Genetic interaction between TTG2 and AtPLC1 reveals a role for phosphoinositide signaling in a co-regulated suite of Arabidopsis epidermal pathways.

The TTG2 transcription factor of Arabidopsis regulates a set of epidermal traits, including the differentiation of leaf trichomes, flavonoid pigment production in cells of the inner testa (or seed coat) layer and mucilage production in specialized cells of the outer testa layer. Despite the fact that TTG2 has been known for over twenty years as an important regulator of multiple developmental pathways, little has been discovered about the downstream mechanisms by which TTG2 co-regulates these epidermal features. In this study, we present evidence of phosphoinositide lipid signaling as a mechanism for the regulation of TTG2-dependent epidermal pathways. Overexpression of the AtPLC1 gene rescues the trichome and seed coat phenotypes of the ttg2-1 mutant plant. Moreover, in the case of seed coat color rescue, AtPLC1 overexpression restored expression of the TTG2 flavonoid pathway target genes, TT12 and TT13/AHA10. Consistent with these observations, a dominant AtPLC1 T-DNA insertion allele (plc1-1D) promotes trichome development in both wild-type and ttg2-3 plants. Also, AtPLC1 promoter:GUS analysis shows expression in trichomes and this expression appears dependent on TTG2. Taken together, the discovery of a genetic interaction between TTG2 and AtPLC1 suggests a role for phosphoinositide signaling in the regulation of trichome development, flavonoid pigment biosynthesis and the differentiation of mucilage-producing cells of the seed coat. This finding provides new avenues for future research at the intersection of the TTG2-dependent developmental pathways and the numerous molecular and cellular phenomena influenced by phospholipid signaling.

Gene expression analysis of drought tolerance and cuticular wax biosynthesis in diploid and tetraploid induced wallflowers.

Whole-genome doubling leads to cell reprogramming, upregulation of stress genes, and establishment of new pathways of drought stress responses in plants. This study investigated the molecular mechanisms of drought tolerance and cuticular wax characteristics in diploid and tetraploid-induced Erysimum cheiri. According to real-time PCR analysis, tetraploid induced wallflowers exhibited increased expression of several genes encoding transcription factors (TFs), including AREB1 and AREB3; the stress response genes RD29A and ERD1 under drought stress conditions. Furthermore, two cuticular wax biosynthetic pathway genes, CER1 and SHN1, were upregulated in tetraploid plants under drought conditions. Leaf morphological studies revealed that tetraploid leaves were covered with unique cuticular wax crystalloids, which produced a white fluffy appearance, while the diploid leaves were green and smooth. The greater content of epicuticular wax in tetraploid leaves than in diploid leaves can explain the decrease in cuticle permeability as well as the decrease in water loss and improvement in drought tolerance in wallflowers. GC‒MS analysis revealed that the wax components included alkanes, alcohols, aldehydes, and fatty acids. The most abundant wax compound in this plant was alkanes (50%), the most predominant of which was C29. The relative abundance of these compounds increased significantly in tetraploid plants under drought stress conditions. These findings revealed that tetraploid-induced wallflowers presented upregulation of multiple drought-related and wax biosynthesis genes; therefore, polyploidization has proved useful for improving plant drought tolerance.

Morphology and chemical composition of Taiwan oil millet (Eccoilopus formosanus) epicuticular wax.

