An ATP-binding cassette subfamily G full transporter is essential for the retention of leaf water in both wild barley and rice

Abstract

Land plants have developed a cuticle preventing uncontrolled water loss. Here we report that an ATP-binding cassette (ABC) subfamily G (ABCG) full transporter is required for leaf water conservation in both wild barley and rice. A spontaneous mutation, eibi1.b, in wild barley has a low capacity to retain leaf water, a phenotype associated with reduced cutin deposition and a thin cuticle. Map-based cloning revealed that Eibi1 encodes an HvABCG31 full transporter. The gene was highly expressed in the elongation zone of a growing leaf (the site of cutin synthesis), and its gene product also was localized in developing, but not in mature tissue. A de novo wild barley mutant named “eibi1.c,” along with two transposon insertion lines of rice mutated in the ortholog of HvABCG31 also were unable to restrict water loss from detached leaves. HvABCG31 is hypothesized to function as a transporter involved in cutin formation. Homologs of HvABCG31 were found in green algae, moss, and lycopods, indicating that this full transporter is highly conserved in the evolution of land plants.

Key physiological and structural innovations were required for plants’ transition from an aqueous to a terrestrial condition. The plant cuticle is an important innovation that plants evolved 450 million years ago during the transition to the land (1). The aerial surface of land plants is protected by the cuticle, a multilayered structure composed of polyester, cutin, and wax. In most species aliphatic cutin monomers are C16 or C18 ω-hydroxylated fatty acids, modified in midchain by additional hydroxy or epoxy groups. In addition, cutin contains glycerol and low amounts of phenolic compounds (2). The cutin matrix is formed by the polymerization of cutin intermediates (3, 4) and is embedded in and covered by wax formed primarily from a mixture of straight-chain C20 to C34 aliphatics (5). Hitherto, at least 47 genes participating in the formation of plant cuticle have been cloned (6). Most are from Arabidopsis thaliana and other dicot plants; a few are from monocot rice and maize (7, 8). Cuticle genes have not been reported as being identified in mutants from wheat and barley, which are important crops.

Members of the diverse and ubiquitous family of ATP-binding cassette (ABC) proteins are involved in the transmembrane transport of various molecules (9–12). ABC subfamily G (ABCG) includes both white-brown complex (WBC) half transporters and pleiotropic drug resistance (PDR) full transporters (13). ABCG full transporters are thought to be specific to plants and fungi, where they participate in defense against pathogens and in resistance to cadmium, antimicrobial terpenoids, and auxinic compounds (14–16). The transport of wax from the epidermal cells of A. thaliana across their plasma membrane is mediated by a pair of ABC half transporters, ABCG11/ABCG12 (11, 17–20), and ABCG11 homodimers have been proposed to be responsible for the export of cutin (3, 21, 22). No ABC full transporter is known to be involved in the formation of cutin.

The naturally occurring drought-hypersensitive wild barley (Hordeum spontaneum Koch) mutant eibi1.b (23) suffers from a particularly severe level of water loss and displays a defective cuticle. This mutant allele also has a pleiotropic effect on the appearance of the leaves, plant stature, fertility, and spike and grain size, and rate of germination (23). The gene has been mapped to the pericentromeric region of chromosome 3H (24) within a 0.11-cM interval defined by the markers BI958842 and Os01g0176800*. The corresponding region on rice chromosome 1 has a physical length of 112.8 kb and contains 16 genes (25). Here, we show that the excessive water loss characteristic of the eibi1.b mutant is associated with a reduced cuticle thickness and a reduced amount of cutin polyester and that the critical mutation lies within an ABCG gene sequence. The conclusions were validated by de novo mutagenesis in both wild barley and rice.

Results

Water Loss in the eibi1 Mutant Leaf Is Related to Cutin Disorganization.

