a maternally deposited endosperm cuticle contributes to the … · its ultimate dispersion. seed...

1218 Plant Physiology®, July 2018, Vol. 177, pp. 1218–1233, www.plantphysiol.org © 2018 American Society of Plant Biologists. All Rights Reserved.

Cuticles and seeds are two cornerstone innovations that appeared early and late, respectively, during ter-restrial plant evolution and that allowed plants to cope with the challenge of a water-deprived environment.

Seeds are specialized plant structures that keep the plant embryo in a desiccated and highly resilient state. Viability and dormancy are the two key physiologi-cal properties of seeds that enable plant dispersal and germination under optimal seasonal conditions, re-spectively. Both are heavily dependent on the seed’s capacity to sustain unavoidable mechanical and oxi-dative damage (Bailly et al., 2008; El-Maarouf-Bouteau and Bailly, 2008; Basbouss-Serhal et al., 2016; Chahtane et al., 2017). In Arabidopsis (Arabidopsis thaliana) ma-ture seeds, the embryo is surrounded by a single-cell endosperm layer, itself surrounded by a seed coat, also called the testa, that faces the outer environment (Fig. 1A).

The development of the Arabidopsis testa is initiat-ed in the ovule after the central and egg cells form the endosperm and the zygote, respectively, upon dou-ble fertilization. As the seed develops, the maternal ovular integument layers surrounding the endo-sperm and embryo undergo a complex developmen-tal transformation. This includes the accumulation of flavonoid-derived tannins, particularly in the ii1 layer in close proximity to the developing endosperm, and the progressive death and compression of the integu-mental layers (Supplemental Fig. S1; Beeckman et al., 2000; Debeaujon et al., 2000, 2007; Windsor et al., 2000). These developmental processes eventually form the mature testa, a hardy and tannin-enriched cover sur-rounding the living endospermic and embryonic tis-sues. Therefore, the mature testa is a dead tissue of maternal origin.

The TRANSPARENT TESTA (TT) genes are a class of genes regulating seed testa development. They encode transcription factors, flavonoid biosynthesis enzymes, and intracellular trafficking regulators (Feinbaum and Ausubel, 1988; Shirley et al., 1992, 1995; Debeaujon et al., 2000; Pourcel et al., 2005; DeBolt et al., 2009; Appelhagen et al., 2011).

tt mutant seeds have structural and pigmentation defects in their testa. These defects are maternally in-herited and are associated with increased seed perme-ability to tetrazolium red salts (Debeaujon et al., 2000; DeBolt et al., 2009; Chen et al., 2014). Higher permea-bility may account for the lower dormancy and viabil-ity of tt mutant seeds. Indeed, it may lead to increased accumulation of oxidative stress in seeds, which has been linked to lower dormancy and viability (Bailly

A Maternally Deposited Endosperm Cuticle Contributes to the Physiological Defects of transparent testa Seeds1[OPEN]

Sylvain Loubéry, Julien De Giorgi, Anne Utz-Pugin, Lara Demonsais, and Luis Lopez-Molina2

Department of Plant Biology and Institute for Genetics and Genomics in Geneva, University of Geneva, CH-1211 Geneva 4, SwitzerlandORCID IDs: 0000-0001-7061-9456 (S.L.); 0000-0002-8131-6335 (J.D.G.); 0000-0003-0463-1187 (L.L.)

Mature dry seeds are highly resilient plant structures where the encapsulated embryo is kept protected and dormant to facilitate its ultimate dispersion. Seed viability is heavily dependent on the seed coat’s capacity to shield living tissues from mechanical and oxidative stress. In Arabidopsis (Arabidopsis thaliana), the seed coat, also called the testa, arises after the differentiation of ma-ternal ovular integuments during seed development. We recently described a thick cuticle tightly embedded in the mature seed’s endosperm cell wall. We show here that it is produced by the maternal inner integument 1 layer and, remarkably, transferred to the developing endosperm. Arabidopsis transparent testa (tt) mutations cause maternally derived seed coat pigmentation de-fects. TT gene products encode proteins involved in flavonoid metabolism and regulators of seed coat development. tt mutants have abnormally high seed coat permeability, resulting in lower seed viability and dormancy. However, the biochemical basis of this high permeability is not fully understood. We show that the cuticles of developing tt mutant integuments have profound structural defects, which are associated with enhanced cuticle permeability. Genetic analysis indicates that a functional proan-thocyanidin synthesis pathway is required to limit cuticle permeability, and our results suggest that proanthocyanidins could be intrinsic components of the cuticle. Together, these results show that the formation of a maternal cuticle is an intrinsic part of the normal integumental differentiation program leading to testa formation and is essential for the seed’s physiological properties.

1This work was supported by grants from the Swiss National Sci-ence Foundation and by the State of Geneva.

2Address correspondence to [email protected] 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.plantphysiol.org) is: Luis Lopez-Molina ([email protected]).

S.L. and L.L.-M. conceived the experiments; S.L., J.D.G., A.U.-P., and L.D. performed the experiments; S.L., J.D.G., L.D., and L.L.-M. analyzed the data; L.D. drew the artwork; S.L. and L.L.-M. wrote the article.

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Plant Physiol. Vol. 177, 2018 1219

et al., 2008; El-Maarouf-Bouteau and Bailly, 2008; Basbouss-Serhal et al., 2016; Chahtane et al., 2017). The higher oxidative stress in tt seeds could be exacerbated by their low levels of flavonoids, which have antioxi-dant properties. Nevertheless, the biochemical basis of the higher permeability of tt mutant seed coats is not fully understood.