MAIN CONCLUSION: Taiwan oil millet has two types of epicuticular wax: platelet wax composed primarily of octacosanol and filament wax constituted essentially by the singular compound of octacosanoic acid. Taiwan oil millet (TOM-Eccoilopus formosanus) is an orphan crop cultivated by the Taiwan indigenous people. It has conspicuous white powder covering its leaf sheath indicating abundant epicuticular waxes, that may contribute to its resilience. Here, we characterized the epicuticular wax secretion in TOM leaf blade and leaf sheath using various microscopy techniques, as well as gas chromatography to determine its composition. Two kinds of waxes, platelet and filaments, were secreted in both the leaf blades and sheaths. The platelet wax is secreted ubiquitously by epidermal cells, whereas the filament wax is secreted by a specific cell called epidermal cork cells. The newly developed filament waxes were markedly re-synthesized by the epidermal cork cells through papillae protrusions on the external periclinal cell wall. Ultrastructural images of cork cell revealed the presence of cortical endoplasmic reticulum (ER) tubules along the periphery of plasma membrane (PM) and ER-PM contact sites (EPCS). The predominant wax component was a C28 primary alcohol in leaf blade, and a C28 free fatty acid in the leaf sheath, pseudopetiole and midrib. The wax morphology present in distinct plant organs corresponds to the specific chemical composition: platelet wax composed of alcohols exists mainly in the leaf blade, whereas filament wax constituted mainly by the singular compound C28 free fatty acids is present abundantly in leaf sheath. Our study clarifies the filament wax composition in relation to a previous study in sorghum. Both platelet and filament waxes comprise a protection barrier for TOM.

GoSTR, a negative modulator of stem trichome formation in cotton.

Trichomes, the outward projection of plant epidermal tissue, provide an effective defense against stress and insect pests. Although numerous genes have been identified to be involved in trichome development, the molecular mechanism for trichome cell fate determination is not well enunciated. Here, we reported GoSTR functions as a master repressor for stem trichome formation, which was isolated by map-based cloning based on a large F2 segregating population derived from a cross between TM-1 (pubescent stem) and J220 (smooth stem). Sequence alignment revealed a critical G-to-T point mutation in GoSTR's coding region that converted codon 2 from GCA (Alanine) to TCA (Serine). This mutation occurred between the majority of Gossypium hirsutum with pubescent stem (GG-haplotype) and G. barbadense with glabrous stem (TT-haplotype). Silencing of GoSTR in J220 and Hai7124 via virus-induced gene silencing resulted in the pubescent stems but no visible change in leaf trichomes, suggesting stem trichomes and leaf trichomes are genetically distinct. Yeast two-hybrid assay and luciferase complementation imaging assay showed GoSTR interacts with GoHD1 and GoHOX3, two key regulators of trichome development. Comparative transcriptomic analysis further indicated that many transcription factors such as GhMYB109, GhTTG1, and GhMYC1/GhDEL65 which function as positive regulators of trichomes were significantly upregulated in the stem from the GoSTR-silencing plant. Taken together, these results indicate that GoSTR functions as an essential negative modulator of stem trichomes and its transcripts will greatly repress trichome cell differentiation and growth. This study provided valuable insights for plant epidermal hair initiation and differentiation research.

Genome-Wide Analysis of the BAHD Family in Welsh Onion and CER2-LIKEs Involved in Wax Metabolism.

BAHD acyltransferases (BAHDs), especially those present in plant epidermal wax metabolism, are crucial for environmental adaptation. Epidermal waxes primarily comprise very-long-chain fatty acids (VLCFAs) and their derivatives, serving as significant components of aboveground plant organs. These waxes play an essential role in resisting biotic and abiotic stresses. In this study, we identified the BAHD family in Welsh onion (Allium fistulosum). Our analysis revealed the presence of AfBAHDs in all chromosomes, with a distinct concentration in Chr3. Furthermore, the cis-acting elements of AfBAHDs were associated with abiotic/biotic stress, hormones, and light. The motif of Welsh onion BAHDs indicated the presence of a specific BAHDs motif. We also established the phylogenetic relationships of AfBAHDs, identifying three homologous genes of CER2. Subsequently, we characterized the expression of AfCER2-LIKEs in a Welsh onion mutant deficient in wax and found that AfCER2-LIKE1 plays a critical role in leaf wax metabolism, while all AfCER2-LIKEs respond to abiotic stress. Our findings provide new insights into the BAHD family and lay a foundation for future studies on the regulation of wax metabolism in Welsh onion.

Brassinosteroid coordinates cell layer interactions in plants via cell wall and tissue mechanics.

