Biosynthesis of barley wax β-diketones: a type-III polyketide synthase condensing two fatty acyl units

Domain: earth_science
Published: 2023-11-10
Editorial outcome: minor_revision

Abstract
The surface coatings of cereal plants are dominated by waxy β-diketones crucial for drought resistance and, therefore, grain yield. Here, barley (Hordeum vulgare) wax analyses reveal β-diketone and associated 2-alkanol ester profiles suggesting a common C16 3-keto acid precursor. Isotope analysis further shows that the major (C31) diketone is synthesized from two plastidial C16 acyl units. Previous studies identified a gene cluster encoding enzymes responsible for β-diketone formation in barley, but left their biochemical functions unknown. Various assays now characterize one of these enzymes as a thioesterase producing long-chain (mainly C16) 3-keto acids, and another one as a polyketide synthase (PKS) condensing the 3-keto acids with long-chain (mainly C16) acyl-CoAs into β-diketones. The two enzymes are localized to the plastids and Endoplasmic Reticulum (ER), respectively, implying substrate transfer between these two sub-cellular compartments. Overall, our findings define a two-step pathway involving an unprecedented PKS reaction leading directly to the β-diketone products.

Introduction
Plants must seal the vast surfaces of their above-ground organs against water loss and pathogens1. They achieve this crucial eco-physiological function with waxes formed in their epidermis and deposited as a thin layer on the surface1,2. The waxes are complex mixtures of aliphatic compounds mostly derived from fatty acid metabolism, with aliphatic chains typically containing 24–34 carbons3. Most wax compounds have oxygen functional groups, predominantly at one chain end but also on mid-chain carbons. The composition of wax mixtures varies substantially between plant species and organs, leading to characteristic distributions of compound classes and chain lengths within each of them3,4. The common wax compounds are synthesized in three stages, involving (i) formation of C16 fatty acyls by plastidial fatty acid synthases (FASs); (ii) further elongation to very-long-chain (VLC) acyl-CoAs by fatty acid elongases (FAEs) in the endoplasmic reticulum (ER); (iii) modification of the carboxyl head groups on pathways involving either reduction to alcohols or decarbonylation to alkanes2.

Many plant species accumulate specialty compounds in their surface waxes, frequently in the form of mid-chain β-diketones. Characteristic VLC β-diketones are found in diverse dicots (e.g., Myrtaceae, Buxaceae, Ericaceae, Caryophyllaceae, and Asteraceae)5–10 and monocots (e.g., Hosta and Vanilla spp.)11,12. The wax β-diketones are especially widespread among the Poaceae10, including crops such as wheat (Triticum durum, T. aestivum)13–15, barley (Hordeum vulgare)8,16, and rye (Secale cereale)17. For example, barley waxes contain predominantly C31 14,16-diketone, together with minor amounts of C33 and C29 diketones8. In many species, β-diketones are accompanied by esters of 2-alkanols thought to be side products of β-diketone formation (Fig. 1A)10,18.

β-Diketones are produced in late vegetative and reproductive growth stages in Poaceae crops, especially in the waxes covering upper leaf sheaths, flag leaves and spikes15,16,19,20. There, they are among the most dynamically regulated wax components during drought conditions21, associated with high water use efficiency and light reflectance22. Therefore, β-diketones are crucial for maintaining grain yield under stress, and their accumulation is a favorable agronomic trait that has been highly selected in crop breeding23. However, the mechanism underlying β-diketone formation is only partially understood.

Early biochemical attempts to address β-diketone synthesis in barley with isotopic tracing suggested 3-ketoacids as key intermediates en route to C31 14,16-diketone (Fig. 1A and Supplementary Fig. S1)8,24–26, but lacked definitive evidence for this assumption. Recently, genetic studies identified metabolic gene clusters involved in β-diketone formation in both wheat and barley, each defined by the presence of three key genes. These genes were predicted to encode a hydrolase/carboxylesterase (named diketone metabolism hydrolase, DMH), a type-III polyketide synthase (PKS) (named diketone metabolism PKS, DMP) and a cytochrome-P450 oxidase (named diketone metabolism CYP450, DMC)19,20. In the process, the barley orthologs were recognized as the Cer-q, Cer-c and Cer-u genes described in earlier genetic studies, respectively20,27. Earlier mutant analyses had shown that the barley Cer-q/HvDMH and Cer-c/HvDMP enzymes were both required for β-diketone formation, and more recent VIGS experiments confirmed the in planta function of the corresponding enzymes also in wheat19,20 Thus, all previous evidence taken together suggested that DMH catalyzes the first step of β-diketone formation leading to the 3-ketoacid intermediate, and preliminary analyses confirmed that barley DMH may indeed intercept fatty acyl elongation intermediates to form 3-ketoacids19. While this provided first evidence for the enzymatic reaction leading to the key intermediate, it did not provide information on the later steps in the pathway.

Two fundamentally different mechanisms for the DMP-catalyzed formation of the diketo group must be considered (Fig. 1A and Supplementary Fig. S1). On the one hand, DMP may catalyze the condensation of 3-ketoacyl-CoA starters and malonyl-CoA extenders10, resembling the activities of most PKSs28. The resulting 3,5-diketoacyl-CoA would require further elongation, likely by FAE-type elongase(s), and head group modification, similar to the pathways leading to common wax compounds. The major β-diketone of barley and wheat waxes, C31 14,16-diketone, may thus be formed via C16 3-ketoacid or C18 3-ketoacid as central pathway intermediate. However, both the nature of the intermediate and the enzymes modifying it in later steps of the predicted pathway remained elusive, despite the characterization of numerous barley β-diketone-deficient mutants. On the other hand, DMP could condense the 3-ketoacid intermediates with common fatty acyl-CoAs to directly form β-diketone products. Such a head-to-head condensation of two pre-formed hydrocarbon chains had been proposed early on29, but various wax formation pathways were since shown to involve incremental build-up of hydrocarbon chains2. This second hypothesis for β-diketone biosynthesis thus diverges substantially from previously known wax formation mechanisms. Furthermore, it also invokes DMP substrates very different from those of most plant PKSs, which prefer aromatic starters and malonyl extenders28,30,31. However, it had been noted that the wheat DMP protein shares sequence similarity with Curcuma longa curcumin synthase (CURS)19, a PKS enzyme known to use an aromatic keto acid extender for head-to-head condensation with a second aromatic acyl32. Here, we aimed to revisit both these fundamental models for hydrocarbon chain formation and to elucidate the overall biosynthetic pathway leading to β-diketones in barley.

Results
To elucidate the β-diketone biosynthesis pathway, we aimed to provide both chemical evidence defining the major pathway intermediates and biochemical evidence characterizing the key enzymes involved.

Homolog and isomer distributions of β-diketones and 2-alkanol esters indicate the major pathway intermediate
In a first set of experiments, we sought to identify all β-diketone homolog and isomer structures accumulating in barley cv. Morex spike wax. To this end, the wax mixture was separated by thin-layer chromatography (TLC), and the fraction containing the β-diketones (Rf 0.56) was analyzed by gas chromatography-mass spectrometry (GC-MS). The β-diketones were identified by characteristic base peaks m/z 10033, while molecular ions and [M-18]+ ions enabled the assignment of overall chain lengths C29 to C33 (Supplementary Fig. S2). Positional isomers for each homolog were assigned based on prominent fingerprint fragments, leading to the identification of C29 12,14- and 14,16-diketone, C31 14,16-diketone and C33 16,18-diketone structures reported before8. In addition, novel even-numbered homologs were identified as C30 14,16-diketone and a mixture of C32 14,16- and C32 15,17-diketones. Analysis of the GC retention times showed that the even-numbered β-diketone homologs had straight hydrocarbon backbones devoid of methyl branches (Supplementary Fig. S3), implying that one of their acyl moieties must be formed from an unbranched, odd-numbered starter of de novo fatty acid synthesis.

GC-MS quantification showed that the spike wax β-diketones comprised 96.4% of C31 14,16-diketone, along with 0.4% C29 12,14-diketone, 0.3% C29 14,16-diketone and 2.6% C33 16,18-diketone (Fig. 2A and Supplementary Fig. S1). The C30 14,16-diketone, C32 14,16-diketone, and C32 15,17-diketone made up 0.1%, 0.2%, and 0.1% of the fraction, respectively. The corresponding TLC fraction of barley flag leaf sheath wax had a very similar β-diketone composition (Supplementary Table S1). Overall, the barely β-diketones comprised predominantly C16 and C18 ketoacyl units (Fig. 1B, m=11 or 13), confirming C16 3-ketoacid and C18 3-ketoacid as most likely pathway intermediates.

To further assess the chain length distributions of key intermediates for β-diketone formation, we analyzed the 2-alkanol esters known to be pathway side-products. In a TLC fraction of barley spike wax (Rf 0.73) both 1-alkanol esters and a series of 2-alkanol esters were identified. Among the 2-alkanol esters, the C33, C35, C36, and C37 homologs accounted for 8.3%, 58.5%, 3.0%, and 30.3%, respectively (Fig. 2B and Supplementary Table S2). Each of the four ester homologs comprised C15 2-alkanol, amounting to 88%, 96%, 100%, and 59%, respectively (Fig. 2B). Across all homologs, the esters contained 84% C15 2-alkanol, along with only 1% C13 and 14% C17 2-alkanols. Similar 2-alkanol ester homolog distributions were observed in barley flag leaf sheath wax (Supplementary Table S3). Based on the general understanding that 2-alkanols are derived from corresponding 3-ketoacids one carbon longer19,24, the high concentration of C15 2-alkanol esters suggests a precursor pool dominated by C16 3-ketoacid rather than C18 3-ketoacid.

