Differential Activation of Partially Redundant Δ9 Stearoyl-ACP Desaturase Genes Is Critical for Omega-9 Monounsaturated Fatty Acid Biosynthesis During Seed Development in Arabidopsis.

TLDR: It is demonstrated that the four desaturases redundantly contribute to storage lipid production during the maturation phase of Arabidopsis thaliana and that the three most highly expressed ones are directly activated by the WRINKLED1 transcription factor.

Abstract: The spatiotemporal pattern of deposition, final amount, and relative abundance of oleic acid (cis-ω-9 C18:1) and its derivatives in the different lipid fractions of the seed of Arabidopsis (Arabidopsis thaliana) indicates that omega-9 monoenes are synthesized at high rates in this organ. Accordingly, we observed that four Δ9 stearoyl-ACP desaturase (SAD)-coding genes (FATTY ACID BIOSYNTHESIS2 [FAB2], ACYL-ACYL CARRIER PROTEIN5 [AAD5], AAD1, and AAD6) are transcriptionally induced in seeds. We established that the three most highly expressed ones are directly activated by the WRINKLED1 transcription factor. We characterized a collection of 30 simple, double, triple, and quadruple mutants affected in SAD-coding genes and thereby revealed the functions of these desaturases throughout seed development. Production of oleic acid by FAB2 and AAD5 appears to be critical at the onset of embryo morphogenesis. Double homozygous plants from crossing fab2 and aad5 could never be obtained, and further investigations revealed that the double mutation results in the arrest of embryo development before the globular stage. During later stages of seed development, these two SADs, together with AAD1, participate in the elaboration of the embryonic cuticle, a barrier essential for embryo-endosperm separation during the phase of invasive embryo growth through the endosperm. This study also demonstrates that the four desaturases redundantly contribute to storage lipid production during the maturation phase.

Figures

Figure 3. Patterns of activity of GUS reporter constructs. Pattern of activity of the ProFAB2:FAB2g:uidA, ProAAD5:AAD5g:uidA, ProAAD1:uidA, ProAAD6:uidA, ProAAD3:uidA, ProAAD2:uidA and ProAAD4:uidA cassettes in young plantlets grown 20 days in vitro, in rosette leaves, in cauline leaves, in inflorescences, in flowers, in early maturing embryos harvested 6, 8, 10, or 12 days after anthesis (DAA), and in seed coat plus endosperm fractions from early maturing seeds aged 10 DAA (from left to right). For histochemical detection of GUS activity in vegetative organs and in flowers, tissues were incubated overnight in a buffer containing 0.2 mM each of potassium ferrocyanide and potassium ferricyanide. For seed fractions (embryos or seed coats plus endosperms attached), tissues were incubated 3 h in a buffer containing 2 mM each of potassium ferrocyanide and potassium ferricyanide. The results for GUS activity were observed on whole-mounted vegetative organs and flowers; microscopy observations of seed fractions were performed using Nomarski optics. Patterns of activity of the ProAAD5:uidA and ProFAB2:uidA cassettes are displayed in Supplemental Figure 2. Bars = 5 mm (vegetative organs and flowers) or 200 µm (seed fractions).

Figure 4. Functional characterization of AADs coding for Δ9 stearoyl-ACP desaturases. The fab2-2 mutant was transformed with different ProFAB2:AADc constructs. For each ProFAB2:AADc cassette tested, three independent transgenic lines (T1-3) were analyzed. Rosette leaves were then subjected to total FA analyses to determine the relative proportion of C18:0 and C16:3. Values are the means and SE of five replicates performed on batches of leaves from distinct plants. Statistical analysis of the data was performed using ANOVA, followed by comparison of means using the Newman-Keuls (SNK) test (P<0.05).

Figure 9. Characterization of oil accumulation in aad seeds. (A) Total fatty acid concentrations in dry seeds determined by gas chromatography. Values are the means and SE of five replicates performed on batches of 20 seeds from five distinct individuals. (B) Total fatty acid concentrations in the different fractions of mature dry seeds determined by gas chromatography. Emb., embryo; Endo., endosperm fraction. Values are the means and SEs of five replicates performed on batches of 20 seeds from five distinct individuals. (C) Concentrations of triacylglycerols and polar lipids of dry seeds. The two lipid fractions were separated by this layer chromatography. Values are the means and SE of five replicates performed on batches of 50 seeds from five distinct individuals. Asterisks indicate significant differences from the wild type according to t-test at *** P<0.001, ** P<0.01, and * P<0.05 respectively.

