Isolation and characterization of the gene HvFAR1 encoding acyl-CoA reductase from the cer-za.227 mutant of barley ( Hordeum vulgare ) and analysis of the cuticular barrier functions

(cid:1) The cuticle is a protective layer covering aerial plant organs. We studied the function of waxes for the establishment of the cuticular barrier in barley ( Hordeum vulgare ). The barley eceriferum mutants cer-za.227 and cer-ye.267 display reduced wax loads, but the genes affected, and the consequences of the wax changes for the barrier function remained unknown. (cid:1) Cuticular waxes and permeabilities were measured in cer-za.227 and cer-ye.267 . The mutant loci were isolated by bulked segregant RNA sequencing. New cer-za alleles were generated by genome editing. The CER-ZA protein was characterized after expression in yeast and Arabidopsis cer4-3 . (cid:1) Cer-za.227 carries a mutation in HORVU5Hr1G089230 encoding acyl-CoA reductase (FAR1). The cer-ye.267 mutation is located to HORVU4Hr1G063420 encoding b -ketoacyl-CoA synthase (KAS1) and is allelic to cer-zh.54 . The amounts of intracuticular waxes were strongly decreased in cer-ye.267 . The cuticular water loss and permeability of cer-za.227 were similar to wild-type (WT), but were increased in cer-ye.267. Removal of epicuticular waxes revealed that intracuticular, but not epicuticular waxes are required to regulate cuticular transpiration. (cid:1) The differential decrease in intracuticular waxes between cer-za.227 and cer-ye.267 , and the removal of epicuticular waxes indicate that the cuticular barrier function mostly depends on the presence of intracuticular waxes.


Introduction
The cuticle forms the apoplastic barrier between aerial plant organs and the environment (Nawrath, 2006).It represents the first boundary of the plant and enhances the resistance against water loss, UV light, and pathogen attack and establishes a selfcleaning surface (Koch et al., 2008;Lee & Suh, 2015).The protective capabilities of the cuticle depend on the presence of waxes which are embedded in (intracuticular) or overlaid onto (epicuticular) the cutin matrix forming crystalline structures on the surface (Nawrath, 2006;Koch et al., 2008).Cuticular waxes are a mixture of very-long-chain fatty acid (VLCFA)-derivatives obtained from plastidial fatty acid de novo biosynthesis, often containing triterpenoids or sterols (Bernard & Joub es, 2013;Batsale et al., 2021).Acyl-CoAs are transported to the endoplasmic reticulum (ER), where they are elongated by the fatty acid elongation (FAE) complex to give rise to VLCFA-CoAs.The FAE complex consists of four enzymes, b-ketoacyl-CoA synthase (KCS), b-ketoacyl-CoA reductase, b-hydroxyacyl-CoA dehydratase, and enoyl-CoA reductase.Different KCS subunits determine the chain length specificity of the FAE complexes (Millar & Kunst, 1997).VLCFA-CoAs are hydrolyzed by thioesterases to release free fatty acids, or they are reduced to aldehydes and decarbonylated to yield odd chain length alkanes, secondary alcohols, and ketones.In a third pathway, VLCFA is reduced to even chain length primary alcohols, which involves the formation of an aldehyde intermediate.Finally, waxes are exported from the plasma membrane, through the cell wall, and incorporated into the cuticle (Kunst & Samuels, 2009).
The identification of wax (eceriferum, cer) mutants was instrumental for the elucidation of the wax biosynthetic pathway.In Arabidopsis thaliana, 21 cer mutants were isolated with altered wax loads or compositions on the stems (Koornneef et al., 1989).Nevertheless, knowledge about the function of the individual wax components and fractions is still incomplete.The accumulation of cuticular waxes under drought for UV irradiation is a common adaptive mechanism for plants (Long et al., 2003;Patwari et al., 2019); therefore, identifying cuticular wax-related genes in crops broadens the scope for breeding strategies to enhance resilience against water loss during drought and heat stress (Mansour et al., 2018).Barley (Hordeum vulgare) is the fourth most cultivated cereal world-wide (www.statistica.com),and an excellent genetic model thanks to the availability of genetic resources, including cultivars, landraces, wild relatives, and mutants (Mascher et al., 2017).Numerous mutants have been isolated, mostly through UV, radioactive, or chemical mutagenesis (Lundqvist & Lundqvist, 1988;Lundqvist, 2014).A total of 1580 cer mutants of barley were isolated and assigned to 79 loci (Lundqvist & Lundqvist, 1988).The cer-za mutation with 78 alleles represents the fourth largest group behind cer-c, cer-q, and cer-u (Lundqvist & Lundqvist, 1988).Less frequent mutations are cer-zh (11 alleles) and cer-ye (5 alleles).Few cer genes in barley have been identified.The cer-c, cer-q, and cer-u loci are a cluster of genes on chromosome 2HS, coding for, respectively, a b-diketone synthase, a lipid/carboxyl transferase, and a P450 protein, and are crucial for the biosynthesis of b-diketones, the major component of the wax bloom on barley spikes and leaf sheaths (Schneider et al., 2016).CER-ZH was identified as HvKCS1, required for fatty acid elongation (Li et al., 2018).Recently, the HvSHINE1 transcription factor involved in the regulation of wax biosynthesis was found to be encoded by CER-X (McAllister et al., 2022), while CER-G and CER-S were characterized as encoding two additional transcription factors, HvYDA1 and HvBRX-Solo, regulating both cuticular integrity and cell patterning (Liu et al., 2022).
To study the relevance of waxes as a cuticular barrier, we selected two cer mutants with a strong reduction in total leaf wax load, cer-za.227and cer-ye.267(Larsson & Svenningsson, 1986;Rost as et al., 2008).Bulked segregant RNA sequencing (BSR-Seq) approaches revealed that cer-za.227contains a mutation in an acyl-CoA reductase (HvFAR1), while cer-ye.267 is allelic to cer-zh.54 which is mutated in HvKCS1.The analysis of different fractions of cuticular waxes revealed that cer-ye.267,but not cerza.227,shows a significantly increased cuticular permeability, which can be attributed to alterations in the amounts of intracuticular waxes.
Seeds of the Arabidopsis thaliana (L.Heynh.)cer4-3 mutant were obtained from Gillian Dean (University of Vancouver, Canada; Rowland et al., 2006).Arabidopsis seeds were surfacesterilized with chlorine gas and cultivated on Murashige and Skoog medium with 1% agarose and 1% sucrose for 2 wk before transfer to soil.Nicotiana benthamiana (Domin) seeds were directly sown into soil.All plants were cultivated in growth chambers at 21°C with 55% relative humidity and 16 h of light (150 lmol m À2 s À1 ).

