line (a

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Pro35Sdual:MYB118 transgenic line exhibiting a strong repression of MYB118

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expression; Barthole et al., 2014), suggesting the up-regulation of MYB115

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expression in the absence of MYB118 (Figure 1S). To further evaluate the effect of

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MYB118 on MYB115 promoter activity, the ProMYB115:uidA construct was

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introduced into the myb118-1 mutant background and the resulting uidA expression

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pattern assayed in seeds aged 10 DAA and 12 DAA (Supplemental Figure 1). The

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proportion of stained seeds and the intensity of GUS staining were drastically

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increased in myb118-1 seeds at both developmental stages, showing the importance

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of MYB118 for repressing MYB115 promoter activity in maturing seeds. 6

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Considering the antagonistic regulation exerted by LEC2 and MYB118 on

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MYB115 expression, we tested whether these regulations were independent of each

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other (Supplemental Figure 2). For this purpose, MYB115 transcript levels were

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quantified by RT-qPCR on cDNA prepared from 10-DAA-old lec2 myb118-1 seeds

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and compared to that of single mutants. MYB115 transcripts levels in seeds of the

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double mutants were not significantly different from that measured in lec2 single

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mutants, demonstrating that the de-repression of MYB115 observed in response to

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the myb118-1 mutation is LEC2-dependent.

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MYB115 and MYB118 are transcriptional regulators that share common targets

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To firmly establish that MYB115 is a functional TF, we first investigated the in

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vivo subcellular localization of the protein with the aid of the green fluorescent protein

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(GFP). A derivative of GFP, mGFP6, was fused to MYB115 cDNA and placed under

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the control of the CaMV dual35S promoter for ubiquitous and high expression. The

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Pro35S:MYB115:GFP construct was transfected into leaves of transgenic Nicotiana

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benthamiana stably expressing RFP fused to histone 2B (RFP-H2B, used as nuclear

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marker; Martin et al., 2009). The Pro35S:MYB118:GFP construct was used as a

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positive control. Confocal imaging of transfected cells showed a co-localization of the

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GFP and RFP signals, demonstrating the nuclear targeting of MYB-GFP fusions

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(Figure 2A).

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In order to determine whether MYB115, like MYB118, possesses transcriptional

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activity, coding regions of the TFs were individually cloned in frame with the GAL4

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DNA binding domain (GAL4-DBD). The constructs thus obtained were introduced into

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the yeast strain AH109, which carries the HIS3 and ADE2 reporter genes under the

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control of heterologous GAL4-responsive upstream activating sequences and

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promoter elements. The expression of these two reporters could be activated in the

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presence of MYB115 or MYB118 fused to GAL4-DBD, thus establishing their ability

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to activate transcription (Figure 2B).

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The predicted DNA-binding domains of MYB115 and MYB118 share 73% amino

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acid identity. In order to test whether MYB115 and MYB118 share common

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transcriptional targets, myb115-1, myb118-1 and myb115-1 myb118-1 lines were

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grown together with wild-type controls and the expression level of target genes of

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MYB118 (Barthole et al., 2014) was measured by RT-qPCR on cDNA prepared from

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10-DAA-old seeds of each line. Repressed targets of MYB118 were first considered 7

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(Figure 2C). Whereas no de-repression of these genes could be detected in myb115-

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1, a significant over-accumulation of corresponding cDNA was measured in the

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double mutant as compared to the myb118-1 single mutant, showing that MYB115

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and MYB118 redundantly repress this set of genes. Conversely, the mRNA level of

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the 2-OXOGLUTARATE-DEPENDENT DIOXYGENASE (ODD) gene, a direct

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inducible target of MYB118, was further decreased in the double mutant as

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compared to myb118-1, suggesting that inducible targets of MYB118 can also be

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shared by the two TFs (Figure 2D). To validate the response of the ODD gene to

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MYB115 deregulation, Pro35Sdual:MYB115 transgenic lines were generated (OE11

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and OE19; Supplemental Figure 3). However, these lines exhibited altered vegetative

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development and were partially sterile, preventing us from analyzing the effect of

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MYB115 overexpression in seeds. Measurements were consequently performed on

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rosette leaves that demonstrated the ability of MYB115 to ectopically activate ODD

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(Figure 2E).

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To test the ability of MYB115 to directly activate ODD expression, we used a

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ProODD:uidA reporter construct in transactivation assays in N. benthamiana (Figure

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2F). The ProBCCP2:uidA construct was used as a negative control. Reporter

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constructs were infiltrated alone or in combination with a vector allowing the

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expression of MYB115, MYB118 (positive control), or MYB107 (negative control) in

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young leaves of N. benthamiana. MYB115, like MYB118, was able to specifically

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activate the ProODD:uidA reporter construct, showing a strong increase in GUS

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activity compared with the reporter alone or the reporter cotransfected with MYB107.

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A similar result was obtained with a reporter construct made of four repeats of the

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TAACGG element fused to the 35S cauliflower mosaic virus minimal promoter

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upstream of the uidA reporter gene. This element was proposed to be the cis-

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regulatory element bound by MYB118 in the promoter sequence of ODD (Barthole et

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al., 2014). All together, these results establish the ability of MYB115 and MYB118 to

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activate common target genes, possibly through the same cis-regulatory element.

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The binding of MYB115 to the ODD promoter sequence was examined in vitro by

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electrophoretic mobility shift assay (EMSA). Purified recombinant MYB115 was

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incubated with a 40-bp promoter fragment containing the TAACGG element and

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binding was determined using a biotin-labeled DNA probe. Addition of MYB115

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resulted in the formation of shifted bands (Figure 2G). The signal intensity increased

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with the concentration of MYB115 in the assay, indicating that the protein binds to the 8

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DNA fragment. The binding was specific since addition of the recombinant WRI1 TF

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did not result in the appearance of shifted bands. Furthermore, in competition

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experiments, addition of increasing amounts of unlabeled promoter fragments

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decreased the binding of MYB115 to the labeled probe. Using EMSA, we finally

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demonstrated the binding of MYB115 to three additional promoter fragments

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containing the TAACGG element and previously shown to be bound by MYB118

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(At5g01030, At3g62230, and At3g12880 promoters; Figure 2H). These results

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confirm that the two TFs bind common cis-regulatory elements.

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Impact of myb115 and myb118 mutations on ω-7 monoene accumulation in

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seeds

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To investigate the function of MYB115 during endosperm maturation and to test

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its redundancy with MYB118, the myb115-1, myb118-1 and myb115-1 myb118-1

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mutants were grown under controlled conditions. Vegetative development of the

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mutants was unaffected. Whole-mount clearing of developing seeds was carried out

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during embryo morphogenesis and early maturation. The structure and early

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development of the three tissues composing the seed were unaffected in the various

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mutant backgrounds considered (Supplemental Figure 4). Likewise, observation of

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14-DAA-old peeled endosperms suggested that the organization of the monolayer of

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endosperm cells was unmodified in the mutant lines. During the course of seed

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maturation, a slight delay could be observed in the elongation and enlargement of

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myb115-1 myb118-1 embryos, that was associated with a moderate decrease of

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mature seed DW with respect to the other genotypes (Supplemental Figure 4).

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Since MYB115 and MYB118 are strongly induced in the maturing endosperm, we

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then evaluated the effects of their mutations on endosperm filling (Figure 3).

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Dissected endosperm and embryo fractions were collected separately during the

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course of seed maturation and total FAs were quantified by gas chromatography.

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Whereas the myb118 mutation led to a significantly increased FA content in the

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endosperm fraction from 12 DAA onward (Figure 3B; Barthole et al., 2014)

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compensated by an equivalent decrease of the embryo FA content (Supplemental

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Figure 5A), no effect of the myb115 mutation on the overall amount of FAs stored in

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the endosperm could be detected.