Plant cell walls can regulate their permeability through three types of modifications: lignification, su-berification, and cutinization (Nawrath et al., 2013). The presence of lignin in seeds was only reported in the hylum region connecting the seed to the mother

plant (Balanzà et al., 2016). Whether lignin plays a ma-jor role in seed permeability to external compounds is poorly understood. In contrast, suberin likely con-tributes to seed coat permeability, since mutant seeds affected in suberin synthesis have increased seed coat permeability and low dormancy (Beisson et al., 2007; Molina et al., 2008; Fedi et al., 2017).

Cutin is found in cuticles, hydrophobic depositions on the plant’s aerial surfaces that limit excessive tran-spiration and regulate the plant’s permeability to outer compounds (Nawrath et al., 2013; Yeats and Rose, 2013; Xue et al., 2017). They are made of a complex

Figure 1. Development of the endosperm cuticle. A, Localization of the endosperm-associated cuticle as described previously (De Giorgi et al., 2015) in mature seeds and TEM micrograph of the cuticle in a mature seed. The drawings are not to scale in order to better visualize the localization of the cuticle with respect to its neighboring elements. Bar = 500 nm. B to F, Sudan Red stainings (middle row) and TEM micrographs (bottom row) of the cuticle in wild-type (Columbia-0 [Col-0]) seeds at the indicat-ed developmental stages. The top row represents the anatomy of seeds at each stage (for details, see Supplemental Fig. S1), and the dashed rectangles indicate the region where the microscopy observations were performed. Cyan dotted lines (middle row) indicate the outer walls of ii1 cells; arrows and brackets indicate the cuticle. c, cuticle; e, endosperm; ii1, inner integument 1 cell; ii1′, inner integument 1′ cell; ii2, inner integument 2 cell; oi1, outer integument 1 cell; oi2, outer integument 2 cell; pcw, primary cell wall. Bars = 10 µm (middle row) and 500 nm (bottom row).

Cuticular Defects in transparent testa Seeds




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assembly of multiple lipids with various degrees of co-valent linkages collectively referred to as cutin, cutan, and waxes. In particular, cuticles contain abundant deposits of polymers of C16 and C18 fatty acid mono-mers referred to as cutin polymers (Beisson et al., 2012; Domínguez et al., 2015). Cuticles also have been impli-cated in plant development, physiology, and defense responses against pathogens (Yeats and Rose, 2013; In-gram and Nawrath, 2017).

Cuticular structures also are present on the devel-oping embryo as well as on integumental seed layers, where their role is less understood. In this context, they

are important to prevent organ fusion, and they may regulate the diffusion of signaling molecules that con-trol seed development (Tanaka et al., 2001; Yang et al., 2008; Moussu et al., 2013; Ingram and Nawrath, 2017).

A thick cuticle tightly associated with the outer side of the mature endosperm was described recently in mature Arabidopsis seeds (De Giorgi et al., 2015). This cuticle is a barrier surrounding and isolating the seed’s living tissues (Fig. 1A). In cutin-deficient bodyguard1 mutant seeds, the endosperm-associated cuticle ex-hibits structural defects (De Giorgi et al., 2015), which are associated with higher endosperm permeability to

Figure 2. Secretion of cuticular components. Electron micrographs of the cuticle are shown in wild-type (Col-0) seeds at the preglobular (A), heart (B), and walking stick (C) stages. Cyan arrows indicate electron-dense material that may contribute to the building up of the cuticle. Magenta arrows indicate organelles of the secretion pathway (endoplasmic reticulum and Golgi apparatus). The dark blue arrow in C (middle image) indicates a secretion vesicle fusing with the plasma membrane and in the process of delivering putative cuticular material to the cell wall. The arrowheads in C indicate stitching artifacts. e, endosperm. Bars = 500 nm.

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Figure 3. Structure and transfer of the cuticle. Electron micrographs of the cuticle are shown in wild-type (Col-0) seeds at the indicated developmental stages. Green arrows indicate the electron-lucent layer of the cuticle, and yellow arrows indicate the endosperm primary cell wall. In B and C, cyan arrows indicate places where the cuticle is closely apposed to the endosperm primary cell wall. The asterisk in C indicates a place where the plasma membrane of an ii1 cell is still in close contact with its primary cell wall and the cuticle. Arrowheads in C and D indicate stitching artifacts. e, endosperm; e pcw, endosperm primary cell wall; t, tannins. Bars = 500 nm.





Toluidine Blue, a dye commonly used to assess cutic-ular permeability (Watanabe et al., 2004; Kurdyukov et al., 2006; Voisin et al., 2009; Denay et al., 2014; De Giorgi et al., 2015; Jakobson et al., 2016). Similar to tt mutants, cutin biosynthesis mutants have low seed vi-ability and dormancy (De Giorgi et al., 2015). However, unlike tt mutants, cutin biosynthesis mutants do not have striking testa developmental defects. Although the cuticle is tightly associated with the endosperm, its cellular origin has not been investigated. Furthermore, whether the cuticle contributes to the high permeability of tt seeds is unknown.

Here, we systematically followed the development of the cuticle throughout seed development. Using a combination of microscopy and permeability experi-ments, we show that the cuticle originates in the mater-nal ii1 layer from where, remarkably, it is transferred to the developing endosperm. We performed a series of microscopy studies to assess the integrity of the endo-sperm-associated cuticle in various tt mutants. We find severe defects in the structure of the cuticle in all of the tt mutant seeds considered. We show that these defects are associated with a marked increased permeability of tt mutant endosperms to Toluidine Blue.

Altogether, our results show that the endosperm- associated cuticle is a maternal structure undergoing dynamic modifications during seed development that are intimately linked to the differentiation of ii1 cells in the testa. Our results raise alternative interpretations of the seed physiology phenotypes associated with tt mutant seeds. Namely, the low seed viability and dor-mancy of tt mutants could originate from a more per-meable endosperm-associated cuticle.