Earlier results suggested that the eibi1.b mutant (Fig. 1A, right plant) has a defective cuticle (23) and therefore cannot retain leaf water in detached leaves as effectively as the wild type can (Fig. 1B). To investigate cuticle structure and composition further, young third leaves of both the mutant and wild-type seedlings were divided into three zones (Fig. 1A), as defined by Richardson et al. (26): the emerged blade (EmBL) lies above the point of emergence, and the nonelongation zone (NEZ) lies below it. The elongation zone (EZ) lies above the point of leaf insertion. Transmission electron microscopy showed that the thickness of the mutant's cuticle was only ∼25% of that of the wild type in all three zones (Fig. 1C). In addition, the quantity of the major cutin monomers (26) of wild barley was reduced to ∼50% in the eibi1.b mutant, but there was little difference between the eibi1.b mutant and wild type in the major wax component, 1-hexacosanol (Fig. 1D). Protrusions of cytoplasm into the vacuole were a feature of the mutant EZ epidermis cells but did not occur in the wild-type epidermis (Fig. 1E) or in the NEZ or EmBL of either the mutant or the wild type. Similar protrusions have been noted in stem epidermis cells of A. thaliana atabcg11 and atabcg12 mutants (11, 18–21). Both of these mutants are unable to export cuticular lipids from the epidermis cells, leading to an accumulation of intracellular lipids which are responsible for the formation of protrusions. On this basis, we hypothesized that Eibi1 may be an ABC transporter gene that is specifically involved in the formation of cutin.

Fig. 1.

The drought sensitivity of the eibi1.b wild barley mutant and its cuticle structure. (A) Wild-type (Left) and eibi1.b (Right) seedlings. The leaf comprises the three regions: the EmBL, the NEZ below the point of emergence (POE), and the EZ above the point of leaf insertion (POLI). (B) Water loss from detached leaves of eibi1 and wild-type plants. (C) Electron micrographs of the cuticle in EmBL, NEZ, and EZ of wild-type plants and the eibi1.b mutant. Arrows indicate the cuticle thickness. (D) Major cutin monomers and wax components in the EmBL of the eibi1.b mutant and wild type. All bars represent mean ± SD (n = 5). Asterisks denote significant differences (P < 0.0001) between wild type and mutant as determined by student's t tests. (E) Epidermis cells with (eibi1.b) or without (wild type) cytoplasmic protrusions into the vacuole. c, cytoplasm; p, protrusions; v, vacuole.

Eibi1 Encodes an ABCG Full Transporter.

The updated map location of Eibi1 (Fig. 2A) reduced the critical genetic interval from 0.11 cM to 0.10 cM, and from 112.8 kb (16 genes) to 76.5 kb (10 genes) in rice. One of these 10 genes encoded an OsABCG31 transporter and so was taken as the candidate for Eibi1. The sequence of the wild-type and mutant alleles revealed a single-nucleotide difference in exon 14, predicted to alter a tryptophan codon into a TAG stop codon (Fig. 2B) and thereby inducing a probable loss of function. The full-length wild-type allele sequence consisted of 11,693 bp arranged into 24 exons, producing a transcript length of 4,799 bp (Fig. 2B) with an ORF of 4,293 bp (1,430 residues) (Fig. S1). The predicted protein sequence of the wild-type allele was highly similar to those of rice (Oryza sativa) OsABCG31/OsPDR6 (an ABCG full transporter) and A. thaliana AtABCG32/AtPDR4 (an ABCG full transporter) (www.ncbi.nlm.nih.gov/Structure and Fig. S2). The Eibi1 gene was named “HvABCG31/HvPDR6” by analogy with the rice gene.

Fig. 2.

