TGMI: an efficient algorithm for identifying pathway regulators through evaluation of triple-gene mutual interaction.

Despite their important roles, the regulators for most metabolic pathways and biological processes remain elusive. Presently, the methods for identifying metabolic pathway and biological process regulators are intensively sought after. We developed a novel algorithm called triple-gene mutual interaction (TGMI) for identifying these regulators using high-throughput gene expression data. It first calculated the regulatory interactions among triple gene blocks (two pathway genes and one transcription factor (TF)), using conditional mutual information, and then identifies significantly interacted triple genes using a newly identified novel mutual interaction measure (MIM), which was substantiated to reflect strengths of regulatory interactions within each triple gene block. The TGMI calculated the MIM for each triple gene block and then examined its statistical significance using bootstrap. Finally, the frequencies of all TFs present in all significantly interacted triple gene blocks were calculated and ranked. We showed that the TFs with higher frequencies were usually genuine pathway regulators upon evaluating multiple pathways in plants, animals and yeast. Comparison of TGMI with several other algorithms demonstrated its higher accuracy. Therefore, TGMI will be a valuable tool that can help biologists to identify regulators of metabolic pathways and biological processes from the exploded high-throughput gene expression data in public repositories.

Overexpression of PsnSuSy1, 2 genes enhances secondary cell wall thickening, vegetative growth, and mechanical strength in transgenic tobacco.

KEY MESSAGE: Two homologs PsnSuSy1 and PsnSuSy2 from poplar played largely similar but little distinct roles in modulating sink strength, accelerating vegetative growth and modifying secondary growth of plant. Co-overexpression of them together resulted in small but perceptible additive effects. Sucrose synthase (SuSy) acts as a crucial determinant of sink strength by controlling the conversion of sucrose into UDP-glucose, which is not only the sole precursor for cellulose biosynthesis but also an extracellular signaling molecule for plants growth. Therefore, modification of SuSy activity in plants is of utmost importance. We have isolated two SuSy genes from poplar, PsnSuSy1 and PsnSuSy2, which were preferentially expressed in secondary xylem/phloem. To investigate their functions, T2 tobacco transgenic lines of PsnSuSy1 and PsnSuSy2 were generated and then crossed to generate PsnSuSy1/PsnSuSy2 dual overexpression transgenic lines. SuSy activities in all lines were significantly increased though PsnSuSy1/PsnSuSy2 lines only exhibited slightly higher SuSy activities than either PsnSuSy1 or PsnSuSy2 lines. The significantly increased fructose and glucose, engendered by augmented SuSy activities, caused the alternations of many physiological, biochemical measures and phenotypic traits that include accelerated vegetative growth, thickened secondary cell wall, and increased stem breaking force, accompanied with altered expression levels of related pathway genes. The correlation relationships between SuSy activities and many of these traits were statistically significant. However, differences of almost all traits among three types of transgenic lines were insignificant. These findings clearly demonstrated that PsnSuSy1 and PsnSuSy2 had similar but little distinct functions and insubstantial additive effects on modulating sink strength and affecting allocation of carbon elements among secondary cell wall components.

Ptr-miR397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa.

Laccases, as early as 1959, were proposed to catalyze the oxidative polymerization of monolignols. Genetic evidence in support of this hypothesis has been elusive due to functional redundancy of laccase genes. An Arabidopsis double mutant demonstrated the involvement of laccases in lignin biosynthesis. We previously identified a subset of laccase genes to be targets of a microRNA (miRNA) ptr-miR397a in Populus trichocarpa. To elucidate the roles of ptr-miR397a and its targets, we characterized the laccase gene family and identified 49 laccase gene models, of which 29 were predicted to be targets of ptr-miR397a. We overexpressed Ptr-MIR397a in transgenic P. trichocarpa. In each of all nine transgenic lines tested, 17 PtrLACs were down-regulated as analyzed by RNA-seq. Transgenic lines with severe reduction in the expression of these laccase genes resulted in an ∼40% decrease in the total laccase activity. Overexpression of Ptr-MIR397a in these transgenic lines also reduced lignin content, whereas levels of all monolignol biosynthetic gene transcripts remained unchanged. A hierarchical genetic regulatory network (GRN) built by a bottom-up graphic Gaussian model algorithm provides additional support for a role of ptr-miR397a as a negative regulator of laccases for lignin biosynthesis. Full transcriptome-based differential gene expression in the overexpressed transgenics and protein domain analyses implicate previously unidentified transcription factors and their targets in an extended hierarchical GRN including ptr-miR397a and laccases that coregulate lignin biosynthesis in wood formation. Ptr-miR397a, laccases, and other regulatory components of this network may provide additional strategies for genetic manipulation of lignin content.