RESULTS

The Endosperm Cuticle Is of Maternal Origin, Arising from ii1 Cells

We previously described a cuticle associated with the outer cell wall of the endosperm in mature Ara-bidopsis seeds (De Giorgi et al., 2015; Fig. 1A). How-ever, despite its tight association with the endosperm, whether the cuticle is produced by endosperm cells was not investigated (De Giorgi et al., 2015). To address this question, we systematically monitored the occur-rence of cuticular structures during seed development using transmission electron microscopy (TEM) and histology techniques. We focused in particular on the ii1 maternal cells because these are in early and per-manent physical proximity to developing endosperm cells, being located on their outer side throughout seed development (for a summary of the organization of the seed and its internal structures during development, see Supplemental Fig. S1).

At the preglobular embryo stage, the endosperm is not yet cellularized (Mansfield and Briarty, 1990). TEM analysis confirmed that no endosperm cells were formed yet; in the developing endosperm, precursor

cell wall and membrane structures were seen aligned in the vicinity of ii1 cells as well as organelles (e.g. chloroplasts and mitochondria; Fig. 1B; Supplemen-tal Fig. S2). At this stage, TEM images clearly revealed the presence of an electron-dense cuticle-like structure on the ii1 cell wall facing the developing endosperm (Fig. 1B; Supplemental Fig. S2). Such a structure also is visible in previous publications (Mansfield and Briarty, 1991; Beeckman et al., 2000). Furthermore, the ii1 cell walls frequently contained dense particles, some of them resembling cutinsomes, suggesting that they could be involved in the building of a cuticle (Fig. 2A; Heredia-Guerrero et al., 2008; Domínguez et al., 2015; Stępiński et al., 2017). The bona fide cuticular nature of this structure was further confirmed by staining with the lipophilic dye Sudan Red 7B (Fig. 1B; Brundrett et al., 1991).

We hypothesized that the endosperm-associated cuticle arises from this ii1-associated cuticle. To test this hypothesis, we followed the development of this cuticle from the preglobular stage throughout seed development.

During the globular and heart stages, dense particles in the cell walls of ii1 cells still could be seen, and the cuticle acquired a globular organization (Figs. 2B and 3, A and B). Moreover, an additional electron-lucent layer of the cuticle started to become visible at these stages, between the electron-dense layer and the endosperm cell wall (Fig. 3, A and B, green arrows). In some in-stances, the ii1 primary cell wall, the cuticle, and the endosperm cell wall were in continuity; in most cells, however, the endosperm cell wall was detached from the cuticle, which was clearly associated with ii1 cells (Figs. 1C, 2, A and B, and 3A).

At the heart stage, the endosperm starts to cellu-larize (Mansfield and Briarty, 1990; Debeaujon et al., 2007), and its cell wall starts to thicken. Remarkably, at this developmental stage, we found that the cuticle be-came more frequently associated with endosperm cells than ii1 cells (Fig. 3B).

At the torpedo and walking stick stages, the con-tinuing development of the cuticle could be inferred from the continuous presence of dense particles in the ii1 cell walls (Fig. 2C). Furthermore, we frequently ob-served organelles of the secretion pathway in ii1 cells (Fig. 2C, magenta arrows), suggesting that these cells were in a state of active secretion. Their secretion ma-chinery may be involved in the production of tannins, but it also could be involved in the secretion of cuticular components.

By the mature embryo stage, the dense particles in ii1 cell walls were no longer observed. Both the elec-tron-lucent and electron-dense layers of the cuticle thickened significantly (Figs. 1E and 3D). The cuticle was fully associated with the endosperm cell wall (Figs. 1E and 3D), suggesting that it is maternally in-herited after being transferred from ii1 cells to endo-sperm cells between the globular and mature embryo stages (Fig. 3). In the last stages of seed development, the electron-dense component of the cuticle became

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Figure 4. tt mature mutant seeds exhibit defective cuticles. A, Sudan Red and Auramine O stainings of the cuticle in wild-type (Col-0, Landsberg erecta [Ler], and Wassilewskija [Ws]), tt4 and tt15 (in the Col-0 background), tt1 and tt5 (in the Ler back-ground), and tt2 (in the Ws background) seeds upon 72 h of paclobutrazol (PAC) imbibition. B, Electron micrographs of the cuticle in wild-type (Col-0 and Ler), tt4, tt5, and tt1 seeds upon 24 h (tt4, tt5, and tt1) or 120 h (Col-0 and Ler) of PAC imbibi-tion. Arrows indicate the cuticle, and arrowheads indicate a missing or aberrant cuticle. e, endosperm; pcw, primary cell wall. Bars = 10 µm (A) and 300 nm (B).





denser; in mature (brown) seeds, the cuticle had its fi-nal appearance, resembling, both in thickness and in texture, the previously described endosperm-associated cuticle (Fig. 1, compare F with A; De Giorgi et al., 2015).

Altogether, these results strongly suggest that the mature endosperm-associated cuticle is of maternal ii1 cellular origin.

tt Mutants Develop a Defective Cuticle with Increased Permeability to Toluidine Blue

TT genes are a set of genes defined by seed coat pigmentation defects in tt mutant seeds. There are

about 20 TT genes that can be broadly described as encoding developmental regulators or proteins in-volved in flavonoid metabolism, including flavonoid synthesis, modification, and transport (Pourcel et al., 2005; Routaboul et al., 2012). tt mutant seeds have abnormally high testa permeability to external com-pounds, such as tetrazolium salt, which leads to lower seed viability and dormancy (Debeaujon et al., 2000). The biophysical basis of these permeability defects is poorly understood. Given the likelihood that the endo-sperm-associated cuticle originates from ii1, and given its previously reported role in regulating permeability to external compounds (Figs. 1–3), we asked whether tt

Figure 5. tt mutant seeds exhibit permeability defects upon testa rupture. Toluidine Blue permeability tests were performed on wild-type or tt mutant seeds upon treatment with PAC (A) or ABA (B). As indicated by the seeds shown in the examples, PAC treatment repressed testa rupture (TR), while ABA treatment induced testa rupture; the arrow in B indicates the exposed endo-sperm and cuticle. tt4 was in the Col-0 background, tt5 and tt1 were in the Ler background, and tt2 was in the Ws background.

