Variation among 532 genomes unveils the origin and evolutionary history of a global insect herbivore.

The diamondback moth, Plutella xylostella is a cosmopolitan pest that has evolved resistance to all classes of insecticide, and costs the world economy an estimated US $4-5 billion annually. We analyse patterns of variation among 532 P. xylostella genomes, representing a worldwide sample of 114 populations. We find evidence that suggests South America is the geographical area of origin of this species, challenging earlier hypotheses of an Old-World origin. Our analysis indicates that Plutella xylostella has experienced three major expansions across the world, mainly facilitated by European colonization and global trade. We identify genomic signatures of selection in genes related to metabolic and signaling pathways that could be evidence of environmental adaptation. This evolutionary history of P. xylostella provides insights into transoceanic movements that have enabled it to become a worldwide pest.

Author Correction: Sexual homomorphism in dioecious trees: extensive tests fail to detect sexual dimorphism in Populus.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Scale and direction of adaptive introgression between black cottonwood (Populus trichocarpa) and balsam poplar (P. balsamifera).

Introgression can introduce novel genetic variation at a faster rate than mutation alone and result in adaptive introgression when adaptive alleles are maintained in the recipient genome over time by natural selection. A previous study from our group demonstrated adaptive introgression from Populus balsamifera into P. trichocarpa in a target genomic region. Here we expand our local ancestry analysis to the whole genome of both parents to provide a comprehensive view of introgression patterns and to identify additional candidate regions for adaptive introgression genomewide. Populus trichocarpa is a large, fast-growing tree of mild coastal regions of the Pacific Northwest, whereas P. balsamifera is a smaller stature tree of continental and boreal regions with intense winter cold. The species hybridize where they are parapatric. We detected asymmetric patterns of introgression across the whole genome of these two poplar species adapted to contrasting environments, with stronger introgression from P. balsamifera to P. trichocarpa than vice versa. Admixed P. trichocarpa individuals contained more genomic regions with unusually high levels of introgression (19 regions) and also the largest introgressed genome fragment (1.02 Mb) compared with admixed P. balsamifera (nine regions). Our analysis also revealed numerous candidate regions for adaptive introgression with strong signals of selection, notably related to disease resistance, and enriched for genes that may play crucial roles in survival and adaptation. Furthermore, we detected a potential overrepresentation of subtelomeric regions in P. balsamifera introgressed into P. trichocarpa and possible protection of sex-determining regions from interspecific gene flow.

Impedance Measures During in vitro Cochlear Implantation Predict Array Positioning.

OBJECTIVE: Improper electrode placement during cochlear implant (CI) insertion can adversely affect speech perception outcomes. However, the intraoperative methods to determine positioning are limited. Because measures of electrode impedance can be made quickly, the goal of this study was to assess the relationship between CI impedance and proximity to adjacent structures. METHODS: An Advanced Bionics CI array was inserted into a clear, plastic cochlea one electrode contact at a time in a saline bath (nine trials). At each insertion depth, response to biphasic current pulses was used to calculate access resistance (Ra), polarization resistance (Rp), and polarization capacitance (Cp). These measures were correlated to actual proximity as assessed by microscopy using linear regression models. RESULTS: Impedance increased with insertion depth and proximity to the inner wall. Specifically, Ra increased, Cp decreased, and Rp slightly increased. Incorporating all impedance measures afforded a prediction model (r = 0.88) while optimizing for sub-mm positioning afforded a model with 78.3% specificity. CONCLUSION: Impedance in vitro greatly changes with electrode insertion depth and proximity to adjacent structures in a predicable manner. SIGNIFICANCE: Assessing proximity of the CI to adjacent structures is a significant first step in qualifying the electrode-neural interface. This information should aid in CI fitting, which should help maximize hearing and speech outcomes with a CI. Additionally, knowledge of the relationship between impedance and positioning could have utility in other tissue implants in the brain, retina, or spinal cord.

Introgression from Populus balsamifera underlies adaptively significant variation and range boundaries in P. trichocarpa.