Cuticular wax analysis
Waxes were analyzed as described previously (Haas & Rentschler, 1984;Zeisler & Schreiber, 2016).Briefly, waxes were extracted from the second leaf of 14-d-old barley plants which were cut at 2 cm from the point of emergence (Richardson et al., 2007), or from stems of 4-wk-old Arabidopsis plants.The plant organs were dipped into chloroform for 10 s, and the internal standard was added (10 lg tetracosane).The waxes were silylated and measured using gas chromatography (GC; Zeisler & Schreiber, 2016).For the selective removal of the epicuticular waxes, barley leaf sections 2 cm in length were treated with collodion (Haas & Rentschler, 1984).The epicuticular waxes were extracted from the strips, and the remaining (intracuticular) waxes were extracted from the stripped leaf.In addition, wax esters were measured by highly sensitive mass spectrometry (MS; Patwari et al., 2019).Briefly, total waxes in chloroform were supplemented with internal standard (18:0ol-17:0), purified using a solid phase extraction and measured by direct infusion MS/MS (Patwari et al., 2019).

Analysis of cuticular permeability and foliar water loss
The entire length of the second leaf of three-leaf stage barley plants was pressure sprayed for 3 s with a solution of 50 lM Metribuzin in 0.1% Brij-L4 (Merck, Darmstadt, Germany).The quantum yield of chlorophyll fluorescence of photosystem II (quantum yield of PSII), a measure for the penetration of the photosynthetic inhibitor into the leaf, was monitored with a pulse-amplitude modulation fluorometer (Walz, Effeltrich, Germany).Leaves were dark-adapted, and the quantum yield of PSII was measured during the treatment in 5-min intervals for 3 h by applying PSII-saturating light flashes.
Foliar water loss was determined as published (Gr€ unhofer et al., 2022).Total water permeance of the leaf (predominantly representing transpiration through the stomata) was measured with an SC-1 Leaf Porometer (Decagon Devices Inc., Pulman, WA, USA).Residual water permeance (representing transpiration through the cuticle) of the detached leaves was gravimetrically measured in constant intervals throughout an incubation at 2% humidity and 25°C.The leaves were finally dried to determine their dry weights.

Determination of contact angles and scanning electron microscopy
Leaf-water drop contact angles were determined with a Drop Shape Analyzer (DSA25; KR € USS, Hamburg, Germany).For imaging epicuticular wax crystals, leaf sections of 14-d-old barley plants were mounted on carbon-coated aluminum stubs and dried in a desiccator.The dry samples were sputter-coated with gold (5 nm; Automatic Sputter Coater; Ingenieurb€ uro Peter Liebscher, Wetzlar, Germany).Images were acquired using a scanning electron microscope (Phenom-World, Eindhoven, the Netherlands; or TESCAN GmbH, Dortmund, Germany).
The coding sequence of CER-ZA was PCR-amplified from the cDNA of Bowman leaves (bn4045, bn4046; Table S2) and ligated into the BamHI/SalI sites of pLH9000-GFP-WSD1 carrying the 35S promoter (Patwari et al., 2019).The pLH9000-GFP-CER-ZA construct, the ER marker (pCB-DsRed-HDEL; Patwari et al., 2019), and pMP19 (Voinnet et al., 2000) were transferred into Agrobacterium GV3101.The Agrobacterium cells were infiltrated into the abaxial side of N. benthamiana leaves and observed with a spinning disc confocal microscope (IX73; Olympus Optical, Tokyo, Japan; DSU filter turret Lambda 10-3, Sutter Instrument, Novato, CA, USA; Linear laser system 400 Series, Oxford Instruments, Oxford, UK).DsRed fluorescence was excited at 561 nm, and its emission was observed with a center wavelength of 607.36 nm.GFP fluorescence was excited at 488 nm, and its emission was filtered with a center wavelength of 525.3 nm.A depth of 290 nm was covered in 30 planes.