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mutants have alterations in the endosperm-associated cuticle (De Giorgi et al., 2015).

In exploratory experiments, we used optical and electron microscopy to assess the structural status of the cuticle in tt mutants. For this analysis, we chose several tt mutants (tt1, tt2, tt4, tt5, and tt15) that were representative of the palette of functions encoded by the TT gene class. TT1 encodes a zinc-finger pro-tein that is part of the regulatory network controlling flavonoid accumulation at the transcriptional level (Appelhagen et al., 2011). TT2 encodes a seed-specific MYB transcription factor that interacts with TT1 and that controls tannin synthesis (Appelhagen et al., 2011). TT4 encodes chalcone synthase, a key flavonoid- synthesizing enzyme (Feinbaum and Ausubel, 1988). TT5 encodes chalcone-flavanone isomerase, also in-volved in flavonoid synthesis (Shirley et al., 1992). TT15 encodes a UDP-Glc:sterol glucosyltransferase sup-posed to regulate proanthocyanidin (PA) accumu-lation in the vacuole (Focks et al., 1999; DeBolt et al., 2009). We stained seed sections using Sudan Red 7B as well as the fluorescent cuticle marker Auramine O (Szczuka and Szczuka, 2003; Buda et al., 2009).

Strikingly, we observed that the cuticle of tt1, tt2, and tt15 mutant seeds seemed either absent or reduced (as judged by the intensity of the staining; Fig. 4A, white arrowheads). Furthermore, TEM examination con-firmed that tt1 mutant seeds had no detectable cuticle (Fig. 4B; Supplemental Fig. S3A). In tt4 and tt5 mutant seeds, a cuticle was detectable (Fig. 4A), but it had an aberrant organization. Indeed, the electron-dense lay-er of the cuticle of tt4 and tt5 mutant seeds was either absent or aberrantly thin when compared with that of wild-type seeds (Fig. 4B; Supplemental Fig. S3B).

These observations indicate that tt mutants have a defective cuticle. To independently test this, we as-sessed the permeability of tt mutant endosperms to Toluidine Blue, as described previously (De Giorgi et al., 2015). Toluidine Blue does not penetrate the tes-ta, which prevents assessing endosperm permeability with this dye. In abscisic acid (ABA)-treated seeds, testa rupture takes place without endosperm rupture, thus directly exposing the endosperm and its cuticle to external compounds (De Giorgi et al., 2015). In con-trast, treating seeds with PAC, an inhibitor of GA syn-thesis, does not rupture their testa or their endosperm.

Figure 6. The endosperm-associated cuticle is of maternal origin. Representative images show Toluidine Blue permeability tests performed on the progeny of crosses between tt1 or tt4 mutant seeds and seeds of their respective wild-type ecotypes (Ler or Col-0, respectively). Whole seeds were incubated in PAC (no testa rupture; A) or in ABA (testa rupture; B) and then incubated in Toluidine Blue (see “Materials and Methods”). Embryos were dissected out of the seeds for imaging.







Therefore, we compared the blue staining of embryos dissected from PAC- and ABA-treated seeds incubated with Toluidine Blue (see “Materials and Methods”).

In PAC-treated seeds, wild-type, tt1, tt2, tt4, and tt5 embryos were not markedly stained with Toluidine Blue (Fig. 5A). This suggests that both wild-type and tt testas have low permeability to Toluidine Blue. In contrast, in ABA-treated seeds, we observed strong blue staining of embryos from tt1, tt2, tt4, and tt5 seeds but not from wild-type seeds (Fig. 5B). These results strongly suggest that tt1, tt2, tt4, and tt5 mutant seeds indeed have a more permeable endosperm-associated cuticle.

Similar to other tt mutants, the seed coat defects of tt1 and tt4 mutants are maternally derived (Debeau-jon et al., 2000). We took advantage of this property to independently evaluate the hypothesis that the cuticle is of maternal origin. We produced hybrid seeds from

reciprocal crosses between wild-type and tt1 or tt4 mu-tants. High Toluidine Blue permeability was observed in hybrid seeds produced by tt1 or tt4 mother plants but not in those produced by wild-type mother plants, whose permeability was similar to that of wild-type seeds (Fig. 6). Furthermore, we used a ProBAN6:BARNASE transgenic line, which specifically expresses the cyto-toxic gene BARNASE in the inner integument cells, in-cluding ii1 cells, to test the contribution of these cells to seed permeability (Debeaujon et al., 2003). Strikingly, ABA-treated ProBAN6:BARNASE seeds exhibited high permeability to Toluidine Blue, further strengthening the conclusion that the cuticle is of maternal origin (Supplemental Fig. S4). We also observed high Tolui-dine Blue permeability in PAC-treated ProBAN6:BARNASE seeds (Supplemental Fig. S4). This indicates that the ProBAN6:BARNASE seed coat has more severe alter-ations than most tt mutants.

Figure 7. Mutant seeds deficient in PA synthesis have increased permeability to Toluidine Blue. A, Toluidine Blue permeabil-ity tests were performed on ABA-treated (ruptured testa) seeds lacking regulators of flavonoid synthesis. B, Schematic of the flavonoid biosynthesis pathway. The three major end products, flavonols, anthocyanins, and PAs, are indicated in boldface colors; the different flavonoid biosynthesis mutants used are indicated next to the arrows. Toluidine Blue permeability tests were performed on ABA-treated (ruptured testa) wild-type (WT) and mutant seeds as indicated. Arrows in the pathway may represent several enzymatic steps. Permeability tests performed on PAC-treated seeds (with unruptured testa) are presented in Supplemental Figure S5.

