Blue-light-enhanced interaction between ZmCRY1s and GL2 modulates epidermal wax composition in Zea mays

Domain: earth_science
Published: 2025-12-16
Editorial outcome: major_revision

Abstract
Cryptochromes (CRYs) are photolyase-like blue-light/ultraviolet-A (UV-A) receptors that regulate diverse aspects of plant growth and development. Maize (Zea mays), a major crop often grown under high UV-B radiation, harbors four copies of CRY. However, whether CRYs in maize have evolved to improve UV tolerance or acquire novel functions remains unclear. In this study, CRISPR-Cas9-engineered Zmcry mutants are used to investigate the functions of ZmCRYs in maize. The findings reveal that ZmCRYs act redundantly in mediating blue light signaling and inhibiting mesocotyl elongation. Furthermore, we demonstrate that ZmCRYs mediate blue light-enhanced UV-B stress tolerance in maize by upregulating the expression of genes involved in the biosynthesis of UV-B stress tolerance-related metabolites, including phenylpropanoids, flavonoids, and fatty acids. Further analyses show that blue light modulates both the accumulation and composition of epidermal waxes, suggesting that blue light enhances epidermal wax accumulation for UV-B stress tolerance. Notably, we identify that ZmCRY1 directly interacts with GLOSSY2 (GL2), a key acyltransferase in very-long-chain fatty acid metabolism, in a blue light-strengthened manner to mediate blue light-promoted C32 aldehyde accumulation, shedding new light on the enigma of the aldehyde-forming pathway in plants. These results highlight the critical roles of ZmCRY1s in mediating blue light-regulated epidermal wax biosynthesis and UV-B tolerance in Zea mays.

Introduction
Cryptochromes (CRYs) are photolyase-like blue-light/UV-A receptors that share high sequence and structural similarity with DNA photolyases1–3, but they lack inherent DNA repair activity, distinguishing them functionally from their photolyase ancestors. These evolutionarily related flavoproteins perform diverse biological functions: DNA photolyase reverses far-UV light (200–300 nm)-induced damage (e.g., growth delay, mutagenesis, and cell death) via blue light (350–450 nm)-dependent photoreactivation across organisms from bacteria to plants and animals3,4, while CRYs regulate growth and development in plants and the circadian clock in both plants and animals5.

In Arabidopsis, the two primary CRY photoreceptors (CRY1 and CRY2) exhibit overlapping yet differentiated functions. They are involved redundantly in mediating photomorphogenesis and thermomorphogenesis, including hypocotyl elongation inhibition6–8. CRY1 also plays critical roles in stomatal opening, balances photosynthesis and defense by mediating pathogen-triggered stomatal closure9, and regulates plant height and shade avoidance responses—functions conserved across Arabidopsis, soybean, and maize10–13. In soybean, CRY1 is further essential for blue light-induced root nodule formation, promoting symbiotic nitrogen fixation in natural ecosystems14. CRY2 is well characterized for its role in regulating floral initiation15–17. A functionally equivalent dominant mutation, CRY2W374A, enhances species adaptation and rapid evolution, potentially serving as a genetic basis for ruderality in Brassicaceae18. CRY2 also participates in thermomorphogenesis, with its protein level regulated by both blue light and ambient temperature6,7. Notably, CRY2 acts in darkness to inhibit root growth, coordinating developmental processes between aboveground and underground organs19. Despite lacking DNA repair activity, CRYs collectively confer resistance to UV-B damage in plants20.

Maize, a major crop cultivated under high light intensity and elevated UV-B radiation, harbors three CRY1 homologs and one CRY2. High-resolution structures of the active photolyase-related (PHR) domain of ZmCRY1a and ZmCRY1c reveal conserved photoactive conformations similar to AtCRYs21,22, suggesting conserved photomorphogenesis roles that may inform breeding for high-density tolerance10,23. However, whether maize CRY paralogs have evolved novel functions or if maize employs alternative strategies to cope with UV-B stress remains unclear.

A primary line of defense against UV-B in terrestrial plants is the epidermal cuticle, where less than 10% of incident UV-B is transmitted24. The epicuticle contains UV-B-screening compounds—including flavonoids, anthocyanins, and phenolic acids, as well as waxes—that synergistically reflect and absorb UV light25–27. Cuticular wax accumulation and composition are modulated by environmental cues such as light wavelength, temperature, and osmotic stress28–33. Light is known to upregulate transcription of genes encoding wax biosynthetic enzymes28,32,34. Genetic analyses combined with weighted gene coexpression network analysis indicate that phytochromes mediate light regulation of epicuticle components and their evolution in land plants31. In faba bean, blue light shifts the dominant primary alcohol in cuticular wax32. However, whether CRYs mediate blue light-regulated wax biosynthesis or directly participate in this process remains unknown.

Cuticular waxes are predominantly composed of very long chain (VLC) aliphatics, derived from fatty acid synthesis and elongation. De novo synthesis of C16–C18 fatty acids occurs in plastids35, catalyzed by fatty acid synthases with chain-length-specific condensing enzymes (KASIII (C2–C4), KASI (C4–C16), and KASII (C16–C18))36–38. These fatty acids are then extended to VLC fatty acids (VLCFAs; ≥ C20) in the endoplasmic reticulum (ER) via multienzyme elongase complexes35,39, with β-ketoacyl-CoA synthases (KCSs) dictating chain-length specificity40,41. Key KCS members—such as KCS5/ECERIFERUM 60 (CER60) and KCS6/CER6—promote elongation beyond C26 (Arabidopsis) or C28, in cooperation with putative BAHD acyltransferases like Arabidopsis CER2, CER2-LIKE, and maize GL2, GL2-LIKE42–46.

VLC Fatty Acyl-CoAs (VLC acyl-CoAs) are processed into wax components via two pathways47: the alcohol-forming pathway (producing even-chain primary alcohols and alkyl esters) and the alkane-forming pathway (producing aldehydes, odd-chain alkanes, secondary alcohols, and ketones)47,48. Alkanes are proposed to derive from VLC aldehydes via decarbonylation, catalyzed by a CER1-CER3 heterodimer complex with cytochrome b5 (CYTB5) as a cofactor49–53. Fatty alcohol oxidase 3 (FAO3) and FAO4b also contribute to aldehyde production by suppressing primary alcohol accumulation54. However, the enzymes responsible for VLC aldehyde formation and the precise mechanism of VLC alkane synthesis remain to be further investigated.

Notably, yeast reconstitution experiments suggest that CER2 and CER2-LIKE enable CER6 to synthesize longer-chain VLCFAs, while subsequent studies suggest the yeast endogenous FAE complex may be associated with this extension46. Based on genetic evidence demonstrating GL2-mediated alterations in VLCFA composition, coupled with yeast reconstitution experiments42–46, it is hypothesized that maize GL2 (and GL2-Like) and Arabidopsis CER2 (and CER2-Like) function in the VLCFA elongation pathway—upstream of aldehyde, alcohol, or hydrocarbon formation—potentially through interaction with the KCS6-composed FAE complex. GLOSSY2 (GL2), a maize putative BAHD acyltransferase, may play a unique role in alkane pathway regulation. Overexpression of GL2 in Arabidopsis cer2-5 mutant or Col-0 induces accumulation of C32 aldehyde, C31 alkane, C31 secondary alcohol, and C31 ketone—components not observed upon overexpression of AtCER2 or maize GL2-LIKE55. Functional divergence between GL2 and GL2-LIKE is further highlighted by their differential dependence on the BAHD catalytic HXXXDX motif: this motif is required for GL2-LIKE activity but not for GL2, indicating evolutionary specialization55. However, the specific biochemical functions of GL2 in fatty acid elongation remain unclear.

This study investigates the roles of ZmCRY1s in mediating blue light-enhanced UV-B tolerance in maize. We show that ZmCRYs regulate transcription of genes involved in the biosynthesis of UV-B-protective metabolites (phenylpropanoids, flavonoids, anthocyanins, and fatty acids56) and modulate epidermal wax accumulation and composition. Furthermore, ZmCRY1 directly interacts with GL2 in a blue light-strengthened manner to promote C32 aldehyde formation, shedding light on VLC aldehyde generation. These findings reveal how blue light organizes UV resistance at the plant surface, potentially enhancing overall UV-B tolerance.

Results
ZmCRYs mediate blue light signaling in maize
To investigate the function of CRYs in maize, we searched maize genome sequence databases and classified the ZmCRY family. Maize CRYs are encoded by a multigene family comprising four members, which are phylogenetically classified as ZmCRY1s (ZmCRY1a, ZmCRY1b, and ZmCRY1c) and ZmCRY2 (Fig. 1a). ZmCRY1a and ZmCRY1b share 92% amino acid sequence identity, indicating a close relationship and likely functional similarity, whereas ZmCRY1c shares 78% and 75% amino acid sequence identity with ZmCRY1a and ZmCRY1b, respectively. Subcellular localization analysis revealed that ZmCRY1a and ZmCRY1b are predominantly nuclear, with minor cytoplasmic presence. In contrast, ZmCRY1c localizes to both compartments, with a significantly higher proportion in the cytoplasm (Supplementary Fig. 1a).

To elucidate their biological functions, we obtained uniform Mutator (Mu) insertion lines of CRY1s from the Maize Genetics Cooperation Stock Center. These lines carry Mu insertions at 1377 bp (CRY1a: UFMu-08910, designated cry1a-mu), −10 bp (CRY1b: UFMu-09028, designated cry1b-mu), and 426 bp (CRY1c: UFMu-08243, designated cry1c-mu) relative to their respective start codons. PCR, RT-PCR, and Sanger sequencing confirmed the presence of Mu elements and their impact on CRY1s expression, with no detectable corresponding CRY1 transcripts in their Mu insertion lines (Supplementary Fig. 1b–d). Phenotypic analysis showed that cry1a-mu and cry1c-mu single mutants exhibit longer mesocotyls than the wild-type W22 inbred line under blue light and white light. The cry1a cry1c-mu double mutant displays even longer mesocotyls than the single mutants and wild type under these light conditions, whereas no significant differences were observed among the Mu insertion lines under red light or in darkness (Supplementary Fig. 1e–h). These results suggest that these CRY1s may function redundantly in mediating blue light signaling in maize.

To generate multiple cry knockout mutants, we used CRISPR-Cas9 technology to target the four cryptochrome genes in the B73 background. Guide RNA 1 (gRNA1) was designed to target conserved regions of ZmCRY1s, and gRNA2 targeted ZmCRY2. We identified Zmcry quadruple mutants (Zmcry-q) with mutations including base deletions in ZmCRY1a (d45, 45-base deletion), ZmCRY1b (d124), ZmCRY1c (d14), as well as a 15-base deletion followed by a 19-base insertion in ZmCRY2 (d15, i19) (Supplementary Fig. 2a). Zmcry-q was backcrossed to B73 and self-pollinated to remove the Cas9 transgene, enabling the isolation of various single, double, and Zmcry1s triple (Zmcry1abc) mutants. Phenotypic analysis under blue light and white light revealed that, compared to wild-type B73, the single mutants Zmcry1a and Zmcry1c exhibit longer mesocotyls. Mesocotyl length increased as the number of functional ZmCRYs decreased across double (Zmcry1ab, Zmcry1ac, Zmcry1bc, Zmcry1acry2), triple (Zmcry1abc), and quadruple (Zmcry-q) mutants, and no significant differences were observed among these genotypes under red light and in darkness (Fig. 1b–e).

We generated overexpression lines of 35S::YFP-ZmCRY1a (CRY1a-OE) and 35S::YFP-ZmCRY1c (CRY1c-OE) in the maize Hi-II background. Three independent transgenic lines were recurrently backcrossed into Chang 7-2 for five generations. Phenotypic analysis showed that all overexpression lines in the Chang 7-2 background exhibit shorter mesocotyls than the wild types specifically under blue light and white light, while no significant differences were observed under red light or in darkness (Supplementary Fig. 2b–f). For phenotypic analysis within a uniform genetic background, randomly selected CRY1a-OE and CRY1c-OE lines were recurrently backcrossed into the B73 background for five generations. These lines also showed shorter mesocotyls than B73 (Fig. 1b–e). Collectively, these results indicate that ZmCRYs function redundantly in mediating blue light signaling to regulate maize photomorphogenesis.

To determine whether the blue light receptor function of ZmCRYs is conserved with that of Arabidopsis CRYs (AtCRYs), we overexpressed ZmCRYs in Arabidopsis. YFP-ZmCRY1s mediated blue light inhibition of hypocotyl elongation in transgenic Arabidopsis seedlings, whereas YFP-ZmCRY2 did not (Supplementary Fig. 3a–d). Photo-excited AtCRY2 undergoes polyubiquitination and subsequent degradation via the 26S proteasome57, and AtCRY1 has recently been shown to degrade in response to high-intensity blue light58. To assess ZmCRYs protein stability under blue light, we constructed ZmCRY-Flag overexpression lines in Arabidopsis, using endogenous AtCRY2 protein and an AtCRY1-Flag overexpression line as controls. Transgenic seedlings were pretreated in darkness for 48 h, then exposed to medium blue light (20 μmol m–2 s–1) or high blue light (100 μmol m–2 s–1) for a 1-h time course. Under medium blue light, ZmCRY2 protein levels decreased similarly to AtCRY2, while ZmCRY1s levels remained unchanged (Supplementary Fig. 3e, f). Under high blue light, ZmCRY1a and ZmCRY1b underwent degradation analogous to AtCRY1, whereas ZmCRY1c remained relatively stable (Supplementary Fig. 3g, h). The stability of ZmCRY1c may be attributed to its predominantly cytoplasmic localization, whereas the E3 ubiquitin ligases Light-Response Bric-a-Brack/Tramtrack/Broads (LRBs) responsible for CRY degradation are nuclear-localized7,58,59. These results suggest that ZmCRY1s and ZmCRY2 are functionally conserved with their Arabidopsis orthologs as blue light receptors.

