Comprehensive expression analyses of the ABCG subfamily reveal SvABCG17 as a potential transporter of lignin monomers in the model C4 grass Setaria viridis.

Although several aspects of lignin metabolism have been extensively characterized, the mechanism(s) by which lignin monomers are transported across the plasma membrane remains largely unknown. Biochemical, proteomic, expression and co-expression analyses from several plant species support the involvement of active transporters, mainly those belonging to the ABC superfamily. Here, we report on the genome-wide characterization of the ABCG gene subfamily in the model C4 grass Setaria viridis and further identification of the members potentially involved in monolignol transport. A total of 48 genes encoding SvABCGs were found in the S. viridis genome, from which 21 SvABCGs were classified as full-size transporters and 27 as half-size transporters. Comprehensive analysis of the ABCG subfamily in S. viridis based on expression and co-expression analyses support a role for SvABCG17 in monolignol transport: (i) SvABCG17 is orthologous to AtABCG29, a monolignol transporter in Arabidopsis thaliana; (ii) SvABCG17 displays a similar expression profile to that of lignin biosynthetic genes in a set of different S. viridis tissues and along the elongating internode; (iii) SvABCG17 is highly co-expressed with lignin-related genes in a public transcriptomic database; (iv) SvABCG17displays particularly high expression in the top of the S. viridis elongating internode, a tissue undergoing active lignification; (v) SvABCG17 mRNA localization coincides with the histochemical pattern of lignin deposition; and (vi) the promoter of SvABCG17 is activated by secondary cell wall-associated transcription factors, especially by lignin-specific activators of the MYB family. Further studies might reveal further aspects of this potential monolignol transporter, including its real substrate specificity and whether it works redundantly with other ABC members during S. viridis lignification.

Transcriptional and metabolic changes associated with internode development and reduced cinnamyl alcohol dehydrogenase activity in sorghum.

The molecular mechanisms associated with secondary cell wall (SCW) deposition in sorghum remain largely uncharacterized. Here, we employed untargeted metabolomics and large-scale transcriptomics to correlate changes in SCW deposition with variation in global gene expression profiles and metabolite abundance along an elongating internode of sorghum, with a major focus on lignin and phenolic metabolism. To gain deeper insight into the metabolic and transcriptional changes associated with pathway perturbations, a bmr6 mutant [with reduced cinnamyl alcohol dehydrogenase (CAD) activity] was analyzed. In the wild type, internode development was accompanied by an increase in the content of oligolignols, p-hydroxybenzaldehyde, hydroxycinnamate esters, and flavonoid glucosides, including tricin derivatives. We further identified modules of genes whose expression pattern correlated with SCW deposition and the accumulation of these target metabolites. Reduced CAD activity resulted in the accumulation of hexosylated forms of hydroxycinnamates (and their derivatives), hydroxycinnamaldehydes, and benzenoids. The expression of genes belonging to one specific module in our co-expression analysis correlated with the differential accumulation of these compounds and contributed to explaining this metabolic phenotype. Metabolomics and transcriptomics data further suggested that CAD perturbation activates distinct detoxification routes in sorghum internodes. Our systems biology approach provides a landscape of the metabolic and transcriptional changes associated with internode development and with reduced CAD activity in sorghum.

Isolation of Promoters and Transcription Factors Involved in the Regulation of Lignin Biosynthesis in Saccharum Species.

Sugarcane bagasse has received attention as a raw material for the production of second-generation ethanol (E2G). However, its use is limited because of the cell wall recalcitrance, mostly conferred by lignin. Recently our knowledge of the genes coding for the enzymes of the lignin biosynthesis pathway has increased; however, still little is known about the transcription factors controlling the expression of these genes in sugarcane. Here we describe protocols to optimize the isolation of the promoters of the lignin biosynthetic genes ShCAD8, ShCOMT and ShF5H and the transcription factors (TFs) ShMYB85 and ShMYB58/63 in Saccharum species. To confirm whether these TFs are able to activate the target promoters, a transactivation assay in BY2 protoplasts of Nicotiana tabacum is also detailed.

A matter of time: regulatory events behind the synchronization of C4 and crassulacean acid metabolism in Portulaca oleracea.