Results
Alterations in water repellence and surface structure in cerza.227and cer-ye.267 After spraying with water, water droplets were not retained on the leaf surfaces of wild-type (WT) barley cultivar Bowman, but accumulated on cer-za.227and cer-ye.267(Fig. 1a).The contact angles of the water droplets on WT Bowman leaves are very large due to the high hydrophobicity of the wax layer, with values > 150°.The contact angles of water droplets on cer-za.227(122.2AE 2.6°) and cer-ye.267(119.9AE 7.4°) were decreased, indicating that the wettability of the mutant leaves was increased.
Scanning electron microscopy revealed that Bowman leaves are densely covered with platelet-shaped wax crystals.The number of crystals was reduced, and the crystals were unevenly distributed on the surfaces of cer-za.227and cer-ye.267(Fig. 1b).The morphology of the crystals of cer-za.227was altered, with fewer platelet-shaped structures accompanying an increase in tubular forms.The structure of wax crystals in cer-ye.267was not altered, although the density of wax crystals appeared lower (Fig. 1).Because cer-za.227and cer-ye.267had similar wax amounts (Fig. 2a), the auto-assembly of waxes into crystals might be affected.
The cer-za.227 mutation is located to HORVU5Hr1G089230 BSR-Seq represents a powerful method to identify mutant genes in small populations (Dong et al., 2018;Wu et al., 2018).The CER-ZA locus was mapped in a F 2 population from a cross of cer-za.227with Bowman (Methods S1).Using BSR-Seq, we identified an elevated mutant allele SNP frequency in the region of 580-660 Mb on chromosome 5, in agreement with previous results (Druka et al., 2011;Bregitzer et al., 2013).This region (Fig. S3) carries 49 genes with high-confidence SNPs compared with the Morex sequence (Mascher et al., 2017).The number of candidate genes was narrowed down by comparing their expression levels between the Bowman control and the cer-za.227bulks, removing genes with similarly abundant RNA reads (Table S3).The analysis of the remainder of the genes for their predicted functions allowed us to identify HOR-VU5Hr1G089230 as a candidate for CER-ZA.Computation of the Δ(SNP-index; Takagi et al., 2013) and the G 0 values (Magwene et al., 2011) of the most likely causal SNPs positioned HORVU5Hr1G089230 among the three most likely genes for CER-ZA.Furthermore, the transcript abundance of HOR-VU5Hr1G089230 was clearly downregulated in the cer-za.227bulk compared with Bowman (log 2 fold change: À2.2; false discovery rate (FDR) 1.704 9 10 À20 ).Fig. 2 Cuticular wax load and composition of cer-za and cer-ye mutant alleles.Waxes were extracted from the leaves, and the wax components were quantified using gas chromatography (GC-FID).(a) Total waxes of the epicuticular (isolated) and intracuticular (remaining) wax fractions, extracted from the leaves of barley cultivars Bowman, cer-za.227,and cer-ye.267.(b) Different wax classes in the epicuticular and intracuticular wax fractions.(d) Wax classes from different cer-za and cer-ye alleles.(c) Total cuticular waxes from cer-za and cer-ye alleles.The wax loads and composition of the cultivar Bowman were very similar to those of Foma (cer-za.232,cer-za.318,cer-ye.267,and cer-ye.582)and Bonus (cer-za.HORVU5Hr1G089230 encodes a protein of 498 amino acids with a calculated mass of 55.95 kDa.According to AHRD prediction, it encodes a FAR likely involved in wax biosynthesis.'NAD_-binding_4' and 'sterile' domains were predicted by Pfam, and a Rossmann fold within the NAD_binding_4 domain (amino acids 11-57) with NADP as potential cofactor was identified using Cofactory.The sterile domain (also known as FAR_C) is shared by members of the FAR family.A transmembrane domain was predicted at the C terminus by TMHMM, in accordance with DeepLoc, which indicated that HORVU5Hr1G089230 is membrane-bound and ER-localized.Therefore, CER-ZA is likely HORVU5Hr1G089230, which is predicted to encode a membrane-bound, ER-localized protein with FAR activity.Protein BLAST searches in barley revealed that HORVU5Hr1G089230 belongs to a family of 22 FAR proteins (Table S4).Sequences of FAR proteins from different plants were used to construct a phylogenetic tree (Fig. S4).HORVU5Hr1G089230 clusters with FAR1 from the grasses Aegilops and Brachypodium.The FAR sequences form four clades with a split of predicted ER-localized FARs from monocots (clade I) or dicots (clade II), and a separation of plastidlocalized FARs into clades III (dicots) and IV (monocots), the latter in agreement with previous results (Zhang et al., 2021).
The BSR-Seq approach revealed 10 SNPs in HOR-VU5Hr1G089230 of cer-za.227compared with Morex_V2 (Fig. 3a; Table S5).Because the sequence around the cer-za.227mutation is presumably derived from Foma, most of the SNPs (1-4 and 6-10) are due to polymorphisms between Foma and Morex.Only SNP5 was specific for cer-za.227.SNP5 caused a guanine deletion at position 584526708 of chromosome 5, associated with a frameshift in exon 4 which causes the exchange of Asn153Ile, a premature stop codon, and the truncation of the polypeptide after 153 amino acids (Fig. 3a,b).Comparison of the RNA-Seq sequence of the other alleles with their respective WT cultivars revealed the presence of further SNPs.The cer-za.318allele displayed the same mutation as cer-za.227;therefore, these two SNPs might be derived from the same mutagenesis event.
The cer-za.232 mutant carried a thymine/adenine exchange at position 584526341 at the exon5/intron5 splicing site after Lys229, leading to the translation of the intron sequence and a premature stop codon after 253 amino acids.Finally, cer-za.173contains a cytosine/adenine exchange at position 584525054, resulting in a Glu331 stop mutation in exon 8.The amino acid sequences of HORVU5Hr1G089230 in Bonus and Morex are identical, while the Foma sequence differs by two amino acids (Asn34Asp and Ala627Tyr).The protein structures (modeled with Alphafold) revealed the presence of a Rossmann fold and are highly similar for all cultivars (www.uniprot.org)(Fig. 3b).The HORVU5Hr1G089230 polypeptides of cer-za.227and cerza.318are truncated, lacking a large part of the C terminus.Similarly, the protein sequences of cer-za.232and cer-za.173are truncated, and the structures are strongly affected (Fig. 3c).

