Histological, chemical and gene expression differences between western redcedar seedlings resistant and susceptible to cedar leaf blight.

Introduction: Western redcedar (Thuja plicata) is an important species in the Cupressaceae both at economic and cultural levels in the Pacific Northwest of North America. In adult trees, the species produces one of the most weathering-resistant heartwoods among conifers, making it one of the preferred species for outdoor applications. However, young T. plicata plants are susceptible to infection with cedar leaf blight (Didymascella thujina), an important foliar pathogen that can be devastating in nurseries and small-spaced plantations. Despite that, variability in the resistance against D. thujina in T. plicata has been documented, and such variability can be used to breed T. plicata for resistance against the pathogen. Objective: This investigation aimed to discern the phenotypic and gene expression differences between resistant and susceptible T. plicata seedlings to shed light on the potential constitutive resistance mechanisms against cedar leaf blight in western redcedar. Methods: The study consisted of two parts. First, the histological differences between four resistant and four susceptible families that were never infected with the pathogen were investigated. And second, the differences between one resistant and one susceptible family that were infected and not infected with the pathogen were analyzed at the chemical (C, N, mineral nutrients, lignin, fiber, starch, and terpenes) and gene expression (RNA-Seq) levels. Results: The histological part showed that T. plicata seedlings resistant to D. thujina had constitutively thicker cuticles and lower stomatal densities than susceptible plants. The chemical analyses revealed that, regardless of their infection status, resistant plants had higher foliar concentrations of sabinene and α-thujene, and higher levels of expression of transcripts that code for leucine-rich repeat receptor-like protein kinases and for bark storage proteins. Conclusion: The data collected in this study shows that constitutive differences at the phenotypic (histological and chemical) and gene expression level exist between T. plicata seedlings susceptible and resistant to D. thujina. Such differences have potential use for marker-assisted selection and breeding for resistance against cedar leaf blight in western redcedar in the future.

OsARF11 Promotes Growth, Meristem, Seed, and Vein Formation during Rice Plant Development.

The plant hormone auxin acts as a mediator providing positional instructions in a range of developmental processes. Studies in Arabidopsis thaliana L. show that auxin acts in large part via activation of Auxin Response Factors (ARFs) that in turn regulate the expression of downstream genes. The rice (Oryza sativa L.) gene OsARF11 is of interest because of its expression in developing rice organs and its high sequence similarity with MONOPTEROS/ARF5, a gene with prominent roles in A. thaliana development. We have assessed the phenotype of homozygous insertion mutants in the OsARF11 gene and found that in relation to wildtype, osarf11 seedlings produced fewer and shorter roots as well as shorter and less wide leaves. Leaves developed fewer veins and larger areoles. Mature osarf11 plants had a reduced root system, fewer branches per panicle, fewer grains per panicle and fewer filled seeds. Mutants had a reduced sensitivity to auxin-mediated callus formation and inhibition of root elongation, and phenylboronic acid (PBA)-mediated inhibition of vein formation. Taken together, our results implicate OsARF11 in auxin-mediated growth of multiple organs and leaf veins. OsARF11 also appears to play a central role in the formation of lateral root, panicle branch, and grain meristems.

Identification of Three Monofunctional Diterpene Synthases with Specific Enzyme Activities Expressed during Heartwood Formation in Western Redcedar (Thuja plicata) Trees.

Upon harvest, Western redcedar (WRC; Thuja plicata) trees have a high incidence and extent of heartwood rot. While monoterpenoids and lignans have been linked to rot resistance in this species, other specialized metabolites, such as diterpenes, are likely to contribute to rot resistance. Here we report the cloning and functional assessment of three putative diterpene synthase (TpdiTPS) genes expressed during heartwood formation in WRC. The predicted proteins of the three genes lack either of the two catalytically independent active sites typical of most diTPS, indicating monofunctional rather than bifunctional activity. To identify potential catalytic activities of these proteins, we expressed them in genetically engineered Escherichia coli strains that produce four potential substrates, geranylgeranyl diphosphate (GGDP), ent, syn, and normal stereoisomers of copalyl diphosphate (CDP). We found that TpdiTPS3 used GGDP to produce CDP. TpdiTPS2 used normal CDP to produce levopimaradiene. TpdiTPS1 showed stereoselectivity as it used normal CDP to produce sandaracopimaradiene and syn-CDP to produce syn-stemod-13(17)-ene. These genes and protein enzymatic activities have not been previously reported in WRC and provide an opportunity to assess their potential roles in heartwood rot resistance in this economically important species.

