The ABCG transporter PEC1/ABCG32 is required for the formation of the developing leaf cuticle in Arabidopsis
Summary
- The cuticle is an essential diffusion barrier on aerial surfaces of land plants whose structural component is the polyester cutin. The PERMEABLE CUTICLE1/ABCG32 (PEC1) transporter is involved in plant cuticle formation in Arabidopsis. The gpat6 pec1 and gpat4 gapt8 pec1 double and triple mutants are characterized. Their PEC1-specific contributions to aliphatic cutin composition and cuticle formation during plant development are revealed by gas chromatography/mass spectrometry and Fourier-transform infrared spectroscopy.
- The composition of cutin changes during rosette leaf expansion in Arabidopsis. C16:0 monomers are in higher abundance in expanding than in fully expanded leaves. The atypical cutin monomer C18:2 dicarboxylic acid is more prominent in fully expanded leaves. Findings point to differences in the regulation of several pathways of cutin precursor synthesis.
- PEC1 plays an essential role during expansion of the rosette leaf cuticle. The reduction of C16 monomers in the pec1 mutant during leaf expansion is unlikely to cause permeability of the leaf cuticle because the gpat6 mutant with even fewer C16:0 monomers forms a functional rosette leaf cuticle at all stages of development.
- PEC1/ABCG32 transport activity affects cutin composition and cuticle structure in a specific and non-redundant fashion.
Introduction
During evolution, land plants developed the cuticle – a hydrophobic structure at the outer surface of the epidermal extracellular matrix that plays essential roles in plant development and physiology, as it represents the interface with other plant organs and the environment. The cuticle fortifies the cell wall protecting against mechanical damage, and functions as a diffusion barrier regulating the flux of molecules between the plant and its environment including water, nutrients, agrochemicals as well as diverse signalling molecules in biotic interactions (Nawrath, 2006; Yeats & Rose, 2013). Its composition is complex comprising solvent-soluble waxes consisting of very long-chain aliphatic molecules and different secondary metabolites including triterpenoids or flavonoids as well as polysaccharides and the insoluble polyester cutin (Jeffree, 2006). Typical cutin components are C16 and C18 fatty acids carrying a hydroxy group in the ω-position and hydroxy and/or epoxy groups on the mid-chain position of the fatty acid. Unsubstituted fatty acids, dicarboxylic acids (DCA) and glycerol as well as low amounts of phenolic compounds, however, are also present (Kolattukudy, 2001). The composition and structure of cutin is species specific and may also differ between organs of the same plant. In Arabidopsis, for example, leaf and stem cutin is rich in highly unsaturated C18:2 DCA, while petal cutin is affluent in 10, 16-dihydroxy C16:0 acid (Li-Beisson et al., 2013). The cuticle is first detected in the globular stage during embryo development, and thereafter is maintained continuously during plant growth and development (Szczuka & Szczuka, 2003; Jeffree, 2006). Extensive changes in the composition and structure of the cuticle during the development of different organs have been reported for, among others, tomato fruits and Clivia miniata leaves (Riederer & Schönherr, 1988; Kosma et al., 2010; Espana et al., 2014). Differences in cutin amount, but not its aliphatic monomer composition have been identified during the maturation of the cuticle in Arabidopsis stems (Suh et al., 2005).