ZmCRYs mediate the blue light-induced transcription of genes involved in UV-B stress tolerance in maize
Current understanding indicates that most CRY-mediated blue light responses in plants are associated with changes in nuclear gene expression. To explore the ZmCRY-dependent blue light-regulated transcriptome, we conducted transcriptome analysis using 7-day-old seedlings of B73 and Zmcry-q grown under long day (LD, 16-h white light/8-h dark) conditions. The seedlings were pretreated in darkness for 48 h, followed by either 1-h blue light (20 μmol m–2 s–1) irradiation or maintenance in darkness. Transcriptome analysis revealed that blue light mediated a total of 5764 differentially expressed genes (DEGs) in the comparison between B73_Blue and B73_Dark (Supplementary Data 1). Venn diagram analysis showed that 45.49% (2,622 genes) of these DEGs observed in wild-type plants under blue light compared to darkness were not differentially expressed in Zmcry-q mutants, indicating that these genes are dependent on the presence of one or more CRY genes (Fig. 2a). A heatmap generated from ZmCRY-mediated DEGs clearly illustrated the blue light-regulated genes mediated by ZmCRYs (Fig. 2a, b).

Blue light regulates various aspects of plant metabolism, development, and morphology by modulating the expression of numerous genes. In addition to the conserved major blue light responses observed in Arabidopsis, Gene Ontology (GO) enrichment analysis revealed several distinct responses in maize, including biosynthesis and metabolic processes of multiple compounds (Supplementary Fig. 4a, b and Supplementary Data 2). This suggests that blue light may promote fatty acid accumulation by enhancing fatty acid biosynthesis while repressing fatty acid β-oxidation. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of blue light-upregulated genes in B73 identified several metabolites associated with UV-B stress tolerance, including those involved in phenylpropanoid biosynthesis, flavonoid biosynthesis, and fatty acid biosynthesis (Fig. 2c and Supplementary Data 3). These results imply that fatty acid accumulation may be an important regulatory target of blue light signaling in maize.

To validate the transcriptome data, we performed quantitative reverse transcription PCR (RT-qPCR) using seedlings transferred from darkness to blue light (20 μmol m–2 s–1) over a 2-h time course. For known blue light signaling pathway regulators detected in the transcriptome, such as the HY5 homolog gene BASIC LEUCINE ZIPPER 61 (bZIP61) and bZIP80, the AtCOP1 homolog gene UBIQUITIN-PROTEIN LIGASE 13 (ZmUPL13) and ZmUPL14, SUPPRESSOR OF PHYTOCHROME A 1 (SPA1)-related 3 and SPA1-related 4, REPRESSOR OF UV-B PHOTOMORPHOGENESIS 2 (ZmRUP2) and CRY3, their expression was upregulated in B73 after blue light irradiation, but this induction was abolished in Zmcry-q mutants (Fig. 2d and Supplementary Fig. 4c). We further verified the expression of genes associated with de novo fatty acid synthesis (fatty acyl-acyl carrier protein (ACP) thioesterase B (FatB), β-ketoacyl-ACP synthase I (KASI), and KASII36,37), those involved in fatty acid elongation (KCS3, KCS4), and genes related to cuticular wax biosynthesis (GL1, GL1-5, and GL4). These genes were induced by blue light, with partial gene induction dependent on CRYs (Fig. 2d and Supplementary Fig. 4d). These findings suggest that blue light may induce fatty acid biosynthesis and wax accumulation through the regulation of gene expression31,32.

Blue light affects the accumulation and the composition of epidermal wax in maize
Given that blue light induces the expression of genes related to fatty acid biosynthesis and wax accumulation via ZmCRYs, we hypothesized that blue light and CRYs might modulate wax accumulation. To test this, we analyzed the effects of different light wavelengths on wax accumulation in maize. Seven-day-old dark-grown seedlings were exposed to various light treatments (UV-B (1 μmol m–2 s–1), blue (20 μmol m–2 s–1), red (20 μmol m–2 s–1), and far-red light (10 μmol m–2 s–1)) for 48 h, after which their epidermal wax compositions were measured. Compared to darkness, all light treatments significantly promoted epidermal wax accumulation (Fig. 3a). The dominant C32 primary alcohol showed a marked increase across all light treatments. Notably, blue light exposure led to a 1.86-fold increase in total cuticular wax, a 0.35-fold decrease in C28 primary alcohol, a 4.56-fold increase in C32 primary alcohol, and the appearance of C32 aldehyde. This finding indicates that the changes in wax composition under blue light are specifically attributed to increased chain lengths of cuticular wax products. Additionally, blue light and UV-B irradiation exerted consistent effects on wax composition. These results indicate that UV-B, blue, red, and far-red light all enhance epidermal wax accumulation, while exerting distinct effects on wax composition.

To determine whether ZmCRYs mediate blue light-regulated wax accumulation, we measured epidermal wax compositions in 7-day-old seedlings of CRY1c-OE, B73, and Zmcry-q mutants grown in either darkness or continuous blue light. No significant difference in total wax content was observed among genotypes under both darkness and blue light conditions, suggesting that blue light-promoted epidermal wax accumulation is not ZmCRY-dependent (Fig. 3b) and that other blue light receptor families may redundantly regulate this process. Compared to darkness, blue light exposure consistently increased total cuticular wax across genotypes: in B73, this was accompanied by a 0.27-fold decrease in C32 fatty acid and a 2.82-fold increase in C32 primary alcohol. C32 aldehyde accumulation was undetectable in any genotype under darkness. Under blue light, however, C32 aldehyde levels were significantly higher in CRY1c-OE line (1.45 ± 0.26 μg cm–2) than in B73 (0.83 ± 0.24 μg cm–2) and much lower in Zmcry-q mutant (0.33 ± 0.03 μg cm–2). These results indicate that the blue light-induced C32 aldehyde biosynthesis is primarily ZmCRY-dependent (Fig. 3b). Furthermore, they suggest that ZmCRYs may mediate blue light responses involved in the formation of very-long-chain (VLC) aldehydes, a process that requires further clarification47.

ZmCRYs enhance the UV-B stress tolerance in maize
Maize is cultivated under high light intensity year-round and thus must cope with elevated UV-B radiation. Our transcriptome analyses support the notion that blue light contributes to UV-B tolerance at the transcriptional level, as previously reported20. To evaluate the potential UV-B protective capacity of the blue light-ZmCRY pathway, we measured the maximum quantum yield of Photosystem II (Fv/Fm) as an indicator of UV-B stress tolerance. For this assay, 10-day-old seedlings of CRY1c-OE, B73, and Zmcry-q grown under LD blue light (20 μmol m–2 s–1) were subjected to two broadband UV-B treatments of varying intensities: (1) high intensity (60 μmol·m–2·s–1) for 10 h, followed by 1-h dark adaptation before Fv/Fm measurement; (2) medium intensity (5 μmol·m–2·s–1) for 6 h, followed by a 2-day recovery period before Fv/Fm measurement and an additional week of growth to assess survival rate. Results showed that Zmcry-q was more sensitive to UV-B stress than B73 under both treatments, while CRY1c-OE exhibited greater tolerance. After 1-h dark adaptation following high-intensity UV-B irradiation, the average Fv/Fm values were 0.561 (B73), 0.681 (CRY1c-OE), and 0.435 (Zmcry-q) (Supplementary Fig. 5b, c). After a 2-day recovery following medium-intensity UV-B irradiation, the average Fv/Fm values were 0.516 (B73), 0.718 (CRY1c-OE), and 0.148 (Zmcry-q) (Fig. 3c, d). Additionally, the average survival rate of CRY1c-OE (90.5%) was significantly higher than that of B73 (61.9%), whereas Zmcry-q showed the lowest survival rate (33.3%) after medium-intensity UV-B irradiation (Supplementary Fig. 5d–f). In the Chang 7-2 background, both CRY1a-OE and CRY1c-OE lines also displayed enhanced UV-B tolerance compared to wild-type Chang 7-2 (Supplementary Fig. 5g, h). Collectively, these findings indicate that ZmCRYs mediate blue light-enhanced UV-B tolerance in maize.

Epidermal wax acts as a primary defense against UV-B radiation33. Blue light modulates both the accumulation and composition of epidermal wax in maize; however, ZmCRYs primarily mediate the blue light-regulated changes in wax composition rather than total wax accumulation. The specific mechanisms by which ZmCRYs influence wax composition, as well as the role of C32 aldehyde or its downstream derivatives in UV-B tolerance, require further investigation.

ZmCRY1 physically interacts with GLOSSY2 in a blue light-enhanced manner
To investigate how ZmCRY influences wax composition and the unique functions of ZmCRY1c due to its distinct cytoplasmic localization, we employed two strategies to identify the ZmCRY1c-interacting proteins under blue light: yeast two-hybrid (Y2H) screening in vitro and in vivo co-immunoprecipitation coupled with mass spectrometry (IP–MS). In the Y2H assay, using ZmCRY1c as bait to screen a maize cDNA library under blue light (20 μmol·m–2·s–1), we sequenced 230 positive clones and identified 87 proteins that interact with CRY1c in yeast (Fig. 4a, b and Supplementary Data 4). Among these, 4 clones encoded various fragments of GLOSSY2 (GL2), a member of the BAHD acyltransferase superfamily involved in the elongation-reductive pathway of very-long-chain fatty acids (VLCFAs). In the IP-MS experiment, we affinity-purified ZmCRY1c complexes from YFP-ZmCRY1c overexpression line (CRY1c-OE) under blue light (20 μmol·m–2·s–1) or in darkness. Analysis of co-purified proteins identified known interactors such as the COP1 homologue ZmUPL13 and the gibberellin receptor GID1L260–62, as well as GL2, which was previously identified via the Y2H assay (Supplementary Data 5). GL2 specifically enhances the elongation of C30 and C32 VLC acyl-CoA63 and indirectly promotes the formation of C32 aldehyde, C31 alkane, C31 secondary alcohol, and C31 ketone55. Consequently, we selected GL2 as a candidate and performed further protein–protein interaction assays to determine whether blue light enhances the interaction between ZmCRY1c and GL2.

Y2H assays revealed that ZmCRY1c and GL2 exhibit a strengthened physical interaction under blue light but a weak interaction in darkness (Fig. 4a). Quantification via β-galactosidase assay showed that the interaction was strengthened only when cells were transferred from darkness to blue light, not when kept in darkness (Fig. 4b). No interaction was detected between GL2 and the flavin-deficient ZmCRY1c variant ZmCRY1cD381A64 (Fig. 4b). ZmCRY1c colocalizes with GL2 in the cytoplasm of Nicotiana benthamiana leaves and maize leaf protoplasts (Fig. 4c, d and Supplementary Fig. 6a, b). We confirmed this interaction in plant cells using bimolecular fluorescence complementation (BiFC) and bimolecular luminescence complementation (BiLC) assays. Strong yellow fluorescent protein (YFP) fluorescence was observed in the cytoplasm of N. benthamiana leaves transiently co-infiltrated with ZmCRY1c-cCFP and nYFP-GL2 constructs, indicating a robust interaction, whereas leaves co-infiltrated with ZmCRY1cD381A-cCFP and nYFP-GL2 showed no interaction (Fig. 4e and Supplementary Fig. 6c). Similarly, strong luminescence was observed in leaves co-infiltrated with ZmCRY1c-nLUC and cLUC-GL2 constructs in BiLC assays (Fig. 4f). Additionally, ZmCRY1a and ZmCRY1b also interacted with GL2 in BiLC assays, while ZmCRY2 showed a weak interaction with GL2 in the BiLC assay (Supplementary Fig. 6d). No interaction was detected between KCS4 and ZmCRY1c in BiFC and BiLC assays (Fig. 4e and Supplementary Fig. 6e). The GL2 homologue, GL2-LIKE, showed a weak interaction with ZmCRY1c, as indicated by faint luminescence in the BiLC assay compared to GL2, but yeast containing ZmCRY1c and GL2-LIKE failed to survive on histidine-deficient (–LWHA) medium in Y2H assays (Supplementary Fig. 6f, g). The in vivo interaction of ZmCRY1c and GL2 was confirmed by co-immunoprecipitation assay. Seedlings pretreated in darkness for 48 h were exposed to blue light for 2 h or kept in darkness before harvesting. Results showed that ZmCRY1c co-precipitated with GL2 in blue light-treated samples, whereas minimal co-precipitation was detected in darkness-maintained samples (Fig. 4g). Collectively, these results reiterate that blue light promotes the interaction between ZmCRY1c and GL2.

The N-terminal PHR domain of ZmCRY1c, which encompasses the chromophore-binding region, was sufficient to mediate the interaction with GL2 in yeast cells (Supplementary Fig. 6h), aligning with the observation that their interaction is blue light-strengthened. Subsequently, Yaqi L. et al. have determined the crystal structure of the constitutively active CRY1c PHR domain (ZmCRY1c-PHRW368A22) in complex with GL2, which provides structural insights into their interaction64.

ZmCRY1 modulates aldehyde accumulation together with GLOSSY2
As described64, the interaction interface between ZmCRY1c-PHRW368A and GL2 primarily involves the α16-α17 loop, α17 of ZmCRY1c (residues 396–419), as well as the α2, β1-β2 loop of GL2 (residues 25–33, 70–76). To validate this, we screened for amino acid site mutations in the Maize EMS-induced Mutant Database (MEMD)65. Among the 14 EMS mutants of GL2, two mutants, gl2V24M and gl2G106D, exhibited a water-beading phenotype similar to that of gl2-ref mutant66 (Fig. 5a, b and Supplementary Fig. 7a). These mutations are located in close proximity to Phe71 of GL2, a residue directly interacting with ZmCRY1c-PHRW368A64 (Supplementary Fig. 7b). To assess the impact of these mutations on protein interaction, we cloned the mutated cDNAs and performed interaction assays. In Y2H assays under blue light, yeast co-transformed with constructs harboring GL2G106D failed to grow on –LWHA medium, indicating that this mutation severely disrupted the interaction with ZmCRY1c (Fig. 5c). In contrast, the GL2V24M mutation had a milder effect on the interaction compared to the wild-type GL2 (Supplementary Fig. 7c). BiLC assays further confirmed this: the luminescent signal from ZmCRY1c-nLUC and cLUC-GL2G106D was faint compared to that with cLUC-GL2, reflecting a significant reduction in interaction strength, while cLUC-GL2V24M showed a milder decrease in interaction with ZmCRY1c-nLUC (Fig. 5d). Additionally, pull-down assays with ZmCRY1c-PHRW368A corroborated these findings: the G106D mutation nearly abolished the interaction, while V24M had a weaker effect (Fig. 5e). Consistent with the defects in protein-protein interaction, these gl2 site mutants displayed epidermal wax compositions comparable to gl2-ref, characterized by reduced levels of major components such as C32 primary alcohol and C32 aldehyde, alongside accumulated C22–C30 VLCFAs (Fig. 5f). These results highlight the importance of the interaction interfaces (INT) of GL2 with ZmCRY1c for GL2’s function.