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Altogether, these results confirm the maternal origin of the endosperm-associated cuticle.

PA Synthesis Is Necessary for Cuticle Integrity

TT1 and TT2 encode seed coat developmental reg-ulators; therefore, it is not surprising that the cuticle of tt1 and tt2 mutant seeds had high permeability to Toluidine Blue (Figs. 5B and 7A), consistent with the severe cuticle alterations observed in our microscopy studies (Fig. 4C). TTG1 and TT16 encode transcription factors that regulate PA accumulation in the seed coat as well as epidermal and endothelial cell fate specifi-cation (Walker et al., 1999; Nesi et al., 2002). Therefore, it was not surprising that ttg1 and tt16 seeds also had high permeability to Toluidine Blue (Fig. 7A). In the case of ttg1, high permeability was also visible in pres-ence of PAC (Supplemental Fig. S5).

On the other hand, TT4 and TT5 encode enzymes that function in the early steps of flavonoid biosynthe-sis, where they control the synthesis of chalcone and naringenin, respectively. The observation that tt4 and tt5 mutants have increased permeability to Toluidine Blue suggests that a functional flavonoid synthesis pathway is necessary for cuticle integrity.

Downstream of naringenin, the flavonoid synthesis pathway trifurcates into the flavonol, anthocyanin, and PA synthesis pathways (Pourcel et al., 2005; Routaboul et al., 2012). To genetically identify the pathway that is functionally necessary for cuticle formation, we exam-ined cuticle permeability to Toluidine Blue in mutant seeds defective in selected flavonoid synthesis genes (Fig. 7B). FLS1 encodes FLAVONOL SYNTHASE1, necessary for the synthesis of flavonols from dihydro-flavonols (Pourcel et al., 2005). fls1 mutants lack flavo-nols but retain the capacity to synthesize anthocyanins and PAs. BANYULS (BAN) encodes a dihydroflavonol reductase-like protein necessary for epicatechin syn-thesis, which is necessary for PA biosynthesis. TT12 encodes a proton antiporter involved in the transpor-tation of PA precursors. ban, tt12, and tt15 mutants are unable to synthesize PAs but can synthesize flavonols and anthocyanins. The cuticle of fls1 mutant seeds dis-played a mild increase in Toluidine Blue permeability relative to the wild type (Fig. 7B). In striking contrast, ban, tt12, and tt15 seeds had a cuticle with markedly increased permeability to Toluidine Blue (Fig. 7B).

PAs turn brown as they oxidize, imparting color to Arabidopsis seeds. Using bright-field microscopy, we observed that the cuticle was brown in wild-type seeds but not in tt4 and tt5 seeds (Supplemental Fig. S6). The brown color also was apparent in our previous report (De Giorgi et al., 2015).

Altogether, these results show that a functional PA synthesis pathway is essential for cuticle integrity. Fur-thermore, they also support the hypothesis that PAs could be components of the endosperm-associated cuticle, as shown previously for the tomato (Solanum lycopersicum) epidermis-associated cuticle, where they

contribute to its biophysical properties (Dominguez et al., 2009; España et al., 2014).

In the course of these experiments, we noticed addi-tional structural features of the mature testa suggesting that the cuticle is integrated in an apoplastic network; this is described and discussed in Supplemental Dis-cussion S1 and Supplemental Figures S7 to S11.

DISCUSSION

Maternal Origin and Transfer of the Endosperm-Associated Cuticle

Here, we describe the development of a maternal cuticle covering the inner side of the ii1 layer during seed development. At early developmental stages (e.g. preglobular), a cuticle is associated with ii1 cells; at this stage, the endosperm cell wall is very thin, and it is seldom seen in contact with the cuticle. Conversely, in mature seeds, a cuticle is clearly associated with the endosperm, and ii1 cells no longer have a primary cell wall. Importantly, we could never observe two inde-pendent cuticles present at the same time between ii1 and endosperm cells. Rather, we were only able to ob-serve one cuticle associated with both an ii1 cell wall and an endosperm cell wall (Fig. 3, B and C). Therefore, and remarkably, these observations show that the cuti-cle is transferred from ii1 cells to endosperm cells (Fig. 3). As ii1 cells undergo programmed cell death during the final steps of testa maturation, their cuticle eventu-ally becomes associated with the endosperm cell wall. Our Toluidine Blue permeability experiment with wild-type × tt hybrid seeds and the ProBAN6:BARNASE transgenic line provides orthogonal evidence for the maternal origin of the endosperm-associated cuticle (Fig. 6; Supplemental Fig. S4).

The developmental phenotypes of tt mutants al-lowed us to distinguish between two phases in the development of the cuticle, approximately before and after the torpedo stage. Indeed, preliminary obser-vations indicate that the cuticle in tt1 and tt4 mutant seeds at heart/torpedo stages is similar to that of wild-type seeds at the same stages (Supplemental Fig. S12). The observation that, in mature tt1 and tt4 seeds, the cuticle is either absent or highly altered suggests that TT genes are necessary for cuticle formation only in this second phase, after the torpedo stage.

During the first phase of cuticle synthesis, granu-lar cuticular components secreted by ii1 cells are seen and the cuticle acquires a globular appearance (Figs. 1 and 2). In the second phase, (1) an electron-lucent layer forms and thickens (Fig. 3, B and C); (2) the elec-tron-dense layer thickens and becomes denser (Fig. 3); and (3) an electron-dense networks forms around ii1 cells, into which the cuticle is integrated (Supplemen-tal Figs. S9–S11). The transition from the first to the second phase coincides with the transfer of the cuticle from ii1 cells to endosperm cells: this opens up the pos-sibility that endosperm cells might regulate the second














phase of cuticle development. However, it is likely that both phases are under the control of ii1 cells. Indeed, it has been shown that TT1 and TT4 are both expressed specifically in ii1 cells during seed development and not in the endosperm (Sagasser et al., 2002; Debeaujon et al., 2003).

