© 2014 Scandinavian Plant Physiology Society, ISSN 0031-9317

Physiologia Plantarum 2014

Involvement of GPI-anchored lipid transfer proteins in the development of seed coats and pollen in Arabidopsis thaliana Monika M. Edstam and Johan Edqvist∗ ¨ ¨ IFM, Linkoping University, Linkoping, SE-581 83, Sweden

Correspondence *Corresponding author, e-mail: [email protected] Received 4 November 2013; revised 6 December 2013 doi:10.1111/ppl.12156

The non-specific lipid transfer proteins (nsLTPs) constitute a large protein family specific for plants. Proteins from the family are found in all land plants but have not been identified in green algae. Their in vivo functions are still disputed although evidence is accumulating for a role of these proteins in cuticle development. In a previous study, we performed a coexpression analysis of glycosylphosphatidylinositol (GPI)-anchored nsLTPs (LTPGs), which suggested that these proteins are also involved in the accumulation of suberin and sporopollenin. Here, we follow up the previous co-expression study by characterizing the phenotypes of Arabidopsis thaliana lines with insertions in LTPG genes. The observed phenotypes include an inability to limit tetrazolium salt uptake in seeds, development of hair-like structures on seeds, altered pollen morphologies and decreased levels of ω-hydroxy fatty acids in seed coats. The observed phenotypes give further support for a role in suberin and sporopollenin biosynthesis or deposition in A. thaliana.

Introduction The first plants conquered land approximately 470 million years ago. The following explosion of land plant species not only transformed the landscape and atmosphere but also set the stage for the subsequent emergence of animals onto land. The first land plants faced numerous challenges that included increased exposure to UV radiation, desiccation and temperature stress. The land plant radiation is marked by a long list of morphological and biochemical innovations and adaptations, such as the biosynthesis of the major extracellular lipid-based polymers and polyesters sporopollenin, cutin and suberin. Cutin is the structural polymer of the epidermal cuticle, the waterproof layer covering primary aerial organs

and which is often the structure first encountered by phytopathogens (Javelle et al. 2011). Cutin is a polyester of C16 and C18 hydroxy fatty acids and glycerol, which in the cuticle is interspersed with and covered by waxes. Suberin is an apoplastic biopolymer that contributes to the control of diffusion of water and solutes across internal root tissues and in periderms (Ranathunge et al. 2011). Suberin is usually described as a heteropolymer with polymeric aliphatic and associated aromatic materials. Long-chain oxygenated fatty acids provide the core suberin polyester. Sporopollenin is found in the exine layer of spore and pollen walls. The exine protects spores and pollen in harsh environments and serves as a barrier against various physical and chemical factors and biological pathogens (Ariizumi and Toriyama 2011). The

Abbreviations – ABC, ATP-binding cassette; AtUSP, A. thaliana UDP-sugar pyrophosphorylase; ER, endoplasmic reticulum; GC–MS, gas chromatography–mass spectrometry; GPAT6, glycerol-3-phosphate acyltransferase 6; GPI, glycosylphosphatidylinositol; LTPGs, glycosylphosphatidylinositol-anchored lipid transfer proteins; MS, Murashige and Skoog; nsLTPs, non-specific lipid transfer proteins; PCR, polymerase chain reaction; SEM, scanning electron microscopy.

Physiol. Plant. 2014

sporopollenin is chemically inert because of insolubility in both aqueous and organic solvents. It is not a homogeneous macromolecule but is instead made up of complex biopolymers derived mainly from saturated precursors such as long-chain fatty acids and long aliphatic chains. The syntheses of sporopollenin, cutin and suberin require at least four processes: the de novo synthesis of polymer precursors, massive secretion from the lipid bilayer to the apoplastic compartment, polymerization and transfer of the precursors through the highly hydrophilic cell wall. Based on the subcellular localization of biosynthetic enzymes, it is likely that the biosynthesis of the polymer compounds starts in the plastids and continues with subsequent steps in the endoplasmic reticulum (ER). Following the synthesis in ER, the polymer precursors should be secreted through the plasma membrane. At present, the best candidates for the channeling of cuticular and suberin components through the plasma membrane are the two closely related ATP-binding cassette (ABC) transporters ABCG12/CER5 and ABCG11/WHITE BROWN COMPLEX11 (WBC11)/DESPERADO (DSO)/CUTICULAR DEFECT AND ORGAN FUSION1 (COF1), located in the plasma membrane of Arabidopsis thaliana (Pighin et al. 2004, Bird et al. 2007, Panikashvili et al. 2007). Although there is no direct experimental evidence that these proteins are involved in the transfer, the respective mutants are significantly affected in wax and cutin deposition and lipid inclusions are found within the epidermal cytoplasm. Once exported, the extremely hydrophobic polymer compounds have to pass through the apoplastic compartment or the highly hydrophilic cell wall. How this is achieved is unknown, but the family of nonspecific lipid transfer proteins (nsLTPs) is a candidate that could be involved in the delivery of sporopollenin and suberin components, wax components and cutin monomers to the apoplastic compartment. There are at least four features that point out the nsLTPs as good candidates for this specific task. First, nsLTPs are abundantly expressed in the epidermis (Sterk et al. 1991). Second, they generally contain a signaling peptide and can be secreted into the apoplast (Edstam et al. 2011). Third, they are small enough to traverse the pores of the cell walls. Finally, the nsLTPs contain a hydrophobic pocket capable of binding longchain fatty acids (Charvolin et al. 1999). The nsLTPs are land plant specific proteins that are encoded by large gene families (Boutrot et al. 2008, Edstam et al. 2011). We have divided the nsLTPs into four major and several minor types according to sequence similarity, intron position and spacing between the cysteine residues (Edstam et al. 2011). In one of

