ERF family transcription factor gene from Arabidopsis thaliana, is expressed in specific cell types of roots, stems and seeds that undergo suberization

Plant Physiology and Biochemistry 46 (2008) 1051–1061

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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy

Research article

AtERF38 (At2g35700), an AP2/ERF family transcription factor gene from Arabidopsis thaliana, is expressed in specific cell types of roots, stems and seeds that undergo suberization Eric Lasserre*, Edouard Jobet, Christel Llauro, Michel Delseny Laboratoire Ge´nome et De´velopement des Plantes, Universite´ de Perpignan, CNRS, IRD; 52 avenue P. Alduy 66860 Perpignan, France

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 February 2008 Accepted 8 July 2008 Available online 19 July 2008

An inverse genetic approach was used to gain insight into the role of AP2/ERF-type transcription factors genes during plant development in Arabidopsis thaliana. Here we show that the expression pattern of AtERF38, which is, among the organs tested, more intensively expressed in mature siliques and floral stems, is closely associated with tissues that undergo secondary cell wall modifications. Firstly, public microarray data sets analysis indicates that AtERF38 is coregulated with several genes involved in secondary wall thickening. Secondly, this was experimentally confirmed in different types of cells expressing a ProAtERF38::GUS fusion: histochemical analysis revealed strong and specific GUS activity in outer integument cells of mature seeds, endodermal cells of the roots in the primary developmental stage and some sclerified cells of mature inflorescence stems. All of these cells are known or shown here to be characterized by a reinforced wall. The latter, which have not been well characterized to date in Arabidopsis and may be suberized, could benefit of the use of AtERF38 as a specific marker. We were not able to detect any phenotype in an insertion line in which ectopic expression of AtERF38 is caused by the insertion of a T-DNA in its promoter. Nevertheless, AtERF28 may be considered as a candidate regulator of secondary wall metabolism in particular cell types that are not reinforced by the typical deposition of lignin and cellulose, but that have at least in common accumulation of suberin-like lipid polyesters in their walls. Ó 2008 Elsevier Masson SAS. All rights reserved.

Keywords: AP2/ERF Coregulation Endoderm Secondary cell wall Seed coat Suberin Transcription factor

1. Introduction The availability of the Arabidopsis thaliana genome sequence revealed the presence of large multigene families putatively encoding more than 1500 transcription factors (TF [1]). The task of unraveling their function is still at its beginning and can be carried out using a variety of reverse genetic methods, including systematic insertional mutagenesis and genome-wide expression profiling technologies such as DNA microarrays. Coexpression patterns across many microarray data sets may indeed reveal networks of genes involved in linked processes, thus providing a relevant starting point for the functional characterization of unknown genes. This approach has previously been used with success to infer relationships between coexpression and function in humans (e.g. [2]) and plants (e.g. [3]). The AP2/ERF family of plant transcriptional regulators is one of the largest, with 147 predicted members in Arabidopsis thaliana according to the most recent study [4]. It is characterized by

* Corresponding author. fax: þ33 4 68 66 84 99. E-mail address: [email protected] (E. Lasserre). 0981-9428/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2008.07.003

a DNA-binding domain first identified in APETALA2 (AP2 [5]) and the Ethylene-Response Factors (ERF [6]). It has been demonstrated that members of this family have important functions in a broad range of biological processes, from growth and development to the response to environmental stimuli ([4] and references therein). However, the biological function of most of them is unknown. Here, we present original data opening the way to the functional characterization of the AtERF38 gene (named according to the nomenclature of Nakano et al. [4], AGI At2g35700). Our primary goal was to identify TF genes controlling seed maturation. In the course of cloning ABI5 [7], we obtained preliminary data indicating that AtERF38, a gene located on an adjacent BAC clone, is expressed at the stage of seed maturation (unpublished data). Depending on the classification, this gene belongs to the group IIId of the ERF subfamily [4] or A-4 of the DREB subfamily [8], both characterized by a single occurrence of the DNA-binding domain. Based on a detailed expression study of this gene, including the analysis of publicly available microarray datasets, we could associate it with the secondary thickening of cell walls in roots, inflorescence stems and seeds. The reinforcement of cell walls occurs in tissues that need to withstand pressure, such as xylem vessels, or to control

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water, solute and gas exchanges, like the seed coat. Secondary wall synthesis is often characterized by a marked increase in the deposition of cellulose, non-cellulosic cross-linking polysaccharides like xylan, and lignin. In some specific cell types, the thickening material can take the form of the apoplastic lipid polyesters suberin or cutin [9]. In Arabidopsis thaliana, several genetic screens for phenotypes like fragile fiber (fra mutants [10]) or inward collapse of xylem cells (irx mutants [11]) allowed the identification of several genes encoding enzymes associated with the biosynthesis and deposition of cellulose, glycans and lignin in the cell wall [3,12–17]. A few transcription factors that regulate these processes have been isolated and characterized [18,19] and some candidates identified in gene lists associated with secondary thickening [3,13,20]. The results presented here indicate that AtERF38 is an interesting candidate gene to encode such a factor in some specific cells of Arabidopsis thaliana. 2. Results 2.1. RT–PCR analysis of AtERF38 expression As a first approach to understanding the biological function of AtERF38, expression analysis was carried out by RT–PCR in selected tissues (Fig. 1A). In these tissues, the maximum accumulation of transcripts was found to occur in siliques from 8 to 11 days after flowering (daf) and in inflorescence stems of mature plants (i.e. 7 weeks old, when the first silique has dried). In our growth conditions, these siliques contained embryos from torpedo to curled cotyledon stages. Based on the fact that the AtERF38 transcripts are most abundant at the stage where those of At2S3, a storage protein gene, begin to accumulate (Fig. 1A), it was interesting to investigate whether this gene could be a regulator of embryo maturation. As a first test, we took advantage of a group of Arabidopsis thaliana mutants known to be affected in several aspects of this developmental stage [21]. ABSCISIC ACID INSENSITIVE3 (ABI3), FUSCA3 (FUS3), LEAFY COTYLEDON1 (LEC1) and LEC2 are transcription factors that constitute a network of local and redundant gene regulation, controlling most aspects of seed maturation, such as accumulation of storage compounds, cotyledon identity, acquisition of desiccation tolerance and dormancy ([21] and references therein). The expression of AtERF38 was therefore investigated at the 8–11 daf stage in the siliques of seven mutants: abi3, fus3, lec2, all combinations of double mutants, the triple mutant and the wild-type control (Fig. 1B). At2S3 was used as a marker of the impact of the mutations studied on the maturation stage of embryo development: its expression is severely reduced in the lines harboring at least the abi3 mutation or in the fus3/lec2 double mutant, less affected in the fus3 background and almost unchanged in lec2, in agreement with previously published results [22]. It is clear in Fig. 1B that the expression af AtERF38 is unaffected in all seven mutant lines. It is therefore very unlikely that this gene is a regulator of embryo maturation. This prompted us to look for another function by identifying co-regulated genes with known function. 2.2. AtERF38 is coregulated with genes involved in secondary cell wall metabolism To identify genes co-expressed with AtERF38, we took advantage of the publicly available microarrays datasets produced in the AtGenExpress project [23], using the Expression Angler tool from the Botany Array Resource ([24], see Section 4). The results are presented in Table 1: 29 genes are listed based on the fact that the Pearson correlation coefficient between their set of expression values across all experiments and that of AtERF38 meet the cut-off value of 0.8. A value of 1 characterizes a perfect