Forward and reverse genetic approaches in Arabidopsis and tomato in recent years have contributed strongly to the elucidation of the biosynthetic pathway of cutin. The latter is divided into three processes: precursor formation, export and assembly (Beisson et al., 2012; Yeats & Rose, 2013). Hydroxy- and epoxy fatty acids are first synthesized at the ER through the concerted action of long-chain acyl-CoA synthetases (LACS) and cytochrome P450-dependent oxidases from the CYP86 and CYP77 subfamilies (Bak et al., 2011). Glycerol-3-phosphate acyltransferases (GPATs) then transfer oxygenated fatty acids to glycerol-3-phosphate. In Arabidopsis GPAT4 and GPAT8 act redundantly in the formation of cutin rich in C18:2 DCA which is accompanied by a total cutin reduction of 90% in rosette leaves of gpat4gpat8 mutants (Li et al., 2007). GPAT6 plays a particular role for the formation of floral organ cutin as the amount of C16 oxygenated fatty acids is reduced by > 90% in the gpat6 mutant (Li-Beisson et al., 2009). Enzymes in the subclade of GPAT4, GPAT6 and GPAT8 transfer oxygenated fatty acids onto the sn2 position of the glycerol-3-phosphate backbone and possess an additional phosphatase activity yielding sn2-monoacylglycerols (2-MAGs) as end products (Yang et al., 2010, 2012). A few cytosolic enzymes have been identified acting in cutin formation including acyltransferases of the BADH family, for example, DEFECTIVE IN CUTICULAR RIDGES (DCR) (Panikashvili et al., 2009). In addition to functioning in cutin synthesis, a number of enzymes such as GPAT6 and DCR are also involved in the formation of related polymers that may impact plant development and fertility, for example, seed coat or pollen formation, respectively (Panikashvili et al., 2009; Li et al., 2012; Chen et al., 2014). Evidence that members of the GDSL-lipase family are cutin synthases came from studies of the tomato cd1 mutant having a 90–95% reduction in cutin (Yang et al., 2012; Yeats et al., 2014). In vitro CD1 transesterifies the oxygenated fatty acid from sn2-mono hydroxyacyl glycerol (2-MHG) molecules to the growing cutin oligomer. This inferred that the 2-MHGs formed by GPATs may be cutin precursors in vivo (Yang et al., 2012). The mechanism of cutin precursor export to the extracellular space has not yet been elucidated although several ABC transporters of the ABCG family play essential roles (Hurlock et al., 2014). Vesicle-mediated trafficking, required for the secretion of cuticular wax, is another process that may also play a role (McFarlane et al., 2014).
ABCG11 and ABCG13 are half transporters involved in cutin formation in Arabidopsis (Bird et al., 2007; Luo et al., 2007; Panikashvili et al., 2007, 2011; Bird, 2008). ABCG11 functions in cutin synthesis as a homodimer or as a heterodimer with yet unknown half transporters (McFarlane et al., 2010). While ABCG11 has a strong impact on cutin formation in several organs including leaves and stems, ABCG13 acts mainly in floral organs. In addition to the clade of ABCG half transporters, the full-size ABCG transporter PEC1/ABCG32 is also required for cutin formation in Arabidopsis. The PEC1/ABCG32 transporter forms a separate subgroup within the family of Pleiotropic Drug Resistance (PDR)-type of ABC transporters in Arabidopsis (Bessire et al., 2011). Close homologues, however, have also been identified in barley and rice, namely, HvABCG31 and OsABCG31. These also act in cuticle formation indicating that the ancestral ABCG32/31 transporter acquired its functions in cuticle formation before the separation of monocots and dicots (Chen et al., 2011). Based on the polar localization towards the plasma membrane of epidermal cells and the reduction of oxygenated cutin monomers in pec1 mutant flowers, PEC1 in Arabidopsis was hypothesized to contribute to export of particular cutin precursors. The initial characterization of the pec1/abcg32 mutant revealed a number of unusual phenotypes. In flowers a good correlation was found between increased cuticle permeability, reduction of certain oxygenated cutin monomers and altered ultrastructure of the cuticle. In expanded rosette leaves, permeability was increased even when significant chemical and ultrastructural changes were difficult to detect (Bessire et al., 2011).
The significance of PEC1 in Arabidopsis cutin formation is elaborated by establishing the relation between PEC1 and different GPAT-family members. PEC1 acts in strong overlap with GPAT6, as the cutin composition of flowers suggested, but also has GPAT6-independent functions. The organ fusion phenotype of the gpat4gpat8pec1 mutant pointed to an essential function of PEC1 in rosette leaf cutin formation during early development. Supporting this deduction the amount and composition of cutin changed during leaf expansion and C16-type aliphatics were more abundant in emerging leaves. The latter were significantly reduced in the pec1 mutant. Ultrastructural features as well as permeability were also more strongly affected in emerging leaves of the pec1 mutant. In the absence of GPAT6, however, a functional cuticle can be formed in leaves emphasizing the importance of PEC1 for the structural arrangement of GPAT6-dependent cutin components.
Material and Methods
Plants and growth conditions
Arabidopsis thaliana (L.) Heynh (accession Col-0) and pec1 mutant alleles have been described previously (Bessire et al., 2011). The gpat6-1 and gpat6-2 mutant alleles as well as the gpat4 gpat8 double mutant were obtained from Frédéric Beisson, CEA-CNRS-Aix Marseille Université, Cadarache (Li-Beisson et al., 2009; Li et al., 2007; Li-Beisson et al., 2009).