Functional characterization of CCR in birch (Betula platyphylla × Betula pendula) through overexpression and suppression analysis.

We cloned a Cinnamoyl-CoA Reductase gene (BpCCR1) from an apical meristem and first internode of Betula platyphylla and characterized its functions in lignin biosynthesis, wood formation and tree growth through transgenic approaches. We generated overexpression and suppression transgenic lines and analyzed them in comparison with the wild-type in terms of lignin content, anatomical characteristics, height and biomass. We found that BpCCR1 overexpression could increase lignin content up to 14.6%, and its underexpression decreased lignin content by 6.3%. Surprisingly, modification of BpCCR1 expression led to conspicuous changes in wood characteristics, including xylem vessel number and arrangement, and secondary wall thickness. The growth of transgenic trees in terms of height was also significantly influenced by the modification of BpCCR1 genes. We discuss the functions of BpCCR1 in the context of a phylogenetic tree built with CCR genes from multiple species.

PtrHAT22, as a higher hierarchy regulator, coordinately regulates secondary cell wall component biosynthesis in Populus trichocarpa.

Homeodomain-leucine zipper (HD-Zip) II transcription factors (TFs) have been reported to play vital roles in diverse biological processes of plants. However, it remains unclear whether HD-Zip II TFs regulate secondary cell wall (SCW) in woody plants. In this study, we performed the functional characterization of a Populus trichocarpa HD-Zip II TF, PtrHAT22, which encodes a nuclear localized transcription repressor predominantly expressing in secondary developing tissues. Overexpression of PtrHAT22 showed arrested growths, including reduced heights and diameters above the ground, small leaves, and decreased biomass. Meanwhile, the contents of lignin, cellulose, and thickness of SCW significantly decreased, whilst the content of hemicellulose obviously increased in PtrHAT22 transgenic poplar. The expressions of some wood-associated TFs and structural genes significantly changed accordingly with the alternations of SCW characteristics in PtrHAT22 transgenic poplar. Furthermore, PtrHAT22 directly repressed the promoter activities of PtrMYB20, PtrMYB28, and PtrCOMT2, and bind two cis-acting elements that were specifically enriched in their promoter regions. Taken together, our results suggested that PtrHAT22, as a higher hierarchy TF like PtrWNDs, exerted coordination regulation of poplar SCW component biosynthesis through directly and indirectly regulating structural genes and different hierarchy TFs of SCW formation network.

Identification of biological pathway and process regulators using sparse partial least squares and triple-gene mutual interaction.

Identification of biological process- and pathway-specific regulators is essential for advancing our understanding of regulation and formation of various phenotypic and complex traits. In this study, we applied two methods, triple-gene mutual interaction (TGMI) and Sparse Partial Least Squares (SPLS), to identify the regulators of multiple metabolic pathways in Arabidopsis thaliana and Populus trichocarpa using high-throughput gene expression data. We analyzed four pathways: (1) lignin biosynthesis pathway in A. thaliana and P. trichocarpa; (2) flavanones, flavonol and anthocyannin biosynthesis in A. thaliana; (3) light reaction pathway and Calvin cycle in A. thaliana. (4) light reaction pathway alone in A. thaliana. The efficiencies of two methods were evaluated by examining the positive known regulators captured, the receiver operating characteristic (ROC) curves and the area under ROC curves (AUROC). Our results showed that TGMI is in general more efficient than SPLS in identifying true pathway regulators and ranks them to the top of candidate regulatory gene lists, but the two methods are to some degree complementary because they could identify some different pathway regulators. This study identified many regulators that potentially regulate the above pathways in plants and are valuable for genetic engineering of these pathways.