the major types, glycosylphosphatidylinositol-anchored lipid transfer protein (LTPG), the transcripts encode a C-terminal signal sequence in addition to the N-terminal one, leading to a posttranslational modification where a glycosylphosphatidylinositol (GPI)-anchor is added to the protein. The GPI-anchor attaches the protein to the extracellular side of the plasma membrane. Rather recently, the isolation and characterization of an A. thaliana mutant disrupted in an LTPG, named LTPG1, revealed reduced alkane accumulation at the plant surface (Debono et al. 2009, Lee et al. 2009), suggesting that this plasma membrane-bound nsLTP is involved in lipid export. The question remains as to whether this nsLTP shuttles across the cell wall to deliver lipids to the cuticle. In a previous investigation (Edstam et al. 2013), we identified the co-expression patterns of LTPG genes in both Oryza sativa and A. thaliana. Gene ontology analyses of the obtained networks suggested roles for LTPGs in the synthesis or deposition of cuticle polymers, suberin and sporopollenin. Here, we follow up the previous investigation by studying the phenotypes of A. thaliana T-DNA mutants with insertions in LTPG genes. We note that some of the analyzed mutants have morphological and metabolic alterations, indicating a role for LTPGs in seed coat and pollen development.

Materials and methods Plant material and growth conditions T-DNA insertion lines were obtained as seeds from The Arabidopsis Information Resource (TAIR) (Appendix S1). All lines are in the Columbia Col-0 background. Two of the lines, ltpg4-1 and ltpg4-2, are in the quartet background, resulting in pollen grains fused together in tetrads (Preuss et al. 1994). Plants were grown either on agar plates, containing half-strength Murashige and Skoog (MS) medium, or in pots on a mixture of vermiculite and soil. Sown seeds were stratified for 3 days in 4◦ C before transfer to growth chamber (22◦ C, 16 h light and 8 h dark). Plants grown on plates were moved to soil after 2 weeks. Identification of mutant lines To identify plants homozygous for the T-DNA insertion, DNA was extracted using the DNeasy Kit (Qiagen, Hilden, Germany) and used as template in a polymerase chain reaction (PCR). Primers complementary to regions flanking the insert were used to detect wild-type or heterozygous plants (Appendix S2). To detect the T-DNA inserts, one flanking primer was used together Physiol. Plant. 2014

with a primer complementary toward the insert. The RNeasy Kit (Qiagen) was used to extract RNA from confirmed homozygous T-DNA insertion mutants, to examine the expression of the genes. The RNA was treated with DNase (Thermo Scientific, Waltham, MA) to remove residual genomic DNA and then used for cDNA synthesis. The reverse transcriptase RevertAid RT (Thermo Scientific) was used according to the manufacturer’s protocol. The cDNA was then used as template in a PCR with primers complementary to the nsLTP sequences (Appendix S2). In the insertion line ltpg5-1, the relative expression, compared to wild-type, was determined using reverse transcriptase polymerase chain reaction (RT-PCR). Four replicates were done. UBC21 encoding an ubiquitin-conjugating enzyme (At5g25760) was used as a reference gene (Czechowski et al. 2005). The PCR reactions were run on a 1% agarose gel supplemented with SYBR Safe (Invitrogen, Carlsbad, CA), and the software GELANALYZER (www.gelanalyzer.com) was used to determine the intensity of the resulting bands. Stress treatments For salt stress, seeds were sown on MS agar plates supplemented with 75 mM NaCl and for osmotic stress the plates were instead supplemented with 150 mM mannitol. Ten days after transfer to the growth chamber, seedling establishment was examined. A seedling was considered as established if both cotyledons were green and fully separated. For dehydration, darkness and cold treatments, the soil-grown plants were exposed to the respective treatment at an age of 5 weeks. For each mutant line and each treatment three plants were used, grown in the same pot together with one wild-type plant. For dehydration, watering was stopped for 14 days and the survival rate was determined. For dark treatment, the plants were placed in a light proof box for 14 days and the survival rate as well as the ability to recover was determined. For cold shock, the plants were incubated at −20◦ C for 30 min and then put back to the growth chamber. The survival rate was determined 2 days after the cold treatment. Cuticle, seed and pollen staining Five weeks old soil-grown plants were used in two different cuticle permeability assays: toluidine staining and chlorophyll leaching (Schnurr et al. 2004, Tanaka et al. 2004). Aqueous toluidine blue O, 0.05%, was used for toluidine staining, and plant parts (both leafs and stems) were dipped into the solution for 2 min. The stain was then removed with water and Physiol. Plant. 2014

