Cloning and characterization of GLOSSY1, a maize gene involved in cuticle membrane and wax production

The cuticle covering the aerial organs of land plants plays a protective role against several biotic and abiotic stresses and, in addition, participates in a variety of plant-insect interactions. Here, we describe the molecular cloning and characterization of the maize (Zea mays) GLOSSY1 (GL1) gene, a component of the pathway leading to cuticular wax biosynthesis in seedling leaves. The genomic and cDNA sequences we isolated differ significantly in length and in most of the coding region from those previously identified. The predicted GL1 protein includes three histidine-rich domains, the landmark of a family of membrane-bound desaturases/hydroxylases, including fatty acid-modifying enzymes. GL1 expression is not restricted to the juvenile developmental stage of the maize plant, pointing to a broader function of the gene product than anticipated on the basis of the mutant phenotype. Indeed, in addition to affecting cuticular wax biosynthesis, gl1 mutations have a pleiotropic effect on epidermis development, altering trichome size and impairing cutin structure. Of the many wax biosynthetic genes identified so far, only a few from Arabidopsis (Arabidopsis thaliana) were found to be essential for normal cutin formation. Among these is WAX2, which shares 62% identity with GL1 at the protein level. In wax2-defective plants, cutin alterations induce postgenital organ fusion. This trait is not displayed by gl1 mutants, suggesting a different role of the maize and Arabidopsis cuticle in plant development.     

The cytochrome P450 enzyme CYP96A15 is the midchain alkane hydroxylase responsible for formation of secondary alcohols and ketones in stem cuticular wax of Arabidopsis

Most aerial surfaces of plants are covered by cuticular wax that is synthesized in epidermal cells. The wax mixture on the inflorescence stems of Arabidopsis (Arabidopsis thaliana) is dominated by alkanes, secondary alcohols, and ketones, all thought to be formed sequentially in the decarbonylation pathway of wax biosynthesis. Here, we used a reverse-genetic approach to identify a cytochrome P450 enzyme (CYP96A15) involved in wax biosynthesis and characterized it as a midchain alkane hydroxylase (MAH1). Stem wax of T-DNA insertional mutant alleles was found to be devoid of secondary alcohols and ketones (mah1-1) or to contain much lower levels of these components (mah1-2 and mah1-3) than wild type. All mutant lines also had increased alkane amounts, partially or fully compensating for the loss of other compound classes. In spite of the chemical variation between mutant and wild-type waxes, there were no discernible differences in the epicuticular wax crystals on the stem surfaces. Mutant stem wax phenotypes could be partially rescued by expression of wild-type MAH1 under the control of the native promoter as well as the cauliflower mosaic virus 35S promoter. Cauliflower mosaic virus 35S-driven overexpression of MAH1 led to ectopic accumulation of secondary alcohols and ketones in Arabidopsis leaf wax, where only traces of these compounds are found in the wild type. The newly formed leaf alcohols and ketones had midchain functional groups on or next to the central carbon, thus matching those compounds in wild-type stem wax. Taken together, mutant analyses and ectopic expression of MAH1 in leaves suggest that this enzyme can catalyze the hydroxylation reaction leading from alkanes to secondary alcohols and possibly also a second hydroxylation leading to the corresponding ketones. MAH1 expression was largely restricted to the expanding regions of the inflorescence stems, specifically to the epidermal pavement cells, but not in trichomes and guard cells. MAH1-green fluorescent protein fusion proteins localized to the endoplasmic reticulum, providing evidence that both intermediate and final products of the decarbonylation pathway are generated in this subcellular compartment and must subsequently be delivered to the plasma membrane for export toward the cuticle.     

核盘菌侵染对拟南芥表皮蜡质结构及化学组成的影响

以野生型拟南芥与蜡质突变体cer1、cer4为试验材料,通过研究核盘菌胁迫对拟南芥茎表皮蜡质结构及组分含量的影响,揭示核盘菌侵染与表皮蜡质的关系。扫描电镜结果显示,野生型拟南芥蜡质晶体以垂直于表面的杆状、块状结构为主;突变体cer1晶体类型以水平的松针状、块状结构为主;突变体cer4蜡质晶体以垂直片层结构为主。核盘菌胁迫下,拟南芥蜡质晶体结构及分布形态发生变化。蜡质层结构在核盘菌胁迫下表现为:杆状、松针状蜡质晶体减少—蜡质晶体熔融—表皮"囊状凸起"—表皮膜层破裂。这些结构变化有利于病菌突破角质层屏障而侵入到植株体内。色质谱分析结果显示:与野生型相比,cer1突变体烷、次级醇、酮类显著减少;cer4突变体表现为一级醇含量减少。接种核盘菌后,野生型拟南芥与蜡质突变体一级醇类显著增加(cer1增加不显著);烷类、次级醇类、酮类含量与蜡质总量均显著减少,表明蜡质前体物质在受到核盘菌胁迫后更多地通过酰基还原途径生成一级醇,从而减少了由脱羰基途径所生成的蜡质组分。核盘菌通过改变表皮蜡质晶体结构与化学组分分泌量来促进侵染。     