Identification of Auxin Response Factor-Encoding Genes Expressed in Distinct Phases of Leaf Vein Development and with Overlapping Functions in Leaf Formation.

Based on mutant phenotypes the MONOPTEROS (MP)/Auxin Response Factor 5 (ARF5) gene acts in several developmental processes including leaf vein development. Since overlapping functions among ARF genes are common, we assessed the related ARF 3-8 and 19 genes for potential overlap in expression during vein development using in-situ hybridization. Like MP/ARF5, ARF3 was expressed in preprocambial and procambial cells. ARF7 was also expressed in procambial cells, close to and during vein differentiation. ARF19 was expressed in differentiating vessel elements. To assess if genes with vein expression have overlapping functions, double mutants were generated. While arf3, 5 and 7 mutants formed leaves normally, double mutant combinations of mp/arf5 with arf3 or arf7 resulted in a breakdown of leaf formation. Instead, novel structures not present in any of the single mutants formed. The results implicate ARF3 and ARF7 in rosette leaf formation and suggest that their functions overlap and act in parallel with MP/ARF5 in this process. The observed vascular expression patterns suggest unique functions (ARF7 and 19) and potentially overlapping functions (ARF3 and 5) in vein development. Since arf3 arf5 double mutants do not form leaves, assessment of their potential combined action in vein development will require the use of conditional mutants.

The gymnosperm cytochrome P450 CYP750B1 catalyzes stereospecific monoterpene hydroxylation of (+)-sabinene in thujone biosynthesis in western redcedar.

Western redcedar (WRC; Thuja plicata) produces high amounts of oxygenated thujone monoterpenoids associated with resistance against herbivore feeding, particularly ungulate browsing. Thujones and other monoterpenoids accumulate in glandular structures in the foliage of WRC. Thujones are produced from (+)-sabinene by sabinol and sabinone. Using metabolite analysis, enzyme assays with WRC tissue extracts, cloning, and functional characterization of cytochrome P450 monooxygenases, we established that trans-sabin-3-ol but not cis-sabin-3-ol is the intermediate in thujone biosynthesis in WRC. Based on transcriptome analysis, full-length complementary DNA cloning, and characterization of expressed P450 proteins, we identified CYP750B1 and CYP76AA25 as the enzymes that catalyze the hydroxylation of (+)-sabinene to trans-sabin-3-ol. Gene-specific transcript analysis in contrasting WRC genotypes producing high and low amounts of monoterpenoids, including a glandless low-terpenoid clone, as well as assays for substrate specificity supported a biological role of CYP750B1 in α- and ß-thujone biosynthesis. This P450 belongs to the apparently gymnosperm-specific CYP750 family and is, to our knowledge, the first member of this family to be functionally characterized. In contrast, CYP76AA25 has a broader substrate spectrum, also converting the sesquiterpene farnesene and the herbicide isoproturon, and its transcript profiles are not well correlated with thujone accumulation.

Transfusion tracheids in the conifer leaves of Thuja plicata (Cupressaceae) are derived from parenchyma and their differentiation is induced by auxin.

PREMISE OF THE STUDY: Conifer leaves are characterized by the differentiation of transfusion tracheids either adjacent to the vascular bundle or away from bundles. Toward uncovering the mechanism regulating this differentiation, we tested the hypotheses that transfusion tracheids differentiate from parenchyma rather than from procambium and that auxin acts as an inducer of this process. • METHODS: Transfusion tracheids were studied at different developmental stages in both dissected and cleared juvenile and mature leaves. Auxin accumulation was induced by application of either auxin to juvenile leaves or of auxin transport inhibitors in lanolin to stems. • KEY RESULTS: Transfusion tracheids originate from parenchyma cells during late stages of leaf development, after the activity of the procambium has ceased. Transfusion tracheids differentiate also in the leaf tip, a region in which there are no procambial cells. Application of either auxin or auxin transport inhibitors resulted in a significant increase in transfusion tracheids in leaves. Disruption of the leaf vascular bundle combined with auxin application resulted in direct differentiation of transfusion tracheids from parenchyma cells; the regeneration of a vascular bundle around the disruption was polar and supports both hypotheses. • CONCLUSIONS: The results provide experimental support for a parenchymatic origin of the transfusion tracheids in a conifer leaf and for auxin acting as an inducer of these cells. Our results suggest a new model in which auxin production in the leaf apex continues after primary tracheids and parenchyma cells have differentiated, and this late auxin flow induces transfusion tracheids from parenchyma cells.