Growth coordination between cell layers is essential for development of most multicellular organisms. Coordination may be mediated by molecular signaling and/or mechanical connectivity between cells, but how genes modify mechanical interactions between layers is unknown. Here we show that genes driving brassinosteroid synthesis promote growth of internal tissue, at least in part, by reducing mechanical epidermal constraint. We identified a brassinosteroid-deficient dwarf mutant in the aquatic plant Utricularia gibba with twisted internal tissue, likely caused by mechanical constraint from a slow-growing epidermis. We tested this hypothesis by showing that a brassinosteroid mutant in Arabidopsis enhances epidermal crack formation, indicative of increased tissue stress. We propose that by remodeling cell walls, brassinosteroids reduce epidermal constraint, showing how genes can control growth coordination between layers by means of mechanics.

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

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

Epidermal injury-induced derepression of key regulator ATML1 in newly exposed cells elicits epidermis regeneration.

Plant cell fate determination depends on the relative positions of the cells in developing organisms. The shoot epidermis, the outermost cell layer of the above-ground organs in land plants, protects plants from environmental stresses. How the shoot epidermis is formed only from the outermost cells has remained unknown. Here we show that when inner leaf mesophyll cells are exposed to the surface, these cells show up-regulation of ATML1, a master regulator for epidermal cell identity in Arabidopsis thaliana. Epidermal cell types such as stomatal guard cells regenerate from young inner-lineage tissues that have a potential to accumulate ATML1 protein after epidermal injury. Surgical analyses indicate that application of pressure to the exposed site was sufficient to inhibit ATML1 derepression in the outermost mesophyll cells, suggesting this process requires pressure release. Furthermore, pharmacological analyses suggest that ATML1 derepression in the outermost mesophyll cells require cortical microtubule formation, MAPK signaling and proteasome activity. Our results suggest that surface-positional cues involving mechanical signaling are used to restrict ATML1 activity to the outermost cells and facilitate epidermal differentiation.

13 CO2 labelling as a tool for elucidating the mechanism of cuticle development: a case of Clusia rosea.

The plant cuticle is an important plant-atmosphere boundary, the synthesis and maintenance of which represents a significant metabolic cost. Only limited information regarding cuticle dynamics is available. We determined the composition and dynamics of Clusia rosea cuticular waxes and matrix using 13 CO2 labelling, compound-specific and bulk isotope ratio mass spectrometry. Collodion was used for wax collection; gas exchange techniques to test for any collodion effects on living leaves. Cutin matrix (MX) area density did not vary between young and mature leaves and between leaf sides. Only young leaves incorporated new carbon into their MX. Collodion-based sampling discriminated between epicuticular (EW) and intracuticular wax (IW) effectively. Epicuticular differed in composition from IW. The newly synthetised wax was deposited in IW first and later in EW. Both young and mature leaves synthetised IW and EW. The faster dynamics in young leaves were due to lower wax coverage, not a faster synthesis rate. Longer-chain alkanes were deposited preferentially on the abaxial, stomatous leaf side, producing differences between leaf sides in wax composition. We introduce a new, sensitive isotope labelling method and demonstrate that cuticular wax is renewed during leaf ontogeny of C. rosea. We discuss the ecophysiological significance of the new insights.

Tree tobacco (Nicotiana glauca) cuticular wax composition is essential for leaf retention during drought, facilitating a speedy recovery following rewatering.

Despite decades of extensive study, the role of cuticular lipids in sustaining plant fitness is far from being understood. We utilized genome-edited tree tobacco (Nicotiana glauca) to investigate the significance of different classes of epicuticular wax in abiotic stress such as cuticular water loss, drought, and light response. We generated mutants displaying a range of wax compositions. Four wax mutants and one cutin mutant were extensively investigated for alterations in their response to abiotic factors. Although the mutations led to elevated cuticular water loss, the wax mutants did not display elevated transpiration or reduced growth under nonstressed conditions. However, under drought, plants lacking alkanes were unable to reduce their transpiration, leading to leaf death, impaired recovery, and stem cracking. By contrast, plants deficient in fatty alcohols exhibited elevated drought tolerance, which was part of a larger trend of plant phenotypes not clustering by a glossy/glaucous appearance in the parameters examined in this study. We conclude that although alkanes have little effect on whole N. glauca transpiration and biomass gain under normal, nonstressed conditions, they are essential during drought responses, since they enable plants to seal their cuticle upon stomatal closure, thereby reducing leaf death and facilitating a speedy recovery.

