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ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) AND ATXR6 are required for the deposition of H3K27me1 and for maintaining genomic stability in Arabidopsis Reduction of ATXR5/6 activity results in activation of DNA damage response genes, along with tissue-specific derepression of transposable elements (TEs), chromocenter decompaction, and genomic instability characterized by accumulation of excess DNA from heterochromatin. How loss of ATXR5/6 and H3K27me1 leads to these phenotypes remains unclear. Here we provide extensive characterization of the atxr5/6 hypomorphic mutant by comprehensively examining gene expression and epigenetic changes in the mutant. We found that the tissue-specific phenotypes of TE derepression and excessive DNA in this atxr5/6 mutant correlated with residual ATXR6 expression from the hypomorphic ATXR6 allele. However, up-regulation of DNA damage genes occurred regardless of ATXR6 levels and thus appears to be a separable process. We also isolated an atxr6-null allele which showed that ATXR5 and ATXR6 are required for female germline development. Finally, we characterize three previously reported suppressors of the hypomorphic atxr5/6 mutant and show that these rescue atxr5/6 via distinct mechanisms, two of which involve increasing H3K27me1 levels.

Significance The plant-specific H3K27me1 methyltransferases ATXR5 and ATXR6 play integral roles connecting epigenetic silencing with genomic stability. However, how H3K27me1 relates to these processes is poorly understood. In this study, we performed a comprehensive transcriptome analysis of tissue- and ploidy-specific expression in a hypomorphic atxr5/6 mutant and revealed that the tissue-specific defects correlate with residual ATXR6 expression. We also determined that ATXR5/6 function is essential for female germline development. Furthermore, we provide a comprehensive analysis of H3K27me1 changes in relation to other epigenetic marks. We also determined that some previously reported suppressors of atxr5/6 may act by restoring the levels of H3K27me1, such as through up-regulation of the ATXR6 transcript in the atxr6 hypomorphic promoter allele., ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) AND ATXR6 are required for the deposition of H3K27me1 and for maintaining genomic stability in Arabidopsis. Reduction of ATXR5/6 activity results in activation of DNA damage response genes, along with tissue-specific derepression of transposable elements (TEs), chromocenter decompaction, and genomic instability characterized by accumulation of excess DNA from heterochromatin. How loss of ATXR5/6 and H3K27me1 leads to these phenotypes remains unclear. Here we provide extensive characterization of the atxr5/6 hypomorphic mutant by comprehensively examining gene expression and epigenetic changes in the mutant. We found that the tissue-specific phenotypes of TE derepression and excessive DNA in this atxr5/6 mutant correlated with residual ATXR6 expression from the hypomorphic ATXR6 allele. However, up-regulation of DNA damage genes occurred regardless of ATXR6 levels and thus appears to be a separable process. We also isolated an atxr6-null allele which showed that ATXR5 and ATXR6 are required for female germline development. Finally, we characterize three previously reported suppressors of the hypomorphic atxr5/6 mutant and show that these rescue atxr5/6 via distinct mechanisms, two of which involve increasing H3K27me1 levels.

Organisms orchestrate cellular functions through transcription factor (TF) interactions with their target genes, although these regulatory relationships are largely unknown in most species. Here we report a high-throughput approach for characterizing TF-target gene interactions across species and its application to 354 TFs across 48 bacteria, generating 17,000 genome-wide binding maps. This dataset revealed themes of ancient conservation and rapid evolution of regulatory modules. We observed rewiring, where the TF sensing and regulatory role is maintained while the arrangement and identity of target genes diverges, in some cases encoding entirely new functions. We further integrated phenotypic information to define new functional regulatory modules and pathways. Finally, we identified 242 new TF DNA binding motifs, including a 70% increase of known Escherichia coli motifs and the first annotation in Pseudomonas simiae, revealing deep conservation in bacterial promoter architecture. Our method provides a versatile tool for functional characterization of genetic pathways in prokaryotes and eukaryotes.

The epigenome orchestrates genome accessibility, functionality, and three-dimensional structure. Because epigenetic variation can impact transcription and thus phenotypes, it may contribute to adaptation. Here, we report 1,107 high-quality single-base resolution methylomes and 1,203 transcriptomes from the 1001 Genomes collection of Arabidopsis thaliana. Although the genetic basis of methylation variation is highly complex, geographic origin is a major predictor of genome-wide DNA methylation levels and of altered gene expression caused by epialleles. Comparison to cistrome and epicistrome datasets identifies associations between transcription factor binding sites, methylation, nucleotide variation, and co-expression modules. Physical maps for nine of the most diverse genomes reveal how transposons and other structural variants shape the epigenome, with dramatic effects on immunity genes. The 1001 Epigenomes Project provides a comprehensive resource for understanding how variation in DNA methylation contributes to molecular and non-molecular phenotypes in natural populations of the most studied model plant.