Generation of cer-za alleles by genome editing
To prove that HORVU5Hr1G089230 is CER-ZA, we mutagenized HORVU5Hr1G089230 by genome editing.Barley Golden Promise was transformed with constructs harboring the Cas9 endonuclease and two sgRNA sequences targeted to PAM sites in exons 5 and 8. Two independent lines, cer-za.2001and cerza.2002,were regenerated.Sequencing of the regions around the PAM site in exon 5 revealed that cer-za.2001 is heteroallelic, with a thymine insertion in one and a thymine deletion in the other allele, while cer-za.2002 is homoallelic, with thymine deletions (Fig. 4a).After spraying with water, water droplets rolled off the leaves of Golden Promise, but they adhered to the leaves of the cer-za mutants obtained by genome editing, similar to cer-za.227(Fig. 4a).The total wax load was decreased from 16.3 lg cm À2 in Golden Promise to 5.7 lg cm À2 in cer-za.2001and 6.7 lg cm À2 in cer-za.2002,and this was mainly based on the decrease in 26:0ol (Fig. 4a).Therefore, cer-za.2001and cer-za.2002display changes in wettability, wax load, and composition analogous tocer-za.227,demonstrating that HORVU5Hr1G089230 is cerza.227.
The AtFAR3/CER4 gene product from Arabidopsis produces 24:0ol and 26:0ol alcohols (Rowland et al., 2006).As a consequence, waxes on the stems of the cer4 mutant are deficient in primary alcohols.To study the in planta function, CER-ZA was introduced into the Arabidopsis cer4-3 mutant under control of the AtCER4 promoter to confer its epidermal expression.Four independent homozygous CER-ZA-expressing cer4-3 plants were selected, and the wax load and composition on the stems were measured (Fig. 4c).The wax ester content was increased in all CER-ZA-cer4-3 lines compared with cer4-3, albeit without reaching WT levels.Concomitantly, the primary alcohol content was doubled in CER-ZA-cer4-3 lines compared with cer4-3, but it also did not reach WT levels.The amounts of alkanes and aldehydes in the transgenic lines were slightly increased compared with WT and cer4-3, while the secondary alcohol contents were unchanged.The expression of CER-ZA in cer4-3 therefore results in the accumulation of primary alcohols, indicating that it harbors FAR activity in planta.
The cer-ye.267 mutation localizes to HORVU4Hr1G063420 and is allelic to cer-zh.54 The computation of the Δ(SNP-index) (Takagi et al., 2013) and the G 0 values (Magwene et al., 2011) for the SNPs between the Bowman and cer-ye.267bulks led to the identification of regions on chromosomes 2H, 4H, and 5H with G 0 values above the threshold (FDR = 0.01; Fig. S5).We focused on chromosome 4 where this mutation was previously reported to be located (Druka et al., 2011).The region of 490-530 Mbp contains seven genes with high-confidence SNPs (Table S6).Combining this approach with the screening for differentially induced genes indicated that HORVU4Hr1G063420 is a likely candidate for cerye.267.This gene covers 2979 bp with an intron-less coding sequence of 1638 bp, encoding a protein of 545 amino acids.
The cer-ye.267sequence harbors a thymine/adenine exchange compared with Morex_V2 and Foma at position 1940 (Fig. 5a), which results in a Phe495Tyr exchange.RNA-Seq analysis of the additional cer-ye alleles revealed that cer-ye.792and cer-ye.1395carry an identical SNP of cytosine/adenine at position 1068 584 528 000 584 527 000 584 525 000 584 524 000 584 526 000 The diagram shows its position on chromosome 5H, the exon/intron structure, the SNPs between cer-za.232and the reference sequence of Morex_V2, the SNPs between cer-za.173and Bonus, and the SNPs between cer-za.232and cer-za.318and Foma.The deletion of G (DG) in cer-za.227and cer-za.318 in exon 8 results in a premature stop codon ( †).The T-to-A exchange at the splicing site after exon 5 in cer-za.232,and the C-to-A exchange in exon 8 of cerza.173,also cause the introduction of premature stop codons.compared with both Morex and Bonus, causing a premature stop codon, and a truncated protein (Fig. 5b).No SNP was found in the HORVU4Hr1G063420 sequence of cer-ye.582compared with Morex or Bonus.Fig. 2 shows that cer-ye.582has the same wax phenotype as cer-ye.267;therefore, cer-ye.267might carry a mutation in the regulatory sequences (promoter or intron), but these sequences were not covered within the RNA-Seq approach.HORVU4Hr1G063420 was previously identified as the gene underlying the cer-zh.54mutation (Li et al., 2018).CER-ZH encodes HvKCS1, and consequently, the cer-zh.54mutant is wax The CER-ZA sequence was expressed in Saccharomyces cerevisiae.Lipids were isolated from cells obtained from 100 ml (OD 600 = 1) and, after conversion into trimethylsilyl ethers, measured using gas chromatography-mass spectrometry (GC-MS) with total ion chromatograms (TIC).Three primary alcohols accumulated in CER-ZA -expressing cells: 16:0ol, 18:0ol, and a very-long-chain alcohol identified as 26:0ol.26:0ol showed the strongest increase.Note that the peak eluting at the retention time of 26:0ol is a contaminant with a different mass spectrum (nd, not detected).Mean AE SD; n = 3; t-test; significant differences to the control (empty vector); **, P ≤ 0.01.(c) Wax analysis of the stems of four independent Arabidopsis thaliana cer4-3 mutant plants expressing CER-ZA from barley.Cuticular waxes were isolated from the stems of transformed cer4-3 plants and measured using GC-MS.deficient and shows the typical adhesion of water droplets on the leaf.To confirm allelism, cer-ye.267and cer-zh.54were crossed, and F 1 plants were analyzed after spraying the leaves with water.A dense accumulation of water droplets was observed on the leaves of the F 1 plants, demonstrating that cer-ye.267and cer-zh.54are allelic (Fig. 5c).
Expression of CER-ZA and CER-YE and the subcellular localization of CER-ZA CER-ZA/HvFAR1 and CER-YE/HvKCS1 are likely expressed in the leaf epidermis, in analogy with numerous genes involved in wax biosynthesis (Suh et al., 2005).The expression was studied using semiquantitative RT-PCR after the isolation of an epidermal strip from the leaves of Bowman, compared with the residual stripped leaf and the roots.Bands of 196 and 238 bp corresponding to CER-ZA and CER-YE, respectively, were observed in the epidermal tissue after electrophoresis, but were absent from the roots or residual leaf material (Fig. 6a).This is consistent with RNA-Seq data (EoRNA database; Milne et al., 2021).CER-ZA/ HvFAR1 (locus BART1_0-p38542) displays low transcript abundance in the roots, apical meristems, spikes, anthers/microspores grains/seeds (including embryo, lemma, palea, lodicule, and rachis), moderate expression in the leaves, and coleoptiles, and high expression in the epidermis.Similarly, CER-YE/HvKCS1 (BART1_0-p30122) is highly expressed in the leaves and epidermis and shows considerable expression in the root maturation zones, lemma, palea and stat-like/multicellular microspores, and low expression in the roots and seeds.Therefore, CER-ZA and CER-YE are highly expressed in the leaf epidermis, while CER-YE is expressed in additional tissues.
CER-ZA was predicted to be ER-localized using DeepLoc.In addition, CER-ZA carries a C-terminal KNKGSV sequence related to the prototypic dilysine ER-retrieval motif KXKXX, also found in the ER-resident fatty acid desaturase FAD3 of Arabidopsis (McCartney et al., 2004).To study its subcellular localization experimentally, the CER-ZA sequence was fused to the C terminus of the green fluorescent protein (GFP) and transiently expressed in N. benthamiana leaves.A DsRed-HDEL construct was coinfiltrated as a marker for the ER.The GFP and DsRed fluorescence were observed using confocal fluorescence microscopy.After co-infiltration, the two signals were observed in a net-like structure in epidermal cells (Fig. 6b).The merged signals revealed a clear overlap, confirming that CER-ZA localizes to the ER.