Positional cloning of eibi1 and its functional assignment. (A) The Eibi1 locus maps to barley chromosome 3H and its ortholog to rice chromosome 1. Numbers to the left of the barley map indicate the number of observed recombination events in a population of 9,070 gametes. Rice and barley orthologs are connected by dashed lines. (B) Exon/intron structure of barley Eibi1. The single-nucleotide difference between the wild-type and the eibi1.b mutant sequence is indicated. The red dashed line indicates the 9-bp deletion in eibi1.c exon 10. Untranslated regions are indicated by an empty box at each end. (C) The γ irradiation-induced eibi1.c mutant and its wild type at the flowering stage. (D) Water-loss test of detached leaves from eibi1.c and OUH602. (E) Exon/intron structure of rice OsABCG31 indicating Tos17 insertions (arrows). (F) Wild-type (one tall seedling) and osabcg31.b mutants (four dwarf seedlings) at the three-leaf stage. (G) Enlarged view of dwarf seedlings in F. (H) Water loss in the detached leaf of the wild type and the mutant. (I) Mutant and wild-type leaves of barley air dried for 1 h and rice air dried for 0.5 h. (J) Toluidine blue staining of leaf segments of mutants and wild types. (Scale bar: 20 mm for wild barley; 5 mm for rice.)

Both forward and reverse genetic approaches then were used to validate the function of Eibi1 in both wild barley and rice. The wild barley accession OUH602 was mutagenized with γ irradiation over its entire life cycle, and M₃ seedlings (pooled progeny of individual M₂ plants) were tested for their capacity to retain leaf water in detached leaves. Four of a set of 28,466 tested M₃ seedlings mimicked the behavior of the eibi1.b mutant (Fig. 2 C, D, I, and J). The Eibi1 sequence present in all four seedlings contained the same deletion of nine nucleotides from exon 10 causing three amino acids missing. This allele has been named “eibi1.c” (Fig. 2B). The reverse genetic approach was applied to rice, taking advantage of a library of established transposon insertions. Two independent transposon Oryza sativa 17 (Tos17) events within OsABCG31 (osabcg31.b and osabcg31.c) were identified (Fig. 2 E–G), and the association between genotype and leaf water retention was tested. The two mutants were both highly sensitive to drought stress (as represented by osabcg31.b in Fig. 2 H and I), and their leaves, along with those of the two wild barley eibi1 mutants, were readily stained by toluidine blue (Fig. 2J), similar to Arabidopsis plants having defective cuticle (27, 28). Characterization of both barley and rice alleles indicated that the ABCG31 gene encoding a full ABCG transporter is involved in the formation of a functional cuticle.

Both Eibi1 Transcript and Product Are Present in the Leaf EZ.

The analysis of Eibi1 expression showed the presence of abundant transcripts in the EZ of the seedling third leaf but only traces in the NEZ and EmBL and none in mature root tissue (Fig. 3 A and B). The gene also was expressed in the developing spike, stem internodes, and nodes and in the germinating plumule and radicle (Fig. S3). In situ hybridization experiments confirmed that the gene product was present in the young leaf but not in the coleoptile (Fig. 3 C and D). The EIBI1 protein was detectable in the EZ of the young leaf but not in the mature leaf in both barley (Fig. 3E) and rice (Fig. 3G). Within the EZ, the protein accumulated not only in the epidermal cells but also in other cell types (Fig. 3 F and H). This pattern of transcription and translation supported our hypothesis that the gene has a function in cutin formation in the epidermal cell layer but suggested additional functions in other cell types also.

Fig. 3.

Eibi1 gene expression and EIBI1 immunolocalization. The Eibi1 expression pattern as revealed by (A) RT-PCR and by (B) quantitative RT-PCR; data shown are means ± SD of three biological replicates. (C) Longitudinal section and (D) cross-section of the young shoot demonstrating gene expression of Eibi1 in the developing leaves of wild barley 23-19, as detected by in situ hybridization with an antisense (Upper image) and sense (Lower image) probe. c, coleoptile; l, leaf. (E–H) Immunofluorescent localization of EIBI1 in cross-sections taken 5–15 mm above the root–shoot junction in wild barley 23-19 (E and F) and rice 'Nipponbare' (G and H) seedlings at the three-leaf stage. The fluorescence signal is shown in red. F and H are enlargements of E and G, respectively. e, epidermis cell; m, mature leaf; y, young leaf.

EIBI1 Is Conserved During the Evolution of Land Plants.

