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Seed quality traits of oilseed rape, Brassica napus ( B. napus ), exhibit quantitative inheritance determined by its genetic makeup and the environment via the mediation of a complex genetic architecture of hundreds to thousands of genes. Thus, instead of single gene analysis, network-based systems genomics and genetics approaches that combine genotype, phenotype, and molecular phenotypes offer a promising alternative to uncover this complex genetic architecture. In the current study, systems genetics approaches were used to explore the genetic regulation of lignin traits in B. napus seeds. Four QTL (qLignin_A09_1, qLignin_A09_2, qLignin_A09_3, and qLignin_C08) distributed on two chromosomes were identified for lignin content. The qLignin_A09_2 and qLignin_C08 loci were homologous QTL from the A and C subgenomes, respectively. Genome-wide gene regulatory network analysis identified eighty-three subnetworks (or modules); and three modules with 910 genes in total, were associated with lignin content, which was confirmed by network QTL analysis. eQTL (expression quantitative trait loci) analysis revealed four cis-eQTL genes including lignin and flavonoid pathway genes, cinnamoyl-CoA-reductase ( CCR1 ), and TRANSPARENT TESTA genes TT4 , TT6 , TT8 , as causal genes. The findings validated the power of systems genetics to identify causal regulatory networks and genes underlying complex traits. Moreover, this information may enable the research community to explore new breeding strategies, such as network selection or gene engineering, to rewire networks to develop climate resilience crops with better seed quality., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 His Majesty the King in Right of Canada.)

Pyramiding multiple resistant genes has been proposed as the most effective way to control wheat rust diseases globally. Identifying the most effective pyramids is challenged by the large pool of rust resistance genes and limited information about their mechanisms of resistance and interactions. Here, using a high-density genetic map, a double haploid population, and multi-rust field testing, we aimed to systematically characterize the most effective gene pyramids for rust resistance from the durable multi-rust resistant CIMMYT cultivar Parula. We revealed that the Parula resistance gene pyramid contains Lr34/Yr18/Sr57 ( Lr34 ), Lr46/Yr29/Sr58 ( Lr46 ), Lr27/Yr30/Sr2 ( Sr2 ), and Lr68 . The efficacy, magnitude of effect, and interactions varied for the three rust diseases. A subpopulation mapping approach was applied to characterize the complex interactions of the resistance genes by controlling for the effect of Lr34 . Using this approach, we found that Lr34 and Lr68 have a strong additive effect for leaf rust, whereas no additive effects were observed for any rusts between Lr34 and Lr46 . Lr34 combined synergistically with Sr12 from Thatcher for stem rust, whereas the additive effect of Lr34 and Sr2 was dependent on the type of rust and environment. Two novel leaf rust quantitative trait loci (QTLs) from Parula were identified in this study, a stable QTL QLr-7BS and QLr-5AS , which showed Lr34 dependent expression. With these findings, we propose combining two to three high-value genes from Canadian wheat (e.g., Sr12 from Thatcher) with a foundational multi-adult plant resistance cassette for desirable and durable resistance to all three rusts in Canadian wheat., Competing Interests: The author(s) declare no conflict of interest.

Fusarium head blight (FHB) resistance is quantitatively inherited, controlled by multiple minor effect genes, and highly affected by the interaction of genotype and environment. This makes genomic selection (GS) that uses genome-wide molecular marker data to predict the genetic breeding value as a promising approach to select superior lines with better resistance. However, various factors can affect accuracies of GS and better understanding how these factors affect GS accuracies could ensure the success of applying GS to improve FHB resistance in wheat. In this study, we performed a comprehensive evaluation of factors that affect GS accuracies with a multi-parental population designed for FHB resistance. We found larger sample sizes could get better accuracies. Training population designed by CDmean based optimization algorithms significantly increased accuracies than random sampling approach, while mean of predictor error variance (PEVmean) had the poorest performance. Different genomic selection models performed similarly for accuracies. Including prior known large effect quantitative trait loci (QTL) as fixed effect into the GS model considerably improved the predictability. Multi-traits models had almost no effects, while the multi-environment model outperformed the single environment model for prediction across different environments. By comparing within and across family prediction, better accuracies were obtained with the training population more closely related to the testing population. However, achieving good accuracies for GS prediction across populations is still a challenging issue for GS application.

Durum wheat is an economically important crop for Canadian farmers. Fusarium head blight (FHB) is one of the most destructive diseases that threatens durum production in Canada. FHB reduces yield and end-use quality and most commonly contaminates the grain with the fungal mycotoxin deoxynivalenol, also known as DON. Serious outbreaks of FHB can occur in durum wheat in Canada, and combining genetic resistance with fungicide application is a cost effective approach to control this disease. However, there is limited variation for genetic resistance to FHB in elite Canadian durum cultivars. To explore and identify useful genetic FHB resistance variation for the improvement of Canadian durum wheat, we assembled an association mapping (AM) panel of diverse durum germplasms and performed genome wide association analysis (GWAS). Thirty-one quantitative trait loci (QTL) across all 14 chromosomes were significantly associated with FHB resistance. On 3BS, a stable QTL with a larger effect for resistance was located close to the centromere of 3BS. Three haplotypes of Fhb1 QTL were identified, with an emmer wheat haplotype contributing to disease susceptibility. The large number of QTL identified here can provide a rich resource to improve FHB resistance in commercially grown durum wheat. Among the 31 QTL most were associated with plant height and/or flower time. QTL 1A.1, 1A.2, 3B.2, 5A.1, 6A.1, 7A.3 were associated with FHB resistance and not associated or only weakly associated with flowering time nor plant height. These QTL have features that would make them good targets for FHB resistance breeding., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Ruan, Zhang, Knox, Berraies, Campbell, Ragupathy, Boyle, Polley, Henriquez, Burt, Kumar, Cuthbert, Fobert, Buerstmayr and DePauw.)

Fusarium head blight (FHB) is a devastating fungal disease of small-grain cereals that results in severe yield and quality losses. FHB resistance is controlled by resistance components including incidence, field severity, visual rating index, Fusarium damaged kernels (FDKs), and the accumulation of the mycotoxin deoxynivalenol (DON). Resistance conferred by each of these components is partial and must be combined to achieve resistance sufficient to protect wheat from yield losses. In this study, two biparental mapping populations were analyzed in Canadian FHB nurseries and quantitative trait loci (QTL) mapped for the traits listed above. Nine genomic loci, on 2AS, 2BS, 3BS, 4AS, 4AL, 4BS, 5AS, 5AL, and 5BL, were enriched for the majority of the QTL controlling FHB resistance. The previously validated FHB resistance QTL on 3BS and 5AS affected resistance to severity, FDK, and DON in these populations. The remaining seven genomic loci colocalize with flowering time and/or plant height QTL. The QTL on 4B was a major contributor to all field resistance traits and plant height in the field. QTL on 4AL showed contrasting effects for FHB resistance between Eastern and Western Canada, indicating a local adapted resistance to FHB. In addition, we also found that the 2AS QTL contributed a major effect for DON, and the 2BS for FDK, while the 5AL conferred mainly effect for both FDK/DON. Results presented here provide insight into the genetic architecture underlying these resistant components and insight into how FHB resistance in wheat is controlled by a complex network of interactions between genes controlling flowering time, plant height, local adaption, and FHB resistance components., (Copyright © 2020 Zhang, Boyle, Brûlé-Babel, Fedak, Gao, Robleh Djama, Polley, Cuthbert, Randhawa, Jiang, Eudes and Fobert.)

