Molecular genetic basis of pod corn (Tunicate maize).

Pod corn is a classic morphological mutant of maize in which the mature kernels of the cob are covered by glumes, in contrast to generally grown maize varieties in which kernels are naked. Pod corn, known since pre-Columbian times, is the result of a dominant gain-of-function mutation at the Tunicate (Tu) locus. Some classic articles of 20th century maize genetics reported that the mutant Tu locus is complex, but molecular details remained elusive. Here, we show that pod corn is caused by a cis-regulatory mutation and duplication of the ZMM19 MADS-box gene. Although the WT locus contains a single-copy gene that is expressed in vegetative organs only, mutation and duplication of ZMM19 in Tu lead to ectopic expression of the gene in the inflorescences, thus conferring vegetative traits to reproductive organs.

MIKC* MADS-box proteins: conserved regulators of the gametophytic generation of land plants.

Land plants (embryophytes) are characterized by an alternation of two generations, the haploid gametophyte and the diploid sporophyte. The development of the small and simple male gametophyte of the flowering plant Arabidopsis (Arabidopsis thaliana) critically depends on the action of five MIKC* group MCM1-AGAMOUS-DEFICIENS-SRF-box (MADS-box) proteins. In this study, these MIKC* MADS-box genes were isolated from land plants with relatively large and complex gametophyte bodies, namely the bryophytes. We found that although the gene family expanded in the mosses Sphagnum subsecundum, Physcomitrella patens, and Funaria hygrometrica, only a single homologue, Marchantia polymorpha MADS-box gene 1 (MpMADS1), has been retained in the liverwort M. polymorpha. Liverworts are the earliest diverging land plants, and so a comparison of MpMADS1 with its angiosperm homologues addresses the molecular evolution of an embryophyte-specific transcription factor over the widest phylogenetic distance. MpMADS1 was found to form a homodimeric DNA-binding complex, which is in contrast to the Arabidopsis proteins that are functional only as heterodimeric complexes. The M. polymorpha homodimer, nevertheless, recognizes the same DNA sequences as its angiosperm counterparts and can functionally replace endogenous MIKC* complexes to a significant extent when heterologously expressed in Arabidopsis pollen. The 11 MIKC* homologues from the moss F. hygrometrica are highly and almost exclusively expressed in the gametophytic generation. Taken together, these findings suggest that MIKC* MADS-box proteins have largely preserved molecular roles in the gametophytic generation of land plants.

The MADS-domain protein MPF1 of Physalis floridana controls plant architecture, seed development and flowering time.

Floral and vegetative development of plants is dependent on the combinatorial action of MADS-domain transcription factors. Members of the STMADS11 subclade, such as MPF1 of Physalis, are abundantly expressed in leaves as well as in floral organs, but their function is not yet clear. Our studies with transgenic Arabidopsis that over-express MPF1 suggest that MPF1 interacts with SOC1 to determine flowering time. However, MPF1 RNAi-mediated knockdown Physalis plants revealed a complex phenotype with changes in flowering time, plant architecture and seed size. Flowering of these plants was delayed by about 20% as compared to wild type. Expression of PFLFY is upregulated in the MPF1 RNAi lines, while PFFT and MPF3 genes are strongly repressed. MPF1 interacts with a subset of MADS-domain factors, namely with PFSOC1 in planta, and with PFSEP3 and PFFUL in yeast, supporting a regulatory role for this protein in flowering. The average size of seeds produced by the transgenic MPF1 RNAi plants is increased almost twofold. The height of these plants is also increased about twofold, but most axillary buds are stunted when compared to controls. Taken together, this suggests that members of the STMADS11 subclade act as positive regulators of flowering but have diverse functions in plant growth.

MPF2-like-a MADS-box genes control the inflated Calyx syndrome in Withania (Solanaceae): roles of Darwinian selection.