Homologs of EIBI1 were found in rice (OsABCG31), Arabidopsis (AtABCG32), Selaginella moellendorffii (Smo 412699), Physcomitrella patens (Pp1s375_5V6.1), and Volvox carteri (Vca 40167) (Table 1). The monocot rice and the dicot Arabidopsis shared 92% and 71% sequence identities with EIBI1, respectively, indicating the conservation of EIBI1 in monocot and dicot plants. The lycopod S. moellendorffii and the moss P. patens shared 59% and 56% sequence identity with EIBI1, respectively, suggesting the conservation of EIBI1 homologs in the conquest of land by plants. The green alga V. carteri also contains an EIBI1 homolog, but the sequence identity is lower, at 35%.

Table 1.

Homologs of HvABCG31

Homolog	Score	E-value	Identity (%)
OsABCG31	2,654	0.0	92 (1309/1430)
AtABCG32	2,114	0.0	71 (1022/1434)
Smo 412699	1,771	0.0	59 (845/1439)
Pp1s375_5V6.1	1,676	0.0	56 (829/1489)
Vca 40167	687	0.0	35 (400/1131)

Homologs of HvABCG31 were found in O. sativa (OsABCG31), A. thaliana (AtABCG32), S. moellendorffii (Smo 412699), P. patens (Pp1s375_5V6.1), and V. carteri (Vca 40167).

Discussion

A combination of positional cloning and forward and reverse genetics has demonstrated that an ABCG full transporter, ABCG31, is required for functional cuticle formation in both barley and rice. The EIBI1 protein appears to be responsible for cuticle formation in barley, because the eibi1.b mutant's leaves form a defective cuticle (23). In the wild-type leaf, the EZ is almost free of cuticular wax, but the EmBL surface is heavily waxed (Fig. S4), showing that wax is deposited gradually as the leaf develops. In contrast, cutin deposition already has been established by the time the EZ is formed (Fig. S5). Richardson et al. (29) have shown similarly that cutin deposition in the epidermis of the barley leaf EZ occurs during the period of cell elongation, whereas wax deposition commences near the distal end of EZ and continues at a more-or-less constant rate into the EmBL. The cuticle of the eibi1.b leaf EZ was characterized by its rather thin structure. This defect was first apparent in the EZ but remained visible in both the NEZ and EmBL (Fig. 1C). The reduced cuticle thickness correlated well with the reduced amount of cutin in the EmBL. The EIBI1 protein was detected exclusively in the EZ (Fig. 3 E and G) where the cutin matrix was formed. These lines of evidence indicated that a likely role for EIBI1 is in cutin matrix formation.

Many mutants that have disorganized cutin and display increased cuticle permeability have been characterized in Arabidopsis. For example, the A. thaliana bodyguard (bdg) mutant readily leaches chlorophyll (30). BDG encodes a member of the α/β-hydrolase fold protein superfamily and is particularly highly expressed in younger tissue. The Arabidopsis defective in cuticle ridges (dcr) mutant, which carries an acyltransferase, is particularly susceptible to salinity and osmotic and drought stress (31). The wild-type DCR protein is proposed to be involved in cutin polymerization during the early stages of leaf expansion (31, 32). In the Arabidopsis aberrant induction of type three genes 1 (att1) mutant, the cuticle is loosely organized, thereby increasing the transpiration rate (33). ATT1 encodes a cytochrome P450 mono-oxygenase, catalyzing fatty acid oxidation for the biosynthesis of extracuticular lipids. The knockout of long-chain acyl-CoA synthetase 2 function that is essential for cutin synthesis results in a cuticle that is highly permeable to solutes and water (28, 34). All these genes involved in cutin formation are expressed in young tissues where the cutin is synthesized. These results as well as the present results demonstrate that a functional cuticle is required for water retention.