Introgression can be an important source of adaptive phenotypes, although conversely it can have deleterious effects. Evidence for adaptive introgression is accumulating but information on the genetic architecture of introgressed traits lags behind. Here we determine trait architecture in Populus trichocarpa under introgression from P. balsamifera using admixture mapping and phenotypic analyses. Our results reveal that admixture is a key driver of clinal adaptation and suggest that the northern range extension of P. trichocarpa depends, at least in part, on introgression from P. balsamifera. However, admixture with P. balsamifera can lead to potentially maladaptive early phenology, and a reduction in growth and disease resistance in P. trichocarpa. Strikingly, an introgressed chromosome 9 haplotype block from P. balsamifera restores the late phenology and high growth parental phenotype in admixed P. trichocarpa. This epistatic restorer block may be strongly advantageous in maximizing carbon assimilation and disease resistance in the southernmost populations where admixture has been detected. We also confirm a previously demonstrated case of adaptive introgression in chromosome 15 and show that introgression generates a transgressive chlorophyll-content phenotype. We provide strong support that introgression provides a reservoir of genetic variation associated with adaptive characters that allows improved survival in new environments.

Overexpression of AtGolS3 and CsRFS in poplar enhances ROS tolerance and represses defense response to leaf rust disease.

Plants respond to pathogens through an orchestration of signaling events that coordinate modifications to transcriptional profiles and physiological processes. Resistance to necrotrophic pathogens often requires jasmonic acid, which antagonizes the salicylic acid dependent biotrophic defense response. Recently, myo-inositol has been shown to negatively impact salicylic acid (SA) levels and signaling, while galactinol enhances jasmonic acid (JA)-dependent induced systemic resistance to necrotrophic pathogens. To investigate the function of these compounds in biotrophic pathogen defense, we characterized the defense response of Populus alba × grandidentata overexpressing Arabidopsis GALACTINOL SYNTHASE3 (AtGolS) and Cucumber sativus RAFFINOSE SYNTHASE (CsRFS) challenged with Melampsora aecidiodes, a causative agent of poplar leaf rust disease. Relative to wild-type leaves, the overexpression of AtGolS3 and CsRFS increased accumulation of galactinol and raffinose and led to increased leaf rust infection. During the resistance response, inoculated wild-type leaves displayed reduced levels of galactinol and repressed transcript abundance of two endogenous GolS genes compared to un-inoculated wild-type leaves prior to the up-regulation of NON-EXPRESSOR OF PR1 and PATHOGENESIS-RELATED1. Transcriptome analysis and qRT-PCR validation also revealed the repression of genes participating in calcium influx, phosphatidic acid biosynthesis and signaling, and salicylic acid signaling in the transgenic lines. In contrast, enhanced tolerance to H2O2 and up-regulation of antioxidant biosynthesis genes were exhibited in the overexpression lines. Thus, we conclude that overexpression of AtGolS and CsRFS antagonizes the defense response to poplar leaf rust disease through repressing reactive oxygen species and attenuating calcium and phosphatidic acid signaling events that lead to SA defense.

Sexual homomorphism in dioecious trees: extensive tests fail to detect sexual dimorphism in Populus †.

The evolution of sexual dimorphism and expansion of sex chromosomes are both driven through sexual conflict, arising from differing fitness optima between males and females. Here, we pair work in poplar (Populus) describing one of the smallest sex-determining regions known thus far in complex eukaryotes (~100 kbp) with comprehensive tests for sexual dimorphism using >1300 individuals from two Populus species and assessing 96 non-reproductive functional traits. Against expectation, we found sexual homomorphism (no non-reproductive trait differences between the sexes), suggesting that gender is functionally neutral with respect to non-reproductive features that affect plant survival and fitness. Combined with a small sex-determining region, we infer that sexual conflict may be effectively stymied or non-existent within these taxa. Both sexual homomorphism and the small sex-determining region occur against a background of strong environmental selection and local adaptation in Populus. This presents a powerful hypothesis for the evolution of dioecious species. Here, we suggest that environmental selection may be sufficient to suppress and stymy sexual conflict if it acts orthogonal to sexual selection, thereby placing limitations on the evolution of sexual dimorphism and genomic expansion of sex chromosomes.