The durum wheat line DT696 is a source of moderate Fusarium head blight (FHB) resistance. Previous analysis using a bi-parental population identified two FHB resistance quantitative trait loci (QTL) on chromosome 5A: 5A1 was co-located with a plant height QTL, and 5A2 with a major maturity QTL. A Genome-Wide Association Study (GWAS) of DT696 derivative lines from 72 crosses based on multi-environment FHB resistance, plant height, and maturity phenotypic data was conducted to improve the mapping resolution and further elucidate the genetic relationship of height and maturity with FHB resistance. The Global Tetraploid Wheat Collection (GTWC) was exploited to identify durum wheat lines with DT696 allele and additional recombination events. The 5A2 QTL was confirmed in the derivatives, suggesting the expression stability of the 5A2 QTL in various genetic backgrounds. The GWAS led to an improved mapping resolution rendering the 5A2 interval 10 Mbp shorter than the bi-parental QTL mapping interval. Haplotype analysis using SNPs within the 5A2 QTL applied to the GTWC identified novel haplotypes and recombination breakpoints, which could be exploited for further improvement of the mapping resolution. This study suggested that GWAS of derivative breeding lines is a credible strategy for improving mapping resolution.

Background: Fusarium head blight (FHB) resistance in the durum wheat breeding gene pool is rarely reported. Triticum turgidum ssp. carthlicum line Blackbird is a tetraploid relative of durum wheat that offers partial FHB resistance. Resistance QTL were identified for the durum wheat cv. Strongfield × Blackbird population on chromosomes 1A, 2A, 2B, 3A, 6A, 6B and 7B in a previous study. The objective of this study was to identify the defense mechanisms underlying the resistance of Blackbird and report candidate regulator defense genes and single nucleotide polymorphism (SNP) markers within these genes for high-resolution mapping of resistance QTL reported for the durum wheat cv. Strongfield/Blackbird population., Results: Gene network analysis identified five networks significantly (P < 0.05) associated with the resistance to FHB spread (Type II FHB resistance) one of which showed significant correlation with both plant height and relative maturity traits. Two gene networks showed subtle differences between Fusarium graminearum-inoculated and mock-inoculated plants, supporting their involvement in constitutive defense. The candidate regulator genes have been implicated in various layers of plant defense including pathogen recognition (mainly Nucleotide-binding Leucine-rich Repeat proteins), signaling pathways including the abscisic acid and mitogen activated protein (MAP) kinase, and downstream defense genes activation including transcription factors (mostly with dual roles in defense and development), and cell death regulator and cell wall reinforcement genes. The expression of five candidate genes measured by quantitative real-time PCR was correlated with that of RNA-seq, corroborating the technical and analytical accuracy of RNA-sequencing., Conclusions: Gene network analysis allowed identification of candidate regulator genes and genes associated with constitutive resistance, those that will not be detected using traditional differential expression analysis. This study also shed light on the association of developmental traits with FHB resistance and partially explained the co-localization of FHB resistance with plant height and maturity QTL reported in several previous studies. It also allowed the identification of candidate hub genes within the interval of three previously reported FHB resistance QTL for the Strongfield/Blackbird population and associated SNPs for future high resolution mapping studies.

UBC13 is required for Lys63-linked polyubiquitination and innate immune responses in mammals, but its functions in plant immunity remain to be defined. Here we used genetic and pathological methods to evaluate roles of Arabidopsis UBC13 in response to pathogens and environmental stresses. Loss of UBC13 failed to activate the expression of numerous cold-responsive genes and resulted in hypersensitivity to low-temperature stress, indicating that UBC13 is involved in plant response to low-temperature stress. Furthermore, the ubc13 mutant displayed low-temperature-induced and salicylic acid-dependent lesion mimic phenotypes. Unlike typical lesion mimic mutants, ubc13 did not enhance disease resistance against virulent bacterial and fungal pathogens, but diminished hypersensitive response and compromised effector-triggered immunity against avirulent bacterial pathogens. UBC13 differently regulates two types of programmed cell death in response to low temperature and pathogen. The lesion mimic phenotype in the ubc13 mutant is partially dependent on SNC1. UBC13 interacts with an F-box protein CPR1 that regulates the homeostasis of SNC1. However, the SNC1 protein level was not altered in the ubc13 mutant, implying that UBC13 is not involved in CPR1-regulated SNC1 protein degradation. Taken together, our results revealed that UBC13 is a key regulator in plant response to low temperature and pathogens., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Fusarium head blight (FHB or scab) caused by Fusarium spp. is a destructive disease of wheat. Since the most effective sources of FHB resistance are typically associated with unfavorable agronomic traits, breeding commercial cultivars that combine desired agronomic traits and a high level of FHB resistance remains a considerable challenge. A better understanding of the molecular mechanisms governing FHB resistance will help to design more efficient and precise breeding strategies. Here, multiple molecular tools and assays were deployed to compare the resistant variety Sumai3 with three regionally adapted Canadian cultivars. Macroscopic and microscopic disease evaluation established the relative level of Type II FHB resistance of the four varieties and revealed that the F. graminearum infection process displayed substantial temporal differences among organs. The rachis was found to play a critical role in preventing F. graminearum spread within spikes. Large-scale, organ-specific RNA-seq at different times after F. graminearum infection demonstrated that diverse defense mechanisms were expressed faster and more intensely in the spikelet of resistant varieties. The roles of plant hormones during the interaction of wheat with F. graminearum was inferred based on the transcriptomic data obtained and the quantification of the major plant hormones. Salicylic acid and jasmonic acid were found to play predominantly positive roles in FHB resistance, whereas auxin and ABA were associated with susceptibility, and ethylene appeared to play a dual role during the interaction with F graminearum., Competing Interests: The authors have declared that no competing interests exist.