The double mutants between different gpat6 and pec1 alleles were identified by a combination of toluidine blue (TB) staining and genotyping of the F2 population of the respective genetic crosses. Here, we present the results of the gpat6-1 pec1-2 and gpat6-2 pec1-3. The triple mutants gpat4 gpat8 pec1-2 and gpat4 gpat8 pec1-3 were identified in the F2 populations from a cross of gpat4 gpat8 and pec1-2 and pec1-3, respectively, by candidate approach and subsequent genotyping. Since both triple mutants had an identical phenotype, this study was mainly performed with gpat4 gpat8 pec1-3.
Short day plants were grown for 10 h at 20°C and 65% humidity under 100 μmol m−2 s−1 of warm white light (colour 830) and for 14 h at 17°C and 80% humidity in the dark. Long-day plants were grown under a 16 h:8 h, light:dark cycle at 20°C under 100 μmol m−2 s−1 of warm white light and 65% humidity.
Assessment of cuticle permeability
Detached rosettes or flowers were stained in 500 mg l−1 toluidine blue (TB)/0.001% Tween 20 solution under gentle agitation and then rinsed thoroughly with water (Tanaka et al., 2004).
Chemical analyses
Fourier transform infrared (FTIR) spectroscopy and subsequent cluster analysis was performed as described previously (Mazurek et al., 2013). Measurements from one petal of six different flowers and six different plants were taken to calculate the spectrotype of each genotype.
For the analysis of the aliphatic polyester composition during development, material was collected from leaf blades of 5-wk-old and 7-wk-old short-day plants with 28–30 and 52–54 leaves, respectively. Leaves to a maximal length of 0.5 cm, corresponding to 4 (leaf # 26–30) and c. 8 leaves (leaf # 45–53) in 5- and 7-wk-old plants, respectively, were used for the class ‘Emerging leaves’. Blades of expanding leaves having a length of 0.5–1.5 cm corresponding to 2 (leaf # 24–25) and 4 leaves (leaf # 41–44) in 5- and 7-wk-old plants were used for the class ‘Expanding leaves 1’. Blades of expanding leaves having a size of 1.5–2.5 cm corresponding to 2 (leaf # 22–23) and 4 leaves (leaf # 37–40) in 5- and 7-wk-old plants, respectively, were used for the class ‘Expanding leaves 2’. Blades of fully expanded leaves of 3–4 cm in length corresponding to leaf # 14–18 and leaf # 25–30 of 5- and 7-wk-old plants, respectively, were used for the class ‘Fully expanded leaves’. For the chemical analysis of entire rosettes, three plants grown under long day conditions for 4–6 wk were pooled. For analysis of floral polyesters, 30 flowers from 4 to 6 plants were used.
Four to six replicates were made from each type. The polyester composition was analysed after transesterification by base-catalysis and following acetylation as described previously (Li-Beisson et al., 2013).
Electron microscopy
For cryo-scanning electron microscopy (cryo-SEM), Arabidopsis flowers were positioned with a mixture of 50% colloidal graphite (Agar Scientific) and 50% Tissue Tek OCT™ at room temperature and then cryofixed. The observations were performed as described previously (Mazurek et al., 2013).
For transmission electron microscopy (TEM), Arabidopsis leaf disc punches (1.5 mm in diameter) and entire flowers were fixed in glutaraldehyde solution (EMS, Hatfield, PA, USA), and 2.5% phosphate buffer (PB 0.1 M pH 7.4) (Sigma, St Louis, MO, USA) during 2 h at room temperature (RT). They were rinsed three times for 5 min in PB buffer and post-fixed in a fresh mixture of osmium tetroxide 1% (EMS) with 1.5% potassium ferrocyanide (Sigma) in PB buffer for 2 h at RT. The samples were then washed two times in distilled water and dehydrated in acetone solution (Sigma) at graded concentrations (30%–40 min; 50%–40 min; 70%–40 min; 100%–3 × 1 h). This procedure was followed by infiltration in Spurr resin (EMS) at graded concentrations (Spurr 1/3 acetone-12 h; Spurr 3/1 acetone-12 h, Spurr 1/1-1 h; Spurr 1/1-2 × 8 h). The Arabidopsis flowers were then dissected in the resin under a binocular. Petals and leaf discs were placed in moulds filled with resin and then polymerized for 48 h at 60°C in an oven. Ultrathin sections (60 nm) were cut on a Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) and picked up on a nickel slot grid 2 × 1 mm (EMS) coated with a polystyrene film (Sigma). Sections were post-stained with 4% uranyl acetate (Sigma) in H2O for 10 min and then rinsed several times with H2O. Afterwards sections were incubated in Reynolds lead citrate in H2O (Sigma) for 10 min and again rinsed several times with H2O. Micrographs were taken with a Philips CM100 TEM (FEI, Eindhoven, the Netherlands) at an acceleration voltage of 80 kV with a TVIPS TemCam-F416 digital camera (TVIPS GmbH, Gauting, Germany).