Sexual epigenetics: gender-specific methylation of a gene in the sex determining region of Populus balsamifera.

Methylation has frequently been implicated in gender determination in plants. The recent discovery of the sex determining region (SDR) of balsam poplar, Populus balsamifera, pinpointed 13 genes with differentiated X and Y copies. We tested these genes for differential methylation using whole methylome sequencing of xylem tissue of multiple individuals grown under field conditions in two common gardens. The only SDR gene to show a marked pattern of gender-specific methylation is PbRR9, a member of the two component response regulator (type-A) gene family, involved in cytokinin signalling. It is an ortholog of Arabidopsis genes ARR16 and ARR17. The strongest patterns of differential methylation (mostly male-biased) are found in the putative promoter and the first intron. The 4th intron is strongly methylated in both sexes and the 5th intron is unmethylated in both sexes. Using a statistical learning algorithm we find that it is possible accurately to assign trees to gender using genome-wide methylation patterns alone. The strongest predictor is the region coincident with PbRR9, showing that this gene stands out against all genes in the genome in having the strongest sex-specific methylation pattern. We propose the hypothesis that PbRR9 has a direct, epigenetically mediated, role in poplar sex determination.

Role of Glycosyltransferases in Pollen Wall Primexine Formation and Exine Patterning.

The pollen cell wall is important for protection of male sperm from physical stresses and consists of an inner gametophyte-derived intine layer and a sporophyte-derived exine layer. The polymeric constituents of the robust exine are termed sporopollenin. The mechanisms by which sporopollenin is anchored onto microspores and polymerized in specific patterns are unknown, but the primexine, a transient cell wall matrix formed on the surface of microspores at the late tetrad stage, is hypothesized to play a key role. Arabidopsis (Arabidopsis thaliana) spongy (spg) and uneven pattern of exine (upex) mutants exhibit defective and irregular exine patterns. SPG2 (synonymous with IRREGULAR XYLEM9-LIKE [IRX9L]) encodes a family GT43 glycosyltransferase involved in xylan backbone biosynthesis, while UPEX1 encodes a family GT31 glycosyltransferase likely involved in galactosylation of arabinogalactan proteins. Imaging of developing irx9l microspores showed that the earliest detectable defect was in primexine formation. Furthermore, wild-type microspores contained primexine-localized epitopes indicative of the presence of xylan, but these were absent in irx9l These data, together with the spg phenotype of a mutant in IRX14L, which also plays a role in xylan backbone elongation, indicate the presence of xylan in pollen wall primexine, which plays a role in exine patterning on the microspore surface. We observed an aberrant primexine and irregular patterns of incipient sporopollenin deposition in upex1, suggesting that primexine-localized arabinogalactan proteins could play roles in sporopollenin adhesion and patterning early in microspore wall development. Our data provide new insights into the biochemical and functional properties of the primexine component of the microspore cell wall.

Gene Expression Patterns of Wood Decay Fungi Postia placenta and Phanerochaete chrysosporium Are Influenced by Wood Substrate Composition during Degradation.