Breeding for Fusarium head blight (FHB) resistance in durum wheat is complicated by the quantitative trait expression and narrow genetic diversity of available resources. High-density mapping of the FHB resistance quantitative trait loci (QTL), evaluation of their co-localization with plant height and maturity QTL and the interaction among the identified QTL are the objectives of this study. Two doubled haploid (DH) populations, one developed from crosses between Triticum turgidum ssp. durum lines DT707 and DT696 and the other between T. turgidum ssp. durum cv. Strongfield and T. turgidum ssp. carthlicum cv. Blackbird were genotyped using the 90K Infinium iSelect chip and evaluated phenotypically at multiple field FHB nurseries over years. A moderate broad-sense heritability indicated a genotype-by-environment interaction for the expression of FHB resistance in both populations. Resistance QTL were identified for the DT707 × DT696 population on chromosomes 1B, 2B, 5A (two loci) and 7A and for the Strongfield × Blackbird population on chromosomes 1A, 2A, 2B, 3A, 6A, 6B and 7B with the QTL on chromosome 1A and those on chromosome 5A being more consistently expressed over environments. FHB resistance co-located with plant height and maturity QTL on chromosome 5A and with a maturity QTL on chromosome 7A for the DT707 × DT696 population. Resistance also co-located with plant height QTL on chromosomes 2A and 3A and with maturity QTL on chromosomes 1A and 7B for the Strongfield × Blackbird population. Additive × additive interactions were identified, for example between the two FHB resistance QTL on chromosome 5A for the DT707 × DT696 population and the FHB resistance QTL on chromosomes 1A and 7B for the Strongfield × Blackbird population. Application of the Single Nucleotide Polymorphic (SNP) markers associated with FHB resistance QTL identified in this study will accelerate combining genes from the two populations., Competing Interests: The authors have declared that no competing interests exist.

Background: Lr16 is a widely deployed leaf rust resistance gene in wheat (Triticum aestivum L.) that is highly effective against the North American Puccinia triticina population when pyramided with the gene Lr34. Lr16 is a seedling leaf rust resistance gene conditioning an incompatible interaction with a distinct necrotic ring surrounding the uredinium. Lr16 was previously mapped to the telomeric region of the short arm of wheat chromosome 2B. The goals of this study were to develop numerous single nucleotide polymorphism (SNP) markers for the Lr16 region and identify diagnostic gene-specific SNP marker assays for marker-assisted selection (MAS)., Results: Forty-three SNP markers were developed and mapped on chromosome 2BS tightly linked with the resistance gene Lr16 across four mapping populations representing a total of 1528 gametes. Kompetitive Allele Specific PCR (KASP) assays were designed for all identified SNPs. Resistance gene analogs (RGAs) linked with the Lr16 locus were identified and RGA-based SNP markers were developed. The diagnostic potential of the SNPs co-segregating with Lr16 was evaluated in a diverse set of 133 cultivars and breeding lines. Six SNP markers were consistent with the Lr16 phenotype and are accurately predictive of Lr16 for all wheat lines/cultivars in the panel., Conclusions: Lr16 was mapped relative to SNP markers in four populations. Six SNP markers exhibited high quality clustering in the KASP assay and are suitable for MAS of Lr16 in wheat breeding programs.

Key Message: SNP markers were developed for the OWBM resistance gene Sm1 that will be useful for MAS. The wheat Sm1 region is collinear with an inverted syntenic interval in B. distachyon. Orange wheat blossom midge (OWBM, Sitodiplosis mosellana Géhin) is an important insect pest of wheat (Triticum aestivum) in many growing regions. Sm1 is the only described OWBM resistance gene and is the foundation of managing OWBM through host genetics. Sm1 was previously mapped to wheat chromosome arm 2BS relative to simple sequence repeat (SSR) markers and the dominant, sequence characterized amplified region (SCAR) marker WM1. The objectives of this research were to saturate the Sm1 region with markers, develop improved markers for marker-assisted selection (MAS), and examine the synteny between wheat, Brachypodium distachyon, and rice (Oryza sativa) in the Sm1 region. The present study mapped Sm1 in four populations relative to single nucleotide polymorphisms (SNPs), SSRs, Diversity Array Technology (DArT) markers, single strand conformation polymorphisms (SSCPs), and the SCAR WM1. Numerous high quality SNP assays were designed that mapped near Sm1. BLAST delineated the syntenic intervals in B. distachyon and rice using gene-based SNPs as query sequences. The Sm1 region in wheat was inverted relative to B. distachyon and rice, which suggests a chromosomal rearrangement within the Triticeae lineage. Seven SNPs were tested on a collection of wheat lines known to carry Sm1 and not to carry Sm1. Sm1-flanking SNPs were identified that were useful for predicting the presence or absence of Sm1 based upon haplotype. These SNPs will be a major improvement for MAS of Sm1 in wheat breeding programs.

Fusarium head blight (FHB) is a serious disease of wheat worldwide. Cultivar resistance to FHB depends on biochemical factors that confine the pathogen spread in spikes. Breeding for cultivar resistance is considered the most practical way to manage this disease. In this study, different spectroscopy and microscopy techniques were applied to discriminate resistance in wheat genotypes against FHB. Synchrotron-based spectroscopy and imaging techniques, including focal plane array infrared and X-ray fluorescence (XRF) spectroscopy were used to understand changes in biochemical and nutrients in rachis following FHB infection. Sumai3 and Muchmore were used to represent resistant and susceptible cultivars to FHB, respectively, in this study. The histological comparison of rachis showed substantial differences in the cell wall thickness between the cultivars after infection. Synchrotron-based infrared imaging emphasized substantial difference in biochemical composition of rachis samples between the two cultivars prior to visible symptoms; in the resistant Sumai3, infrared bands representing lignin and hemicellulose were stronger and more persistent compared to the susceptible cultivar. These bands may be the candidates of biochemical markers for FHB resistance. Focal plane array infrared imaging (FPA) spectra from the rachis epidermis and vascular bundles revealed a new band (1710 cm(-1)) related to the oxidative stress on the susceptible cultivar only. XRF spectroscopy data revealed differences in nutrients composition between cultivars, and between controls and inoculated samples, with substantial increases observed for Ca, K, Mn, Fe, Zn, and Si in the resistant cultivar. These nutrients are related to cell wall stability, metabolic process, and plant defense mechanisms such as lignification pathway and callose deposition. The combination of cell wall composition and lignification plays a role in the mechanism of type II host resistance to FHB. Biochemical profiling using the synchrotron-based spectroscopy holds potential for screening wheat genotypes for FHB resistance.

Metabolic changes in spikelets of wheat varieties FL62R1, Stettler, Muchmore and Sumai3 following Fusarium graminearum infection were explored using NMR analysis. Extensive 1D and 2D 1H NMR measurements provided information for detailed metabolite assignment and quantification leading to possible metabolic markers discriminating resistance level in wheat subtypes. In addition, metabolic changes that are observed in all studied varieties as well as wheat variety specific changes have been determined and discussed. A new method for metabolite quantification from NMR data that automatically aligns spectra of standards and samples prior to quantification using multivariate linear regression optimization of spectra of assigned metabolites to samples' 1D spectra is described and utilized. Fusarium infection-induced metabolic changes in different wheat varieties are discussed in the context of metabolic network and resistance.