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The effect of the myb mutations on the FA composition of the oil stored in the

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endosperm fraction was then examined. A focus was put on monoenes of the ω-7 9

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series that were previously reported to be highly abundant in the endosperm oil of

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mature Arabidopsis seeds (Penfield et al., 2004; Li et al., 2006). Before addressing

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the role of the MYB TFs in the control of oil FA composition, we first characterized the

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accumulation of ω-7 FAs in the two zygotic tissues of wild-type seeds (Supplemental

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Figure 6). In the endosperm fraction, ω-7 FAs were massively deposited between 9

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and 14 DAA. In mature seeds, they accounted for more than 20 Mol% of total FAs,

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with paullinic acid representing the more abundant species of the ω-7 series of FAs.

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The pattern of ω-7 FA accumulation was strikingly different in the embryo, with a later

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storage of ω-7 FA species, a decreased abundance of these monoenes (they

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represent only 2 Mol% of total FAs in dry embryos), and a predominance of vaccenic

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acid over paullinic acid. We then evaluated the effect of the myb mutations on the

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accumulation of ω-7 FAs in the endosperm (Figure 3C). The myb118 mutation

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yielded a sharp decrease in the proportion of ω-7 FAs stored in this compartment.

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Reversion of this phenotype could be obtained by introgression of a wild-type copy of

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the MYB118 gene into the mutant background (Figure 3D). Whereas the single

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myb115 mutation did not affect the accumulation of ω-7 FAs, an aggravated

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phenotype could be detected in the double myb115-1 myb118-1 mutant with respect

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to the myb118-1 background, denoting partially redundant functions of MYB115 and

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MYB118 in the control of ω-7 FA biosynthesis in the endosperm (Figure 3C). The

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endosperm tissue comprises different territories and the expression patterns of

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MYB115 and MYB118 were not similar in this tissue. MYB118 exhibited a high and

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homogeneous induction throughout the endosperm (Barthole et al., 2014), whereas

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the induction level of MYB115 was weaker and less homogeneous, the promoter

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activity of MYB115 being particularly intense in the chalazal endosperm (see above).

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We therefore measured the ω-7 FA contents of subfractions of the endosperm

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compartment in mature dry seeds. The peripheral endosperm was separated from

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the basal endosperm (comprising both the chalazal and micropylar endosperms) and

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the collected subfractions were analyzed separately by gas chromatography (Figure

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3E-G). The results obtained suggest that MYB115 and MYB118 redundantly control

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ω-7 FA production in all endosperm territories, with the action of MYB118

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predominating over that of MYB115 everywhere. A slight but reproducible negative

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effect of the myb mutations on ω-7 FA synthesis was also observed in the embryo

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(Supplemental Figure 5). 10

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Identification of two acyl-ACP desaturases transcriptionally activated by

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MYB115 and MYB118

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In order to identify the ∆9 PADs responsible for ω-7 FA biosynthesis in seeds of

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Arabidopsis, we used an RT-qPCR approach and examined the expression profiles

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of the seven Arabidopsis genes predicted to encode AADs (named AAD1/SAD1 to

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AAD6/SAD6, plus FAB2) in the search for positively regulated targets of MYB115 and

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MYB118. Target mRNAs were quantified in maturing myb mutant seeds (10 DAA)

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and in rosette leaves of transgenic lines overexpressing MYB118 (lines OE1 and

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OE2) or MYB115 (lines OE11 and OE19). Two AAD genes, namely AAD2 and AAD3,

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were strongly downregulated in myb mutant seeds with corresponding mRNA levels

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correlating with the reduced ω-7 FA contents in these seeds (Figure 4A). Conversely,

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MYB115 and MYB118 were able to ectopically activate these two putative target

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genes (Figure 4B,C).

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To confirm the ability of the two MYBs to directly activate AAD2 and AAD3

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expression, we used reporter ProAAD2:uidA and ProAAD3:uidA constructs in

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transactivation assays in N. benthamiana leaves. The ProBCCP2:uidA construct was

322

used as a negative control. Reporter constructs were infiltrated alone or in

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combination with vectors allowing the expression of MYB115, MYB118, or MYB107

324

(negative control) in young leaves (Figure 4D). MYB115 and MYB118 were able to

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specifically activate ProAAD2:uidA and ProAAD3:uidA reporter constructs, showing a

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strong increase in GUS activity compared with the reporters alone or the reporters

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cotransfected with MYB107.

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We finally characterized the expression patterns of AAD2 and AAD3 in

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developing seeds. The reporter constructs previously used for transient activation

330

assays in N. benthamiana were stably introduced in Arabidopsis and resulting uidA

331

expression patterns were assayed. For each construct tested, GUS staining was

332

specifically observed in the maturing endosperm (Figure 4E,F). Then, RT-qPCR

333

experiments carried out with cDNA prepared from dissected seed fractions confirmed

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the tissue specificity of AAD2 and AAD3 expression, with corresponding mRNA

335

levels peaking 14 DAA in the endosperm fraction (Figure 4G). Expression patterns of

336

AAD2 and AAD3 were consistent with the positive transcriptional regulation exerted

337

both by MYB115 and MYB118 on these genes.

338 11

339

Role of AAD2 and AAD3 in the biosynthesis of ω-7 monoenes in seeds

340

In order to demonstrate that transcriptional activation of AAD2 and AAD3 is

341

sufficient to trigger the biosynthesis of ω-7 monoenes, we transiently overexpressed

342

these desaturases in leaves of N. benthamiana. FA composition was analyzed in

343

transformed leaves five days after infiltration and revealed a significant enrichment in

344

ω-7 monoenes in this material (Figure 5A). This ectopic stimulation of ω-7 FA

345

synthesis was specific since overexpression of other Arabidopsis AAD isoforms

346

(AAD1 or AAD6) had no effect on the biosynthesis of these FA species. To confirm

347

these results, Pro35Sdual:AAD2 and Pro35Sdual:AAD3 constructs were stably

348

introduced in Arabidopsis transgenic lines. First, RT-qPCR experiments carried out

349

with cDNAs prepared from rosette leaves demonstrated the efficient overexpression

350

of the transgenes in the selected lines (Figure 5B). Then, total FA analyses carried

351

out with leaf material confirmed the ability of AAD2 and AAD3 to ectopically induce ω-

352

7 FA biosynthesis.

353

To firmly establish that transcriptional activation of AAD2 and AAD3 is

354

responsible for ω-7 FA biosynthesis in the endosperm of Arabidopsis seeds, a

355

collection of aad2 and aad3 T-DNA insertion alleles (all in Col-0 background) was

356

obtained and characterized at the molecular level (Figure 5C). The mutants were

357

grown under controlled conditions. Their mature dry seeds were dissected and seed

358

fractions were subjected to biochemical analyses. Determinations of total FA

359

compositions demonstrated that the aad mutations negatively affected ω-7 FA

360

accumulation in the endosperm fraction of seeds (Figure 5D). For each pair of

361

mutants considered, an allelism test was carried out. No complementation could be

362

observed in the F1 hybrid seeds (Supplemental Figure 7), demonstrating that the

363

mutated aad alleles of each pair were allelic and ascertaining the link between the T-

364

DNA insertions and the oil phenotype characterized. Ultimately, the aad2-3 aad3-3

365

double mutant was obtained. Only traces of ω-7 FAs could be detected in the

366

endosperm fraction of the double mutant (Figure 5D), demonstrating that AAD2 and

367

AAD3 are the major contributors to ω-7 FA biosynthesis in this tissue, the relative

368

contributions of the two isoforms being proportional to their respective induction

369

levels (Figure 4G).