UNLABELLED: Identification of the specific genes and enzymes involved in the fungal degradation of lignocellulosic biomass derived from feedstocks with various compositions is essential to the development of improved bioenergy processes. In order to elucidate the effect of substrate composition on gene expression in wood-rotting fungi, we employed microarrays based on the annotated genomes of the brown- and white-rot fungi, Rhodonia placenta (formerly Postia placenta) and Phanerochaete chrysosporium, respectively. We monitored the expression of genes involved in the enzymatic deconstruction of the cell walls of three 4-year-old Populus trichocarpa (poplar) trees of genotypes with distinct cell wall chemistries, selected from a population of several hundred trees grown in a common garden. The woody substrates were incubated with wood decay fungi for 10, 20, and 30 days. An analysis of transcript abundance in all pairwise comparisons highlighted 64 and 84 differentially expressed genes (>2-fold, P < 0.05) in P. chrysosporium and P. placenta, respectively. Cross-fungal comparisons also revealed an array of highly differentially expressed genes (>4-fold, P < 0.01) across different substrates and time points. These results clearly demonstrate that gene expression profiles of P. chrysosporium and P. placenta are influenced by wood substrate composition and the duration of incubation. Many of the significantly expressed genes encode "proteins of unknown function," and determining their role in lignocellulose degradation presents opportunities and challenges for future research. IMPORTANCE: This study describes the variation in expression patterns of two wood-degrading fungi (brown- and white-rot fungi) during colonization and incubation on three different naturally occurring poplar substrates of differing chemical compositions, over time. The results clearly show that the two fungi respond differentially to their substrates and that several known and, more interestingly, currently unknown genes are highly misregulated in response to various substrate compositions. These findings highlight the need to characterize several unknown proteins for catalytic function but also as potential candidate proteins to improve the efficiency of enzymatic cocktails to degrade lignocellulosic substrates in industrial applications, such as in a biochemically based bioenergy platform.

Functional network analysis of genes differentially expressed during xylogenesis in soc1ful woody Arabidopsis plants.

Many plant genes are known to be involved in the development of cambium and wood, but how the expression and functional interaction of these genes determine the unique biology of wood remains largely unknown. We used the soc1ful loss of function mutant - the woodiest genotype known in the otherwise herbaceous model plant Arabidopsis - to investigate the expression and interactions of genes involved in secondary growth (wood formation). Detailed anatomical observations of the stem in combination with mRNA sequencing were used to assess transcriptome remodeling during xylogenesis in wild-type and woody soc1ful plants. To interpret the transcriptome changes, we constructed functional gene association networks of differentially expressed genes using the STRING database. This analysis revealed functionally enriched gene association hubs that are differentially expressed in herbaceous and woody tissues. In particular, we observed the differential expression of genes related to mechanical stress and jasmonate biosynthesis/signaling during wood formation in soc1ful plants that may be an effect of greater tension within woody tissues. Our results suggest that habit shifts from herbaceous to woody life forms observed in many angiosperm lineages could have evolved convergently by genetic changes that modulate the gene expression and interaction network, and thereby redeploy the conserved wood developmental program.

Genomic and functional approaches reveal a case of adaptive introgression from Populus balsamifera (balsam poplar) in P. trichocarpa (black cottonwood).

Natural hybrid zones in forest trees provide systems to study the transfer of adaptive genetic variation by introgression. Previous landscape genomic studies in Populus trichocarpa, a keystone tree species, indicated genomic footprints of admixture with its sister species Populus balsamifera and identified candidate genes for local adaptation. Here, we explored the patterns of introgression and signals of local adaptation in P. trichocarpa and P. balsamifera, employing genome resequencing data from three chromosomes in pure species and admixed individuals from wild populations. Local ancestry analysis in admixed P. trichocarpa revealed a telomeric region in chromosome 15 with P. balsamifera ancestry, containing several candidate genes for local adaptation. Genomic analyses revealed signals of selection in certain genes in this region (e.g. PRR5, COMT1), and functional analyses based on gene expression variation and correlations with adaptive phenotypes suggest distinct functions of the introgressed alleles. In contrast, a block of genes in chromosome 12 paralogous to the introgressed region showed no signs of introgression or signatures of selection. We hypothesize that the introgressed region in chromosome 15 has introduced modular or cassette-like variation into P. trichocarpa. These linked adaptive mutations are associated with a block of genes in chromosome 15 that appear to have undergone neo- or subfunctionalization relative to paralogs in a duplicated region on chromosome 12 that show no signatures of adaptive variation. The association between P. balsamifera introgressed alleles with the expression of adaptive traits in P. trichocarpa supports the hypothesis that this is a case of adaptive introgression in an ecologically important foundation species.

Evolutionary Quantitative Genomics of Populus trichocarpa.