Mutant wax and carbon isotope analyses test elongation of the 3-ketoacid intermediate
To test whether β-diketone formation requires ER-localized VLC acyl elongation, we analyzed the wax of flag leaf sheaths of the emr1 mutant lacking the core enzyme of the FAE complex responsible for wax precursor elongation, β-ketoacyl-CoA synthase 6 (KCS6/CER6). The mutant wax mixture showed a distinct elongation phenotype, with chain length distributions shifted to shorter homologs in comparison with the corresponding wild type (H. vulgare cv. Ingrid). All compound classes comprising chain lengths longer than C24 in the wild type (alkanes, 1-alkanols, 1-alkanol esters, and alkyl guaiacols34) had profiles shifted towards shorter homologs in the mutant (Supplementary Fig. S4), similar to previous reports on the leaf wax of these lines35. However, the C31 β-diketones and hydroxy-β-diketones dominating the flag leaf sheath wax were found in similar concentrations in both lines. Formation of their carbon backbones is thus independent of the major wax ER-elongation enzyme, and our findings do not support elongation of a ketoacid precursor towards β-diketones.

Plant lipids have characteristic 13C depletion patterns known to encode information on growth conditions as well as biosynthetic mechanisms36. In particular, previous studies revealed differential isotope incorporation into wax compound classes and chain lengths in various biosynthesis steps37–39. Therefore, we aimed to analyze the isotope composition of β-diketones by GC coupled with isotope ratio mass spectrometry (IRMS), and to compare it with the isotope compositions of wax constituents formed along known biosynthesis pathways. The latter (alkanes and esters) had δ13C values positively correlated with the amounts of carbon incorporated during plastidial fatty acid synthesis (r2=0.60) (Fig. 2C). The β-diketones (>95% C31 14,16-diketone) had δ13C −35.3 ± 0.3‰, a value fitting the trend of all other compound classes only if assuming 100% plastidial carbon content in the β-diketone. Thus, our isotope analysis further corroborates that β-diketone formation does not involve elongation of 3-ketoacid intermediates by ER-residing FAE enzymes (c.f. Fig. 1 and Supplementary Fig. S1).

Thioesterase activity and chain length preference of barley DMH (HvDMH)
To further establish the chain length distribution of the β-diketone synthesis intermediates, we analyzed the product profiles of the barley DMH enzyme (HvDMH) thought to catalyze the first reaction of the pathway. GC-MS analyses of E. coli expressing HvDMH identified saturated and monounsaturated C13–C17 2-ketones and 2-alkanols, along with C12–C16 3-hydroxyfatty acids (Fig. 3A and Supplementary Fig. S5). The 2-alkanol and 2-ketone series were dominated by respective unsaturated C15 as well as saturated C13 and C15 compounds (Fig. 3B, C). Further MS analysis of dimethyldisulfide (DMDS) adducts identified the unsaturated 2-ketones as isomers with double bonds mainly at the ω−7 position (Supplementary Fig. S6). The series of 3-hydroxyacids comprised predominantly saturated C14 and C16 as well as unsaturated C16 compounds (Fig. 3D). None of the 2-ketones and 2-alkanols could be detected in corresponding empty-vector controls, while the concentrations of all 3-hydroxyacids were increased ~twofold from controls to HvDMH-expressing lines. As 2-ketones and 2-alkanols are likely formed by decarboxylation of respective 3-ketoacids and 3-hydroxyacids one carbon longer, our results imply that HvDMH generated predominantly C16 3-ketoacid and 3-hydroxyacid in E. coli. These HvDMH chain length preferences matched our previous results showing that the wax β-diketones and 2-alkanol esters originated mainly from C16 ketoacyls.

To compare the chain length profiles of 3-keto-functionalized products with those of the available E. coli acyl pools, the latter were assessed by fatty acid methyl ester (FAME) analysis. The total lipids of the HvDMH expressors and empty-vector control lines had similar acyl profiles, with saturated C12–C18 fatty acyls peaking at C16 and corresponding unsaturated acyl peaking at C18 (Fig. 3E).

To corroborate the 3-ketoacyl thioesterase activity of HvDMH, we performed in vitro assays similar to those reported for characterization of tomato methylketone synthase40,41. In these coupled assays, purified E. coli acyl-carrier-protein (EcACP), E. coli malonyl-CoA:ACP transferase (EcFabD) and Mycobacterium tuberculosis 3-ketoacyl-ACP synthase (MtFabH) were used to convert malonyl-CoA substrate into malonyl-ACP and, together with C14 acyl-CoA as second substrate, further into C16 3-ketoacyl-ACP. After incubation of this assay mixture with HvDMH, lipids were extracted and derivatized using elevated temperatures to decarboxylate the thioesterase product, sensitive 3-ketoacid, into corresponding 2-ketones for reliable quantification. In the assay product mixture, C15 2-ketone was identified based on GC retention time (Fig. 4A) and MS fragmentation patterns (Fig. 4B) identical to those of an authentic standard. Control in vitro assays with boiled HvDMH protein yielded small amounts of C15 2-ketone (Fig. 4C), while further control assays lacking MtFabH contained no detectable 2-ketone. Taken together, these assays thus defined a background of C15 2-ketone formed by hydrolysis of C16 3-ketoayl-ACP and decarboxylation of the resulting free 3-ketoacid, independent of HvDMH activity. Assays with the intact HvDMH enzyme contained significantly more C15 2-ketone than the boiled-enzyme controls, thus showing the thioesterase activity of the enzyme (Fig. 4C). Overall, our assays demonstrated that HvDMH has in vitro thioesterase activity catalyzing the hydrolysis of 3-ketoacyl-ACP into free 3-ketoacid intermediates of β-diketone biosynthesis.

Biochemical function of the barley DMP (HvDMP)
Based on all the evidence suggesting C16 3-ketoacid as central intermediate of the β-diketone pathway, our next goal was to test its role as a substrate of the ensuing step and, thus, to establish the biochemical activity of HvDMP. However, the expression of HvDMH in yeast (Saccharomyces cerevisiae) did not lead to the production of the ketoacid, so the compound had to be supplemented exogenously. When C16 3-ketoacid was fed to yeast expressing HvDMP, three compounds were detected which were not present in controls expressing empty vector or lacking substrate. Based on their GC-MS characteristics, the major product was identified as C31 14,16-diketone, along with C29 14,16-diketone and a monounsaturated C31 14,16-diketone (Fig. 5A, B). Thus, HvDMP proved active in yeast, likely condensing C16 3-ketoacid directly with C16 fatty acyl to form C31 14,16-diketone.

To corroborate the biochemical activity of HvDMP, the recombinant enzyme was assayed in vitro with various substrate combinations. Incubation with C16 ketoacid and C16 fatty acyl-CoA gave a single product, identified as C31 14,16-diketone (Fig. 5B, C). In contrast, corresponding assays of HvDMP with varying molar ratios of C16 ketoacid and malonyl-CoA yielded no detectable β-diketone or triketide/tetraketide products. Incubation of the enzyme with C16 fatty acyl-CoA and malonyl-CoA in diverse concentrations also did not give detectable products. Together, these findings confirm that HvDMP uses a ketoacid instead of malonyl-CoA extenders for condensation with a fatty acyl-CoA.

To test the HvDMP-catalyzed condensation of the two substrates further, we fed per-deuterated C16 fatty acid together with C16 ketoacid to yeast expressing the enzyme. The resulting lipids contained unlabeled C31 14,16-diketone together with a novel compound with shorter GC retention time (Fig. 5D). The MS characteristics (Fig. 5E and Supplementary Fig. S7) unambiguously identified this compound as C31 14,16-diketone with one normal (C13H27) and one per-deuterated hydrocarbon tail (C15D31) originating from the labeled fatty acid. The formation and exact structure of this product indicated the direct incorporation of the entire acyl moiety into the β-diketone product and further confirmed that HvDMP catalyzed its condensation with ketoacid also in vivo.

To test whether HvDMP condensed entire ketoacid and fatty acyl-CoA units, we assayed the enzyme with substrates having unusual odd-numbered acyl chains. In one experiment, feeding C15 fatty acid and C16 ketoacid to yeast expressing HvDMP yielded three compounds absent from empty-vector controls, the major compound being C31 14,16-diketone and the two minor ones C29 14,16-diketone and C31 14,16-diketone (Fig. 5F, G). In a similar assay, C17 ketoacid and C14 fatty acid were fed to HvDMP-expressing yeast, resulting in the formation of C30 14,16-diketone as a major product together with C31 15,17-diketone, C32 16,18-diketone and C33 15,17-diketone (Fig. 5G, H). The finding that each of these assays yielded several products sharing one acyl moiety, taken together with the structure of respective major β-diketone products, further confirmed that HvDMP catalyzed the direct condensation of fatty acyl-CoA and ketoacid substrates.