The Chinese lantern, which is the inflated calyx syndrome (ICS) of Physalis, is formed by MPF2 in the presence of the plant hormones, cytokinin and gibberellin. MPF2 knockdown mutants of Physalis have small leaves, no ICS, and are male sterile, thus, revealing three MPF2-related functions. Of the close relatives of Physalis, Tubocapsicum has only a rudimentary calyx, whereas others, like the Withania species, have ICS. From all Withania samples tested, two classes of MPF2-like orthologs, MPF2-like-A and MPF2-like-B, were isolated, whereas only the latter class was obtained from tetraploid Tubocapsicum. Though distinct differences can be observed between MPF2-like-A and MPF2-like-B proteins, that is MPF2-like-A proteins have an aberrant structure in that they have a three amino acid deletion in their C-domain and an eight amino acid extension at the C-terminal end, MPF2-like-A genes are phylogenetically closer to the Physalis MPF2-like genes. Unlike MPF2-like-B, the overexpression of MPF2-like-A in Arabidopsis revealed extra large sepals thus suggesting that MPF2-like-A genes are very likely responsible for the ICS formation in Withania. This correlated with the expression pattern of MPF2-like-A in vegetative and flower tissues, whereas MPF2-like-B is expressed only in vegetative tissues of Withania. In Tubocapsicum, however, MPF2-like-B RNA is detectable in all tissues tested. Finally, positive Darwinian selection was observed in the branch leading to Physalis MPF2-like and Withania MPF2-like-A proteins, followed by purifying selection once the trait had evolved. By contrast, purifying selection was detected for all other MPF2-like proteins tested. The contribution of the MPF2-like gene duplication to subfunctionalization is discussed.

Evolution of MIR168 paralogs in Brassicaceae.

BACKGROUND: In plants, expression of ARGONAUTE1 (AGO1), the catalytic subunit of the RNA-Induced Silencing Complex responsible for post-transcriptional gene silencing, is controlled through a feedback loop involving the miR168 microRNA. This complex auto-regulatory loop, composed of miR168-guided AGO1-catalyzed cleavage of AGO1 mRNA and AGO1-mediated stabilization of miR168, was shown to ensure the maintenance of AGO1 homeostasis that is pivotal for the correct functioning of the miRNA pathway. RESULTS: We applied different approaches to studying the genomic organization and the structural and functional evolution of MIR168 homologs in Brassicaeae. A whole genome comparison of Arabidopsis and poplar, phylogenetic footprinting and phylogenetic reconstruction were used to date the duplication events originating MIR168 homologs in these genomes. While orthology was lacking between Arabidopsis and poplar MIR168 genes, we successfully isolated orthologs of both loci present in Arabidopsis (MIR168a and MIR168b) from all the Brassicaceae species analyzed, including the basal species Aethionema grandiflora, thus indicating that (1) independent duplication events took place in Arabidopsis and poplar lineages and (2) the origin of MIR168 paralogs predates both the Brassicaceae radiation and the Arabidopsis alpha polyploidization. Different phylogenetic footprints, corresponding to known functionally relevant regions (transcription starting site and double-stranded structures responsible for microRNA biogenesis and function) or for which functions could be proposed, were found to be highly conserved among MIR168 homologs. Comparative predictions of the identified microRNAs also indicate extreme conservation of secondary structure and thermodynamic stability. CONCLUSION: We used a comparative phylogenetic footprinting approach to identify the structural and functional constraints that shaped MIR168 evolution in Brassicaceae. Although their duplication happened at least 40 million years ago, we found evidence that both MIR168 paralogs have been maintained throughout the evolution of Brassicaceae, most likely functionally as indicated by the extremely high conservation of functionally relevant regions, predicted secondary structure and thermodynamic profile. Interestingly, the expression patterns observed in Arabidopsis indicate that MIR168b underwent partial subfunctionalization as determined by the experimental characterization of its expression pattern provided in this study. We found further evolutionary evidence that pre-miR168 lower stem (the RNA-duplex structure adjacent to the miR-miR* stem) is significantly longer than animal lower stems and probably plays a relevant role in multi-step miR168 biogenesis.