Background: Fusarium head blight (FHB), a scab principally caused by Fusarium graminearum Schw., is a serious disease of wheat. The purpose of this study is to evaluate the potential of combining synchrotron based phase contrast X-ray imaging (PCI) with Fourier Transform mid infrared (FTIR) spectroscopy to understand the mechanisms of resistance to FHB by resistant wheat cultivars. Our hypothesis is that structural and biochemical differences between resistant and susceptible cultivars play a significant role in developing resistance to FHB., Results: Synchrotron based PCI images and FTIR absorption spectra (4000-800 cm(-1)) of the floret and rachis from Fusarium-damaged and undamaged spikes of the resistant cultivar 'Sumai3', tolerant cultivar 'FL62R1', and susceptible cultivar 'Muchmore' were collected and analyzed. The PCI images show significant differences between infected and non-infected florets and rachises of different wheat cultivars. However, no pronounced difference between non-inoculated resistant and susceptible cultivar in terms of floret structures could be determined due to the complexity of the internal structures. The FTIR spectra showed significant variability between infected and non-infected floret and rachis of the wheat cultivars. The changes in absorption wavenumbers following pathogenic infection were mostly in the spectral range from 1800-800 cm(-1). The Principal Component Analysis (PCA) was also used to determine the significant chemical changes inside floret and rachis when exposed to the FHB disease stress to understand the plant response mechanism. In the floret and rachis samples, PCA of FTIR spectra revealed differences in cell wall related polysaccharides. In the florets, absorption peaks for Amide I, cellulose, hemicellulose and pectin were affected by the pathogenic fungus. In the rachis of the wheat cultivars, PCA underlines significant changes in pectin, cellulose, and hemicellulose characteristic absorption spectra. Amide II and lignin absorption peaks, persistent in the rachis of Sumai3, together with increased peak shift at 1245 cm(-1) after infection with FHB may be a marker for stress response in which the cell wall compounds related to pathways for lignification are increased., Conclusions: Synchrotron based PCI combined with FTIR spectroscopy show promising results related to FHB in wheat. The combined technique is a powerful new tool for internal visualisation and biomolecular monitoring before and during plant-microbe interactions to understand both the differences between cultivars and their different responses to disease stress.

Background: HIGH-LEVEL EXPRESSION OF SUGAR INDUCIBLE GENE2 (HSI2), also known as VAL1, is a B3 domain transcriptional repressor that acts redundantly with its closest relative, HSI2-LIKE1 (HSL1), to suppress the seed maturation program following germination. Mutant hsi2 hsl1 seedlings are arrested early in development and differentially express a number of abiotic stress-related genes. To test the potential requirement for HSI2 during abiotic stress, hsi2 single mutants and plants overexpressing HSI2 were subjected to simulated drought stress by withholding watering, and characterized through physiological, metabolic and gene expression studies., Results: The hsi2 mutants demonstrated reduced wilting and maintained higher relative water content than wild-type after withholding watering, while the overexpressing lines displayed the opposite phenotype. The hsi2 mutant displayed lower constitutive and ABA-induced stomatal conductance than wild-type and accumulated lower levels of ABA metabolites and several osmolytes and osmoprotectants following water withdrawal. Microarray comparisons between wild-type and the hsi2 mutant revealed that steady-state levels of numerous stress-induced genes were up-regulated in the mutant in the absence of stress but down-regulated at visible wilting. Plants with altered levels of HSI2 responded to exogenous application of ABA and a long-lived ABA analog, but the hsi2 mutant did not show altered expression of several ABA-responsive or ABA signalling genes 4 hr after application., Conclusions: These results implicate HSI2 as a negative regulator of drought stress response in Arabidopsis, acting, at least in part, by regulating transpirational water loss. Metabolic and global transcript profiling comparisons of the hsi2 mutant and wild-type plants do not support a model whereby the greater drought tolerance observed in the hsi2 mutant is conferred by the accumulation of known osmolytes and osmoprotectants. Instead, data are consistent with mutants experiencing a relatively milder dehydration stress following water withdrawal.

During the plant immune response, large-scale transcriptional reprogramming is modulated by numerous transcription (co) factors. The Arabidopsis basic leucine zipper transcription factors TGA1 and TGA4, which comprise the clade I TGA factors, have been shown to positively contribute to disease resistance against virulent strains of the bacterial pathogen Pseudomonas syringae. Despite physically interacting with the key immune regulator, NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1), following elicitation with salicylic acid (SA), clade I function was shown to be largely independent of NPR1. Unlike mutants in NPR1, tga1-1 tga4-1 plants do not display reductions in steady-state levels of SA-pathway marker genes following treatment with this phenolic signaling metabolite or after challenge with virulent or avirulent P. syringae. By exploiting bacterial strains that have limited capacity to suppress Arabidopsis defence responses, the present study demonstrates that tga1-1 tga4-1 plants are compromised in basal resistance and defective in several apoplastic defence responses, including the oxidative burst of reactive oxygen species, callose deposition, as well as total and apoplastic PATHOGENESIS-RELATED 1 (PR-1) protein accumulation. Furthermore, analysis of npr1-1 and the tga1-1 tga4-1 npr1-1 triple mutant indicates that clade I TGA factors act substantially independent of NPR1 in mediating disease resistance against these strains of P. syringae. Increased sensitivity to the N-glycosylation inhibitor tunicamycin and elevated levels of endoplasmic reticulum (ER) stress marker genes encoding ER-resident chaperones in mutant seedlings suggest that loss of apoplastic defence responses is associated with aberrant protein secretion and implicate clade I TGA factors as positive regulators of one or more ER-related secretion pathways.

DIR1 is a lipid transfer protein (LTP) postulated to complex with and/or chaperone a signal(s) to distant leaves during Systemic Acquired Resistance (SAR) in Arabidopsis. DIR1 was detected in phloem sap-enriched petiole exudates collected from wild-type leaves induced for SAR, suggesting that DIR1 gains access to the phloem for movement from the induced leaf. Occasionally the defective in induced resistance1 (dir1-1) mutant displayed a partially SAR-competent phenotype and a DIR1-sized band in protein gel blots was detected in dir1-1 exudates suggesting that a highly similar protein, DIR1-like (At5g48490), may contribute to SAR. Recombinant protein studies demonstrated that DIR1 polyclonal antibodies recognize DIR1 and DIR1-like. Homology modeling of DIR1-like using the DIR1-phospholipid crystal structure as template, provides clues as to why the dir1-1 mutant is rarely SAR-competent. The contribution of DIR1 and DIR1-like during SAR was examined using an Agrobacterium-mediated transient expression-SAR assay and an estrogen-inducible DIR1-EGFP/dir1-1 line. We provide evidence that upon SAR induction, DIR1 moves down the leaf petiole to distant leaves. Our data also suggests that DIR1-like displays a reduced capacity to move to distant leaves during SAR and this may explain why dir1-1 is occasionally SAR-competent.

Transcriptional reprogramming during induction of salicylic acid (SA)-mediated defenses is regulated primarily by NPR1 (NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1), likely through interactions with TGA bZIP transcription factors. To ascertain the contributions of clade I TGA factors (TGA1 and TGA4) to defense responses, a tga1-1 tga4-1 double mutant was constructed and challenged with Pseudomonas syringae and Hyaloperonospora arabidopsidis. Although the mutant displayed enhanced susceptibility to virulent P. syringae, it was not compromised in systemic acquired resistance against this pathogen or resistance against avirulent H. arabidopsidis. Microarray analysis of nonelicited and SA-treated plants indicated that clade I TGA factors regulate fewer genes than NPR1. Approximately half of TGA-dependent genes were regulated by NPR1 but, in all cases, the direction of change was opposite in the two mutants. In support of the microarray data, the NPR1-independent disease resistance observed in the autoimmune resistance (R) gene mutant snc1 is partly compromised by tga1-1 tga4-1 mutations, and a triple mutant of clade I TGA factors with npr1-1 is more susceptible than either parent. These results suggest that clade I TGA factors are required for resistance against virulent pathogens and avirulent pathogens mediated by at least some R gene specificities, acting substantially through NPR1-independent pathways.