Results
PEC1 knockout exaggerates the phenotypes of the gpat6 mutant in cutin formation and fertility
The amount of aliphatic cutin monomers that require GPAT6 activity for their incorporation into cutin was reduced in the flowers of the pec1 mutant. Thus, gpat6 pec1 double mutants were generated to investigate whether GPAT6 acts epistatically to PEC1 and may be involved in the export of GPAT6-dependent cutin precursors (Li-Beisson et al., 2009; Bessire et al., 2011). The FTIR analyses and chemical cutin analysis showed that gpat6-2 has less cutin than gpat6-1 under our experimental conditions, thus the latter is a weaker gpat6 allele (Fig. 1b). Different alleles of the pec1 mutant have identical phenotypes (Fig. 1b). Thus, when distinction is not necessary they are simply called pec1.
A comparison of the cutin monomer composition of the gpat6 and pec1 single mutants with the respective gpat6 pec1 double mutants revealed that most cutin monomers were more strongly reduced in the gpat6 mutant as in the pec1 mutant. Their amounts were not significantly lower in gpat6 pec1 double mutants. This suggests that GPAT6 acts mainly epistatic to PEC1 as shown in Fig. 1(a,b) for gpat6-2 pec1 and for gpat6-1 pec1 in Supporting Information Fig. S1(a). However, the incorporation of unsaturated ω-hydroxy C18 acids, that is, octadecatrieneoic acid (18:3 ω-OH), ω-hydroxy octadecadieneoic acid (18:2 ω-OH) and ω-hydroxy octadecenoic acid (18:1 ω-OH) into cutin was more strongly reduced in gpat6 pec1 double mutants than in the single mutants. In double mutants with the gpat6-1 allele, a reduction of α, ω-hexadecanedioic acid (16:0 DCA) and ω-hydroxy hexadecanoic acid (16:0 ω-OH) was also significant (Fig. S1a). This indicated that PEC1 acts partially independently from GPAT6. In addition, the amounts of aliphatic esters in the petals were calculated based on the band vibration of the stretching C-H and the (C=O) vibrations measured by FTIR microspectroscopy. Additive contributions of GPAT6 and PEC1 to the total amount of aliphatic esters in the petals could be identified in both the gpat6-1 pec1-2 double mutant as well as the gpat6-2 pec1-3 in comparison with their respective parental lines (Fig. 1b). This confirms that PEC1 acts partially independent of GPAT6 and is not only involved in the export of GPAT6-dependent precursors.
The stronger impairments in cutin deposition in gpat6-1 pec1 double mutant petals were also reflected in a higher permeability of the petal cuticle (Fig. S1b). Interestingly, the fertility was also lower in the gpat6-2 pec1 double mutant than in both the gpat6 mutant alleles as quantified based on seed number per silique or silique length (Figs 2, S2). Careful investigation also showed a slightly reduced fertility in the pec1 mutant (Fig. 2). These results indicate that PEC1 may also have a function in reproduction that is at least partially independent of GPAT6. Interestingly, different attempts to generate pec1 dcr double mutants failed since neither plants that were double homozygous dcr pec1 nor homozygous for one and heterozygous for the other genotype could be obtained. These results also indicate an essential role of PEC1 during development.
PEC1 knockout enhances cuticular deficiencies of the gpat4 gpat8 mutant
We studied the relationship between PEC1 and both GPATs that are redundantly involved in the formation of leaf and stem cutin formation, that is, GPAT4 and GPAT8. The pec1 mutant exhibited only minor reductions in the amounts of cuticular lipids of expanded rosette leaves (Li et al., 2007; Bessire et al., 2011). The gpat4 gpat8 pec1 triple mutant grew slower and was dwarfed in comparison with the parental lines. In addition, many fusions were formed among different rosette leaves (Fig. 3). To perform chemical analysis of the cutin composition, triple mutant plants were cultured longer than the parental lines until the leaf number was similar to the control plants at harvest. Despite the pronounced changes in the growth habit of the gpat4 gpat8 pec1 triple mutants vs the parental lines, the amount of cutin and its composition in the rosettes of the triple mutant was similar to the one in the gpat4 gpat8 double mutant, except in two minor monomers, that is, 9(10),16-dihydroxy hexadecanoic acid (16:0 diOH) and (18:3 ω-OH) that were more strongly reduced in both parental lines (Fig. S3). In contrast to the rosettes, the cutin composition of the entire flowers of the gpat4 gpat8 pec1 was similar to the pec1 mutant (Fig. S4a). Analysis of both the stretching (C-H) vibrations and the C=O band vibration characteristic for aliphatic polyesters by Fourier-transform infrared spectroscopy identified a slightly lower polyester content in the petals of the gpat4 gpat8 pec1 mutant as in the pec1 mutant (Fig. S4b).