Forest trees generally show high levels of local adaptation and efforts focusing on understanding adaptation to climate will be crucial for species survival and management. Here, we address fundamental questions regarding the molecular basis of adaptation in undomesticated forest tree populations to past climatic environments by employing an integrative quantitative genetics and landscape genomics approach. Using this comprehensive approach, we studied the molecular basis of climate adaptation in 433 Populus trichocarpa (black cottonwood) genotypes originating across western North America. Variation in 74 field-assessed traits (growth, ecophysiology, phenology, leaf stomata, wood, and disease resistance) was investigated for signatures of selection (comparing QST-FST) using clustering of individuals by climate of origin (temperature and precipitation). 29,354 SNPs were investigated employing three different outlier detection methods and marker-inferred relatedness was estimated to obtain the narrow-sense estimate of population differentiation in wild populations. In addition, we compared our results with previously assessed selection of candidate SNPs using the 25 topographical units (drainages) across the P. trichocarpa sampling range as population groupings. Narrow-sense QST for 53% of distinct field traits was significantly divergent from expectations of neutrality (indicating adaptive trait variation); 2,855 SNPs showed signals of diversifying selection and of these, 118 SNPs (within 81 genes) were associated with adaptive traits (based on significant QST). Many SNPs were putatively pleiotropic for functionally uncorrelated adaptive traits, such as autumn phenology, height, and disease resistance. Evolutionary quantitative genomics in P. trichocarpa provides an enhanced understanding regarding the molecular basis of climate-driven selection in forest trees and we highlight that important loci underlying adaptive trait variation also show relationship to climate of origin. We consider our approach the most comprehensive, as it uncovers the molecular mechanisms of adaptation using multiple methods and tests. We also provide a detailed outline of the required analyses for studying adaptation to the environment in a population genomics context to better understand the species' potential adaptive capacity to future climatic scenarios.

A role for OVATE FAMILY PROTEIN1 (OFP1) and OFP4 in a BLH6-KNAT7 multi-protein complex regulating secondary cell wall formation in Arabidopsis thaliana.

Formation of secondary walls is a complex process that requires the coordinated and developmentally regulated expression of secondary wall biosynthetic genes. In Arabidopsis thaliana, a transcriptional network orchestrates the biosynthesis and deposition of the main SCW components in xylem and fiber cells. It was recently reported that interacting TALE homeodomain proteins BEL-LIKE HOMEODOMAIN6 (BLH6) and KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) negatively regulate secondary cell wall formation in the interfascicular fibers of Arabidopsis inflorescence stems. Members of the Arabidopsis OVATE FAMILY PROTEIN (OFP) family of transcriptional regulators have been shown to physically interact in yeast with various KNAT and BLH proteins, forming a proposed TALE-OFP protein interaction network. This study presents molecular and genetic data indicating that OFP1 and OFP4, previously reported to interact with TALE homeodomain proteins, enhance the repression activity of BLH6, supporting a role for these OFPs as components of a putative multi-protein transcription regulatory complex containing BLH6 and KNAT7.

Comparative analysis of plant carbohydrate active enZymes and their role in xylogenesis.

BACKGROUND: Carbohydrate metabolism is a key feature of vascular plant architecture, and is of particular importance in large woody species, where lignocellulosic biomass is responsible for bearing the bulk of the stem and crown. Since Carbohydrate Active enZymes (CAZymes) in plants are responsible for the synthesis, modification and degradation of carbohydrate biopolymers, the differences in gene copy number and regulation between woody and herbaceous species have been highlighted previously. There are still many unanswered questions about the role of CAZymes in land plant evolution and the formation of wood, a strong carbohydrate sink. RESULTS: Here, twenty-two publically available plant genomes were used to characterize the frequency, diversity and complexity of CAZymes in plants. We find that a conserved suite of CAZymes is a feature of land plant evolution, with similar diversity and complexity regardless of growth habit and form. In addition, we compared the diversity and levels of CAZyme gene expression during wood formation in trees using mRNA-seq data from two distantly related angiosperm tree species Eucalyptus grandis and Populus trichocarpa, highlighting the major CAZyme classes involved in xylogenesis and lignocellulosic biomass production. CONCLUSIONS: CAZyme domain ratio across embryophytes is maintained, and the diversity of CAZyme domains is similar in all land plants, regardless of woody habit. The stoichiometric conservation of gene expression in woody and non-woody tissues of Eucalyptus and Populus are indicative of gene balance preservation.