Previous studies have shown that overexpression of GL2 in Arabidopsis induces the production of C32 aldehyde, C31 alkane, C31 secondary alcohol, and C31 ketone55. To determine if blue light regulates GL2-mediated C32 aldehyde accumulation, we generated GL2 overexpression lines in the B73 background and selected three independent lines for wax composition analysis (Supplementary Fig. 8a). Epidermal waxes were measured in 7-day-old seedlings grown in darkness or under blue light (20 μmol m–2 s–1). Results showed that C32 aldehyde accumulation in GL2 overexpression lines was significantly induced by blue light but undetectable in darkness, whereas C32 primary alcohol induction in GL2 overexpression lines was light-independent (Fig. 6a). This indicates that blue light regulates GL2’s function in promoting C32 aldehyde accumulation.

To verify whether C32 aldehyde accumulation in CRY1c-OE lines depends on GL2, we analyzed YFP-ZmCRY1c overexpression lines in gl2-ref mutant background (CRY1c-OE/gl2-ref) (Supplementary Fig. 8b). Seven-day-old dark-grown seedlings were irradiated with blue light (20 μmol m–2 s–1) for 48 h, after which epidermal waxes were measured. The results showed that C32 aldehyde accumulation in CRY1c-OE line was abolished in the absence of functional GL2, demonstrating that ZmCRY1c regulates C32 aldehyde accumulation in a GL2-dependent manner (Fig. 6b).

To evaluate the UV-B tolerance of wax-deficient gl2 variants and GL2’s contribution to CRY-mediated UV-B tolerance, 10-day-old seedlings of B73, gl2 variants, CRY1c-OE, and CRY1c-OE/gl2-ref grown under LD blue light (20 μmol m–2 s–1) were treated with high-intensity broadband UV-B light (60 μmol m–2 s–1) for 10 h, followed by 1-h dark adaptation before Fv/Fm measurement (Supplementary Fig. 8c, d). Separately, these genotypes grown under LD white light (50 μmol m–2 s–1) were treated with medium-intensity broadband UV-B light (5 μmol m–2 s–1) for 6 h per day over 2 consecutive days, with a subsequent 2-day recovery period prior to Fv/Fm measurement (Fig. 6c, d). Results revealed that gl2 variants were more sensitive to UV-B stress than B73 under both treatment regimens. Additionally, the absence of functional GL2 attenuated the enhanced UV-B tolerance of CRY1c-OE; however, CRY1c-OE/gl2-ref still exhibited slightly greater resistance than B73 (Fig. 6c, d and Supplementary Fig. 8c, d). These findings underscore the multifaceted regulation of UV-B tolerance by CRY20.

GLOSSY2 plays dual roles in fatty acid elongation and wax maturation through competitive partner interactions
GL2/CER2 proteins function uniquely as cofactors with KCS6 in the biochemical and physiological processes of elongating VLC acyl-CoAs up to C3442–45. We hypothesized that ZmCRY1c might enhance the interactions between these proteins to facilitate VLC acyl-CoA elongation. To test this, we performed BiLC assays; no luminescence was observed in leaves co-infiltrated with ZmCRY1c-nLUC and cLUC-KCS6 constructs in BiLC assays (Supplementary Fig. 8e). Furthermore, co-expression of ZmCRY1c with KCS6-nLUC and cLUC-GL2 reduced the chemiluminescent signal; a similar reduction was observed when ZmKCS6 was co-expressed with ZmCRY1c-nLUC and cLUC-GL2 (Fig. 6e, f). These results suggest that ZmCRY1c and KCS6 may compete for binding to GL2 rather than forming a ternary complex. Collectively, these findings indicate that GL2 interacts with KCS6 to promote VLCFA elongation in darkness. Under blue light, activated ZmCRY1c can outcompete KCS6 for GL2 interaction, thereby enhancing aldehyde accumulation.

Our working model proposes that blue light-activated ZmCRY1 enhances phenylpropanoid biosynthesis, flavonoid biosynthesis, fatty acid biosynthesis, and cuticle synthesis by promoting the transcription of genes encoding related enzymes, including FatB, KASI, KASII, KCS, GL1, GL1-5, and GL4. Additionally, ZmCRY1 directly interacts with GL2 in a blue light-enhanced manner to facilitate C32 aldehyde accumulation (Fig. 6g). In this way, blue light affects the accumulation of phenylpropanoids, flavonoids, and cuticular waxes, collectively enhancing the UV-B tolerance of maize through the coordinated regulation of metabolite biosynthesis and epidermal barrier modification.

Methods
Plant materials and growth conditions
Maize (Zea mays ssp. mays) inbred line B73, Chang 7-2, W22, and hybrid line Hi-II were used. To generate maize transformants overexpressing ZmCRYs, the full-length coding sequences of ZmCRYs amplified from B73 leaves were cloned into pB7WGY2 vector69. For GL2 overexpression in the B73 background, the full-length coding sequence of GL2 was subcloned into pEGAD-Myc vector. For CRISPR-Cas9-engineered Zmcry mutants in B73 background, gRNA1 and gRNA2 were cloned into pBUE411-2gR vector. All resulting constructs were delivered into Agrobacterium tumefaciens strain EHA105, and maize transformation was performed by Weimi Biotechnology (Jiangsu) Co., Ltd. Uniform Mu insertion lines of CRY1s (UFMu-08910, UFMu-09028, and UFMu-08243) were obtained from the Maize Genetics Cooperation Stock Center. glossy2 alleles (mutant IDs: EMS4-1836a9, EMS4-0eb086, EMS4-02f442, EMS4-0eb085, EMS4-0eb084, EMS4-02f441, EMS4-0eb071, EMS4-0eb070, EMS4-02f427, EMS4-02f426, EMS4-02f425, EMS4-02f423, EMS4-0eb06f) were acquired from the Maize EMS-induced Mutant Database (MEMD; http://maizeems.qlnu.edu.cn/). The glossy2-ref mutant was previously described63,66. CRY1c-OE/gl2-ref was generated by crossing CRY1c-OE (in B73 background) with glossy2-ref mutant.

For Arabidopsis experiments, the Columbia (Col-0) ecotype served as the wild type. AtCRY1-Flag overexpression line was previously described9. Full-length coding sequences of ZmCRYs, cloned in pB7WGY2 (carrying YFP tag) and pCambia1300 (carrying Flag tag), were transformed into Col-0 background.

For high intensity UV-B stress analysis in maize (Supplementary Figs. 5b, c and 8c, d), plants were grown at 28°C under LD conditions with blue light (20 μmol m–2 s–1) for 10 days. On day 11, they were treated with (+UV) or without (−UV) broadband UV-B (60 μmol m–2 s–1) for 10 h, then allowed to 1-h dark adaptation before measuring the maximal quantum yield of PSII (Fv/Fm).

For experiments in Fig. 3c, d, plants were grown at 28°C under LD conditions with blue light (20 μmol m–2 s–1) for 10 days. On day 11, they were treated with (+UV) or without (−UV) broadband UV-B (5 μmol m–2 s–1) for 6 h per day over 2 consecutive days, then allowed to recover for 2 days before measuring Fv/Fm; for survival rate analysis (Supplementary Fig. 5d–f), plants were grown for an additional week post-treatment. For each biological replicate, eight seedlings were planted per pot, and the number of surviving plants was recorded (three independent biological replicates were performed).

For experiments in Fig. 6c, d, plants were grown under LD conditions with white light (50 μmol m–2 s–1) for 10 days. They were treated with (+UV) or without (−UV) broadband UV-B (5 μmol m–2 s–1) for 6 h per day over 2 consecutive days, then allowed to recover for 2 days before measuring Fv/Fm.

For different light treatments, blue light (460 ± 40 nm), red light (675 ± 40 nm), far-red light (740 ± 40 nm), and narrowband UV-B (310 ± 10 nm) were generated using LEDs manufactured by Qiding Optoelectronic (Shanghai) Co., Ltd. Broadband UV-B was provided by TL 40W/12 UV fluorescent tubes (Philips). The intensities of white light, blue light, red light, and far-red light were measured with an ILT1400 Radiometer Photometer, while UV-B light was measured with a LUYOR-340 UV Light Meter.

RNA-seq and transcriptome analysis
For RNA-seq experiments, B73 and zmcry-q seedlings were grown for 7 days under LD (white light, 50 μmol m–2 s–1), pretreated with 48-h darkness, followed by transfer to blue light (20 μmol m–2 s–1) for 1 h. The second leaves were harvested, and total RNA was isolated using RNAiso Plus (Takara). Three biological replicates were independently prepared. RNA purification, reverse transcription, library construction, and sequencing were performed at Shanghai Majorbio Biopharm Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturer’s instructions. RNA-seq transcriptome libraries were prepared following Illumina® Stranded mRNA Prep, Ligation protocol (San Diego, CA) using 1 μg of total RNA. The sequencing libraries were run on the NovaSeq X Plus platform (PE150) using NovaSeq Reagent Kit. The raw paired-end reads were trimmed and quality-controlled by fastp70 with default parameters. Then clean reads were separately aligned to reference genome with orientation mode using HISAT271 software. The mapped reads of each sample were assembled by StringTie72 in a reference-based approach. To identify DEGs (differentially expressed genes) between different samples, the expression level of each transcript was calculated according to the transcripts per million reads (TPM) method. RSEM73 was used to quantify gene abundances. Differential expression analysis was performed using the DESeq274. DEGs with |log2 FC| ≥ 1 and FDR < 0.05 were considered to be significantly differentially expressed genes. GO functional enrichments were analyzed at https://geneontology.org. KEGG pathway enrichments were analyzed on the online platform of Majorbio Cloud Platform (https://cloud.majorbio.com/).

Phylogenetic analysis
Amino acid sequences were downloaded from UniProt (https://www.uniprot.org) and aligned using MUltiple Sequence Comparison by Log-Expectation (MUSCLE) in the MEGA7 software with the default settings for protein multiple alignment. Evolutionary distances were computed using Poisson correction analysis. The bootstrap method with 1000 replicates for phylogeny testing was used.

Yeast two-hybrid assay (Y2H)
For the Y2H library screening, the coding sequence of ZmCRY1c was fused in-frame with the GAL4 DNA binding domain (BD) of the bait vector pBridge (Clontech). The resulting constructs were transformed into Y2HGold competent yeast cells. The Zea mays whole transcriptome cDNA library, cloned into the prey vector pGADT7, was constructed by OE Biotech (Shanghai). The cDNA was reverse transcribed from an equal mixture of mRNA extracted from various tissues, including roots, stems, sheaths, leaves (at the VE, V2, and V6 stages), ears, and tassels. The cDNA library contained a total of 1.28 × 10⁷ Colony-Forming Units (CFUs). This library was transformed into Y187 competent yeast cells. The Y2H library screening assays were performed using yeast mating according to the manufacturer’s instructions (Matchmaker User’s Manual, Clontech, California). Screening plates were incubated (colony side facing downward) at 30°C under continuous blue light (20 μmol m–2 s–1) for 3–5 days. Survived yeast clones were sequenced and subjected to a second round of point-to-point validation.

For the point-to-point assays, the sequences encoding ZmCRY1c-PHR (a.a. 1–500), ZmCRY1c-CCE (a.a. 501–688), GL2, GL2G106D, GL2V24M were cloned into pBridge (Clontech). The resulting constructs were transformed into Y2HGold competent yeast cells. The sequences encoding ZmCRY1c-PHR, ZmCRY1c-CCE, GL2, GL2G106D, GL2V24M, GL2-LIKE were cloned into pGADT7. The resulting constructs were transformed into Y187 competent yeast cells. The Y2H assays were performed using yeast mating according to the manufacturer’s instructions (Matchmaker user’s manual, Clontech, California). P53/T was a positive interaction control (provided by Takara Bio).

Immunoprecipitation–mass spectrometry (IP–MS)
Samples (2.5 g) of ZmCRY1c-OE and B73 (7-day-old seedlings grown under LD, pretreated with 48-h darkness followed by 2-h blue light, or maintained in darkness) were collected. A total of 4 samples were analyzed: Sample 1: ZmCRY1c-OE under blue light (biological repeat 1); Sample 2: ZmCRY1c-OE under blue light (biological repeat 2); Sample 3: B73 under blue light; Sample 4: ZmCRY1c-OE in darkness. Sample 3 and 4 served as no-transgene control and no-blue-light control, respectively. The frozen samples were ground into powder in liquid nitrogen and transferred to a mortar with 2 mL of extraction buffer A (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 5 mM MgCl2, 10% v/v glycerol, 0.5% v/v NP40, 5 mM DTT, 1 mM PMSF, 1× protease inhibitor cocktail). After grinding, the homogenate was incubated on a rotary mixer for 30 minutes (4°C, 35 rpm) and centrifuged at 14,000 g for 10 minutes. The supernatant was collected and centrifuged again at 14,000 g for 10 minutes. Then, 3 mL of buffer B (buffer A without NP40) was added and incubated with Anti-GFP Affinity agarose beads (Smart-Life-sciences #SA070005, 25 μL, pre-washed with buffer B twice) on a rotary mixer for 30 minutes (room temperature, 15 rpm). After incubation, the beads were washed 6 times with washing buffer C (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 5 mM MgCl2, 0.2% v/v NP40, 5 mM DTT, 1 mM PMSF, 1× protease inhibitor cocktail) and 6 times with buffer D (buffer C without NP40). The resulting beads were eluted with buffer E (2% SDS, 100 mM Tris pH 8.0, 10 mM TCEP, 50 mM CAA).

