Genetic manipulation of putrescine biosynthesis reprograms the cellular transcriptome and the metabolome.

BACKGROUND: With the increasing interest in metabolic engineering of plants using genetic manipulation and gene editing technologies to enhance growth, nutritional value and environmental adaptation, a major concern is the potential of undesirable broad and distant effects of manipulating the target gene or metabolic step in the resulting plant. A comprehensive transcriptomic and metabolomic analysis of the product may shed some useful light in this regard. The present study used these two techniques with plant cell cultures to analyze the effects of genetic manipulation of a single step in the biosynthesis of polyamines because of their well-known roles in plant growth, development and stress responses. RESULTS: The transcriptomes and metabolomes of a control and a high putrescine (HP) producing cell line of poplar (Populus nigra x maximowiczii) were compared using microarrays and GC/MS. The HP cells expressed an ornithine decarboxylase transgene and accumulated several-fold higher concentrations of putrescine, with only small changes in spermidine and spermine. The results show that up-regulation of a single step in the polyamine biosynthetic pathway (i.e. ornithine → putrescine) altered the expression of a broad spectrum of genes; many of which were involved in transcription, translation, membrane transport, osmoregulation, shock/stress/wounding, and cell wall metabolism. More than half of the 200 detected metabolites were significantly altered (p ≤ 0.05) in the HP cells irrespective of sampling date. The most noteworthy differences were in organic acids, carbohydrates and nitrogen-containing metabolites. CONCLUSIONS: The results provide valuable information about the role of polyamines in regulating nitrogen and carbon use pathways in cell cultures of high putrescine producing transgenic cells of poplar vs. their low putrescine counterparts. The results underscore the complexity of cellular responses to genetic perturbation of a single metabolic step related to nitrogen metabolism in plants. Combined with recent studies from our lab, where we showed that higher putrescine production caused an increased flux of glutamate into ornithine concurrent with enhancement in glutamate production via additional nitrogen and carbon assimilation, the results from this study provide guidance in designing transgenic plants with increased nitrogen use efficiency, especially in plants intended for non-food/feed applications (e.g. increased biomass production for biofuels).

Using next generation transcriptome sequencing to predict an ectomycorrhizal metabolome.

BACKGROUND: Mycorrhizae, symbiotic interactions between soil fungi and tree roots, are ubiquitous in terrestrial ecosystems. The fungi contribute phosphorous, nitrogen and mobilized nutrients from organic matter in the soil and in return the fungus receives photosynthetically-derived carbohydrates. This union of plant and fungal metabolisms is the mycorrhizal metabolome. Understanding this symbiotic relationship at a molecular level provides important contributions to the understanding of forest ecosystems and global carbon cycling. RESULTS: We generated next generation short-read transcriptomic sequencing data from fully-formed ectomycorrhizae between Laccaria bicolor and aspen (Populus tremuloides) roots. The transcriptomic data was used to identify statistically significantly expressed gene models using a bootstrap-style approach, and these expressed genes were mapped to specific metabolic pathways. Integration of expressed genes that code for metabolic enzymes and the set of expressed membrane transporters generates a predictive model of the ectomycorrhizal metabolome. The generated model of mycorrhizal metabolome predicts that the specific compounds glycine, glutamate, and allantoin are synthesized by L. bicolor and that these compounds or their metabolites may be used for the benefit of aspen in exchange for the photosynthetically-derived sugars fructose and glucose. CONCLUSIONS: The analysis illustrates an approach to generate testable biological hypotheses to investigate the complex molecular interactions that drive ectomycorrhizal symbiosis. These models are consistent with experimental environmental data and provide insight into the molecular exchange processes for organisms in this complex ecosystem. The method used here for predicting metabolomic models of mycorrhizal systems from deep RNA sequencing data can be generalized and is broadly applicable to transcriptomic data derived from complex systems.

Using deep RNA sequencing for the structural annotation of the Laccaria bicolor mycorrhizal transcriptome.