370 371

Molecular determination of substrate specificity of AAD2 and AAD3

12

372

Previous studies have established the importance of the side chains of the eight

373

amino acid residues lining the bottom part of the substrate channel of AADs for

374

determining the substrate specificity of the enzymes (Cahoon et al., 1997). The

375

channel of the archetype ∆9 SAD is deep enough to accommodate C18:0 substrates,

376

thus forming ω-9 monoenes (Supplemental Figure 8). A shorter channel is more

377

adapted to C16:0 substrates, yielding a ∆9 PAD activity and the production of ω-7

378

monoenes. These eight amino acid residues were first identified thanks to the crystal

379

structure of a ∆9 SAD from R. communis (Lindqvist et al., 1996). They appear to be

380

well conserved among the SAD isoforms characterized so far in higher plants, as in

381

the Arabidopsis FAB2 protein (Supplemental Figure 8). Alignments of amino acid

382

sequences

383

(http://swissmodel.expasy.org/) (Arnold et al., 2006; Bordoli et al., 2009) and the SAD

384

from R. communis (pdb code: 1AFR; Lindqvist et al., 1996) as a template allowed

385

identification of the corresponding amino acid residues in the AAD2 and AAD3

386

sequences and a proposed model for the substrate channel of these enzymes

387

(Supplemental Figure 8). These analyses revealed three divergent residues with

388

respect to the SAD archetype. To test the importance of these residues in

389

determining the specific PAD activity of AAD2 and AAD3, amino acid substitutions

390

were realized by site-directed mutagenesis in the sequence of FAB2 so as to

391

introduce these residues, alone or in combination, in a SAD sequence. Modified

392

versions of FAB2 were then transiently expressed in leaves of N. benthamiana and

393

the production of ω-7 FAs was monitored in order to detect PAD activities. Plasmids

394

allowing the expression of the Arabidopsis plastidial enolase or that of AAD2 were

395

used

396

overexpression of FAB2 was not sufficient to trigger a significant accumulation of ω-7

397

FAs in transformed leaves, mutagenized versions of FAB2 harboring a T219F

398

mutation, alone or in combination with other substitutions, significantly stimulated the

399

biosynthesis of ω-7 FAs, denoting a PAD activity (Figure 5E). These results strongly

400

suggested that the Phe residue lining the bottom part of the substrate channel of

401

AAD2 and AAD3 (at position 226 or 216, respectively) plays a key role in determining

402

the substrate specificity of these isoforms, most probably by reducing the depth of

403

this channel thanks to its bulky lateral chain.

as

and

3D

negative

structure

and

modeling

positive

using

controls,

404

13

the

SWISS-MODEL

respectively.

Whereas

server

ectopic

405 406 407

DISCUSSION

408 409

Reserve accumulation in maturing seeds is finely regulated. Developmental

410

regulators ensure that the corresponding biosynthetic pathways are activated during

411

the transition phase between embryogenesis and seed maturation, then shut down in

412

late maturing and germinating seeds. Superimposed on this temporal pattern of

413

regulation, spatial control elements regulate the differential partitioning of reserves

414

between seed compartments. Arabidopsis seeds indeed consist of two compartments

415

accumulating storage compounds, namely the endosperm and the embryo, the latter

416

accounting for about 90% of total seed reserves. Early during seed formation,

417

developmental programs establish the embryo as the preponderant tissue within

418

these exalbuminous seeds. Then, transcriptional repressors of the maturation

419

program expressed in the endosperm during the maturation phase further reinforce a

420

differential partitioning of reserves between zygotic tissues (Barthole et al., 2014).

421

Beyond this differential partitioning, the fine characterization of Arabidopsis seeds

422

has revealed the strikingly different compositions of the reserves stored in the two

423

compartments, with endosperm oil exhibiting for instance dramatically increased

424

proportions of the economically important ω-7 monounsaturated FAs (Penfield et al.,

425

2004). Here we demonstrate that the two closely related MYB115 and MYB118 TFs,

426

which are transcriptionally induced by LEC2 in the endosperm at the onset of seed

427

maturation, activate the transcription of two PAD genes, namely AAD2 and AAD3, in

428

this tissue. Transcriptional activation of these two isoforms is necessary and sufficient

429

to promote ω-7 FA biosynthesis in the endosperm. Taken together, these data allow

430

a better understanding of how FA metabolism is developmentally regulated at the

431

spatiotemporal level, adding another level of complexity to the regulatory network

432

controlling reserve accumulation in maturing Arabidopsis seeds.

433 434

Transcriptional activation of MYB115 in the maturing endosperm

435

Detailed characterization of the MYB115 expression pattern based on

436

complementary approaches like RT-qPCR and promoter:GUS analyses established

437

the endosperm-specific induction of this gene in early maturing seeds. These data

438

are fully consistent with the results of transcriptome analyses of developmental series 14

439

of seeds and seed tissues microdissected by laser-capture (Le et al., 2010; Day et

440

al., 2008; Belmonte et al., 2013). They suggest that the closely related MYB115 and

441

MYB118 genes, both induced in the maturing endosperm, may have similar cis-

442

regulatory elements in their promoter sequences. Their respective expression

443

patterns are not strictly identical though. If accumulation of the two mRNA

444

populations dramatically increases at the onset of seed maturation, before

445

decreasing during the course of seed maturation, induction level of MYB115 is much

446

weaker. Then, expression of MYB115 is not restricted to seeds since the activity of

447

the MYB115 promoter is also detected in pollen grains. These observations reflect

448

the ongoing divergent evolution of the promoter sequences of the two paralogs.

449

The pattern of MYB115 promoter activity matches MYB115 mRNA accumulation

450

both at the spatial and temporal levels; therefore MYB115 tissue-specific expression

451

is probably largely controlled at the transcriptional level. Interestingly, the data

452

presented in this study establish that MYB115, like MYB118, is a target of the master

453

regulator LEC2. This common transcriptional activation of the two TFs by LEC2,

454

together with the negative feedback regulation exerted by MYB118 on LEC2

455

expression (Barthole et al., 2014) provides an interesting mechanism explaining how

456

the myb118 mutation can be partially compensated by overexpression of MYB115 in

457

the myb118 mutant background (Figure 6). At the temporal level, the examination of

458

the expression profiles of LEC2, MYB118, and MYB115 is consistent with a

459

transcriptional activation of the MYB TFs by the master regulator. At the spatial level

460

however, the lack of induction of the two MYBs in the embryo is striking considering

461

that LEC2 is expressed in the embryo too (Kroj et al., 2003; Barthole et al., 2014). To

462

reconcile these apparent discrepancies, one has to postulate the existence of

463

endosperm-specific factors directing the expression of MYB115 and MYB118 in this

464

tissue, or that of embryo specific repressors counteracting LEC2 in the embryo.

465

Endosperm development appears to be predominantly under epigenetic control

466

(Berger, 2003; Sun et al., 2010); therefore, it would be interesting to further

467

investigate how these controls affect oil metabolism in the maturing endosperm

468

(Fatihi et al., 2013), possibly through dedicated transcriptional regulators such as

469

MYB115 and MYB118.

470 471

MYB115 and MYB118 have common transcriptional targets

15

472

MYB118 was previously shown to antagonistically regulate distinct gene

473

networks. Whereas several maturation-related genes are repressed by the TF in the

474

maturing endosperm, MYB118 directly triggers the expression of several endosperm-

475

induced genes (Barthole et al., 2014). The data presented in this study demonstrate

476

that sets of target genes are shared by MYB115 and MYB118 in each of the two

477

subcircuits (e.g., GRP19, SM3, and CRUL, or ODD, AAD2, and AAD3). These

478

observations are consistent with a recent report by Zhang et al. (2015) depicting how

479

the two conserved TFs co-control expression of genes encoding enzymes of the

480

benzoyloxy glucosinolate pathway in seeds. Expression analyses supported by yeast

481

one-hybrid assays have shown that several actors (AOP3, BZO1, SCPL17) of this

482

newly evolved biosynthetic pathway are transcriptionally repressed by the two MYBs.