The eluted proteins were digested using a modified filter-aided sample preparation (FASP) method75: briefly, eluted proteins were added to 200 μL of UA buffer (8 M urea and 50 mM Tris-HCl, pH 8.0), loaded onto a 10 kDa ultrafiltration centrifuge tube (Sartorius), and centrifuged at 14,000 g for 30 min (repeated twice). Samples were subjected to reductive alkylation by adding 100 μL of UA buffer containing 10 mM TCEP and 20 mM CAA, followed by shaking at 650 rpm for 30 min. Then, 300 μL of UA buffer was added, and the mixture was centrifuged at 14,000 g for 30 min (repeated twice). Next, 300 μL of 50 mM NH4HCO3 solution was added, followed by centrifugation at 14,000 g for 20 min (repeated twice). 2 μL of trypsin solution (1 μg of trypsin in 100 μL of 100 mM NH4HCO3) was added, and the samples were shaken at 650 rpm at 37°C for 12 h. The digests were collected by centrifugation at 14,000 × g for 20 min, and the filtrate was desalted using C18 cartridges (Thermo Scientific) after adding 0.1% trifluoroacetic acid (TFA). The peptide concentration was determined using the Pierce Quantitative Colorimetric Peptide Assay kit (Thermo Scientific). Samples were analyzed by timsTOF Pro2 (Bruker). MS data were acquired as shotgun proteomics (IP-MS) and analyzed using Paser ver 2023 software. The raw data were searched against the Maize_UP000007305_4577 Reference proteome database from Uniprot (https://www.uniprot.org/proteomes/UP000007305) and processed as described methods76.

BiFC and subcellular localization assays
In the BiFC assay, constructs expressing ZmCRY1c or GL2 fused to the C-terminus of CFP (a.a. 155–238) or N-terminus of YFP (a.a. 1–172) were transformed into Agrobacterium strain GV3101. For subcellular localization assays, constructs expressing ZmCRY1a, ZmCRY1b, or ZmCRY1c fused to eCFP were transformed into Agrobacterium strain GV3101. For co-localization assays in Nicotiana benthamiana, constructs expressing ZmCRY1c or GL2 fused to CFP or YFP were transformed into Agrobacterium strain GV3101. Overnight agrobacterial cultures were collected by centrifugation, resuspended in MES buffer (pH 5.6) to an OD of 0.6, and incubated at room temperature for 2 h before infiltration. Using a 1-mL syringe (without the metal needle), we gently press-infiltrated the Agrobacterium suspensions into the healthy leaves of 3-week-old N. benthamiana. Plants were maintained under LD white light conditions for 2 days after infiltration.

For the co-localization assay in maize protoplasts, constructs expressing ZmCRY1c or GL2 fused to GFP or CFP were transformed into E. coli MC1601. High-concentration constructs (1 to 2 μg/μL) were used for subsequent steps. Protoplasts were transformed with these constructs, cultured at 22°C for 14–16 h, then collected by centrifugation at 100 g for 2 min. Most of the supernatant was removed, and fluorescence signals were observed using a confocal microscope (Leica TSC SP8 STED 3X).

BiLC assays
ZmCRY1a, ZmCRY1b, ZmCRY1c; GL2, GL2V24M, and GL2G106D were fused to sequences encoding either the C- or N-terminus of firefly luciferase. These constructs were transformed into Agrobacterium strain GV3101. After infiltration into N. benthamiana leaves, plants were maintained under LD white light conditions for 2 days. The leaves were infiltrated with luciferin solution (1 mM luciferin and 0.01% Triton X-100), and images were captured using a CCD camera 5 min later.

Pull-down assays
The genes encoding ZmCRY1c-PHRW368A, along with GL2, GL2V24M, or GL2G106D, were cloned into pET-duet vector, with a 6×His tag at the N-terminus of GL2 or GL2 point mutations. E. coli BL21 (DE3) strain was used for protein expression. The bacteria were cultured at 37°C, 200 rpm, then induced with 0.25 mM isopropyl β-D-thiogalactopyranoside (IPTG) for 12 h. Bacterial cells were collected, resuspended in buffer A (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM TCEP) supplemented with 1 mM PMSF, lysed using a high-pressure cell disruptor at 700 bar, and centrifuged at 20,000 g for 1 h. The supernatant was incubated with Ni-NTA beads (Qiagen) at 4°C for 1 h. After incubation, the Ni-NTA beads with bound proteins were washed with buffer A supplemented with 25 mM imidazole. The proteins were eluted using buffer A supplemented with 250 mM imidazole, then concentrated and further purified using a Superdex-200 10/300 column.

Immunoblot
Immunoblot was carried out as described previously. For anti-ZmCRY1c-CCE and anti-GL2 polyclonal antibody production, the coding sequence of ZmCRY1c-CCE and full-length GL2 were cloned into pET28a vector (Novagen). Protein expression and purification were carried out as described in pull-down assays. Rabbit immunization with the purified target proteins were performed by Orizymes Biotechnology (Shanghai) Co., Ltd. Briefly, two New Zealand rabbits were used for the preparation of polyclonal antibodies. Freund’s complete adjuvant was used for the first injection, while subsequent immunizations used Freund’s incomplete adjuvant. The injection doses for each rabbit were 250 μg, 500 μg, and 500 μg of protein on days 0, 14, and 35, respectively. The rabbit serum was harvested on day 49. The anti-ZmCRY1c-CCE and anti-GL2 antibodies were used at a dilution of 1:3000. The anti-GFP (Abicode, #M0802-3a) and anti-Flag (Smart-Lifescience, #SLAB01C) monoclonal antibodies were used at 1:3000.

GC-MS analysis of wax composition
Wax extraction and gas chromatography-mass spectrometry (GC-MS) analyses were performed according to the described methods with some modifications76. In different assays, the horizontally expanding parts of the second leaves (2–4 cm from the tip) were taken, and 4 leaves were collected as one replicate and immediately immersed in 10 mL of chloroform with 20 μg of N-tetracosane as an internal standard for 30–60 s. The sample leaves were photographed, and the leaf area of each sample was calculated using Adobe Photoshop 2020. The extracts were then allowed to evaporate to dryness in a fume hood and subsequently transferred to a 2 mL tube. The dried extracts were dissolved in 60 μL of pyridine and derivatized by adding 60 μL of N,N-bis(trimethylsilyl)trifluoroacetamide (BSTFA; Macherey-Nagel) and incubating at 70°C for 1 h. The derivatized samples were filtered into a new sample vial and analyzed using gas chromatography with a flame ionization detector (Agilent GC 7890 Technologies) and GC-MS (Agilent gas chromatograph coupled to an Agilent MSD 5977B quadrupole mass selective detector). GC was performed using an HP-5MS column (Agilent, 30 m × 0.25 mm i.d., 0.25 μm film thickness, 5% phenyl methyl siloxane stationary phase). Helium (purity > 99.999%) was used as the carrier gas at a constant flow rate of 1.0 mL/min. The GC oven temperature was programmed as follows: an initial temperature of 50°C for 2 min, ramped at 5°C/min to 300°C, and held at 300°C for 15 min. The mass range was recorded from m/z 50 to m/z 700. The electron energy was maintained at 70 eV, while the source and quad temperatures were kept at 230°C and 150°C, respectively. Peaks were identified by comparing with standards and using the National Institute of Standards and Technology library (NIST, version 14). The peak areas were determined by cumulative scoring, and the contents of each ingredient were calculated by comparing with the C24 standard. The wax load per unit leaf area was calculated based on the sample leaf area. Three replicates were conducted for each experiment.