Transgenic Arabidopsis plants expressing a fungal cutinase show alterations in the structure and properties of the cuticle and postgenital organ fusions

A major structural component of the cuticle of plants is cutin. Analysis of the function of cutin in vivo has been limited because no mutants with specific defects in cutin have been characterized. Therefore, transgenic Arabidopsis plants were generated that express and secrete a cutinase from Fusarium solani f sp pisi. Arabidopsis plants expressing the cutinase in the extracellular space showed an altered ultrastructure of the cuticle and an enhanced permeability of the cuticle to solutes. In addition, pollen could germinate on fully differentiated leaves of cutinase-expressing plants but not on control leaves. These differences coincided with strong postgenital organ fusions. The junctions of the fusions contained pectic polysaccharides. As fused organs grew apart from each other, organ deformations and protrusions of epidermal cells developed at positions with high mechanical stress. These results demonstrate that an intact cutin layer not only is important for plant-environment interactions but also prevents fusions between different plant organs and is therefore necessary for normal epidermal differentiation and organ formation.     

Epidermal cell interactions: a case for local talk

Epidermal cell interactions play an important role in reproduction, creating variation in floral structure and modulating gametophyte selection and growth during pollination. Recent progress in understanding the processes by which epidermal cells become responsive to contact, indicate that the developmental quiescence of the epidermis is regulated by a set of genes, some of which modulate the permeability properties of the cell wall and cuticle. Research on mutants of Arabidopsis has revealed a mechanistic uniformity between two processes involving epidermis-mediated interactions that had hitherto been considered to be biologically distinct: postgenital organ fusion and pollen hydration.     

ABC-type transporters and cuticle assembly: Linking function to polarity in epidermis cells

The aerial organs of plants are covered with a cuticle, a continuous layer overlaying the outermost cell walls of the epidermis. The cuticle is composed of two major classes of the lipid biopolymers: cutin and waxes, collectively termed cuticular lipids. Biosynthesis and transport of cuticular lipids occur predominantly in the epidermis cells. In the transport pathway, cuticular lipids are exported from their site of biosynthesis in the ER/plastid to the extracellular space through the plasma membrane and cell wall. Growing evidence suggests that ATP-binding cassette (ABC) transporters are implicated in transport of cuticular lipids across the plasma membrane of epidermal cells. The Arabidopsis ABC-type transporter protein CER5 (WBC12) was reported to act as a wax monomers transporter. In recent works, our group and others showed that a CER5-related protein, DESPERADO (DSO/WBC11), is required for cutin and wax monomers transport through the plasma membrane of Arabidopsis epidermis cells. Unlike the cer5 mutant, DSO loss-of-function had a profound effect on plant growth and development, particularly dwarfism, postgenital organ fusions, and altered epidermal cell differentiation. The partially overlapping function of CER5 and DSO and the fact that these proteins are half-size ABC transporters suggest that they might form a hetero-dimeric complex while transporting wax components. An intriguing observation was the polar localization of DSO in the distal part of epidermis cells. This polar expression might be explained by DSO localization within lipid rafts, specific plasma membrane microdomains which are associated with polar protein expression. In this review we suggest possible mechanisms for cuticular lipids transport and a link between DSO function and polar expression. Furthermore, we also discuss the subsequent transport of cuticular constituents through the hydrophobic cell wall and the possible involvement of lipid transfer proteins in this process.Key words: ABC transporter, cuticular lipids, polar expression, plasma membrane, epidermis     

Organ fusion and defective cuticle function in a lacs1 lacs2 double mutant of Arabidopsis