correlation and AtERF38 against itself is indicated in Table 1 to remind us of this fact. Several lines of evidence clearly link most of the listed genes to secondary cell wall metabolism. The most striking is that mutations in six of these genes give rise to an irregular xylem (irx) phenotype. Their names are indicated in the last column of Table 1. These mutants display collapsed xylem cells in the mature inflorescence stem because their weakened walls do not resist the negative pressure of water flow [11]. Three of them, irx1, 3 and 5, are particularly interesting because they correspond to mutations in the cellulose synthase genes CesA8, 7 and 4 respectively. The irx phenotype is caused in that case by a deficit in cellulose deposition [12]. This result clearly orientates the putative function of the coregulated genes towards secondary cell wall metabolism: among the ten genes of the CesA family three, CesA8, 7 and 4, are involved in cellulose deposition in secondary walls. Three other genes of the same family, namely CesA1, 3 and 6, form a complex in primary cell wall biosynthesis [25]. None of the latter appears in the list of the genes coregulated with AtERF38, even after decreasing the Pearson correlation coefficient cut-off value (data not shown). The second evidence is that ANAC012 (At1g32770), another gene listed as coregulated with AtERF38, has recently been shown to negatively regulate xylary fiber development in Arabidopsis thaliana [19]. The expression of this gene in xylem had previously been experimentally confirmed [20,26]. Apart from the six IRX genes and ANAC012, thirteen other genes appearing in the list have been attributed a putative role in secondary wall structure or vascular tissue function based on genome-wide expression profiling. In Table 1, these genes are reported as core xylem-specific [26], secondary cell wall gene [13], coregulated with CesA4, 7 and 8 genes [3] or vascular tissue function gene [20]. Among them, the expression of ANAC073 (At4g28500) in xylem has been experimentally confirmed [26]. Among the remaining nine genes, At3g59010, whose expression is most closely correlated with that of AtERF38, putatively encodes a pectinesterase and is thus annotated as involved in cell wall modification. Taken together, these results led us to look for clues about the function of AtERF38 in tissues that undergo secondary thickening. Based on the RT–PCR analysis presented in Fig. 1, we focused our attention on seeds and inflorescence stems.

2.3. The promoter of AtERF38 drives GUS expression in the cells of the outer integuments of mature seeds Based on the fact that AtERF38 is most highly expressed in seeds among the tissues we tested (Fig. 1A) but is unlikely to have a role in embryo maturation (Fig. 1B), we first focused our analysis on the seed coat. This tissue, composed of five cell layers and which differentiates primarily from cells of the ovule integuments, undergoes a dramatic differentiation process during which cells of both outer integument layers produce a thick secondary wall [27,28]. Fig. 2 clearly shows that the seeds of a transgenic line carrying a ProAtERF38::GUS construct display GUS activity specifically in the outer integument. Firstly, whole tissue observations (8– 11 daf) reveal a thin area of blue coloration all around the seed (Fig. 2A) that is located in the seed coat but not in the embryo (Fig. 2B). Secondly, semi-thin sections confirm this observation (Fig. 2C) and show more precisely that GUS activity is specifically located in both layers of the outer integument (Fig. 2D and E). Fig. 2E is a detailed view of these areas in a section stained with toluidine blue. It clearly shows that the cells of the outer layer have a thickened outer periclinal wall (white arrowheads) forming the columella, that the cells of the inner layer have a thickened inner periclinal wall (black

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Fig. 1. RT–PCR analysis of AtERF38 expression. RT was carried out on total RNA and cDNAs produced and amplified as indicated in Section 4. AtEF1a was used as a constitutive control to normalize the amount of cDNA between samples. (A) Total RNA was extracted from the indicated tissues on mature plants (seven weeks-old, first silique dry) and dry seeds without siliques. At2S3 and AtEm6 were used as markers of storage protein accumulation and desiccation stages, respectively. The images presented were obtained after transfer to membrane, hybridization and signal acquisition with a blot imaging system. daf, days after flowering. The graphs present the quantification of the signals (arbitrary unit) obtained for AtERF38, At2S3 and AtEm6 after normalization to AtEF1a signal. Note that the maximum expression level for At2S3 and AtEm6 is approximately ten times higher than the one for AtERF38. (B) Total RNA was extracted from siliques from 8 to 11 daf collected on the following mutants: a: abi3; f: fus3; l: lec2; al, af, fl, afl: the corresponding double and triple mutants. At2S3 was used as a control of the impact of the mutations on gene expression. The images presented are photos of ethidium bromide-stained gels.

arrowheads), and that both cell types specifically display GUS activity. 2.4. The promoter of AtERF38 drives GUS expression in some sclerified cells of inflorescence stems The strong expression of AtERF38 detected by RT–PCR in inflorescence stems (Fig. 1) prompted us to carry out a histological analysis of GUS expression in this tissue of the ProAtERF38::GUS line. Among samples collected at the base of the floral stem from 7- to 8week-old plants, promoter activity is detected in cells that have a rectangular shape and are organized in interrupted rows forming

bundles (Fig. 3A). On a radial axis, these cells are located between the phloem and the cortex. However, they are not present at the level of the interfascicular arc (Fig. 3B–D), thus delimiting the phloem. Interestingly, they display clear features of sclerification. First, their walls autofluoresce under UV light (Fig. 3D–F), demonstrating the presence of aromatics, and are stained by Sudan IV (Fig. 3G and H), indicating the presence of lipid polyesters and routinely used to reveal suberin. Suberin is an apoplastic lipid polyester that is deposited at distinct locations during plant growth. Its functions are to seal off the entire plant or one of its tissues against loss of water and solutes and to contribute to the strength of the cell wall [29–31]. The current structural view is that