Portulaca species can switch between C4 and crassulacean acid metabolism (CAM) depending on environmental conditions. However, the regulatory mechanisms behind this rare photosynthetic adaptation remain elusive. Using Portulaca oleracea as a model system, here we investigated the involvement of the circadian clock, plant hormones, and transcription factors in coordinating C4 and CAM gene expression. Free-running experiments in constant conditions suggested that C4 and CAM gene expression are intrinsically connected to the circadian clock. Detailed time-course, drought, and rewatering experiments revealed distinct time frames for CAM induction and reversion (days versus hours, respectively), which were accompanied by changes in abscisic acid (ABA) and cytokinin metabolism and signaling. Exogenous ABA and cytokinins were shown to promote and repress CAM expression in P. oleracea, respectively. Moreover, the drought-induced decline in C4 transcript levels was completely recovered upon cytokinin treatment. The ABA-regulated transcription factor genes HB7, NFYA7, NFYC9, TT8, and ARR12 were identified as likely candidate regulators of CAM induction following this approach, whereas NFYC4 and ARR9 were connected to C4 expression patterns. Therefore, we provide insights into the signaling events controlling C4-CAM transitions in response to water availability and over the day/night cycle, highlighting candidate genes for future functional studies in the context of facultative C4-CAM photosynthesis.

The sugarcane ShMYB78 transcription factor activates suberin biosynthesis in Nicotiana benthamiana.

KEY MESSAGE: A sugarcane MYB present in the culm induces suberin biosynthesis and is involved both with fatty acid and phenolics metabolism. Few transcription factors have been described as regulators of cell wall polymers deposition in C4 grasses. Particularly, regulation of suberin biosynthesis in this group of plants remains poorly understood. Here, we showed that the sugarcane MYB transcription factor ShMYB78 is an activator of suberin biosynthesis and deposition. ShMYB78 was identified upon screening genes whose expression was upregulated in sugarcane internodes undergoing suberization during culm development or triggered by wounding. Agrobacterium-mediated transient expression of ShMYB78 in Nicotiana benthamiana leaves induced the ectopic deposition of suberin and its aliphatic and aromatic monomers. Further, the expression of suberin-related genes was induced by ShMYB78 heterologous expression in Nicotiana benthamiana leaves. ShMYB78 was shown to be a nuclear protein based on its presence in sugarcane internode nuclear protein extracts, and protoplast transactivation assays demonstrated that ShMYB78 activates the promoters of the sugarcane suberin biosynthetic genes ß-ketoacyl-CoA synthase (ShKCS20) and caffeic acid-O-methyltransferase (ShCOMT). Our results suggest that ShMYB78 may be involved in the transcriptional regulation of suberin deposition, from fatty acid metabolism to phenylpropanoid biosynthesis, in sugarcane internodes.

In silico guided structural and functional analysis of genes with potential involvement in resistance to coffee leaf rust: A functional marker based approach.

Physiology-based differentiation of SH genes and Hemileia vastatrix races is the principal method employed for the characterization of coffee leaf rust resistance. Based on the gene-for-gene theory, nine major rust resistance genes (SH1-9) have been proposed. However, these genes have not been characterized at the molecular level. Consequently, the lack of molecular data regarding rust resistance genes or candidates is a major bottleneck in coffee breeding. To address this issue, we screened a BAC library with resistance gene analogs (RGAs), identified RGAs, characterized and explored for any SH related candidate genes. Herein, we report the identification and characterization of a gene (gene 11), which shares conserved sequences with other SH genes and displays a characteristic polymorphic allele conferring different resistance phenotypes. Furthermore, comparative analysis of the two RGAs belonging to CC-NBS-LRR revealed more intense diversifying selection in tomato and grape genomes than in coffee. For the first time, the present study has unveiled novel insights into the molecular nature of the SH genes, thereby opening new avenues for coffee rust resistance molecular breeding. The characterized candidate RGA is of particular importance for further biological function analysis in coffee.

Genome-wide characterization of the laccase gene family in Setaria viridis reveals members potentially involved in lignification.