Figure 8. Observation of the developing and germinating embryos of aad5-1 fab2-2 and aad1 fab2 mutants. (A) Observation of embryo elongation and folding in developing seeds. Whole mounts of early maturing seeds (6, 8, and 10 days after anthesis) were observed with Nomarski optics. Isolated arrowheads indicate contact sites between cotyledons and the surrounding tissues in seeds aged 8 days after anthesis. Doted arrows denote embryo folding in seeds aged 10 days after anthesis. (B) Evaluation of toluidine blue permeability in etiolated seedlings grown for 4 days in the dark. (C) Evaluation of toluidine blue permeability in seedlings grown for 10 days under day/night alternation. Bars = 100 µm.

Figure 1. Accumulation of ω-9 fatty acids and derivatives in seeds of the Arabidopsis accession Col-0. (A) Time-course analysis of C18:1 and derivatives accumulation in developing seeds. Total fatty acids were analyzed by gas chromatography. Yellow to red stacked bars represent C18:1 and derivatives. Blue segments represent other fatty acids. Values are the means of five replicates performed on batches of 20 to 40 seeds from distinct individuals. (B) Amounts of C18:1 and derivatives in the different fractions of mature dry seeds. Emb., embryo; Endo., endosperm fraction. Values are the means of five replicates performed on batches of 20 seeds from distinct individuals. (C-D) Relative proportions of C18:1 and derivatives in the main lipid classes contained in seeds. Lipids were extracted from dry seeds. Concentrations of polar lipids (PLs) and triacylglycerols (TAGs) separated by thin-layer chromatography are expressed on a seed dry weight (DW) basis and indicated in the center of the circular charts (C). The concentration of polyester monomers (D) is expressed on a dry weight basis of delipidated residue (DR). The circular charts represent the fatty acid composition (in Mol %) of each lipid class. Values are the means of five replicates performed on batches of seeds from distinct individuals. DCA, dicarboxylic fatty acid; ωOH, ω-hydroxyl fatty acid.

Figure 2. Patterns of mRNA accumulation among the AAD multigene family. (A-B) Analysis of relative mRNA accumulation of AADs was performed in developing seeds (A), in embryo (Emb.) and endosperm fractions (Endo.) dissected from seeds aged 12 or 16 DAA (B). The results obtained are standardized to the constitutive 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. DAA, days after anthesis.

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Dierential Activation of Partially Redundant ∆9
Stearoyl-ACP Desaturase Genes Is Critical for Omega-9
Monounsaturated Fatty Acid Biosynthesis During Seed
Development in Arabidopsis
Sami Kazaz, Guillaume Barthole, Frédéric Domergue, Hasna Ettaki,
Alexandra To, Damien Vasselon, Delphine de Vos, Katia Belcram, Loic
Lepiniec, Sébastien Baud
To cite this version:
Sami Kazaz, Guillaume Barthole, Frédéric Domergue, Hasna Ettaki, Alexandra To, et al.. Dieren-
tial Activation of Partially Redundant ∆9 Stearoyl-ACP Desaturase Genes Is Critical for Omega-9
Monounsaturated Fatty Acid Biosynthesis During Seed Development in Arabidopsis. The Plant cell,
American Society of Plant Biologists (ASPB), 2020, 32 (11), pp.3613-3637. �10.1105/tpc.20.00554�.
�hal-02996686�

1
Differential*Activation*of*Partially*Redundant*∆9*Stearoyl-ACP*
Desaturase*Genes*Is*Critical*for*Omega-9*Monounsaturated*
Fatty*Acid*Biosynthesis*During*Seed*Development*
in*Arabidopsis*
Sami Kazaz
a
, Guillaume Barthole
a
, Frédéric Domergue
b,c
, Hasna Ettaki
a
, Alexandra
To
a
, Damien Vasselon
a
, Delphine De Vos
a
, Katia Belcram
a
, Loïc Lepiniec
a
, and
Sébastien Baud
a
a
Institut Jean-Pierre Bourgin, INRAE, CNRS, AgroParisTech, Université Paris-
Saclay, 78000 Versailles, France
b
Université de Bordeaux, Laboratoire de Biogenèse Membranaire, UMR 5200,
Villenave d’Ornon, France
c
CNRS, Laboratoire de Biogenèse Membranaire, UMR 5200, Villenave d’Ornon,
France
Running title
Roles of Δ9 stearoyl-ACP desaturases in seeds
Corresponding author e-mail
sebastien.baud@inrae.fr
One sentence summary
Oleic acid and derivatives synthesized by four Δ9 stearoyl-ACP desaturases, FAB2,
AAD1, AAD5, and AAD6, play critical roles during Arabidopsis embryo development
and seed maturation.
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 (sebastien.baud@inrae.fr). 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