Chain length preference of HvDMP for fatty acyl-CoA and 3-ketoacid substrates
Four experiments were performed to test the chain length preferences of HvDMP. In a first set of assays, we supplemented yeast expressing HvDMP with equal molar amounts of C14, C16, or C18 ketoacids and quantified the resulting β-diketone products. Feeding of C14 3-ketoacid led to a β-diketone mixture comprising mainly C27 12,14-diketone, whereas C16 3-ketoacid feeding afforded mainly C31 14,16-diketone, and C18 3-ketoacid mainly C33 16,18-diketone (Fig. 6A, B). Thus, the HvDMP preferences for acyl-CoA substrates depended on the 3-ketoacid present, resulting in condensation of C14 3-ketoacid mainly with C14 acyl-CoA, C16 3-ketoacid with C16 acyl-CoA, and C18 3-ketoacid with C14 and C16 acyl-CoAs.

To gauge the effect of fatty acyl chain lengths available in the in vivo assays, we quantified the fatty acyls in the total lipid mixture of the yeast lines (as FAMEs). The acyl pools of control yeast (without substrate feeding) comprised mainly monounsaturated C16 and C18 chains (together 88%), along with saturated C12–C18 acyls dominated by C16 (8%) and C18 (3%) (Fig. 6C). Compared with this profile of available substrates, HvDMP greatly preferred saturated over unsaturated acyl substrates in our assays, and to some degree C14 acyls over the more abundant C16 and C18 homologs (Supplementary Fig. S8A).

The preference of HvDMP for certain acyl-CoA chain lengths was assessed in a second in vivo experiment where we supplemented yeast expressing the enzyme with different fatty acid homologs. Preliminary tests showed that co-feeding of C16 3-ketoacid with either C11 or C17 fatty acids yielded the same β-diketones as controls lacking exogenous acids. However, co-feeding of C16 3-ketoacid with a mixture of C12–C15 fatty acids led to production of C27–C31 β-diketones (Fig. 6D). The products had a near-symmetric chain length distribution centered at C29, including substantial amounts of two even-numbered β-diketones, C28 and C30 (Fig. 6E). In contrast to the controls lacking exogenous fatty acids, unsaturated β-diketones could not be detected in this assay. Fatty acid profiling of the total yeast lipids confirmed substantially increased amounts of C12–C15 acyls in the complemented assays (Fig. 6F). Overall, HvDMP thus condensed C16 3-ketoacid with fatty acyls ranging from C12 to C16, but preferring C13/C14 acyls (Supplementary Fig. S8B).

Finally, the interdependence between the HvDMP 3-ketoacid and acyl substrate chain length preferences was explored using in vitro assays with defined, limiting substrate concentrations. In one set of experiments, recombinant HvDMP was incubated with a mixture containing one molar equivalent each of C14, C16, and C18 ketoacids and 1.5 equivalents of a particular fatty acyl-CoA (C12, C14, or C16). In all three assays, over 60% of the β-diketone products were formed by incorporation of C14 3-ketoacid substrate, accompanied by 20% of C16 3-ketoacid condensation products and circa 10% of C18 ketoacid products (Fig. 6G, H). The HvDMP enzyme, thus, had a strong preference for C14 3-ketoacid, irrespective of acyl-CoA co-substrate chain length.

In a final set of experiments, the acyl-CoA preference of HvDMP was tested in competition assays with limited amounts of 3-ketoacid co-substrates, where the enzyme was incubated with a mixture of C12, C14 and C16 fatty acyl-CoAs (1:1:1 molar ratio) and either C14, C16, or C18 3-ketoacid (1.5 molar equivalents). In all three assays, C14 fatty acyl-CoA substrate was slightly preferred over C16 fatty acyl-CoA, and less than 7% of the β-diketones originated from C12 acyl-CoA (Fig. 6I, J). Overall, the in vitro assays thus showed that HvDMP preferentially condensed C14 3-ketoacid with C14 or C16 fatty acyl-CoA to form C27 or C29 14,16-diketone, respectively.

Subcellular compartmentation of the β-diketone-forming enzymes
Based on our biochemical results showing that only two enzymes, HvDMH and HvDMP, were required for β-diketone formation, we aimed to localize the biosynthetic pathway across the cellular compartments. For this, we first determined the subcellular localization of HvDMH by confocal microscopy of barley protoplasts transiently expressing green fluorescent protein (GFP) fusion constructs. The resulting GFP fluorescence signal matched the chlorophyll autofluorescence patterns within the cells (Fig. 7A–D), indicating that this enzyme resided in the chloroplast. In contrast, transient expression of GFP-HvDMP fusion proteins in tobacco leaves showed a reticulate pattern and colocalized with ER-specific marker HDEL-RFP (Fig. 7E–G), suggesting that the HvDMP protein resides in the ER.

Methods
Chemicals
All chemicals were obtained from Sigma-Aldrich, unless specified otherwise below.

Plant materials and growth conditions
H. vulgare cv. Morex (NGB23015) seeds were obtained from the Nordic Genetic Resource Center (Alnarp, Sweden), seeds of H. vulgare cv. Ingrid and emr1 were kindly provided by Dr. U. Schaffrath (Technische Hochschule Aachen, Germany). Seeds were germinated on moist filter paper in Petri dishes at room temperature, then transferred to soil (Sunshine Mix 5, Sun Gro) in 10-l pots. Plants were grown in a growth chamber under 16-h light/21°C, 8-h dark/19°C cycles. For barley protoplast preparation, barley cv. Morex seeds were germinated on moist filter paper in Petri dishes, and plantlets were transferred to soil in small pots covered with plastic domes and kept in the growth chamber for 10 days under the conditions described above. Tobacco (Nicotiana benthamiana) plants grown under the same conditions for 1 month were used for leaf transient expression.

RNA isolation, reverse transcription, plasmid construction, and transformation
Flag leaf sheaths and spikes of H. vulgare cv. Morex were excised and immediately frozen in liquid nitrogen, total RNA was extracted using PureLink RNA mini kit (Invitrogen), and genomic DNA was removed by on-column DNase digestion with PureLink DNase (Invitrogen) following the manufacturer’s protocol. In total, 1 µg of resulting total RNA and Oligo(dT) primer (Invitrogen) was used to synthesize first-strand complementary DNA (cDNA) with SuperScript III Reverse Transcriptase (Invitrogen). The resulting cDNA served as a template for gene cloning.

E. coli expression vectors pET28-HvDMH, pET28-HvDMP and pET28-TEVH were kindly provided by Dr. A. Aharoni (Weizmann Inst., Rehovot, Israel). The expression cassette contains an N-terminal 6xHis-tag followed by a TEV cleavage site and the open reading frame (ORF) of the target gene. To construct E. coli expression vectors pET28a-EcACP, pET28a-EcFabD and pET28a-MtFabH, EcACP was amplified from E. coli (DH 5α) with Phusion polymerase (NEB) using primers EcACP-NdeI-F/EcACP-BamHI-R (Supplementary Table S4). The EcFabD and MtFabH sequences were synthesized by Twist Bioscience and further amplified using primers EcFabD-NdeI-F/EcFabD-BamHI-R and MtFabH-NdeI-F/MtFabH-BamHI-R, respectively. The PCR products were purified and inserted into pET28a vector and in frame with N-terminal 6xHis-tag followed by a thrombin cleavage site. E. coli transformed with target vector were screened on lysogeny broth (LB) plates containing 50 µg/ml kanamycin, and verified by colony PCR, restriction digestions, and Sanger sequencing. The confirmed expression vectors were transformed into E. coli BL21 (DE3) competent cells (Invitrogen) according to the manufacturer’s protocol, and the presence of the gene insert was verified by colony PCR. Three to four independent transformants of each E. coli line were selected for further in vivo assay or protein purification.

To construct plant expression vectors, the HvDMH and HvDMP coding regions were amplified from H. vulgare cv. Morex flag leaf sheath cDNA with primers HvDMH-F/HvDMH-R and HvDMP-F/HvDMP-R, respectively (Supplementary Table S4). PCR products were ligated to Gateway entry vector pCR8/GW/TOPO (Invitrogen) according to the manufacturer’s protocol, and the presence of the target sequences was verified by colony PCR and Sanger sequencing. LR reactions were performed with LR Clonase II enzyme mix (Invitrogen) to transfer target genes from the entry vector to the destination vector for in-frame C-terminal or N-terminal fusion with GFP. Positive transformants were identified by colony PCR and Sanger sequencing. The resulting constructs were transformed into Agrobacterium tumefaciens GV3101 competent cells by electroporation, screened on LB plates containing proper antibiotics, and verified by colony PCR. Three verified transformants were cultured for tobacco transient expression.