In developing cutinized tissues, it is crucial to en-sure that the cuticle remains continuous as cells grow and divide. This is achieved by direct cuticle inheri-tance by the daughter cells. In such cases, epidermal cells divide in an anticlinal manner and their cuticle is inherited and shared between their daughter cells. An exception to this model is embryonic de novo syn-thesis. For example, it has been shown in Arabidopsis, rice (Oryza sativa), and soybean (Glycine max) that em-bryonic cuticles are deposited de novo at the surface of young embryos (Chamberlin et al., 1994; Mariani et al., 1999; Moussu et al., 2013; Ingram and Nawrath, 2017). Here, we describe a model of cuticle transmission during development in which a cuticle is transmitted

from one cell to another that is juxtaposed to it. A striking feature of this mode of inheritance of the cuticle is that the transmission happens between cells that are not of the same type, nor of the same lineage, nor even of the same genetic endowment (i.e. from diploid integu-mental cells to triploid endosperm cells).

The Endosperm-Associated Cuticle Might Originate in the Epidermal Nucellar Cells of the Ovule Primordium: An Additional Transfer?

In a preliminary attempt to uncover the earliest pos-sible origin of the cuticle, we examined prefertiliza-tion samples. We observed an electron-dense cuticle covering the nucellar epidermal cells (Supplemental Fig. S13). In contrast, inner integument primordia cells were not cutinized, except in some locations in close proximity to the nucellus epidermis. In such places, a thin cuticle could be seen at the surface of the inner integument (Supplemental Fig. S13, cyan arrows).

Figure 8. Model describing the formation and successive transfers of a maternal cuticle. Schematic drawings illustrate the for-mation of the endosperm-associated cuticle present in mature seeds. Top images represent whole organs, and bottom images are magnified views of the insets. Drawings were not made to scale in order to better visualize the localization of the cuticle with respect to its neighboring elements. A, In developing ovules, a cuticle primarily covers the nucellus epidermis; the inner side of inner integument primordia also is cutinized, but this cuticle is thinner than the nucellar one. Upon ovule development and degeneration of the nucellus, both cuticles together become the inner integument cuticle. B, After fertilization, this cuticle is found covering the inner cell wall of ii1 cells. During seed development, the primary cell walls of endosperm cells become closely apposed to the cuticle. By the end of seed maturation, ii1 cells undergo cell death and the cuticle becomes an extension of the endosperm cell wall. At this stage it is integrated in an apoplastic network that surrounds ii1 cells. ii1′, inner integument 1′ cell; ii2, inner integument 2 cell; oi1, outer integument 1 cell; oi2, outer integument 2 cell.

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Upon the completion of ovule maturation and fertil-ization, the nucellus eventually degenerates, while the fusion of the central cell and the sperm nuclei produces the endosperm (for review, see Ingram, 2017). As a con-sequence, after fertilization, both the nucellus cuticle and the inner integument cuticle are predicted to be lo-calized between the endosperm and ii1 cells, where the precursor of the endosperm cuticle is located (Fig. 8). This suggests that the endosperm cuticle may be in-herited from the inner integuments but also from the nucellar epidermis after its degeneration.

Altogether, we speculate that the endosperm cuticle could be the result of a double transfer: first, it would originate at the surface of nucellar epidermal cells, which would transmit it to ii1 cells (with some contri-bution from de novo synthesis at the surface of ii1 cells during the growth of the inner integument primor-dia); later, during seed development, inner integument

cells, in turn, would transmit the cuticle to endosperm cells (Fig. 8).

Alternative Interpretation of the High Permeability of tt Mutant Testa

Seed dormancy and viability are perhaps the most important physiological traits of mature seeds. Both traits rely on the testa’s biophysical permeability to ex-ternal compounds, and in particular on its capacity to limit oxidative damage of the embryo’s living tissues by limiting oxygen entry (Bailly et al., 2008; El-Maarouf- Bouteau and Bailly, 2008; Chahtane et al., 2017). The low seed viability and dormancy of the tt mutants is usually ascribed to the high permeability of the tt mu-tant testas, which, in turn, has been attributed to their low tannin content and structural defects (Debeaujon et al., 2000). However, the biophysical basis of their high permeability is not clear. Concerning the role of flavo-noids, the tannin depositions in seeds are not continu-ous structures that seal the seed’s living tissues from the outer environment. Rather, they appear as patches in the seed coat, corresponding to the cell bodies from which their synthesis was initiated during seed devel-opment (Supplemental Fig. S1). Thus, whether tannins by themselves play a significant role in limiting testa permeability appears uncertain.

Our permeability and microscopy observations showed that the endosperm-associated cuticle is de-fective in tt mutants. Genetic analyses pinpointed the importance of a functional PA synthesis pathway for cuticle integrity (Fig. 7B). Previous reports have shown that the syntheses of flavonoids and cuticle components are biochemically linked, both depending on products of the phenylpropanoid pathway (Vogt, 2010); moreover, in tomato, flavonoids are components

Table I. Ecotypes and mutants used in the studyThe wild-type ecotypes used in the study are Col-0, Ler, and Ws.

The table indicates the backgrounds of the mutant and transgenic lines used in the study.

Mutant or Transgenic Line Background

ban-1 WsProBAN6:BARNASE Wsfls1 Wstt1-1 Lertt2-3 Wstt4-11 Col-0tt5-1 Lertt12-1 Wstt15 Col-0tt16-1 Wsttg1-1 Ler

Table II. Sample size for microscopy dataThe number of seeds and cells that were analyzed for each genotype and stage presented in the article

are shown. No. Cells for TEM data refers to the number of ii1 cells examined in each case. Dashes indicate conditions that are not documented in the article.