483

Interestingly, the TAACGG element present as a part of the in vivo MYB118-binding

484

site, that may consequently belong to type 1 MYB-binding sites (pAACnG, where p

485

indicates T or C, and n indicates any nucleotide; Romero et al., 1998; Prouse and

486

Campbell, 2012) was identified both in positively and in negatively regulated targets

487

of the MYB TFs under study (Barthole et al., 2014; Zhang et al., 2015). These

488

observations raise the question of (i) the molecular mechanisms specifying the type

489

of regulation exerted by the TFs and of (ii) the identity of the unknown actors

490

participating in these regulations. In the same line, a detailed structure-function study

491

using in vitro techniques like SELEX or Biacore, or in vivo chromatin

492

immunoprecipitation experiments, would be useful to fully characterize the respective

493

DNA-binding matrixes of MYB115 and MYB118 in order to (i) better characterize the

494

spectrum of putative targets of the TFs and (ii) to determine whether these matrixes

495

are identical or not. The amino acid identity shared by the predicted DNA-binding

496

domains of MYB115 and MYB118 (73%) falls within the range of values obtained for

497

other MYBs previously shown to act redundantly. For instance, DNA-binding domains

498

of TT2/MYB123 (At5g35550) and MYB5 (At3g13540) share 72% amino acid identity

499

(Xu et al., 2014). Some of the data presented in this article (e.g., induction levels of

500

target genes in stable Pro35Sdual:MYB lines) indicate that the two MYBs exhibit

501

different behaviors in the presence of certain targets, implying that their binding

502

specificity may have started diverging. The hypothesis according to which these

503

divergences are such that MYB115 and MYB118 only share a subset of targets could

504

explain why the effects of the myb115 and myb118 mutations are additive for some

505

phenotypes (e.g., production of ω-7 monounsaturated FAs or some glucosinolates) 16

506

and not for some others (e.g., total amount of oil stored in the endosperm). However,

507

diverging binding specificities are not the only driving force for differentiating sets of

508

targets between related TFs: evolution of protein-protein interactions between protein

509

partners of a transcriptional complex can also play a key role. In this regard,

510

complementary studies would now be required to characterize the transcriptional

511

complexes involving MYB115 and MYB118.

512 513

Transcriptional control of ω-7 monounsaturated FA biosynthesis

514

The most common monoenes in land plants are of the ω-9 series, as oleic acid.

515

Unlike the ω-9, ω-7 monoenes like palmitoleic acid and its elongation products

516

vaccenic acid and paullinic acid are relatively rare. Plant oils containing ω-7

517

monoenes, though uncommon, are enriched in seeds or fruits of non-crop species

518

like cat’s claw vine (Doxantha unguis-cati), macadamia, or sea buckthorn (Hippophae

519

rhamnoides) (Bondaruk et al., 2007; Fatima et al., 2012). Oils enriched in ω-7 FA

520

were also described in seeds of the Brassicaceae family, but this enrichment solely

521

concerned the endosperm (Penfield et al., 2004; Li et al., 2006). The transcriptional

522

activation cascade described in this study (Figure 6) allows us to understand how two

523

∆9 PADs specifically induced in the maturing endosperm of Arabidopsis seeds,

524

namely AAD2 and AAD3, confer its peculiar FA composition to the oil stored in this

525

tissue. Further work will be required to fully elucidate the molecular mechanisms

526

underpinning the transcriptional activation of these two desaturases by MYB115 and

527

MYB118 and to precisely identify the cis-elements required for this activation.

528

Interestingly, MYB118 exerts antagonistic control on different actors of oil

529

metabolism: while repressing the overall amount of oil stored in the endosperm, this

530

TF promotes the biosynthesis of ω-7 monoenes over that of ω-9, raising the question

531

of the biological function of these molecular species in the endosperm. As for the ω-7

532

FAs present at low levels in the embryo, their biosynthesis may result from a very

533

limited, though detectable, activation of the above-mentioned transcriptional

534

regulatory cascade in this tissue.

535

The position of the unsaturation within the aliphatic chains of monounsaturated

536

FAs contributes to the physicochemical properties of acyl lipids derived from these

537

FAs. The specific functions of ω-7 containing lipids in the plant cell, if any, remain

538

unknown. Under standard growth conditions, development of the aad2 aad3 double

17

539

mutant is unaffected, but the remnant ω-7 FAs present in this genetic background

540

may impair the detection of phenotypes. Complementary studies, possibly requiring

541

the preparation of new multiple mutants for the AAD genes, will be essential to

542

address this question. Regardless of their function in plants, these ω-7 have uses for

543

a number of industrial applications. Biodiesel s produced from plant oils with high ω-7

544

content have superior functional properties (Wu et al., 2012). The ω-7 FAs also have

545

considerable potential as a feedstock for the production of 1-octene by metathesis

546

chemistry (Meier, 2009), 1-octene representing a high-demand feedstock mainly

547

used to make linear low-density polyethylene. Vegetable oils enriched in ω-7 FAs

548

have finally been ascribed a number of beneficial health properties. In animals,

549

adipose tissues use lipokines such as palmitoleic acid to communicate with distant

550

organs and regulate systemic metabolic homeostasis (Cao et al., 2008). There is

551

growing evidence that palmitoleic acid plays a key role in the pathophysiology of

552

insulin resistance in humans, increasing muscle response to insulin (Stefan et al.,

553

2010). Palmitoleic acid is also a candidate anti-melanogenic agent (Yoon et al.,

554

2010). Aside from above-mentioned nutritional functions, palmitoleic acid also has

555

anti-oxidant,

556

Pharmaceutical companies have already developed foods and nutraceuticals for

557

health purposes enriched in ω-7 FAs, often sourced from species exhibiting low

558

yields and poor agronomic properties such as sea buckthorn. In recent years,

559

biotechnical approaches have been implemented to develop specialized high-yielding

560

platforms through the metabolic engineering of oilseed crops (Bondaruk et al., 2007;

561

Nguyen et al., 2015). Findings regarding the control of ω-7 FA biosynthesis in the

562

plant cell may provide new interesting tools for the development of new strategies for

563

ω-7 FA production.

anti-microbial,

and

anti-aging

properties

(Wu

et

al.,

2012).

564 565

AAD2 and AAD3 are ∆9 palmitoyl-ACP desaturases

566

The Arabidopsis thaliana genome contains seven related genes coding for

567

predicted acyl-ACP desaturases, previously named FAB2 and SAD1 to SAD6

568

(Kachroo et al., 2007). FAB2, the best-characterized member of the family, encodes

569

a ∆9 SAD (producing ω-9 FAs) essential for plant development and defense signaling

570

(Lightner et al., 1994; Kachroo et al., 2001). Despite recent advances in the

571

characterization of this multigene family (Klinkenberg et al., 2014), the functions of

18

572

most members of the family have long remained elusive. In the light of the research

573

presented in this article, it appears that two isoforms have diverged from the

574

archetype SAD and exhibit a ∆9 PAD activity responsible for the biosynthesis of ω-7

575

monoenes. We therefore propose to adopt a new nomenclature and to rename the

576

already published SAD1-6 genes ACYL-ACP DESATURASE1-6 (AAD1-6), this

577

general designation encompassing the variety of in planta activities exhibited by the

578

corresponding isoforms. The ∆9 PAD activities of AAD2 and AAD3 are responsible

579

for the production of ω-7 monoenes stored in the endosperm of Arabidopsis seeds.