The cuticular phenotypes of petals were further investigated in the different genotypes. The gpat4 gpat8 pec1 triple mutant stained more strongly than the pec1 and gpat4 gpat8 mutants after short TB incubation times (Fig. 4a). In addition, the flowers often showed abnormalities in petal number and increased formation of organ fusions. Cryo-SEM studies revealed that the nanoridges of the epidermal cells in the petals were shallower and more unorganized in the gpat4 gpat8 mutant than in WT (Fig. 4b). Nanoridges of the pec1 mutant had the same size and depth as in WT, but covered only the upper part of the conical cells similar to previous studies (Bessire et al., 2011). Interestingly, the nanoridges in the gpat4 gpat8 pec1 triple mutant showed both features, that is, shallow, unorganized nanoridges that covered only the upper part of the conical cells (Fig. 4b).
In summary, in leaves and petals, the gpat4 gpat8 pec1 triple mutant exhibited stronger cuticle-associated impairments than the parental lines including organ fusion, nanoridge formation and enhanced cuticle permeability – this could not always be correlated with the amount or composition of aliphatic cutin monomers in mature organs.
Cutin composition changes during leaf expansion in Arabidopsis
The aliphatic cutin monomer composition was investigated during leaf expansion in Arabidopsis WT and the pec1 mutant plants grown under short-day conditions. Residue-bound lipids of emerging rosette leaves contained much higher amounts of 1,16-hexadecanedioic acid (16:0 DCA) and 16:0 diOH (4.6 and 2.8 μg mm−2, respectively) than fully expanded WT leaves (0.7 and 0.05 μg mm−2, respectively) (Figs 5a, S5). Thus, the amount of typical C16 monomers per square mm diminished by 85–98% during leaf expansion. A few minor C18-monomers with low desaturation, that is, 1,18-octadecanedioic acid (18:0 DCA) and ω-hydroxy octadenoic acid (18:1 FA) showed a similar behaviour.
By contrast, 1, 18-octadecenedioic acid (18:2 DCA), the most abundant monomer in WT leaves, decreased quickly during the initial expansion of the emerging leaves to the expanding leaf stage 1 (from 9.3 to c. 6.0 μg mm−2). It then remained constant during further leaf expansion. Thus, C18:2 DCA was reduced overall by only 30% during the entire period of leaf expansion (Figs 5b, S5). Other highly desaturated monomers such as 18:3 ω-OH and 18:2 ω-OH showed the same pattern of abundance. Thus, oxygenated cutin monomers have two different profiles in their abundances during leaf expansion.
Expanding leaves from 5-wk-old rosettes were also analysed because these results might reflect differences in the cutin composition of leaves as a function of rosette age. Similar differences in monomer composition have been observed in 5-wk-old rosettes as in 7-wk-old rosettes (Fig. S6). This indicates that the compositional differences reflect compositional changes during leaf ontogeny.
In addition, the total amount of oxygenated cutin monomers per surface area also changed during leaf expansion. It was higher in emerging leaves than in fully expanded leaves. A particularly strong (50%) drop in the amount of extractable cutin monomers per surface area occurred between the emerging leaf stage and the first quickly expanding leaf stage (Fig. S5). Several different processes may contribute to such a decrease, including redistribution of cutin monomers over a larger surface area, crosslinking in domains of the polyester, that is, the monomers cannot be extracted anymore by transesterification or regulated turnover of monomers during development. Furthermore, normalization of the leaf area that did not consider the surface of the trichomes, which are more densely distributed in emerging leaves than in the other stages of leaf expansion. Although the composition of oxygenated fatty acids strongly changes, the total amount of oxygenated fatty acids remains relatively stable from 1.5 cm long expanding leaves to fully expanded leaves (Fig. S5).