Background Transcription factors (TFs) play a key role in regulating plant development and response to environmental stimuli. While most genes revert to single copy after whole genome duplication (WGD) event, transcription factors are retained at a significantly higher rate. Little is known about how TF duplicates have diverged in their expression and regulation, the answer to which may contribute to a better understanding of the elevated retention rate among TFs. Results Here we assessed what features may explain differences in the retention of TF duplicates and other genes using Arabidopsis thaliana as a model. We integrated 34 expression, sequence, and conservation features to build a linear model for predicting the extent of duplicate retention following WGD events among TFs and 19 groups of genes with other functions. We found that TFs was the least well predicted, demonstrating the features of TFs are substantially deviated from duplicate genes in other function groups. Consistent with this, the evolution of TF expression patterns and cis-regulatory cites favors the partitioning of ancestral states among the resulting duplicates: one “ancestral” TF duplicate retains most ancestral expression and cis-regulatory sites, while the “non-ancestral” duplicate is enriched for novel regulatory sites. By modeling the retention of ancestral expression and cis-regulatory states in duplicate pairs using a system of differential equations, we found that TF duplicate pairs in a partitioned state are preferentially maintained. Conclusions These TF duplicates with asymmetrically partitioned ancestral states are likely maintained because one copy retains ancestral functions while the other, at least in some cases, acquires novel cis-regulatory sites that may be important for novel, adaptive traits. Electronic supplementary material The online version of this article (10.1186/s12862-019-1398-z) contains supplementary material, which is available to authorized users.

While genome assembly projects have been successful in a number of haploid or inbred species, one of the current main challenges is assembling non-inbred or rearranged heterozygous genomes. To address this critical need, we introduce the open-source FALCON and FALCON-Unzip algorithms (https://github.com/PacificBiosciences/FALCON/) to assemble Single Molecule Real-Time (SMRT®) Sequencing data into highly accurate, contiguous, and correctly phased diploid genomes. We demonstrate the quality of this approach by assembling new reference sequences for three heterozygous samples, including an F1 hybrid of the model species Arabidopsis thaliana, the widely cultivated V. vinifera cv. Cabernet Sauvignon, and the coral fungus Clavicorona pyxidata that have challenged short-read assembly approaches. The FALCON-based assemblies were substantially more contiguous and complete than alternate short or long-read approaches. The phased diploid assembly enabled the study of haplotype structures and heterozygosities between the homologous chromosomes, including identifying widespread heterozygous structural variations within the coding sequences.

Broad-scale protein-protein interaction mapping is a major challenge given the cost, time, and sensitivity constraints of existing technologies. Here, we present a massively multiplexed yeast two-hybrid method, CrY2H-seq, which uses a Cre recombinase interaction reporter to intracellularly fuse the coding sequences of two interacting proteins and next-generation DNA sequencing to identify these interactions en masse. We applied CrY2H-seq to investigate sparsely annotated Arabidopsis thaliana transcription factors interactions. By performing ten independent screens testing a total of 36 million binary interaction combinations, and uncovering a network of 8,577 interactions among 1,453 transcription factors, we demonstrate CrY2H-seq's improved screening capacity, efficiency, and sensitivity over those of existing technologies. The deep-coverage network resource we call AtTFIN-1 recapitulates one-third of previously reported interactions derived from diverse methods, expands the number of known plant transcription factor interactions by three-fold, and reveals previously unknown family-specific interaction module associations with plant reproductive development, root architecture, and circadian coordination.

SUMMARY Single-cell transcriptome profiling of heterogeneous tissues can provide high-resolution windows into developmental dynamics and environmental responses, but its application to plants has been limited. Here, we used the high-throughput Drop-seq approach to profile >12,000 cells from Arabidopsis roots. This identified numerous distinct cell types, covering all major root tissues and developmental stages, and illuminated specific marker genes for these populations. In addition, we demonstrate the utility of this approach to study the impact of environmental conditions on developmental processes. Analysis of roots grown with or without sucrose supplementation uncovers changes in the relative frequencies of cell types in response to sucrose. Finally, we characterize the transcriptome changes that occur across endodermis development and identify nearly 800 genes with dynamic expression as this tissue differentiates. Collectively, we demonstrate that single-cell RNA-seq can be used to profile developmental processes in plants and show how they can be altered by external stimuli., Graphical Abstract, In Brief The application of single-cell transcriptome profiling to plants has been limited. Shulse et al. performed Drop-seq on Arabidopsis roots, generating a transcriptional resource for >12,000 cells across major populations. This revealed marker genes for distinct cell types, cell frequency changes resulting from sucrose addition, and genes dynamically regulated during development.