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Table 1 List of the genes coregulated with AtERF38 Coregulated genes

Pearson’s correlation coefficient

At2g35700 1.000 At3g59010 0.884 At3g59845 0.856 At5g06930 0.850 At1g22480 0.850 At2g31930 0.848 At3g50220 0.842 At1g27380 0.841

Expression dataa

Protein function or similarity

Gene ontology annotation Biological process

Cellular component

AP2-type transcription factor Pectinesterase family NADP-dependent oxidoreductase Unknown Plastocyanin-like domaincontaining protein Unknown Unknown function (DUF579) P21-Rho-binding domain (RIC2)

Regulation of transcription, DNA-dependent Cell wall modification Response to oxidative stress

Nucleus

Electron transport

Chloroplast Anchored to membrane

Pollen tube growth

Anchored to membrane

Regulation of transcription, DNA-dependent

Nucleus

At3g15050 0.837

Calmodulin-binding family

Chloroplast

At2g44745 0.836

WRKY family transcription Regulation of transcription, factor DNA-dependent Glycosyltransferase Biosynthesis family 8

Chloroplast, nucleus Chloroplast

At2g41610 0.834

Unknown

At1g32770 0.832

No apical meristem family Development (ANAC012)

At1g07120 0.832 At1g27440 0.832

Unknown Exostosin family

At5g17420 0.828

Cellulose synthase (CesA7/ Cellulose biosynthesis, IRX3) secondary cell wall biosynthesis

COBRA family (COBL4/ IRX6)

At5g60490 0.822

Fasciclin-like Cell adhesion arabinogalactan protein (FLA12) No apical meristem family Development (ANAC073)

At2g29130 0.818

At5g03170 0.818

At4g28380 0.817 At1g09440 0.817

Cellulose biosynthesis, secondary cell wall biosynthesis

Core xylem-specific gene Secondary cell wall gene Co-regulated with CesA4, 7 and 8 genes Core xylem-specific gene Vascular tissue function Core xylem-specific gene Co-regulated with CesA4, 7 and 8 genes

Core xylem-specific gene Co-regulated with CesA4, 7 and 8 genes Core xylem-specific gene Co-regulated with CesA4, 7 and 8 genes Expressed in xylem [20,26] Core xylem-specific gene Vascular tissue function

Membrane

At5g15630 0.826

At3g16920 0.819

Core xylem-specific gene

Core xylem-specific gene

myb family transcription factor (MYB103)

At4g28500 0.820

This paper

Cell wall

At1g63910 0.841

At3g18660 0.834

Function or mutant phenotype

Plasma membrane

Plasma membrane

Anchored to membrane

Negative regulator of xylary fiber development [19]

Core xylem-specific gene Co-regulated with CesA4, 7 and 8 genes Core xylem-specific gene

Irregular Xylem (irx3 [12])

Secondary cell wall gene Co-regulated with CesA4, 7 and 8 genes Core xylem-specific gene

Irregular Xylem (irx6 [13])

Secondary cell wall gene Co-regulated with CesA4, 7 and 8 genes

Expressed in procambium, xylem [26] Core xylem-specific gene Co-regulated with CesA4, 7 and 8 genes Vascular tissue function Glycoside hydrolase family Cell wall catabolism, response Endomembrane Secondary cell wall gene 19 (chitinase) to other organism system Laccase/diphenol oxidase Endomembrane Core xylem-specific gene system Co-regulated with CesA4, 7 and 8 genes Fasciclin-like Cell adhesion Anchored to Expressed in sclerenchyma cells in Irregular Xylem (irx13 [3]) arabinogalactan protein membrane floral stem and siliques [44] (FLA11) Core xylem-specific gene Co-regulated with CesA4, 7 and 8 genes Leucine-rich repeat family, Endomembrane extensin family system Protein kinase family Protein amino acid Endomembrane phosphorylation system

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Table 1 (continued ) Coregulated genes

Pearson’s correlation coefficient

At1g80170 0.815 At5g54690 0.810

Gene ontology annotation Biological process

Cellular component

Glycosyl hydrolase family 28 (polygalacturonase) Glycosyl transferase family 8

Carbohydrate metabolism

Endomembrane Core xylem-specific gene system Core xylem-specific gene Secondary cell wall gene Co-regulated with CesA4, 7 and 8 genes Plasma Core xylem-specific gene membrane Secondary cell wall gene Co-regulated with CesA4, 7 and 8 genes Cytoplasm

Carbohydrate biosynthesis

At4g18780 0.809

Cellulose synthase (CesA8/ Cellulose biosynthesis, IRX1) secondary cell wall biosynthesis

At2g40120 0.807

Protein kinase family

At5g44030 0.803

a

Expression dataa

Protein function or similarity

Protein amino acid phosphorylation Cellulose synthase (CesA4/ Cellulose biosynthesis, IRX5) secondary cell wall biosynthesis

Plasma membrane

Core xylem-specific gene Secondary cell wall gene Co-regulated with CesA4, 7 and 8 genes

Function or mutant phenotype

Irregular Xylem (irx8 [3,14])

Irregular Xylem (irx1 [12])

Irregular Xylem (irx5 [15])

Core xylem-specific gene: [26]; Secondary cell wall gene: [13]; Co-regulated with CesA4, 7 and 8 genes: [3]; Vascular tissue function: [20].

