[Show abstract][Hide abstract] ABSTRACT: Fatty acids in seeds affect seed germination and seedling vigour and fatty acid composition determines the quality of seed oil. In this study, quantitative trait locus (QTL) mapping of fatty acid and transcript abundance was integrated with gene network analysis to unravel the genetic regulation of seed fatty acid composition in a Brassica rapa doubled haploid population from a cross between a yellow sarson oil-type and a black seeded pak choi. The distribution of major QTLs for fatty acids showed a relationship with the fatty acid types: linkage group A03 for mono-unsaturated fatty acids (MUFAs), A04 for saturated fatty acids (SFAs) and A05 for poly-unsaturated fatty acids (PUFAs). Using a genetical genomics approach, expression QTL (eQTL) hotspots were found at major fatty acid QTLs on linkage groups A03, A04, A05 and A09. An eQTL-guided gene co-expression network of lipid metabolism related genes showed major hubs at the genes BrPLA2-ALPHA, BrWD-40, a number of seed storage protein genes and a transcription factor BrMD-2, suggesting essential roles for these genes in lipid metabolism. Three sub-networks were extracted for the economically important and most abundant fatty acids erucic-, oleic-, linoleic- and linolenic acid. Network analysis, combined with comparison of genome positions of cis- or trans- eQTLs with fatty acid QTLs, allowed identification of candidate genes for genetic regulation of these fatty acids. The generated insights in the genetic architecture of fatty acid composition and underlying complex gene regulatory networks in B. rapa seeds are discussed.
No preview · Article · Oct 2015 · Plant physiology
[Show abstract][Hide abstract] ABSTRACT: 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. In the current study, Fourier transform infrared (FTIR) spectroscopy (4000–800 cm−1 ) was used of bulk samples of florets and rachises to better understand the mechanism of FHB resistance. Three cultivars, resistant (‘Sumai3’), moderately resistant (‘FL62R1’) and susceptible (‘Muchmore’) to FHB, were compared. Changes in absorption spectra following inoculation were observed mostly between 3400–800 cm−1 , and this range of FTIR spectra was analysed using integrated area of absorption bands to identify differences among the cultivars, and between diseased and healthy florets and rachises. Marked differences were observed in association with amide I, aromatic, carbonyl ester, phosphate, CH2 and functional groups between infected and non-infected spikes. In the rachis of resistant ‘Sumai3’, the bands of 1460 cm−1 , 1650 cm−1 and 1615–1590 cm−1 representing CH2, amide I and aromatics, respectively, were persistent after infection at 4 and 10 days post-inoculation compared with those from the other two cultivars. These bands may be candidate biochemical markers for FHB resistance. The presence of these bands was more consistent in the rachis than in floret. The histological comparison of rachis showed many differences in the cell wall of the wheat cultivars before and after infection. It is concluded that FTIR spectroscopy can be a useful method in understanding the biochemical modes of action to FHB based on metabolic changes.
[Show abstract][Hide abstract] ABSTRACT: 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. 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 PCI images revealed significant differences between infected and non-infected rachis of different wheat cultivars. 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 FTIR spectra were analyzed using Principal Component Analysis (PCA) to determine the difference between diseased and healthy florets or rachises of wheat spikes for two time periods (4 and 10 Days After Inoculation (DAI)). The 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 significant differences in cell wall related proteins and 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. The combined technique may be used as a supplemental tool to other commonly used techniques such as optical or electron microscopy, proteomics, and RNA sequencing in investigating the resistance mechanisms of wheat cultivars to FHB.
Full-text · Article · Nov 2014 · BMC Plant Biology
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Aug 2012 · Molecular Plant-Microbe Interactions
[Show abstract][Hide abstract] ABSTRACT: Nowadays, it is possible to collect expression levels of a set of genes from a set of biological samples during a series of time points. Such data have three dimensions: gene-sample-time (GST). Thus they are called 3D microarray gene expression data. To take advantage of the 3D data collected, and to fully understand the biological knowledge hidden in the GST data, novel subspace clustering algorithms have to be developed to effectively address the biological problem in the corresponding space.
We developed a subspace clustering algorithm called Order Preserving Triclustering (OPTricluster), for 3D short time-series data mining. OPTricluster is able to identify 3D clusters with coherent evolution from a given 3D dataset using a combinatorial approach on the sample dimension, and the order preserving (OP) concept on the time dimension. The fusion of the two methodologies allows one to study similarities and differences between samples in terms of their temporal expression profile. OPTricluster has been successfully applied to four case studies: immune response in mice infected by malaria (Plasmodium chabaudi), systemic acquired resistance in Arabidopsis thaliana, similarities and differences between inner and outer cotyledon in Brassica napus during seed development, and to Brassica napus whole seed development. These studies showed that OPTricluster is robust to noise and is able to detect the similarities and differences between biological samples.