the plants were checked for colored spots, indicating increased cuticle permeability. For the chlorophyll leaching experiment, rosette leaves were weighed and immersed in 5 ml 80% ethanol. At several time points (10 min, 30 min, 60 min and 24 h), a 100 μl aliquot was removed and analyzed with a Nanodrop spectrophotometer (NanoDrop ND-1000 Spectrophotometer, Thermo Scientific). Absorbance was measured at 664 and 647 nm, corresponding to chlorophyll a and b. The equation 7.93 (A664) + 19.53 (A647) was used to calculate the total amount of leached chlorophyll in the solution (Lolle et al. 1997). For ruthenium red staining, the seeds from wild type and mutants were immersed in water for 1 h, to start the imbibition, and then stained in 0.05% ruthenium red (in deionized water) for 1 h (Western et al. 2000). The stained seeds were observed with a stereomicroscope. To analyze the permeability of seed coats, the seeds were weighed and put into packages of Miracloth and incubated in 1% aqueous tetrazolium (2,3,5triphenyltetrazolium chloride) for 15, 30 or 48 h in darkness at 30◦ C (Debeaujon et al. 2000). Three replicates were used for each seed line. After incubation, the seeds were rinsed with water and photographed using a Dino-Lite Pro digital microscope (AnMo Electronics Corporation, Hsinchu, Taiwan). The seeds were then transferred to 2 ml microcentrifuge tubes together with 200 μl absolute ethanol. To obtain the formazans, seeds were ground with a TissueLyser (Qiagen) for 2 × 40 s and immediately centrifuged for 3 min at 15 000 g . The supernatant was recovered and the concentration of formazans was measured at 485 nm using a Nanodrop spectrophotometer. Significant changes in the values from insertion lines compared to wild-type were calculated using Student’s two-tailed t -test. Alexander staining procedure was used to examine if pollen grains from mutant plants were viable or not (Alexander 1969). The Alexander stain was prepared by mixing 10 ml of 95% ethanol, 1 ml of Malachite green (1% solution in 95% ethanol), 50 ml of deionized water, 25 ml of glycerol, 5 ml of acid fuchsin (1% solution in water), 0.5 ml of Orange G (1% solution in water) and 4 ml of glacial acetic acid. The volume was adjusted to 100 ml with deionized water. Whole flowers were fixed in ethanol:chloroform:acetic acid (6:3:1) for 2 h before staining. Samples were stained with Alexander stain and heated to just below boiling for 30 s, rinsed and observed with a microscope [Nikon Elipse 80i microscope (Nikon, Tokyo, Japan) equipped with the digital camera Nikon DS-Fi1].

Suberin analysis Seed coat suberin was extracted and depolymerized according to a well-established protocol (Molina et al. 2006). Briefly, the seeds were ground with mortar and pestle in liquid nitrogen and then transferred to a tube with boiling isopropanol. After 10 min, the samples were allowed to cool down and were then ground again, this time with an Ultra Turrax (IKA, Staufen, Germany). This was followed by several steps of washing with different solvents (Molina et al. 2006) to extract seed lipids from the seed coats. After the last extraction step, the seed coat residues were filtered and dried in a vacuum desiccator for several days. Internal standards (C17:0 methyl ester and pentadecalactone) were added and suberin was depolymerized through methanolysis with sodium methoxide. The obtained fatty acid methyl esters were derivatized with N,OBis(trimethylsilyl)trifluoroacetamide (BSTFA) before gas chromatography–mass spectrometry (GC–MS). The GC–MS was performed using a DB-5 capillary column (FactorFour from Varian, Palo Alto, CA) with helium carrier gas. Splitless injection mode was used and the oven was preheated to 100◦ C. After injection, the temperature was maintained at 100◦ C for 1 min and then increased by 10◦ C every minute, up to 280◦ C. At 280◦ C, the temperature was maintained for 20 min. Manual integration was used to estimate the peak areas. Relative peak areas were calculated after removal of internal standards. Scanning electron microscopy Seeds and pollen were covered by 10 nm platinum before observation with a JSM 6320F or a JSM 830 scanning electron microscope (JEOL, Tokyo, Japan).

Results Characterization of LTPG insertion lines The complete set of A. thaliana LTPG genes was identified and characterized in a previous study (Edstam et al. 2013). In that study, we also systematically examined microarray data to investigate the coexpression patterns of the LTPGs. Fourteen LTPG genes could be arranged in three expression modules that were named as AtI, AtII and AtIII. The genes LTPG1, LTPG2 and LTPG6 were placed in the module AtI. LTPG5, LTPG15, LTPG16, LTPG17 , LTPG20 , LTPG22 and LTPG30 were set to AtII, whereas LTPG3, LTPG4, LTPG23 and LTPG26 were located to AtIII. Starting from these modules, we generated extended networks with Arabidopsis genes co-expressed with the modules. Gene