To construct yeast expression vectors, the coding sequence of long-chain acyl-CoA synthetase 1 (LACS1) that enhances yeast lipid uptake61 was amplified from Arabidopsis stem cDNA using primers LACS1-BamHI-F and LACS1-SalI-R and inserted into the pESC-Trp yeast expression vector in-frame with C-terminal MYC epitope tag, for expression under control of the galactose-inducible promoter GAL1, resulting in construct pESC-Trp-GAL1::LACS1-MYC. HvDMP was sub-cloned from pGWB6-35Spro::GFP-HvDMP using primers HvDMP-EcoRI-F and HvDMP-SpeI-R and spliced into pESC-Trp-GAL1::LACS1-MYC vector, C-terminally in-frame with FLAG epitope tag, resulting in construct pESC-Trp-GAL1::LACS1-MYC-GAL10::HvDMP-FLAG. E. coli transformed with either vector were screened on LB plates containing 100 µg/ml ampicillin, and verified by colony PCR, restriction digestions and Sanger sequencing. The resulting expression vectors were transformed into yeast strain INVSc1 (MATα/MATa his3Δ1/his3Δ1 leu2/leu2 trp1-289/trp1-289 ura3-52/ura3-52) as described62, and the resulting transformants were screened on yeast minimal medium plates lacking the appropriate amino acids. The transformed yeast colonies were verified by colony PCR. Three to four verified yeast transformants were selected for further in vivo assays.

Chemical synthesis of standards and substrates
C31 14,16-diketone (hentriacontane-14,16-dione). In all, 1 g of hexadecanol was stirred at room temperature with 2.5 g of Dess-Martin reagent periodinane in 30 ml CH2Cl2. Upon completion of the oxidation (monitored by TLC), the reagent was quenched with aqueous Na2S2O3 solution. The mixture was extracted with CH2Cl2, and the combined organic fractions were washed with aqueous NaHCO3 solution, dried with Na2SO4 and concentrated in vacuo. The product, hexadecanal, was purified by column chromatography (CHCl3) and verified by GC-MS analysis.

In all, 0.7 g 2-pentadecanone was stirred at 0°C with 0.1 M lithium diisopropylamide in 11.5 ml tetrahydrofuran (THF) for 10 min, then 2 ml THF containing 0.8 g hexadecanal were added drop-wise, and after 1 h the reaction was quenched with 5 ml 20% H2SO4. The mixture was extracted with CH2Cl2, and the combined organic solutions were washed, dried and concentrated described as above. The product, 16-hydroxyhentriacontan-14-one, was purified by column chromatography (1:1 hexane:CHCl3) and verified by GC-MS analysis.

In all, 0.5 ml 1 M oxalyl chloride in CH2Cl2 was added drop-wise to 150 µl dimethyl sulfoxide (DMSO) in 2 ml CH2Cl2 under stirring at −78°C. After gas evolution had ceased, 0.2 g 16-hydroxyhentriacontan-14-one in 3 ml CH2Cl2 were added drop-wise, the reaction mixture was allowed to warm to room temperature, and then 0.3 ml Et3N was added. The resulting solid was re-dissolved by adding 5 ml water, and organic products were extracted with CH2Cl2. The combined organic phases were washed first with diluted HCl and then with water, dried with Na2SO4, and concentrated. The resulting product, hentriacontane-14,16-dione, was purified by column chromatography (3:1 hexane:CHCl3) and verified by MS analysis.

Other diketone homologs were synthesized following the same protocol from starting materials with respective chain lengths.

C21 2-alkanol (2-heneicosanol). In all, 91 mg of LiAlH4 were added to a solution of 500 mg 2-heneicosanone in 10 ml CH2Cl2. The reaction was quenched after 1 h by slowly adding 2 ml of 1 N HCl, and products were recovered by extraction with CHCl3. Organic fractions were combined, washed with saturated aqueous NaHCO3, dried with Na2SO4, and concentrated under vacuum. The final product was purified by column chromatography (CHCl3).

C14–C18 3-ketoacids. C16 3-ketoacid methyl ester was synthesized as described previously63, with small modifications: 0.45 g myristic acid was dissolved in 3 ml CH2Cl2 containing 0.4 g N,N’-dicyclohexylcarbodiimide. Separately, 1.9 g 4-dimethylaminopyridine was added to 20 ml pyridine containing 1.6 g Meldrum’s acid. Both mixtures were stirred at room temperature for 15 min before combining them. After overnight stirring, the solvent was evaporated under vacuum and the solid re-dissolved in 20 ml methanol supplemented with two drops of concentrated H2SO4. The mixture was refluxed overnight, and product was extracted with Et2O. The combined organic fractions were washed with aqueous NaHCO3 solution, distilled water and finally saturated NaCl, the organic phase removed and dried with Na2SO4, and the solvent evaporated in vacuo. The resulting solid was purified by column chromatography (CHCl3) and product purity verified by GC-MS analysis.

In all, 0.5 g C16 3-ketoacid methyl ester was dissolved in glacial acetic acid supplemented with concentrated HCl, and the mixture stored at 10°C. Prior to each experiment, product was obtained by extraction with Et2O. The organic phase was washed with deionized water, and the solvent evaporated under vacuum. GC-MS analysis showed that the crude product contained non-hydrolyzed C16 3-ketoacid methyl ester and C16 3-ketoacid as main products, accompanied by small amounts of the decarboxylation product, C15 2-ketone. This mixture was separated by flash column chromatography (CHCl3), to yield pure C16 3-ketoacid for use in biochemical assays (while C16 3-ketoacid methyl ester was recovered for further hydrolysis).

The same protocols were used to synthesize C14, C17, and C18 3-ketoacids.

Enzyme assays
E. coli in vivo assay. Three independent E. coli BL21 (DE3) colonies each carrying pET28-TEVH or pET28-HvDMH were inoculated into 1 ml LB liquid medium with kanamycin and precultured at 37°C overnight. Then, each broth was diluted 1:100 in 100 ml LB liquid medium supplemented with kanamycin, cultivated at 37°C to OD600 ~0.6, supplemented with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce the expression of recombinant protein, and incubated at 22°C overnight. Then, the OD were measured for normalization against cell numbers and E. coli cells collected by centrifugation for lipid analysis.

Yeast in vivo assay. Three independent yeast colonies each of lines expressing pESC-Trp-GAL1::LACS1-MYC or pESC-Trp-GAL1::LACS1-MYC-GAL10::HvDMP-FLAG were inoculated into 2 ml appropriate liquid minimal medium supplemented with 2% glucose and incubated at 28°C overnight. The precultures were then expanded to 30 ml and cultivated for 18 h. The resulting yeast cells were collected by centrifugation and transferred into liquid minimal medium supplemented with 2% galactose to induce the expression of target genes and cultivated at 28°C for 16 h. Then, yeast cells were transferred to 30 ml liquid minimal medium based on phosphate buffer (20 mM Na2HPO4/NaH2PO4, 300 mM NaCl, pH 7.4) and containing 2% galactose. Enzyme substrates (Supplementary Table S5) dissolved in ethanol were added (0.22 mM final concentrations of fatty acids and 3-ketoacids), and the yeast cells were cultivated for another 24 h at 22°C. Finally, the OD600 of the resulting yeast were measured for normalization against cell numbers, and cells were harvested by centrifugation for further product analysis.

Protein purification and in vitro assay. E. coli BL21 (DE3) colonies carrying pET28a-EcACP, pET28a-EcFabD, pET28a-MtFabH, pET28-HvDMH or pET28-HvDMP were inoculated into 5 ml LB liquid medium with kanamycin and precultured at 37°C overnight. E. coli precultures were diluted 1:100 in LB liquid medium supplemented with kanamycin and cultivated at 37°C to OD600 ~0.6, and 0.5 mM IPTG was added to induce expression of recombinant protein. After incubating at 16°C overnight (E. coli expressing MtFabH were incubated at 37°C for 3 h), E. coli cells were collected by centrifugation (10,000 × g, 4°C). All following steps of protein purification were performed using phosphate-based buffers (20 mM Na2HPO4/NaH2PO4, 300 mM NaCl with imidazole, pH 7.4) supplemented with 1% Triton X-100, and equilibration buffer was supplemented with 0.1 mM phenylmethylsulfonyl fluoride (PMSF) as protease inhibitor. E. coli pellets were resuspended in equilibration buffer and lysed by sonication at 4°C. The recombinant EcACP, EcFabD, MtFabH, HvDMH, and HvDMP proteins were obtained by Ni-NTA affinity purification with HisPur Ni-NTA resin (Invitrogen) following the manufacturer’s protocol. Respective recombinant proteins were eluted in phosphate buffer and dialyzed three times for 7 h in dialysis buffer (0.1 M Na2HPO4/NaH2PO4, 2.5 mM dithiothreitol (DTT), 10 mM MgCl2, 0.025% Triton X-100, pH 7.4) at 4°C. The purity of recombinant proteins was verified by SDS-PAGE, and the protein concentration was determined by protein assay using the Bradford method with Protein Assay Dye Reagent Concentrate (Bio-Rad). The obtained proteins were used immediately in enzyme assays.

HvDMH in vitro assays were performed as described previously40,41. In brief, recombinant EcACP (1.0 mg), EcFabD (100 µg) and MtFabH (100 µg) were coincubated in 2 ml 1,3-bis(tris(hydroxymethyl)methylaminopropane (20 mM, pH 7.0) containing malonyl-CoA (0.2 mM) and C14 acyl-CoA (0.2 mM) at 37°C for 5 h. 500 µg of HvDMH were added, and the reaction was incubated for an additional 2 h. The reaction was quenched by one drop of 2 mM HCl with tetracosane (3 µg) supplemented as an internal standard.