Two half transporters involved in cutin secretion, ABCG11 and ABCG13, have been identified in Arabidopsis. abcg11 readily leaches chlorophyll (18) and reacts strongly to toluidine blue staining (19). The wild-type allele ABCG11/WBC11 is expressed in the cotyledons as well as in seedling root tips and young leaves (18, 19). Abcg13 cuticle-related phenotypes, including interorgan postgenital fusions and a significant reduction in flower cutin monomers, were restricted to flowers. ABCG13 is highly expressed in flowers (35). The function of HvABCG31 is more similar to that of AtABCG11 than to that of AtABCG13. AtABCG11 is involved in both wax deposition and cutin loading (17–20), whereas HvABCG31 is related to cutin formation but not wax deposition. The effect of mutation on cuticle permeability is more pronounced in hvabcg31 than in atabcg11, as indicated by the ratio of chlorophyll extraction from mutant to that from wild type after 1 h incubation in 80% ethanol: 5.2 for hvabcg31 and 2.6 for atabcg11 (23, 18). Therefore, HvABCG31 is unique for cutin secretion.

A few mutants that have disorganized cutin have been characterized in the monocot. The Sorghum bicolor brown midrib2 (bm2; previously “bm22”) mutants show a reduced cuticle thickness and increased water loss (36, 37). However, the BM2 gene has not been isolated. The maize glabrous1 (gl1) mutations affect cuticular wax biosynthesis and have a pleiotropic effect on epidermis development, altering trichome size and impairing cutin structure (38). In the gl1 mutant, cuticle thickness is reduced by about 50%, and the cuticle proper is almost absent. However, the cuticle permeability to chlorophyll is not affected by the gl1 mutations. GL1 expression is evident in juvenile and adult leaves and in floral organs. The predicted GL1 protein includes three histidine-rich domains, the landmark of a family of membrane-bound desaturases/hydroxylases, including fatty acid-modifying enzymes, but the precise biological function of GL1 remains poorly characterized. The rice wilted dwarf and lethal 1 (wdl1) mutant is a dwarf and dies at the seedling stage because of increased rates of water loss (39). WDL1 encodes a lipase. The increased water loss from wdl1 mutant leaves is associated with loose packing of the cuticle and irregular thickness of the cell wall. However, no significant changes are found either in the total amount of each monomer or in the levels of lipid polymers, including cutin and other covalently bound lipids. The biological function of WDL1 remains unknown. In contrast, evidence has been presented for the association of the HvABCG31 full transporter with water retention in the leaf of wild barley and rice and for a likely role of HvABCG31 in cutin matrix formation.

The deposition of cutin and wax may be under independent control in monocots during leaf emergence. EIBI1 functions in the formation of a functional cutin matrix rather than in wax deposition (Fig. 1D). It is possible that EIBI1 has a direct role in the secretion of monomers for the cutin matrix; however, an indirect function in the formation of the cuticle cannot be excluded. In the A. thaliana eceriferum (cer) 3/wax2/yore-yore/faceless pollen-1 and cer1-1 mutants, wax deposition is defective, and the plants suffer a high rate of water loss, but neither cutin loading nor its composition are compromised (40–42). Similarly, in the cer5 mutant (CER5 is the half transporter ABCG12), the plants produce significantly lower levels of all wax constituents, and thin linear inclusions are formed in stem epidermis cells (11).

EIBI1 and its rice ortholog OsABCG31 may play additional roles in plant development. Loss-of-function mutants in both wild barley (eibi1.b and eibi1.c) and rice (osabcg31.b and osabcg31.c) are less able to retain leaf water than their respective wild types. However, shoot development was compromised much more heavily in the rice mutants than in wild barley mutants. The rice mutants attained a plant height of just 3–8 cm and did not develop beyond the four- to five-leaf stage; whereas the two barley mutants attained a height of about two-thirds that of the wild type and were able to complete their life cycle through to maturity. Further study of the function of OsABCG31 is necessary to understand the seedling lethality of osabcg31.b and osabcg31.c mutants. An example of orthologous mutants showing different phenotypes is presented by ONION1 (ONI1) in rice, a gene that encodes a fatty acid elongase; in addition to its effect on long-chain fatty acid synthesis, it is required for normal shoot development (43). The oni1 mutant, like its A. thaliana equivalent fiddlehead (fdh), forms organ fusions and is specifically expressed in the outermost cell layer of the shoot apical meristem. However, unlike fdh, oni1 is seedling lethal. The functional difference between these two related genes has been suggested to indicate an additional role for very long-chain fatty acids in rice development or a lack of partial redundancy in rice.

