Department of Botany, Sri Krishnadevaraya University, Anantapuram 515 003, Andhra Pradesh, India
Received date: 12 September 2013 Revised date: 20 September 2013 Accepted date: 25 September 2013
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Plant surfaces are ensheathed by cuticular wax, amorphous intra-cuticular embedded in cutin polymer and crystalloid epi-cuticular that imparts a whitish appearance, confers drought resistance by reducing stomatal transpiration and also protects from U.V Radiation, phytophagous insects etc. Very long chain fatty acids acts as precursors for cuticular wax bio-synthesis. Wax bio-synthesis begins with fatty acid synthesis in the plastid (de novo synthesis of C16 and C18) and elongation of fatty acids in endoplasmic reticulum (C20 – C34) by four distinct enzymes 3-ketoacyl-CoA synthase, 3-ketoacyl-CoA reductase, 3-hydroxacyl-CoA dehydratase, trans-2,3-enoyl-CoA reductase (KCS, KCR, HCD, ECR). The KCS, a fatty acid elongase, determines the chain length and substrate specificity of the condensation reaction, a rate limiting step and the subsequent elongated products alkanes, aldehydes, primary alcohols, secondary alcohols, ketones and wax esters. 21 KCS genes were annotated in Arabidopsis thaliana Genome of which some KCSs were identified involved in cuticle formation (CER6) (CUT1), KCS1, KCS2, (DAISY), KCS20 and FDH).The current review will focus on the bio-chemical, genetic and molecular approaches on KCSs genes, predominantly KCS1 in plants particularly useful in identifying and characterizing gene products involved in wax bio-synthesis, secretion and function for developing transgenic crops that combat various stresses. INTRODUCTION
Cuticular wax, elongation, ensheathed, 3-ketoacyl-CoA synthase, precursors
Cuticular wax is a special features developed by plants to seal the aerial organs and to tolerate various biotic and abiotic stresses.Epicuticular wax is the first line of defense to prevent non-stomatal harmful water loss [forming a continuous hydrophobic barrier obstructing water loss from plant organs].Other functions in plant are protection from Ultraviolet [UV] light by reflection [24,45], restrict the attachment and growth of insects  increased plant resistance against pathogens like bacteria and fungi and reduces water deposition on the surface of the plant thus reducing retention of dust, pollen and air pollutants  and plays a role in plant–insect  and plant–pathogen interactions . Reduction in the amount of epicuticular wax on plant surface showed increased rates of transpiration. Brushing waxes off the excised leaves significantly increased the rate of water loss .Similarly, leaves of rice [Oryza sativa], dipped for two seconds in chloroform to remove epicuticular waxes, and exhibited more than a two fold increase in cuticular conductance to water vapor compared to control leaves . Leaf glaucousness [waxinees] is a characteristic referred to as a plant adaptation to drought . Cuticular wax is a general term for a complex mixture of hydrophobic compounds including homologous series of very long chain aliphatic lipids [fatty acids, alcohols, aldehydes, alkanes, ketones and wax esters] which are all derived from VLCFAs by the fatty acid elongase mechanism.
The Fatty Acid Elongase mechanism involves four successive reactions catalyzed by four distinct enzymes [KCS, KCR, HCD and ECR] organized in an endoplasmic reticulum [ER]-associated complex, the acyl-CoA elongases.