ontology analyses of the obtained networks suggested that the LTPGs in the AtI-module would primarily be involved with synthesis or deposition of cuticular lipids, the AtII-module with suberin and the AtIII-module with sporopollenin. To functionally characterize as many as possible of the 14 Arabidopsis LTPGs allocated to the co-expression modules, all publically available single T-DNA insertion lines were identified. We obtained one insertion line for LTPG1, three for LTPG2, one for LTPG3, three for LTPG4, one for LTPG5, three for LTPG6, two for LTPG15, two for LTPG20 and one for LTPG22 (Appendix S1). At first, we analyzed the genomic DNA in order to find insertion lines that were homozygous for the T-DNA insertion. Lines where we failed to identify plants homozygous for the T-DNA insertion were removed from the investigation, while seeds from homozygous plants were sown and used further on. Next, the expression of the genes carrying T-DNA insertions was investigated on a transcriptional level. Lines which did not show a knockout or knockdown effect of the T-DNA insert were not used in the remaining experiments. After these selective steps, we were left with one insertion line for LTPG1, two for LTPG2, one for LTPG3, two for LTPG4, one for LTPG5 and two for LTPG6. The insertion line ltpg1-1 is a complete knockout of LTPG1, with the insertion located to the first intron (Fig. 1). Two different lines with an insertion in the gene LTPG2 were used, ltpg2-1 and ltpg2-2 (Fig. 1) where ltpg2-1 has the insertion in intron 2 and ltpg2-2 in exon 3. Both these lines showed a complete knockout of the LTPG2 expression. In the line ltpg3-1, the insertion is found in the first exon, which led to a complete knockout of the gene (Fig. 1). Two different lines were used for LTPG4, ltpg4-1 and ltpg4-2. In both cases, the insertion is in intron 2 (Fig. 1). The transcript analysis revealed that although the first exon of the gene is expressed in both the lines, the expression of the 3 region of the open reading frame was abolished in both mutant lines (Fig. 1B). This would mean that the GPI-anchor signal is missing from the polypeptides formed after the translation of the transcripts. For ltpg5-1, the insertion is located in the 5 untranslated region, leading to a knockdown effect on the expression of the gene (Fig. 1C). Two different T-DNA insertion lines were used for LTPG6, ltpg6-1 and ltpg6-2. In ltpg6-1, the insertion is located in exon 3, whereas it is in exon 2 for ltpg6-2 (Fig. 1A). As in the case with ltpg4-1 and ltpg4-2, the two lines still had an expression of the first exon, but the transcripts were truncated and translated proteins would be without the GPI-anchoring signal. Physiol. Plant. 2014

A

B

C

Fig. 1. The structure and expression of the T-DNA insertion lines. (A) Schematic structures of the genes, with exons as boxes and introns as lines. The approximate sites for the insertions are indicated with triangles. Primer-binding sites are indicated with arrows. (B) The expression of the LTPG genes in the knockout lines and wild type. UBC21 encoding an ubiquitin-conjugating enzyme (At5g25760) was used to confirm a functional cDNA synthesis. (C) Relative expression of LTPG5 in the line ltpg5-1 and in the wild-type Arabidopsis thaliana. The expression level in ltpg5 is less than 60% of that in the wild type. P = 0.04 (calculated using Student’s two-tailed t-test). UBC21 encoding an ubiquitin-conjugating enzyme (At5g25760) was used as a reference gene.

Reduced fertility observed in some of the LTPG insertion lines The growth and development of the mutant lines were followed during normal and alternative conditions, such as dark treatment, salinity stress, drought stress and cold stress. In most cases, ocular inspection of the mutant lines did not reveal any phenotypical changes in comparison to the wild type. An interesting observation was, however, the increase in early aborted seeds and infertile ovules in ltpg6-1 (Fig. 2) and several of the other mutant lines (ltpg2-1, ltpg2-2, ltpg4-1, ltpg4-2 and ltpg6-2) (data not shown) during standard growth conditions.

An increased salt permeability in the seeds from several LTPG mutants Next, we continued with a series of histochemical and microscopic analyses to deduce any possible Physiol. Plant. 2014

Fig. 2. A silique from wild-type Arabidopsis thaliana is compared to one from ltpg6-1 that has a reduced seed set due to unfertilized ovules. A similar phenotype was observed for ltpg2-1, ltpg2-2, ltpg4-1, ltpg4-2 and ltpg6-2.

abnormalities caused by the insertion in an LTPGencoding gene. Staining with Toluidine Blue O did not reveal increased cuticle permeability in any of the mutant lines. Furthermore, the chlorophyll leaching experiment, which is another approach to study the cuticle permeability, failed to show any significant

A

B

Fig. 3. Results from the tetrazolium assay. (A) Seeds from wild-type Arabidopsis thaliana and four different T-DNA insertion lines after 48 h of tetrazolium staining. A few wild-type seeds are weakly stained, but not to the same extent as seeds from the insertion lines. (B) Measurements of the absorbance at 485 nm, corresponding to the concentration of formazans in the seeds. The formazans are formed when a tetrazolium salt penetrates the seed coat and reaches the embryo. The values are based on three replicates, where the error bars show the standard deviations and an asterisk indicates values that significantly differ from those of the wild type (P < 0.01, calculated using Student’s two-tailed t-test).

changes between wild type and any of the mutant lines. Thus, the mutations studied here seem not to affect the permeability of the A. thaliana cuticle. After investigating the cuticle, we turned to perform a detailed inspection of the seeds from the mutant lines. First, germination tests revealed that none of the seeds showed a reduced germination rate. Ruthenium red stains pectin that is secreted from seeds during imbibition. Staining with ruthenium red did not reveal any defects in pectin excretion in seeds of any of the knockout or knockdown lines (not shown). However, when the seeds were incubated in a solution of tetrazolium salt, several of the mutant lines (ltpg2-2, ltpg3-1, ltpg4-1, ltpg4-2, ltpg5-1, ltpg6-1 and ltpg6-2) were found to have an inability to restrict the salt uptake (Fig. 3). Unusual hair-like outgrowths in the seeds from LTPG mutants ltpg4-1 and ltpg5-1 Scanning electron microscopy (SEM) was applied to detect possible alterations in the seed morphology of