PEC1 knockout predominantly affects cutin composition as well as cuticle structure and properties in emerging leaves
The cutin composition of the pec1 mutant was evaluated in comparison with WT during leaf expansion in plants grown under short-day conditions. Most of the residue-bound aliphatic components changed during leaf expansion in the pec1 mutant similar to WT plants (Fig. S5). However, specific oxygenated fatty acids – in particular the oxygenated C16-monomers 16:0 DCA and 16:0 diOH – were reduced in emerging leaves to c. 50% in the pec1 mutant vs the WT (Fig. 6a,b). Changes in C18:2 were insignificant (Fig. 6c). In addition, highly unsaturated ω-hydroxy acids, that is, 18:2 ω-OH and 18:3 ω-OH were significantly reduced in the pec1 mutant (Fig. S5). Interestingly, the differences between pec1 and WT were gradually less prominent at later stages of leaf expansion. Because the cutin of emerging leaves is relatively rich in C16-monomers, their decrease resulted in an overall reduction in the oxygenated fatty acids of 25% in the cutin of emerging leaves in the pec1 mutant. This reduction was c. 10% in the expanded leaves of the 7-wk-old rosettes (Fig. 6d).
The alterations in cutin composition and the amount of pec1 were more prominent early during leaf expansion than in fully expanded leaves. Thus, the ultrastructure of the cuticle was investigated by TEM in emerging leaves of the pec1 mutant and WT (Fig. 7a). The WT cuticle could be visualized as a thin, regularly formed electron-opaque layer with little or no reticulation. By contrast, the cuticle of the pec1 plants was thinner, interrupted and with a more irregular reticulation as in WT plants at this stage of development. To assess the functionality of the cuticle as a diffusion barrier, entire rosettes were stained with TB for different time periods. Intriguingly, the permeability of the expanding leaves of the pec1 mutant was highly increased vs fully compared with the expanded leaves (Fig. 7b). This matched the growth-stage specific defects in cutin formation. Interestingly, the cuticle of gpat6 mutants is not permeable in expanding leaves, while that of pec1 mutants is permeable (Fig. S7b). This is despite the fact that pec1 mutants and gpat6 mutants have similar amounts of cutin, and C16-monomers are more strongly reduced in gpat6 than in pec1 in expanding leaves (Fig. S7a).
Discussion
PEC1 is required for cutin formation independent of a specific GPAT-dependent pathway
The construction of various double and triple mutants between pec1 and different gpat mutants revealed further insights into the function of PEC1 in cutin formation. Despite the significant overlap of PEC1 function with the GPAT6-dependent pathway, two minor monomers, 18:2 ω-OH and 18:3 ω-OH, were specifically reduced in leaves and flowers of gpat6 pec1 mutants. This shows that PEC1 is not associated directly with the GPAT6 pathway. The same fatty acids were also more strongly reduced in rosette leaves of the gpat4 gpat8 pec1 triple mutant than of the gpat4 gpat8 double mutant. This indicates the particular relevance of PEC1 for the incorporation of these polyunsaturated ω-hydroxy C18 acids into cutin. The potential relevance of these minor monomers for the cuticular structure, however, has not been addressed in this study. The notion that PEC1 acts independently from GPAT6 was further substantiated by the fact that in petals the total amount of aliphatic esters was lower in both gpat6 pec1 double mutants than in their parental lines. The small additive contributions to the content of the pertinent highly unsaturated C18:2 hydroxy acids in rosettes indicate that PEC1 acts autonomously from GPAT4 and GPAT8. In entire flowers, however, additive contributions to cutin monomer content were insignificant. This might arise from differences in cutin composition in different parts of floral organs as well as from the small sample size and high biological variability or increased crosslinking of the polymer. FTIR spectroscopy on petals, however, gives evidence for additive contributions of PEC1, GPAT4 and GPAT8 to cutin amount of floral organs. These findings exemplify that in certain conditions FTIR spectroscopy is more powerful than the more generally used transesterification reaction combined with GC-MS.
PEC1 is necessary early during organ development
Although the knockout of PEC1 in the gpat4 gpat8 background did not affect cutin amount in mature rosettes the frequent formation of fusions among these leaves occurred. Organ fusions have been observed in a number of other cuticle mutants and are generally associated with defective cuticle formation during early organ development (Nawrath, 2006; Voisin et al., 2009). Cutin composition and the amount in organ fusion mutants could not always be linked to the function of the affected enzyme, for example, in lacerata (lcr), fiddlehead (fdh) and bodyguard (bdg) mutants because hypothesized compensation reactions occurred leading to higher cutin contents than in WT plants (Kurdyukov et al., 2006; Voisin et al., 2009). During the present studies, however, no compensatory effects were seen. Enhanced cuticle permeability in petals and stronger abnormalities in floral organ formation including petal distortions and altered petal number were also observed in the gpat6 pec1 double mutants as well as in the gpat4 gpat8 pec1 triple mutant. These observations support the notion that the transport activity of PEC1 is required for cutin formation and cuticle structure during the early development of given organs.