Comparative interrogation of the developing xylem transcriptomes of two wood-forming species: Populus trichocarpa and Eucalyptus grandis.

Wood formation is a complex developmental process governed by genetic and environmental stimuli. Populus and Eucalyptus are fast-growing, high-yielding tree genera that represent ecologically and economically important species suitable for generating significant lignocellulosic biomass. Comparative analysis of the developing xylem and leaf transcriptomes of Populus trichocarpa and Eucalyptus grandis together with phylogenetic analyses identified clusters of homologous genes preferentially expressed during xylem formation in both species. A conserved set of 336 single gene pairs showed highly similar xylem preferential expression patterns, as well as evidence of high functional constraint. Individual members of multi-gene orthologous clusters known to be involved in secondary cell wall biosynthesis also showed conserved xylem expression profiles. However, species-specific expression as well as opposite (xylem versus leaf) expression patterns observed for a subset of genes suggest subtle differences in the transcriptional regulation important for xylem development in each species. Using sequence similarity and gene expression status, we identified functional homologs likely to be involved in xylem developmental and biosynthetic processes in Populus and Eucalyptus. Our study suggests that, while genes involved in secondary cell wall biosynthesis show high levels of gene expression conservation, differential regulation of some xylem development genes may give rise to unique xylem properties.

High-resolution genetic mapping of allelic variants associated with cell wall chemistry in Populus.

BACKGROUND: QTL cloning for the discovery of genes underlying polygenic traits has historically been cumbersome in long-lived perennial plants like Populus. Linkage disequilibrium-based association mapping has been proposed as a cloning tool, and recent advances in high-throughput genotyping and whole-genome resequencing enable marker saturation to levels sufficient for association mapping with no a priori candidate gene selection. Here, multiyear and multienvironment evaluation of cell wall phenotypes was conducted in an interspecific P. trichocarpa x P. deltoides pseudo-backcross mapping pedigree and two partially overlapping populations of unrelated P. trichocarpa genotypes using pyrolysis molecular beam mass spectrometry, saccharification, and/ or traditional wet chemistry. QTL mapping was conducted using a high-density genetic map with 3,568 SNP markers. As a fine-mapping approach, chromosome-wide association mapping targeting a QTL hot-spot on linkage group XIV was performed in the two P. trichocarpa populations. Both populations were genotyped using the 34 K Populus Infinium SNP array and whole-genome resequencing of one of the populations facilitated marker-saturation of candidate intervals for gene identification. RESULTS: Five QTLs ranging in size from 0.6 to 1.8 Mb were mapped on linkage group XIV for lignin content, syringyl to guaiacyl (S/G) ratio, 5- and 6-carbon sugars using the mapping pedigree. Six candidate loci exhibiting significant associations with phenotypes were identified within QTL intervals. These associations were reproducible across multiple environments, two independent genotyping platforms, and different plant growth stages. cDNA sequencing for allelic variants of three of the six loci identified polymorphisms leading to variable length poly glutamine (PolyQ) stretch in a transcription factor annotated as an ANGUSTIFOLIA C-terminus Binding Protein (CtBP) and premature stop codons in a KANADI transcription factor as well as a protein kinase. Results from protoplast transient expression assays suggested that each of the polymorphisms conferred allelic differences in the activation of cellulose, hemicelluloses, and lignin pathway marker genes. CONCLUSION: This study illustrates the utility of complementary QTL and association mapping as tools for gene discovery with no a priori candidate gene selection. This proof of concept in a perennial organism opens up opportunities for discovery of novel genetic determinants of economically important but complex traits in plants.

Genetic differentiation of the regional Plutella xylostella populations across the Taiwan Strait based on identification of microsatellite markers.