suberin is made of a polyaromatic domain deriving from ferulic acid and embedded in the primary cell wall, covalently linked to a glycerol-based polyaliphatic domain located between the wall and the plasma membrane [30,31]. It is therefore likely that the floral stem cells that express AtERF38 are suberized. Secondly, some characteristics of cell death are visible, i.e. a shrunken protoplasm that separates from the wall, leaving an empty space (Fig. 3G and I). It is finally important to note that the xylem cells, that are well known to make a thick secondary wall and that also display autofluorescence due to the deposition of lignin (Fig. 3E), do not express AtERF38 (Fig. 3B,D,G–I). 2.5. The promoter of AtERF38 drives GUS expression in endodermal cells of young roots While no AtERF38 mRNAs had been detected by RT–PCR in roots of 7-week-old plants removed from soil (Fig. 1), in the course of this analysis GUS activity was also detected in the roots of 10-day-old in vitro-grown plantlets. As in inflorescence stems, the blue staining is detected in long, interrupted rows of cells, both in the main and secondary roots (Fig. 3J–L), that are located immediately around the stele (Fig. 3J and K). Transverse sections demonstrated that the promoter activity is specifically localized in the endodermal cells because these cells are located between the stele and the cortex (Fig. 3N), are stained in red by Sudan IV (Fig. 3M) that reveals the aliphatic domain of suberin and display autofluorescence under UV (Fig. 3O and P). In contrast to the stem, suberization of the endodermis of roots in the primary developmental stage in Arabidopsis has previously been described [32]. Finally, similar to the floral stem, AtERF38 is not expressed in the xylem cells of the stele (Fig. 3M and N). Consistent with these observations, the specific accumulation of AtERF38 mRNA in root endodermal cells is confirmed by analysis of the expression map of the whole root [33]. In this study, individual cells from the Arabidopsis root were fractionated and a genomewide expression analysis undertaken at the cell-type level. At all three stages of root development, AtERF38 mRNA is preferentially detected in the endodermis (Fig. 4). 2.6. A T-DNA insertion line in the promoter of AtERF38 does not display any visual nor histological phenotype To further investigate the function of AtERF38, we identified and characterized a T-DNA insertion line in the Salk Institute collection

(SALK_000610 line, see Section 4). The structure of the insertion is described in Fig. 5. The insert is an inverted tandem repeat localized 239 bp upstream from the start codon (Fig. 5). In the wild type line, AtERF38 is expressed in siliques and floral stems (Fig. 1) but not in leaves (Fig. 5B). However, in the SALK_000610 line, RT–PCR analysis established that it is strongly expressed in leaves, both in hemizygous and heterozygous genotypes (Fig. 5B). This demonstrates that the insertion does not cause a knock-out but ectopic expression of the gene, most probably under the control of the 35S promoter located near the left border of the pROK2 T-DNA used to make the Salk collection. Based on the expression pattern of the AtERF38 gene, we investigated whether the misexpression of this gene in the SALK_000610 line could induce developmental defects commonly associated with a function in secondary wall synthesis. However, no such effects were observed (data not shown): we did not detect any change in wall thickness or autofluorescence in any cells of leaves, roots or inflorescence stem. No collapsed xylem or a tendency for the stem to recline was observed either. Finally, given the GUS activity in the seed coat, we compared the germination rate between the SALK_000610 line and the corresponding wild-type line but no significant difference could be detected.

3. Discussion 3.1. In silico analysis of expression data link AtERF38 to secondary wall metabolism Microarray data analysis indicated that AtERF38 is coregulated with genes involved in secondary cell wall metabolism. A first analysis of the genes listed in Table 1 indicated that this association may not be very close because previous genome-wide microarray analyses, aimed at identifying genes involved in secondary cell wall formation and that indeed identified such genes, did not list the AtERF38 gene. This absence of AtERF38 might be due to the statistically-defined threshold values that are generally applied to the microarray datasets to consider that a gene is expressed over background and that were applied in the papers cited here [3,13,20,26]. This leads to a restriction of the probe sets taken into consideration. Thus, potential significant variations that are the basis of the comparisons to evaluate whether genes are coregulated may actually be considered as differences in the background noise and overlooked. This prevents

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Fig. 2. Histological analysis of the promoter activity of AtERF38 in seeds. All samples are seeds removed from 8 to 11 daf siliques, a stage at which AtERF38 is the more highly expressed. (A, B) Whole-mount observations showing GUS activity in the seed coat but not in the embryo (in B, seeds were dissected by crushing them between the slide and the cover slip). (C–E) Semi-thin longitudinal sections (5 mm). C: Promoter activity is specifically visible in the cells of the outer integument. D is a close-up view of the framed area in C and confirms that the GUS activity is located in the two cell layers composing the outer integument. On the toluidine-stained section shown in E, the arrowheads indicate the thickened tangential cell walls that characterize the cells into which the promoter activity is evident. em, embryo; sc, seed coat; oi, outer integument; ii, inner integument; en, endothelium; al, endosperm aleurone; ol, outer layer of the outer integument; il, inner layer of the outer integument; mu, mucilage. Bars ¼ 10 mm.

genes with low average expression levels, like AtERF38, from being accurately analyzed. The Expression Angler tool does not select the probe sets a priori but allows the user to manually examine the expression levels in the output [24]. This probably has the disadvantage of listing more false positives but allows the discovery of looser but still informative relationships, such as that between AtERF38 and genes involved in secondary wall metabolism that we report here.

3.2. AtERF38 is expressed in cells that undergo secondary wall thickening The association between AtERF38 expression and secondary metabolism of cell walls was experimentally confirmed: the histological analysis of its promoter activity revealed localized expression in very specific cell types that undergo a thickening of their wall. This occurs differently depending on the cell types. In

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Fig. 3. Histological analysis of the promoter activity of AtERF38 in roots and inflorescence stems. (A–I) Tissue from the base of the inflorescence stem of seven weeks-old plants. (A) Whole-mount of a piece of inflorescence stem. Promoter activity is evident in cells of rectangular shape forming bundles of interrupted rows. (B–I) transverse sections. (B) Hand-cut section. GUS activity is located in cells between the phloem and the cortex, delimiting the phloem, but not at the level of the interfascicular arc. (C–I) Semi-thin (4 mm) sections. (C) Toluidine blue-stained sample. GUS activity is located in two large and flattened cells outside both extremities of the phloem. D, E and F are images of the same sample observed under bright field to visualize GUS activity in cells located outside the phloem (D) or UV epifluorescence to visualize autofluorescence of lignified or suberized cell walls (E). (F) Composite image of D and E to show that the blue color indicating the GUS activity is located into these cells, different from the xylem, that similarly display autofluorescence. Note that the blue autofluorescence signal has been artificially changed to yellow for clarity. (G, H) Sudan IV-stained sections. Promoter activity is evident into cells stained in red, indicating the presence of a suberized secondary wall. (I) Promoter activity is evident into a dying cell characterized by a plasmolyzed cytoplasm leaving an empty space. Such a feature is also visible in G (arrowheads). (J–P) tissue from the main root of ten days-old plantlets grown in vitro. (J–L) Whole-mount views of a main (J, K, L) and secondary (J) root showing the general organization of the cells displaying GUS activity. (M–P) Transverse sections. (M) Sudan IV-stained section confirming that the promoter activity is located in the suberized endodermal cells. N, O and P are images of the same sample observed under bright field to visualize GUS activity in cells located between the cortex and the stele (N) or UV epifluorescence to visualize autofluorescence of the suberin of the endodermal cells (O). (P) Composite image of N and O to show that the blue color revealing the GUS activity is located into the endodermal cells. xy, xylem; ph, phloem; co, cortex; en, endodermis; pi, pith; st, stele. Bars ¼ 10 mm (C, D, G, H, I, N, M) or 50 mm (A, B, J, K).