580

Using a quantitative trait loci (QTL) approach followed by genetic analyses, Bryant

581

and coworkers (2016) have also identified AAD2 and AAD3 as the major desaturases

582

synthesizing ω-7 monoenes in the endosperm. These observations are consistent

583

with previous results from Kachroo et al. (2007), who assayed the enzymatic

584

activities and substrate specificities of several Arabidopsis ∆9 AADs produced in E.

585

coli. These in vitro data already pointed out the preference of DES3/AAD3 for C16:0

586

substrates, yielding a PAD activity. The enzymatic characterization of AAD from

587

Arabidopsis (Kachroo et al., 2007) and other plant species (Cahoon et al., 1998;

588

Rodriguez Rodriguez et al., 2015) then unraveled that, beyond marked preferences

589

of the desaturases for substrates of a given chain length, the enzymes can also

590

desaturate slightly shorter or longer chains, although with a reduced affinity. The

591

traces of ω-7 FAs detected in aad2 aad3 seeds may consequently originate from the

592

activity of SADs like FAB2, which is also expressed in maturing seeds.

593

A group of eight residues lining the bottom part of the substrate channels of

594

AADs was previously shown to set constraints on the chain lengths of FA substrates,

595

thus determining the substrate specificity of the enzymes (Cahoon et al., 1997;

596

Cahoon et al., 1998). Despite the functional divergence of some of its founding

597

members, the Arabidopsis AADs still share a high degree of amino acid sequence

598

similarity and a common structural fold (Kachroo et al., 2007). Interestingly, three of

599

the eight amino acid residues determining substrate-specificity distinguish AAD2 and

600

AAD3 from the archetype SAD structure. The results of our site-directed mutagenesis

601

experiments point out the importance of one of the divergent amino acids identified,

602

namely Phe-226 in AAD2 or -216 in AAD3, for conferring a PAD activity to the

603

desaturases. The bulky lateral chain of this Phe residue may reduce the depth of the

604

substrate pocket, thus favoring the binding of shorter C16:0-ACP substrate. Beyond

19

605

the predominant role played by this residue, the two other divergent residues

606

identified in AAD2 and AAD3 may also contribute to create the substrate profile

607

displayed by these isoforms. A ∆9 PAD from cat’s claw vine was previously cloned

608

and characterized (Cahoon et al., 1998). Despite similar enzymatic activities, the ∆9

609

PAD from Arabidopsis and cat’s claw vine do not have identical substrate channels.

610

In the case of cat’s claw vine, only one residue diverged from the archetype SAD

611

sequence: replacement of a Leu by a Trp residue at the extremity of the channel was

612

presented as the major determinant of substrate specificity for this isoform (Cahoon

613

et al., 1998). During the evolution of AADs, the diversification of which was probably

614

favored by the emergence of multigene families within the genome of higher plants,

615

∆9 PAD activities may therefore have appeared independently in different species.

616

The prevalence of structural rules (e.g., presence of a hydrophobic residue with a

617

bulky lateral chain lining the bottom part of the substrate channel) over highly

618

conserved consensus sequences for determining the substrate specificity of PAD

619

enzymes was further established by metabolic engineering based on combinatorial

620

saturation mutagenesis and logical redesign of desaturases (Whittle and Shanklin,

621

2001; Cahoon et al., 1997; Cahoon and Shanklin, 2000). These approaches have

622

allowed tailoring several original PAD enzymes by reducing the ability of the

623

substrate pocket of SAD enzymes to accommodate the longer 18:0-ACP substrate.

624

Most of the combinations of amino acid substitutions yielding such a result involved

625

the replacement of a hydrophobic residue lining the pocket by a bulkier and still

626

hydrophobic residue.

627

In conclusion, these results exemplify how transcriptional regulations significantly

628

contribute to the differential regulation of FA and oil metabolism in the two zygotic

629

tissues comprising maturing seeds of Arabidopsis, leading to different oil

630

compositions in these adjacent compartments. In the maturing endosperm, two

631

closely related MYB TFs activated by LEC2, namely MYB118 and MYB115, trigger in

632

turn the expression of the AAD2 and AAD3 genes, that code for ∆9 PADs responsible

633

for the biosynthesis of the ω-7 monounsaturated FAs stored at high levels in the

634

endosperm oil. Specialization of oil metabolism in the endosperm arose both from the

635

emergence of an original structure of the substrate channel of one of the members of

636

the AAD family, and from the concomitant set up of a complex transcriptional

637

regulatory cascade able to precisely control the spatiotemporal expression of this

20

638

desaturase. Further work will be required to decipher the upstream molecular

639

mechanisms that allow this cascade to be specifically activated in the endosperm.

640 641

METHODS

642 643

Plant material and growth conditions

644

Arabidopsis thaliana seeds of the Col-0 accession were obtained from the

645

Arabidopsis thaliana resource center for genomics at the Institut Jean-Pierre Bourgin

646

(http://www-ijpb.versailles.inra.fr/) and T-DNA mutant lines (aad2-3, N670942; aad2-

647

4, N584160; aad3-3, N567280; aad3-4, N825777) were ordered from the NASC

648

(http://arabidopsis.info). Lec2-10, lec2-11, myb115-1 and myb118-1 T-DNA mutant

649

lines were described previously in Barthole et al. (2014). Plants were cultured as

650

described in Baud et al. (2007a). Dexamethasone induction experiments using the

651

Pro35S:LEC2:GR construct were carried out as described by Santos Mendoza et al.

652

(2005). To sample embryo and endosperm fractions, seeds excised from siliques

653

were dissected using a scalpel and dissecting tweezers under an optical glass

654

binocular magnifier. Material used for RNA extraction was frozen in liquid nitrogen

655

immediately after harvest, and then stored at -80°C.

656 657

Molecular characterization of T-DNA mutants

658

Plant genomic DNA flanking the T-DNA border of the mutants were amplified by PCR

659

(Supplemental Table 1) and sequenced to confirm the flanking sequence tags

660

identified. Homozygous lines were then isolated for further characterization. RT-PCR

661

analyses were ultimately carried out to analyze gene expression in mutant

662

backgrounds (Supplemental Table 2).

663 664

Constructs and plant transformation

665

The sequences of primers used for DNA amplification are indicated in Supplemental

666

Table 3.

667

Construction of the ProMYB115:uidA transgene: region -998 to -1 bp relative to the

668

MYB115 translational start codon was amplified with the proofreading Pfu Ultra DNA

669

polymerase (Stratagene) from Col-0 genomic DNA. The PCR product was introduced

670

by BP recombination into the pDONR207 entry vector (Invitrogen) and transferred

671

into the destination vector pBI101-R1R2-GUS (Baud et al., 2007b) by LR 21

672

recombination. The resulting binary vector was electroporated into Agrobacterium

673

tumefaciens C58C1 strain and used for agroinfiltration of flower buds of Arabidopsis

674

(Bechtold et al., 1993). Primary transformants were selected on MS medium

675

containing kanamycin (50 mg.l-1) and transferred to soil for further characterization:

676

23 independent transgenic lines were analyzed.

677

Construction of the ProAAD3:uidA transgene: a similar procedure was adopted.

678

Region -2,022 to -1 bp relative to the AAD3 translational start codon was cloned; 12

679

independent transgenic lines were analyzed.

680

Construction of the ProAAD2:uidA transgene: a similar procedure was adopted.

681

Region -1,000 to -1 bp relative to the AAD2 translational start codon was cloned into

682

the pGWB3 vector (Nakagawa et al., 2007); 19 independent transgenic lines were

683

analyzed.