Cuticle formation during leaf expansion in Arabidopsis
The cutin of Arabidopsis rosette leaves undergoes extensive changes in both amount and composition during leaf expansion. 18:2 DCA is the most abundant monomer based on leaf area in all stages of rosette leaf expansion, representing c. 40% of the oxygenated polyester monomers in emerging leaves and 70% in fully expanded ones. The cutin of emerging leaves consists of c. 35% oxygenated C16-monomers, but of expanded leaves only 10%. The monomers that steadily decrease in their proportion during leaf expansion were typical cutin monomers, such as 16:0 diOH, that can be found in many species, including Solanum lycopersicum and Vicia faba (Kolattukudy, 2001). Those that decreased only in the first expansion period were the highly desaturated ones typical for the Brassicacea (Bonaventure et al., 2004; Franke et al., 2005; Molina et al., 2006). These two patterns of incorporation of oxygenated monomers into cutin during leaf expansion suggest that the synthesis of precursors containing oxygenated cutin monomers of no or low unsaturation (common monomers) is differently regulated than that of precursors containing oxygenated polyunsaturated fatty acids (Brassicaceae-typical monomers).
C16:0 diOH decreased steadily during rosette leaf expansion, by 98% based on leaf area and also when based on leaf number indicating a potential crosslinking and thus unavailability for transesterification or a turnover of cutin. A decrease of the total amount of extractable cutin monomers was observed in the oldest stem segments of Arabidopsis (Suh et al., 2005). In the present experiments a particularly strong drop (50%) of all oxygenated cutin monomers occurred, however, already between the emerging leaf stage vs fast expanding leaf stage 1 that most logically might be explained by a redistribution process of cutin monomers over a larger area. Comparison of the ultrastructure of the cuticle of emerging rosette leaves with fully expanded leaves of WT supports the idea of a reorganization process occurring during this time since the cuticle becomes visually more heterogeneous as well as thinner (Fig. S8). During this process the WT rosette leaf cuticle retains its barrier function being impermeable at all stages of expansion (Bessire et al., 2011). Modification of the ultrastructure during cuticle maturation has also been also been observed in other species including Clivia miniata, Utricularia laterifolia and several species of spruce trees (reviewed in Jeffree, 2006). The pro-cuticle is in all species simpler and has often a more homogeneous appearance, while mature cuticles have diverse ultrastructures. Cuticle development conceivably includes complex structural and compositional modification of the cutin polymer, including redistribution, turnover and crosslinking.
A detailed study of cutin monomer composition during leaf expansion is available for C. miniata (Riederer & Schönherr, 1988). In contrast to our findings in Arabidopsis, in C. miniata leaves all of the oxygenated fatty acids as well as the total cutin content increased during cuticle maturation after leaf expansion had ceased (Riederer & Schönherr, 1988). Similar to C. miniata the cutin of the early developing cuticle (the so-called pro-cuticle) in Arabidopsis is enriched in oxygenated C16-monomers.
PEC1 plays a particular role in leaf cuticle expansion
In the pec1 mutant the composition of aliphatic cutin monomers was strongly affected in emerging, quickly expanding leaves rich in C16 cutin monomers, but gradually became less marked as expansion slowed. At the same time highly unsaturated C18 monomers that are incorporated PEC1-independently became more abundant in the cutin. These changes during early leaf expansion correlate well with changes in the ultrastructure of the cuticle of the pec1 mutant as well as its higher permeability to TB in emerging leaves compared with fully expanded leaves. Although PEC1 contributes only weakly to the cutin composition of fully expanded rosette leaves the cuticles of such leaves in the pec1 mutant are permeable (Bessire et al., 2011). This indicates that the cutin composition and structure early during cutin formation influences cutin structure in mature leaves.
The gpat6 mutants have a stronger reduction in the amount of several pertinent monomers than the pec1 mutant yet, surprisingly, the cuticle permeability of rosette leaves is unaffected. Several hypotheses can be formulated to explain these phenomena. First, the aliphatic cutin monomers 18:2 ωOH and 18:3 ωOH are reduced in the pec1 mutant, but not in the gpat6 mutant. This may play a particular role for cutin structure. Second, not only the presence of C16-type cutin monomers but also their structural arrangement may be required for the formation of a functional leaf cuticle. Third, the structural arrangement of cutin monomers with waxes depends on cutin composition. The notion that not the amount but the structural arrangement of aliphatic cuticular components is essential for the formation of a functional diffusion barrier has been discussed previously (reviewed in Schreiber, 2010; Nawrath et al., 2013). PEC1 plays a critical, non-redundant role in this process, when GPAT6-dependent C16:0 monomers are present.