The first step of fatty acid elongation is the condensation by the KCS [or condensing enzyme] of a long chain acyl-CoA with a malonyl-Co A. The 3-ketoacyl-CoA is then reduced by a KCR to 3-hydroxyacyl-CoA. It is then HCD to form a trans-2, 3-enoyl-CoA. The final elongation step is the reduction by the ECR which yields a two carbon acyl Co A. Considering the condensing activity; several genes have been identified as encoding the KCS. The first one, FAE1, was isolated from the Arabidopsis thaliana mutant fae1 and characterized as the seed-specific KCS gene responsible for the synthesis of VLCFA present in storage lipids . In the Arabidopsis genome, 21 FAE1-like genes are present . Only eight of them have been demonstrated to encode proteins able to catalyze VLCFA production [4,40,54]. These KCS enzymes have different substrate specificities suggesting that multiple fatty acid elongases exist in plants. Three KCSs have been shown to be involved in the formation of the cuticle in Arabidopsis: CER6 [9,18,36], KCS1  and FDH [42,59].Complete loss of function of CER6 or KCS1 leads to the accumulation of wax derivatives with 24 carbons suggesting that these two KCSs are involved in the biosynthesis of C26 and longer VLCFAs for the production of epicuticular waxes. In addition, loss of function of FDH [FIDDLEHEAD] results in ectopic organ fusions suggesting a role of this KCS in organ development. Another KCS named HIC has been described as a negative regulator of stomatal development that responds to CO2 concentration . The 3D structure of the KCS proteins using the model generated for the KCS1 protein, analyzed the subcellular localization in tobacco cells by confocal microscopy of 7 KCS proteins as well as that of the two reductases of the acyl-CoA elongase complex, KCR and ECR. Twenty-one KCS genes have been previously identified in the Arabidopsis genome .
Adopted from Je´roˆme Joube`s et al., , Blacklock and Jaworski 1  ; 2, Costaglioli et al.,  ; 3, Efremova et al.,  ;4, Fiebig et al.  ;5, Ghanevati and Jaworski .,  6, Ghanevati and Jaworski ; 7, Gray et al., ; 8, Hooker et al., ; 9, James and Dooner ; 10, James et al., ; 11, Kunst et al., ; 12, Kunst and Clemens ; 13, Lolle et al.,  ; 14, Millar and Kunst ; 15, Millar et al., ; 16, Paul et al., ; 17, Pruitt et al., ; 18, Rossak et al., ; 19, Stasolla et al., ; 20, Todd et al., ; 21, Trenkamp et al., .
KCS 1 is a condensing enzyme involved in the critical fatty acid elongase process. [ACESSION NO: #AT1G01120]. A cDNA encoding KCS was identified in Arabidopsis thaliana. The recombinant protein was expressed in yeast and its function as a fatty acid elongase condensing enzyme confirmed. The analysis of T-DNA tagged kcs-1 mutant demonstrated the in vivo involvement of KCS-1 in very long chain fatty acid and wax bio-synthesis in vegetative tissues. It elongates C18:0-Co A through C26:0-Co A and is more active towards saturated fatty acids. Molecular weight of polypeptide is 58.0 kDa [from nucleotide sequence]  .
A long chain 2, 3, 4-saturated fatty acyl co A + malonyl co A ↔ a long chain 3 oxalo acyl co A + CO2 + Co enzyme A. The structure of KCS1 shows that out of 8 sub classes [α, β, γ, δ, ζ, ε, η, θ] KCS1 belongs to the sub class δ [showing similarity to KCS13, KCS14]. By using the ARAMEMNON plant membrane protein database server [http://aramemnon.botanik.unikoeln. de/index.ep] [Schwacke R] , identified KCS1 anchored to the membrane with two trans -membrane domains [residues 44-66 & 85-103]. Proposed model of KCS1 is triangular shape with prominent arm made of 2 parallel α helices [A1 and A2] linked to the protein by a complex network of coils. The size, shape and position of this arm make it suitable for anchoring to the membrane. The body of the protein shows α-β-α-α [4 layered] structure. Amino acid sequence of KCS is CYS258 HIS 454 ASN458 .of which “Active Cystine residue” is the acyl acceptor in the first step of condensation and creates electronic environment resembling in acyl chain elongation reaction. A fourthresidue that is a serine at position 317 has been demonstrated to be crucial for the activity of the FAE1 enzyme in Brassica napus . Volume and spatial organization of KCS1, putative binding pocket is compatible with docking of malonyl co A and acyl chain alpha carbons. The chromosomal locations of KCS1 genes in relation to the segmental duplication history were analyzed and found that KCS1, KCS13 of δ subclass have evolved from an older segmental duplication with a good statistical significance . Localization of GFP-KCS fusion protein in Endoplasmic reticulum modeling shows accessible putative phosphorylation sites, predictions have been made using at high stringency and mapped on the models to check accessibility.