ltpg4-1, ltpg4-2, ltpg5-1, ltpg6-1 and ltpg6-2. These lines were selected for the analysis because of their inability to limit the tetrazolium uptake in seeds. About 30% of the seeds from ltpg4-1 and ltpg4-2 appeared shrunken and deformed (Fig. 4B). More strikingly, peculiar hair-like outgrowths were seen on seeds from lines ltpg4-1, ltpg4-2 and ltpg5-1 (Fig. 4C, D). These odd outgrowths were never found on seeds from the wild type (Fig. 4A). The investigation was continued with an analysis of the lipid polyester monomers of the seed coats from some of the LTPG insertion lines. Only the lines with the largest increase in salt permeability were examined: ltpg4-1, ltpg4-2, ltpg6-1 and ltpg6-2. The lipid analysis revealed two large changes compared with wild type. First, there is an increase in unsubstituted fatty acids in the knockout lines, from 10% in wild type to around 15% in the mutant lines (Fig. 5A). Second, there is a large decrease in ω-hydroxy fatty acids, from almost 35% in wild type to around 25% in the mutant lines. Among the unsubstituted fatty acids, the largest difference between the mutant lines and the wild type is the increase of C20:0, C22:0 and C24:0 in the mutant lines (Fig. 5B). Among the ω-hydroxy fatty acids, the largest decrease is seen for 24-hydroxytetracosanoic acid (C24ωOH), which is reduced from 25% in the wild type to below 20% in the mutant lines (Fig. 5C). The ω-hydroxy fatty acids are known to be important constituents of suberin. Defects in the pollen morphology of some of the LTPG mutants Both the genes, LTPG3 and LTPG6, are abundantly expressed in pollen (Edstam et al. 2013). Therefore, we examined the corresponding mutants regarding pollen defects. Alexander staining of pollen grains from the ltpg3-1, ltpg4-1 and ltpg4-2 lines did not show any indications of dead pollen grains (not shown).We further applied SEM to inspect if there were any morphological changes on the surfaces of the pollen grains. Strikingly, pollen grains from all three knockout lines were deformed or collapsed (Fig. 6B–D). Such morphological alterations were never found for pollen grains from the wild-type line (Fig. 6A).

Discussion In a previous study on the co-expression of A. thaliana and O. sativa LTPG genes, we put forward the hypothesis that these proteins are involved in the synthesis of cuticular wax and lipid polyesters such as cutin, suberin and sporopollenin (Edstam et al. 2013). A role of LTPGs in the synthesis of cuticle components has also been Physiol. Plant. 2014

A

B

C

D

Fig. 4. SEM pictures of Arabidopsis thaliana seeds. (A) A seed from wild-type A. thaliana, showing regular pattern of cell walls and a columella in each cell. (B) A seed from the T-DNA insertion line ltpg4-1 showing a much more irregular pattern. Seeds from the insertion lines ltpg4-2 (C) and ltpg5-1 (D) with hair-like outgrowths.

suggested from experiments. For instance, decreased LTPG1 expression in A. thaliana resulted in that less wax was loaded on the stem surface (Debono et al. 2009). However, when LTPG1 was disrupted in another study, there were no significant alterations found for the wax load (Lee et al. 2009). Rather, they demonstrated a 10% reduction in the C29 alkane (nonacosane), a major component of cuticular waxes in stems and siliques. More recently, it was shown that LTPG2 is functionally redundant or overlapping with LTPG1 as the C29 alkane and the wax load was reduced in stems and siliques with about 10% in an LTPG2 insertion mutant (Kim et al. 2012). Seeds from several of the ltpg -lines analyzed here showed an increased permeability to tetrazolium salt. This indicates a malfunctioning seed coat. Morphological seed coat alterations were also shown for several of the ltpg -lines where peculiar hair-like outgrowths were observed on the seeds. Seed hairs are not normally found in A. thaliana but are seen on the seeds of some other plants. The most well-known example is probably the cotton fibers that are single-celled seed hairs. Interestingly, several nsLTPs are abundantly and exclusively expressed in cotton fiber cells (Ma et al. 1997, Orford and Timmis 2000, Wu et al. 2006). It has been suggested that the function of nsLTPs in cotton fiber development is related to the deposition of cutin (Orford and Timmis 2000). It was recently shown that overexpressing or co-expressing the cotton genes GhRDL1 and GhMYB2 in A. thaliana activated hair production Physiol. Plant. 2014

in 4–8% of the seeds from transformed plants (Guan et al. 2011). The function of GhRDL1 is unknown, but it is highly expressed in fiber cells. GhMYB2 is encoding a MYB transcription factor. The odd outgrowths that we have observed on seeds from LTPG mutants resemble the A. thaliana seed hairs obtained by Guan et al. (2011). Further analysis of the lipid composition in the seed coat revealed a reduction in ω-hydroxylated fatty acids, suggesting a reduced suberization in the seed coat of these mutants. Seemingly, the alterations in salt permeability and seed coat morphology in the ltpg -lines can be explained by a deficiency in the suberin synthesis and accumulation. The elongation of seed hairs in A. thaliana results from the elongation of columella cells, which consists of cell wall materials (Guan et al. 2011). In the wild-type seeds, the columella cell elongation would normally be blocked by the rigid seed coat. However, the reduced levels of suberin in the ltpg seed coat may allow for the elongation of the columella cells to form the seed hairs. To our knowledge, we have in this study presented the first experimental results that suggest a role for nsLTPs in suberin biosynthesis. The ω-hydroxylated fatty acid monomers of suberin are synthesized from fatty acids by cytochrome P450s. The hydroxylation process ¨ probably takes place in the ER (Hofer et al. 2008). The polymer precursors should then be secreted through the plasma membrane. The channeling of the polymer precursors through the plasma membrane in A. thaliana