ROXY1 and ROXY2 are CC-type floral glutaredoxins with redundant functions in Arabidopsis (Arabidopsis thaliana) anther development. We show here that plants lacking the basic leucine-zipper transcription factors TGA9 and TGA10 have defects in male gametogenesis that are strikingly similar to those in roxy1 roxy2 mutants. In tga9 tga10 mutants, adaxial and abaxial anther lobe development is differentially affected, with early steps in anther development blocked in adaxial lobes and later steps affected in abaxial lobes. Distinct from roxy1 roxy2, microspore development in abaxial anther lobes proceeds to a later stage with the production of inviable pollen grains contained within nondehiscent anthers. Histological analysis shows multiple defects in the anther dehiscence program, including abnormal stability and lignification of the middle layer and defects in septum and stomium function. Compatible with these defects, TGA9 and TGA10 are expressed throughout early anther primordia but resolve to the middle and tapetum layers during meiosis of pollen mother cells. Several lines of evidence suggest that ROXY promotion of anther development is mediated in part by TGA9 and TGA10. First, TGA9 and TGA10 expression overlaps with ROXY1/2 during anther development. Second, TGA9/10 and ROXY1/2 operate downstream of SPOROCYTELESS/NOZZLE, where they positively regulate a common set of genes that contribute to tapetal development. Third, TGA9 and TGA10 directly interact with ROXY proteins in yeast and in plant cell nuclei. These findings suggest that activation of TGA9/10 transcription factors by ROXY-mediated modification of cysteine residues promotes anther development, thus broadening our understanding of how redox-regulated TGA factors function in plants.

TGA2 and NONEXPRESSER OF PR GENES1 (NPR1) are activators of systemic acquired resistance (SAR) and of the SAR marker gene pathogenesis-related-1 (PR-1) in Arabidopsis thaliana. TGA2 is a transcriptional repressor required for basal repression of PR-1, but during SAR, TGA2 recruits NPR1 as part of an enhanceosome. Transactivation by the enhanceosome requires the NPR1 BTB/POZ domain. However, the NPR1 BTB/POZ domain does not contain an autonomous transactivation domain; thus, its molecular role within the enhanceosome remains elusive. We now show by gel filtration analyses that TGA2 binds DNA as a dimer, tetramer, or oligomer. Using in vivo plant transcription assays, we localize the repression domain of TGA2 to the N terminus and demonstrate that this domain is responsible for modulating the DNA binding activity of the oligomer both in vitro and in vivo. We confirm that the NPR1 BTB/POZ domain interacts with and negates the molecular function of the TGA2 repression domain by excluding TGA2 oligomers from cognate DNA. These data distinguish the NPR1 BTB/POZ domain from other known BTB/POZ domains and establish its molecular role in the context of the Arabidopsis PR-1 gene enhanceosome.

Background: Seed oil accumulates primarily as triacylglycerol (TAG). While the biochemical pathway for TAG biosynthesis is known, its regulation remains unclear. Previous research identified microsomal diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) as controlling a rate-limiting step in the TAG biosynthesis pathway. Of note, overexpression of DGAT1 results in substantial increases in oil content and seed size. To further analyze the global consequences of manipulating DGAT1 levels during seed development, a concerted transcriptome and metabolome analysis of transgenic B. napus prototypes was performed., Results: Using a targeted Brassica cDNA microarray, about 200 genes were differentially expressed in two independent transgenic lines analyzed. Interestingly, 24-33% of the targets showing significant changes have no matching gene in Arabidopsis although these represent only 5% of the targets on the microarray. Further analysis of some of these novel transcripts indicated that several are inducible by ABA in microspore-derived embryos. Of the 200 Arabidopsis genes implicated in lipid biology present on the microarray, 36 were found to be differentially regulated in DGAT transgenic lines. Furthermore, kinetic reverse transcriptase Polymerase Chain Reaction (k-PCR) analysis revealed up-regulation of genes encoding enzymes of the Kennedy pathway involved in assembly of TAGs. Hormone profiling indicated that levels of auxins and cytokinins varied between transgenic lines and untransformed controls, while differences in the pool sizes of ABA and catabolites were only observed at later stages of development., Conclusion: Our results indicate that the increased TAG accumulation observed in transgenic DGAT1 plants is associated with modest transcriptional and hormonal changes during seed development that are not limited to the TAG biosynthesis pathway. These might be associated with feedback or feed-forward effects due to altered levels of DGAT1 activity. The fact that a large fraction of significant amplicons have no matching genes in Arabidopsis compromised our ability to draw concrete inferences from the data at this stage, but has led to the identification of novel genes of potential interest.

Developing seeds of Brassica napus contain significant levels of ABA and products of oxidation at the 7'- and 9'-methyl groups of ABA, 7'- and 9'-hydroxy ABA, as well stable products of oxidation of the 8'-methyl group, phaseic acid and dihydrophaseic acid. To probe the biological roles of the initially formed hydroxylated compounds, we have compared the effects of supplied ABA and the hydroxylated metabolites in regulating oil synthesis in microspore-derived embryos of B. napus, cv Hero that accumulate long chain fatty acids. Uptake into the embryos and metabolism of each of the hormone metabolites was studied by using deuterium labeled analogs. Supplied ABA, which was rapidly metabolized, induced expression of oleosin and fatty acid elongase genes and increased the accumulation of triacylglycerols and very long chain fatty acids. The metabolites 7'- and 9'-hydroxy ABA had similar effects, with the 9'-hydroxy ABA having even greater activity than ABA. The principal catabolite of ABA, 8'-hydroxy ABA, also had hormonal activity and led to increased oil synthesis but induced the genes weakly. These results indicate that all compounds tested could be involved in lipid synthesis in B. napus, and may have hormonal roles in other ABA-regulated processes.

ABSTRACT Systemic acquired resistance (SAR) is an induced defense response that confers long-lasting protection against a broad range of microbial pathogens. Here we show that treatment of Brassica napus plants with the SAR-inducing chemical benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) significantly enhanced resistance against virulent strains of the bacterial pathogen Pseudomonas syringae pv. maculicola and the fungal pathogen Leptosphaeria maculans. Localized preinoculation of plants with an avirulent strain of P. syringae pv. maculicola also enhanced resistance to these pathogens but was not as effective as BTH treatment. Single applications of either SAR-inducing pretreatment were effective against P. syringae pv. maculicola, even when given more than 3 weeks prior to the secondary challenge. The pretreatments also led to the accumulation of pathogenesis-related (PR) genes, including BnPR-1 and BnPR-2, with higher levels of transcripts observed in the BTH-treatment material. B. napus plants expressing a bacterial salicylate hydroxylase transgene (NahG) that metabolizes salicylic acid to catechol were substantially compromised in SAR and accumulated reduced levels of PR gene transcripts when compared with untransformed controls. Thus, SAR in B. napus displays many of the hallmarks of classical SAR including long lasting and broad host range resistance, association with PR gene activation, and a requirement for salicylic acid.

NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) regulates systemic acquired resistance (SAR) in Arabidopsis thaliana, and current models propose that after treatment with salicylic acid (SA), Cys-82 and Cys-216 of NPR1 are reduced, leading to nuclear import. The interaction of nucleus-localized NPR1 with TGA transcription factors results in the activation of defense genes, including the SAR marker PATHOGENESIS-RELATED-1 (PR-1), and the deployment of SAR. Little is known about how TGA factors or NPR1 regulate transcription or whether a TGA-NPR1 complex forms on DNA. We show that TGA2 and NPR1 are recruited to PR-1 independently of each other and of SA treatment. Consistent with the result that a triple knockout in TGA2/5/6 derepresses PR-1, in vivo plant transcription assays revealed that TGA2 is not an autonomous transcription activator but is a transcriptional repressor in both untreated and SA-treated cells. However, after stimulation with SA, TGA2 is incorporated into a transactivating complex with NPR1, forming an enhanceosome that requires the core of the NPR1 BTB/POZ domain (residues 80 to 91) and the oxidation of NPR1 Cys-521 and Cys-529. These Cys residues are found in a new type of transactivation domain that we term Cys-oxidized. These data further our understanding of the mechanism by which TGA2 and NPR1 activate Arabidopsis PR-1.

Changes in gene expression during systemic acquired resistance (SAR) require the phenolic signaling molecule salicylic acid (SA) and are modulated by the interaction between the NON EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) protein and members of the TGA family of transcription factors. In the past two years, the activities of NPR1 and of the TGA factors TGA1 and TGA4 have been shown to be modulated by SA-induced oxidoreduction (redox) modifications of key cysteine residues. Reduction of two conserved cysteines in NPR1 leads to its monomerization and nuclear localization, which is required for the activation of PATHOGENESIS-RELATED(PR) genes. Reduction of conserved cysteines in TGA1 and TGA4 enables their interaction with NPR1, which acts as a redox-sensitive cofactor in stimulating TGA1 DNA-binding activity. The identity of the redox mediators that are involved in regulating NPR1 and TGA factors is unknown. However, a novel thioredoxin interacts with the disease resistance protein Cf-9 and modulates Cf-9-dependent signaling. These results begin to provide a mechanistic understanding of how SAR is regulated by redox conditions.

To date there have been few systematic studies comparing the results of transcript profiling from different microarray platform technologies. We evaluated in detail two different Arabidopsis thaliana microarray platforms: our own Genomic Amplicon arrays and the Qiagen long oligonucleotide arrays designed by Operon; furthermore, we cross-validated these arrays against the Affymetrix AG and ATH1 GeneChips. Data were obtained from all three platforms in each of two separate experiments; (1) at 2 h and (2) 8 h following a salicylic acid treatment applied to both wild-type and npr1-3 mutant plants. A total of 20 hybridizations were performed, analyzing the expression of 26,814 unique locus IDs. We demonstrate that intensity rank is a key variable that affects both inter-platform and cross-platform reproducibility. Although general agreement between platform technologies is low, data derived from high signal intensities (90th percentile) can correlate as well between differing platforms as replicates within the same platform (r=0.4-0.7). We also show that the identification of differentially expressed genes by significance analysis of microarrays is influenced by signal intensity and that overlap between significant gene lists from different platform technologies was as high as 67% when low intensity values were removed. Validation of 41 genes by Northern blot hybridization showed that all platform technologies performed well, qualitatively confirming 83-100% of differential gene expression. Our results suggest that the potential for the broad integration of microarray data from different platforms and laboratories is promising.

The Arabidopsis genome contains six NPR1-related genes. Given the pivotal role played by NPR1 in controlling salicylic acid (SA)-mediated gene expression and disease resistance, functional characterization of other family members appears to be justified. Reverse genetics was used to analyze the role of one NPR1-like gene, which we called NPR4. The NPR4 protein shares 36% identity with NPR1 and interacts with the same spectrum of TGA transcription factors in yeast two-hybrid assays. Plants with T-DNA insertions in NPR4 are more susceptible to the virulent bacterial pathogen Pseudomonas syringe pv. tomato DC3000. This phenotype is complemented by expression of the wild type NPR4 coding region. As determined by the parasite reproduction, the npr4-1 mutant is more susceptible to the fungal pathogen Erysiphe cichoracearum, but does not differ markedly from wild type in its interaction with virulent and avirulent strains of the oomycete Peronospora parasitica. In leaves of wild-type plants, NPR4 mRNA levels increase following pathogen challenge or SA treatment, and decrease rapidly following methyl jasmonic acid (MeJA) treatment. Transcripts of the pathogenesis-related (PR) genes PR-1, PR-2, and PR-5 are only marginally reduced in the npr4-1 mutant following pathogen challenge or SA treatment. This reduction of PR gene expression is more pronounced when leaves are challenged with the bacterial pathogen following SA treatment. Expression of the jasmonic acid-dependent pathway marker gene PDF1.2 is compromised in npr4-1 leaves following application of MeJA or a combination of SA and MeJA. These results indicate that NPR4 is required for basal defense against pathogens, and that it may be implicated in the cross-talk between the SA- and JA-dependent signaling pathways.

Various data mining techniques combined with sequence motif information in the promoter region of genes were applied to discover functional genes that are involved in the defense mechanism of systemic acquired resistance (SAR) in Arabidopsis thaliana. A series of K-Means clustering with difference-in-shape as distance measure was initially applied. A stability measure was used to validate this clustering process. A decision tree algorithm with the discover-and-mask technique was used to identify a group of most informative genes. Appearance and abundance of various transcription factor binding sites in the promoter region of the genes were studied. Through the combination of these techniques, we were able to identify 24 candidate genes involved in the SAR defense mechanism. The candidate genes fell into 2 highly resolved categories, each category showing significantly unique profiles of regulatory elements in their promoter regions. This study demonstrates the strength of such integration methods and suggests a broader application of this approach.

Transcriptional reprogramming is critical for plant disease resistance responses; its global control is not well understood. Salicylic acid (SA) can induce plant defense gene expression and a long-lasting disease resistance state called systemic acquired resistance (SAR). Plant-specific "Whirly" DNA binding proteins were previously implicated in defense gene regulation. We demonstrate that the potato StWhy1 protein is a transcriptional activator of genes containing the PBF2 binding PB promoter element. DNA binding activity of AtWhy1, the Arabidopsis StWhy1 ortholog, is induced by SA and is required for both SA-dependent disease resistance and SA-induced expression of an SAR response gene. AtWhy1 is required for both full basal and specific disease resistance responses. The transcription factor-associated protein NPR1 is also required for SAR. Surprisingly, AtWhy1 activation by SA is NPR1 independent, suggesting that AtWhy1 works in conjunction with NPR1 to transduce the SA signal. Our analysis of AtWhy1 adds a critical component to the SA-dependent plant disease resistance response.