The functions of KCS1 were, it is involved in the formation of the cuticle,  involved in the bio-synthesis of C26 and long VLCFAs [very long chain fatty acid synthesis] for the production of epi-cuticular waxes, fatty acid biosynthesis pathway, fatty acid elongase activity, lipid biosynthesis process, transferase activity [transfer of acyl groups other than aminoacyl group] Very long chain fatty acid metabolic process. Activity of KCS1 in-vitro condensation assay of isolated [HIS] 6KCS gene expression is ubiquitous in roots, stem and epidermis .
KCS1 is the most expressive gene for different abiotic stresses among all the members of KCS family [KCS1 to KCS21] duly supported by the evidences from bio-chemical, genomic and molecular approaches in various plants.
Wax Bio-synthesis pathways exemplification has been possible due to the development of complex techniques like gas chromatography and mass spectrometry that allowed for the identification of wax components and exquisite genetic and bio-chemical studies using radio labeled tracers. As there is limited knowledge on enzymes involved in wax bio-synthesis regarding the association of these enzymes with membranes, insufficient amount of epidermal tissue for enzyme purification, enzyme activity assay substrates being insoluble in aqueous buffers are not commercially available. Genetic approaches alone were initially not successful in revealing the identity of loci involved in wax production. By studying wax biosynthesis in Arabidopsis twenty-two mutants were identified, called the eceriferum mutants [cer], displaying varying degrees of waxlessness cer1- cer10 are very glossy and cer 11- cer22 are less so, with altered epicuticular wax patterns compared to the wild type . Homologous mutations to eceriferum were also found in other plants, e.g. in barley, where the locus is also termed eceriferum. Barley is the most extensively studied species, with 85 identified cer loci. In maize and Brassica napus the mutants are named glossy, referring to the leaves‟ glossy surface. Scanning electron microscopy revealed that the epi-cuticular wax crystals on these mutants are absent or exhibit particular morphological changes. Chemical analyses demonstrated that mutants have a lower wax load or altered chemical composition. Thus, biochemical functions of the mutated gene products are difficult to predict from the alterations in wax compositions from the visual screens but only mutants with significant decreases in epicuticular crystalloid accumulation would be isolated in the screen with lesions in regulatory genes, or genes affecting epidermal development. Mutants affecting individual biosynthetic enzymes would be difficult to find due to their more subtle biochemical phenotypes. Interestingly the KCS genes, that take part in the control of VLCFA biosynthesis, show different tissue specific expression patterns by both molecular and histo-chemical tests. The varying expression levels of KCS isoforms in different tissues suggest the specific roles of these enzymes . The examination of the eceriferum mutants also resulted in the discovery of the importance of the operation of the KCS gene family. Since the expression of these genes is essential for the production of VLCFAs, their inhibition results in the block of wax biosynthesis  Yephremov et al . Made a list of the candidate genes involved in wax biosynthesis, suggesting that the synthesized molecules by the elongase complexes are commonly modified, and therefore their proper constitution is crucial for the possibilities of later modifications.