As the outermost layer on aerial tissues of the primary plant body, the cuticle plays important roles in plant development and physiology. The major components of the cuticle are cutin and cuticular wax, both of which are composed primarily of fatty acid derivatives synthesized in the epidermal cells. Long-chain acyl-CoA synthetases (LACS) catalyze the formation of long-chain acyl-CoAs and the Arabidopsis genome contains a family of nine genes shown to encode LACS enzymes. LACS2 is required for cutin biosynthesis, as revealed by previous investigations on lacs2 mutants. Here, we characterize lacs1 mutants of Arabidopsis that reveals a role for LACS1 in biosynthesis of cuticular wax components. lacs1 lacs2 double-mutant plants displayed pleiotropic phenotypes including organ fusion, abnormal flower development and reduced seed set; phenotypes not found in either of the parental mutants. The leaf cuticular permeability of lacs1 lacs2 was higher than that of either lacs1 or lacs2 single mutants, as determined by measurements of chlorophyll leaching from leaves immersed in 80% ethanol, staining with toluidine blue dye and direct measurements of water loss. Furthermore, lacs1 lacs2 mutant plants are highly susceptible to drought stress. Our results indicate that a deficiency in cuticular wax synthesis and a deficiency in cutin synthesis together have compounding effects on the functional integrity of the cuticular barrier, compromising the ability of the cuticle to restrict water movement, protect against drought stress and prevent organ fusion.     

Arabidopsis LTPG Is a Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Required for Export of Lipids to the Plant Surface

Plant epidermal cells dedicate more than half of their lipid metabolism to the synthesis of cuticular lipids, which seal and protect the plant shoot. The cuticle is made up of a cutin polymer and waxes, diverse hydrophobic compounds including very-long-chain fatty acids and their derivatives. How such hydrophobic compounds are exported to the cuticle, especially through the hydrophilic plant cell wall, is not known. By performing a reverse genetic screen, we have identified LTPG, a glycosylphosphatidylinositol-anchored lipid transfer protein that is highly expressed in the epidermis during cuticle biosynthesis in Arabidopsis thaliana inflorescence stems. Mutant plant lines with decreased LTPG expression had reduced wax load on the stem surface, showing that LTPG is involved either directly or indirectly in cuticular lipid deposition. In vitro 2-p-toluidinonaphthalene-6-sulfonate assays showed that recombinant LTPG has the capacity to bind to this lipid probe. LTPG was primarily localized to the plasma membrane on all faces of stem epidermal cells in the growing regions of inflorescence stems where wax is actively secreted. These data suggest that LTPG may function as a component of the cuticular lipid export machinery.     

Chemically Induced Cuticle Mutation Affecting Epidermal Conductance to Water Vapor and Disease Susceptibility in Sorghum bicolor (L.) Moench

Analysis of Sorghum bicolor bloomless (bm) mutants with altered epicuticular wax (EW) structure uncovered a mutation affecting both EW and cuticle deposition. The cuticle of mutant bm-22 was about 60% thinner and approximately one-fifth the weight of the wild-type parent P954035 (WT-P954035) cuticles. Reduced cuticle deposition was associated with increased epidermal conductance to water vapor. The reduction in EW and cuticle deposition increased susceptibility to the fungal pathogen Exserohilum turcicum. Evidence suggests that this recessive mutation occurs at a single locus with pleiotropic effects. The independently occurring gene mutations of bm-2, bm-6, bm-22, and bm-33 are allelic. These chemically induced mutants had essentially identical EW structure, water loss, and cuticle deposition. Furthermore, 138 F2 plants from a bm-22 x WT-P954035 backcross showed no recombination of these traits. This unique mutation in a near-isogenic background provides a useful biological system to examine plant cuticle biosynthesis, physiology, and function.     

Fiddlehead: an Arabidopsis mutant constitutively expressing an organ fusion program that involves interactions between epidermal cells.

In most circumstances plant epidermal cells do not respond to surface contact with adjacent plant parts. We have identified and characterized a mutant of Arabidopsis thaliana, designated fiddlehead, where lateral appendages of the shoot fuse with one another. While fusion between floral organs is most frequent, leaf fusions also occur. Using scanning and transmission electron microscopy, we show that adhesion takes place between epidermal cells and does not involve cytoplasmic union. We also show that the frequency of organ fusion is dictated by organ proximity. In wildtype Arabidopsis, postgenital fusion takes place exclusively in the gynoecium, whereas in the fiddlehead mutant, this program becomes expressed constitutively. The existence of such a mutant demonstrates that postgenital fusion is a genetically distinct program superimposed upon other aspects of gynoecial development in Arabidopsis.     