A

B

C

Fig. 5. The lipid composition of the seed coat suberin layer in the T-DNA insertion lines ltpg4 and ltpg6 and wild type. Error bars denote 95% confidence intervals. (A) All investigated lipids, those of the same type grouped together. (B) The unsubstituted fatty acid monomers. (C) The 1,ω-dicarboxylic fatty acids, ω-hydroxy fatty acids, alcohols and some members from the group ‘other’ (ferulate, sinapate and squalene). Unidentified substances are not shown.

is probably mediated by ABC transporters, such as ABCG12/CER5 and ABCG11/WBC11/DSO /COF1. The extracellular localization of nsLTPs suggests that they could be involved in the transport of the polyester compounds through the apoplast or cell wall. The GPIanchored LTPGs are likely attached to the apoplastic side of the plasma membrane. On the apoplastic side, the LTPGs could receive the suberin components from the plasma membrane-bound ABC transporters and then deliver the lipid to the suberization sites, such as the Casparian strips in the root endodermis or the seed coat. Thus, the LTPGs could form a necessary link connecting the plasma membrane with the lipid polyester synthesis sites. Similar to what we have found for several ltpg lines, ugt80A2- and ugt80B1-lines have an inability to limit tetrazolium salt uptake, a drastic decrease in

aliphatic suberin and cutin-like polymers and produce seeds with an altered morphology (DeBolt et al. 2009). UGT80A2 and UGT80B1 encode uracil-diphosphate (UDP)-glucose:sterol glucosyltransferase. This enzyme is involved in the synthesis of steryl glucosides that together with acyl steryl glucosides are abundant constituents of the membranes of higher plants. DeBolt et al. (2009) proposed that steryl glucosides and acyl steryl glucosides are necessary membrane components required for efficient lipid polyester precursor trafficking or export into the apoplast in plant seeds. The phenotypical relationship between ugt - and ltpg -lines lends support to our suggestion that LTPGs are involved in the trafficking of lipid polyester precursors. SEM revealed that ltpg3-1, ltpg4-1 and ltpg4-2 had morphologically defective pollen grains. Similar phenotypes have also been observed for several other mutants. One example is the mutant line with a T-DNA insertion in the gene encoding A. thaliana UDP-sugar pyrophosphorylase (AtUSP) (Schnurr et al. 2006). These pollen grains were non-viable, thus no homozygotes were found. The collapsed appearance was shown to be because of the absence of an intine, the inner wall of a pollen grain. The intine is rich in cellulose and pectin, but not lipids. It is therefore unlikely that this is the cause of the collapsed pollen grains in ltpg3-1, ltpg4-1 and ltpg4-2. Another mutation showing collapsed pollen grains in A. thaliana is that of glycerol-3-phosphate acyltransferase 6 (GPAT6), a gene involved in glycerolipid biosynthesis (Gimeno and Cao 2008, Li et al. 2012). The T-DNA insertion line gpat6 has decreased fertility, flawed exine and defective tapetum, in addition to the collapsed pollen grains. GPAT6 has previously been shown to play a role in cutin synthesis, indicating a connection between this gene and the nsLTPs, where GPAT6 is involved in the synthesis and the nsLTPs in the subsequent transport (Pollard et al. 2008). The synthesis of sporopollenin requires the delivery of the sporopollenin precursors from tapetum cells to the microspores. It was previously suggested that nsLTPs are involved in this translocation of the sporopollenin precursors and the first evidence for this was recently presented (Huang et al. 2013). Huang et al. showed that Type III/C nsLTPs were located on the pollen exine and that they were affecting the pollen intine and the pollen susceptibility to dehydration damage. However, they did not find any visual changes of the pollen exine or structure. Our observation of altered pollen morphologies in ltpg lines is the first experimental evidence that supports an involvement of nsLTPs in exine formation in A. thaliana. Physiol. Plant. 2014

A

B

C

D

Fig. 6. SEM pictures of Arabidopsis thaliana pollen grains. In comparison to wt (A), the pollen grains from ltpg3-1 (B), ltpg4-1 (C) and ltpg4-2 (D) show a deformed and collapsed phenotype.

In two of the ltpg -lines with increased seed coat permeability, we could detect mRNA expressed from the genes with T-DNA insertions. In both cases, the expressed transcripts lack the codons that translate into a GPI-anchor signal sequence. It seems that the anchor is crucial for the function of the proteins and possibly required for normal suberin accumulation in seed coats. We have previously showed that alternative splicing occurs within some LTPG -transcripts, where the GPIanchor signal is lost in one of the putative isoforms of the protein (Edstam et al. 2013). Our data presented here indicate that there are different functions of anchored and anchorless LTPG isoforms. It was interesting to analyze our investigations of the insertion lines in the context of our previous co-expression profiling (Edstam et al. 2013). There we placed LTPG1, LTPG2 and LTPG6 in the co-expression module AtI with an involvement in cuticle development. As mentioned above, a connection to cuticle biosynthesis has already been experimentally verified for LTPG1 and LTPG2. In this study, we expected that also ltpg6-1 and ltpg6-2 would show phenotypes related to defects in the cuticle. However, this was not found in our assays. As we had previously observed that the AtI-genes were abundantly expressed in most tissues above ground, it was relevant to test AtI-insertion lines also for aberrant seed phenotypes. Our results for ltpg6-1 and ltpg6-2 Physiol. Plant. 2014