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Fig. 4. Graphical representation of Root Microarray Data for AtERF38. The tissues tested are: lrc, lateral root cap; epi, epidermis; cort-endo, endodermis and cortex; endo, endodermis. The developmental stage 1 is represented by tissues closest to the root tip, stage 3 by tissues furthest from the root tip. The numbers in the y-axis are the raw values of the hybridization signals extracted from the Root Microarray Data [33].

roots and inflorescence stems, AtERF38 is specifically expressed in cells that are characterized by the presence of both the aromatic and aliphatic components of suberin [30,31]. In roots, they correspond to the endodermis cells that have actually been shown to be suberized in arabidopsis [32]. However, GUS activity was not detected in xylem cells of roots or floral stems. Moreover, xylem cells in which cellulose and lignin deposition occurs typically display a blue color after staining with toluidine blue, as we observed (Fig. 3C), while cells that express AtERF38 in the inflorescence stem do not (Fig. 3C). In seeds, promoter activity is restricted to the outer integument cells layers, which are known to produce thick cell walls [27,28]. These latter cells have not been clearly described as suberized but rather as accumulating unstructured material in their walls [34]. However, analysis of the lipid polyester

Fig. 5. Structural characterization of the SALK610 insertion line. (A) the insert (blackstriped rectangle) is an inverted tandem repeat localized 239 bp upstream the start codon. The arrows at the top of the black squares indicates the 35S promoter of the pROK2 T-DNA. The AtERF38 open reading frame is represented by a black rectangle, the 50 and 30 untranslated regions by a thick black line. TATA, TATA box; ERF, ERF domain; LB, left border. (B) RT–PCR analysis of the expression of AtERF38 in the leaves of the SALK610 line and the corresponding WT line showing that the insertion does not cause a knock-out but the ectopic expression of the gene.

composition of Arabidopsis thaliana seeds identified aliphatic monomers which are entirely associated with the seed coat and are collectively characteristic of suberin [35]. Moreover, the expression pattern of AtERF38 presents some striking similarities with that of the GPAT5 gene that encodes an acyltransferase required for the synthesis of aliphatic suberin in the seed coat and roots [36]. GUS expression driven by both promoters is indeed revealed as a patchy pattern in the main and lateral roots of young plantlets, in cells that closely surround the stele (probably the endoderm as demonstrated for AtERF38). In mature seeds before desiccation, GUS staining is present in the seed coat but not in the embryo. Based on expression data, AtERF28 may then be considered as a candidate for regulation of secondary wall metabolism in particular cell types that are not reinforced by the typical deposition of lignin and cellulose. These cells may have in common suberization or at least accumulation of suberin-like lipid polyesters in their walls. It is nevertheless unlikely that AtERF38 is a major regulator of suberin biosynthesis since the list of coregulated genes does not include genes encoding enzymes such as fatty acid elongases, acyltransferases or cytochrome P450 monooxygenases that have been reported to be critical to this biosynthetic pathway [31]. The fact that the gene list contains a putative pectinesterase gene (the best correlated expression profile) and a putative polygalacturonase gene may indicate that AtERF38 could be related with primary cell wall changes before or concomitantly with suberization. The fact that AtERF38 is described as being coregulated with genes involved in cellulose biosynthesis or which are active in xylem but is not expressed in xylem or in cells whose walls are reinforced by cellulose may appear contradictory. But it is important to note that the resolution level of the expression data that we obtained from microarrays is most often organs or their components at best, but not the cell-type. Thus, the coregulation between AtERF38 and the CesA genes involved in secondary cell wall metabolism is the consequence of expression in similar organs at similar developmental stages. Our experimental data indeed demonstrate that the promoter of AtERF38 is active in organ parts in which cellulose deposition and lignification occur. However, the genes that have been found to be coregulated on this basis are not expressed in the same cell types of a given organ, which probably explains why the correlation coefficient is low. This is clear when analyzing the only available microarray data that were obtained at the cell-type resolution level, called ‘‘the expression map of the root’’ [33]: CesA7 and 8 are mainly expressed in the stele [13] whereas AtERF38 is preferentially expressed in the endodermis (Fig. 4). The latter was confirmed by our promoter activity studies (Fig. 3M–P). Apart from the expression data reported above, no hypothesis can be inferred from the unique insertion line identified to date (SALK_000610). Its molecular characterization revealed that the AtERF38 gene is not knocked out but ectopically expressed. For three NAC transcription factors, this misregulation had a phenotypic impact. The ectopic expression of NST1 and NST2 causes ectopic thickening of secondary walls in various aboveground tissues [18]. On the other hand, ectopic overexpression of ANAC012 suppresses secondary wall deposition in the xylary fiber [19]. However, this apparently does not cause defects or abnormalities in secondary wall formation in the case of AtERF38. This may be explained by the fact that the AtERF38 gene does not encode a limiting factor in the tissues where it is normally expressed and increasing its level has no effect. In the tissues where it is not normally expressed, AtERF38 alone may not be sufficient to have an impact on secondary wall thickening, probably because its regulatory partners are not present. However, a deeper examination, based for instance on

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biochemical analysis of the cell walls composition, may unravel subtle differences that are not accessible through the relatively coarse type of analysis we have carried out. Knock-out or RNAi lines would be of use to check whether it is necessary but this approach may be limited by the fact that the AP2/ERF family of transcription factors contains many members (147, based on the analysis by Nakano et al [4]) that could compensate the inactivation of one or a couple of them.