A novel approach for measuring membrane permeability for organic compounds via surface plasmon resonance detection.

Well-known methods for measuring permeability of membranes include static or flow diffusion chambers. When studying the effects of organic compounds on plants, the use of such model systems allows to investigate xenobiotic behavior at the cuticular barrier level and obtain an understanding of the initial penetration processes of these substances into plant leaves. However, the use of diffusion chambers has disadvantages, including being time-consuming, requiring sampling, or a sufficiently large membrane area, which cannot be obtained from all types of plants. Therefore, we propose a new method based on surface plasmon resonance imaging (SPRi) to enable rapid membrane permeability evaluation. This study presents the methodology for measuring permeability of isolated cuticles for organic compounds via surface plasmon resonance detection, where the selected model analyte was the widely used pesticide metazachlor. Experiments were performed on the cuticles of Ficus elastica, Citrus pyriformis, and an artificial PES membrane, which is used in passive samplers for the detection of xenobiotics in water and soils. The average permeability for metazachlor was 5.23 × 10-14 m2 s-1 for C. pyriformis, 1.34 × 10-13 m2 s-1 for F. elastica, and 7.74 × 10-12 m2 s-1 for the PES membrane. We confirmed that the combination of a flow-through diffusion cell and real-time optical detection of transposed molecules represents a promising method for determining the permeability of membranes to xenobiotics occurring in the environment. This is necessary for determining a pesticide dosage in agriculture, selecting suitable membranes for passive samplers in analytics, testing membranes for water treatment, or studying material use of impregnated membranes.

Conserved signalling components coordinate epidermal patterning and cuticle deposition in barley.

Faced with terrestrial threats, land plants seal their aerial surfaces with a lipid-rich cuticle. To breathe, plants interrupt their cuticles with adjustable epidermal pores, called stomata, that regulate gas exchange, and develop other specialised epidermal cells such as defensive hairs. Mechanisms coordinating epidermal features remain poorly understood. Addressing this, we studied two loci whose allelic variation causes both cuticular wax-deficiency and misarranged stomata in barley, identifying the underlying genes, Cer-g/ HvYDA1, encoding a YODA-like (YDA) MAPKKK, and Cer-s/ HvBRX-Solo, encoding a single BREVIS-RADIX (BRX) domain protein. Both genes control cuticular integrity, the spacing and identity of epidermal cells, and barley's distinctive epicuticular wax blooms, as well as stomatal patterning in elevated CO2 conditions. Genetic analyses revealed epistatic and modifying relationships between HvYDA1 and HvBRX-Solo, intimating that their products participate in interacting pathway(s) linking epidermal patterning with cuticular properties in barley. This may represent a mechanism for coordinating multiple adaptive features of the land plant epidermis in a cultivated cereal.

Proteomic Profiling of Plant and Pathogen Interaction on the Leaf Epidermis.

The plant epidermis is the first line of plant defense against pathogen invasion, and likely contains important regulatory proteins related to the plant-pathogen interaction. This study aims to identify the candidates of these regulatory proteins expressed in the plant epidermis. We performed comparative proteomic studies to identify rapidly and locally expressed proteins in the leaf epidermis inoculated with fungal phytopathogen. The conidia solutions were dropped onto the Arabidopsis leaf surface, and then, we collected the epidermal tissues from inoculated and mock-treated leaves at 4 and 24 hpi. The label-free quantification methods showed that expressions of Arabidopsis proteins, which are related to defense signals, such as BAK1, MKK5, receptor-like protein kinases, transcription factors, and stomatal functions, were rapidly induced in the epidermal tissues of inoculated leaves. In contrast, most of them were not differentially regulated by fugal inoculation in the whole leaves. These findings clearly indicate that epidermal proteomics can monitor locally expressed proteins in inoculated areas of plant tissues. We also identified the 61 fungal proteins, including effector-like proteins specifically expressed on the Arabidopsis epidermis. Our new findings suggested that epidermal proteomics is useful for understanding the local expressions of plant and fungal proteins related to their interactions.