The microRNA regulated SBP-box genes SPL9 and SPL15 control shoot maturation in Arabidopsis.

Throughout development the Arabidopsis shoot apical meristem successively undergoes several major phase transitions such as the juvenile-to-adult and floral transitions until, finally, it will produce flowers instead of leaves and shoots. Members of the Arabidopsis SBP-box gene family of transcription factors have been implicated in promoting the floral transition in dependence of miR156 and, accordingly, transgenics constitutively over-expressing this microRNA are delayed in flowering. To elaborate their roles in Arabidopsis shoot development, we analysed two of the 11 miR156 regulated Arabidopsis SBP-box genes, i.e. the likely paralogous genes SPL9 and SPL15. Single and double mutant phenotype analysis showed these genes to act redundantly in controlling the juvenile-to-adult phase transition. In addition, their loss-of-function results in a shortened plastochron during vegetative growth, altered inflorescence architecture and enhanced branching. In these aspects, the double mutant partly phenocopies constitutive MIR156b over-expressing transgenic plants and thus a major contribution to the phenotype of these transgenics as a result of the repression of SPL9 and SPL15 is strongly suggested.

SBP-domain transcription factors as possible effectors of cryptochrome-mediated blue light signalling in the moss Physcomitrella patens.

Cryptochromes are blue light absorbing photoreceptors found in many organisms and involved in numerous developmental processes. At least two highly similar cryptochromes are known to affect branching during gametophytic development in the moss Physcomitrella patens. We uncovered a relationship between these cryptochromes and the expression of particular members of the SBP-box genes, a plant specific transcription factor family. Transcript levels of the respective moss SBP-box genes, all belonging to the LG1-subfamily, were found to be dependent, albeit not exclusively, on blue light. Moreover, disruptant lines generated for two moss representatives of this SBP-box gene subfamily, both showed enhanced caulonema side branch formation, a phenotype opposite to that of the ppcry1a/1b double disruptant line. In this report we show that PpCRY1a and PpCRY1b act negatively on the transcript levels of several related moss SBP-box genes and that at least PpSBP1 and PpSBP4 act as negative regulators of side branch formation.

MADS-complexes regulate transcriptome dynamics during pollen maturation.

BACKGROUND: Differentiation processes are responsible for the diversity and functional specialization of the cell types that compose an organism. The outcome of these processes can be studied at molecular, physiologic, and biochemical levels by comparing different cell types, but the complexity and dynamics of the regulatory processes that specify the differentiation are largely unexplored. RESULTS: Here we identified the pollen-specific MIKC* class of MADS-domain transcription factors as major regulators of transcriptome dynamics during male reproductive cell development in Arabidopsis thaliana. Pollen transcript profiling of mutants deficient in different MIKC* protein complexes revealed that they control a transcriptional switch that directs pollen maturation and that is essential for pollen competitive ability. We resolved the functional redundancy among the MIKC* proteins and uncovered part of the underlying network by identifying the non-MIKC* MADS-box genes AGL18 and AGL29 as downstream regulators of a subset of the MIKC* MADS-controlled genes. CONCLUSION: Our results provide a first, unique, and compelling insight into the complexity of a transcription factor network that directs cellular differentiation during pollen maturation, a process that is essential for male reproductive fitness in flowering plants.

Evolution of the inflated calyx syndrome in Solanaceae.

