Spatial identification of transcripts and biological processes in laser micro-dissected sub-regions of waterlogged corn roots with altered expression of phytoglobin.

Over-expression of the corn phytoglobin ZmPgb1.2 increases tolerance to waterlogging, while suppression of ZmPgb1.2 compromises plant growth. To unravel compartment-specific transcriptional changes evoked by ZmPgb1.2 during hypoxia, laser micro-dissected sub-regions from waterlogged roots of WT and ZmPgb1.2 overexpressing [ZmPgb1.2(S)] plants were probed for global transcriptional analysis using next generation RNA sequencing. These sub-regions included compartments within the meristematic, elongation, and maturation zone. Of the 149 genes differentially expressed by the up-regulation of ZmPgb1.2, 78 occurred within the meristematic region and included genes involved in jasmonic acid synthesis and response, ascorbic acid metabolism, and ethylene signalling. The ZmPgb1.2 regulation of these genes, discussed in the context of known functions of Pgbs, was further validated by monitoring their expression in meristematic cells of waterlogged roots suppressing ZmPgb1.2. Of the 27 genes differentially expressed by the over-expression of ZmPgb1.2 in the elongation zone, pyruvate kinase and alcohol dehydrogenase showed an expression pattern correlated to the level of ZmPgb1.2 in the tissue. The transcriptional induction of these two enzymes in hypoxic domains of the elongation zone over-expressing ZmPgb1.2 suggests the activation of the fermentation pathway which might be required to sustain metabolic flux and production of ATP in support of cell elongation.

The biocontrol agent Pseudomonas chlororaphis PA23 primes Brassica napus defenses through distinct gene networks.

BACKGROUND: The biological control agent Pseudomonas chlororaphis PA23 is capable of protecting Brassica napus (canola) from the necrotrophic fungus Sclerotinia sclerotiorum via direct antagonism. While we have elucidated bacterial genes and gene products responsible biocontrol, little is known about how the host plant responds to bacterial priming on the leaf surface, including global changes in gene activity in the presence and absence of S. sclerotiorum. RESULTS: Application of PA23 to the aerial surfaces of canola plants reduced the number of S. sclerotiorum lesion-forming petals by 91.1%. RNA sequencing of the host pathogen interface showed that pretreatment with PA23 reduced the number of genes upregulated in response to S. sclerotiorum by 16-fold. By itself, PA23 activated unique defense networks indicative of defense priming. Genes encoding MAMP-triggered immunity receptors detecting flagellin and peptidoglycan were downregulated in PA23 only-treated plants, consistent with post-stimulus desensitization. Downstream, we observed reactive oxygen species (ROS) production involving low levels of H2O2 and overexpression of genes associated with glycerol-3-phosphate (G3P)-mediated systemic acquired resistance (SAR). Leaf chloroplasts exhibited increased thylakoid membrane structures and chlorophyll content, while lipid metabolic processes were upregulated. CONCLUSION: In addition to directly antagonizing S. sclerotiorum, PA23 primes the plant defense response through induction of unique local and systemic defense networks. This study provides novel insight into the effects of biocontrol agents applied to the plant phyllosphere. Understanding these interactions will aid in the development of biocontrol systems as an alternative to chemical pesticides for protection of important crop systems.

Transcriptome analysis of the Brassica napus-Leptosphaeria maculans pathosystem identifies receptor, signaling and structural genes underlying plant resistance.

The hemibiotrophic fungal pathogen Leptosphaeria maculans is the causal agent of blackleg disease in Brassica napus (canola, oilseed rape) and causes significant loss of yield worldwide. While genetic resistance has been used to mitigate the disease by means of traditional breeding strategies, there is little knowledge about the genes that contribute to blackleg resistance. RNA sequencing and a streamlined bioinformatics pipeline identified unique genes and plant defense pathways specific to plant resistance in the B. napus-L. maculans LepR1-AvrLepR1 interaction over time. We complemented our temporal analyses by monitoring gene activity directly at the infection site using laser microdissection coupled to quantitative PCR. Finally, we characterized genes involved in plant resistance to blackleg in the Arabidopsis-L. maculans model pathosystem. Data reveal an accelerated activation of the plant transcriptome in resistant host cotyledons associated with transcripts coding for extracellular receptors and phytohormone signaling molecules. Functional characterization provides direct support for transcriptome data and positively identifies resistance regulators in the Brassicaceae. Spatial gradients of gene activity were identified in response to L. maculans proximal to the site of infection. This dataset provides unprecedented spatial and temporal resolution of the genes required for blackleg resistance and serves as a valuable resource for those interested in host-pathogen interactions.

Tissue-specific laser microdissection of the Brassica napus funiculus improves gene discovery and spatial identification of biological processes.