BACKGROUND: Accurate structural annotation is important for prediction of function and required for in vitro approaches to characterize or validate the gene expression products. Despite significant efforts in the field, determination of the gene structure from genomic data alone is a challenging and inaccurate process. The ease of acquisition of transcriptomic sequence provides a direct route to identify expressed sequences and determine the correct gene structure. METHODOLOGY: We developed methods to utilize RNA-seq data to correct errors in the structural annotation and extend the boundaries of current gene models using assembly approaches. The methods were validated with a transcriptomic data set derived from the fungus Laccaria bicolor, which develops a mycorrhizal symbiotic association with the roots of many tree species. Our analysis focused on the subset of 1501 gene models that are differentially expressed in the free living vs. mycorrhizal transcriptome and are expected to be important elements related to carbon metabolism, membrane permeability and transport, and intracellular signaling. Of the set of 1501 gene models, 1439 (96%) successfully generated modified gene models in which all error flags were successfully resolved and the sequences aligned to the genomic sequence. The remaining 4% (62 gene models) either had deviations from transcriptomic data that could not be spanned or generated sequence that did not align to genomic sequence. The outcome of this process is a set of high confidence gene models that can be reliably used for experimental characterization of protein function. CONCLUSIONS: 69% of expressed mycorrhizal JGI "best" gene models deviated from the transcript sequence derived by this method. The transcriptomic sequence enabled correction of a majority of the structural inconsistencies and resulted in a set of validated models for 96% of the mycorrhizal genes. The method described here can be applied to improve gene structural annotation in other species, provided that there is a sequenced genome and a set of gene models.

Transcriptomic comparison in the leaves of two aspen genotypes having similar carbon assimilation rates but different partitioning patterns under elevated [CO2].

This study compared the leaf transcription profiles, physiological characteristics and primary metabolites of two Populus tremuloides genotypes (clones 216 and 271) known to differ in their responses to long-term elevated [CO2] (e[CO2]) at the Aspen free-air CO2 enrichment site near Rhinelander, WI, USA. The physiological responses of these clones were similar in terms of photosynthesis, stomatal conductance and leaf area index under e[CO2], yet very different in terms of growth enhancement (0-10% in clone 216; 40-50% in clone 271). Although few genes responded to long-term exposure to e[CO2], the transcriptional activity of leaf e[CO2]-responsive genes was distinctly different between the clones, differentially impacting multiple pathways during both early and late growing seasons. An analysis of transcript abundance and carbon/nitrogen biochemistry suggested that the CO2-responsive clone (271) partitions carbon into pathways associated with active defense/response to stress, carbohydrate/starch biosynthesis and subsequent growth. The CO2-unresponsive clone (216) partitions carbon into pathways associated with passive defense (e.g. lignin, phenylpropanoid) and cell wall thickening. This study indicates that there is significant variation in expression patterns between different tree genotypes in response to long-term exposure to e[CO2]. Consequently, future efforts to improve productivity or other advantageous traits for carbon sequestration should include an examination of genetic variability in CO2 responsiveness.

Comparative proteomic analysis of Botrytis cinerea secretome.

Botrytis cinerea (B. cinerea) is a filamentous fungus infecting more than 200 plant species, causing significant economic losses worldwide. Secreted proteins are released as an initial response of the fungus to its plant host. We report the use of a high-throughput LC-MS/MS approach to analyze B. cinerea BO5.10 secreted proteins. Secretions were collected from fungus grown on a solid substrate of cellophane membrane while mock infecting media supplemented with the extract of full red tomato, ripened strawberry or Arabidopsis leaf extract. Overall, 89 B. cinerea proteins were identified from all growth conditions. Sixty proteins were predicted to contain a SignalP motif indicating the extracellular location of the proteins. Seven proteins were observed in all the growth conditions implying a constitutive nature of their secretion. Identified in the secretions were transport proteins, proteins well-characterized for carbohydrate metabolism, peptidases, oxidation/reduction, and pathogenicity factors that provide important insights into how B. cinerea may use secreted proteins for plant infection and colonization.

Cold induced Botrytis cinerea enolase (BcEnol-1) functions as a transcriptional regulator and is controlled by cAMP.