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program calculates the Pearson correlation coefficients for expression of all gene expression vectors as compared to that of the AGI number entered (At2g35700 here). A file is created containing the expression data and the correlation coefficients that meet the cut-off criterion entered. The AtGenExpress Tissue data set was used in this study, with a correlation coefficient cut-off range from 0.8 to 1, a value of 1 characterizing a perfect correlation. 4.2. Plant material and growth conditions

3.3. What are the cells of the inflorescence stem that express the AtERF38 gene? Promoter activation of AtERF38, reported by GUS activity, was detected in cells that are located at the base of mature inflorescence stems (Fig. 3A–I) but not in the middle or at the top (not shown). The walls of these cells autofluoresce under UV and are stained by an aliphatic suberin dye. Based on this developmental stage, their radial position (the most internal layer of the cortex, adjacent to the phloem) and their final death, they may correspond to endodermal cells that become sclerified. The fate of these cells has not yet been described in Arabidopsis thaliana. Some dead cells called fibersclereids in the primary phloem or phloem fibers have been characterized in approximately the same area of the floral stem [37] but they were not described as suberized and depicted as very short in longitudinal section, which is not the case here (Fig. 3A). In a thorough review on vascular development in Arabidopsis [38], the authors state that endodermal cells adjacent to the interfascicular fiber can redifferentiate into secondary phloem and xylem but that those adjacent to the primary phloem, precisely the cells we describe here, cannot. Their fate is not described further. The observations reported here indicate that this fate may be suberization and death. However, several structural features of these cells are reminiscent of phellem cells [30,39]: they are rectangular in shape, radially flattened and form interrupted tangential rows grouped in bundles localized outside of the phloem (Fig. 3A–I), as described for the organization of phellogen or cork cambium cells. They may ultimately be suberized and die (Fig. 3G–I) as phellem or cork cells do. We have observed this to occur systematically at the same cellular and tissular locations, thus corresponding to a normal developmental process rather than to the random or accidental sclerification of some phloem or endodermal cells that has previously been described [37,38]. It is known that the pericycle cells of the hypocotyl of Arabidopsis thaliana can give rise to a cork cambium that produces a periderm consisting largely of cork cells [40]. This has not been described in the inflorescence stem. However, neither the autofluorescence nor Sudan IV staining that characterize the cells described in this paper were detected above the interfascicular arcs, indicating that they do not form a sheath like cork does in woody species. Moreover, suberization is normally associated with cells that derive from a distinct cambium [30] and we did not detect such a cambial activity in that area, confirming the statement of Ye et al. [38] reported above. Nevertheless, the origin of these cells is still questionable and their characterization would necessitate a deeper histological analysis than that presented in this paper. The AtERF38 gene might be very helpful in such studies as a specific marker of these cells.

The Col0 ecotype of Arabidopsis thaliana was used as the wildtype strain. The T-DNA insertion line SALK_000610 [41] was obtained from the Nottingham Arabidopsis Seed Stock Centre (Nottingham, UK). The seed maturation mutants (i.e. abi3, lec2, fus3, the three combinations of double mutants and the triple mutant) and the corresponding control line have previously been described [21,22] and were obtained from F. Parcy. Plants were usually grown in vitro on 0,7% plant-agar plates containing half-strength MS salts, Gamborg’s B5 vitamins and 10 g L1 of saccharose, at 21  C under constant illumination. When needed, selection was applied by adding 50 mg L1 of kanamycin to plates without saccharose. Seedlings were then transferred to soil (3/4 soil and 1/4 vermiculite) and grown at 21  C under a light/darkness regime of 16 h/8 h. 4.3. RT–PCR analysis RNA was extracted using an Invisorb Spin Plant RNA Minikit (Invitek), following the manufacturer’s instructions, except that the last traces of contaminating DNA were removed by on-column digestion with a RNase-Free DNase (Qiagen) during 30 min at 37  C before the last washing step of the spin columns. Total absence of DNA was then checked by PCR on each RNA sample before RT. Five micrograms of total RNA were used for the synthesis of the firststrand cDNA using an oligodT primer following the protocol of the ProSTARÔ First-Strand RT–PCR kit (Stratagene). An aliquot of 2 ml of first strand cDNA was added to 25 ml of PCR mix. Specific amplifications were obtained with the following primers: T20F21F (TAG ACC AGC TAG TGC CGA CC) and T20F21R (AGG AAG GGT TCT TCA TAG GG) generate a band of 247 bp for AtERF38; 2S3F (GCA CGA TGA TCA TGA GTG TTG C) and 2S3R (TTT AGT TTA GCT AAC CGT ACG C) generate a band of 208 bp for At2S3; EM6F (CCA AGA CCT AAA TCA AAC TTC C) and EM6R (AAA CGA GAC ACT TTA CGT AGA CG) generate a band of 167 bp for AtEm6; EF1F1 (CTG CTA ACT TCA CCT CCC AG) and EF1R (TGG TGG GTA CTC AGA GAA GG) generate a band of 251 bp for EF1a. The PCR conditions were as follows: 21 cycles at 92  C for 30 s, 58  C for 30 s and 72  C for 30 s. They were set to keep the amplification of AtERF38 and EF1a cDNAs in the linear phase. For the first experiment, each amplicon has been sequenced to confirm the identity of the sequence and the specificity of the amplification. They were then transferred to a Hybondþ nylon membrane (GE Healthcare Life Sciences) and submitted to hybridization with the corresponding 32P-labeled amplicon using standard protocols to amplify the signals and further confirm the identity of the amplified sequences for each experiment (Fig. 1A) or simply stained with ethidium bromide (Fig. 1B). The radioactive signals were acquired with a Storm PhosphorImager and the ImageQuant 5.1 software (GE HealthCare Life Sciences).

4. Methods

4.4. Construction of the ProAtERF38::GUS line

4.1. In silico analysis of AtERF38 expression

The promoter is a fragment of 2444 bp that includes the 22 first codons of AtERF38 and most of the intergenic sequence between At2g35690 and Atg35700 (AtERF38), obtained by PCR on genomic DNA using the following primers: B1proT20F21F (GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT AAT TGG CTT TTT TCG ATC C) and B2proT20F21R (GGG GAC CAC TTT GTA CAA GAA AGC TGG GTA TTT

Genes co-expressed with AtERF38 were identified using the Expression Angler tool (http://bbc.botany.utoronto.ca/ntools/cgibin/ntools_expression_angler.cgi) from the Botany Array Resource [24]. Based on many microarray datasets that can be selected, this