The plant hormone auxin is perceived by a family of F-box proteins called the TIR1/AFBs. Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6. Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling by promoting the degradation of the Aux/IAA transcriptional repressors. In this report, we demonstrate that both AFB4 and AFB5 also function as auxin receptors based on in vitro assays. We also provide genetic evidence that AFB4 and AFB5 are targets of the picloram family of auxinic herbicides in addition to indole-3-acetic acid. In contrast to previous studies we find that null afb4 alleles do not exhibit obvious defects in seedling morphology or auxin hypersensitivity. We conclude that AFB4 and AFB5 act in a similar fashion to other members of the family but exhibit a distinct auxin specificity.

Genotype is the primary determinate of phenotype. During the past two decades, however, there has been an emergent recognition of the epigenotype, a separate layer of heredity distinct from the primary DNA sequence that can have profound effects on phenotype. The epigenotype is a collection of chemical modifications to the DNA and nucleosomes in conjunction with noncoding RNA transcripts, and together these epigenetic marks act as a potent and expansive regulatory system for controlling gene expression. In this review, we discuss our current understanding of variation in epigenotype in the model plant Arabidopsis and how allelic differences attributable to epigenetic changes, or epialleles, can affect phenotype. We discuss examples of epialleles that have been created in the laboratory and others that have been identified in natural populations, because these two models provide complementary information regarding the genetic pathways, mechanisms of transmission, and biological and evolutionary context for the role of the epigenotype in phenotypic variation.

The plant hormone auxin is perceived by a family of F-box proteins called the TIR1/AFBs. Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6 (Parry et al. 2009). Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling by promoting the degradation of the Aux/IAA transcriptional repressors (Dharmasiri et al. 2005; Parry et al. 2009). In this report we demonstrate that both AFB4 and AFB5 also function as auxin receptors based on in vitro assays. We also provide genetic evidence that both AFB4 and AFB5 are targets of the picloram family of auxinic herbicides. In contrast to previous studies we find that null afb4 alleles do not exhibit obvious defects in seedling morphology or auxin hypersensitivity. We conclude that AFB4 and AFB5 act in a similar fashion to other members of the family but exhibit a distinct auxin specificity.

SummaryDeciphering the multiple layers of epigenetic regulation that control transcription is critical to understanding how plants develop and respond to their environment. Using sequencing-by-synthesis technology we directly sequenced the cytosine methylome (methylC-seq), transcriptome (mRNA-seq), and small RNA transcriptome (smRNA-seq) to generate highly integrated epigenome maps for wild-type Arabidopsis thaliana and mutants defective in DNA methyltransferase or demethylase activity. At single-base resolution we discovered extensive, previously undetected DNA methylation, identified the context and level of methylation at each site, and observed local sequence effects upon methylation state. Deep sequencing of smRNAs revealed a direct relationship between the location of smRNAs and DNA methylation, perturbation of smRNA biogenesis upon loss of CpG DNA methylation, and a tendency for smRNAs to direct strand-specific DNA methylation in regions of RNA-DNA homology. Finally, strand-specific mRNA-seq revealed altered transcript abundance of hundreds of genes, transposons, and unannotated intergenic transcripts upon modification of the DNA methylation state.

The T-DNA sequence-indexed mutant collections contain insertional mutants for most Arabidopsis thaliana genes and have played an important role in plant biology research for almost two decades. By providing a large source of mutant alleles for in vivo characterization of gene function, this resource has been leveraged thousands of times to study a wide range of problems in plant biology. Our primary goal in this chapter is to provide a general guide to strategies for the effective use of the data and materials in these collections. To do this, we provide a general introduction to the T-DNA insertional sequence-indexed mutant collections with a focus on how best to use the available data sources for good line selection. As isolation of a homozygous line is a common next step once a potential disruption line has been identified, the second half of the chapter provides a step-by-step guide for the design and implementation of a T-DNA genotyping pipeline. Finally, we describe interpretation of genotyping results and include a troubleshooting section for common types of segregation distortions that we have observed. In this chapter we introduce both basic concepts and specific applications to both new and more experienced users of the collections for the design and implementation of small- to large-scale genotyping pipelines.