Botrytis cinerea is a necrotrophic fungal plant pathogen that can survive, grow and infect crops under cold stress. In an attempt to understand the molecular mechanisms leading to cold tolerance of this phytopathogen, we identified an enolase, BcEnol-1. BcEnol-1 encodes a 48 kDa protein that shows high identity to yeast, Arabidopsis and human enolases (72, 63 and 63%, respectively). Northern analysis confirms that an increase in transcript abundance of BcEnol-1 was observed when B. cinerea mycelium was shifted from 22 to 4 degrees C. In order to understand its regulation during cold stress, BcEnol-1 expression was studied in B. cinerea mutants viz Deltabcg1 (mutant of B. cinerea for bcg1), Deltabcg3 (mutant of B. cinerea for bcg3) and Deltabac (mutant of B. cinerea for adenylate cyclase). A decrease in enolase expression in these mutants was observed during cold stress suggesting enolase activation by a cAMP mediated cascade. Expression of enolase was restored with the exogenous addition of cAMP to the Deltabac mutant. Recombinant enolase protein was also found to bind to the promoter elements of transcripts belonging to the Zinc-C(6) protein family and calpain like proteases. Based on these results we conclude that enolase from Botrytis is cold responsive, influenced by cAMP and acts putatively as a transcriptional regulator.

High efficiency poplar transformation.

With the completion of the poplar tree genome database, Populus species have become one of the most useful model systems for the study of woody plant biology. Populus tremuloides (quaking aspen) is the most wide-spread tree species in North America, and its rapid growth generates the most abundant wood-based biomass out of any other plant species. To study such beneficial traits, there is a need for easier and more efficient transformation procedures that will allow the study of large numbers of tree genes. We have developed transformation procedures that are suitable for high-throughput format transformations using either Agrobacterium tumefaciens to produce transformed trees or Agrobacterium rhizogenes to generate hairy roots. Our method uses Agrobacterium inoculated aspen seedling hypocotyls followed by direct thidiazuron (TDZ)-mediated shoot regeneration on selective media. Transformation was verified through beta-glucuronidase (GUS) reporter gene expression in all tree tissues, PCR amplification of appropriate vector products from isolated genomic DNA, and northern hybridization of incorporated and expressed transgenes. The hairy root protocol follows the same inoculation procedures and was tested using GUS reporter gene integration and antibiotic selection. The benefit of these procedures is that they are simple and efficient, requiring no maintenance of starting materials and allowing fully formed transgenic trees (or hairy roots) to be generated in only 3-4 months, rather than the 6-12 months required by more traditional methods. Likewise, the fact that the protocols are amenable to high-throughput formats makes them better suited for large-scale functional genomics studies in poplars.

Identification of PTM5 protein interaction partners, a MADS-box gene involved in aspen tree vegetative development.

In a past article, our lab described the identification and characterization of a novel vegetative MADS-box gene from quaking aspen trees, Populus tremuloides MADS-box 5 (PTM5). PTM5 was shown to be a member of the SOC1/TM3 class of MADS-box genes with a seasonal expression pattern specific to developing vascular tissues including the vascular cambium, the precursor to all woody branches, stems, and roots. Since the proper function of MADS-box proteins is dependent on specific interactions with other regulatory proteins, we further examined PTM5 protein-protein interactions as a means to better understand its function. Through yeast two-hybrid analyses, it was demonstrated that, like other SOC1/TM3 class proteins, PTM5 is capable of interacting with itself as well as other MADS-box proteins from aspen. In addition, yeast two-hybrid library screening revealed that PTM5 interacts with two non-MADS proteins, an actin depolymerizing factor (PtADF) and a novel leucine-rich repeat protein (PtLRR). In situ RNA localization was used to verify the overlapping expression patterns of these genes, and transgenic studies showed that over-expression of PTM5 in aspen causes alterations in root vasculature and root biomass development consistent with the cell growth and expansion functions of related ADF and LRR genes. These results suggest that the interaction of vegetative MADS-box genes with specific protein cofactors is a key step in the mechanisms that control woody tissue development in trees.

SEP-class genes in Populus tremuloides and their likely role in reproductive survival of poplar trees.