Very-long-chain fatty acids [VLCFAs] are important functional components of various lipid classes, including cuticular lipids in the higher plant epidermis and lipid-derived second messengers. FAE1, the seed-specific β-ketoacyl-CoA synthase [KCS] catalyzes the first rate-limiting step in VLCFA biosynthesis. Misexpression of FAE1 changes the VLCFAs in different classes of lipids. In Arabidopsis, mutations in the FATTY ACID ELONGATION [FAE1] gene result in reduced levels of very long chain fatty acids in seed [20,30,33]. The genes encoding FAE1 and its homologs have been cloned from Arabidopsis , Jojoba  and Brassica napus . Ghanevati M et al., performed site directed mutagenesis experiments on the Arabidopsis FAE1 decipher the importance of Cys and His as residues in condensing enzyme catalysis, shown that Cys-223 and four His residues are essential for the enzyme activity. Extensive characterization of FAE1and the related KCS1 has confirmed that these genes encode condensing enzymes [12,46,53]. The FAE1- like gene family appears to be unique to plants.
In Garden nasturtium [Tropaeolum majus] seeds the fatty acid elongase involved in producing significant amounts of erucic acid [22:1] [70%–75%] of total fatty acid , and accumulates trierucin as the predominant triacylglycerol [TAG] in its seed oil . Using a degenerate primers approach, a cDNA of a putative embryo FAE was obtained, containing a 1,512-bp open reading frame encoding a protein of 504 amino acids. A genomic clone of the nasturtium FAE gene was isolated and sequence analyses indicated the absence of introns, restricted to the embryo[ from Northern hybridization], encoded by a small multigene family [from Southern hybridization]. The cDNA into two different heterologous chromosomal backgrounds [Arabidopsis and tobacco [Nicotiana tabacum]] under the control of a seed-specific [napin] promoter and the tandem 35S promoter for functional confirmation. Seed-specific expression resulted in an 8-fold increase in erucic acid proportions in Arabidopsis seed oil, while constitutive expression in transgenic tobacco tissue resulted in increased proportions of VLCFAs indicate that the nasturtium FAE gene encodes a condensing enzyme involved in the biosynthesis of VLCFAs, utilizing monounsaturated and saturated acyl substrates. With its strong and unique preference for elongating 20:1-CoA, FAE gene product is used for directing or engineering increased synthesis of erucic acid.
In Jojoba manipulation of the microsomal fatty acyl-Co A elongation pathway converts high erucic acid rapeseed oil rich in VLCFAs [used as an industrial feedstock] into canola [edible oil] devoid of VLCFAs significant for agriculture. The cDNA was cloned from developing Jojoba embryos [involved in microsomal fatty acid elongation] which is homologous to the Arabidopsis[FAEI] gene encoding KCS.The characterization and transformation of low erucic acid rapeseed with the Jojoba cDNArestored KCS activity for developing embryos and altered the transgenic seed oil composition to contain high levels ofVLCFAs .
In Jojoba a gene encoding the KCS was isolated from developing seeds of Lunaria annua, Brassicaceae [rich in nervonic acid] complimented the canola [Brassica napus].Mutation of this gene knocked out the production of very long chain fatty acids in the seed oil. The influence of the Jojoba KCS substrate preferences was evident from the appearance of up to 5% nervonic acid in the transgenic oil nervonic acid present in jojoba oil but found at very low levels in rapeseed oil . Expression of the Lunaria KCS in transgenic Brassica resulted in rapeseed oils with greater than 25% nervonic acid.