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CAC TCT TCT CTC CGT CGT C). The underlined sections of the primers are the AttB1 and AttB2 sequences, respectively, used to clone this fragment into the Gateway donor vector pDONR 221 (Invitrogen) by BP cloning. The promoter was then transferred into the binary Gateway destination vector pKGWFS7 (http://www.psb. ugent.be/gateway/, [42] by LR cloning. BP and LR cloning were done following the manufacturer’s instructions (Invitrogen), except that the volume of each product and of the reaction was divided by 2. The construct was introduced into A. tumefaciens GV3101 and used to transform Arabidopsis Col0 plants using the floral dip method [43]. Ten independent transgenic lines were obtained. Based on the transmission of kanamycin resistance, we selected transgenic plants with only one locus of T-DNA insertion at the homozygous stage. The results obtained with one representative line are presented. 4.5. Histological analysis of GUS expression Tissue was vacuum infiltrated for 10 min with staining solution (100 mM Tris–HCl buffer pH 7.0, 0,1% Triton-X-100, 0,5 mM potassium ferrocyanide, 0,5 mM potassium ferricyanide, 1 mm Xgluc) and incubated at 37  C for 24 h. Samples were then fixed and decolored into 70% ethanol. For whole-mount observations, samples were further decolored in Hoyer’s solution (arabic gum 7.5 g, chloral hydrate 100 g, glycerol 16.7% in 30 ml H2O). For semithin sections, the plant material was dehydrated through a graded ethanol series, embedded in Technovit 7100 resin (Heraeus Kulzer, Wehrheim, Germany) and sectioned using a LEICA1130 Biocut microtome (Leica Microsystems, Wetzlar, Germany). The preparations were mounted in 5% glycerol and observed under a Zeiss axioplan microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with a digital Leica DC 300FX camera. Unstained samples were observed using Nomarski optics. The autofluorescence of lignin and suberin was examined under epifluorescence illumination using the Zeiss filter set 49 (excitation: G365, beamsplitter: FT 395, emission: BP 445/450). For the staining of suberin, sections were incubated in a saturated solution of Sudan IV in 70% ethanol for 5 min and rinsed in 70% ethanol. Toluidine blue staining was performed by immersion of the section in a 0,02% solution in water for 5 min and then brief rinsing in water. Images were treated with the GIMP2.2 software (http://www.gimp.org/). 4.6. Genotyping and structural analysis of the SALK_000610 line The SALK_000610 line was identified via two FST sequences (SALK_000610.54.00 and SALK_000610.52.35) that matched to the promoter AtERF38 using the T-DNA express tool (http://signal.salk. edu/cgi-bin/tdnaexpress). The structure of the T-DNA insertion, as indicated in Fig. 5, was partly solved by a combination of PCR and sequencing. The following primers were used: LBb1 (CAA CGA GAG AGA GAG AGA TCG AG) matches to a region located near the left border of the T-DNA and is oriented outwards. The T20F21F2 (CAA CGA GAG AGA GAG AGA TCG AG) hybridization site is located in the promoter of AtERF28, 21 bp upstream the insertion site. T20F21R is described above. Homozygous lines for the insertion were characterized by two bands of 914 bp (LBb1 and T20F21R) and 207 bp (LBb1 and T20F21F2). The hemizygous lines displayed an additional band of 852 bp (T20F21F2 and T20F21R). The corresponding WT line was identified in the progeny of an hemizygous line and only display the 852 bp band. The structure of the junction between the two inverted repeats of the T-DNA could not be solved with primers matching the right border, probably because of deletions and/or rearrangements that could explain the lack, in our hands, of a clear kanamycin resistance of the T3 lines. RT–PCR analysis of AtERF38 expression in these lines was carried out using the T20F21F and T20F21R primers (see RT–PCR analysis section).

Acknowledgments This work was supported by the Centre National de la Recherche Scientifique. We thank J. Guilleminot for her precious help with microscopic analysis and F. Parcy for the gift of the abi3, fus3, lec2 mutants and their combinations.

References [1] J.L. Riechmann, Transcriptional regulation: a genomic overview.http://www. aspb.org/publications/arabidopsis/ 2002doi: 101199/tab.0085,. [2] H. Lee, A. Hsu, J. Sajdak, J. Qin, P. Pavlidis, Coexpression analysis of human genes across many microarray data sets, Genome Res. 14 (2004) 1085–1094. [3] S. Persson, H. Wei, J. Milne, G.P. Page, C.R. Somerville, Identification of genes required for cellulose synthesis by regression analysis of public microarray data sets, Proc. Natl Acad. Sci. U.S.A. 102 (2005) 8633–8638. [4] T. Nakano, K. Suzuki, T. Fujimura, H. Shinshi, Genome-wide analysis of the ERF gene family in Arabidopsis and rice, Plant Physiol. 140 (2006) 411–432. [5] K.D. Jofuku, B.G. den Boer, M. Van Montagu, J.K. Okamuro, Control of Arabidopsis flower and seed development by the homeotic gene APETALA2, Plant Cell 6 (1994) 1211–1225. [6] M. Ohme-Takagi, H. Shinshi, Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element, Plant Cell 7 (1995) 173–182. [7] C. Carles, N. Bies-Etheve, L. Aspart, K. Le´on-Kloosterziel, M. Koornneef, M. Echeverria, M. Delseny, Regulation of Arabidopsis thaliana Em genes: role of ABI5, Plant J 30 (2002) 373–383. [8] Y. Sakuma, Q. Liu, J.G. Dubouzet, H. Abe, K. Shinozaki, K. Yamaguchi-Shinozaki, DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression, Biochem. Biophys. Res. Commun. 290 (2002) 998–1009. [9] N. Carpita, M. McCann, The cell wall, in: B.B. Buchanan, W. Gruissem, R.L. Jones (Eds.), Biochemistry and Molecular Biology of Plants, American Society of Plant Biologists, Rockville, MD, 2000, pp. 52–108. [10] R. Zhong, D.H. Burk, Z.H. Ye, Fibers a model for studying cell differentiation, cell elongation, and cell wall biosynthesis, Plant Physiol 126 (2001) 477–479. [11] S.R. Turner, N. Taylor, L. Jones, Mutations of the secondary cell wall, Plant Mol. Biol. 47 (2001) 209–219. [12] S.R. Turner, C.R. Somerville, Collapsed xylem phenotype of Arabidopsis identifies mutants deficient in cellulose deposition in the secondary cell wall, Plant Cell 9 (1997) 689–701. [13] D.M. Brown, L.A.H. Zeef, J. Ellis, R. Goodacre, S.R. Turner, Identification of novel genes in Arabidopsis involved in secondary cell wall formation using expression profiling and reverse genetics, Plant Cell 17 (2005) 2281–2295. [14] S. Persson, K.H. Caffall, G. Freshour, M.T. Hilley, S. Bauer, P. Poindexter, M.G. Hahn, D. Mohnen, C. Somerville, The Arabidopsis irregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan, which are essential for secondary cell wall integrity, Plant Cell 19 (2007) 237–255. [15] N.G. Taylor, R.M. Howells, A.K. Huttly, K. Vickers, S.R. Turner, Interactions among three distinct cesA proteins essential for cellulose synthesis, Proc. Natl Acad. Sci. U.S.A. 100 (2003) 1450–1455. [16] R. Zhong, M.J. Pen˜a, G. Zhou, C.J. Nairn, A. Wood-Jones, E.A. Richardson, W.H.3. Morrison, A.G. Darvill, W.S. York, Z. Ye, Arabidopsis fragile fiber8, which encodes a putative glucuronyltransferase, is essential for normal secondary wall synthesis, Plant Cell 17 (2005) 3390–3408. [17] L. Jones, A.R. Ennos, S.R. Turner, Cloning and characterization of irregular xylem4 (irx4): a severely lignin-deficient mutant of Arabidopsis, Plant J. 26 (2001) 205–216. [18] N. Mitsuda, M. Seki, K. Shinozaki, M. Ohme-Takagi, The NAC transcription factors NST1 and NST2 of Arabidopsis regulate secondary wall thickenings and are required for anther dehiscence, Plant Cell 17 (2005) 2993–3006. [19] J. Ko, S.H. Yang, A. Park, O. Lerouxel, K. Han, ANAC012 a member of the plantspecific NAC transcription factor family, negatively regulates xylary fiber development in Arabidopsis thaliana, Plant J. 50 (2007) 1035–1048. [20] C. Zhao, J.C. Craig, H.E. Petzold, A.W. Dickerman, E.P. Beers, The xylem and phloem transcriptomes from secondary tissues of the arabidopsis roothypocotyl, Plant Physiol 138 (2005) 803–818. [21] A. To, C. Valon, G. Savino, J. Guilleminot, M. Devic, J. Giraudat, F. Parcy, A network of local and redundant gene regulation governs Arabidopsis seed maturation, Plant Cell 18 (2006) 1642–1651. [22] T. Kroj, G. Savino, C. Valon, J. Giraudat, F. Parcy, Regulation of storage protein gene expression in Arabidopsis, Development 130 (2003) 6065–6073. [23] M. Schmid, T.S. Davison, S.R. Henz, U.J. Pape, M. Demar, M. Vingron, B. Scho¨lkopf, D. Weigel, J.U. Lohmann, A gene expression map of Arabidopsis thaliana development, Nat. Genet. 37 (2005) 501–506. [24] K. Toufighi, S. Brady, R. Austin, E. Ly, N.J. Provart, The Botany Array Resource: e-northerns, expression angling, and promoter analyses, Plant J. 43 (2005) 153–163. [25] N.A. Eckardt, Cellulose synthesis takes the cesA train, Plant Cell 15 (2003) 1685–1687. [26] J. Ko, E.P. Beers, K. Han, Global comparative transcriptome analysis identifies gene network regulating secondary xylem development in Arabidopsis thaliana, Mol. Gen. Genomics 276 (2006) 517–531.