Ethylene influences a number of processes in Arabidopsis (Arabidopsis thaliana) through the action of five receptors. In this study, we used high-resolution, time-lapse imaging to examine the long-term effects of ethylene on growing, etiolated Arabidopsis seedlings. These measurements revealed that ethylene stimulates nutations of the hypocotyls with an average delay in onset of over 6 h. The nutation response was constitutive in ctr1-2 mutants maintained in air, whereas ein2-1 mutants failed to nutate when treated with ethylene. Ethylene-stimulated nutations were also eliminated in etr1-7 loss-of-function mutants. Transformation of the etr1-7 mutant with a wild-type genomic ETR1 transgene rescued the nutation phenotype, further supporting a requirement for ETR1. Loss-of-function mutations in the other receptor isoforms had no effect on ethylene-stimulated nutations. However, the double ers1-2 ers2-3 and triple etr2-3 ers2-3 ein4-4 loss-of-function mutants constitutively nutated in air. These results support a model where all the receptors are involved in ethylene-stimulated nutations, but the ETR1 receptor is required and has a contrasting role from the other receptor isoforms in this nutation phenotype. Naphthylphthalamic acid eliminated ethylene-stimulated nutations but had no effect on growth inhibition caused by ethylene, pointing to a role for auxin transport in the nutation phenotype.

Ethylene signaling in plants is mediated by a family of ethylene receptors related to bacterial two-component regulators. Expression in yeast of ethylene-binding domains from the five receptor isoforms from Arabidopsis thaliana and five-receptor isoforms from tomato confirmed that all members of the family are capable of high-affinity ethylene-binding activity. All receptor isoforms displayed a similar level of ethylene binding on a per unit protein basis, while members of both subfamily I and subfamily II from Arabidopsis showed similar slow-release kinetics for ethylene. Quantification of receptor-isoform mRNA levels in receptor-deficient Arabidopsis lines indicated a direct correlation between total message level and total ethylene-binding activity in planta. Increased expression of remaining receptor isoforms in receptor-deficient lines tended to compensate for missing receptors at the level of mRNA expression and ethylene-binding activity, but not at the level of receptor signaling, consistent with specialized roles for family members in receptor signal output.

Responses to the plant hormone ethylene are mediated by a family of five receptors in Arabidopsis that act in the absence of ethylene as negative regulators of response pathways. In this study, we examined the rapid kinetics of growth inhibition by ethylene and growth recovery after ethylene withdrawal in hypocotyls of etiolated seedlings of wild-type and ethylene receptor-deficient Arabidopsis lines. This analysis revealed that there are two phases to growth inhibition by ethylene in wild type: a rapid phase followed by a prolonged, slower phase. Full recovery of growth occurs approximately 90 min after ethylene removal. None of the receptor null mutations tested had a measurable effect on the two phases of growth inhibition. However, loss-of-function mutations in ETR1, ETR2, and EIN4 significantly prolonged the time for recovery of growth rate after ethylene was removed. Plants with an etr1-6;etr2-3;ein4-4 triple loss-of-function mutation took longer to recover than any of the single mutants, while the ers1;ers2 double mutant had no effect on recovery rate, suggesting that receiver domains play a role in recovery. Transformation of the ers1-2;etr1-7 double mutant with wild-type genomic ETR1 rescued the slow recovery phenotype, while a His kinase-inactivated ETR1 construct did not. To account for the rapid recovery from growth inhibition, a model in which clustered receptors act cooperatively is proposed.

Ethylene signaling in plants is mediated by a family of receptors related to bacterial two-component histidine kinases. Of the five members of the Arabidopsis ethylene receptor family, members of subfamily I (ETR1 and ERS1) contain completely conserved histidine kinase domains, whereas members of subfamily II (ETR2, EIN4, and ERS2) lack conserved residues thought to be necessary for kinase activity. To examine the role of the conserved histidine kinase domain in receptor signaling, ers1;etr1 loss-of-function double mutants were generated. The double mutants exhibited a severe constitutive ethylene response phenotype consistent with the negative regulator model for receptor function. The adult ers1-2;etr1-6 and ers1-2;etr1-7 phenotypes included miniature rosette size, delayed flowering, and both male and female sterility, whereas etiolated-seedling responses were less affected. Chimeric transgene constructs in which the ETR1 promoter was used to drive expression of cDNAs for each of the five receptor isoforms were transferred into the ers1-2;etr1-7 double-mutant plants. Subfamily I constructs restored normal growth, whereas subfamily II constructs failed to rescue the double mutant, providing evidence for a unique role for subfamily I in receptor signaling. However, transformation of either the ers1-2;etr1-6 or ers1-2;etr1-7 mutant with a kinase-inactivated ETR1 genomic clone also resulted in complete restoration of normal growth and ethylene responsiveness in the double-mutant background, leading to the conclusion that canonical histidine kinase activity by receptors is not required for ethylene receptor signaling.