684

Construction of the Pro35Sdual:MYB115, Pro35Sdual:AAD, and Pro35Sdual:FAB2

685

transgenes: the procedure adopted was similar to that described for the construction

686

of Pro35Sdual:MYB107 and Pro35Sdual:MYB118 transgenes (Barthole et al., 2014).

687

Construction of the Pro35Sdual:FAB2m1-7 transgenes: mutations on the FAB2 cDNA

688

cloned in pDONR207 were performed with the QuickChange Site Directed

689

Mutagenesis kit (Agilent) according to the manufacturer’s instructions. Primers used

690

are presented in Supplemental Table 4. Mutagenized cDNAs were then transferred in

691

pMDC32 as described above.

692

Construction

693

DBD:MYB118 transgenes: MYB115 and MYB118 cDNAs previously cloned into the

694

pDONR207 entry vector were transferred into a modified pDEST32 (carrying a

695

kanamycin-resistance gene) for GAL4-DBD fusion (Invitrogen).

696

Construction of the Pro35Sdual:MYB115:mGFP6 and Pro35Sdual:MYB118:mGFP6

697

transgenes: cDNAs without STOP codon were amplified with the proofreading Pfu

698

Ultra DNA polymerase (Stratagene) from a mixture of seed cDNA (Col-0 accession).

699

The PCR products were introduced by BP recombination into the pDONR207 entry

700

vector (Invitrogen) and transferred into the destination vector pMDC83 (Curtis and

701

Grossniklaus, 2003) by LR recombination.

702

Construction of the ProMYB118:MYB118 transgene was previously described in

703

Barthole et al. (2014).

of

the

ProADH1:GAL4-DBD:MYB115

704 705

RNA analyses 22

and

ProADH1:GAL4-

706

RNA extraction, reverse transcription, RT-PCR and real-time RT quantitative PCR

707

were carried out as previously described (Baud et al., 2004). The sequences of

708

primers used for RT-PCR and real-time RT-qPCR are indicated in Supplemental

709

Tables 2 and 5. Purity of the different seed fractions sampled was assessed as

710

described in Barthole et al. (2014). Briefly, marker genes for each of the fractions

711

sampled, namely ZHOUPI (endosperm-specific) and At2g23230 (embryo-specific)

712

were quantified on cDNA prepared from these fractions, thus confirming that no

713

significant contamination occurred between fractions.

714 715

Lipid analyses

716

Total FA analyses were performed as previously described (Li et al., 2006) on leaf

717

disks from agroinfiltrated N. benthamiana, or on pools of Arabidopsis seeds or seed

718

fractions. The endosperm tissue was analyzed with the seed coat attached as in

719

Penfield et al. (2004). However, this procedure did not bias our evaluation of the

720

endosperm oil content since the integuments of the seed do not accumulate storage

721

compounds and in fact undergo programmed cell death early during maturation

722

(Beeckman et al., 2000; Li et al., 2006).

723 724

Electrophoretic mobility shift assays

725

The expression plasmid was constructed by transferring MYB115 cDNA from the

726

pDONR207 to the expression vector pETG10A (http://www.embl-hamburg.de/). The

727

resulting vector was electroporated into E. coli RosettaBlue(DE3)pLysS strain

728

(Novagen)

729

thiogalactopyranoside (IPTG) in LB buffer, cells were grown over night at 20°C. Cell

730

lysis and protein purification were performed as previously described (Baud et al.,

731

2009). To prepare DNA probes, complementary biotin-labeled (at the 5’ end)

732

oligonucleotides (Eurofins MWG Operon) were annealed. For DNA-binding assays,

733

MYB115 recombinant protein was incubated with 30 fmol probe in binding buffer (20

734

mM Tris-HCl pH 8, 250 mM NaCl, 2 mM MgCl2, 1% glycerol (v/v), 1 mg.ml-1 BSA, 1

735

mM DTT, 85 ng µl-1 poly(dI-dC)). For competition assays, the unbiotinylated

736

competitor was incubated briefly with the recombinant protein before the biotinylated

737

probe was added. After addition of the biotinylated probe, reactions were incubated

738

30 min at room temperature, then fractionated at 4°C by 6% PAGE. Electrophoretic

739

transfer to nylon membrane and detection of the biotin-labeled DNA were carried out

for

expression.

After

induction

23

by

1

mM

isopropyl-b-D-

740

according to the manufacturer’s instructions (Chemiluminescent Nucleic Acid

741

Detection Module, PIERCE) using an ImageQuant LAS 4000 system (GE

742

Healthcare).

743 744

Microscopy

745

Histochemical detection of GUS activity and bright-field microscopy observations

746

were carried out as described in Baud et al. (2007a). Leaves of N. benthamiana were

747

imaged with a Zeiss LSM710 confocal microscope as described in Miart et al. (2014).

748 749

Accession Numbers

750

Sequence data from this article can be found in the EMBL/GenBank data libraries

751

under accession numbers: AAD1/SAD1, At5g16240; AAD2/SAD2, At3g02610;

752

AAD3/SAD3,

At5g16230;

AAD4/SAD4,

753

AAD6/SAD6,

At1g43800;

BCCP2,

754

At5g60390; FAB2/SSI2, At2g43710; GRP19, At5g07550; LEC2, At1g28300;

755

MYB107, At3g02940; MYB115, At5g40360; MYB118, At3g27785; ODD, At1g04380;

756

SM3, At2g25890.

At3g02620;

At5g15530;

AAD5/SAD5,

CRUL,

At3g02630;

At1g03890;

EF1αA4,

757 758

Supplemental Data

759

Supplemental Figure 1. Characterization of the regulation of MYB115 by MYB118.

760

Supplemental Figure 2. Characterization of the LEC2-dependent de-repression of

761

MYB115 in myb118-1.

762

Supplemental Figure 3. Characterization of MYB115 overexpressing lines.

763

Supplemental Figure 4. Characterization of myb115-1, myb118-1, and myb115-1

764

myb118-1 seed development.

765

Supplemental Figure 5. Impact of myb115 and myb118 mutations on ω-7 fatty acid

766

accumulation in the embryo of developing seeds.

767

Supplemental Figure 6. Time course analysis of ω-7 monounsaturated fatty acid

768

accumulation in Arabidopsis seeds.

769

Supplemental Figure 7. Complementation tests among the aad mutants.

770

Supplemental Figure 8. Complementary information for the sequences, structures,

771

and functions of acyl-ACP desaturases in Arabidopsis.

772

Supplemental Table 1. Primers used for molecular characterization of T-DNA

773

insertions. 24

774

Supplemental Table 2. Primers used for characterizing gene expression by RT-PCR

775

(as displayed in Figure 5C).

776

Supplemental Table 3. Primers used for construct preparation.

777

Supplemental Table 4. Primers used for site-directed mutagenesis experiments.

778

Supplemental Table 5. Primers used for quantitative RT-PCR.

779 780 781

ACKNOWLEDGMENTS

782

We thank D. Kosma for critical reading of the article, D. de Vos, C. Boulard, A. Wilch,

783

and O. Grandjean (Observatoire du Végétal, INRA-IJPB) for their technical

784

assistance. This work was supported by the French National Research Agency

785

(SOLAR, grant no. ANR-10-GENM-009) and by the Research Executive Agency

786

(TRIANON, grant no. PIEF-GA-2013-625204). The IJPB benefits from the support of

787

the Labex Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS). The confocal

788

equipment used in this study was partly financed by the Ile-de-France Region.