In addition to its major site of expression in the developing leaf, the Eibi1 transcript or translation product was present in other parts of the plant, including radicle (Fig. 3 F and H and Fig. S3), implying that cutin synthesis and water conservation are not the sole function of the EIBI1 protein. The function of Eibi1 in radicle is not clear. The AtABCG11 involved in cutin and wax secretion also is expressed in roots (17).

ABCG transporters have rather diverse functions. Spirodella polyrhiza SpTUR2 and rice OsPDR9 both play a role in the response to abiotic stress (44, 45). ABCG full transporter genes also have been identified as being involved in volatile compound production and rhizosphere signaling (46, 47). A. thaliana penetration 3/ABCG36 participates in pathogen defense, the removal of cadmium, the transport of an auxinic metabolite, root hair development, and cotyledon expansion (48). In bread wheat, a specific resistance against the foliar fungal pathogen leaf rust is encoded by leaf rust resistance (Lr) gene Lr34, a gene producing an ABCG full transporter. This gene is active in the adult plant, particularly at the tips and margins of the final leaf (49). In parallel to our studies on HvABCG31 in barley, its closest homolog in Arabidopsis, AtABCG32, has been studied by characterizing the Arabidopsis permeable cuticle 1 (pec1) mutant (50). Similar to our results, knockout mutations in AtABCG32 also led to a permeable cuticle, because the pec1 mutant exhibited rapid calcofluor white and toluidine blue staining of the cell wall underneath the cuticle, increased water loss from rosettes that were cut at their hypocotyls, and increased herbicide sensitivity. The amounts of oxygenated cutin monomers in pec1 flowers were reduced and coincided with ultrastructural changes in the cuticle and with the appearance of lipidic inclusions in epidermal cells in pec1 petals. Thus, AtABCG32 represents the true ortholog of HvABCG31, and its function in the formation of a functional cuticle, most likely by exporting cutin monomers from epidermal cells, is conserved among monocot and dicot plants. However, leaf permeability is much more increased in hvabcg31 and osabcg31 than in atabcg32, indicating the importance of ABCG31 in monocot leaf water retention.

The present study identifies the function of a monocot full-length ABCG in plant cutin formation. This work has important implications for the study of land plant evolution. Indeed, HvABCG31 homologs were identified in the early land plants such as moss and lycopod and even in green algae V. carteri (Table 1). One may infer that V. carteri has a cutin-like layer, although experimental evidence is not available. Genes involved in the synthesis of cutin monomers, such as Cytochrome P450 (Cyp) 86, Cyp703, and Cyp704 are found in the P. patens genome (51). Pp1s375_5V6.1, the homolog of HvABCG31, may be required for the secretion of cutin monomers to form cutin matrix in P. patens. Lycopod, Arabidopsis, and rice are known to have cutin matrix whose formation may require the homologs of HvABCG31. It may be concluded that EIBI1 is highly conserved in the evolution of land plants.

In this study we present evidence that the HvABCG31 full transporter is associated with water retention in the leaf of wild barley and rice and probably has a role, perhaps by secreting cutin monomers, in the formation of cutin matrix in monocots. Although it is clear that the loss of function of HvABCG31 generates a defective cutin matrix, an important future experiment will be to assay directly the substrate specificity of HvABCG31. This work underlines the importance of an intact cuticle and its structural polyester in protecting leaves from uncontrolled water loss and the crucial evolution of HvABCG31 homologs in plant land colonization, a major milestone in life's terrestrial evolution.

Materials and Methods

Plant Materials.