Mutation of cer-ye.267, but not cer-za.227, affects the function of the cuticular barrier
To study the consequences of the wax changes for the barrier function, the permeation properties of the cuticles of cer-za.227and cer-ye.267were measured after treatment with the photosynthetic inhibitor Metribuzin (Fig. 7a).The quantum yield of PSII was used as a measure for the penetration rate of Metribuzin across the cuticular barrier.After the application of Metribuzin to the leaves, the quantum yield of PSII decreased within 25 min from 0.7 to c. 0.48 in Bowman and cer-za.227,indicating that the penetration rates were similar.The quantum yield of PSII drastically declined in cer-ye.267reaching values as low as c. 0.25, then it slowly recovered to c. 0.4, but was always lower than in Bowman and cer-za.227.Therefore, Metribuzin penetrated the cuticle of cer-ye.267much faster than that of Bowman or cer-za.227.
Water loss through the epidermis can be explained by transpiration through stomata and by water evaporation through the cuticle.Under normal conditions with open stomata, transpiration through the stomata is predominant.Therefore, the total water permeance of a leaf is mostly caused by transpiration through the stomata.The total water permeance was determined by measuring the water flow for the adaxial and abaxial leaf sites with a porometer.The total water permeance did not differ between Bowman, cer-za.227,and cer-ye.267,indicating that transpiration through the stomata was not affected (Fig. 7b).This was in agreement with the finding that stomatal sizes and densities were very similar to Bowman as revealed by microscopic inspection of the leaf surfaces.Next, the water permeance of detached leaves was measured and plotted against the relative water deficit (RWD; Fig. S6).The water permeance was initially high for all lines, but then rapidly declined due to stomatal closure to reach final values of residual permeance P min , which were c. 95% lower.The P min , which is a proxy for the cuticular transpiration rate, was calculated from the averages of the measurements after stomatal closure in the range of RWD of 0.15-0.55(Figs 7c, S6).The P min of cer-za.227(2.14 AE 0.56 nm s À1 ) was slightly but not significantly higher compared with Bowman (1.63 AE 0.18 nm s À1 ; 95% significance level, t-test, P = 0.086), whereas P min of cer-ye.267(2.35 AE 0.35 nm s À1 ; P = 0.0005) was even higher and significantly increased compared with Bowman.Therefore, the penetration rate of Metribuzin and the cuticular transpiration are not significantly changed in cer-za.227,but both are compromised in cer-ye.267.
To study the roles of intracuticular and epicuticular waxes for water permeability directly, the water loss of a barley leaf was recorded starting 1 h after detachment.At this time, the stomata were closed, and the residual water loss is a measure of cuticular transpiration.After one additional h, the epicuticular waxes were removed with collodion, and the measurements were continued.The rate of water loss did not change after removal of the epicuticular waxes (Fig. 7d).Therefore, cuticular transpiration rates do not depend on the presence of epicuticular waxes.Fig. 7(e) shows an electron micrograph of the leaf surface with the removal of epicuticular waxes on the left side, leaving behind the layer of intracuticular waxes, while the right side remained untreated.This experiment demonstrates that intracuticular waxes rather than epicuticular waxes are crucial for the cuticular barrier function of barley.