789 790 791

AUTHORS CONTRIBUTIONS

792

A.T., G.T., and M.M. performed the research and analyzed the data, L.L. designed

793

the research and analyzed the data, M.A. T.-P., G.B. and S.B. designed and

794

performed the research, analyzed the data, and wrote the article.

795 796

25

797

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Figure 1. Expression pattern and transcriptional regulation of MYB115. (A-C) Analysis of relative mRNA accumulation of MYB115 was performed in different plant organs (A), in developing seeds (B), and in developmental series of endosperm and embryo fractions (C). The results obtained are standardized to the EF1αA4 (EF) gene expression level. Values are the means and SE of three to six replicates carried out on cDNA dilutions obtained from three independent mRNA extractions. Cl, cauline leaves; Fl, flowers; ND, not detected; Rl, rosette leaves; Ro, roots; St, stems.(D-N) Pattern of activity of the ProMYB115:uidA cassette in flowers (D), in stamens (E), in pollen grains (F), in developing seeds harvested 6 (G), 8 (H), 10 (I) or 12 (J) DAA, and in early maturing embryos harvested 6 (K), 8 (L), 10 (M) or 12 (N) DAA. For histochemical detection of GUS activity, tissues were incubated overnight in a buffer containing 0.2 mM each of potassium ferrocyanide and potassium ferricyanide. The results for GUS activity were observed on whole-mounted inflorescences; microscopy observations of stamens, pollen grains, seeds and excised embryos were performed using Nomarski optics. Bars = 1 mm in (D), 50 μm in (E), 20 μm in (F), 100 μm in (G-J), and 50 μm in (K-N). (O) Accumulation of MYB115 mRNA in lec2 mutant seeds was quantified 10 DAA by RT-qPCR and presented as the percentage of the EF1αA4 (EF) gene expression. Values are the means and SE of three to six replicates carried out on three independent cDNA preparations obtained from batches of seeds dissected from four to five siliques. The three silique sets were harvested on distinct individuals. ***, Significant difference from WT according to t-test, P < 0.001. (P) Accumulation of MYB115 mRNA in leaves of transgenic Pro35S:LEC2:GR, Pro35S:TTG1:GR (negative control), or wild-type 10-day-old plants cultured in vitro on a medium with (+ DEX) or without (-DEX) 10-5 M dexamethasone for two additional weeks, was quantified by RT-qPCR and presented as percentage of the EF1αA4 (EF) gene expression. Values are the means and SE of three to four replicates carried out on three independent cDNA preparations. (Q) Time-course analysis of MYB115 mRNA accumulation in leaves of 10-day-old Pro35S:LEC2:GR plants transferred to a growth medium containing 10-5 M dexamethasone (induction) and grown two weeks in vitro. Accumulation of mRNA was determined by RT-qPCR and presented as the percentage of the EF1αA4 (EF) gene expression. Values are the means and SE of three to six replicates carried out on three independent cDNA preparations. (R) Transgenic ProMYB115:uidA x Pro35S:LEC2:GR seedlings were transferred 10 days after germination on a dexamethasone-containing medium (10-5 M; + DEX). Rosette leaves were analyzed two weeks after induction. For histochemical detection of GUS activity, tissues were incubated overnight in a buffer containing 0.2 mM each of potassium ferrocyanide and potassium ferricyanide. Bars = 0.2 cm. (S) Accumulation of MYB115 mRNA in myb118 mutant seeds (myb118-1 and OE3) was quantified 10 DAA by RTqPCR and presented as the percentage of the EF1αA4 (EF) gene expression. Values are the means and SE of three replicates carried out on cDNA dilutions obtained from three independent mRNA extractions. OE3, Pro35Sdual:MYB118 transgenic line exhibiting a strong repression of MYB118 expression (Barthole et al., 2014); WT, wild type (Col-0). ***, **: Significant difference from WT according to t-test at P < 0.001 and 0.01, respectively. DAA, days after anthesis; WT, wild type.

Figure 2. Functional characterization of MYB118 and MYB115. (A) Confocal micrographs showing localization of MYB115:GFP and MYB118:GFP fusion proteins in transgenic Nicotiana benthamiana plants expressing RFP:H2B (Martin et al., 2009). Plants were co-infiltrated with the Pro35Sdual:MYB:GFP construct and a vector allowing the expression of the p19 protein of tomato bushy stunt virus (TBSV) that prevents the onset of post-transcriptional gene silencing (Voinnet et al., 2003). GFP was observed 4 days after infiltration. Bar = 50 µm. (B) Transcriptional activity of MYB115 and MYB118. MYB115 and MYB118 coding sequences were cloned in frame with the GAL4 DNA-binding domain (DBD). The fusion constructs were introduced into reporter yeast containing the HIS3 and ADE2 reporter genes, before being plated on appropriate media to maintain the expression of the vectors (SD-Leu) and to test the activation of the HIS3 (SD-Leu-His) or HIS3 and ADE2 reporter genes (SD-Leu-His-Ade). Data presented are representative from the results obtained for eight independent colonies. SD, synthetic drop-out medium. (C,D) RT-qPCR analysis of transcript abundance for negatively (C) and positively (D) regulated targets of MYB118 in cDNA prepared from wild-type (WT; Col-0) and mutant seeds harvested 10 days after anthesis. Values are the means and SE of 12 replicates carried out on cDNA dilutions obtained from three independent mRNA extractions. ***, **, and *: Significant difference from wild type (WT) according to t-test at P < 0.001, 0.01, and 0.05, respectively. (E) RT-qPCR analysis of transcript abundance in cDNA prepared from rosette leaves of Pro35Sdual:MYB115 lines (OE11 and OE19). Values are the means and SE of nine replicates carried out on cDNA dilutions obtained from three independent mRNA extractions. ** and * indicate significant difference from wild type (WT) according to ttest at P < 0.01 and 0.05, respectively. (F) Transactivation assay in leaves of N. benthamiana. Schematic representations of the reporter constructs used are presented. Open boxes indicate TAACGG elements and the closed box represents the 35S cauliflower mosaic virus minimal promoter. Pro:uidA reporter constructs alone or in combination with a vector allowing the expression of MYB115, MYB118 or MYB107 (negative control) were co-infiltrated in young leaves of N. benthamiana with a vector allowing the expression of the p19 protein. Leaf discs were assayed for GUS activity three days after infiltration. Tissues were incubated 17 h in a buffer containing 2 mM each of potassium ferrocyanide and potassium ferricyanide. Representative discs (diameter = 0.8 cm) are presented. TAACGG concatemer, promoter sequence made of a concatemer of TAACGG elements separated by ten nucleotides (Barthole et al., 2014). (G) Binding of MYB115 to the proximal upstream region of ODD. Electrophoretic mobility shift assay (EMSA) of a probe covering a region from -240 to -200 upstream from the ATG codon of ODD with increasing amounts of MYB115 (‘+’ = 0.5 µg, ‘++’ = 1.5 µg). WRI1 was used as a negative control. Competition of MYB115 binding was carried out in the presence of 75-, 100-, and 200-fold amounts of the unlabeled ProODD(-240 to -200 bp) fragment. Position of free probe (open arrowhead) and the shifted bands (closed arrowhead) are indicated. (H) Binding of MYB115 to the proximal upstream regions of targets of MYB118, namely At5g01030, At3g62230, and At3g12880. The promoter sequence of the ODD gene (At1g04380) was used as a positive control. For each gene considered, the promoter region covered by the probe is indicated between brackets.