MAIN CONCLUSION: Five laccase genes are potentially involved in developmental lignification in the model C4 grass Setaria viridis and their different tissue specificities suggest subfunctionalization events. Plant laccases are copper-containing glycoproteins involved in monolignol oxidation and, therefore, their activity is essential for lignin polymerization. Although these enzymes belong to large multigene families with highly redundant members, not all of them are thought to be involved in lignin metabolism. Here, we report on the genome-wide characterization of the laccase gene family in the model C4 grass Setaria viridis and further identification of the members potentially involved in monolignol oxidation. A total of 52 genes encoding laccases (SvLAC1 to SvLAC52) were found in the genome of S. viridis, and phylogenetic analyses showed that these genes were heterogeneously distributed among the characteristic six subclades of the family and are under relaxed selective constraints. The observed expansion in the total number of genes in this species was mainly caused by tandem duplications within subclade V, which accounts for 68% of the whole family. Comparative phylogenetic analyses showed that the expansion of subclade V is specifically observed for the Paniceae tribe within the Panicoideae subfamily in grasses. Five SvLAC genes (SvLAC9, SvLAC13, SvLAC15, SvLAC50, and SvLAC52) fulfilled the criteria established to identify lignin-related candidates: (1) phylogenetic proximity to previously characterized lignin-related laccases from other species, (2) similar expression pattern to that observed for lignin biosynthetic genes in the S. viridis elongating internode, and (3) high expression in S. viridis tissues undergoing active lignification. In addition, in situ hybridization experiments not only confirmed that these selected SvLAC genes were expressed in lignifying cells, but also that their expression showed different tissue specificities, suggesting subfunctionalization events within the family. These five laccase genes are strong candidates to be involved in lignin polymerization in S. viridis and might be good targets for lignin bioengineering strategies.

Differentiation of Tracheary Elements in Sugarcane Suspension Cells Involves Changes in Secondary Wall Deposition and Extensive Transcriptional Reprogramming.

Plant lignocellulosic biomass, mostly composed of polysaccharide-rich secondary cell walls (SCWs), provides fermentable sugars that may be used to produce biofuels and biomaterials. However, the complex chemical composition and physical structure of SCWs hinder efficient processing of plant biomass. Understanding the molecular mechanisms underlying SCW deposition is, thus, essential to optimize bioenergy feedstocks. Here, we establish a xylogenic culture as a model system to study SCW deposition in sugarcane; the first of its kind in a C4 grass species. We used auxin and brassinolide to differentiate sugarcane suspension cells into tracheary elements, which showed metaxylem-like reticulate or pitted SCW patterning. The differentiation led to increased lignin levels, mainly caused by S-lignin units, and a rise in p-coumarate, leading to increased p-coumarate:ferulate ratios. RNAseq analysis revealed massive transcriptional reprogramming during differentiation, with upregulation of genes associated with cell wall biogenesis and phenylpropanoid metabolism and downregulation of genes related to cell division and primary metabolism. To better understand the differentiation process, we constructed regulatory networks of transcription factors and SCW-related genes based on co-expression analyses. Accordingly, we found multiple regulatory modules that may underpin SCW deposition in sugarcane. Our results provide important insights and resources to identify biotechnological strategies for sugarcane biomass optimization.

Correction to: High throughput transcriptome analysis of coffee reveals prehaustorial resistance in response to Hemileia vastatrix infection.

All the transcriptome sequencing data mentioned in the original article is publicly available at the National Center of Biotechnology Information (NCBI).

The lignin toolbox of the model grass Setaria viridis.

KEY MESSAGE: The core set of biosynthetic genes potentially involved in developmental lignification was identified in the model C4 grass Setaria viridis. Lignin has been recognized as a major recalcitrant factor negatively affecting the processing of plant biomass into bioproducts. However, the efficient manipulation of lignin deposition in order to generate optimized crops for the biorefinery requires a fundamental knowledge of several aspects of lignin metabolism, including regulation, biosynthesis and polymerization. The current availability of an annotated genome for the model grass Setaria viridis allows the genome-wide characterization of genes involved in the metabolic pathway leading to the production of monolignols, the main building blocks of lignin. Here we performed a comprehensive study of monolignol biosynthetic genes as an initial step into the characterization of lignin metabolism in S. viridis. A total of 56 genes encoding bona fide enzymes catalyzing the consecutive ten steps of the monolignol biosynthetic pathway were identified in the S. viridis genome. A combination of comparative phylogenetic studies, high-throughput expression analysis and quantitative RT-PCR analysis was further employed to identify the family members potentially involved in developmental lignification. Accordingly, 14 genes clustered with genes from closely related species with a known function in lignification and showed an expression pattern that correlates with lignin deposition. These genes were considered the "core lignin toolbox" responsible for the constitutive, developmental lignification in S. viridis. These results provide the basis for further understanding lignin deposition in C4 grasses and will ultimately allow the validation of biotechnological strategies to produce crops with enhanced processing properties.

High throughput transcriptome analysis of coffee reveals prehaustorial resistance in response to Hemileia vastatrix infection.