In rapeseed [Brassica napus], the two elongation steps from oleoyl-CoA [C18:1-CoA] to erucic acid are each controlled by alleles at two loci, E1 and E2 tightly linked to the FAE1 gene loci. Advances in the biochemistry of the seed elongases and the cloning of the loci controlling the erucic acid content, the nature of the mutations that characterize the agriculturally important LEAR trait remain obscure. So the comparison of the component elongase activities in rapeseed varieties that vary in erucic acid content showed that an absence of KCS is associated with variation in the sequence of the Bn-FAE1 gene. Low erucic acid rapeseed [LEAR] a near absence of VLCFAs in the seed oil lack L-ketoacyl-CoA synthase activity, acyl-CoA and ATP-dependent elongation activities in microsomes. High erucic acid rapeseed [HEAR] having high molecular mass acyl-CoA elongase complex in solubilised microsomes [from Size exclusion chromatography]. Transcripts for the Bn-FAE1 genes were detected in LEAR embryos, immunoblots using antisera raised against the L-ketoacyl-CoA synthase indicated an absence of this protein. Comparison of the deduced amino acid sequences of immature embryo cDNAs reveals that LEAR alleles of Bn-FAE1 encode variant L- ketoacyl-CoA synthase proteins. So, the coding region of LEAR was overexpressed for the development of transgenic seeds with significant increase in erucic acid. To develop low level oferucic acid in rapeseeds by intron-spliced hairpin RNA, an inverted repeat unit of a partial BnFAE1.1gene interrupted by a spliceable intron cloned into pCAMBIA3301, and a seed-specific [Napin] promoter used to control the transcription of the transgene. Four transgenic plants harboring a single copy of transgene were generated. Expression of endogenous BnFAE1.1 gene in developing T3 seeds was significantly reduced. In mature T3 seeds, erucic acid was decreased by 60.8 to 99.1% compared with wild type seeds, and accounted for 0.36 to 15.56% of total fatty acids. The level of eicosenoic acid was also greatly decreased. The expression of endogenous BnFAE1.1 was efficiently silenced by the designed RNAi silencer, causing a significant down-regulation in the level of erucic acid. Therefore, the RNAi-mediated post-transcriptional silencing of FAE1 gene to reduce oleic acid in rapeseeds was an efficient method to breed some new Brassica napus lines.
In Cotton several genes encoding putative KCSs were significantly up-regulated during early cotton fiber development. GhCER6 cDNA containing an open reading frame of 1479 bp, encoding a protein of 492 amino acid residues homologous to the Arabidopsis condensing enzyme CER6, was isolated and cloned. In situ hybridization results demonstrated that GhCER6 mRNA was detected only in the elongating wild-type cotton fiber cells.When GhCER6 was transformed to the Saccharomyces cerevisiae elo3 deletion mutation strain, deficient in the production of 26-carbon fatty acids displayed a very slow growth phenotype, the mutant cells were found to divide similarly compared with those of the wild-type cells, heterologous expression of GhCER6 restored the viability of the Sacchromyces cerevisiae haploid elo2 and elo3 double-deletion strain. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis showed that GhCER6 was enzymatically active since the yeast elo2 and elo3 double-deletion mutant expressing the cotton gene produced verylong- chain fatty acids that are essential for cell growth suggesting that GhCER6 encodes a functional KCS .
In wheat TaCer6 was firstly cloned by rapid amplification of cDNA ends [RACE] and identified as a tissue-specific gene. To determine if environmental factors such as drought and low temperature induce TaCer6 transcription, examination of the effects of these factors on TaCer6 in two wheat cultivars was done, demonstrating that light was essential for TaCer6 transcription, salt stress inhibited TaCer6 expression, application of salicylic acid enhanced TaCer6 transcripts accumulation. Polyethylene glycol [PEG 6000] and abscisic acid increased the expression of TaCer6 more in the drought and cold tolerant cultivar Jinmai47 than in non-tolerant cultivar Shi4185. Low temperature increased TaCer6 transcription in Jinmai47 while decreased it in Shi4185 Cer6 encoding KCS catalyzes biosynthesis of VLCFA, which play an important role in holding water from astomatous dissipating, cell elongation, or fertility [32,36,43]. In Arabidopsis Cer6 mutant, wax load was reduced to 6 - 7 % of wild-type levels and most of pollens were sterile .In tomato LeCer6 mutant with leaf and fruit deficient in n-alkanes and aldehydes with chain lengths beyond C30, cuticular water loss was about four times larger than in wild tomato [32,56]. Application of lignoceric acid [C24:0] in ovule culture medium to cotton lintless mutant GhCer6 could overcome the inhibition of fiber elongation [43,44]. The isolation and expression of wheatCer6-like KCS cDNA, which was named as TaCer6 [EU159697] .