Accession 23-19 of wild barley, the progenitor of the eibi1.b mutant (23), is maintained at the Institute of Evolution, University of Haifa. Grains of the commercial barley 'Morex' were provided by Andy Kleinhofs, Washington State University, Pullman, WA. The wild barley accession OUH602 was obtained from the Institute of Plant Science and Resources, Okayama University, Japan. The rice cultivar 'Nipponbare' with its Tos17 insertion mutants osabcg31.b and osabcg31.c was obtained from the National Institute of Agrobiological Sciences, Japan.

Leaf Water Loss Assay.

Caryopses of the eibi1.b mutant and wild-type 23-19 were sown in 2.5-L pots filled with commercial compost, and the plants were grown in a greenhouse at about 60% relative humidity, 25 °C/19 °C (day/night) under natural light. At the three-leaf stage (Fig. 1A), the developing third leaf was sampled for cuticle analysis and RNA extraction, and the second leaf was used for the leaf water loss assay. Both the wild-type ‘Nipponbare’ and the two Tos17-insertion mutants osabcg31.b and osabcg31.c (segregated from heterozygous plants) were grown in a growth chamber at 70% relative humidity with daily cycles of 13 h light (intensity 200 μmol m⁻² s⁻¹) at 30 °C and 11 h dark at 25 °C. The leaf water loss assay used an ∼4-cm-long fragment of the distal end of the leaf; these fragments first were allowed to dry at room temperature, abaxial side up, on tissue paper. The weight of each leaf fragment was measured over a time series. Leaf water loss was expressed as a proportion of the original fresh weight. The assay also was performed on the F₂ progeny of the cross 'Morex' × eibi1.b mutant. Seedlings from this population were grown on moistened filter paper until the first seedling leaf was fully expanded; then the distal 2 cm was removed for the assay, and an additional 1-cm fragment was harvested to obtain DNA. The drying assay was carried out on leaf fragments placed abaxial side up for 1 h on tissue paper at room temperature (23).

Transmission Electron Microscopy.

Leaf three was fixed in 2% (vol/vol) glutaraldehyde at room temperature for 2 h, followed by 1% (wt/vol) OsO₄ at 4 °C overnight. The samples then were dehydrated through an ethanol series and infiltrated with Spurr's standard epoxy resin. The embedded samples were sectioned (70 nm thick) and examined using a TEM-1230 (JEOL) device.

Analysis of Cutin and Wax Content.

The distal 6 cm (the EmBL region) of leaf three of the eibi1.b mutants and the wild-type plants was removed from the growing leaf three. A pool of 11 leaf segments per line was replicated five times. The area of the leaf material was measured from digital images, and the material then was immersed in chloroform and agitated for 20 s. The extract was dried for derivation and GC-MS/flame ionization detector (FID) analysis as previously described (52). After the wax extraction, the remaining leaf matter was delipidated, depolymerized, and derivatized with N,O-bis(trimethylsilyl) trifluoroacetamide to allow GC-MS/FID analysis as described previously (28).

Fine Mapping of Eibi1.

The locus was already known to lie in a 0.11-cM region of chromosome 3H, cosegregating with the EST loci BF626522, CJ579262, AV918546, and BJ480900 and flanked by CD902914 and BI958842 (24, 25). Further gene sequences in this region were obtained by assuming synteny with a segment of rice chromosome 1. A mapping population of 4,535 ‘Morex’ × eibi1.b F₂ individuals was used to delimit the eibi1 locus.

Sequence Polymorphism in Eibi1.

The 'Morex' allele of Eibi1 was obtained from the gene copy present on the BAC clone M280G24, selected from the full 'Morex' library (Clemson University Genomic Institute, Clemson, SC) on the basis of positive amplification with primers directed against the EST sequence CJ579262. The ‘Morex’ genomic contig containing Eibi1 was sequenced by primers walking on this clone. Both ends of the gene were determined by a full-length cDNA sequencing using a GeneRacer Kit (Invitrogen). Based on the ‘Morex’ Eibi1 sequence, primers were designed for sequencing of eibi1.b, eibi1.c, and wild types by PCR of overlapping fragments.