The cistrome is the complete set of transcription factor (TF) binding sites (cis-elements) in an organism, while an epicistrome incorporates tissue-specific DNA chemical modifications and TF-specific chemical sensitivities into these binding profiles. Robust methods to construct comprehensive cistrome and epicistrome maps are critical for elucidating complex transcriptional networks that underlie growth, behavior, and disease. Here, we describe DNA affinity purification sequencing (DAP-seq), a high-throughput TF binding site discovery method that interrogates genomic DNA with in-vitro-expressed TFs. Using DAP-seq, we defined the Arabidopsis cistrome by resolving motifs and peaks for 529 TFs. Because genomic DNA used in DAP-seq retains 5-methylcytosines, we determined that >75% (248/327) of Arabidopsis TFs surveyed were methylation sensitive, a property that strongly impacts the epicistrome landscape. DAP-seq datasets also yielded insight into the biology and binding site architecture of numerous TFs, demonstrating the value of DAP-seq for cost-effective cistromic and epicistromic annotation in any organism.

Epigenetic information, which may affect an organism’s phenotype, can be stored and stably inherited in the form of cytosine DNA methylation. Changes in DNA methylation can produce meiotically stable epialleles that affect transcription and morphology, but the rates of spontaneous gain or loss of DNA methylation are unknown. We examined spontaneously occurring variation in DNA methylation in Arabidopsis thaliana plants propagated by single-seed descent for 30 generations. We identified 114,287 CG single methylation polymorphisms and 2485 CG differentially methylated regions (DMRs), both of which show patterns of divergence compared with the ancestral state. Thus, transgenerational epigenetic variation in DNA methylation may generate new allelic states that alter transcription, providing a mechanism for phenotypic diversity in the absence of genetic mutation.

Summary The large collections of Arabidopsis thaliana sequence-indexed T-DNA insertion mutants are among the most important resources to emerge from the sequencing of the genome. Several laboratories around the world have used the Arabidopsis reference genome sequence to map T-DNA flanking sequence tags (FST) for over 325 000 T-DNA insertion lines. Over the past decade, phenotypes identified with T-DNA-induced mutants have played a critical role in advancing both basic and applied plant research. These widely used mutants are an invaluable tool for direct interrogation of gene function. However, most lines are hemizygous for the insertion, necessitating a genotyping step to identify homozygous plants for the quantification of phenotypes. This situation has limited the application of these collections for genome-wide screens. Isolating multiple homozygous insert lines for every gene in the genome would make it possible to systematically test the phenotypic consequence of gene loss under a wide variety of conditions. One major obstacle to achieving this goal is that 12% of genes have no insertion and 8% are only represented by a single allele. Generation of additional mutations to achieve full genome coverage has been slow and expensive since each insertion is sequenced one at a time. Recent advances in high-throughput sequencing technology open up a potentially faster and cost-effective means to create new, very large insertion mutant populations for plants or animals. With the combination of new tools for genome-wide studies and emerging phenotyping platforms, these sequence-indexed mutant collections are poised to have a larger impact on our understanding of gene function.

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are abundant endogenous small RNAs (smRNAs) that control transcript expression through posttranscriptional gene silencing. Here, we show that concomitant loss of XRN4/EIN5, a 5'-3' exoribonuclease, and ABH1/CBP80, a subunit of the mRNA cap binding complex, results in Arabidopsis plants manifesting myriad developmental defects. We find that ABH1/CBP80 is necessary to obtain proper mature miRNA levels, which suggests this protein affects the miRNA-mediated RNA silencing pathway. Additionally, we show that XRN4/EIN5 affects the levels of a smRNA class that is processed from both sense and antisense strands of approximately 130 endogenous transcripts that apparently are converted to double-stranded RNA (dsRNA) and subsequently processed. We find that the parent transcripts of these smRNAs accumulate in an uncapped form upon loss of XRN4/EIN5, which suggests that uncapped endogenous transcripts can become smRNA biogenesis substrates. Overall, our results reveal unexpected connections between RNA metabolism and silencing pathways.