For HvDMP in vitro assays, 50 µg of purified recombinant protein was incubated in 3 ml 50 mM phosphate buffer (pH 7.4) containing 2.5 mM DTT, 10 mM MgCl2 and 0.025% Triton X-100 with the substrate combinations (Supplementary Table S6) at room temperature for 14 h with gentle shaking. The reaction was quenched by adding 200 µl 10% H2SO4, and the reaction products were extracted immediately for lipid analysis.

Lipid analysis
Wax analysis. Flag leaf sheaths and spikes were excised from H. vulgare cv. Morex plants grown 85 d after germination. Surface waxes were extracted by submerging tissues twice for 30 s in CHCl3, extracts were combined, and the solvent was evaporated under gentle N2 flow. Samples were transferred into GC vials and derivatized with 10 µl bis-N,O-trimethylsilyltrifluoroacetamide (BSTFA) in 10 µl pyridine at 72°C for 45 min. The reagents were evaporated under gentle N2 flow, and the product was dissolved in 200 µl CHCl3. The derivatized wax mixtures were then analyzed on a Gas Chromatography system (6890N, Agilent) equipped with HP-1 capillary column (30 m × 0.32 mm i.d., 1-µm film thickness, Agilent) coupled with Mass Selective Detector (5793N, Agilent) using cool-on-column injection and the oven program: 50°C held for 2 min, raised by 40°C min−1 to 200°C and held for 2 min, raised by 3°C min−1 to 320°C and held for 30 min.

E. coli lipid analysis. E. coli pellets were washed with distilled water, collected by centrifugation and allowed to air-dry. Known quantities of C21 2-ketone, C21 2-alkanol and C20 2-hydroxyacid methyl ester were added to each sample for quantification of respective compound classes, E. coli cells were resuspended in 1 ml methanol containing 0.5 M sulfuric acid and 2% (v/v) 2,2-dimethoxypropane, and incubated at 80°C for 3 h. Then 2.5% NaCl solution (w/v) was added, and the mixture was extracted three times with hexane, and the combined organic phases were concentrated under N2 flow and derivatized as described above. The final products were dissolved in CHCl3 and analyzed by GC-MS as above, but with the following oven program: 50°C held for 2 min, raised by 40°C min−1 to 100°C and held for 2 min, raised by 3°C min−1 to 320°C and held for 5 min. The correction factor for quantifying 3-hydroxyacids was experimentally determined using authentic C16 3-hydroxyacid and C20 2-hydroxyacid standards.

Yeast lipid analysis. Overall, 30 ml-yeast cultures were split into 25 ml for β-diketone analysis and 5 ml for fatty acid profiling, and cells were collected by centrifugation, washed with distilled water, and allowed to air-dry. For product analysis, C35 14,16-diketone was supplemented to yeast pellets as internal standard. Yeast cells were resuspended in 3 ml distilled water and lysed with 0.5 mm glass beads (BioSpec) by vertexing. 2 ml saturated NaCl solution was added, and total lipids were extracted three times with CHCl3, the combined organic phases were dried with Na2SO4 and filtered, and the solvent was evaporated under vacuum. The resulting lipids were transferred into GC vials, derivatized, and analyzed by GC-MS as described for wax analysis. All diketones were quantified using m/z 100 selected-ion traces. For fatty acid profiling, yeast cells were resuspended in 1 ml methanol containing 0.5 M sulfuric acid and 2% (v/v) 2,2-dimethoxypropane. The transmethylation reaction was performed as described above, and products were extracted with hexane, derivatized as previously described, and analyzed by GC-MS as described for E. coli lipid analysis.

In vitro product analysis. Samples from HvDMH in vitro assay were extracted three times with hexane, and the combined organic phases were concentrated under N2, derivatized and analyzed by GC-MS as described for wax analysis. Samples of the HvDMP in vitro reaction mixture were prepared in the same way, but extracting twice with CHCl3 and drying under vacuum.

Carbon isotope analysis
Wax was extracted from H. vulgare cv. Morex flag leaf sheaths as described above and fractionated by TLC (CHCl3: hexane (1:1); 0.25 mm layer thickness; Analtech). TLC bands were stained with primuline (1% in acetone) and visualized under UV light, target fractions were scratched from the plate and extracted with CHCl3, and fraction purities were verified by GC-MS analysis. For the two fractions containing esters/alkanes and β-diketones, compound-specific carbon isotope compositions were determined using GC-IRMS as described before64. In brief, a trace GC system equipped with Rxi-5ms column (30 m × 0.25 mm, film thickness 0.25 µm, Restek) and a programmable temperature vaporizing injector operated in solvent split mode was connected via a GC Isolink to a combustion furnace at 1000°C and via a Conflo IV interface further to a Delta V Plus isotope ratio mass spectrometer (Thermo Scientific). The CO2 reference gas was analyzed daily with a standard deviation of <0.06‰ for instrument stability and linearity. Six reference peaks of CO2 bracketed analyte peaks during the course of a GC-IRMS run; two were used for standardization, the rest were used to monitor stability. Samples were interspersed with an external standard (A6 mix) obtained from Dr. A. Schimmelmann, Indiana University, Bloomington, containing 15 alkanes (C16 to C30) with δ13C values ranging from −33.34 to −26.15‰ in order to establish a multi-point correlation, with which data were normalized to the Vienna Pee Dee Belemnite (VPDB) carbon isotopic scale.

Barley protoplast and tobacco leaf transient expression and microscopy
Two-week-old H. vulgare cv. Morex seedlings were used for protoplast preparation and transformation following the method described previously65. pGWB5-35Spro::HvDMH-GFP was used for protoplast transient expression. Transformed protoplasts were incubated for 16–18 h and imaged by an Olympus FV1000 multiphoton confocal laser scanning microscope equipped with 405 nm, 473 nm, and 559 nm lasers. Transient expression of pGWB6-35Spro::GFP-HvDMP and ER-specific marker p35S:HDEL-RFP (obtained from Drs. M. Schuetz and L. Samuels, UBC, Vancouver) were in N. benthamiana was performed as described previously66 and imaged with the same microscope. GFP and RFP were at 488 nm and 561 nm, respectively, and fluorescence of GFP, RFP, and chlorophyll was detected at 525 nm, 595–625 nm, and 660–750 nm, respectively. ImageJ 1.51r software was used for image processing.