Genotype StageTEM Histology

No. Seeds No. CellsNo. Seeds Sudan Red

No. Seeds Auramine O

No. Seeds Unstained

Col-0 Preglobular 3 37 6 – –Globular 3 22 11 – –

Heart 6 33 7 – –Torpedo 3 27 – – –

Walking stick 4 35 12 – –Mature embryo 4 32 9 – –

Brown seed 2 7 11 12 –Imbibed seed 8 30 120 41 43

Ler Imbibed seed 3 25 32 43 9Ws Imbibed seed – – 26 18 –tt1 Heart/torpedo 1 12 – – –

Imbibed seed 3 23 33 60 17tt2 Imbibed seed – – 19 10 –tt4 Heart/torpedo 1 8 – – –

Imbibed seed 4 22 20 57 43tt5 Imbibed seed 2 5 11 10 –tt15 Imbibed seed – – 18 20 –






of cuticles, influencing their synthesis and function (Dominguez et al., 2009; España et al., 2014; Outchk-ourov et al., 2018). The unambiguous identification of flavonoids as components of tomato cuticles was facil-itated by the fact that the tomato cuticle can be isolated physically. In contrast to the tomato cuticle, the Ara-bidopsis endosperm-associated cuticle cannot be iso-lated in sufficient amounts at present, precluding any biochemical analysis of its composition. An alternative approach to assess the presence of flavonoids in Ara-bidopsis cuticles is mass spectrometry imaging (Dong et al., 2016a,b). This also is very challenging. Indeed, the spatial resolution of this technique appears to be limited by the size of the ionization beam, which can go down to a few micrometers at best in order to study lipid composition (Dong et al., 2016a). Given that the endosperm-associated cuticle is approximately 300 nm thick, and given that it is immediately adjacent to the flavonoid-rich ii1 cells, mass spectrometry imaging would prove itself unable to unambiguously ascribe any chemical composition to this cuticle without in-curring contamination from the flavonoids contained in ii1 cells.

Here, we have provided visual evidence that PAs could indeed be components of the endosperm-associated cuticle in Arabidopsis seeds (Supplemental Fig. S6). Thus, our results suggest that the abnormal permeability of tt seeds originates from defects in their endosperm- associated cuticle, which could be due to their low PA content.

However, it cannot be excluded that PAs also exert a signaling function in the seed coat, regulating cuticle development. Consistent with this possibility, it has been shown that PAs regulate auxin signaling and ox-idative stress responses, which, in turn, could regulate cuticle development in Arabidopsis seeds (Doughty et al., 2014; Watkins et al., 2014).

Finally, the role of the cuticle during seed develop-ment is unknown. However, given its localization (i.e. inside a seed, which itself is inside a silique), two pu-tative roles seem particularly relevant among the pan-el of documented functions of cuticles (for review, see Shepherd and Griffiths, 2006; Ingram and Nawrath, 2017). First, one of the general functions of cuticles is to prevent organ fusion during the growth of adjacent organs (Sieber et al., 2000; Tanaka et al., 2001). In this scope, the cuticle could play a role in preventing fusion between endosperm and integumental cells. Second, the cuticle may prevent the diffusion of developmental signals promoting integumental cell death to the en-dosperm. Indeed, in the course of seed development, integumental cells secrete caspase-like proteins that trigger cell death in the different testa layers (Nakaune et al., 2005; Andème Ondzighi et al., 2008). Thus, it may be of paramount importance for the survival of the seed that the endosperm does not perceive these death signals; the cuticle might be the diffusion barrier ensuring that, indeed, this does not happen. Further research is needed to address these questions.

In the course of this study, we noticed two structural features of the cuticle that are worth mentioning. First, the cuticle has two gaps at the micropylar and chala-zal poles (Supplemental Figs. S7 and S8); while these gaps do not impair the permeability barrier function of the cuticle, they might play a role in seed development and seed germination (for details, see Supplemental Discussion S1). Second, the mature cuticle is integrated in an apoplastic network in the testa, part of which (if not its entirety) is of cuticular nature (Supplemental Figs. S9–S11; Supplemental Discussion S1).

MATERIALS AND METHODS

Plant Material and Growth Conditions

tt1-1 was described by Sagasser et al. (2002), tt2-3 by Nesi et al. (2001), and tt5-1 by Peer et al. (2001). Arabidopsis (Arabidopsis thaliana) seeds were provided by Maarten Koornneef and Roman Ulm. tt4-11 (SALK_020583) and tt15 (SALK_103581.54.50), both having a T-DNA exon insertion, were obtained from the Nottingham Arabidopsis Stock Centre. ban-1, tt12-1, tt16-1, fls1, ttg1-1, and the ProBAN6:BARNASE transgenic line were kindly provided by Isabelle Debeaujon and Loïc Lepiniec.

For each particular experiment presented, the seed material used (i.e. the wild-type seed material in the appropriate ecotype background and mutant seed material) was harvested on the same day from plants grown under iden-tical environmental conditions. Dry siliques were obtained around 8 weeks after planting.

Ecotype abbreviations and wild-type backgrounds for mutant and trans-genic lines are summarized in Table I. The sample size for each condition stud-ied by histology or by TEM is indicated in Table II.

Histology

Histology was performed as described previously (De Giorgi et al., 2015). Briefly, fixation was done overnight at 4°C in phosphate buffer, pH 7.2, with 4% (v/v) formaldehyde and 0.25% (v/v) glutaraldehyde. Dry seeds were punctured with a fine needle at the start of fixation, and all samples were infiltrated under vacuum. Samples were then embedded in pellets of 1.5% (w/v) agarose, dehydrated in a graded ethanol series, cleared in Neoclear, and embedded in paraffin. Twelve-micrometer-thick sections were cut with a Cut 4050 microtome (MicroTec), placed on SuperFrost slides (Roth), deparaffinized with Neoclear, and rehydrated with water. Staining with Sudan Red 7B was performed as described (Brundrett et al., 1991), using 0.1% (w/v) Sudan Red 7B in a 1:1 (v/v) mix of polyethylene glycol 400 and 90% (v/v) glycerol, incu-bated for 15 min; sections were then washed in water and mounted in glyc-erol. For double Calcofluor White/Auramine O stainings, sections were first incubated for 1 min in 0.01% (w/v) Calcofluor White in water, then washed in water and incubated for 5 min in 0.001% (w/v) Auramine O in water; finally, they were washed in PBS and mounted in PBS with glycerol (1:1, v/v).