Discussion
The cuticle establishes a barrier between the plant and the environment, providing essential protection against abiotic and biotic stresses.The composition of the cuticular wax layer has been studied in detail, but the contribution of the different wax fractions to the barrier functions remains unclear.The two barley mutants cer-za.227 and cer-ye.267reveal strong decreases in total wax load, but unique changes in wax composition and the epicuticular and intracuticular distribution.The CER-ZA gene was identified as HORVU5Hr1G089230, encoding HvFAR1, while the mutation in cer-ye.267 is allelic to cer-zh.54,which corresponds to HORVU4Hr1G063420, encoding HvKCS1.In contrast to cer-za.227, the cer-ye.267mutation affected the barrier functions, which are therefore presumably established by unique properties in wax distribution rather than a decrease in total wax load.

Identification of the CER-ZA and CER-YE genes
The gene HORVU5Hr1G089230 was identified as CER-ZA by BSR-Seq and confirmed by the generation of novel cer-za alleles.CER-ZA represents the first FAR characterized in barley.A total of 22 and eight FAR sequences were annotated in barley and Arabidopsis, respectively (Table S4; Aarts et al., 1997;Doan et al., 2009Doan et al., , 2012)).FAR enzymes were characterized in wheat and Brachypodium (Wang et al., 2015b(Wang et al., , 2018;;Chai et al., 2018).Expression in yeast confirmed that CER-ZA harbors alcoholforming FAR activity similar to AtFAR3/CER4 (Rowland et al., 2006).CER-ZA localizes to the ER, in analogy with the FARs in Arabidopsis (Rowland et al., 2006) and wheat (Wang et al., 2015b;Chai et al., 2018).Some FARs, including AtFAR1, AtFAR4, and AtFAR5, are expressed in the leaves and roots and therefore also contribute to suberin biosynthesis (Domergue et al., 2010).CER-ZA is highly expressed in the leaf epidermis, but not in the roots (Fig. 6a), similar to AtFAR3/CER4, suggesting that the two proteins are functional orthologs.The expression of CER-ZA in the Arabidopsis cer4-3 mutant led to increases in the amounts of primary alcohols and wax esters, however without reaching WT levels; therefore, CER-ZA partially restores the wax deficiency of cer4-3, similar to TaFAR2, TaFAR3, TaFAR3, TaFAR5, BdFAR1, BdFAR2, and BdFAR3 from wheat and Brachypodium (Wang et al., 2015b(Wang et al., , 2016(Wang et al., , 2018)).Possibly, the heterologous FARs from monocots localize to different subcompartments of the ER, resulting in an incomplete complementation of cer4 (Wang et al., 2015a).
Wax composition of cer-za.227and cer-ye.267 The cer-za.227 and cer-ye.267mutations decreased the total wax load by c. 70% (Fig. 2), in agreement with previous results using cer-za.126and cer-zh.54(Rost as et al., 2008;Li et al., 2018).The amounts of epicuticular waxes were strongly decreased.Intracuticular waxes were less affected in cer-za.227,and they were strongly decreased in cer-ye.267(Fig. 2).The most prominent change in the two mutants was the decline in the amount of 26:0ol.In cer-za.227, the amounts of aldehydes (26:0al) and wax esters were decreased, while alkanes (mostly C33) accumulated.Primary alcohols and wax esters are derived from the reductive pathway, whose initial step is catalyzed by FAR (Haslam et al., 2017).A restriction in the production of primary alcohols aldehydes, esters, alkanes and acids, were decreased in cer-ye.267, in analogy with cer-zh.54(Larsson & Svenningsson, 1986;Li et al., 2018).The cer-ye.267 and cer-zh.54lines carry mutations in HvKCS1, involved in acyl chain elongation.The two mutants displayed changes in chain length distribution of wax esters, because long-chain esters were decreased compared with short ones (Fig. S1j; Li et al., 2018).It is possible that the HvKCS1 activity is redundant with other HvKCS proteins in barley (Li et al., 2018).Furthermore, the amount of alkanes was decreased in cer-ye.267rather than increased as in cer-za.267.Although these two plants carry mutations in genes of wax biosynthetic enzymes, they result in a different distribution between intracuticular and epicuticular waxes.This might be caused by differences in the wax composition, which might alter their auto-assembly and affect the ratio of intracuticular to epicuticular waxes (Koch et al., 2008).