Statistics and reproducibility
Microsoft Excel (version 2108) and GraphPad Prism 10 software were used for statistical analysis. Two-tailed Student’s t tests were used to assess significant differences between samples in different assays or wax analyses. Details are provided in the captions of the corresponding figures. All experiments and assays were repeated independently two to three times with similar results.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Sun et al. reported the discovery of the biosynthetic pathway of barley wax beta-diketones involved in a hydrolase HvDMH and a type III polyketide synthase (PKS), HvDMP. Through in vivo enzyme assays using yeast and in vitro assays, the authors showed that this type III PKS harbors novel beta-diketones-producing PKS activities and HvDPH has been also suggested to be a hydrolase. It is thus strongly speculated that both enzymes are indeed involved in the biosynthesis. The HvDPH activity is also novel as the type III PKS. Overall, the topic of the study is interesting, and the quality of the experimental data is generally good. However, some points should be more reliable to support authors conclusion and additional studies are needed to publish it in Nature Communications.\n1. A major weakness of the paper is that the in vivo biochemical function of HvDMP in the plants is not clearly demonstrated. It is known that in vitro activities of plant type III PKSs sometimes differ from their major activities in the heterologous expression system. To support the main claim “HvDMP is involved in the biosynthesis of barley beta-diketides”, the authors need to demonstrate HvDMP indeed harbors the claimed activities in the plant. For example, this can be demonstrated by VIGS experiment in barley or its tissue culture, which may include callus, to knockdown expression of HvDMP. The transient expression of HvDMP in Nicotiana may be also available for this. This can be also said to HvDMH as well.\n2. HvDMH has been concluded to hydrolyze 3-ketoacyl-ACPs to produce 3-ketoacid type. However, it is unclear from the present study only. To support this, the authors need to demonstrate HvDMH indeed harbors the claimed activities in vitro or equivalent methods.\n3. Substrate preference of MvDMP: To understand the enzyme mechanism, a basic steady-state kinetic analysis is required. If the authors want to compare the relative activity (%) of each product (or for each substrate), at least, values of the catalytic efficiency (kcat/Km) or maximal velocity (Vmax) of each enzyme should be provided. Otherwise, to confirm the contribution of ligand-binding, the authors can provide experimental protein-ligand binding results using Isothermal titration calorimetry (ITC) or Surface Plasmon Resonance (SPR).\n4. In the discussion, authors mentioned that “Only two plant PKSs, CURS and CUS, had previously been known to catalyze reaction somewhat similar to HvDMP”. However, this description is mistake. As the similar type of type III PKSs, HsPKS3 from Huperzia serrata, alkylquinolone synthase (AQS) from Evodia rutaecarpa, and diarylpentanoid-producing polyketide synthase (PECPS) from Aquilaria sinensis have been also reported. The description and sequential discussion should be corrected.\n5. In the proposed HvDMP reaction mechanism, Ser200 and Glu196 have been mentioned as the residues involved in the proton donor for the catalytic Cys. However, to the best of my knowledge, such mechanism has been never proposed to the type III PKS: The His residue in the catalytic triad has been suggested as such residue. To propose this mechanism, more explanation why authors could propose this should be provided.", "reviewer_2": "The authors identified the β-diketone biosynthesis pathway in barley by biochemical characterizations of two enzymes DMH and DMP from previous identified gene cluster. DMH is a thioesterase producing long-chain (mainly C16), and DMP is a rare polyketide synthase (PKS) condensing the 3-ketoacids with long-chain (mainly C16) acyl-CoAs into β-diketones, which are functionally similar to known curcuminoid synthase. The manuscript is very easy to understand and there is nothing new for natural products biosynthetic field. This significance of this paper is that the waxy natural product is related with the surface coatings of cereal plants barley and the authors demonstrated the details of the PKS mediated key step biosynthesis. Anyway, additional modification may be needed to improve the manuscript as listed below before acceptance.\nMajor comments\n1. Page 4: the description “While this provided first evidence for the enzymatic reaction leading to the key intermediate, it did not provide information on the later steps in the pathway.” is not proper. Ref 19 identified that DMP in the gene cluster participated in the β-diketone biosynthesis by gene silencing experiments, and provided information about that DMP showed significant sequence similarity to curcuminoid synthase, which catalyzes the head-to-head condensation of two acyl chains. Please make some suitable changes.\n2. Page 10: The C-terminal HDEL enables proteins to be localized in the endoplasmic reticulum (ER). Why use the marker HDEL-RFP, not the RFP-HDEL? In addition, please analyze the localization signal peptide of DMP. Pro35S: GFP-HvDMP was lost.\nMinor comments\n3. Figure 2A, please provide the complete histogram information and the standard deviation of C31 diketone.\n4. Page 17: “µ” in “1 µg of resulting total RNA” need to be Italic, “N” in “an N-terminal 6xHis-tag followed” need to be Italic.\n5. The formats of ref 3, 20, 33, 34, 35, 36, 37, 40 and 50 need to be checked.", "reviewer_3": "Review of the article Biosynthesis of barley wax β-diketones: a type-III polyketide synthase condensing two fatty acyl units. Sun et al.\nThis is a well written, well organized, and highly informative paper. The investigation of barley β-diketones was among the first genetic models for the study of plant wax biochemical genetics, and the presence of barley’s structurally unique β-diketone class of compounds has been a focus of much interest since the 1970s beginning especially with the work of Penelope von Wettstein-Knowles. Previous publications indicate that the B-diketones themselves are important in plant environmental stress adaptation (especially for plant tolerance to drought, and supra-optimal solar radiation and heat), and thereby constitute an ecologically important class of waxes, and define overall a class of wax compounds with high potential for modification toward improving crop stress tolerance.\nThe authors apply a straightforward scientific approach that elucidates a novel metabolic pathway being employed by barley (and likely other plants that synthesize β-diketones) for the synthesis of the β-diketones and its derivatives the hydroxy β-diketones as well as the related compounds 2-alkanol esters. A key finding is that the 3-ketoacids are a central intermediary in these pathways, and that the barley derived enzymes Diketone Metabolism Hydrolase (DMH) and Diketone Metabolism Polyketide Synthase (DMP) are together sufficient to generate the β-diketones from plastidial acyl precursors. The authors have a strong publication record involving the chemical analysis of plant waxes, as well as related metabolic engineering studies, and the methods used in this paper are sound.\nThe authors provide strong evidence that the biosynthesis of β-diketones and hydroxy β-diketones are produced in a way that it does not utilize the previously described sequential 2-carbon-based elongation steps catalyzed by the Fatty Acid Elongase (FAE) pathway, and that their synthesis does not require the endoplasmic reticulum (ER)-localized β-ketoacyl-CoA synthase (KCS6). The authors demonstrated the emr1 mutant deficient in KCS6 was able to produce β-diketones with normal chain length, even though other wax constituents on emr1 were much shorter, and so β-diketone synthesis in barley does not require that same elongation pathway. The authors present evidence that there is not another β-diketone parallel elongation pathway, citing a previous article showing that fatty acyl chain elongation inhibitors do not inhibit elongation of β-diketones and hydroxy β-diketones, indicating that β-diketone synthesis does not involve the previously describes ER based FAE. Further, carbon isotope studies by the authors of this paper using GC coupled with isotope-ratio mass spectrometry revealed that the β-diketones on barley are composed of all plastid generated carbons (enriched in 13C), and not the ER associated carbons wherein only about 50% of the carbons would be plastidial if the chains were further elongated using the FAE pathway mechanism (wherein 13C composition would be low). The authors tested these 13C ratios against other wax classes in barley, providing strong evidence to support a novel synthesis that does not involve ER elongation by FAE.\nInstead, the authors provide strong evidence for an alternative and novel very long chain β-diketone synthesis pathway that involves Diketone Metabolism Hydrolase (DMH) that has a thioesterase function and generates fatty acyl derivatives with functional groups on the 3rd carbon, and a Diketone Metabolism Polyketide Synthase (DMP) that performs a decarboxylative condensation of 3-ketoacid acyl chains and fatty acyl-CoA to make the β-diketones. E. coli and yeast expression studies were used to demonstrate both HvDMH and HvDMP preferred substrate classes and chain length specificity. It’s notable that the yeast system expressing HvDMH could not make the 3-ketoacids substrate for use by HvDMP in synthesis of β-diketones, and so that is why exogenous 3-ketoacids needed to be added to test HvDMP activity. As such, this study does not directly show that HvDMH catalyzes the thioesterase reaction leading to 3-ketoacyls as would be necessary in the plant system for β-diketone production. The authors speculate that HvDMH might not interact properly (as it would in planta) with the yeast FAS complexes, or that it requires specific ACP isoforms and discriminates against yeast ACPs, which are reasonable speculations by these authors. However, HvDMH expressed in E. coli did hydrolyze substrates to make acyl products having 3-carbon functional groups, which provided evidence that HvDMH likely generates the 3-ketoacyls in planta. It is known that E. coli has very low relative amounts of saturated acyl substrates overall (relative to unsaturated), which may explain why detectable amounts of the specific 3-ketoacyls were not generated by HvDMH in the E. coli system.\nStudies in the yeast system using deuterated C16 acids showed that HvDMP could condense (with decarboxylation) a deuterated C16 fatty acid with 3-ketoacids to make β-diketones, and so this then shows that HvDMP has the capacity to combine two long chain substrates together. HvDMP’s ability to condense fatty acyl-CoAs with ketoacid substrates was further confirmed in yeast experiments using uncommon odd chain length substrates. Further in vitro/yeast studies tested chain length specificity of HvDMP to find that the HvDMP enzyme prefers the C14 3-ketoacid, and either the C14 or C16 fatty acyl-CoA. But notably HvDMP does in fact readily accepts the C16 3-ketoacid. The fact the C16 3-ketoacids and C16 fatty acyl-CoAs are by far the dominant substrate pool available in planta explains why the C31 14,16-diketones are the dominant homologues found on the surface of wild-type barley sheaths, being 96% of the waxes. The functions described here for HvDMP are thus consistent with the β-diketone wax profiles observed on barley.