Samples were examined with an Eclipse 80i widefield microscope (Nikon) equipped with a 40× Plan Fluor NA 0.75 lens, differential interference contrast optics, and a Digital Sight DS-Fi1 color CCD camera (Nikon). Fluorescence ex-citation was done with an Intensilight C-HGFI mercury vapor lamp (Nikon); Calcofluor White was examined using a 4′,6-diamino-phenylindole filter set (excitation, 352–402 nm; emission, 417–477 nm), and Auramine O was exam-ined using a CFP filter set (excitation, 426–450 nm; emission, 467–499 nm). For Supplemental Figure S9, Auramine O was examined using an SP5 confocal microscope (Leica) equipped with a 63× PlanApo NA 1.4 oil lens, an argon la-ser with excitation at 458 nm, a HyD detector with emission collection between 483 and 513 nm, and using a pixel size of 100 nm; a maximal projection of a few planes was performed and displayed.

TEM

TEM was performed as described previously (De Giorgi et al., 2015). Briefly, seeds were delicately punctured with a fine needle and fixed overnight at 4°C in 2.5% (v/v) glutaraldehyde and 0.01% (v/v) Tween-20 in 100 mm sodium

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cacodylate, pH 7, after vacuum infiltration. After a primary postfixation in 1.5% (v/v) osmium tetroxide for 2 h at 4°C and a secondary postfixation in 1% (w/v) uranyl acetate for 1 h at 4°C, seeds were embedded in pellets of 1.5% (w/v) agarose, dehydrated in a graded ethanol series, and embedded in Epon 812. Then, 85-nm ultra-thin sections were cut using a UCT microtome (Leica), stained with 2.5% (w/v) uranyl acetate and Reynolds lead citrate, and finally observed with a Tecnai G2 Sphera (FEI) at 120 kV, equipped with a high-resolution digital camera.

Image Treatment and Analysis

Images were analyzed using the software Fiji (Schindelin et al., 2012). For both histology and TEM, tiling and stitching were used to obtain high- resolution large fields of view. Tiling was performed manually, and stitching was done using the Fiji MosaicJ plugin (Thévenaz and Unser, 2007). When Auramine O simple stainings were shown, images were colorized using the Fire look-up table in order to provide a larger dynamic range and a higher quality of visualization of cuticular signals (or the absence of signal). When Auramine O/Calcofluor White double stainings were shown, Auramine O and Calcofluor White were colorized with a green and a magenta look-up table, respectively.

Figures were mounted using Illustrator CS 11.0.0 (Adobe).

Toluidine Blue Permeability Tests

Seeds were plated on Murashige and Skoog medium supplemented with 0.8% (w/v) Bacto-Agar (Applichem) and 5 μm ABA or 10 μm PAC. At 24 to 48 h after the start of imbibition, they were transferred to microcentrifuge tubes containing a solution of 0.05% (w/v) Toluidine Blue, 5 μm ABA, or 10 μm PAC, and they were incubated for an additional 12 h. Embryos were then dissected out of the seeds and imaged using an S6D stereomicroscope (Leica) equipped with an MC120HD CCD camera (Leica).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AT1G34790 (TT1), AT5G35550 (TT2), AT5G13930 (TT4), AT3G55120 (TT5), AT5G08640 (FLS1), AT1G61720 (BAN), AT5G24520 (TTG1), AT3G59030 (TT12), AT1G43620 (TT15), and AT5G23260 (TT16).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Summary of developing seed and seed coat anat-omy.

Supplemental Figure S2. TEM image of the endosperm before cellular-

ization.

Supplemental Figure S3. Cuticular defects in tt mature mutant seeds.

Supplemental Figure S4. A transgenic line expressing BARNASE in inner

integuments cells displays high seed permeability.

Supplemental Figure S5. Permeability tests on PAC-treated (unruptured

testa) seeds affected in flavonoid biosynthesis.

Supplemental Figure S6. The cuticle is brown in wild-type seeds but not in

flavonoid-deficient mutant seeds.

Supplemental Figure S7. Cuticular gaps in developing seeds.

Supplemental Figure S8. Gaps in the mature seed cuticle at the micropylar

and chalazal poles.

Supplemental Figure S9. The endosperm cuticle in mature seeds is inte-

grated in an apoplastic network of the testa.

Supplemental Figure S10. High-resolution view of the cuticle and the ap-

oplastic network of the testa in mature seeds.

Supplemental Figure S11. The cuticle does not form a network until the

mature embryo stage.

Supplemental Figure S12. Developing tt1 and tt4 mutant seeds appear to

have a normal cuticle.

Supplemental Figure S13. Cuticles in developing ovules.

Supplemental Discussion S1.

ACKNOWLEDGMENTS

We thank the reviewers for insightful comments. We thank Maarten Koorn-neef and Roman Ulm for providing mutant seed material. We are especially indebted to Isabelle Debeaujon, Loïc Lepiniec, and Steve Penfield for provid-ing mutant seed material during the revision stages of the article. We thank Mayumi Iwasaki for critical reading of the article and Mayumi Iwasaki and Urszula Piskurewicz for helpful discussions.

Received May 8, 2018; accepted May 22, 2018; published May 30, 2018.

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a maternally deposited endosperm cuticle contributes to the … · its ultimate dispersion. seed...

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