demonstrate that AtI-genes are involved not only in cuticle development but also play a role in the seed coat suberization. It is important to note that the cuticle composition was never investigated here. Mainly because of that the obtained phenotypes turned our focus to seed coats and pollen at an early stage of the study. Hence, further experiments are needed to clarify the role of LTPG6 in cuticle development. Seven LTPGs had been placed in the module AtII with connections to suberin synthesis. We obtained insertion lines for four of these genes, but only one (ltpg5-1) was confirmed as a knockdown and thus used in the investigations. As expected, ltpg5-1 showed increased seed coat permeability, but the difference was not among the largest and hence it was not selected for suberin analysis. Unlike the other LTPGs, the genes in AtII show a strong expression in roots, and it will be of great interest to perform detailed investigations of root development and to measure the root suberin content of ltpg5-1. Both LTPG3 and LTPG4, together with LTPG23 and LTPG26, were placed in the co-expression module AtIII related to sporopollenin. The present results strengthen the interpreted function in sporopollenin synthesis for these genes. Unfortunately, no insertion lines for LTPG23 and LTP26 were available and their involvement in this process could not be examined. It was more unexpected that ltpg4-1 and ltpg4-2 would also show phenotypes indicating deficient

seed coats. Seemingly, the function of the AtIII-genes is not restricted to pollen development. Rather, the data suggest that there are functional overlaps between the co-expression modules AtI, AtII and AtIII. To summarize, we have in this study provided experimental support for the hypothesis that nsLTPs are required for the biosynthesis of the lipid polymers suberin and sporopollenin. A role in the apoplastic transport of polymer precursor seems likely for the nsLTPs. Further investigations are required to conclusively pinpoint the exact function of these proteins in the process of polymer synthesis.

Authors’ contributions M. M. E. designed, performed, analyzed and wrote research. J. E. designed, analyzed and wrote research. Acknowledgements – The authors thank Dylan Kosma for kindly providing the protocol for suberin analysis. The authors are also grateful to Elke Schweda and Johan Dahl´en for assistance with the GC–MS, and thank Kristina Blomqvist for comments on the manuscript. This work ¨ was financially supported by Carl Tryggers Stiftelse for Vetenskaplig Forskning.

References Alexander MP (1969) Differential staining of aborted and nonaborted pollen. Stain Technol 44: 117–122 Ariizumi T, Toriyama K (2011) Genetic regulation of sporopollenin synthesis and pollen exine development. Annu Rev Plant Biol 62: 437–460 Bird D, Beisson F, Brigham A, Shin J, Greer S, Jetter R, Kunst L, Wu X, Yephremov A, Samuels L (2007) Characterization of Arabidopsis ABCG11/WBC11, an ATP binding cassette (ABC) transporter that is required for cuticular lipid secretion. Plant J 52: 485–498 Boutrot F, Chantret N, Gautier MF (2008) Genome-wide analysis of the rice and Arabidopsis non-specific lipid transfer protein (nsLtp) gene families and identification of wheat nsLtp genes by EST data mining. BMC Genomics 9: 86 Charvolin D, Douliez JP, Marion D, Cohen-Addad C, Pebay-Peyroula E (1999) The crystal structure of a wheat nonspecific lipid transfer protein (ns-LTP1) complexed with two molecules of phospholipid at 2.1 A resolution. Eur J Biochem 264: 562–568 Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139: 5–17 Debeaujon I, Leon-Kloosterziel KM, Koornneef M (2000) Influence of the testa on seed dormancy, germination, and longevity in Arabidopsis. Plant Physiol 122: 403–414