In intact tomato [Lycopersicon esculentum] fruits are used, due to their astomatous surface, as a novel integrative approach to investigate composition, function and relationship of wax amounts and compositions of tomato were manipulated before measuring unbiased cuticular transpiration. First, successive mechanical and extractive wax removal steps allowed the selective modification of epi cuticular wax layer consisting of very long chain aliphatics and intra cuticular wax layers containing large quantities of triterpenoids. Second, applying reverse genetic techniques, a loss of function mutation with a transposon insertion in a very long chain fatty acid elongase b- ketoacyl Co A synthase .
Apathogen related tomato line called dfd [delayed fruit deterioration] described as a cutin-overexpressing cultivar, shown that it displays a higher level of water retention than the control cultivar, „Ailsa Craig‟. This effect is possibly due to the altered composition and greater amount of cuticular components in dfd tomatoes .
In apple [Malus domestica Borkh.] wax production is an important aspect of fruit development. To develop a fresh, shiny apple the analysis of expression levels of genes based on gene expression data from Arabidopsis thaliana, some genes in apple were assigned for analysis, EST sequences of apple used to design specific primers for RT-PCR experiments, by isolating intact RNA from different apple tissues and performing RT-PCR reaction in obtaining expression patterns using actin primer for the detection of intron containing genomic DNA, and tubulin primers as internal controls in RT-PCR experiments found tissue specific differences between the expression patterns of candidate genes, showing that KCS1 and KCS4 are overexpressed in the skin tissue, having skin-specific expression in apple fruit that are potentially involved in wax-biosynthesis .
ABA regulates the expression of many genes, including those encoding oil-body proteins in microspore-derived and zygotic embryos of Brassica napus [16,17,55,57] . MYB96 transcription factor, the abscisic acid [ABA]-inducible plays a role in drought resistance, also regulates cuticular wax biosynthesis by binding directly to the promoters of genes encoding fatty acid elongating enzymes, like KCS1, KCS2, KCS6 and KCR1 that constitute a rate-limiting step in cuticular wax biosynthesis, downstream events are also activated and cuticular waxes are accumulated to a high level in the leaves and stems of the myb96-1D mutant. These observations indicate that the MYB96 transcription factor serves as a molecular web that incorporates drought stress signals to promote fatty acid elongation in Cuticular wax biosynthesis. In myb96-1D mutant KCS, KCR, ECR and 3-hydroxyacyl Co A dehydratase [PAS2] are upregulated, CER3 [ECERIFERUM 3], CYP96A15/MAH1 [CYTOCHROME P450 96 A1] involved in decarbonylation pathway and CER4 and Wax ester synthase/diacylglycerol acyl transferase [WSD1] functions in acyl reduction pathway. ABC transporter that exports cutin monomers and waxes, are also up regulated in the mutant, indicating that MYB96 regulates both cuticular wax biosynthesis and transport .
The emphasis to the plant cuticular wax is a reflection of its significant contribution to plant biology. Wax exterior to plant is the result of the metabolic activity of epidermis, its examination has contributed valuable information about the chemical diversity of wax composition within a single species and amongst taxa. The pioneering contributions of Joubes , Millar [36,37], Kunst [28,29] and others have shown us the basic wax biochemical pathways. The isolation of mutants in Arabidopsis and other plant species, combined with biochemical analyses of mutant phenotypes, resulted in identification of gene products involved in wax production, and cloning of a number of wax biosynthetic genes. Not all genes involved in the biochemistry, regulation and transport of wax to the plant surface have been identified by genetic approaches. The challenge now is to take the genomic tools available to find genes whose functions are essential, redundant, or more subtle, like KCS1 and investigate their specific contributions to cuticular wax production during the life cycle of the plant, develop transgenic crops for various stress tolerances.
CS greatly acknowledges the DBT, DST and UGC, Government of India, New Delhi for financial support.