The tomato transcription factor Pti4, an ethylene-responsive factor (ERF), interacts physically with the disease resistance protein Pto and binds the GCC box cis element that is present in the promoters of many pathogenesis-related (PR) genes. We reported previously that Arabidopsis plants expressing Pti4 constitutively express several GCC box-containing PR genes and show reduced disease symptoms compared with wild-type plants after inoculation with Pseudomonas syringae pv tomato or Erysiphe orontii. To gain insight into how genome-wide gene expression is affected by Pti4, we used serial analysis of gene expression (SAGE) to compare transcripts in wild-type and Pti4-expressing Arabidopsis plants. SAGE provided quantitative measurements of >20,000 transcripts and identified the 50 most highly expressed genes in Arabidopsis vegetative tissues. Comparison of the profiles from wild-type and Pti4-expressing Arabidopsis plants revealed 78 differentially abundant transcripts encoding defense-related proteins, protein kinases, ribosomal proteins, transporters, and two transcription factors (TFs). Many of the genes identified were expressed differentially in wild-type Arabidopsis during infection by Pseudomonas syringae pv tomato, supporting a role for them in defense-related processes. Unexpectedly, the promoters of most Pti4-regulated genes did not have a GCC box. Chromatin immunoprecipitation experiments confirmed that Pti4 binds in vivo to promoters lacking this cis element. Potential binding sites for ERF, MYB, and GBF TFs were present in statistically significantly increased numbers in promoters regulated by Pti4. Thus, Pti4 appears to regulate gene expression directly by binding the GCC box and possibly a non-GCC box element and indirectly by either activating the expression of TF genes or interacting physically with other TFs.

The Arabidopsis NPR1 protein is essential for regulating salicylic acid-dependent gene expression during systemic acquired resistance. NPR1 interacts differentially with members of the TGA class of basic domain/Leu zipper transcription factors and regulates their DNA binding activity. Here, we report that although TGA1 does not interact with NPR1 in yeast two-hybrid assays, treatment with salicylic acid induces the interaction between these proteins in Arabidopsis leaves. This phenomenon is correlated with a reduction of TGA1 Cys residues. Furthermore, site-directed mutagenesis of TGA1 Cys-260 and Cys-266 enables the interaction with NPR1 in yeast and Arabidopsis. Together, these results indicate that TGA1 relies on the oxidation state of Cys residues to mediate the interaction with NPR1. An intramolecular disulfide bridge in TGA1 precludes interaction with NPR1, and NPR1 can only stimulate the DNA binding activity of the reduced form of TGA1. Unlike its animal and yeast counterparts, the DNA binding activity of TGA1 is not redox regulated; however, this property is conferred by interaction with the NPR1 cofactor.

To study flower development in the model legume Lotus japonicus, a population of transgenic plants containing a maize transposable element (Ac) in their genome was screened for floral mutants. One mutation named proliferating floral organs (pfo) causes plants to produce a large number of sepal-like organs instead of normal flowers. It segregates as a single recessive Mendelian locus, and causes sterility. Scanning electron microscopy revealed that pfo affects the identity, number and arrangement of floral organs. Sepal-like organs form in the first whorl, and secondary floral meristems are produced in the next whorl. These in turn produce sepal-like organs in the first whorl and floral meristems in the second whorl, and the process is reiterated. Petals and stamens are absent while carpels are either absent or reduced. The pfo phenotype was correlated with the presence of an Ac insertion yielding a 1.6-kb HindIII restriction fragment on Southern blots. Both the mutant phenotype and this Ac element are unstable. Using the transposon as a tag, the Pfo gene was isolated. Conceptual translation of Pfo predicts a protein containing an F-box, with high overall similarity to the Antirrhinum FIMBRIATA, Arabidopsis UNUSUAL FLORAL ORGANS and Pisum sativum Stamina pistilloida proteins. This suggests that Pfo may regulate floral organ identity and meristem determinacy by targeting proteins for ubiquitination.

Three D-cyclin genes are expressed in the apical meristems of snapdragon (Antirrhinum majus). The cyclin D1 and D3b genes are expressed throughout meristems, whereas cyclin D3a is restricted to the peripheral region of the meristem, especially the organ primordia. During floral development, cyclin D3b expression is: (a) locally modulated in the cells immediately surrounding the base of organ primordia, defining a zone between lateral organs that may act as a developmental boundary; (b) locally modulated in the ventral petals during petal folding; and (c) is specifically repressed in the dorsal stamen by the cycloidea gene. Expression of both cyclin D3 genes is reduced prior to the cessation of cell cycle activity, as judged by histone H4 expression. Expression of all three D-cyclin genes is modulated by factors that regulate plant growth, particularly sucrose and cytokinin. These observations may provide a molecular basis for understanding the local regulation of cell proliferation during plant growth and development.

The Arabidopsis NPR1 gene is essential in activating systemic, inducible plant defense responses. To gain a better understanding of NPR1 function, we conducted a yeast two-hybrid screening procedure and identified a differential interaction between NPR1 and all known members of the Arabidopsis TGA family of basic leucine zipper transcription factors. In the electrophoretic mobility shift assay, NPR1 substantially increased the binding of TGA2 to its cognate promoter element (as-1) as well as to a positive salicylic acid-inducible element (LS7) and a negative element (LS5) in the promoter of the pathogenesis-related PR-1 gene. Proteins encoded by npr1 mutants interacted poorly with TGA2 and did not substantially increase TGA2 binding to the as-1, LS5, or LS7 elements, thus establishing a link between the loss of disease resistance and the loss of TGA2 interaction and NPR1-enhanced DNA binding. Coupled with observations that the DNA binding activity of TGA factors is deregulated in npr1 plants, the results suggest that NPR1-mediated DNA binding of TGA2 is critical for activation of defense genes.

Cyclin-dependent kinases (CDKs) are important regulators of the eukaryotic cell division cycle. To study protein-protein interactions involving plant CDKs, the Arabidopsis thaliana Cdc2aAt was used as bait in the yeast two-hybrid system. Here we report on the isolation of ICK2, and show that it interacts with Cdc2aAt, but not with a second CDK from Arabidopsis, Cdc2bAt. ICK2 contains a carboxy-terminal domain related to that of ICK1, a previously described CDK inhibitor from Arabidopsis, and to the CDK-binding domain of the mammalian inhibitor p27Kip1. Outside of this domain, ICK2 is distinct from ICK1, p27Kip1, and other proteins. At nanogram levels (8 nM), purified recombinant ICK2 inhibits p13Suc1-associated histone H1 kinase activity from Arabidopsis tissue extracts, demonstrating that it is a potent inhibitor of plant CDK activity in vitro. ICK2 mRNA was present in all tissues analysed by Northern hybridization, and its distribution was distinct from that of ICK1. These results demonstrate that plants possess a family of differentially regulated CDK inhibitors that contain a conserved carboxy terminal but with distinct amino terminal regions.