An apple long-chain acyl-CoA synthase, MdLACS1, enhances biotic and abiotic stress resistance in plants.

Epidermal waxes are part of the outermost hydrophobic structures of apples and play a significant role in enhancing apple resistance and improving fruit quality. The biosynthetic precursors of epidermal waxes are very long-chain fatty acids (VLCFAs), which are made into different wax components through various wax synthesis pathways. In Arabidopsis thaliana, the AtLACS1 protein can activate the alkane synthesis pathway to produce very long-chain acyl CoAs (VLC-acyl-CoAs), which provide substrates for wax synthesis, from VLCFAs. The apple protein MdLACS1, encoded by the MdLACS1 gene, belongs to the AMP-binding superfamily and has long-chain acyl coenzyme A synthase activity, but its function in apple remains unclear. Here, we identified MdLACS1 in apple (Malus × domestica) and analyzed its function. Our results suggest that MdLACS1 promotes wax synthesis and improves biotic and abiotic stress tolerance, which were directly or indirectly dependent on wax. Our study further refines the molecular mechanism of wax biosynthesis in apples and elucidates the physiological function of wax in resistance to external stresses. These findings provide candidate genes for the synergistic enhancement of apple fruit quality and stress tolerance.

Two Arabidopsis MYB-SHAQKYF transcription repressors regulate leaf wax biosynthesis via transcriptional suppression on DEWAX.

Plant cuticular wax accumulation limits nonstomatal transpiration and is regulated by external environmental stresses. DEWAX (DECREASE WAX BIOSYNTHESIS) plays a vital role in diurnal wax biosynthesis. However, how DEWAX expression is controlled and the molecular mechanism of wax biosynthesis regulated by the diurnal cycle remains largely unknown. Here, we identified two Arabidopsis MYB-SHAQKYF transcription factors, MYS1 and MYS2, as new regulators in wax biosynthesis and drought tolerance. Mutations of both MYS1 and MYS2 caused significantly reduced leaf wax, whereas overexpression of MYS1 or MYS2 increased leaf wax biosynthesis and enhanced drought tolerance. Our results demonstrated that MYS1 and MYS2 act as transcription repressors and directly suppress DEWAX expression via ethylene response factor-associated amphiphilic repression motifs. Genetic interaction analysis with DEWAX, SPL9 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9), and CER1 (ECERIFERUM 1) in wax biosynthesis and under drought stresses demonstrated that MYS1 and MYS2 act upstream of the DEWAX-SPL9 module, thus regulating CER1 expression. Expression analysis suggested that the diurnal expression pattern of DEWAX is partly regulated by MYS1 and MYS2. Our findings demonstrate the roles of two unidentified transcription repressors, MYS1 and MYS2, in wax biosynthesis and provide insights into the mechanism of diurnal cycle-regulated wax biosynthesis.

A novel regulatory complex mediated by Lanata (Ln) controls multicellular trichome formation in tomato.

Trichomes that originate from plant aerial epidermis act as mechanical and chemical barriers against herbivores. Although several regulators have recently been identified, the regulatory pathway underlying multicellular trichome formation remains largely unknown in tomato. Here, we report a novel HD-ZIP IV transcription factor, Lanata (Ln), a missense mutation which caused the hairy phenotype. Biochemical analyses demonstrate that Ln separately interacts with two trichome regulators, Woolly (Wo) and Hair (H). Genetic and molecular evidence demonstrates that Ln directly regulates the expression of H. The interaction between Ln and Wo can increase trichome density by enhancing the expression of SlCycB2 and SlCycB3, which we previously showed are involved in tomato trichome formation. Furthermore, SlCycB2 represses the transactivation of the SlCycB3 gene by Ln and vice versa. Our findings provide new insights into the novel regulatory network controlling multicellular trichome formation in tomato.