E. Lasserre et al. / Plant Physiology and Biochemistry 46 (2008) 1051–1061 [27] G. Haughn, A. Chaudhury, Genetic analysis of seed coat development in Arabidopsis, Trends Plant Sci. 10 (2005) 472–477. [28] J.B. Windsor, V.V. Symonds, J. Mendenhall, A.M. Lloyd, Arabidopsis seed coat development: morphological differentiation of the outer integument, Plant J. 22 (2000) 483–493. [29] C. Nawrath, The biopolymers cutin and suberin.http://www.aspb.org/ publications/arabidopsis/ 2002doi: 101199/tab.0021,. [30] M. Bernards, Demystifying suberin, Can. J. Bot. 80 (2002) 227–240. [31] R. Franke, L. Schreiber, Suberinda biopolyester forming apoplastic plant interfaces, Curr. Opin. Plant Biol. 10 (2007) 252–259. [32] R. Franke, I. Briesen, T. Wojciechowski, A. Faust, A. Yephremov, C. Nawrath, L. Schreiber, Apoplastic polyesters in arabidopsis surface tissuesda typical suberin and a particular cutin, Phytochemistry 66 (2005) 2643–2658. [33] K. Birnbaum, D.E. Shasha, J.Y. Wang, J.W. Jung, G.M. Lambert, D.W. Galbraith, P.N. Benfey, A gene expression map of the Arabidopsis root, Science 302 (2003) 1956–1960. [34] T. Beeckman, R. De Rycke, R. Viane, D. Inze´, Histological study of seed coat development in Arabidopsis thaliana, J. Plant Res. 113 (2000) 139–148. [35] I. Molina, G. Bonaventure, J. Ohlrogge, M. Pollard, The lipid polyester composition of Arabidopsis thaliana and Brassica napus seeds, Phytochemistry 67 (2006) 2597–2610.

1061

[36] F. Beisson, Y. Li, G. Bonaventure, M. Pollard, J.B. Ohlrogge, The acyltransferase GPAT5 is required for the synthesis of suberin in seed coat and root of Arabidopsis, Plant Cell 19 (2007) 351–368. [37] M. Altamura, M. Possenti, A. Matteucci, S. Baima, I. Ruberti, G. Morelli, Development of the vascular system in the inflorescence stem of Arabidopsis, New Phytol 151 (2001) 381–389. [38] Z. Ye, G. Freshour, M.G. Hahn, D.H. Burk, R. Zhong, Vascular development in Arabidopsis, Int. Rev. Cytol. 220 (2002) 225–256. [39] R.P. Sabba, E.C. Lulai, Histological analysis of the maturation of native and wound periderm in potato (Solanum tuberosum L.) tuber, Ann. Bot. 90 (2002) 1–10. [40] N. Chaffey, E. Cholewa, S. Regan, B. Sundberg, Secondary xylem development in Arabidopsis: a model for wood formation, Physiol. Plant 114 (2002) 594–600. [41] J.M. Alonso, A.N. Stepanova, T.J. Leisse, Genome-wide insertional mutagenesis of Arabidopsis thaliana, Science 301 (2003) 653–657. [42] M. Karimi, D. Inze, A. Depicker, Gateway vectors for agrobacterium-mediated plant transformation, Trends Plant Sci. 7 (2002) 193–195. [43] S.J. Clough, A. Bent, Floral dip: a simplified method for agrobacterium-mediated transformation of Arabidopsis thaliana, Plant J. 16 (1998) 735–743. [44] S. Ito, Y. Suzuki, K. Miyamoto, J. Ueda, I. Yamaguchi, AtFLA11 a fasciclin-like arabinogalactan-protein, specifically localized in sclerenchyma cells, Biosci. Biotech. Biochem. 69 (2005) 1963–1969.