Figure 3. Impact of myb115 and myb118 mutations on the accumulation of ω-7 fatty acids in the endosperm fraction of developing seeds. (A) Schematic representation of mature Arabidopsis seeds. (B,C) Time-course analysis of total fatty acid (FA) content (A) and relative proportions of ω-7 FAs (cis-ω-7 C18:1 and cis-ω-7 C20:1) (B) in whole endosperm fractions dissected from wild-type and mutant seeds. Values are the means and SE of five replicates performed on batches of 20 to 40 seeds from five distinct plants. ***, **, and *: Significant difference according to t-test at P < 0.001, 0.01, and 0.05, respectively. (D) ω-7 FA (cis-ω-7 C18:1 and cis-ω-7 C20:1) content of whole endosperm fractions dissected from wild-type, myb118-1, or complemented mature dry seeds. Values are the means and SE of five replicates performed on batches of 20 seeds from five distinct plants. (E-G) ω-7 fatty acid content (cis-ω-7 C18:1 and cis-ω-7 C20:1) of whole endosperm fractions (E), peripheral endosperm fractions (F), and basal endosperm fractions (G) dissected from mature seeds. Values are the means and SE of five replicates performed on batches of 40 seeds from five distinct plants. . ***: Significant difference according to t-test at P < 0.001.

Figure 4. AAD genes induced by MYB115 and MYB118. (A-C) RT-qPCR analysis of transcript abundance in cDNA prepared from myb115-1, myb118-1, and myb115-1 myb118-1 mutant seeds harvested 10 DAA (A), from rosette leaves of Pro35Sdual:MYB118 lines (OE1 and OE2) (B), or from rosette leaves of Pro35Sdual:MYB115 lines (OE11 and OE19) (C). Values are the means and SE of 3 to 9 replicates carried out on cDNA dilutions obtained from three (B,C) or four (A) independent mRNA extractions. ***, **, and *: Significant difference from wild type (WT) according to t-test at P < 0.001, 0.01, and 0.05, respectively. (D) Transactivation assay in leaves of Nicotiana benthamiana. Pro:uidA reporter constructs alone or in combination with a vector allowing the expression of MYB115, MYB118 or MYB107 (negative control) were coinfiltrated in young leaves of N. benthamiana with a vector allowing the expression of the p19 protein. Leaf discs were assayed for GUS activity three days after infiltration. Tissues were incubated 4 h in a buffer containing 2 mM each of potassium ferrocyanide and potassium ferricyanide. Representative discs (diameter = 0.8 cm) are presented. (E,F) Pattern of activity of the ProAAD2:uidA (E) and ProAAD3:uidA (F) cassettes in developing seeds harvested 8, 10 or 12 DAA (main pictures, from left to right), and in early maturing embryos harvested 8, 10 or 12 DAA (secondary small pictures, from left to right). For histochemical detection of GUS activity, tissues were incubated overnight in a buffer containing 2 mM each of potassium ferrocyanide and potassium ferricyanide. Microscopy observations of seeds and excised embryos were performed using Nomarski optics. Bars = 100 µm. (G) Analysis of relative mRNA accumulation of AAD2 and AAD3 was performed in developmental series of endosperm and embryo fractions. The results obtained are standardized to the EF1αA4 (EF) gene expression level. Values are the means and SE of three to six replicates carried out on cDNA dilutions obtained from three independent mRNA extractions. Endo., endosperm fraction. DAA, days after anthesis.

Figure 5. Functional characterization of AAD2 and AAD3. (A) Transient expression assay in leaves of Nicotiana benthamiana. Pro35Sdual:AAD constructs were coinfiltrated in young leaves of N. benthamiana with a vector allowing the expression of the p19 protein. Leaf discs harvested five days after infiltration were subjected to total fatty acid (FA) analyses to determine the relative proportion of ω-7 FAs (cis-ω-7 C18:1) in this material. Values are the means and SE of ten replicates performed on batches of 2 disks from two to three distinct plants. ***: Significant difference from control according to ttest at P < 0.001. (B) Stable overexpression of AAD2 and AAD3 in transgenic A. thaliana lines. RT-qPCR analysis of transcript abundance in cDNA prepared from rosette leaves of the transgenic lines were first carried out to assess efficient overexpression of the transgenes (upper panel). Values are the means and SE of three replicates carried out on cDNA dilutions obtained from three independent mRNA extractions. Rosette leaves were then subjected to total FA analyses to determine the relative proportion of ω-7 FAs (cis-ω-7 C18:1) in this material (lower panel). Values are the means and SE of ten replicates performed on batches of two leaf tips from ten distinct plants. ***, **: Significant difference from WT according to t-test at P < 0.001 and 0.01, respectively. (C) Molecular characterization of aad2 and aad3 mutants. Structure of the AAD2 and AAD3 genes showing the position of T-DNA insertions in aad2-3, aad2-4, aad3-3, and aad3-4 are presented. For each T-DNA insertion considered, confirmed flanking sequence tag(s) are anchored in the gene structure and represented by vertical bar(s). Closed boxes represent exons and open boxes untranslated regions (UTRs). Accumulation of AAD2 and AAD3 mRNA in wild-type and corresponding mutant backgrounds was studied by reverse transcriptase polymerase chain reaction (RT-PCR) on developing seeds harvested 16 days after anthesis. EF1αA4 (EF) gene expression was used as a constitutive control. Primers used for this study are indicated by arrows (see Supplemental Table 2). (D) Relative proportion of ω-7 FAs (cis-ω-7 C18:1 and cis-ω-7 C20:1) in endosperm fractions dissected from wildtype and aad mature dry seeds. Values are the means and SE of five replicates performed on batches of 20 seeds from five distinct plants. ***: Significant difference from WT according to t-test at P < 0.001. (E) Site-directed mutagenesis experiments followed by transient expression assays in leaves of N. benthamiana. Constructs allowing the expression of the plastidial enolase (At1g74030; negative control), FAB2, AAD2, and mutagenized versions of FAB2 (FAB2m1-7) were co-infiltrated in young leaves of N. benthamiana with a vector allowing the expression of the p19 protein. Leaf discs harvested five days after infiltration were subjected to total FA analyses to determine the relative proportion of ω-7 FAs (cis-ω-7 C18:1) in this material. Values are the means and SE of 20 replicates performed on batches of 2 disks from four distinct plants. Statistical analyses of the data were performed using a variance analysis (ANOVA), followed by a comparison of means using the Newman–Keuls (SNK) test (P < 0.05). A schematic view of the eight residues lining the bottom part of the substrate pockets of the desaturases assayed is presented (see also Supplemental Figure 8). WT, wild type (Col-0).

Figure 6. Model for the regulation of ω-7 monoene fatty acid production in Arabidopsis seeds. MYB115 and MYB118 are induced in the endosperm at the onset of the maturation phase. The master regulator LEC2 activates their transcription, and MYB118 exerts negative feedback regulation on LEC2. MYB115 and MYB118 coordinately trigger the transcription of two soluble ∆9 palmitoyl-ACP desaturases, namely AAD2 and AAD3. These enzymes catalyze the synthesis of ω-7 monounsaturated fatty acids, which are accumulated at high levels in the oil stored within the endosperm. The transcriptional negative regulatory loop involving LEC2 and MYB118 was previously described in Barthole et al. (2014).

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Transcriptional Activation of Two Palmitoyl-ACP ∆9 Desaturase Genes by MYB115 and MYB118 is Critical for Biosynthesis of Omega-7 Monounsaturated Fatty Acid in the Endosperm of Arabidopsis Seeds Manuel Adrian Troncoso-Ponce, Guillaume Barthole, Geoffrey Tremblais, Alexandra To, Martine Miquel, Loic Lepiniec and Sébastien Baud Plant Cell; originally published online September 28, 2016; DOI 10.1105/tpc.16.00612 This information is current as of September 28, 2016 Supplemental Data

http://www.plantcell.org/content/suppl/2016/09/28/tpc.16.00612.DC1.html

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