Movement of individuals through events, such as storms or crop transportation, may affect survival and distribution of insect pests, as well as population genetic structure at a regional scale. Understanding what factors contribute to gene flow in pest populations remains very important for sustainable pest management. The diamondback moth (Plutella xylostella) is an insect pest well known for its capacity of moving over short to long distances. Here, we used newly isolated microsatellite markers to analyze the genetic structure of nine populations across the Taiwan Strait of China (Taiwan and Fujian). A total of 12,152 simple sequence repeats (SSRs) were initially identified from the P. xylostella transcriptome (~94 Mb), with an average of 129 SSRs per Mb. Nine SSRs were validated to be polymorphic markers, and eight were used for this population genetic study. Our results showed that the P. xylostella populations could be divided into distinct two clusters, which is likely due to the year-round airflows in this region. A pattern of isolation by distance among the local populations within Fujian was found, and may be related to vegetable transportation. Considering the complexity of the P. xylostella population genetic structure from local and regional to global levels, we propose that developing ecologically sound strategies for managing this pest will require knowledge of the link between behavioral and population ecology and its genetic structure.

The biosynthesis, composition and assembly of the outer pollen wall: A tough case to crack.

The formation of the durable outer pollen wall, largely composed of sporopollenin, is essential for the protection of the male gametophyte and plant reproduction. Despite its apparent strict conservation amongst land plants, the composition of sporopollenin and the biosynthetic pathway(s) yielding this recalcitrant biopolymer remain elusive. Recent molecular genetic studies in Arabidopsis thaliana (Arabidopsis) and rice have, however, identified key genes involved in sporopollenin formation, allowing a better understanding of the biochemistry and cell biology underlying sporopollenin biosynthesis and pollen wall development. Herein, current knowledge of the biochemical composition of the outer pollen wall is reviewed, with an emphasis on enzymes with characterized biochemical activities in sporopollenin and pollen coat biosynthesis. The tapetum, which forms the innermost sporophytic cell layer of the anther and envelops developing pollen, plays an essential role in sporopollenin and pollen coat formation. Recent studies show that several tapetum-expressed genes encode enzymes that metabolize fatty acid derived compounds to form putative sporopollenin precursors, including tetraketides derived from fatty acyl-CoA starter molecules, but analysis of mutants defective in pollen wall development indicate that other components are also incorporated into sporopollenin. Also highlighted are the many uncertainties remaining in the development of a sporopollenin-fortified pollen wall, particularly in relation to the mechanisms of sporopollenin precursor transport and assembly into the patterned form of the pollen wall. A working model for sporopollenin biosynthesis is proposed based on the data obtained largely from studies of Arabidopsis, and future challenges to complete our understanding of pollen wall biology are outlined.

BEL1-LIKE HOMEODOMAIN6 and KNOTTED ARABIDOPSIS THALIANA7 interact and regulate secondary cell wall formation via repression of REVOLUTA.

The TALE homeodomain transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) is part of a regulatory network governing the commitment to secondary cell wall biosynthesis of Arabidopsis thaliana, where it contributes to negative regulation of this process. Here, we report that BLH6, a BELL1-LIKE HOMEODOMAIN protein, specifically interacts with KNAT7, and this interaction influences secondary cell wall development. BLH6 is a transcriptional repressor, and BLH6-KNAT7 physical interaction enhances KNAT7 and BLH6 repression activities. The overlapping expression patterns of BLH6 and KNAT7 and phenotypes of blh6, knat7, and blh6 knat7 loss-of-function mutants are consistent with the existence of a BLH6-KNAT7 heterodimer that represses commitment to secondary cell wall biosynthesis in interfascicular fibers. BLH6 and KNAT7 overexpression results in thinner interfascicular fiber secondary cell walls, phenotypes that are dependent on the interacting partner. A major impact of the loss of BLH6 and KNAT7 function is enhanced expression of the homeodomain-leucine zipper transcription factor REVOLUTA/INTERFASCICULAR FIBERLESS1 (REV/IFL1). BLH6 and KNAT7 bind to the REV promoter and repress REV expression, while blh6 and knat7 interfascicular fiber secondary cell wall phenotypes are suppressed in blh6 rev and knat7 rev double mutants, suggesting that BLH6/KNAT7 signaling acts through REV as a direct target.