\nFinally, the authors localized both HvDMH and HvDMP using confocal microscopy, barley protoplast, and GFP constructs, showing the HvDMH enzyme was localized in the chloroplasts and HvDMP in ER membranes. They proposed a quite reasonable model for substrate transfer (and related reaction mechanisms) in the biosynthetic pathway.\nThey also describe for the first time unbranched even chain length β-diketones, likely derived from an initial C3 rather than the more common C2 moiety. They then noted that the locations of the functional groups on these even chain β-diketones are consistent with a main finding of this report that the C16 3-ketoacids are a major intermediate in this β-diketone pathway.\nAlthough not necessary for publication in the opinion of this reviewer (as will be described below), additional evidence that HvDMH directly catalyzed synthesis of the 3-ketoacyls in yeast or E. coli (or another system) would have helped strengthen the narrative that two enzymes are sufficient for β-diketone synthesis. Notwithstanding, that the E. coli studies showed that HvDMH produced closely related 3-carbon modified acyl derivatives does provide good evidence that the HvDMH does in fact generate 3-ketoacyls in planta. Regardless, the presentation of only indirect evidence of HvDMH function in 3-ketoacyl synthesis does not detract from the overall main discoveries of this paper, which is the determination that the HvDMP enzyme generates the β-diketones, and that this is done using a novel reaction that does not involve the well-known sequential C2 FAE elongation steps, but instead uses HvDMP to perform a decarboxylative condensation reaction of two long chain plastidial derived substrates. There are no previous reports of such a condensation reaction of long chain acyl compounds in plants, and this is the first report of the synthesis of very long chain aliphatic compounds that does not involve C2 elongation steps.\nThe findings here represent novel and remarkable discoveries on their own and are clearly supported by the data and its interpretation.\nSome minor edits requested:\nUnder carbon isotope analysis in methods, lines 2 and 3, recommend simply rewording ass “were stained with””.\nRemove the word “at” in line 15 for Fig 1 caption.\nIn Fig 2A, the green colors in the legend are a bit difficult to align to the bars. Making the colors more different will improve the ease of interpretation. The colors in Fig 2 C are also difficult to visualize. Point symbols should be enlarged"}, "author_response": "Reviewer #1 (Remarks to the Author):\nSun et al. reported the discovery of the biosynthetic pathway of barley wax beta-diketones involved in a\nhydrolase HvDMH and a type III polyketide synthase (PKS), HvDMP. Through in vivo enzyme assays using\nyeast and in vitro assays, the authors showed that this type III PKS harbors novel beta-diketones-\nproducing PKS activities and HvDPH has been also suggested to be a hydrolase. It is thus strongly\nspeculated that both enzymes are indeed involved in the biosynthesis. The HvDPH activity is also novel\nas the type III PKS. Overall, the topic of the study is interesting, and the quality of the experimental data\nis generally good. However, some points should be more reliable to support authors conclusion and\nadditional studies are needed to publish it in Nature Communications.\n1. A major weakness of the paper is that the in vivo biochemical function of HvDMP in the plants is not\nclearly demonstrated. It is known that in vitro activities of plant type III PKSs sometimes differ from their\nmajor activities in the heterologous expression system. To support the main claim “HvDMP is involved in\nthe biosynthesis of barley beta-diketides”, the authors need to demonstrate HvDMP indeed harbors the\nclaimed activities in the plant. For example, this can be demonstrated by VIGS experiment in barley or its\ntissue culture, which may include callus, to knockdown expression of HvDMP. The transient expression\nof HvDMP in Nicotiana may be also available for this. This can be also said to HvDMH as well.\nWe agree that the in planta activity of the enzymes must be addressed and apologize for neglecting this\nin the original version of the manuscript. Indeed, detailed and repeated analyses of respective barley\nmutants had already firmly established the central roles of both enzymes, DMH and DMP, in the\nformation of beta-diketones. Corresponding VIGS experiments independently confirmed the in central\nrole of both enzymes also in beta-diketone biosynthesis in wheat. Given the high genetic similarity of\nboth plant species, the in planta function of both enzymes on this pathway had thus been firmly\nestablished prior to our work, and there was consequently no need for us to repeat respective mutant\nanalyses. However, it was important to reference the previous work on this, and we have added this\ninformation to the Introduction section of our manuscript.\n2. HvDMH has been concluded to hydrolyze 3-ketoacyl-ACPs to produce 3-ketoacid type. However, it is\nunclear from the present study only. To support this, the authors need to demonstrate HvDMH indeed\nharbors the claimed activities in vitro or equivalent methods.\nWe thank the reviewer for this suggestion and have since performed the necessary experiments to test\nthe HvDMH activity in vitro. A new figure (fig 4) presents the additional results, and a newly added\nparagraph in the Results section on HvDMH explains the findings.\n3. Substrate preference of MvDMP: To understand the enzyme mechanism, a basic steady-state kinetic\nanalysis is required. If the authors want to compare the relative activity (%) of each product (or for each\nsubstrate), at least, values of the catalytic efficiency (kcat/Km) or maximal velocity (Vmax) of each\nenzyme should be provided. Otherwise, to confirm the contribution of ligand-binding, the authors can\nprovide experimental protein-ligand binding results using Isothermal titration calorimetry (ITC) or\nSurface Plasmon Resonance (SPR).\nWe agree that a kinetic characterization of the enzymes would help to quantify specificities. However,\ndue to the highly lipophilic nature of the substrates, their concentrations in respective assays cannot be\ncontrolled and varied enough for proper measurements of kinetic parameters. Therefore, kinetics of no\nenzymes handling long-chain aliphatics have been reported to date, and substrate specificities were\nassessed qualitatively at best. For example, most previous work on long-chain or very-long-chain acyl\ncondensing enzymes tended to report only product specificities but not substrate specificities. In\ncomparison with this precedence, our in vitro characterizations and competition assays stretch the limits\nfar beyond the previous methods for assessing relative activities on various substrates. However, we\nunderstand that biochemists outside the acyl lipid field may have different expectations regarding enzyme\ncharacterization. Therefore, we have toned down our descriptions and interpretations of the various\nexperiments into this, for example by replacing “specificity” with “preference”.\n4. In the discussion, authors mentioned that “Only two plant PKSs, CURS and CUS, had previously been\nknown to catalyze reaction somewhat similar to HvDMP”. However, this description is mistake. As the\nsimilar type of type III PKSs, HsPKS3 from Huperzia serrata, alkylquinolone synthase (AQS) from Evodia\nrutaecarpa, and diarylpentanoid-producing polyketide synthase (PECPS) from Aquilaria sinensis have\nbeen also reported. The description and sequential discussion should be corrected.\nThanks for pointing this out. We have added a paragraph discussing these enzymes, and illustrated the\nreactions they catalyze either in planta or in vitro in a new supplementary figure (fig. S9).\n5. In the proposed HvDMP reaction mechanism, Ser200 and Glu196 have been mentioned as the\nresidues involved in the proton donor for the catalytic Cys. However, to the best of my knowledge, such\nmechanism has been never proposed to the type III PKS: The His residue in the catalytic triad has been\nsuggested as such residue. To propose this mechanism, more explanation why authors could propose\nthis should be provided.\nOur hypothetical reaction mechanism closely follows those proposed by Morita et al 2010 (PNAS 107,\n19778-83) and Morita et al 2019 (JBC 294, 15121-36) for the CUS enzyme, since it catalyzes a head-to-\nhead condensation fairly similar to ours. We understand that, in their model, the His in the catalytic\ntriad is assumed to protonate the CoA leaving group of the starter acyl and to deprotonate the catalytic\ncysteine after the reaction cycle. However, the re-protonation of the catalytic (via water) at the end of\nthe catalytic cycle had in many cases not been shown for related enzyme mechanisms. It is plausible to\ninvoke a combination of nearby Ser/Glu for this proton flow, but we agree that this part of our original\nmechanism was highly speculative. To avoid confusion, we deleted respective details of the mechanism,\nand the new version now matches the mechanisms proposed in various previous papers to the best of\nour knowledge.\nReviewer #2 (Remarks to the Author):\nThe authors identified the β-diketone biosynthesis pathway in barley by biochemical characterizations of\ntwo enzymes DMH and DMP from previous identified gene cluster. DMH is a thioesterase producing\nlong-chain (mainly C16), and DMP is a rare polyketide synthase (PKS) condensing the 3-ketoacids with\nlong-chain (mainly C16) acyl-CoAs into β-diketones, which are functionally similar to known curcuminoid\nsynthase. The manuscript is very easy to understand and there is nothing new for natural products\nbiosynthetic field. This significance of this paper is that the waxy natural product is related with the\nsurface coatings of cereal plants barley and the authors demonstrated the details of the PKS mediated\nkey step biosynthesis. Anyway, additional modification may be needed to improve the manuscript as\nlisted below before acceptance.\nMajor comments\n1. Page 4: the description “While this provided first evidence for the enzymatic reaction leading to the\nkey intermediate, it did not provide information on the later steps in the pathway.” is not proper. Ref 19\nidentified that DMP in the gene cluster participated in the β-diketone biosynthesis by gene silencing\nexperiments, and provided information about that DMP showed significant sequence similarity to\ncurcuminoid synthase, which catalyzes the head-to-head condensation of two acyl chains. Please make\nsome suitable changes.\nWe have added text to the Introduction passage focusing on the sequence of steps on the pathway and\nthe ketoacid as a key intermediate, to explain the previous mutant/gene silencing results (see also\nresponse to reviewer #1, point 1).\nWe have also modified the Introduction passage detailing previous knowledge/hypotheses for the DMP\nenzyme, to emphasize that the sequence similarity between the wheat DMP and Curcuma longa CURS\nproteins had been noticed prior to our work.\n2. Page 10: The C-terminal HDEL enables proteins to be localized in the endoplasmic reticulum (ER). Why\nuse the marker HDEL-RFP, not the RFP-HDEL? In addition, please analyze the localization signal peptide\nof DMP. Pro35S: GFP-HvDMP was lost.\nBoth HDEL-RFP and RFP-HDEL have been used as ER marker, to our knowledge with equal success. Based\non amino acid sequence prediction, HvDMP does not contain a strong signal peptide.