We have isolated a constitutive promoter sequence, tCUP, from tobacco by T-DNA tagging using a promoterless GUS-nos3' reporter gene construct. The T-DNA integration event produced a translational fusion with the GUS gene that is expressed widely in organs, at both the mRNA and enzyme activity levels. In tobacco transformed with a tCUP-GUS-nos3' gene, GUS specific activity in leaves was within a range of values similar to those of plants transformed with the widely used constitutive promoter gene fusion, CaMV 35S promoter-GUS-nos3'. Characteristics of the tCUP promoter sequence differ from those of other plant constitutive promoters; for instance, the tCUP sequence lacks a TATA box. Transcription initiates at a single site within the tCUP sequence which is similar to a transcriptional start site consensus sequence determined for plant genes. The tCUP promoter is cryptic as RNA accumulation at the transcriptional start site is not detected in untransformed tobacco. Thus, tCUP is the first example of a cryptic, constitutive promoter isolated from plants. The tCUP-GUS-nos3' gene fusion produced GUS activity in tissues of all species tested suggesting that tCUP may utilize fundamental transcription mechanisms found in plants.

cdc2 and several related genes encode the catalytic subunits of cyclin-dependent kinases, which have been implicated in a number of cellular processes, including control of cell division. As a first step in exploring their function in plants, we isolated four cdc2-related genes from Antirrhinum. Two genes, cdc2a and cdc2b, encode proteins that contain a perfectly conserved PSTAIRE motif characteristic of cdc2 homologs, whereas the products of the two remaining genes, cdc2c and cdc2d, appear to represent a new subclass of proteins that have so far only been identified in plants. Transcripts of these novel genes were localized in isolated cells dispersed throughout actively dividing regions of the inflorescence. This localization is consistent with accumulation that is specific to particular phases of the cell cycle. Correlating cell labeling with nuclear condensation and double-labeling experiments using cdc2 and histone H4 as probes indicated that cdc2c transcripts accumulate during S phase as well as during the G2 and M transition, whereas cdc2d expression was specific to the G2 and M phases. All cells labeled with cdc2d also contained cdc2c label, Indicating that expression of cdc2d completely overlapped with that of cdc2c. Transcripts of cdc2a and cdc2b were detected in all cells within actively dividing regions, but at levels that were only slightly higher than those observed in nondividing areas. These transcripts did not appear to accumulate in a cell cycle-specific fashion. The genes cdc2a and cdc2b were able to partially complement a yeast cdc2 mutation, although all four genes appeared to interfere with the sizing mechanism of yeast cells. We propose that plants contain at least two classes of cdc2-related genes that differ in structure, expression, and perhaps function.

T-DNA tagging with a promoterless beta-glucuronidase (GUS) gene generated a transgenic Nicotiana tabacum plant that expressed GUS activity only in developing seed coats. Cloning and deletion analysis of the GUS fusion revealed that the promoter responsible for seed coat specificity was located in the plant DNA proximal to the GUS gene. A 3.3 kb fragment corresponding to the insertion site was isolated from untransformed plants. No long open reading frames were detected in this region. Northern blots and RNase protection assays failed to detect transcripts from this region in untransformed plants. Furthermore, the insertion site was situated within the N. tomentosiformis genome of the allotetraploid species N. tabacum, in a region which is not conserved within the genus Nicotiana. It is concluded that seed coat-specific GUS expression in this transgenic plant resulted from T-DNA insertion next to a cryptic promoter. These results suggest that at least some of the fusions generated to marker genes in promoter trapping studies are not associated with conventional gene promoters. The possibility that similar insertion events play a role in gene evolution is discussed.

Transcripts from five cell cycle related genes accumulate in isolated cells dispersed throughout the actively dividing regions of plant meristems. We propose that this pattern reflects gene expression during particular phases of the cell division cycle. The high proportion of isolated cells suggests that synchrony between daughter cells is rapidly lost following mitosis. This is the first time that such a cell specific expression pattern has been described in a higher organism. Counterstaining with a DNA specific dye revealed that transcripts from three genes (two mitotic cyclins and a cdc2-like gene) accumulate during part of interphase and early mitosis whereas transcripts from a histone H4 gene are preferentially detected only in interphase cells. Double labelling for cyclin and histone H4 transcripts confirms that these genes are expressed in different cells, and therefore at different phases of the cell cycle. The results suggest that transcriptional regulation of cell cycle related genes may be important in controlling cell division in plants, and that these genes are useful markers for identifying cells at specific phases of the cell cycle within plant meristems.

A binary vector, pPRF120, was designed to detect T-DNA insertions within transcriptionally active areas of the plant genome. Linked to the right-border repeat, the vector contains a promoterless beta-glucuronidase (GUS) gene which can, upon integration into chromosomes, be activated by cis-acting regulatory elements. The vector also incorporates a chimeric marker gene conferring resistance to kanamycin to ensure recovery of gene fusions regardless of the extent of their tissue-specific or developmentally regulated expression, and to permit analysis of the frequency of plants which express the promoterless reporter. Approximately 1000 transgenic tobacco plants harboring pPRF120 were regenerated. Analysis of 52 individuals indicated that more than 80% contain single, intact copies of the T-DNA, regardless of their ability to express the promoterless GUS gene. Screening of leaf tissue from the 1000 pPRF120 transformants revealed that ca. 5% of the plants contained GUS activity. Fluorogenic and histological GUS assays were used to visualize and quantify tissue- and cell-specific gene expression. The potential usefulness of pPRF120 in comparison to other vectors designed to generate in vivo gene fusions is discussed.

The specificity between the sym-2 gene bred into certain cultivars of pea (Pisum sativum L.) and the nodX gene, present only rarely in isolates of Rhizobium leguminosarum, can be exploited to preempt competition or nodulation blocking by a Rhizobium strain indigenous to a soil environment. The principle is to isolate an R. leguminosarum strain prevalent in a locale, convert it into a strain that will nodulate a desirable pea cultivar carrying sym-2 by establishing nodX in it, and then use the resulting Rhizobium strain with the pea cultivar carrying sym-2. To accomplish this, we first constructed a transposon Tn5 derivative called Tn5nodX and an efficient delivery vehicle that is suicidal in R. leguminosarum. We tested the potential utility of the system in greenhouse experiments. The results are encouraging enough to warrant extensive experiments under field conditions.

The minimal replicon of the incompatibility N group plasmid pCU1 is contained within a 2-kb DNA region of the plasmid. The ability of this region and of the deletion derivatives thereof, that are capable of autonomous maintenance, to direct polypeptide synthesis was examined. Two proteins of 27 and 5.5 kDa are encoded by the minimal replicon. Polypeptide chain-terminating mutations within the predicted open reading frame for the 27-kDa polypeptide abolished the synthesis of this polypeptide and also the Escherichia coli polA-independence phenotype of the pCU1 replicon. However, these mutations did not affect the autonomous replication ability of the pCU1 replicon in wild-type E. coli and the expression of incompatibility towards the parental plasmid.