2
ABSTRACT
The spatiotemporal pattern of deposition, final amount, and relative abundance of
oleic acid (cis-ω-9 C18:1) and its derivatives in the different lipid fractions of seeds
suggested a preeminent role of Δ9 stearoyl-ACP desaturases (SADs) during seed
development. Accordingly, four SAD-coding genes (FAB2, AAD5, AAD1, and AAD6)
are transcriptionally induced in this organ. The three most highly expressed ones are
directly activated by the WRINKLED1 transcription factor. The thorough
characterization of a collection of 30 simple, double, triple, and quadruple mutants
affected in SAD-coding genes then revealed major roles played by these
desaturases at various stages of seed development. First, production of oleic acid by
FAB2 and AAD5 appears to be critical at the onset of embryo morphogenesis.
Double homozygous plants from crossing fab2 and aad5 could never be obtained,
and further investigations revealed that the double mutation resulted in the arrest of
embryo development before the globular stage. During later stages, these two SADs,
together with AAD1, participate in the elaboration of the embryonic cuticle, a barrier
essential for embryo-endosperm separation during the phase of invasive embryo
growth through the endosperm. This study finally demonstrates that the four
desaturases redundantly contribute to storage lipid production during the maturation
phase.

3
INTRODUCTION
In spermatophyta, seed production interrupts the life cycle and allows survival and
dispersion of plants (Bewley, 1997). To establish the next generation, seeds package
together genetic material from the species and nutrients essential to fuel post-
germinative seedling establishment. In Angiosperms, seeds originate from the
fertilized ovule: the double fertilization of the embryo sac initiates the development of
two zygotes, the diploid embryo and the triploid endosperm. Several layers of
maternal origin derived from the ovular integuments and referred to as the seed coat
protect these zygotic tissues. The coordinated growth of these three tissues leads to
the formation of seeds. This developmental process is classically divided into two
main phases: embryo morphogenesis and maturation (Vicente-Carbajosa and
Carbonero, 2005). The early morphogenetic phase of embryogenesis establishes the
basic plant body organization of the embryo (Laux and Jürgens, 1997). The
maturation phase which follows consists of the accumulation of reserve compounds
and the preparation of the embryo for desiccation and dormancy (Baud et al., 2008).
The maturation process is developmentally controlled by a complex network of
transcription factors induced at the onset of this phase and named master regulators
of seed maturation (Lepiniec et al., 2018).
Main seed reserves consist of storage proteins, carbohydrates, and storage lipids.
Tissue localization and relative proportions of these compounds vary greatly
depending on the species considered, but a source of nitrogen and a source of
carbon are usually associated. Exalbuminous seeds of Arabidopsis thaliana
accumulate seed storage proteins and triacylglycerols (TAGs) (Baud et al., 2002).
The residual endosperm in mature Arabidopsis seeds consists in a thin peripheral
cell layer. Reserves are massively stored in a central enlarged embryo structure.
Beyond the strong imbalance observed between the amounts of reserves
accumulated in the two zygotes, the composition of these reserves was also shown
to be clearly different (Barthole et al., 2014). In the embryo, the vast majority of fatty
acids (FAs) found in TAGs consist of oleic acid (cis-ω-9 C18:1) and its elongated and
polyunsaturated derivatives, whereas the relative abundance of ω-7
monounsaturated FAs (MUFAs) is very low. All the FAs present in the embryo were
also detected in the endosperm, but the latter contains much higher proportions of ω-