Our analysis showed that OPTricluster generally outperforms other well known clustering algorithms such as the TRICLUSTER, gTRICLUSTER and K-means; it is robust to noise and can effectively mine the biological knowledge hidden in the 3D short time-series gene expression data.
[Show abstract][Hide abstract] ABSTRACT: Systemic Acquired Resistance (SAR) is an induced resistance response to pathogens, characterized by the translocation of a long-distance signal from induced leaves to distant tissues to prime them for increased resistance to future infection. DEFECTIVE in INDUCED RESISTANCE 1 (DIR1) has been hypothesized to chaperone a small signaling molecule to distant tissues during SAR in Arabidopsis.
DIR1 promoter:DIR1-GUS/dir1-1 lines were constructed to examine DIR1 expression. DIR1 is expressed in seedlings, flowers and ubiquitously in untreated or mock-inoculated mature leaf cells, including phloem sieve elements and companion cells. Inoculation of leaves with SAR-inducing avirulent or virulent Pseudomonas syringae pv tomato (Pst) resulted in Type III Secretion System-dependent suppression of DIR1 expression in leaf cells. Transient expression of fluorescent fusion proteins in tobacco and intercellular washing fluid experiments indicated that DIR1's ER signal sequence targets it for secretion to the cell wall. However, DIR1 expressed without a signal sequence rescued the dir1-1 SAR defect, suggesting that a cytosolic pool of DIR1 is important for the SAR response.
Although expression of DIR1 decreases during SAR induction, the protein localizes to all living cell types of the vasculature, including companion cells and sieve elements, and therefore DIR1 is well situated to participate in long-distance signaling during SAR.
[Show abstract][Hide abstract] ABSTRACT: The architecture of the Brassica napus genome is marked by its evolutionary origins. The genome of B. napus was formed from the hybridization of two closely related diploid Brassica species, both of which evolved from an hexaploid ancestor. The extensive whole genome duplication events in its near and distant past result in the allotetraploid genome of B. napus maintaining multiple copies of most genes, which predicts a highly complex and redundant transcriptome that can confound any expression analyses. A stringent assembly of 142,399 B. napus expressed sequence tags allowed the development of a well-differentiated set of reference transcripts, which were used as a foundation to assess the efficacy of available tools for identifying and distinguishing transcripts in B. napus; including microarray hybridization and 3' anchored sequence tag capture. Microarray platforms cannot distinguish transcripts derived from the two progenitors or close homologues, although observed differential expression appeared to be biased towards unique transcripts. The use of 3' capture enhanced the ability to unambiguously identify homologues within the B. napus transcriptome but was limited by tag length. The ability to comprehensively catalogue gene expression in polyploid species could be transformed by the application of cost-efficient next generation sequencing technologies that will capture millions of long sequence tags.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Vast amount of data in various forms have been accumulated through many years of functional genomic research throughout the
world. It is a challenge to discover and disseminate knowledge hidden in these data. Many computational methods have been
developed to solve this problem. Taking analysis of the microarray data as an example, we spent the past decade developing
many data mining strategies and software tools. It appears still insufficient to cover all sources of data. In this paper,
we summarize our experiences in mining microarray data by using two plant species, Brassica napus and Arabidopsis thaliana, as examples. We present several successful stories and also a few lessons learnt. The domain problems that we dealt with
were the transcriptional regulation in seed development and during defense response against pathogen infection.
[Show abstract][Hide abstract] ABSTRACT: We studied defense mechanism of the Arabidopsis thaliana subjected to Salicylic Acid (SA) treatment for 0, 1, and 8 hours using a broader application of the frequent itemset approach. Four genotypes of the plant were used in this study, Columbia wild type, mutant npr1-3, double mutant tga1 tga4 and triple mutant tga2 tga5 tga6. We defined the major patterns of transcription regulation governing pathogen defense mechanism, thereby creating a model of the Systemic Acquired Resistance (SAR) at three time points. The temporal model describes the relationships among the regulators and defines groups of genes that are subject to similar regulation. The results obtained offered a first glimpse into the temporal pattern of the transcription regulatory network during SAR in Arabidopsis thaliana. We found that most of the genes that responded to SA challenge are in fact dependent on one or more of the NPR1 and TGA factors tested in this study.