ZmEREB46, a maize ortholog of Arabidopsis WAX INDUCER1/SHINE1, is involved in the biosynthesis of leaf epicuticular very-long-chain waxes and drought tolerance.

The aerial surfaces of plants are covered by a layer of cuticular wax that is composed of long-chain hydrocarbon compounds for protection against adverse environmental conditions. The current study identified a maize (Zea mays L.) APETALA2/ethylene-responsive element-binding protein (AP2/EREBP)-type transcription factor, ZmEREB46. Ectopic expression of ZmEREB46 in Arabidopsis increased the accumulation of epicuticular wax on the leaves and enhanced the drought tolerance of plants. The amounts of C24/C32 fatty acids, C32/C34 aldehydes, C32/C34 1-alcohols and C31 alkanes in zmereb46 (ZmEREB46 knockout mutant) leaves were reduced. The amount of leaf total epicuticular wax decreased approximately 50% in zmereb46. Compared to wild-type LH244 leaves, the cuticle permeability of zmereb46 leaves was increased, which resulted from decreased epicuticular wax load and a thinner cuticle layer. ZmEREB46 had transcriptional activation activity and directly bound to promoter regions of ZmCER2, ZmCER3.2 and ZmKCS1. The zmereb46 seedlings also exhibited reduced drought tolerance. These results, and the observations in ZmEREB46-overexpressing lines, suggest that ZmEREB46 is involved in cuticular metabolism by influencing the biosynthesis of very-long-chain waxes and participates in the cutin biosynthesis pathway. These results are helpful to further analyze the regulatory network of wax accumulation in maize.

Regulatory mechanisms underlying cuticular wax biosynthesis.

Plants are sessile organisms that have developed hydrophobic cuticles that cover their aerial epidermal cells to protect them from terrestrial stresses. The cuticle layer is mainly composed of cutin, a polyester of hydroxy and epoxy fatty acids, and cuticular wax, a mixture of very-long-chain fatty acids (>20 carbon atoms) and their derivatives, aldehydes, alkanes, ketones, alcohols, and wax esters. During the last 30 years, forward and reverse genetic, transcriptomic, and biochemical approaches have enabled the identification of key enzymes, transporters, and regulators involved in the biosynthesis of cutin and cuticular waxes. In particular, cuticular wax biosynthesis is significantly influenced in an organ-specific manner or by environmental conditions, and is controlled using a variety of regulators. Recent studies on the regulatory mechanisms underlying cuticular wax biosynthesis have enabled us to understand how plants finely control carbon metabolic pathways to balance between optimal growth and development and defense against abiotic and biotic stresses. In this review, we summarize the regulatory mechanisms underlying cuticular wax biosynthesis at the transcriptional, post-transcriptional, post-translational, and epigenetic levels.

Fatty alcohol oxidase 3 (FAO3) and FAO4b connect the alcohol- and alkane-forming pathways in Arabidopsis stem wax biosynthesis.

The alcohol- and alkane-forming pathways in cuticular wax biosynthesis are well characterized in Arabidopsis. However, potential interactions between the two pathways remain unclear. Here, we reveal that mutation of CER4, the key gene in the alcohol-forming pathway, also led to a deficiency in the alkane-forming pathway in distal stems. To trace the connection between the two pathways, we characterized two homologs of fatty alcohol oxidase (FAO), FAO3 and FAO4b, which were highly expressed in distal stems and localized to the endoplasmic reticulum. The amounts of waxes from the alkane-forming pathway were significantly decreased in stems of fao4b and much lower in fao3 fao4b plants, indicative of an overlapping function for the two proteins in wax synthesis. Additionally, overexpression of FAO3 and FAO4b in Arabidopsis resulted in a dramatic reduction of primary alcohols and significant increases of aldehydes and related waxes. Moreover, expressing FAO3 or FAO4b led to significantly decreased amounts of C18-C26 alcohols in yeast co-expressing CER4 and FAR1. Collectively, these findings demonstrate that FAO3 and FAO4b are functionally redundant in suppressing accumulation of primary alcohols and contributing to aldehyde production, which provides a missing and long-sought-after link between these two pathways in wax biosynthesis.