\nWe tried to co-express GFP-HvDMP and ER marker HDEL-RFP in barley protoplasts, but could not find\ncells harboring both these constructs. This was likely due to low transformation efficiencies combined\nwith difficulties in preparing barley protoplasts. We repeated this experiment three times, but without\nsuccess, before moving to tobacco transient expression to confirm the sub-cellular localization of\nHvDMP.\nMinor comments\n3. Figure 2A, please provide the complete histogram information and the standard deviation of C31\ndiketone.\nThanks for alerting us that this figure was difficult to read. We have improved the color contrasts to\nbetter emphasize the numbers showing the exact column height and variability. We hope this will help\nreaders understand that the y-axis is cut at 4%, thus truncating the bar for C diketone. We chose this\n31\ntruncation of the axis/bar to make the other bars better visible, since the main purpose of the figure is\nto illustrate the amounts of the minor homologs/isomers. (Introducing a y-axis break is not an option\nsince it would greatly deceive the eye on the relative bar heights of minor versus major components).\n4. Page 17: “µ” in “1 µg of resulting total RNA” need to be Italic, “N” in “an N-terminal 6xHis-tag\nfollowed” need to be Italic.\nWe would be happy to italicize respective letters, but to the best of our knowledge the Greek sign for\n“micro” and the N/C terminus designations of proteins are typically shown in normal font.\n5. The formats of ref 3, 20, 33, 34, 35, 36, 37, 40 and 50 need to be checked.\nDone\nReviewer #3 (Remarks to the Author):\nReview of the article Biosynthesis of barley wax β-diketones: a type-III polyketide synthase condensing\ntwo fatty acyl units. Sun et al.\nThis is a well written, well organized, and highly informative paper. The investigation of barley β-\ndiketones was among the first genetic models for the study of plant wax biochemical genetics, and the\npresence of barley’s structurally unique β-diketone class of compounds has been a focus of much\ninterest since the 1970s beginning especially with the work of Penelope von Wettstein-Knowles.\nPrevious publications indicate that the B-diketones themselves are important in plant environmental\nstress adaptation (especially for plant tolerance to drought, and supra-optimal solar radiation and heat),\nand thereby constitute an ecologically important class of waxes, and define overall a class of wax\ncompounds with high potential for modification toward improving crop stress tolerance.\nThe authors apply a straightforward scientific approach that elucidates a novel metabolic pathway being\nemployed by barley (and likely other plants that synthesize β-diketones) for the synthesis of the β-\ndiketones and its derivatives the hydroxy β-diketones as well as the related compounds 2-alkanol esters.\nA key finding is that the 3-ketoacids are a central intermediary in these pathways, and that the barley\nderived enzymes Diketone Metabolism Hydrolase (DMH) and Diketone Metabolism Polyketide Synthase\n(DMP) are together sufficient to generate the β-diketones from plastidial acyl precursors. The authors\nhave a strong publication record involving the chemical analysis of plant waxes, as well as related\nmetabolic engineering studies, and the methods used in this paper are sound.\nThe authors provide strong evidence that the biosynthesis of β-diketones and hydroxy β-diketones are\nproduced in a way that it does not utilize the previously described sequential 2-carbon-based elongation\nsteps catalyzed by the Fatty Acid Elongase (FAE) pathway, and that their synthesis does not require the\nendoplasmic reticulum (ER)-localized β-ketoacyl-CoA synthase (KCS6). The authors demonstrated the\nemr1 mutant deficient in KCS6 was able to produce β-diketones with normal chain length, even though\nother wax constituents on emr1 were much shorter, and so β-diketone synthesis in barley does not\nrequire that same elongation pathway. The authors present evidence that there is not another β-\ndiketone parallel elongation pathway, citing a previous article showing that fatty acyl chain elongation\ninhibitors do not inhibit elongation of β-diketones and hydroxy β-diketones, indicating that β-diketone\nsynthesis does not involve the previously describes ER based FAE. Further, carbon isotope studies by the\nauthors of this paper using GC coupled with isotope-ratio mass spectrometry revealed that the β-\ndiketones on barley are composed of all plastid generated carbons (enriched in 13C), and not the ER\nassociated carbons wherein only about 50% of the carbons would be plastidial if the chains were further\nelongated using the FAE pathway mechanism (wherein 13C composition would be low). The authors\ntested these 13C ratios against other wax classes in barley, providing strong evidence to support a novel\nsynthesis that does not involve ER elongation by FAE.\nInstead, the authors provide strong evidence for an alternative and novel very long chain β-diketone\nsynthesis pathway that involves Diketone Metabolism Hydrolase (DMH) that has a thioesterase function\nand generates fatty acyl derivatives with functional groups on the 3rd carbon, and a Diketone\nMetabolism Polyketide Synthase (DMP) that performs a decarboxylative condensation of 3-ketoacid acyl\nchains and fatty acyl-CoA to make the β-diketones. E. coli and yeast expression studies were used to\ndemonstrate both HvDMH and HvDMP preferred substrate classes and chain length specificity. It’s\nnotable that the yeast system expressing HvDMH could not make the 3-ketoacids substrate for use by\nHvDMP in synthesis of β-diketones, and so that is why exogenous 3-ketoacids needed to be added to\ntest HvDMP activity. As such, this study does not directly show that HvDMH catalyzes the thioesterase\nreaction leading to 3-ketoacyls as would be necessary in the plant system for β-diketone production. The\nauthors speculate that HvDMH might not interact properly (as it would in planta) with the yeast FAS\ncomplexes, or that it requires specific ACP isoforms and discriminates against yeast ACPs, which are\nreasonable speculations by these authors. However, HvDMH expressed in E. coli did hydrolyze\nsubstrates to make acyl products having 3-carbon functional groups, which provided evidence that\nHvDMH likely generates the 3-ketoacyls in planta. It is known that E. coli has very low relative amounts\nof saturated acyl substrates overall (relative to unsaturated), which may explain why detectable\namounts of the specific 3-ketoacyls were not generated by HvDMH in the E. coli system.\nStudies in the yeast system using deuterated C16 acids showed that HvDMP could condense (with\ndecarboxylation) a deuterated C16 fatty acid with 3-ketoacids to make β-diketones, and so this then\nshows that HvDMP has the capacity to combine two long chain substrates together. HvDMP’s ability to\ncondense fatty acyl-CoAs with ketoacid substrates was further confirmed in yeast experiments using\nuncommon odd chain length substrates. Further in vitro/yeast studies tested chain length specificity of\nHvDMP to find that the HvDMP enzyme prefers the C14 3-ketoacid, and either the C14 or C16 fatty acyl-\nCoA. But notably HvDMP does in fact readily accepts the C16 3-ketoacid. The fact the C16 3-ketoacids\nand C16 fatty acyl-CoAs are by far the dominant substrate pool available in planta explains why the C31\n14,16-diketones are the dominant homologues found on the surface of wild-type barley sheaths, being\n96% of the waxes. The functions described here for HvDMP are thus consistent with the β-diketone wax\nprofiles observed on barley.\nFinally, the authors localized both HvDMH and HvDMP using confocal microscopy, barley protoplast, and\nGFP constructs, showing the HvDMH enzyme was localized in the chloroplasts and HvDMP in ER\nmembranes. They proposed a quite reasonable model for substrate transfer (and related reaction\nmechanisms) in the biosynthetic pathway.\nThey also describe for the first time unbranched even chain length β-diketones, likely derived from an\ninitial C3 rather than the more common C2 moiety. They then noted that the locations of the functional\ngroups on these even chain β-diketones are consistent with a main finding of this report that the C16 3-\nketoacids are a major intermediate in this β-diketone pathway.\nAlthough not necessary for publication in the opinion of this reviewer (as will be described below),\nadditional evidence that HvDMH directly catalyzed synthesis of the 3-ketoacyls in yeast or E. coli (or\nanother system) would have helped strengthen the narrative that two enzymes are sufficient for β-\ndiketone synthesis. Notwithstanding, that the E. coli studies showed that HvDMH produced closely\nrelated 3-carbon modified acyl derivatives does provide good evidence that the HvDMH does in fact\ngenerate 3-ketoacyls in planta. Regardless, the presentation of only indirect evidence of HvDMH\nfunction in 3-ketoacyl synthesis does not detract from the overall main discoveries of this paper, which\nis the determination that the HvDMP enzyme generates the β-diketones, and that this is done using a\nnovel reaction that does not involve the well-known sequential C2 FAE elongation steps, but instead\nuses HvDMP to perform a decarboxylative condensation reaction of two long chain plastidial derived\nsubstrates. There are no previous reports of such a condensation reaction of long chain acyl compounds\nin plants, and this is the first report of the synthesis of very long chain aliphatic compounds that does\nnot involve C2 elongation steps.\nWe thank the reviewer for their very detailed comments! The remark above that our study “does not\ndirectly show that HvDMH catalyzes the thioesterase reaction” as well as the comments here on\nadditional evidence prompted us to perform further in vitro experiments to assay the HvDMH activity\n(see also response to reviewer #1, point 2). A new figure (fig 4) shows respective results, and an\nadditional paragraph in the Results section on HvDMH explains the findings.\nThe findings here represent novel and remarkable discoveries on their own and are clearly supported by\nthe data and its interpretation.\nSome minor edits requested:\nUnder carbon isotope analysis in methods, lines 2 and 3, recommend simply rewording ass “were\nstained with””.\nDone\nRemove the word “at” in line 15 for Fig 1 caption.\nDone\nIn Fig 2A, the green colors in the legend are a bit difficult to align to the bars. Making the colors more\ndifferent will improve the ease of interpretation. The colors in Fig 2 C are also difficult to visualize. Point\nsymbols should be enlarged\nThanks for alerting us that both these figure panels were difficult to read. We have changed the colors\nand symbol sizes to help readers distinguish and properly assign data points according to the legend."}, {"round": 2, "reviewer_comments": {"reviewer_1": "This reviewer is satisfied with the additional experiments and revised manuscript. This\nmanuscript is now suitable for publication in Nature Communications.", "reviewer_2": "The authors addressed the reviewers' concerns. Publish as it is.", "reviewer_3": "This reviewer was concerned that the study did not directly show that HvDMH catalyzes the\nthioesterase reaction leading to 3-ketoacyls as would be necessary in the plant system for β-\ndiketone production. Nevertheless, this reviewer felt that the original submission contained\nstrong evidence that HvDMH did in fact have such activity as claimed. To strengthen the\nauthors conclusions about HvDMH activity, the authors performed additional in vitro\nexperiments to assess and confirm HvDMH function. The authors have adequately\naddressed this concern."}, "author_response": ""}]

Decision letter