Statistical analysis
Phenotype analysis and gene expression analysis were assessed as described in the figure legends. P values were calculated using two-sided Student’s t-tests or one-way ANOVA with Tukey’s multiple comparisons test in GraphPad Prism 9 and are shown in bar graphs or source data.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript presents a series of carefully conducted set of experiments that show the intriguing interactions between the\nCRY1c blue light receptor and GL2, a protein in maize that is hypothesized to function in very long chain fatty acid\nelongation in the ER, thereby impacting the types of cuticular wax products that are made. The mechanism by which GL2 in\nmaize or CER2 in Arabidopsis is still not well understood. This manuscript provides interesting new insights into how GL2\nitself may be regulated by blue light. A series of different techniques (yeast-2-hybrid, IP-MS, BiFC) demonstrate that GL2 and\nCRY1c can interact, and that this may specifically enhance cuticular aldehyde synthesis. This manuscript greatly adds to our\nunderstanding of the regulation of cuticular wax biosynthesis and provides more context relative to the role of GL2, and is an\nimpactful addition to the field. The manuscript could benefit from additional consideration in a few key areas provided below.\nSpecific comments are also listed by line number.\nMajor themes/suggestions:\n1. Readability: The manuscript needs to be carefully edited for grammar and correct tense/form of different words. There are\nmany instances in which the incorrect form of a word is used (for example, in line 54, “reversal” should be “reverse”). The\nintended meaning of some sentences may be obscured, so careful editing is required.\n2. Introduction: The introduction could be improved with some reorganization. The first paragraph presents interesting\nbackground information about CRY1 and CRY2. However, it is not organized by protein or by theme and instead bounces\nbetween CRY1 and CRY2. Also, from this paragraph it is unclear whether CRY1 and CRY2 actually have photolyase\nactivity, as it is stated toward the end of this section that these proteins do not have DNA repair activity. The introduction also\ndoes not include information about the hypothesized functions of GL2 (and GL2-Like) in maize and CER2 (and CER2-Like)\nin Arabidopsis in very long chain fatty acid elongation. See specific comments/suggestions below.\n3. Methods: The methods do not include sufficient details. The methods for RNAseq data processing and quantification as\nwell as the tool/package used for DEG analysis should be provided. It is difficult to understand the methods for Y2H and the\nblue light treatment as written. The Immunoprecipitation–Mass Spectrometry (IP–MS) section should present information\nabout the antibodies used and describe (or cite) the methods for timsTOF Pro 2 specifically. The BiFC section does not\nprovide information about split reporters. The pull-down assay section does not provide the methods for the actual pull-down\nexperiment, including the antibody used and other details. Information provided about antibodies in the Immunoblot section\nshould include type of antibody (polyclonal vs. monoclonal), how they were produced, etc. In “Analysis of wax composition”,\nthe GC-FID method (oven temperature method), column information, etc. should be included. Limits of detection and\nquantification should also be included.\n4. Connections between transcriptome results and role of CRYs in cuticle biosynthesis: It is unclear how the transcriptome\nwork that is presented in the first part of the manuscript is linked to the CRY-GL2 interactions in the second part of the\nmanuscript. It is not clearly stated whether the only cuticle-related genes that were differentially expressed in the\ntranscriptome analyses were the ones that were noted in the Results (two KCSs and genes involved in de novo fatty acid\nsynthesis). Were gl2 or gl2-like differentially expressed? These too are genes involved in very long chain fatty acid\nbiosynthesis. In the second half of the manuscript, KCS6 was shown to interact with CRY1c. Was KCS6 differential\nexpressed in the RNAseq analysis? In a similar vein, the gene encoding KCS4 was shown to be differentially expressed\n(Figure 2d), might it also interact with CRYc1? It would also be helpful to know whether genes in the reductive or\ndecarbonylative pathways of cuticular wax biosynthesis, or genes related to cutin biosynthesis, were differentially\nexpressed.\n5. Physical interactions between CRYs and cuticle-related proteins: The interactions between CRY1c and GL2 are\nintriguing. It is not clear whether interactions between CRY1 and GL2-Like were tested and found not to interact or if GL2-\nLike was not pursued. Are the residues in GL2 that are thought to interact with CRY2 (and that yield a glossy and water\nbeading phenotype when mutated) conserved in GL2-LIKE? Although GL2-LIKE is not expressed in seedlings, it is\nexpressed in other tissues and therefore should be considered for its potential interactions with Cry1c. Has it been tested\nwhether and of the CRY1s and KCS6 physically interact? This seems like a formal possibility that should be tested or at\nleast addressed in the text. Also, was CRY2 tested?\n6. Figures: The formatting of the bar graphs and color coding of the data points only make the data hard to see in many\ncases. Cuticular wax graphs in Figures 3, 5 and 6 are very hard to interpret. Each genotype is represented by a filled or open\ncircle of different colors (many having similar shades), and it is hard to distinguish open from closed circles. The bars\nthemselves are all either white or gray. It would be easier to track genotypes if each bar were colored separately instead of\nthe data points. This comment also applies to similarly formatted graphs plotting different types of data (e.g. Figure 3d).\nSpecific comments, by line:\nLine 46. A word is needed after “aldehyde-forming”. It is unclear what is meant by the “enigma of aldehyde-forming”.\nLine 85: “blue light regulated-wax biosynthesis” is mentioned in this sentence. It should be expanded upon with relevant\nresearch cited as this is the focus of the paper.\nLines 102-103: This statement, “However, the VLC-aldehyde forming enzyme and VLC-alkane synthesis remain largely\nunknown” is over-stated and should be modified or deleted. The following paragraph presents information about the\nCER1/CER3 complex that produces hydrocarbons in Arabidopsis.\nLines 110-117: This section is a bit misleading relative to GL2. The text should clearly state that it is hypothesized that GL2\nand GL2-LIKE function in the fatty acid elongation pathway (upstream of aldehyde, alcohol or hydrocarbon formation),\nbased on the observations that VLCFA composition differs in gl2 mutants compared to wildtype. Also, it is unclear how\nBAHD-acyltransferase activity would function in fatty acid elongation. Work on CER2 and CER2-LIKE in Arabidopsis should be\ndescribed as well as the one maize GL2 paper that is cited (there is a 2nd one that could also be cited). Although some\nappropriate papers are cited in this section, details about GL2 (or CER2) and its role in fatty acid elongation are not\nmentioned and should be.\nLines 133-135: the type of sequence identify should be noted. Is it amino acid or DNA sequence?\nLines 186-187: It is not clear how the AtCRY1 protein was monitored in the ZmCRY expressing Arabidopsis lines.\nLine 189: The statement “while ZmCRY1c was stable” is not supported by Supplemental Figure 3g. Relative protein level\nchanges drastically at each time point (not stable) and no error bars are presented in this plot or in panel e. Without\nreplication of these experiments, it is difficult to see the patterns (or lack thereof). While “stability” is better supported in\nmedium blue light (Supplemental Figure 3e), the text in this section should be reevaluated relative Supplemental Figure 3g\nand stability of ZmCRY1c.\nLine 195: The title of this section should be modified. The second “the” may need to be deleted.\nLine 202: “regulated” doesn’t seem to be the correct word here. “mediated” might work.\nLines 203-204: Presenting the rationale for the statement, “A Venn diagram analysis indicated 203 that 45.49% (2622) of\nthese DEGs were potentially dependent on ZmCRYs.”, would be helpful. Presumably, the authors are concluding that these\n2622 genes that are differentially expressed in wildtype plants between dark and blue light but not in cry-quadruple mutants,\nare therefore dependent on the presence of one or more cry genes.\nLines 228-233: The genes listed here are mostly involved in de novo fatty acid biosynthesis and not necessarily related to\ncuticular waxes. Kcs4 is known to be involved in cuticular wax biosynthesis and reference 30 could be cited here to\nstrengthen the last sentence of this section. If other genes involved in cuticle synthesis are differentially expressed (for\nexample those in the decarbonylative or reductive pathways), these should be shown to make a stronger case.\nLines 235-236: Fatty acids (even VLCFAs) play different roles in the cell (e.g. components of sphingolipids) and are not only\nprecursors for or themselves are cuticular waxes. This statement is not very convincing and could be reframed. The data that\nfollows indeed does show compelling differences in cuticular wax composition.\nLines 240-245: The differences in total cuticular wax accumulation and in specific metabolite differences under exposure to\nblue light could be better highlighted by presenting in either fold-change or percentage increase/decrease.\nLines 253-258: The authors might also note whether there were statistical differences in C32 fatty acid, as the C32 fatty-acyl\nCoA would be a precursor to either the C32 fatty acid or C32 fatty aldehyde and therefore differences in accumulation might\nalso occur in the C32 fatty acid (or alcohol).\nLines 269-270: I am not sure how to interpret “B73 showed 58.1% resistance from 0.77 to 0.447, 269 CRY1c-OE showed\n94.87% resistance from 0.767 to 0.727”. It is unclear what is meant by 58.1% resistance or 94.87% resistance. It seems that it\nis simply saying that the 0.727 value is ~95% of the 0.767 value, but the importance of that result is not clear. A similar\nstatement in lines 272-273 should also be rephrased for clarity.\nLine 283: If referring to the protein, Glossy2 should be written as GLOSSY2.\nLine 296: “the newly identified GL2” makes it seem like GL2 is a newly identified protein in this study, which is not the case.\nThis should be rephrased.\nLine 297: In the statement, “GL2 specifically enhances the elongation process between chain lengths of C30 and C32”, the\ntext needs to clearly state that it is the elongation of the very long chain acyl-CoA. The promotion of “the formation of C32\naldehyde, C31 alkane, C31 secondary alcohol, and C31 ketone” is therefore likely indirect.\nLines 323-325: The methods for this yeast-2-hybrid experiment with only the PHR domain need to be added to the Y2H\nsection of the Methods.\nLines 326-327: There is a brief statement that there is a crystal structure of GL2 interacting with the CRY1c PHR domain with\nno additional information provided (including the significance of the ZmCRY1c-PHR W368A variant). The cited reference\nprovides only a title and no journal information and was not found in a Google Scholar search. This reference should be\nupdated, and this should be carefully discussed in the Discussion.\nLine 334: What specific allele does “gl2” refer to here and elsewhere below (e.g. line 350)? Does this refer to the gl2-\nSalamini line that is often used as the reference allele? This should be clarified, as the gl2-EMS alleles that also show a\nglossy and a water beading phenotype would also be “gl2 alleles”.\nLines 359-353: Do these variants of the Gl2 gene that show aberrant cuticular wax compositions also show differences if\nFv/Fm? If so, that would more directly relate the CRY-mediated resistance to UV-B to the cuticle.\nLines 365-367: It is not clear what gl2 mutant was used for this experiment.\nLine 389: The authors did not seem to test epicuticular waxes per se, and so here “epicuticle” may not be correct. Simply\nstating “cuticular waxes” might be more accurate.\nLine 485: Please clarify the nature of the cDNA library, including what tissue or tissues were represented, the number of\ngenes in the library, etc. A reference should be provided if this is a commercial library or was previously used in other\npublished papers.\nLine 547-549: This statement about silylation is not a complete sentence.\nSupplemental Figure 4d: The specific KCS should be noted (which number?).\nSupplemental Table 4: Please include gene names with each of the Zm IDs so that protein annotation is clear.", "reviewer_2": "In this paper , Zhao et al investigate the function of the 4 identified cryptochromes in Zea Mays. They generated\ncombinatorial mutant using CRISPR Cas9 technology and firstly characterized their photomorphogenic phenotype, showing\ntheir redundant role on regulation of mesocotyl elongation under blue light, with Cry2 playing a minor role.\nTo further understand the fonctions of ZmCrys and in particular ZmCry1c (which seem to accumulate in the cytosol), they\ncompare the transcriptome of the quadruple mutant to WT, and analyse the interactome of Cry1c (by Y2H and Co-IP/MS).\nThey focus on the blue light regulation of wax accumulation and composition and show that CRYs control this metabolism\nnot only at the transcriptional levels by inducing the expression of genes coding for enzymes related to this metabolism, but\nalso ZmCry1c could more directly regulate the activity of at least one enzyme of the pathway, GL2, via direct interaction.\nThis is an interesting finding as it indicates the potential function of photoreceptor on metabolism via direct interaction with\nenzymes of the pathway. The experiments are nicely described and the conclusions seem in general supported by the data.\nHowever, I have some concern about the proposed link between UV-B tolerance and GL2 activity/wax composition\nregulation by CRY1. It is clear from previous studies and from their own data that CRY contribute to UV-B tolerance in\ndifferent species. However, how GL2 activity/wax composition contribute to UV-B tolerance is not clear. An important\nexperiment will be to test the UV-B tolerance of GL2 variants impaired in the interaction with ZmCry1c and gl2 mutant (in WT\nand in Overexpressor line of CRY1c).\nOther minor comments concern the writing, in particular the introduction and missing references in different parts of the\nmanuscript.\n-The paragraph on CRY function needs some corrections/precisions.\nFor example :\nline 57 : “In Arabidopsis, two CRY photoreceptors, CRY1 primarily facilitate the plant photomorphogenesis, while CRY2\nregulate the floral initiation“ : CRY1 and CRY2 play redundant function in e.g. photomorphogenesis, so please correct the\nsentence to not restrict CRY2 function to flowering.\n“The functionally equivalent dominant mutations in CRY2 boost the adaptation and rapid evolution of species, which may\nserve as a common genetic basis for ruderality in Brassicaceae 1 “ : To me it s not clear which “equivalent dominant\nmutations “ they refer to, and the rest of the sentence is also very cryptic.\nLine 67 : “CRY1s regulate the plant height and shade avoidance in soybean and maize” : please mention also Arabidopsis\nand the corresponding references, e.g. Keller et al. 2011; Pedmale et al., 2016.\n-“UV-B stress tolerance related metabolites including phenylpropanoid, flavonoid, anthocynin and fatty acid biosynthesis” :\nAny references indicating that fatty acid biosynthesis play a role in UV tolerance ?\n-“Epidermal wax functions as the initial defense against UV-B radiation.” Could you please also add a reference ?\n-Line 325 “Subsequently, the crystal structure of the constitutive active CRY1c PHR domain (ZmCRY1c-PHRW368A 20) in\ncomplex with GL2 was determined by Yaqi L., et al.60 “ which corresponds in the reference list to :\nLiu, Y. et al. Crystal structure of a plant cryptochrome photo signaling complex (2025).\nI cannot find the reference, the journal and link are missing. This reference is also cited as source for the absence of\ninteraction between Cry 1 and GL2 in Arabidopsis, however again we can not check this paper.\nLegends\nplease revise the sentence “Lowercase letters indicate statistically significant differences “-> different letters indicate\nstatistically significant differences (or common letters indicate no statistically significant difference)\nMaterial and Methods : Survival rate: please specify how this was quantitatively determined", "reviewer_3": "Cryptochromes are UV-A / Blue light photoreceptors that bare resemblance to photolyses from which they are evolutionary\nderived.