Species that express the inflated calyx syndrome (ICS) are found in several genera of the Solanaceae. The MADS-box protein MPF2, together with the plant hormones cytokinin and gibberellin, has been shown to be responsible for this trait in Physalis floridana. We have used sequence data from 114 species belonging to 35 genera to construct a molecular phylogeny of Solanaceae. Apart from the 2 Witheringia species analyzed, species within a given genus cluster together on the resulting cladogram. Witheringia solanacea is embedded within the Physalinae, but Witheringia coccoloboides is placed basal to the Iochrominae. The ICS trait seems to be of multiple origins both within the Solanaceae and the Physaleae. Surprisingly, expression of MPF2-like genes in floral organs appears to be plesiomorphic in both the Physaleae and the Capsiceae. Some species in these tribes that show neither ICS nor calyx accrescence fail to express the MPF2-like gene in floral organs. Among those that do express this gene in the calyx are the species Capsicum baccatum, Lycianthes biflora, Tubocapsicum anomalum, W. solanacea, and Vassobia breviflora, all of which form small calyces that do not respond to externally applied hormones. The plesiomorphic nature of MPF2-like gene expression in the calyx of the Physaleae and Capsiceae raises the possibility that originally ICS also was actually a plesiomorphic character in these 2 groups. However, this trait might have undergone changes in a number of species due to secondary loss of components in ICS formation, like hormone response of calyx development. These findings are discussed in an evolutionary context of a molecular pathway leading to ICS.

Comparative analysis of the SBP-box gene families in P. patens and seed plants.

To come to a better understanding of the evolution and function of the SBP-box transcription factor family in plants, we identified, isolated and characterized 13 of its members from the moss Physcomitrella patens. For the majority of the moss SBP-box genes, clear orthologous relationships with family members of flowering plants could be established by phylogenetic analysis based on the conserved DNA-binding SBP-domain, as well as additional synapomorphic molecular characters. The P. patens SBP-box genes cluster in four separable groups. One of these consists exclusively of moss genes; the three others are shared with family members of Arabidopsis and rice. Besides the family defining DNA-binding SBP-domain, other features can be found conserved between moss and other plant SBP-domain proteins. An AHA-like motif conserved from the unicellular alga Chlamydomonas reinhardtii to flowering plants, was found able to promote transcription in a heterologous yeast system. The conservation of a functional microRNA response element in the mRNA of three of the moss SBP-box genes supports the idea of an ancient origin of microRNA dependent regulation of SBP-box gene family members. As our current knowledge concerning the roles of SBP-box genes in plant development is scarce and the model system P. patens allows targeted mutation, the material we isolated and characterized will be helpful to generate the mutant phenotypes necessary to further elucidate these roles.

The MADS-domain protein PPM2 preferentially occurs in gametangia and sporophytes of the moss Physcomitrella patens.

To date, the function of MADS-domain transcription factors in non-seed plants remains largely elusive, although a number of genes have been isolated and characterized from a variety of species. In our study we analyzed PPM2, a classical MIKC-type MADS-box gene from the moss Physcomitrella patens, taking advantage of the unique technical properties Physcomitrella offers in terms of efficient homologous recombination. We determined mRNA and protein distribution and performed targeted disruption of the genomic locus for functional analysis of PPM2. Despite weak ubiquitous expression, PPM2 protein is mostly found in male and female gametangia and basal parts of developing sporophytes. Therefore, PPM2 seems to function in both the haploid and the diploid phase of the moss life cycle. This situation reflects an evolutionary transition state of gene recruitment from an ancestral gametophytic generation into a derived sporophytic generation which became dominating in tracheophytes. However, a knock-out of the PPM2 gene did not cause visible phenotypical changes in the respective structures. The implications of our findings for the understanding of the evolutionary history of MADS-box transcription factors in plants are discussed.

ROSINA (RSI) is part of a CACTA transposable element, TamRSI, and links flower development to transposon activity.

ROSINA (RSI) was isolated as a DNA binding factor able to bind to the CArG-box present in the promoter of the MADS-box gene DEFICIENS of Antirrhinum majus. The mosaic nature of RSI and its multi-copy presence in the A. majus genome indicated that RSI could be a part of a mobile genetic element. Here we show that RSI is a part of a CACTA transposable element system of A. majus, named TamRSI, which has evolved and is still evolving within the terminal inverted repeats (TIRs) of this CACTA transposon. Interestingly, RSI is always found in opposite orientation with respect to the transcription of a second gene present within the CACTA transposon, which encodes a putative TRANSPOSASE (TNP). This structural configuration has not yet been described for any member of the CACTA transposons superfamily. Internal deletion derivatives of the TamRSI produce aberrant RSI transcripts (RSI-ATs) that carry parts of the RSI RNA fused to parts of the TNP RNA. In addition, an intriguing seed phenotype shown by RNAi transgenic lines generated to silence RSI, relate TamRSI to epigenetic mechanisms and associate the control of flower development to transposon activity.