Towards the transport activity of PEC1
The goal of this study was to learn more about the transport function of PEC1 in the formation of cutin and its impact on cuticle function. PEC1 was tightly associated with the formation of C16-rich cutin and not with the formation of C18:2 DCA-rich cutin in leaves and floral organs. This suggests that PEC1 may be directly or indirectly involved in the export of molecules containing C16-monomers. No evidence was found that PEC1 is specifically required for the export of simple aliphatic C16-precursors including mono 2-MHGs. Increasing evidence, on the contrary, implies that the non-redundant function of the PEC1 transport activity lies in facilitating the incorporation of aliphatic C16-monomers in the cuticle into the particular 3D structure necessary for building a functional cuticle. The nature of molecules with such functions is still unknown. They might be, for example, more complex cutin precursors containing largely, but not exclusively GPAT6-dependent aliphatic moieties in addition to non-aliphatic moieties. Modifications of the ultrastructure in the cuticle layer of the pec1 mutant at the interface between the cuticle and the cell wall were reported (Bessire et al., 2011) pointing to the incorporation of PEC1-dependent molecules into the polysaccharide enriched, cuticular layer. It cannot be excluded that PEC1 activity affects directly only the incorporation of non-aliphatic components at the cuticle/cell wall interface and changes in the aliphatic cutin composition are only indirect. The highly reproducible and genetically manipulable cutin monomer composition of pec1 mutants of different genetic backgrounds, however, argues against the latter possibility.
During this study FTIR microspectroscopy proved useful for the direct comparison of plants of many different genotypes. The analysis revealed that the gpat6-2 pec1 mutant had the lowest amount of cutin of all mutants analysed (Fig. S9). Because the contributions of ester bonds from other cell wall components, for example, methylesterified pectin, becomes more prominent with decreasing amounts of cutin, the absolute cutin content is most likely even lower in such plants than calculated (Mazurek et al., 2013). A separate analysis of the aliphatic and ester contributions indicated that severe blocks in cutin formation were not compensated by the formation of other aliphatic polymers lacking ester bonds (Villena et al., 1999). Crosslinking of cutin with itself or to the cell wall, however, could not be evidenced. Neither could information on the chemical nature of the molecules missing in the pec1 cuticle of Arabidopsis leaves and flowers be obtained. The characterization of the function of PEC1/ABCG32 homologues in plants with organs having more substantial cuticles (e.g. the tomato) allowing cutin analysis by partial depolymerization might give additional insights into the structural components that are incorporated into the extracellular matrix by PEC1/ABCG32 and ultimately lead to the identification of its substrate.
Perspectives
Fundamental changes in cutin structure and composition occur during development. These developmental changes have not been considered during the isolation and characterization of Arabidopsis mutants in prior studies. The detailed characterization of the pec1 mutant led to the identification of organ- and development-specific functions of PEC1 in cutin synthesis as well as the formation of a functional cuticle in Arabidopsis. The identification of cuticle mutants at different developmental stages and the re-evaluation of previously identified cuticle mutants during plant development is therefore necessary. This information will aid in the understanding of the complex process of cuticle development and maturation. The results also underscore that the aliphatic cutin monomer composition per se is not the main determining factor in the formation of a functional cuticle. Understanding the structural arrangement of the precursors within the cutin as well as other cuticular components and their role in building a functional cuticle remains a challenging task in the future.
Acknowledgements
We thank Sébastien Desarzens, Guillaume Gremion, Delphine Le Roux and Sylvain Escande for their technical assistance. We thank the Swiss Plant Science Web for their support of FTIR within the frame of the Bio-molecular Analysis Platform, and the FBM-UNIL for generous support of the Electron Microscopy Facility. We thank Frédéric Beisson, CNRS/CEA-IBEB, France, for supplying us with the seeds of the different gpat mutants, Willy Blanchard, EMF, UNIL, for the preparation of the EM artwork and Penny von Wettstein-Knowles, University of Copenhagen, Denmark, for critical reading of the manuscript. C.N. was supported by the Swiss National Science Foundation (grant 31003A_125009) as well as the Herbette Foundation, UNIL, and S.M. by the Swiss Plant Science Web.