\nThis manuscript starts from the observation that Zea mays has four CRY genes, annotated here as CRY1a-c and CRY2.\nThe\nmanuscript studies if these CRYs are involved in blu light-mediated UV-B responses at the level of photo protective) waxes.\nIt has been shown previously in Arabidopsis that CRYs are indeed involved in blue light-mediated UV-B responses, that in\nArabidopsis occur through the UV-B photoreceptor UVR-8 primarily.\nDespite this overlap with existing knowledge in Arabidopsis, the manuscript does explore this topic further and shows how\nthe GLOSSY2 (GL2) protein is required for CRY1 to modulate wax synthesis.\nThere are a number of elements that I would suggest the authors to consider.\n1. It might be good to highlight better what the innovative elements of this manuscript are. Since CRY-mediated detection of\nB light is already known to modulate UV-B responsiveness, it is important to have this very clear.\n2. You made a variety of knockout and OE lines for the different CRYs, but their phenotypes are described quite\nrudimentarily. At the very minimum it is necessary to also show dark and white light controls for the materials in Fig 1b,c,d\nand Supplemental Figs1 e-g, and Suppl Fig2b,c,e\nThe same applies for Suppl Fig 3a-c: include dark and white light controls.\n3. You start from the question why there are several CRY1 copies in maize. However, you focus purely on the UV-B\nprotection. There are many more roles that cryptochromes have and they may be differential between the different copies. If\nyou define the scope so broadly (which I think is a good idea), then it would be helpful to also scan a wider range of cantons.\nSince you have the lines made already, it should not be very difficult to do so. Obvious aspects to check would include\ncircadian rhythms (of gene expression), DNA binding ability (since they're derived from photolyases), plant development.\nIn your\n4. The subheading results in line 283 states that CRY1-GL2 interaction is blue light-dependent. Since the co-IP shows that\nthe interaction also occurs in darkness, but much less, I would be a bit more careful. Blue light seems to strengthen the\ninteraction, but is not fully mandatory. In case of a photoreceptor this is a relevant distinction.\n5. With respect to BiFC assays in Fig4, the negative control of free nYFP (not fused to a protein) is not ideal. Since you have\nthe the mutant CRY1 version involved this is not a big problem. You could have chosen a different protein to fuse it to\nthough.\nIn addition, have you verified that the different constructs are expressed equally well in your assays? If mutant CRY1 is for\nsome reason expressed more weakly than WT CRY1, your results might look similar to what you see now, but for a very\ndifferent reason (and conclusion).\n6. Phenotyping of UV-B resistance should be improved. You used only three replicates (judging from the graph in Fig3d)\nfor Fv/Fm quantification, and show photo's of only two leaves for visual inspection (Fig. 3c). Since these data are needed to\nshow the functionality of your study, they are very important and should be done more elaborately. In these assays I would\nrecommend including gl2 mutant as well.\n7. The big unknown, or absentee in your manuscript, is the UV-B sensor itself. Your summarising Figure 6d also omits a UV-\nB sensor. of course, this sensor is known from Arabidopsis: UVR-8. It is not clear to me why UVR-8 was not included in your\nstudy. Since the stress that you pose on your plants for which you expect the Blue-CRY1-GL2-Wax pathway to be relevant,\nis UV-B and this is sensed through UVR-8 to activate photoprotective responses, this would be a crucial element in your\nstudy.\n8. Information about biological replication (numbers, how were they collected, etc) is often missing. This would best be\nprovided in the methods and in the figure captions.\nMinor: There are quite some typo's in your manuscript. They do not make it difficult to read at all, but it would be good to fix\nthem. I would assume a spell checker can detect most of them."}, "author_response": "RESPONSE: We appreciate greatly these excellent points raised by the reviewer!\nMajor themes/suggestions:\n1. Readability: The manuscript needs to be carefully edited for grammar and correct tense/form\nof different words. There are many instances in which the incorrect form of a word is used (for\nexample, in line 54, “reversal” should be “reverse”). The intended meaning of some sentences\nmay be obscured, so careful editing is required.\nRESPONSE: Thanks a million! It is revised, the manuscript is carefully edited.\n2. Introduction: The introduction could be improved with some reorganization. The first\nparagraph presents interesting background information about CRY1 and CRY2. However, it is\nnot organized by protein or by theme and instead bounces between CRY1 and CRY2. Also,\nfrom this paragraph it is unclear whether CRY1 and CRY2 actually have photolyase\nactivity, as it is stated toward the end of this section that these proteins do not have DNA repair activity.\nThe introduction also does not include information about the hypothesized functions of GL2\n(and GL2-Like) in maize and CER2 (and CER2-Like) in Arabidopsis in very long chain fatty\nacid elongation. See specific comments/suggestions below.\nRESPONSE: Thanks a million! As suggested, we’ve reorganized the CRY1/CRY2 background\nin the introduction thematically by protein (lines 59–72), clarified that CRYs lack photolyase\nactivity (lines 51–53), and added information on the hypothesized roles of maize GL2 (and\nGL2-Like) and Arabidopsis CER2 (and CER2-Like) in very long chain fatty acid elongation\n(lines 110–124).\n3. Methods: The methods do not include sufficient details. The methods for RNAseq data\nprocessing and quantification as well as the tool/package used for DEG analysis should be\nprovided. It is difficult to understand the methods for Y2H and the blue light treatment as\nwritten. The Immunoprecipitation–Mass Spectrometry (IP–MS) section should present\ninformation about the antibodies used and describe (or cite) the methods for timsTOF Pro 2\nspecifically. The BiFC section does not provide information about split reporters. The pull-\ndown assay section does not provide the methods for the actual pull-down experiment,\nincluding the antibody used and other details. Information provided about antibodies in the\nImmunoblot section should include type of antibody (polyclonal vs. monoclonal), how they\nwere produced, etc. In “Analysis of wax composition”, the GC-FID method (oven temperature\nmethod), column information, etc. should be included. Limits of detection and quantification\nshould also be included.\nRESPONSE: Thank you very much for your thorough feedback on the Methods section. We have\naddressed the gaps in detail as follows:\nFor RNA-seq analysis, we have added comprehensive details on data processing, quantification,\nand the specific tools/packages used for differential expression analysis, with relevant citations\n(lines 515–521).\nThe Y2H assay protocol and blue light treatment procedures have been clarified and expanded\n(lines 539–544).\nIn the IP-MS section, we have included information on the antibodies used (lines 561–563) and\ndetailed the timsTOF Pro 2 methodology, with relevant citations (lines 568–582).\nThe BiFC section now specifies the split reporters utilized (lines 584–586).\nFor the pull-down assay, we have added step-by-step methods, including antibody details and\nexperimental parameters (lines 611–619).\nImmunoblot antibody information has been expanded to include type (polyclonal/monoclonal),\nproduction methods, and other relevant characteristics (lines 621–632).\nThe \"Analysis of wax composition\" section now includes complete GC-FID parameters (oven\ntemperature program, column specifications) as well as limits of detection and quantification\n(lines 634–657).\n4. Connections between transcriptome results and role of CRYs in cuticle biosynthesis: It is\nunclear how the transcriptome work that is presented in the first part of the manuscript is linked\nto the CRY-GL2 interactions in the second part of the manuscript. It is not clearly stated whether\nthe only cuticle-related genes that were differentially expressed in the transcriptome analyses\nwere the ones that were noted in the Results (two KCSs and genes involved in de novo fatty\nacid synthesis). Were gl2 or gl2-like differentially expressed? These too are genes involved in\nvery long chain fatty acid biosynthesis. In the second half of the manuscript, KCS6 was shown\nto interact with CRY1c. Was KCS6 differential expressed in the RNAseq analysis? In a similar\nvein, the gene encoding KCS4 was shown to be differentially expressed (Figure 2d), might it\nalso interact with CRYc1? It would also be helpful to know whether genes in the reductive or\ndecarbonylative pathways of cuticular wax biosynthesis, or genes related to cutin biosynthesis,\nwere differentially expressed.\nRESPONSE: Thanks a million! We’ve addressed the connections as follows:\nAdditional cuticle-related DEGs from RNA-seq (including KCS3, KCS4, GL1, GL4, GL1-5)\nwere validated by RT-qPCR; these genes are blue light-upregulated but CRY-independent (line\n235-240 and Fig. 2d, Fig. S4d). GL2, GL2-Like, and KCS6 showed no significant differential\nexpression in transcriptome data.\nThe interaction between KCS4 and CRY1c was tested using BiFC and BiLC assays but no\ninteraction was detected. The results were added in Fig. 4d and Fig. S6e. CRY1c does not\ninteract with KCS6. We added a BiLC assay in the supplymentary Fig. 8c to make a clear\nannouncement. Our results suggest that ZmCRY1c and KCS6 may compete to interact with GL2.\n5. Physical interactions between CRYs and cuticle-related proteins: The interactions between\nCRY1c and GL2 are intriguing. It is not clear whether interactions between CRY1 and GL2-\nLike were tested and found not to interact or if GL2-Like was not pursued. Are the residues in\nGL2 that are thought to interact with CRY2 (and that yield a glossy and water beading\nphenotype when mutated) conserved in GL2-LIKE? Although GL2-LIKE is not expressed in\nseedlings, it is expressed in other tissues and therefore should be considered for its potential\ninteractions with Cry1c. Has it been tested whether and of the CRY1s and KCS6 physically\ninteract? This seems like a formal possibility that should be tested or at least addressed in the\ntext. Also, was CRY2 tested?\nRESPONSE: Thanks a million! We’ve addressed this as follows:\nGL2 interaction residues are conserved in GL2-LIKE, which showed weak interaction with\nZmCRY1c in BiLC but not Y2H assays (line 329-332, and supplymentary Fig.6f, g).\nA new BiLC assay added in supplymentary Fig. 8c confirms no CRY1c-KCS6 interaction (line\n396-398). Additionally, our results showed that ZmCRY2 showed a weak interaction with GL2\nin the BiLC assay (supplymentary Fig. 6d).\n6. Figures: The formatting of the bar graphs and color coding of the data points only make the\ndata hard to see in many cases. Cuticular wax graphs in Figures 3, 5 and 6 are very hard to\ninterpret. Each genotype is represented by a filled or open circle of different colors (many\nhaving similar shades), and it is hard to distinguish open from closed circles. The bars\nthemselves are all either white or gray. It would be easier to track genotypes if each bar were\ncolored separately instead of the data points. This comment also applies to similarly formatted\ngraphs plotting different types of data (e.g. Figure 3d).\nRESPONSE: Thanks a million! They are changed\nSpecific comments, by line:\nLine 46. A word is needed after “aldehyde-forming”. It is unclear what is meant by the “enigma\nof aldehyde-forming”.\nRESPONSE: We change it to “enigma of the aldehyde-forming pathway”, thanks.\nLine 85: “blue light regulated-wax biosynthesis” is mentioned in this sentence. It should be\nexpanded upon with relevant research cited as this is the focus of the paper.\nRESPONSE: Thanks a million! It was expanded as suggested (line 86-91).\nLines 102-103: This statement, “However, the VLC-aldehyde forming enzyme and VLC-alkane\nsynthesis remain largely unknown” is over-stated and should be modified or deleted. The\nfollowing paragraph presents information about the CER1/CER3 complex that produces\nhydrocarbons in Arabidopsis.\nRESPONSE: Thanks a million! It was deleted.\nLines 110-117: This section is a bit misleading relative to GL2. The text should clearly state\nthat it is hypothesized that GL2 and GL2-LIKE function in the fatty acid elongation pathway\n(upstream of aldehyde, alcohol or hydrocarbon formation), based on the observations that\nVLCFA composition differs in gl2 mutants compared to wildtype. Also, it is unclear how\nBAHD-acyltransferase activity would function in fatty acid elongation. Work on CER2 and\nCER2-LIKE in Arabidopsis should be described as well as the one maize GL2 paper that is\ncited (there is a 2nd one that could also be cited). Although some appropriate papers are cited\nin this section, details about GL2 (or CER2) and its role in fatty acid elongation are not\nmentioned and should be.\nRESPONSE: Thanks a million! It is changed as suggested (line 110-124).\nLines 133-135: the type of sequence identify should be noted. Is it amino acid or DNA sequence?\nRESPONSE: Thanks a million! It is changed. It is amino acid.\nLines 186-187: It is not clear how the AtCRY1 protein was monitored in the ZmCRY expressing\nArabidopsis lines.\nRESPONSE: Thanks a million! An AtCRY1-Flag OE line was used for monitoring the AtCRY1\nprotein. The information is added (line 186-189, 489).\nLine 189: The statement “while ZmCRY1c was stable” is not supported by Supplemental Figure\n3g. Relative protein level changes drastically at each time point (not stable) and no error bars\nare presented in this plot or in panel e. Without replication of these experiments, it is difficult\nto see the patterns (or lack thereof). While “stability” is better supported in medium blue light\n(Supplemental Figure 3e), the text in this section should be reevaluated relative Supplemental\nFigure 3g and stability of ZmCRY1c.\nRESPONSE: Thanks a million! New experiment replicates were added for these analyses\n(supplymentary Fig.3f, h and associated source data).\nLine 195: The title of this section should be modified. The second “the” may need to be deleted.\nRESPONSE: It is deleted as suggested, thanks.\nLine 202: “regulated” doesn’t seem to be the correct word here. “mediated” might work.\nRESPONSE: It is changed, thanks.\nLines 203-204: Presenting the rationale for the statement, “A Venn diagram analysis indicated\n203 that 45.49% (2622) of these DEGs were potentially dependent on ZmCRYs.”, would be\nhelpful. Presumably, the authors are concluding that these 2622 genes that are differentially\nexpressed in wildtype plants between dark and blue light but not in cry-quadruple mutants, are\ntherefore dependent on the presence of one or more cry genes.\nRESPONSE: They have been modified as suggested, thanks.\nLines 228-233: The genes listed here are mostly involved in de novo fatty acid biosynthesis and\nnot necessarily related to cuticular waxes. Kcs4 is known to be involved in cuticular wax\nbiosynthesis and reference 30 could be cited here to strengthen the last sentence of this section.\nIf other genes involved in cuticle synthesis are differentially expressed (for example those in\nthe decarbonylative or reductive pathways), these should be shown to make a stronger case.\nRESPONSE: Thanks a million! The citation is added. Additionally, we have validated the\ndifferentially expressed cuticle synthesis-related genes identified in RNA-seq via RT-qPCR (line\n235-240 and Fig. 2d, supplymentary Fig. 4d).\nLines 235-236: Fatty acids (even VLCFAs) play different roles in the cell (e.g. components of\nsphingolipids) and are not only precursors for or themselves are cuticular waxes. This statement\nis not very convincing and could be reframed. The data that follows indeed does show\ncompelling differences in cuticular wax composition.\nRESPONSE: Thanks a million! They are modified as suggested, genes involved in cuticle\nsynthesis differentially expressed in RNA-seq are validated by RT-qPCR (Fig. 2d, line 235-240).\nLines 240-245: The differences in total cuticular wax accumulation and in specific metabolite\ndifferences under exposure to blue light could be better highlighted by presenting in either fold-\nchange or percentage increase/decrease.\nRESPONSE: Thanks a million! The fold-changes are added as suggested (line 250-252).\nLines 253-258: The authors might also note whether there were statistical differences in C32\nfatty acid, as the C32 fatty-acyl CoA would be a precursor to either the C32 fatty acid or C32\nfatty aldehyde and therefore differences in accumulation might also occur in the C32 fatty acid\n(or alcohol).