Identification of genes in Thuja plicata foliar terpenoid defenses.

Thuja plicata (western redcedar) is a long-lived conifer species whose foliage is rarely affected by disease or insect pests, but can be severely damaged by ungulate browsing. Deterrence to browsing correlates with high foliar levels of terpenoids, in particular the monoterpenoid α-thujone. Here, we set out to identify genes whose products may be involved in the production of α-thujone and other terpenoids in this species. First, we generated a foliar transcriptome database from which to draw candidate genes. Second, we mapped the storage of thujones and other terpenoids to foliar glands. Third, we used global expression profiling to identify more than 600 genes that are expressed at high levels in foliage with glands, but can either not be detected or are expressed at low levels in a natural variant lacking foliar glands. Fourth, we used in situ RNA hybridization to map the expression of a putative monoterpene synthase to the epithelium of glands and used enzyme assays with recombinant protein of the same gene to show that it produces sabinene, the monoterpene precursor of α-thujone. Finally, we identified candidate genes with predicted enzymatic functions for the conversion of sabinene to α-thujone. Taken together, this approach generated both general resources and detailed functional characterization in the identification of genes of foliar terpenoid biosynthesis in T. plicata.

SHORT INTERNODES/STYLISH genes, regulators of auxin biosynthesis, are involved in leaf vein development in Arabidopsis thaliana.

Leaves depend on highly developed venation systems to collect fixed carbon for transport and to distribute water. We hypothesized that local regulation of auxin biosynthesis plays a role in vein development. To this effect, we assessed the role of the SHORT INTERNODES/STYLISH (SHI/STY) gene family, zinc-finger transcription factors linked to regulation of auxin biosynthesis, in Arabidopsis thaliana leaf vein development. Gene functions were assessed by a combination of high-resolution spatio-temporal expression analysis of promoter-marker lines and phenotypic analysis of plants homozygous for single and multiple mutant combinations. The SHI/STY genes showed expression patterns with variations on a common theme of activity in incipient and developing cotyledon and leaf primordia, narrowing to apices and hydathode regions. Mutant analysis of single to quintuple mutant combinations revealed dose-dependent defects in vein patterning affecting multiple vein traits, most notably in cotyledons. Here we demonstrate that local regulation of auxin biosynthesis is an important aspect of leaf vein development. Our findings also support a model in which auxin synthesized at the periphery of primordia affects vein development.

Multiple MONOPTEROS-dependent pathways are involved in leaf initiation.

Initiation of leaves at the flanks of the shoot apical meristem occurs at sites of auxin accumulation and pronounced expression of auxin-inducible PIN-FORMED1 (PIN) genes, suggesting a feedback loop to progressively focus auxin in concrete spots. Because PIN expression is regulated by auxin response factor activity, including MONOPTEROS (MP), it appeared possible that MP affects leaf formation as a positive regulator of PIN genes and auxin transport. Here, we analyze a novel, completely leafless phenotype arising from simultaneous interference with both auxin signaling and auxin transport. We show that mp pin1 double mutants, as well as mp mutants treated with auxin-efflux inhibitors, display synergistic abnormalities not seen in wild type regardless of how strongly auxin transport was reduced. The synergism of abnormalities indicates that the role of MP in shoot meristem organization is not limited to auxin transport regulation. In the mp mutant background, auxin transport inhibition completely abolishes leaf formation. Instead of forming leaves, the abnormal shoot meristems dramatically increase in size, harboring correspondingly enlarged expression domains of CLAVATA3 and SHOOTMERISTEMLESS, molecular markers for the central stem cell zone and the complete meristem, respectively. The observed synergism under conditions of auxin efflux inhibition was further supported by an unrestricted PIN1 expression in mp meristems, as compared to a partial restriction in wild-type meristems. Auxin transport-inhibited mp meristems also lacked detectable auxin maxima. We conclude that MP promotes the focusing of auxin and leaf initiation in part through pathways not affected by auxin efflux inhibitors.

Conifers have a unique small RNA silencing signature.