The three primary tissue systems of the funiculus each undergo unique developmental programs to support the growth and development of the filial seed. To understand the underlying transcriptional mechanisms that orchestrate development of the funiculus at the globular embryonic stage of seed development, we used laser microdissection coupled with RNA-sequencing to produce a high-resolution dataset of the mRNAs present in the epidermis, cortex, and vasculature of the Brassica napus (canola) funiculus. We identified 7761 additional genes in these tissues compared with the whole funiculus organ alone using this technology. Differential expression and enrichment analyses were used to identify several biological processes associated with each tissue system. Our data show that cell wall modification and lipid metabolism are prominent in the epidermis, cell growth and modification occur in the cortex, and vascular tissue proliferation and differentiation occur in the central vascular strand. We provide further evidence that each of the three tissue systems of the globular stage funiculus are involved in specific biological processes that all co-ordinate to support seed development. The identification of genes and gene regulators responsible for tissue-specific developmental processes of the canola funiculus now serves as a valuable resource for seed improvement research.

Chalazal seed coat development in Brassica napus.

The chalazal seed coat (CZSC) is a maternal subregion adjacent to the funiculus which serves as the first point of entry into the developing seed. This subregion is of particular interest in Brassica napus (canola) because of its location within the seed and its putative contribution to seed filling processes. In this study, the CZSC of canola was characterized at an anatomical and molecular level to (i) describe the cellular and subcellular features of the CZSC throughout seed development, (ii) reveal cellular features of the CZSC that relate to transport processes, (iii) study gene activity of transporters and transcriptional regulators in the CZSC subregion over developmental time, and (iv) briefly investigate the contribution of the A and C constituent genomes to B. napus CZSC gene activity. We found that the CZSC contains terminating ends of xylem and phloem as well as a mosaic of endomembrane and plasmodesmatal connections, suggesting that this subregion is likely involved in the transport of material and information from the maternal tissues of the plant to other regions of the seed. Laser microdissection coupled with quantitative RT-PCR identified the relative abundance of sugar, water, auxin and amino acid transporter homologs inherited from the constituent genomes of this complex polyploid. We also studied the expression of three transcription factors that were shown to co-express with these biological processes providing a preliminary framework for the regulatory networks responsible for seed filling in canola and discuss the relationship of the CZSC to other regions and subregions of the seed and its role in seed development.

Transcriptome atlas of the Arabidopsis funiculus--a study of maternal seed subregions.

The funiculus anchors the structurally complex seed to the maternal plant, and is the only direct route of transport for nutrients and maternal signals to the seed. While our understanding of seed development is becoming clearer, current understanding of the genetics and cellular mechanisms that contribute to funiculus development is limited. Using laser microdissection combined with global RNA-profiling experiments we compared the genetic profiles of all maternal and zygotic regions and subregions during seed development. We found that the funiculus is a dynamic region of the seed that is enriched for mRNAs associated with hormone metabolism, molecular transport, and metabolic activities corresponding to biological processes that have yet to be described in this maternal seed structure. We complemented our genetic data with a complete histological analysis of the funiculus from the earliest stages of development through to seed maturation at the light and electron microscopy levels. The anatomy revealed signs of photosynthesis, the endomembrane system, cellular respiration, and transport within the funiculus, all of which supported data from the transcriptional analysis. Finally, we studied the transcriptional programming of the funiculus compared to other seed subregions throughout seed development. Using newly designed in silico algorithms, we identified a number of transcriptional networks hypothesized to be responsible for biological processes like auxin response and glucosinolate biosynthesis found specifically within the funiculus. Taken together, patterns of gene activity and histological observations reveal putative functions of the understudied funiculus region and identify predictive transcriptional circuits underlying these biological processes in space and time.

Predicting transcriptional circuitry underlying seed coat development.

Filling, protection, and dispersal of angiosperm seeds are largely dependent on the development of the maternally derived seed coat. The development of the seed coat in plants such as Arabidopsis thaliana and Glycine max (soybean) is regulated by a complex network of genes and gene products responsible for the establishment and identity of this multicellular structure. Recent studies support the hypothesis that the structure, development, and function of the seed coat are under the control of transcriptional regulators that are specified in space and time. Furthermore, these transcriptional regulators can act in combination to orchestrate the expression of large gene sets. We discuss the underlying transcriptional circuits of the seed coat sub-regions through the interrogation of large-scale datasets, and also provide some ideas on how the identification and analysis of these datasets can be further improved in these two model oilseed systems.

Transcriptional circuitry underlying seed coat development in Arabidopsis.

We analyzed two sub-regions of the maternal seed coat, chalazal (CZSC) and distal (SC), using transcriptomic and histological analyses in the model plant Arabidopsis thaliana. Hierarchical clustering analysis showed that the CZSC and SC are transcriptionally distinct, though the two sub-regions are more similar during early stages of seed development. Robust statistical and network analysis revealed novel roles for both sub-regions during the course of the seed lifecycle and provides insight into the regulatory circuitry underlying these poorly studied sub-regions of the seed. Data show many of the processes that characterize the SC including starch deposition during the morphogenesis phase, and mucilage deposition and cell wall thickening during the maturation phase, are either absent or expressed to a much lesser extent in the CZSC. We further analyzed the CZSC in detail and show that this sub-region is likely involved in the control of information into the seed from the maternal plant and that some of these processes are predicted to operate through the activity of bZIP transcription factors through the G-box DNA sequence motif.