Gene Expression Analysis.

RNA was extracted, using the TRIzol reagent (Invitrogen), separately from the EZ, NEZ, and EmBL of young third leaves harvested from eibi1.b mutant and wild-type 23-19 plants at the three-leaf stage, as well as from the spike, peduncle, the first node, and the flag leaf when the plants had reached the heading stage, and from the plumule and radicle of the germinating grain. The first cDNA strand was synthesized using SuperScript II (Invitrogen). The 5′ and 3′ ends of the gene were identified by means of a GeneRacer kit (Invitrogen). RT-PCR and full-length cDNA sequencing were carried out using gene-specific primers (Table S1). Transcript levels of Eibi1 and Actin2 (the reference gene) were determined by quantitative real-time RT-PCR as described by Yamaji and Ma (53). This assay was performed in a 20-μL reaction volume containing 2 μL 1:5 diluted cDNA, 200 nM of each gene-specific primer, and SYBR Premix Ex Taq (Takara Bio) using an Applied Biosystems 7500 device.

De Novo eibi1 Alleles.

Wild barley OUH602 plants were exposed to γ irradiation at 0.47–0.92 Gy/d over their whole life cycle, by means of an 88.8 TBq ⁶⁰Co source at the Institute of Radiation Breeding, National Institute of Agrobiological Sciences, Japan. M₃ seedlings were used to identify novel eibi1 mutants by searching for the eibi1 mutant phenotype, quick dehydration of a detached leaf. The identified putative eibi1 mutants were sequenced for the full Eibi1 gene.

Rice Tos17 Insertion Mutants.

The rice ortholog of the putative barley Eibi1 sequence was identified by BLAST analysis. This sequence then was entered as a search query in the rice Tos17 insertion mutant database (tos.nias.affrc.go.jp).

Toluidine Blue Staining.

Segments of young third leaves of wild barley 23-19, the eib1.b and eib1.c mutants, 'Nipponbare,' and the rice mutants osabcg31.b and osabcg31.c were immersed for 30 min in 0.05% (wt/vol) aqueous toluidine blue (Solarbio) filtered through a fiber medium filter (pore diameter 0.2 mm; Sartorius Stedim Biotech). The leaf segments then were rinsed in water and photographed using a digital camera.

In Situ Hybridization.

A 200-bp DNA fragment comprising part of Eibi1 exon 2 and part of exon 3 was amplified from a cDNA template using primers AJ535049F852 and ABCcontig7F644 (Table S1) and was cloned into the pCR4-TOPO vector (Invitrogen). After digestion by EcoRI, the insert was subcloned into the pBluescript II KS(+) vector (Stratagene). NotI was used to release the insert, and T3 RNA polymerase was used to generate both an antisense probe and a sense probe. In situ hybridization was conducted following Komatsuda et al. (54).

Immunostaining of EIBI1 Protein.

The synthetic oligopeptide SGDAEQFFRRIRARFDAVH (positions 87–105 of EIBI1) was injected into rabbits to obtain an antibody recognizing EIBI1. The cross-section of the basal part of wild barley 23-19 and ‘Nipponbare’ seedlings at the three-leaf stage was challenged with a 1:300 diluted anti-EIBI1 antibody as described previously (53). The signal from the secondary antibody (Alexa Fluor 555 goat anti-rabbit IgG; Molecular Probes) was observed by fluorescence microscopy.

Alignment of the HvABCG31 Homologs.

The conserved domains (CD) search tool (www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) was used to obtain the HvABCG31 homologs in rice and Arabidopsis. The HvABCG31 protein sequence was used to search the proteomes of S. moellendorffii, P. patens, and V. carteri using the BLAST function available at the PHYTOZOME version 7.1 (http://www.phytozome.net/search.php). The closest match was selected as the homolog of HvABCG31 for each species. The alignment of the homologs was conducted with the multiple sequence alignment tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&BLAST_SPEC=blast2seq&LINK_LOC=blasttab&LAST_PAGE=blastn&BLAST_INIT=blast2seq).