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7 MUFAs, that represent more than 20 mol % of total FAs in this compartment
(Penfield et al., 2004; Li et al., 2006). MYB115 and MYB118, two transcription factors
of the MYB family induced in the endosperm at the onset of the maturation phase,
were recently shown to control the amount and composition of the reserves stored in
this tissue (Barthole et al., 2014; Troncoso-Ponce et al., 2016a).
In plants, de novo synthesis of FAs is localized in plastids and uses end products
of the glycolysis as carbon source (Harwood, 1996; Troncoso-Ponce et al., 2016b).
TAGs, which are formed from glycerol and FAs, are assembled within the
endoplasmic reticulum (ER) and stored in dedicated structures, the oil bodies (Bates
et al., 2013). Oil production, like other maturation-related programs, is controlled at
the transcriptional level by the master regulators of seed maturation, either directly or
indirectly depending on the target genes considered (Baud et al., 2007b; Baud and
Lepiniec, 2010). A regulator of the APETALA2/ethylene-responsive element binding
(AP2/EREBP) family induced by master regulators, named WRINKLED1 (WRI1),
was shown to play a key role in the transcriptional activation of FA biosynthetic genes
at the onset of the maturation phase (Cernac et al., 2004; Marchive et al., 2014).
Upon binding to AW cis-regulatory elements present in the promoter sequences of
these genes (Maeo et al., 2009), WRI1 recruits the Mediator complex and, in turn,
the Pol II complex to initiate their transcription (Kim et al., 2016), thus promoting
sustained rates of FA production.
Plastidial production of long-chain saturated FAs is performed in a stepwise
manner by the type II FA synthase. Stromal soluble acyl-acyl carrier protein
desaturases (AADs) can introduce a carbon-carbon double bond within these
saturated acyl chains to form cis-MUFAs (Shanklin and Cahoon, 1998). Depending
on their regio- and substrate specificity, AADs synthesize cis-MUFAs differing by the
position of the double bond within their aliphatic chain. While Δ9 stearoyl-ACP
desaturases (SADs) efficiently desaturate stearic acid (C18:0) to form oleic acid, Δ9
palmitoyl-ACP desaturases (PADs) prefer palmitic acid (C16:0) and mainly produce
palmitoleic acid (cis-ω-7 C16:1) (Cahoon et al., 1998). In seeds of Arabidopsis, oleic
acid can be elongated in the ER by the acyl-CoA elongase complex, yielding ω-9
very-long-chain MUFAs like gondoic acid (cis-ω-9 C20:1) and erucic acid (cis-ω-9
C22:1), or further desaturated by membrane-bound FA desaturases to form linoleic
acid (C18:2) and α-linolenic acid (C18:3). Palmitolic acid is also efficiently elongated

Frequently asked questions

Q1. What are the contributions in "Differential activation of partially redundant ∆9 stearoyl-acp desaturase genes is critical for omega-9 monounsaturated fatty acid biosynthesis during seed development in arabidopsis" ?

In this paper, two transcription factors of the MYB family induced in the endosperm at the onset of the maturation phase, were recently shown to control the amount and composition of reserve reserves stored in a central enlarged embryo structure. 

Q2. What is the purpose of the HAL?

Desaturase Genes Is Critical for Omega-9 Monounsaturated Fatty Acid Biosynthesis During Seed Development in ArabidopsisSami Kazaza, Guillaume Bartholea, Frédéric Domergueb,c, Hasna Ettakia, Alexandra Toa, Damien Vasselona, Delphine De Vosa, Katia Belcrama, Loïc Lepinieca, andSébastien BaudaaInstitut Jean-Pierre Bourgin, INRAE, CNRS, AgroParisTech, Université ParisSaclay, 78000 Versailles, France bUniversité de Bordeaux, Laboratoire de Biogenèse Membranaire, UMR 5200, Villenave d’Ornon, France cCNRS, Laboratoire de Biogenèse Membranaire, UMR 5200, Villenave d’Ornon, FranceRoles of Δ9 stearoyl-ACP desaturases in seedsCorresponding author e-mail sebastien.baud@inrae.frOleic acid and derivatives synthesized by four Δ9 stearoyl-ACP desaturases, FAB2, AAD1, AAD5, and AAD6, play critical roles during Arabidopsis embryo development and seed maturation. 

Q3. Who is responsible for the distribution of materials integral to the findings presented in this article?

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 (sebastien.baud@inrae.fr).