PFMAGO, a MAGO NASHI-like factor, interacts with the MADS-domain protein MPF2 from Physalis floridana.

MADS-domain proteins serve as regulators of plant development and often form dimers and higher order complexes to function. Heterotopic expression of MPF2, a MADS-box gene, in reproductive tissues is a key component in the evolution of the inflated calyx syndrome in Physalis, but RNAi studies demonstrate that MPF2 has also acquired a role in male fertility in Physalis floridana. Using the yeast 2-hybrid system, we have now identified numerous MPF2-interacting MADS-domain proteins from Physalis, including homologs of SOC1, AP1, SEP1, SEP3, AG, and AGL6. Among the many non-MADS-domain proteins recovered was a homolog of MAGO NASHI, a highly conserved RNA-binding protein known to be involved in many developmental processes including germ cell differentiation. Two MAGO genes, termed P. floridana mago nashi1 (PFMAGO1) and PFMAGO2, were isolated from P. floridana. Both copies were found to be coexpressed in leaves, fruits, and, albeit at lower level, also in roots, stems, and flowers. DNA sequence analysis revealed that, although the coding sequences of the 2 genes are highly conserved, they differ substantially in their intron and promoter sequences. Two-hybrid screening of a Physalis expression library with both PFMAGO1 and PFMAGO2 as baits yielded numerous gene products, including an Y14-like protein. Y14 is an RNA-binding protein that forms part of various "gene expression machines." The function of MPF2 and 2 PFMAGO proteins in ensuring male fertility and evolution of calyx development in Physalis is discussed.

Hormonal control of the inflated calyx syndrome, a morphological novelty, in Physalis.

The 'Chinese lantern' phenotype or inflated calyx syndrome (ICS)--inflated sepals encapsulating the mature berry of Physalis floridana--is a morphological novelty within the Solanaceae. ICS is associated with heterotopic expression of MPF2, which codes for a MADS-box transcription factor otherwise involved in leaf formation and male fertility. In accordance with this finding, the MPF2 promoter sequence differs significantly from that of its orthologue STMADS16 in the related Solanum tuberosum, which does not exhibit ICS. However, heterotopic expression of MPF2 is not sufficient for ICS formation in P. floridana- fertilization is also important. Here we report that the hormones cytokinin and gibberellin are essential for ICS formation. MPF2 controls sepal cell division, but the resulting cells are small. Calyx size increases substantially only if gibberellin and cytokinin are available to promote cell elongation and further cell division. Transient expression of appropriate MPF2-/STMADS16-GFP fusions in leaf tissues in the presence of hormones revealed that cytokinin, but not gibberellin, facilitated transport of the transcription factor into the nucleus. Furthermore, an ICS-like structure can be induced in transgenic S. tuberosum by ectopic expression of STMADS16 and simultaneous treatment with cytokinin and gibberellin. Strikingly, transgenic Arabidopsis ectopically expressing solanaceous MPF2-like proteins display enhanced sepal growth when exposed to cytokinin only, while orthologous proteins from non-solanaceous plants did not require cytokinin for this function. These data are incorporated into a detailed model for ICS formation in P. floridana.