\nRESPONSE: Thanks a million! They are added as suggested (line 262-265).\nLines 269-270: I am not sure how to interpret “B73 showed 58.1% resistance from 0.77 to\n0.447, 269 CRY1c-OE showed 94.87% resistance from 0.767 to 0.727”. It is unclear what is\nmeant by 58.1% resistance or 94.87% resistance. It seems that it is simply saying that the 0.727\nvalue is ~95% of the 0.767 value, but the importance of that result is not clear. A similar\nstatement in lines 272-273 should also be rephrased for clarity.\nRESPONSE: Thanks a million! As suggested, we have rephrased these statements for clarity\nand added experimental replicates to strengthen the results (line 278-281).\nLine 283: If referring to the protein, Glossy2 should be written as GLOSSY2.\nRESPONSE: It is changed as suggested, thanks.\nLine 296: “the newly identified GL2” makes it seem like GL2 is a newly identified protein in\nthis study, which is not the case. This should be rephrased.\nRESPONSE: Thanks a million! We’ve rephrased it to: “…, as well as GL2, which was previously\nidentified via Y2H assay” (line304-307).\nLine 297: In the statement, “GL2 specifically enhances the elongation process between chain\nlengths of C30 and C32”, the text needs to clearly state that it is the elongation of the very long\nchain acyl-CoA. The promotion of “the formation of C32 aldehyde, C31 alkane, C31 secondary\nalcohol, and C31 ketone” is therefore likely indirect.\nRESPONSE: Thanks a million! As suggested, we’ve revised it to: “GL2 specifically enhances\nthe elongation of C30 and C32 VLC acyl-CoA and indirectly promotes the formation of C32\naldehyde, C31 alkane, C31 secondary alcohol, and C31 ketone.”\nLines 323-325: The methods for this yeast-2-hybrid experiment with only the PHR domain\nneed to be added to the Y2H section of the Methods.\nRESPONSE: Thanks a million! They are added as suggested (line545-552).\nLines 326-327: There is a brief statement that there is a crystal structure of GL2 interacting\nwith the CRY1c PHR domain with no additional information provided (including the\nsignificance of the ZmCRY1c-PHR W368A variant). The cited reference provides only a title\nand no journal information and was not found in a Google Scholar search. This reference should\nbe updated, and this should be carefully discussed in the Discussion.\nRESPONSE: As suggested, the back-to-back submitted paper is available on preprint Biorxiv\nlink:https://www.biorxiv.org/content/10.1101/2025.08.06.668860v1 Thanks!\nLine 334: What specific allele does “gl2” refer to here and elsewhere below (e.g. line 350)?\nDoes this refer to the gl2-Salamini line that is often used as the reference allele? This should be\nclarified, as the gl2-EMS alleles that also show a glossy and a water beading phenotype would\nalso be “gl2 alleles”.\nRESPONSE: The “gl2” allele referenced here (and in line349-350) refers to the gl2-ref allele,\nas described in Zheng, J. et al. (2019; Plant J 97, 530-542. https://doi.org/10.1111/tpj.14140).\nWe have updated the text to “gl2-ref” for clarity. Thanks!\nLines 359-353: Do these variants of the Gl2 gene that show aberrant cuticular wax compositions\nalso show differences if Fv/Fm? If so, that would more directly relate the CRY-mediated\nresistance to UV-B to the cuticle.\nRESPONSE: The gl2 variants do show differences in the Fv/Fm measurement; the assay result\nhas been added in Fig. 6c. Thanks!\nLines 365-367: It is not clear what gl2 mutant was used for this experiment.\nRESPONSE: The gl2 mutant used in this experiment is the gl2-ref allele, as described in Zheng,\nJ. et al. (2019; Plant J 97, 530-542. https://doi.org/10.1111/tpj.14140). We have updated the\ntext to “gl2-ref” for clarity. Thanks!\nLine 389: The authors did not seem to test epicuticular waxes per se, and so here “epicuticle”\nmay not be correct. Simply stating “cuticular waxes” might be more accurate.\nRESPONSE: It is changed as suggested, thanks.\nLine 485: Please clarify the nature of the cDNA library, including what tissue or tissues were\nrepresented, the number of genes in the library, etc. A reference should be provided if this is a\ncommercial library or was previously used in other published papers.\nRESPONSE: It is added as suggested, thanks.\nLine 547-549: This statement about silylation is not a complete sentence.\nRESPONSE: It is rephrased, thanks.\nSupplemental Figure 4d: The specific KCS should be noted (which number?).\nRESPONSE: The gene names of the KCSs have been corrected as follows: Zm00001eb167740\n(KCS3) and Zm00001eb344070 (KCS4). These, along with other genes involved in cuticle\nsynthesis that were differentially expressed in RNA-seq, were validated by RT-qPCR, as shown\nin Fig. 2d, thanks.\nSupplemental Table 4: Please include gene names with each of the Zm IDs so that protein\nannotation is clear.\nRESPONSE: They are added as suggested, thanks.\nReviewer #2:\nIn this paper , Zhao et al investigate the function of the 4 identified cryptochromes in Zea Mays.\nThey generated combinatorial mutant using CRISPR Cas9 technology and firstly characterized\ntheir photomorphogenic phenotype, showing their redundant role on regulation of mesocotyl\nelongation under blue light, with Cry2 playing a minor role.\nTo further understand the fonctions of ZmCrys and in particular ZmCry1c (which seem to\naccumulate in the cytosol), they compare the transcriptome of the quadruple mutant to WT, and\nanalyse the interactome of Cry1c (by Y2H and Co-IP/MS). They focus on the blue light\nregulation of wax accumulation and composition and show that CRYs control this metabolism\nnot only at the transcriptional levels by inducing the expression of genes coding for enzymes\nrelated to this metabolism, but also ZmCry1c could more directly regulate the activity of at\nleast one enzyme of the pathway, GL2, via direct interaction. This is an interesting finding as it\nindicates the potential function of photoreceptor on metabolism via direct interaction with\nenzymes of the pathway. The experiments are nicely described and the conclusions seem in\ngeneral supported by the data. However, I have some concern about the proposed link between\nUV-B tolerance and GL2 activity/wax composition regulation by CRY1. It is clear from\nprevious studies and from their own data that CRY contribute to UV-B tolerance in different\nspecies. However, how GL2 activity/wax composition contribute to UV-B tolerance is not clear.\nAn important experiment will be to test the UV-B tolerance of GL2 variants impaired in the\ninteraction with ZmCry1c and gl2 mutant (in WT and in Overexpressor line of CRY1c).\nRESPONSE: We appreciate greatly these excellent points raised by the reviewer! The Fv/Fm\nmeasurements for the suggested lines have been added in Fig. 6c to address the link between\nGL2 activity/wax composition to UV-B tolerance.Thanks for your guidance.\nOther minor comments concern the writing, in particular the introduction and missing\nreferences in different parts of the manuscript.\n-The paragraph on CRY function needs some corrections/precisions.\nFor example :\nline 57 : “In Arabidopsis, two CRY photoreceptors, CRY1 primarily facilitate the plant\nphotomorphogenesis, while CRY2 regulate the floral initiation“ : CRY1 and CRY2 play\nredundant function in e.g. photomorphogenesis, so please correct the sentence to not restrict\nCRY2 function to flowering.\nRESPONSE: Thanks a million! It is corrected as suggested (line 59-73).\n“The functionally equivalent dominant mutations in CRY2 boost the adaptation and rapid\nevolution of species, which may serve as a common genetic basis for ruderality in Brassicaceae\n1 “ : To me it s not clear which “equivalent dominant mutations “ they refer to, and the rest of\nthe sentence is also very cryptic.\nRESPONSE: Thanks a million! It is modified to “A functionally equivalent dominant mutation,\nCRY2W374A, enhances species adaptation and rapid evolution, potentially serving as a genetic\nbasis for ruderality in Brassicaceae”. Additionally, we have rephrased this CRY1/CRY2\nbackground paragraph thematically by protein for better clarity (lines 59–73).\nLine 67 : “CRY1s regulate the plant height and shade avoidance in soybean and maize” : please\nmention also Arabidopsis and the corresponding references, e.g. Keller et al. 2011; Pedmale et\nal., 2016.\nRESPONSE: Thanks a million! We have revised it to: “CRY1 also plays…, and regulates the\nplant height and shade avoidance responses–functions conserved across Arabidopsis, soybean\nand maize.” (line 61-64)\n-“UV-B stress tolerance related metabolites including phenylpropanoid, flavonoid, anthocynin\nand fatty acid biosynthesis” : Any references indicating that fatty acid biosynthesis play a role\nin UV tolerance ?\nRESPONSE: As noted in Shoaib, N. et al. (2024; Plant Cell Environ 47, 387-407.\nhttps://doi.org/10.1111/pce.14774), plants up-regulate specific metabolites—including\nunsaturated fatty acids—during acclimation to UV-B radiation and drought stress, with these\nmetabolites contributing to environmental stress protection. However, the authors also note that\nresearch on fatty acid content, composition, and unsaturation levels under combined drought\nand UV-B stress remains limited, and the specific role of unsaturated fatty acids in mitigating\nsuch combined stress is not yet clear. we have cited this reference as suggested. thanks!\n-“Epidermal wax functions as the initial defense against UV-B radiation.” Could you please\nalso add a reference ?\nRESPONSE: Thanks a million! It is added.\n-Line 325 “Subsequently, the crystal structure of the constitutive active CRY1c PHR domain\n(ZmCRY1c-PHRW368A 20) in complex with GL2 was determined by Yaqi L., et al.60 “ which\ncorresponds in the reference list to :\nLiu, Y. et al. Crystal structure of a plant cryptochrome photo signaling complex (2025).\nI cannot find the reference, the journal and link are missing. This reference is also cited as\nsource for the absence of interaction between Cry 1 and GL2 in Arabidopsis, however again we\ncan not check this paper.\nRESPONSE: As suggested, the back-to-back submitted paper is available on preprint Biorxiv\nlink:https://www.biorxiv.org/content/10.1101/2025.08.06.668860v1 Thanks!\nLegends\nplease revise the sentence “Lowercase letters indicate statistically significant differences “->\ndifferent letters indicate statistically significant differences (or common letters indicate no\nstatistically significant difference)\nRESPONSE: It is changed as suggested, thanks.\nMaterial and Methods : Survival rate: please specify how this was quantitatively determined\nRESPONSE: It is added as suggested (line 495-498), thanks.\nReviewer #3:\nCryptochromes are UV-A / Blue light photoreceptors that bare resemblance to photolyses from\nwhich they are evolutionary derived. This manuscript starts from the observation that Zea mays\nhas four CRY genes, annotated here as CRY1a-c and CRY2. The manuscript studies if these\nCRYs are involved in blu light-mediated UV-B responses at the level of photo protective) waxes.\nIt has been shown previously in Arabidopsis that CRYs are indeed involved in blue light-\nmediated UV-B responses, that in Arabidopsis occur through the UV-B photoreceptor UVR-8\nprimarily. Despite this overlap with existing knowledge in Arabidopsis, the manuscript does\nexplore this topic further and shows how the GLOSSY2 (GL2) protein is required for CRY1 to\nmodulate wax synthesis.\nRESPONSE: We appreciate greatly these excellent points raised by the reviewer!\nThere are a number of elements that I would suggest the authors to consider.\n1. It might be good to highlight better what the innovative elements of this manuscript are.\nSince CRY-mediated detection of B light is already known to modulate UV-B responsiveness,\nit is important to have this very clear.\nRESPONSE: Thank you for this suggestion! As recommended, we have added a section in the\ndiscussion (lines 416–423) to highlight the innovative aspects of our work. Specifically, we\ncontrast our findings with previous studies—where CRY1, phyB, and UVR8 redundantly\nregulate UV-B tolerance via CRY1-mediated induction of RUP1/2 expression—by emphasizing\nour novel discovery: blue light-activated maize CRYs modulate wax biosynthesis (both\ntranscriptionally and directly interacts with wax biosynthesis enzyme) to organize UV-B stress\nresistance at the plant’s outermost barrier, thereby enhancing UV-B tolerance. We appreciate\nyour guidance!\n2. You made a variety of knockout and OE lines for the different CRYs, but their phenotypes\nare described quite rudimentarily. At the very minimum it is necessary to also show dark and\nwhite light controls for the materials in Fig 1b,c,d and Supplemental Figs1 e-g, and Suppl\nFig2b,c,e\nThe same applies for Suppl Fig 3a-c: include dark and white light controls.\nRESPONSE: They are added as suggested (Fig 1b-e, Supplemental Fig 1 e-h, Supplemental\nFig 2 c-f, Supplemental Fig 3 a-d), thanks.\n3. You start from the question why there are several CRY1 copies in maize. However, you focus\npurely on the UV-B protection. There are many more roles that cryptochromes have and they\nmay be differential between the different copies. If you define the scope so broadly (which I\nthink is a good idea), then it would be helpful to also scan a wider range of cantons. Since you\nhave the lines made already, it should not be very difficult to do so. Obvious aspects to check\nwould include circadian rhythms (of gene expression), DNA binding ability (since they're\nderived from photolyases), plant development.\nRESPONSE: We appreciate greatly this excellent point raised by the reviewer! In fact, we are\ncurrently checking additional agronomic traits associated with cry in maize, including\nchloroplast development, flowering, high-density tolerance, drought-stress tolerance and yield.\nHowever, given these investigations are still in their preliminary stages, we have chosen not to\nincorporate them into this manuscript. To align with the current scope, we have revised the\nopening question to “whether CRYs in maize have evolved to improve UV tolerance or acquire\nnovel functions remains unclear” accordingly. Your insightful advice has been invaluable in\nstrengthening the clarity and focus of our work. Thank you!\n4. The subheading results in line 283 states that CRY1-GL2 interaction is blue light-dependent.\nSince the co-IP shows that the interaction also occurs in darkness, but much less, I would be a\nbit more careful. Blue light seems to strengthen the interaction, but is not fully mandatory. In\ncase of a photoreceptor this is a relevant distinction.\nRESPONSE: Thanks a million! We changed the subheading to “ZmCRY1 physically interacts\nwith GLOSSY2 in a blue light-enhanced manner” to accurately reflect that blue light enhances\nrather than strictly enables the interaction. We have also updated related sentences, including\nthe manuscript title, for consistency.\n5. With respect to BiFC assays in Fig4, the negative control of free nYFP (not fused to a protein)\nis not ideal. Since you have the the mutant CRY1 version involved this is not a big problem.\nYou could have chosen a different protein to fuse it to though.\nIn addition, have you verified that the different constructs are expressed equally well in your\nassays? If mutant CRY1 is for some reason expressed more weakly than WT CRY1, your results\nmight look similar to what you see now, but for a very different reason (and conclusion).\nRESPONSE: Thanks a million! As recommended, KCS4 is added as a negative control as\nsuggested (Fig. 4e). Since we have antibodies anti-CRY1c-CCE and anti-GL2, a western is\nadded to show the expression level of CRY1c, CRY1cD381A and GL2 in the BiFC assay\n(Supplementary Fig. 6c).\n6. Phenotyping of UV-B resistance should be improved. You used only three replicates (judging\nfrom the graph in Fig3d) for Fv/Fm quantification, and show photo's of only two leaves for\nvisual inspection (Fig. 3c). Since these data are needed to show the functionality of your study,\nthey are very important and should be done more elaborately. In these assays I would\nrecommend including gl2 mutant as well.\nRESPONSE: Thanks for this important suggestion. We have enhanced the UV-B resistance\nphenotyping as follows: In Fig.3d, additional replicates have been included for the\nmeasurement of Fv/Fm, with each data point collected from two leaves per replicate to minimize\nindividual variation. As recommended, Fv/Fm measurements of gl2 mutants have also been\nadded in Fig. 6c.\n7. The big unknown, or absentee in your manuscript, is the UV-B sensor itself. Your\nsummarising Figure 6d also omits a UV-B sensor. of course, this sensor is known from\nArabidopsis: UVR-8. It is not clear to me why UVR-8 was not included in your study. Since\nthe stress that you pose on your plants for which you expect the Blue-CRY1-GL2-Wax pathway\nto be relevant, is UV-B and this is sensed through UVR-8 to activate photoprotective responses,\nthis would be a crucial element in your study.