KEY MESSAGE: We provide a transcriptional profile of coffee rust interaction and identified putative up regulated resistant genes Coffee rust disease, caused by the fungus Hemileia vastatrix, is one of the major diseases in coffee throughout the world. The use of resistant cultivars is considered to be the most effective control strategy for this disease. To identify candidate genes related to different mechanism defense in coffee, we present a time-course comparative gene expression profile of Caturra (susceptible) and Híbrido de Timor (HdT, resistant) in response to H. vastatrix race XXXIII infection. The main objectives were to obtain a global overview of transcriptome in both interaction, compatible and incompatible, and, specially, analyze up-regulated HdT specific genes with inducible resistant and defense signaling pathways. Using both Coffea canephora as a reference genome and de novo assembly, we obtained 43,159 transcripts. At early infection events (12 and 24 h after infection), HdT responded to the attack of H. vastatrix with a larger number of up-regulated genes than Caturra, which was related to prehaustorial resistance. The genes found in HdT at early hours were involved in receptor-like kinases, response ion fluxes, production of reactive oxygen species, protein phosphorylation, ethylene biosynthesis and callose deposition. We selected 13 up-regulated HdT-exclusive genes to validate by real-time qPCR, which most of them confirmed their higher expression in HdT than in Caturra at early stage of infection. These genes have the potential to assist the development of new coffee rust control strategies. Collectively, our results provide understanding of expression profiles in coffee-H. vastatrix interaction over a time course in susceptible and resistant coffee plants.

Co-expression network analysis reveals transcription factors associated to cell wall biosynthesis in sugarcane.

Sugarcane is a hybrid of Saccharum officinarum and Saccharum spontaneum, with minor contributions from other species in Saccharum and other genera. Understanding the molecular basis of cell wall metabolism in sugarcane may allow for rational changes in fiber quality and content when designing new energy crops. This work describes a comparative expression profiling of sugarcane ancestral genotypes: S. officinarum, S. spontaneum and S. robustum and a commercial hybrid: RB867515, linking gene expression to phenotypes to identify genes for sugarcane improvement. Oligoarray experiments of leaves, immature and intermediate internodes, detected 12,621 sense and 995 antisense transcripts. Amino acid metabolism was particularly evident among pathways showing natural antisense transcripts expression. For all tissues sampled, expression analysis revealed 831, 674 and 648 differentially expressed genes in S. officinarum, S. robustum and S. spontaneum, respectively, using RB867515 as reference. Expression of sugar transporters might explain sucrose differences among genotypes, but an unexpected differential expression of histones were also identified between high and low Brix° genotypes. Lignin biosynthetic genes and bioenergetics-related genes were up-regulated in the high lignin genotype, suggesting that these genes are important for S. spontaneum to allocate carbon to lignin, while S. officinarum allocates it to sucrose storage. Co-expression network analysis identified 18 transcription factors possibly related to cell wall biosynthesis while in silico analysis detected cis-elements involved in cell wall biosynthesis in their promoters. Our results provide information to elucidate regulatory networks underlying traits of interest that will allow the improvement of sugarcane for biofuel and chemicals production.

Full-length enriched cDNA libraries and ORFeome analysis of sugarcane hybrid and ancestor genotypes.

Sugarcane is a major crop used for food and bioenergy production. Modern cultivars are hybrids derived from crosses between Saccharum officinarum and Saccharum spontaneum. Hybrid cultivars combine favorable characteristics from ancestral species and contain a genome that is highly polyploid and aneuploid, containing 100-130 chromosomes. These complex genomes represent a huge challenge for molecular studies and for the development of biotechnological tools that can facilitate sugarcane improvement. Here, we describe full-length enriched cDNA libraries for Saccharum officinarum, Saccharum spontaneum, and one hybrid genotype (SP803280) and analyze the set of open reading frames (ORFs) in their genomes (i.e., their ORFeomes). We found 38,195 (19%) sugarcane-specific transcripts that did not match transcripts from other databases. Less than 1.6% of all transcripts were ancestor-specific (i.e., not expressed in SP803280). We also found 78,008 putative new sugarcane transcripts that were absent in the largest sugarcane expressed sequence tag database (SUCEST). Functional annotation showed a high frequency of protein kinases and stress-related proteins. We also detected natural antisense transcript expression, which mapped to 94% of all plant KEGG pathways; however, each genotype showed different pathways enriched in antisense transcripts. Our data appeared to cover 53.2% (17,563 genes) and 46.8% (937 transcription factors) of all sugarcane full-length genes and transcription factors, respectively. This work represents a significant advancement in defining the sugarcane ORFeome and will be useful for protein characterization, single nucleotide polymorphism and splicing variant identification, evolutionary and comparative studies, and sugarcane genome assembly and annotation.