Plants produce small RNAs to negatively regulate genes, viral nucleic acids, and repetitive elements at either the transcriptional or post-transcriptional level in a process that is referred to as RNA silencing. While RNA silencing has been extensively studied across the different phyla of the animal kingdom (e.g., mouse, fly, worm), similar studies in the plant kingdom have focused primarily on angiosperms, thus limiting evolutionary studies of RNA silencing in plants. Here we report on an unexpected phylogenetic difference in the size distribution of small RNAs among the vascular plants. By extracting total RNA from freshly growing shoot tissue, we conducted a survey of small RNAs in 24 vascular plant species. We find that conifers, which radiated from the other seed-bearing plants approximately 260 million years ago, fail to produce significant amounts of 24-nucleotide (nt) RNAs that are known to guide DNA methylation and heterochromatin formation in angiosperms. Instead, they synthesize a diverse population of small RNAs that are exactly 21-nt long. This finding was confirmed by high-throughput sequencing of the small RNA sequences from a conifer, Pinus contorta. A conifer EST search revealed the presence of a novel Dicer-like (DCL) family, which may be responsible for the observed change in small RNA expression. No evidence for DCL3, an enzyme that matures 24-nt RNAs in angiosperms, was found. We hypothesize that the diverse class of 21-nt RNAs found in conifers may help to maintain organization of their unusually large genomes.

Identification of genes expressed in vascular tissues using NPA-induced vascular overgrowth in Arabidopsis.

The genetic basis of vascular differentiation and function is relatively poorly understood, partly due to the difficulty of screening for mutants defective in internal vascular tissues. Here we present an approach based on a predicted increase in vascular-related gene expression in response to an auxin transport inhibitor-induced vascular overgrowth. We used microarray analyses to identify 336 genes that were up-regulated > or =2-fold in shoot tissues of Arabidopsis thaliana showing vascular overgrowth. Promoter-marker gene fusions revealed that 38 out of 40 genes with > or =4-fold up-regulation in vascular overgrowth tissues had vascular-related expression in transgenic Arabidopsis plants. Obtained expression patterns included cambial tissues and differentiating xylem, phloem and fibers. A total of 15 genes were found to have vascular-specific expression patterns in the leaves and/or inflorescence stems. This study provides empirical evidence of the efficiency of the approach and describes for the first time the in situ expression patterns of the majority of the assessed genes.

Dynamics of MONOPTEROS and PIN-FORMED1 expression during leaf vein pattern formation in Arabidopsis thaliana.

Genetic evidence links the Arabidopsis MONOPTEROS (MP) and PIN-FORMED1 (PIN1) genes to the patterning of leaf veins. To elucidate their potential functions and interactions in this process, we have assessed the dynamics of MP and PIN1 expression during vascular patterning in Arabidopsis leaf primordia. Both genes undergo a dynamic process of gradual refinement of expression into files one to two cells wide before overt vascular differentiation. The subcellular distribution of PIN1 is also gradually refined from a non-polar distribution in isodiametric cells to strongly polarized in elongated procambial cells and provides an indication of overall directions of auxin flow. We found evidence that MP expression can be activated by auxin exposure and that PIN1 as well as DR5::GUS expression is defective in mp mutant leaves. Taken together the results suggest a feedback regulatory loop that involves auxin, MP and PIN1 and provide novel experimental support for the canalization-of-auxin-flow hypothesis.

Auxin signaling in Arabidopsis leaf vascular development.

A number of observations have implicated auxin in the formation of vascular tissues in plant organs. These include vascular strand formation in response to local auxin application, the effects of impaired auxin transport on vascular patterns and suggestive phenotypes of Arabidopsis auxin response mutants. In this study, we have used molecular markers to visualize auxin response patterns in developing Arabidopsis leaves as well as Arabidopsis mutants and transgenic plants to trace pathways of auxin signal transduction controlling the expression of early procambial genes. We show that in young Arabidopsis leaf primordia, molecular auxin response patterns presage sites of procambial differentiation. This is the case not only in normal development but also upon experimental manipulation of auxin transport suggesting that local auxin signals are instrumental in patterning Arabidopsis leaf vasculature. We further found that the activity of the Arabidopsis gene MONOPTEROS, which is required for proper vascular differentiation, is also essential in a spectrum of auxin responses, which include the regulation of rapidly auxin-inducible AUX/IAA genes, and discovered the tissue-specific vascular expression profile of the class I homeodomain-leucine zipper gene, AtHB20. Interestingly, MONOPTEROS activity is a limiting factor in the expression of AtHB8 and AtHB20, two genes encoding transcriptional regulators expressed early in procambial development. Our observations connect general auxin signaling with early controls of vascular differentiation and suggest molecular mechanisms for auxin signaling in patterned cell differentiation.