\nRESPONSE: We appreciate greatly this point raised by the reviewer! We fully agree that the\nUV-B sensor UVR8, a well-characterized regulator of UV-B-induced photoprotection in\nArabidopsis and other plants, is a key consideration.\nIn our investigations, we also find that UVR8 is conserved as a UV-B photoreceptor in maize\nand plays a prominent role in UV-enhanced wax accumulation, with nearly half of this process\nbeing UVR8-dependent. However, to maintain focus on the specific mechanism by which blue\nlight-activated maize CRYs modulate wax biosynthesis for UV stress resistance, we have\ndedicated the UVR8 mediate pathway of UV-enhanced wax accumulation to a separate study,\nwhich is currently under submission. Thanks!\n8. Information about biological replication (numbers, how were they collected, etc) is often\nmissing. This would best be provided in the methods and in the figure captions.\nRESPONSE: They are added as suggested, thanks.\nMinor: There are quite some typo's in your manuscript. They do not make it difficult to read at\nall, but it would be good to fix them. I would assume a spell checker can detect most of them.\nRESPONSE: Thanks for your kind advice, the typos are corrected."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised version of this manuscript carefully incorporated a majority of the suggestions and concerns posed in my initial review. The introduction has been reorganized and the whole manuscript edited to improve clarity and readability. I appreciate the additional experiments that are now presented as supplemental data for GL2-LIKE and other aspects of the study. The modifications to the presentation of cuticular wax data aid in my ability to view and interpret the data. I list below a few specific minor modifications that could be considered. This manuscript presents what I consider to be novel findings that substantially forward our understanding of the regulation of cuticular wax biosynthesis and its relationship to blue light.\nLine 86: “Light is known to upregulate transcription of wax biosynthetic enzymes”. Formally, this text should state something like “…known to upregulate transcription of genes encoding wax biosynthetic enzymes”.\nLines 99-100: “cooperation with BAHD acyltransferases like Arabidopsis CER2, CER2-LIKE, and maize GL2, GL2-LIKE”. It is my understanding that these proteins have not been proven to be BAHD acyltransferases with the predicted acyltransferase activity, they have been labeled as such based on sequence similarity. This could be modified to “sequence-homologous to…” or “putative BAHD acyltransferases”, “in the BAHD protein family”, etc.\nLine 102: Formally, it the Very Long Chain Fatty Acyl-CoA (not the VLCFA) that is the precursor for the alcohol and alkane forming pathways.\nLines 110-112: This sentence is difficult to interpret, “Notably, yeast reconstitution experiments suggest that CER2, CER2-LIKE, or OsCER2 enabled CER6 to synthesize longer-chain VLCFAs, while subsequent studies suggest the yeast endogenous FAE complex may be associated with this extension”. Both CER2 and OsCER2 are mentioned, which presumably represent CER2 genes from different organisms (one being rice and the other not being stated). Perhaps saying CER2 and CER2-LIKE is sufficient. The organism(s) could also be noted.\nLines 235-238: It would be helpful to categorize this list of differentially regulated genes into de novo fatty acid synthesis-related, fatty acid elongation and cuticular wax biosynthesis-related genes.\nLines 254-255: There is an opportunity here to specify that the changes in composition are specific to increased chain lengths of cuticular wax products under blue light exposure.\nLines 421-422: In “…light-activated maize CRYs mediate blue light-regulated wax biosynthesis and directly participate in the wax biosynthetic process…” I’m not sure that it has been shown that CRY directly participates in wax biosynthesis. This sentence could be clarified to state the direct step in which CRY participates in the biosynthesis pathway.\nLine 424: In “RNA-seq analysis indicates that maize exhibits more extensive regulation of compound biosynthesis or metabolic processes by blue light compared to Arabidopsis”, this might be an overstatement. This could be expanded to state similar studies in Arabidopsis that show fewer genes differentially regulated under blue light.\nLines 431-436: Consider citing relevant figures and data, as done for other statements earlier in the Discussion.\nLine 437-438: Authors should consider rephrasing “a BAHD family acyltransferase”. Acyltransferase activity to my knowledge has not been confirmed for GL2, GL2-LIKE or CER2.\nLines 462-269: A reported role for GL2 and CER2 is in elongation of the VLCFA-CoA precursors of both the alkane- and alcohol-forming pathways. Is it therefore possible that the role of CRY1s and GL2 to facilitate C32 aldehyde accumulation is indirect? Or perhaps there is another binding partner (one of the many others the authors identified as interacting with CRY1s) that interacts with GL2 and CRY1 together that facilitates the increased conversion to aldehyde. There might be room for a bit more discussion in this section. The Discussion might also mention briefly that this interaction seems specific to GL2 and not GL2-LIKE and why this might be.", "reviewer_2": "In this revised version, Zhao et al. have improved their manuscript and have satisfactorily addressed my previous comments.\nI still have a few questions:\nRegarding the conditions used for the UV-B stress tolerance assays, why was Fv/Fm measured only after 48 hours of recovery? Why did you assess Fv/Fm after two days with two 6-hour UV treatments, instead of directly after a few hours of BB-UV-B exposure? If the BL-induced modification of cuticle composition enhances UV absorbance, a difference in Fv/Fm should already be detectable after a few hours of BB-UV-B, which directly damages PSII. If additional recovery time is required before a difference becomes evident, this would rather suggest that the variation in Fv/Fm is due to differences in PSII repair capacity or in the sensitivity of damaged PSII to ambient visible light, since the cuticle can also influence the absorbance/reflectance of visible light. It would therefore be valuable to include Fv/Fm data after the first 6 hours of BB-UV-B treatment.\nFinally, I also noticed the absence of technical details about the lamps used for light treatments—particularly for Nb-UV-B and BB-UV-B—including the method used to measure BB intensity (e.g., what device was employed), it should be provided.", "reviewer_3": "The authors have take great care of the all the suggestions made by me and the other reviewers. It is a great source of new information and tools in maize, and a beautiful story to read.\nI have no remaining comments."}, "author_response": "RESPONSE: We would like to express our sincere gratitude for the excellent and constructive points raised by the reviewer! Your advice has greatly contributed to improving the accuracy, clarity, and readability of our manuscript.\nLine 86: “Light is known to upregulate transcription of wax biosynthetic enzymes”. Formally, this text should state something like “…known to upregulate transcription of genes encoding wax biosynthetic enzymes”.\nRESPONSE: Thanks a million! It is changed as suggested (line 85).\nLines 99-100: “cooperation with BAHD acyltransferases like Arabidopsis CER2, CER2-LIKE, and maize GL2, GL2-LIKE”. It is my understanding that these proteins have not been proven to be BAHD acyltransferases with the predicted acyltransferase activity, they have been labeled as such based on sequence similarity. This could be modified to “sequence-homologous to…” or “putative BAHD acyltransferases”, “in the BAHD protein family”, etc.\nRESPONSE: It is modified as “putative BAHD acyltransferase” (lines 99)\nLine 102: Formally, it the Very Long Chain Fatty Acyl-CoA (not the VLCFA) that is the precursor for the alcohol and alkane forming pathways.\nRESPONSE: Thanks a million! It is changed as suggested (line 101).\nLines 110-112: This sentence is difficult to interpret, “Notably, yeast reconstitution experiments suggest that CER2, CER2-LIKE, or OsCER2 enabled CER6 to synthesize longer-chain VLCFAs, while subsequent studies suggest the yeast endogenous FAE complex may be associated with this extension”. Both CER2 and OsCER2 are mentioned, which presumably represent CER2 genes from different organisms (one being rice and the other not being stated). Perhaps saying CER2 and CER2-LIKE is sufficient. The organism(s) could also be noted.\nRESPONSE: Thanks a million! As suggested, it is changed to “Notably, yeast reconstitution experiments suggest that CER2 and CER2-LIKE enable CER6 to synthesize longer-chain VLCFAs, while subsequent studies suggest the yeast endogenous FAE complex may be associated with this extension” (line 110–112).\nLines 235-238: It would be helpful to categorize this list of differentially regulated genes into de novo fatty acid synthesis-related, fatty acid elongation and cuticular wax biosynthesis-related genes.\nRESPONSE: Thanks a million! As suggested, it is modified as “We further verified the expression of genes associated with de novo fatty acid synthesis (fatty acyl-acyl carrier protein (ACP) thioesterase B (FatB), β-ketoacyl-ACP synthase I (KAS I), and KAS II 36,37), those involved in fatty acid elongation (KCS3, KCS4), and genes related to cuticular wax biosynthesis (GL1, GL1-5, and GL4).” (line 235–238).\nLines 254-255: There is an opportunity here to specify that the changes in composition are specific to increased chain lengths of cuticular wax products under blue light exposure.\nRESPONSE: It is added as suggested, thanks. (line252–253)\nLines 421-422: In “…light-activated maize CRYs mediate blue light-regulated wax biosynthesis and directly participate in the wax biosynthetic process…” I’m not sure that it has been shown that CRY directly participates in wax biosynthesis. This sentence could be clarified to state the direct step in which CRY participates in the biosynthesis pathway.\nRESPONSE: Thanks a million! As suggested, we’ve clarified it to: “…light-activated maize CRYs mediate blue light-regulated wax biosynthesis and directly interact with GL2 to facilitate C32 aldehyde accumulation (Fig. 6g) …” (line433–435).\nLine 424: In “RNA-seq analysis indicates that maize exhibits more extensive regulation of compound biosynthesis or metabolic processes by blue light compared to Arabidopsis”, this might be an overstatement. This could be expanded to state similar studies in Arabidopsis that show fewer genes differentially regulated under blue light.\nRESPONSE: Thanks a million! As suggested, we’ve rephrased it to: “RNA-seq analysis reveals that blue light regulates compound biosynthesis and metabolic processes in maize, whereas similar studies in Arabidopsis show fewer genes differentially regulated under blue light” (line438–440).\nLines 431-436: Consider citing relevant figures and data, as done for other statements earlier in the Discussion.\nRESPONSE: It is added as suggested, thanks.\nLine 437-438: Authors should consider rephrasing “a BAHD family acyltransferase”. Acyltransferase activity to my knowledge has not been confirmed for GL2, GL2-LIKE or CER2.\nRESPONSE: They are rephrased as “putative BAHD family acyltransferase” in the text (lines 99,117,451)\nLines 462-269: A reported role for GL2 and CER2 is in elongation of the VLCFA-CoA precursors of both the alkane- and alcohol-forming pathways. Is it therefore possible that the role of CRY1s and GL2 to facilitate C32 aldehyde accumulation is indirect? Or perhaps there is another binding partner (one of the many others the authors identified as interacting with CRY1s) that interacts with GL2 and CRY1 together that facilitates the increased conversion to aldehyde. There might be room for a bit more discussion in this section. The Discussion might also mention briefly that this interaction seems specific to GL2 and not GL2-LIKE and why this might be.\nRESPONSE: Thanks a million! As suggested, we have expanded the discussion to: “GL2 and GL2-LIKE, which cooperate with KCS6, are recognized as major factors involved in the elongation of VLCFA-CoA precursors longer than C28 in both the alkane- and alcohol-forming pathways. In this study, we show that blue light-activated ZmCRY1s interact with GL2, whereas ZmCRY1c and KCS6 do not form a complex but instead compete for binding to GL2 (Fig. 6e, f; Supplementary Fig. 8c). Furthermore, GL2-LIKE exhibited a weaker interaction with ZmCRY1c compared to GL2 (Supplementary Fig. 6f, g). It has been reported that overexpression of GL2—but not GL2-Like—induces the formation of C32 aldehyde, C31 alkane, C31 secondary alcohol, and C31 ketone. Additionally, the BAHD catalytic HXXXDX motif is required for GL2-LIKE activity but not for GL2. All these results hint that the CRY1-GL2 complex may indirectly facilitate the VLCFA-CoA biosynthetic flux toward aldehyde synthesis in maize, potentially by recruiting additional binding partners, and this effect appears to be specific to GL2 rather than GL2-LIKE. These findings shed new light on the VLC aldehyde generation, although the precise underlying molecular mechanisms require further elucidation.” (line476–489)\nReviewer #2 (Remarks to the Author):\nIn this revised version, Zhao et al. have improved their manuscript and have satisfactorily addressed my previous comments.\nI still have a few questions:\nRegarding the conditions used for the UV-B stress tolerance assays, why was Fv/Fm measured only after 48 hours of recovery? Why did you assess Fv/Fm after two days with two 6-hour UV treatments, instead of directly after a few hours of BB-UV-B exposure? If the BL-induced modification of cuticle composition enhances UV absorbance, a difference in Fv/Fm should already be detectable after a few hours of BB-UV-B, which directly damages PSII. If additional recovery time is required before a difference becomes evident, this would rather suggest that the variation in Fv/Fm is due to differences in PSII repair capacity or in the sensitivity of damaged PSII to ambient visible light, since the cuticle can also influence the absorbance/reflectance of visible light. It would therefore be valuable to include Fv/Fm data after the first 6 hours of BB-UV-B treatment.\nRESPONSE: Thanks for this constructive comment! As suggested, we did new experiments, we increased the broadband UV-B intensity to 60 μmol·m−2·s−1 and extending the irradiation duration to 10 hours, which enhanced UV-B-induced damage, and measured the Fv/Fm immediately after UV-B exposure with 1-hour dark adaptation, ensuring that the detected Fv/Fm variations primarily reflect UV-B-induced damage rather than subsequent repair (Supplementary Fig. 5b, c and Supplementary Fig. 8c, d). Additionally, to further resolve the issue of indistinct leaf phenotypes when assessing Fv/Fm, we have included Fv/Fm imaging data to visually illustrate differences in PSII activity (Supplementary Fig. 5b and Supplementary Fig. 8c) (line 275-290 and 391-399).\nFinally, I also noticed the absence of technical details about the lamps used for light treatments—particularly for Nb-UV-B and BB-UV-B—including the method used to measure BB intensity (e.g., what device was employed), it should be provided.\nRESPONSE: Thanks a million! As suggested, we added the information to the “Plant materials and growth conditions” section: “For different light treatments, blue light (460 ± 40 nm), red light (675 ± 40 nm), far-red light (740 ± 40 nm), and narrowband UV-B (310 ± 10 nm) were generated using LEDs manufactured by Qiding Optoelectronic (Shanghai) Co., Ltd. Broadband UV-B was provided by TL 40W/12 UV fluorescent tubes (Philips). The intensities of white light, blue light, red light, and far-red light were measured with an ILT1400 Radiometer Photometer, while UV-B light was measured with a LUYOR-340 UV Light Meter.” (line527–532).\nReviewer #3 (Remarks to the Author):\nThe authors have take great care of the all the suggestions made by me and the other reviewers. It is a great source of new information and tools in maize, and a beautiful story to read.\nI have no remaining comments.\nRESPONSE: We thanks for all the excellent and constructive points raised by the reviewer!."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have addressed my comments and concerns. This manuscript describes a carefully conducted study that advances our understanding of the regulation and execution of cuticular wax biosynthesis.", "reviewer_2": "The authors have carefully addressed all the suggestions made by me and the other reviewers. Congratulations and thank you for this nice story. I don’t have any other comments."}, "author_response": ""}]

Decision letter