DeBolt S, Scheible WR, Schrick K, Auer M, Beisson F, Bischoff V, Bouvier-Nave P, Carroll A, Hematy K, Li Y, Milne J, Nair M, Schaller H, Zemla M, Somerville C (2009) Mutations in UDP-glucose:sterol glucosyl transferase in Arabidopsis cause transparent testa phenotype and suberization defect in seeds. Plant Physiol 151: 78–87 Debono A, Yeats TH, Rose JK, Bird D, Jetter R, Kunst L, Samuels L (2009) Arabidopsis LTPG is a glycosylphosphatidylinositol-anchored lipid transfer protein required for export of lipids to the plant surface. Plant Cell 21: 1230–1238 Edstam MM, Viitanen L, Salminen TA, Edqvist J (2011) Evolutionary history of the non-specific lipid transfer proteins. Mol Plant 4: 947–964 ¨ A, Wennergren U, Edqvist Edstam MM, Blomqvist K, Eklof J (2013) Coexpression patterns indicate that GPI-anchored non-specific lipid transfer proteins are involved in accumulation of cuticular wax, suberin and sporopollenin. Plant Mol Biol 83: 625–649 Gimeno RE, Cao J (2008) Thematic review series: glycerolipids. Mammalian glycerol-3-phosphate acyltransferases: new genes for an old activity. J Lipid Res 49: 2079–2088 Guan X, Lee JJ, Pang M, Shi X, Stelly DM, Chen ZJ (2011) Activation of Arabidopsis seed hair development by cotton fiber-related genes. PLoS One 6: 21301 ¨ Hofer R, Briesen I, Beck M, Pinot F, Schreiber L, Franke R (2008) The Arabidopsis cytochrome P450 CYP86A1 encodes a fatty acid omega-hydroxylase involved in suberin monomer biosynthesis. J Exp Bot 59: 2347–2360 Huang MD, Chen TL, Huang AH (2013) Abundant type III lipid transfer proteins in Arabidopsis tapetum are secreted to the locule and become a constituent of the pollen exine. Plant Physiol 163: 1218–1229 Javelle M, Vernoud V, Rogowsky PM, Ingram GC (2011) Epidermis: the formation and functions of a fundamental plant tissue. New Phytol 189: 17–39 Kim H, Lee SB, Kim HJ, Min MK, Hwang I, Suh MC (2012) Characterization of glycosylphosphatidylinositolanchored lipid transfer protein 2 (LTPG2) and overlapping function between LTPG/LTPG1 and LTPG2 in cuticular wax export or accumulation in Arabidopsis thaliana. Plant Cell Physiol 53: 1391–1403 Lee SB, Go YS, Bae HJ, Park JH, Cho SH, Cho HJ, Lee DS, Park OK, Hwang I, Suh MC (2009) Disruption of glycosylphosphatidylinositol-anchored lipid transfer protein gene altered cuticular lipid composition, increased plastoglobules, and enhanced susceptibility to infection by the fungal pathogen Alternaria brassicicola. Plant Physiol 150: 42–54 Li XC, Zhu J, Yang J, Zhang GR, Xing WF, Zhang S, Yang ZN (2012) Glycerol-3-phosphate acyltransferase 6 (GPAT6) is important for tapetum development in

Physiol. Plant. 2014

Arabidopsis and plays multiple roles in plant fertility. Mol Plant 5: 131–142 Lolle SJ, Berlyn GP, Engstrom EM, Krolikowski KA, Reiter WD, Pruitt RE (1997) Developmental regulation of cell interactions in the Arabidopsis fiddlehead-1 mutant: a role for the epidermal cell wall and cuticle. Dev Biol 189: 311–321 Ma DP, Liu HC, Tan H, Creech RG, Jenkins JN, Chang YF (1997) Cloning and characterization of a cotton lipid transfer protein gene specifically expressed in fiber cells. Biochim Biophys Acta 1344: 111–114 Molina I, Bonaventure G, Ohlrogge J, Pollard M (2006) The lipid polyester composition of Arabidopsis thaliana and Brassica napus seeds. Phytochemistry 67: 2597–2610 Orford SJ, Timmis JN (2000) Expression of a lipid transfer protein gene family during cotton fiber development. Biochim Biophys Acta 1483: 275–284 Panikashvili D, Savaldi-Goldstein S, Mandel T, Yifhar T, Franke RB, Hofer R, Schreiber L, Chory J, Aharoni A (2007) The Arabidopsis DESPERADO/AtWBC11 transporter is required for cutin and wax secretion. Plant Physiol 145: 1345–1360 Pighin JA, Zheng H, Balakshin LJ, Goodman IP, Western TL, Jetter R, Kunst L, Samuels AL (2004) Plant cuticular lipid export requires an ABC transporter. Science 306: 702–704 Pollard M, Beisson F, Li Y, Ohlrogge JB (2008) Building lipid barriers: biosynthesis of cutin and suberin. Trends Plant Sci 13: 236–246 Preuss D, Rhee SY, Davis RW (1994) Tetrad analysis possible in Arabidopsis with mutation of the QUARTET (QRT) genes. Science 264: 1458–1460 Ranathunge K, Schreiber L, Franke R (2011) Suberin research in the genomics era – new interest for an old polymer. Plant Sci 180: 399–413

Edited by H.-T. Cho

Physiol. Plant. 2014

Schnurr J, Shockey J, Browse J (2004) The acyl-CoA synthetase encoded by LACS2 is essential for normal cuticle development in Arabidopsis. Plant Cell 16: 629–642 Schnurr JA, Storey KK, Jung HJ, Somers DA, Gronwald JW (2006) UDP-sugar pyrophosphorylase is essential for pollen development in Arabidopsis. Planta 224: 520–532 Sterk P, Booij H, Schellekens GA, Van Kammen A, De Vries SC (1991) Cell-specific expression of the carrot EP2 lipid transfer protein gene. Plant Cell 3: 907–921 Tanaka T, Tanaka H, Machida C, Watanabe M, Machida Y (2004) A new method for rapid visualization of defects in leaf cuticle reveals five intrinsic patterns of surface defects in Arabidopsis. Plant J 37: 139–146 Western TL, Skinner DJ, Haughn GW (2000) Differentiation of mucilage secretory cells of the Arabidopsis seed coat. Plant Physiol 122: 345–356 Wu Y, Machado AC, White RG, Llewellyn DJ, Dennis ES (2006) Expression profiling identifies genes expressed early during lint fiber initiation in cotton. Plant Cell Physiol 47: 107–127

Supporting Information Additional Supporting Information may be found in the online version of this article: Appendix S1. T-DNA insertion lines used in this study. Appendix S2. Synthetic oligonucleotides used in this study.