The miRNA156/157 recognition element in the 3' UTR of the Arabidopsis SBP box gene SPL3 prevents early flowering by translational inhibition in seedlings.

miRNAs are a class of versatile small RNAs that control gene expression post-transcriptionally, governing many facets of plant cell functions. They interact with their target mRNA at a site of sequence complementarity and modulate their expression levels. Here, we provide evidence, based on transient assays and stable transgenic lines, that the 3' UTR of the Arabidopsis SBP box gene SPL3 contains a functional miRNA-responsive element (MRE) that is complementary to miR156 and miRNA157. Seedlings of transgenic lines constitutively over-expressing an SPL3 transgene either carrying an unaltered or a disrupted MRE accumulate considerable levels of SPL3 transcripts. However, while the unaltered MRE UTR does not allow the expression of detectable levels of SPL3 protein, the altered MRE does. Translational inhibition thus provides an important mechanism for miRNA-mediated post-transcriptional repression of SPL3. As a consequence of precocious translation of the constitutively expressed SPL3 transgene, due to the absence of a functional MRE, plants exhibit very early flowering in addition to frequent morphological changes.

SPL8, a local regulator in a subset of gibberellin-mediated developmental processes in Arabidopsis.

Gibberellins (GAs) are important plant growth regulators, regulating many plant developmental processes, including seed germination, root and stem elongation, rosette expansion, floral induction and anther development. The diverse effects of GAs on plant development make it critical to maintain an appropriate endogenous GA level and a fine-tuned GA signalling. Some global regulators in GA signalling have been identified but little is known about genes specifically involved in local implementation of GA signalling. Here we report that the Arabidopsis thaliana SBP-box gene SQUAMOSA-PROMOTER-BINDING-PROTEIN-LIKE8 (SPL8) acts as a local regulator in a subset of GA-dependent developmental processes. Previous knowledge holds that SPL8 is involved in reproductive development as deduced from its loss-of-function phenotype (Unte et al. (2003) Plant Cell 15:1009-1019). We now determined that constitutive overexpression of SPL8 affects fertility due to non-dehiscent anthers, likely resulting from a constitutive GA response, suggesting a positive role of SPL8 in GA-mediated anther development. On the other hand, SPL8 gain- and loss-of-function mutants showed opposite responses to GA and its biosynthetic inhibitor paclobutrazol (PAC) with respect to seed germination and root elongation during the seedling stage. Genes involved in GA biosynthesis and signalling are transcriptionally affected by altered SPL8 expression. Our study uncovered a tissue-dependent regulatory role for SPL8 in the response to GA signalling in plant development.

MIKC* MADS-protein complexes bind motifs enriched in the proximal region of late pollen-specific Arabidopsis promoters.

The genome of Arabidopsis (Arabidopsis thaliana) encodes over 100 MADS-domain transcription factors, categorized into five phylogenetic subgroups. Most research efforts have focused on just one of these subgroups (MIKC(c)), whereas the other four remain largely unexplored. Here, we report on five members of the so-called Mdelta or Arabidopsis MIKC* (AtMIKC*) subgroup, which are predominantly expressed during the late stages of pollen development. Very few MADS-box genes function in mature pollen, and from this perspective, the AtMIKC* genes are therefore highly exceptional. We found that the AtMIKC* proteins are able to form multiple heterodimeric complexes in planta, and that these protein complexes exhibit a for the MADS-family unusual and high DNA binding specificity in vitro. Compared to their occurrence in promoters genome wide, AtMIKC* binding sites are strongly overrepresented in the proximal region of late pollen-specific promoters. By combining our experimental data with in silico genomics and pollen transcriptomics approaches, we identified a considerable number of putative direct target genes of the AtMIKC* transcription factor complexes in pollen, many of which have known or proposed functions in pollen tube growth. The expression of several of these predicted targets is altered in mutant pollen in which all AtMIKC* complexes are affected, and in vitro germination of this mutant pollen is severely impaired. Our data therefore suggest that the AtMIKC* protein complexes play an essential role in transcriptional regulation during late pollen development.

Wax-deficient anther1 is involved in cuticle and wax production in rice anther walls and is required for pollen development.