Wax crystal formation and self-cleaning effect
Primary alcohols in the epicuticular waxes play an important role for the formation of plate-shaped crystals, which render the surface super-hydrophobic (Koch et al., 2006).Bowman leaves are densely covered with plate-like crystals.The number of crystals was reduced in cer-za.228and cer-ye.267,which both displayed decreased amounts of 26:0ol, in agreement with previous results for cer-za.126and cer-zh.54(Rost as et al., 2008;Li et al., 2018).The crystals provide an efficient self-cleaning effect, resulting in decreased water droplet angles and compromised water repellence.Therefore, deficiency in epicuticular wax in cer-za.227and cer-ye.267affects their self-cleaning properties and water repellence.
Water loss and cuticular permeability in cer-za.227and cer-ye.267 The intracuticular waxes in cer-za.227are decreased to c. 60% of Bowman levels, and the remaining amount was still sufficient to establish a functional barrier for Metribuzin and to maintain a slightly but not significantly increased cuticular transpiration rate (Fig. 7).The amount of intracuticular waxes in cer-ye.267 is decreased to c. 46% of Bowman levels, and the permeability for Metribuzin and the cuticular transpiration were significantly increased (Fig. 7).Direct measurements of residual water loss of a leaf after collodion treatment demonstrated that intracuticular rather than epicuticular waxes are relevant for establishing the cuticular transpiration barrier (Fig. 7d,e).These results are in good agreement with previous findings, which showed that cuticular barrier functions are primarily established by intracuticular waxes (Jetter & Riederer, 2016;Zeisler & Schreiber, 2016;Zeisler-Diehl et al., 2018).No simple and linear relationship between cuticular barrier properties and wax amounts was found for different species (Riederer & Schreiber, 2001), or for the same species (Sadler et al., 2016;Gr€ unhofer et al., 2022).The cuticular permeability remained unchanged or it increased in a set of seven cuticle mutants of Arabidopsis, with decreases or increases in wax or cutin amounts.Therefore, the coordinated deposition and the semicrystalline arrangement of the highly ordered wax barrier within the cuticle might have been disturbed in the mutants with increased permeabilities.This could also be the reason for the altered cuticular permeability of cer-ye.267(Fig. 7).In contrast to the total wax load, alkanes and esters were proposed to contribute to the establishment of a functional transport barrier (Larsson & Svenningsson, 1986;W. Wang et al., 2015;Bueno et al., 2019;Li et al., 2020;Wu et al., 2022).The amount of alkanes was increased in cer-za.227,possibly contributing to the barrier function, but it was decreased in cerye.267(Fig. S2).Although the wax ester content was decreased, this effect was restricted to the epicuticular waxes.The cer-ye.267 and cer-zh.54mutants showed a shift in chain length distribution of wax esters (Fig. S1j; Li et al., 2018) possibly contributing to the modulation of cuticular permeability.Conversely, the strong reduction in the amounts of primary alcohols (26:0ol) did not affect the cuticular barrier function in cer-za.227,and it therefore cannot be the cause for increased permeability and cuticular transpiration in cer-ye.267.Instead, primary alcohols, which are abundant in Poaceae, presumably fulfill other functions in crystal formation and water repellence and light reflection (Fig. S2; Koch et al., 2006).
In conclusion, the two barley lines cer-za.227and cer-ye.267,which carry mutations in two genes involved in wax biosynthesis, HvFAR1 and HvKCS1, respectively, reveal a strong reduction in total wax amounts, distinct changes in composition, and amounts of intracuticular to epicuticular waxes.These changes caused an increase in cuticular permeability (Fig. 7a) in cer-ye.267,but not in cer-za.227.Therefore, the decrease in cuticular barrier function in cer-ye.267compared with cer-za.227 is presumably explained by the differences in wax composition and a reduction in intracuticular waxes.The precise role of the individual wax lipids for the establishment of the (intra)cuticular barrier, however, remains to be discovered.

Fig. 1
Fig. 1 Wettability and surface structures of barley cultivars Bowman, cer-za.227and cer-ye.267.(a) Water droplets accumulate on the surfaces of cerza.227and cer-ye.267leaves but roll off the leaf surface of the Bowman cultivar.(b) Scanning electron microscopy of leaf surfaces reveals a dense coverage with plate-shaped crystalline waxes on Bowman leaves, but a low number of wax crystals on cer-za.227and cer-ye.267leaves.Bars: (b) 10 lm; (insets) 8 lm.