Agrobacterium transfer DNA (T-DNA) is an effective plant mutagen that has been used to create sequence-indexed T-DNA insertion lines in Arabidopsis thaliana as a tool to study gene function. Creating T-DNA insertion lines requires a dependable method for locating the site of insertion in the genome. In this protocol, we describe an adapter ligation-mediated PCR method that we have used to screen a mutant library and identify over 150,000 T-DNA insertional mutants; the method can also be applied to map individual mutants. The procedure consists of three steps: a restriction enzyme-mediated ligation of an adapter to the genomic DNA; a PCR amplification of the T-DNA/genomic DNA junction with primers specific to the adapter and T-DNA; and sequencing of the T-DNA/genomic junction to enable mapping to the reference genome. In most cases, the sequenced genomic region extends to the T-DNA border, enabling the exact location of the insert to be identified. The entire process takes 2 weeks to complete.

Ethylene signaling in plants is mediated by a family of ethylene receptors related to bacterial two-component regulators. Expression in yeast of ethylene-binding domains from the five receptor isoforms from Arabidopsis thaliana and five-receptor isoforms from tomato confirmed that all members of the family are capable of high-affinity ethylene-binding activity. All receptor isoforms displayed a similar level of ethylene binding on a per unit protein basis, while members of both subfamily I and subfamily II from Arabidopsis showed similar slow-release kinetics for ethylene. Quantification of receptor-isoform mRNA levels in receptor-deficient Arabidopsis lines indicated a direct correlation between total message level and total ethylene-binding activity in planta. Increased expression of remaining receptor isoforms in receptor-deficient lines tended to compensate for missing receptors at the level of mRNA expression and ethylene-binding activity, but not at the level of receptor signaling, consistent with specialized roles for family members in receptor signal output.

bidopsis genome reveal more than 1000 genes encoding cell-wall-related proteins encompassing precursor-gen erating enzymes, synthases and glycosyl transferases, structural proteins, and a host of enzymes involved in polysaccharide modification and depolymerization (Carpita et al. 2001). An equal number of unannotated genes are suspected of encoding proteins that function in wall assembly or disassembly. Proteomics approaches that select for polypeptides secreted to the exterior of the plasma membrane reveal sequences of many proteins whose function is completely unknown (Borderies et al. 2003). Complete genome sequences of model fungi, ani mals and plants provide a powerful means to identify proteins that are unique to plants (Gutierrez et al. 2004). Other strategies to identify unannoted genes as wall-re lated include database profiling of the expression of genes closely associated with genes of known function (Perrin et al. 2001) and global expression of genes asso ciated with secondary wall induction during trans-dif ferentiation of Zinnia mesophyll cells into tracheary elements in vitro (Milioni et al. 2002). Two types of walls are found among angiosperms. For extensive review of the differences between grass species and all other angiosperms with respect to poly mer structure, architecture, and biosynthesis, see McC ann and Roberts (1991) and Carpita and Gibeaut (1993). Briefly, "Type I" walls, which are found in dicots and the non-commelinoid monocots, contain about equal amounts of cellulose and cross-linking xyloglucans, with minor amounts of arabinoxylans, glucomannans, and galacto-glucomannans. This strong framework is embedded in a pectin matrix of homogalacturonans and rhamnogalacturonan I that controls several physiologi cal properties, such as wall porosity, charge density, and microfibril spacing. A variant of homogalacturonan, called rhamnogalacturonan II, is decorated with side groups containing many rare sugars and linkage struc tures and forms boron di-diester dimers that function in

Publisher Summary Arabidopsis detects the hormone through a family of ethylene receptors consisting of ETR1 and its four isoforms. These ethylene receptors are composed of a hydrophobic N-terminal domain that has a high affinity for ethylene, followed by a cytoplasmic GAF-like domain and a C-terminal domain that is related to bacterial two component signal transducers. ETR1 is the only receptor isoform that contains all the necessary conserved residues and domains typically required for two component signaling. The ETR1 and ERS1 histidine kinase domain and ETR1 response regulator domains do interact with CTR1, a Raf-like kinase required for the suppression of ethylene responses, suggesting some role for these domains in signaling even if it is not based on kinase activity. A similar interaction between a response regulator and MAPKKK in the osmotic response pathway in yeast and evidence concerning two-component signaling in plant cytokinin responses are discussed to help evaluate a possible role for ETR1 two-component signaling in plant ethylene responses.