In vegetative leaf tissues, cuticles including cuticular waxes are important for protection against nonstomatal water loss and pathogen infection as well as for adaptations to environmental stress. However, their roles in the anther wall are rarely studied. The innermost layer of the anther wall (the tapetum) is essential for generating male gametes. Here, we report the characterization of a T-DNA insertional mutant in the Wax-deficient anther1 (Wda1) gene of rice (Oryza sativa), which shows significant defects in the biosynthesis of very-long-chain fatty acids in both layers. This gene is strongly expressed in the epidermal cells of anthers. Scanning electron microscopy analyses showed that epicuticular wax crystals were absent in the outer layer of the anther and that microspore development was severely retarded and finally disrupted as a result of defective pollen exine formation in the mutant anthers. These biochemical and developmental defects in tapetum found in wda1 mutants are earlier events than those in other male-sterile mutants, which showed defects of lipidic molecules in exine. Our findings provide new insights into the biochemical and developmental aspects of the role of waxes in microspore exine development in the tapetum as well as the role of epicuticular waxes in anther expansion.

Genetic and biochemical evidence for involvement of HOTHEAD in the biosynthesis of long-chain alpha-,omega-dicarboxylic fatty acids and formation of extracellular matrix.

In plants, extracellular matrix polymers built from polysaccharides and cuticular lipids have structural and protective functions. The cuticle is found to be ten times thinner in Arabidopsis thaliana (L.) Heynh than in many other plants, and there is evidence that it is unusual in having a high content of alpha-,omega-dicarboxylic fatty acids (FAs) in its polyesters. We designated the new organ fusion mutant hth-12 after it appeared to be allelic to adhesion of calyx edges (ace) and hothead (hth), upon molecular cloning of the gene by transposon tagging. This mutant is deficient in its ability to oxidize long-chain omega-hydroxy FAs to omega-oxo FAs, which results in leaf polyesters in decreased alpha-,omega-dicarboxylic FAs and increased omega-hydroxy FAs. These chemical phenotypes lead to disorder of the cuticle membrane structure in hth-12. ACE/HTH is a single-domain protein showing sequence similarity to long-chain FA omega-alcohol dehydrogenases from Candida species, and we hypothesize that it may catalyze the next step after cytochrome P450 FA omega-hydroxylases in the omega-oxidation pathway. We show that ACE/HTH is specifically expressed in epidermal cells. It appears very likely therefore that the changes in the amount of alpha-,omega-dicarboxylic FAs in hth-12 reflect the different composition of cuticular polyesters. The ACE/HTH gene is also expressed in root epidermal cells which do not form a polyester membrane on the exterior surface, thereby making it possible that the end products of the pathway, alpha-,omega-dicarboxylic FAs, are generally required for the cross-linking that ensures the integrity of the outer epidermal cell wall.

The epidermis-specific extracellular BODYGUARD controls cuticle development and morphogenesis in Arabidopsis.

The outermost epidermal cell wall is specialized to withstand pathogens and natural stresses, and lipid-based cuticular polymers are the major barrier against incursions. The Arabidopsis thaliana mutant bodyguard (bdg), which exhibits defects characteristic of the loss of cuticle structure not attributable to a lack of typical cutin monomers, unexpectedly accumulates significantly more cell wall-bound lipids and epicuticular waxes than wild-type plants. Pleiotropic effects of the bdg mutation on growth, viability, and cell differentiation are also observed. BDG encodes a member of the alpha/beta-hydrolase fold protein superfamily and is expressed exclusively in epidermal cells. Using Strep-tag epitope-tagged BDG for mutant complementation and immunolocalization, we show that BDG is a polarly localized protein that accumulates in the outermost cell wall in the epidermis. With regard to the appearance and structure of the cuticle, the phenotype conferred by bdg is reminiscent of that of transgenic Arabidopsis plants that express an extracellular fungal cutinase, suggesting that bdg may be incapable of completing the polymerization of carboxylic esters in the cuticular layer of the cell wall or the cuticle proper. We propose that BDG codes for an extracellular synthase responsible for the formation of cuticle. The alternative hypothesis proposes that BDG controls the proliferation/differentiation status of the epidermis via an unknown mechanism.