Mutants in Arabidopsis thaliana Altered in Epicuticular Wax and Leaf Morphology

We report eight new mutants in Arabidopsis thaliana possessing altered leaf morphology and epicuticular wax. These were isolated from a T-DNA-mutagenized population using a visual screen for altered leaf reflectance, i.e. increased glaucousness or glossiness. The mutants were placed into three distinct classes based on alterations in overall plant morphology: knobhead (knb), bicentifolia (bcf), and wax. The four knb mutants formed callus-like growths in the axillary region of the rosette leaves and apical meristem, the two bcf mutants produced hundreds of narrow leaves, and the two wax mutants had leaves and stems that were more glossy than wild type and organs that fused during early development. Leaves of knb and bcf were more glaucous and abnormally shaped than wild type. Epicuticular wax crystals over knb and bcf leaf surfaces (where none were present on wild type) likely contributed to their more glaucous appearance. In contrast, the glossy appearance of the wax mutants was associated with a reduced epicuticular wax load on both leaves and stems. One representative from each phenotypic class was selected for detailed analyses of epicuticular wax chemistry. All three lines, knb1, bcf1, and wax1, had dramatic alterations in the total amounts and relative proportions of their leaf epicuticular wax constituents.     

Cytological and biochemical analysis of COF1, an Arabidopsis mutant of an ABC transporter gene

In transposon-tagged lines of Arabidopsis we found two new mutants, cof1-1 and cof1-2 (cuticular defect and organ fusion), that show the phenotype of wilting when grown in soil, organ fusion of rosette leaves and infertility. Toluidine blue testing and scanning electron microscopy observation revealed that these mutants had cuticular defects in the stems and adult leaves, but not in cotyledones. Transmission electron microscopy observation revealed thinner cuticle layers in the mutants, and cuticular materials interspersed between the two fused epidermal layers were observed in the mutant rosette leaves. These two mutants had a transposon insertion in the coding regions of WBC11, which was classified as a member of ABC transporter genes in Arabidopsis. WBC11 showed high sequence similarity to CER5 (also called WBC12), which was involved in cuticular lipid export. Gas chromatographic analysis revealed that C29 alkane extracted from the stem surface of cof1 mutants was reduced whereas C29 ketone was accumulated, which was different from the case of cer5 mutants. While cer5 mutants had fairly normal morphology, cof1 mutants had pleiotropic phenotypes so that COF1/WBC11 could have important roles different from those of CER5/WBC12. We also found that C29 alkane was accumulated in the intracellular extract of cof1 mutants, suggesting a function for WBC11 in the direct transport of lipid molecules. Pollen observation showed that mutant pollen grains were irregularly shaped. The function of COF1/WBC11 in lipid transport for the construction of cuticle layers and pollen coats for normal organ formation is discussed.     

Pleiotropic phenotypes of the sticky peel mutant provide new insight into the role of CUTIN DEFICIENT2 in epidermal cell function in tomato

Plant epidermal cells have evolved specialist functions associated with adaptation to stress. These include the synthesis and deposition of specialized metabolites such as waxes and cutin together with flavonoids and anthocyanins, which have important roles in providing a barrier to water loss and protection against UV radiation, respectively. Characterization of the sticky peel (pe) mutant of tomato (Solanum lycopersicum) revealed several phenotypes indicative of a defect in epidermal cell function, including reduced anthocyanin accumulation, a lower density of glandular trichomes, and an associated reduction in trichome-derived terpenes. In addition, pe mutant fruit are glossy and peels have increased elasticity due to a severe reduction in cutin biosynthesis and altered wax deposition. Leaves of the pe mutant are also cutin deficient and the epicuticular waxes contain a lower proportion of long-chain alkanes. Direct measurements of transpiration, together with chlorophyll-leaching assays, indicate increased cuticular permeability of pe leaves. Genetic mapping revealed that the pe locus represents a new allele of CUTIN DEFICIENT2 (CD2), a member of the class IV homeodomain-leucine zipper gene family, previously only associated with cutin deficiency in tomato fruit. CD2 is preferentially expressed in epidermal cells of tomato stems and is a homolog of Arabidopsis (Arabidopsis thaliana) ANTHOCYANINLESS2 (ANL2). Analysis of cuticle composition in leaves of anl2 revealed that cutin accumulates to approximately 60% of the levels observed in wild-type Arabidopsis. Together, these data provide new insight into the role of CD2 and ANL2 in regulating diverse metabolic pathways and in particular, those associated with epidermal cells.