Fig. 3
Fig. 3 cer-za mutations are associated with SNPs in the gene HORVU5Hr1G089230.(a) Representation of the HORVU5Hr1G089230 locus.The diagram shows its position on chromosome 5H, the exon/intron structure, the SNPs between cer-za.232and the reference sequence of Morex_V2, the SNPs between cer-za.173and Bonus, and the SNPs between cer-za.232and cer-za.318and Foma.The deletion of G (DG) in cer-za.227and cer-za.318 in exon 8 results in a premature stop codon ( †).The T-to-A exchange at the splicing site after exon 5 in cer-za.232,and the C-to-A exchange in exon 8 of cerza.173,also cause the introduction of premature stop codons.(b) Protein sequences based on SNPs identified busing BSR-Seq for the different cer-za alleles in comparison with Morex, Foma, and Bonus.The amino acid sequences of Morex, Bowman, and Bonus are identical.Foma differs from this sequence by two amino acids.Bars indicate the degree of identity between all sequences at a given position.(c) Protein models calculated by Alphafold.Proteins of cerza.227,cer-za.318,cer-za.232,and cer-za.173are truncated, lacking considerable parts of the C-terminal structural elements.
Fig. 3 cer-za mutations are associated with SNPs in the gene HORVU5Hr1G089230.(a) Representation of the HORVU5Hr1G089230 locus.The diagram shows its position on chromosome 5H, the exon/intron structure, the SNPs between cer-za.232and the reference sequence of Morex_V2, the SNPs between cer-za.173and Bonus, and the SNPs between cer-za.232and cer-za.318and Foma.The deletion of G (DG) in cer-za.227and cer-za.318 in exon 8 results in a premature stop codon ( †).The T-to-A exchange at the splicing site after exon 5 in cer-za.232,and the C-to-A exchange in exon 8 of cerza.173,also cause the introduction of premature stop codons.(b) Protein sequences based on SNPs identified busing BSR-Seq for the different cer-za alleles in comparison with Morex, Foma, and Bonus.The amino acid sequences of Morex, Bowman, and Bonus are identical.Foma differs from this sequence by two amino acids.Bars indicate the degree of identity between all sequences at a given position.(c) Protein models calculated by Alphafold.Proteins of cerza.227,cer-za.318,cer-za.232,and cer-za.173are truncated, lacking considerable parts of the C-terminal structural elements.

Fig. 4
Fig.4CER-ZA gene product harbors acyl-CoA reductase activity.(a) The eceriferum mutant plants cer-za.2001and cer-za.2002generated by genome editing.Alignment of the DNA sequences around the protospacer adjacent motif (PAM, blue) in exon 5 of HORVU5Hr1G089230.Sequences of barley cultivars Morex, Golden Promise, and the alleles of the two mutant lines.The sequence of cer-za.2001shows an insertion (I, green) of a T in one allele and a deletion (D, orange) of a T the other one.The sequence of cer-za.2002 is homoallelic with a deletion of a T. The water droplets adhere to the leaves of the cer-za alleles obtained by genome editing (shown here: cer-za.2001), in contrast to Golden Promise.Total amount and composition of cuticular waxes on the leaves of cer-za.227,cer-za.2001,and cer-za.2002.Only single measurements of waxes of the genome-edited lines cer-za.2001and cer-za.2002were possible due to the limitation of leaf material.(b) The CER-ZA sequence was expressed in Saccharomyces cerevisiae.Lipids were isolated from cells obtained from 100 ml (OD 600 = 1) and, after conversion into trimethylsilyl ethers, measured using gas chromatography-mass spectrometry (GC-MS) with total ion chromatograms (TIC).Three primary alcohols accumulated in CER-ZA -expressing cells: 16:0ol, 18:0ol, and a very-long-chain alcohol identified as 26:0ol.26:0ol showed the strongest increase.Note that the peak eluting at the retention time of 26:0ol is a contaminant with a different mass spectrum (nd, not detected).Mean AE SD; n = 3; t-test; significant differences to the control (empty vector); **, P ≤ 0.01.(c) Wax analysis of the stems of four independent Arabidopsis thaliana cer4-3 mutant plants expressing CER-ZA from barley.Cuticular waxes were isolated from the stems of transformed cer4-3 plants and measured using GC-MS.The panels show total wax coverage and the distribution of wax classes.Mean AE SD; n = 3; ANOVA; different letters indicate significant differences; P < 0.05.

ÓFig. 5
Fig. 5 cer-ye mutations are caused by SNPs in the gene HORVU4Hr1G063420.(a) Representation of the HORVU4Hr1G063420 locus.The diagram shows the position on chromosome 4H, the exon/intron structure, and the SNPs between cer-ye.267,cer-ye.792,and cer-za.1395and the reference sequence of Morex_V2.The cer-ye.267 mutation of T/A causes an amino acid exchange of Phe/Tyr.The cer-ye.792 and cer-ye.1395mutants carry the same mutation of C/A resulting in a premature stop codon ( †).(b) Protein sequences based on SNPs identified using BSR-Seq for Morex and the different cer-za alleles, cer-ye.267,cer-ye.582,cer-ye.1395,and cer-ye.792.The amino acid sequences of barley cultivars Morex, Bowman, and Bonus are identical.Bars indicate the degree of identity between all sequences.The position of the amino acid exchange Phe/Typr in cer-ye.267 is depicted with a red arrow.(c) Allelism test for cer-ye.267(BW136) and cer-zh.54(Bonus).The two mutants cer-ye.267 and cer-zh.54were crossed, and leaves from the plants of the F 1 generation were sprayed with water to determine the wettability of the leaf surface.No water droplets adhere to leaves of Bowman (shown as control) or Bonus.

Fig. 6 Fig. 7
Fig. 6 Expression analysis of barley cultivars CER-ZA and CER-YE and subcellular localization of CER-ZA.(a) Expression of CER-ZA and CER-YE was compared using a semiquantitative RT-PCR of RNA isolated from epidermal strips of leaves, the stripped leaf, and the root tissue.The ACT2 gene was used as the control.RT-PCR bands were separated using agarose gel electrophoresis and stained with Midori green.(b) Subcellular localization of CER-ZA.The CER-ZA sequence was fused to the C terminus of GFP, and the GFP-CER-ZA construct expressed in Nicotiana benthamiana leaves together with the endplasmic reticulum (ER) marker DsRed-HDEL.Epidermal leaf cells were observed using confocal fluorescence microscopy.
The panels show total wax coverage and the distribution of wax classes.Mean AE SD; n = 3; ANOVA; different letters indicate significant differences; P < 0.05.