One of the most important processes to the survival of a species is its ability to reproduce. In plants, SEPALLATA-class MADS-box genes have been found to control the development of the inner whorls of flowers. However, while much is known about floral development in herbaceous plants, similar systems in woody trees remain poorly understood. Populus tremuloides (trembling aspen) is a widespread North American tree having important economic value, and its floral development differs from that of well-studied species in that the flowers have only two whorls and are truly unisexual. Sequence based analyses indicate that PTM3 (Populus tremuloides MADS-box 3), and a duplicate gene PTM4, are related to the SEPALLATA1-and 2-class of MADS-box genes. Another gene, PTM6, is related to SEP3, and each of these genes has a counterpart in the poplar genomic database along with additional members of the A, B, C, D, and E-classes of MADS-box genes. PTM3/4 and 6 are expressed in all stages of male and female aspen floral development. However, PTM3/4 is also expressed in the terminal buds, young leaves, and young stems. In situ RNA localization identified PTM3/4 and 6 transcripts predominantly in the inner, sexual whorl, within developing ovules of female flowers and anther primordia of male flowers. Tree researchers often use heterologous systems to help study tree floral development due to the long juvenile periods found in most trees. We found that the participation of PTM3/4 in floral development is supported by transgenic experiments in both P. tremuloides and heterologous systems such as tobacco and Arabidopsis. However, phenotypic artifacts were observed in the heterologous systems. Together the results suggest a role for poplar SEP-class genes in reproductive viability.

Isolation and analysis of a symbiosis-regulated and Ras-interacting vesicular assembly protein gene from the ectomycorrhizal fungus Laccaria bicolor.

• A yeast two-hybrid library prepared from Laccaria bicolor × Pinus resinosa mycorrhizas was screened using a LbRAS clone, previously characterized, as a bait to isolate LbRAS interacting signaling-related genes from L. bicolor. • Using this method, a novel line of Ras-interacting yeast two-hybrid mycorrhizal (Rythm) clones were isolated and analysed for their symbiosis-regulation. One such clone identified (RythmA) had homology to Ap180-like vesicular proteins. • Sequence homology and parsimony-based phylogenetic analysis showed its relatedness to Ap180-like proteins from other systems. DNA analysis suggested that L. bicolor had one or two copies of the RythmA gene. • An RNA analysis showed that the expression of RythmA could be detected 36 h after interaction with the host, which follows the expression of Lbras. Immunolocalization of LbRAS near dolipore septum of the fungal cells in the Hartig net area suggests that RythmA protein may be involved in the transport of signaling proteins such as LbRAS.

Characterization of PTM5 in aspen trees: a MADS-box gene expressed during woody vascular development.

The vascular component of trees possesses some of the most specialized processes active in the formation of roots, stems, and branches, and its wood component continues to be of primary importance to our daily lives. The molecular mechanisms of wood development, however, remain poorly understood with few well-characterized regulatory genes. We have identified a vascular tissue-specific MADS-box gene, Populus tremuloides MADS-box 5 (PTM5) that is expressed in differentiating primary and secondary xylem and phloem. Phylogenetic analysis has shown that PTM5 is a member of the SOC1/TM3 class of MADS-box genes. Temporal expression analysis of PTM5 in staged vascular cambium and other tissues indicated that PTM5 expression is seasonal and is limited to spring wood formation and rapidly expanding floral catkins. Spatial expression analysis using in situ hybridization revealed that PTM5 expression is localized within a few layers of differentiating vascular cambium and xylem tissues as well as the vascular bundles of expanding catkins. Since many MADS-box genes are known to act as transcription factors, these results suggest that the coordinated expression of PTM5 with other vascular developmental genes may be a hallmark of the complex events that lead to the formation of the woody plant body.

Differential expression of a malate synthase gene during the preinfection stage of symbiosis in the ectomycorrhizal fungus Laccaria bicolor.

• The ectomycorrhiza is a symbiotic organ formed between a filamentous fungus and a plant root, mainly tree roots. Root colonization involves significant shifts in gene expression resulting in metabolic and structural changes in the fungus, including growth toward the plant root, penetration and establishment of the symbiotic organ. • The preinfection stage of the association is crucial as changes that occur throughout mycorrhiza formation are set in motion. Using an in vitro system for identifying preinfection stage symbiosis-regulated genes from the Laccaria bicolor-Pinus resinosa interaction we have identified a malate synthase from L. bicolor (Lb-MS). • The glyoxylate pathway, of which malate synthase is an enzyme, acts as a tricarboxylic acid pathway bypass generating four-carbon compounds for biosynthesis. While it is anticipated that malate synthase would be a part of the genetic and metabolic machinery of any fungus, Lb-MS is of interest because it is symbiosis regulated. • Lb-MS is regulated through interaction between the fungus and the host, by glucose and by the presence of other carbon sources in the medium. Its proposed role in the symbiosis is in the utilization of two carbon compounds formed from catabolic processes in early interaction facilitating hyphal net growth.