Gene network analysis of poplar root transcriptome in response to drought stress identifies a PtaJAZ3PtaRAP2.6-centered hierarchical network.

Using time-series transcriptomic data from poplar roots undergoing polyethylene glycol (PEG)-induced drought stress, we built a genetic network model of the involved putative molecular responses. We found that the network resembled a hierarchical structure. The highest hierarchical level in this structure is occupied by 9 genes, which we called superhubs because they were primarily connected to 18 hub genes, which are then connected to 2,934 terminal genes. We were only able to regenerate transgenic plants overexpressing two of the superhubs, suggesting that the majority of the superhubs might interfere with the regeneration process and did not allow recovery of transgenic plants. The two superhubs encode proteins with closest homology to JAZ3 and RAP2.6 genes of Arabidopsis and were consequently named PtaJAZ3 and PtaRAP2.6. PtaJAZ3 and PtaRAP2.6 overexpressing transgenic lines showed a significant increase in both root elongation and lateral root proliferation and these responses were specific for the drought stress conditions and were highly correlated with the levels of overexpression of the transgenes. Several lines of evidence suggest of regulatory interactions between the two superhubs. Both superhubs were significantly induced by methyl jasmonate (MeJA). Because jasmonate signaling involves ubiquitin-mediated proteasome degradation, treatment with proteasome inhibitor abolished the MeJA induction for both genes. PtaRAP2.6 was upregulated in PtaJAZ3 transgenics but PtaJAZ3 expression was not affected in the PtaRAP2.6 overexpressors. The discovery of the two genes and further future insights into the associated mechanisms can lead to improved understanding and novel approaches to regulate root architecture in relation to drought stress.

Functional Characterization of Populus PsnSHN2 in Coordinated Regulation of Secondary Wall Components in Tobacco.

Wood formation is a biological process during which the most abundant lignocellulosic biomass on earth is produced. Although a number of transcription factors have been linked to the regulation of wood formation process, none of them has been demonstrated to be a higher hierarchical regulator that coordinately regulates secondary wall biosynthesis genes. Here, we identified a Populus gene, PsnSHN2, a counterpart of the Arabidopsis AP2/ERF type transcription factor, SHINE2. PsnSHN2 is predominantly expressed in xylem tissues and acted evidently as a high hierarchical transcriptional activator. Overexpression of PsnSHN2 in tobacco significantly altered the expression of both transcription factors and biosynthesis genes involved in secondary wall formation, leading to the thickened secondary walls and the changed cell wall composition. The most significant changes occurred in the contents of cellulose and hemicellulose that increased 37% and 28%, respectively, whereas the content of lignin that decreased 34%. Furthermore, PsnSHN2 activated or repressed the promoter activities of transcription factors involved in secondary wall biosynthesis and bound to five cis-acting elements enriched in the promoter regions of these transcription factors. Taken together, our results suggest PsnSHN2 coordinately regulate secondary wall formation through selective up/down-regulation of its downstream transcription factors that control secondary wall formation.

Overexpression of poplar xylem sucrose synthase in tobacco leads to a thickened cell wall and increased height.

Sucrose synthase (SuSy) is considered the first key enzyme for secondary growth because it is a highly regulated cytosolic enzyme that catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose. Although SuSy enzymes preferentially functions in the direction of sucrose cleavage at most cellular condition, they also catalyze the synthetic reaction. We isolated a gene that encodes a SuSy from Populus simonii×Populus nigra and named it PsnSuSy2 because it shares high similarity to SuSy2 in Populus trichocarpa. RT-PCR revealed that PsnSuSy2 was highly expressed in xylem, but lowly expressed in young leaves. To characterize its functions in secondary growth, multiple tobacco overexpression transgenic lines of PnsSuSy2 were generated via Agrobacterium-mediated transformation. The PsnSuSy2 expression levels and altered wood properties in stem segments from the different transgenic lines were carefully characterized. The results demonstrated that the levels of PsnSuSy2 enzyme activity, chlorophyll content, total soluble sugars, fructose and glucose increased significantly, while the sucrose level decreased significantly. Consequently, the cellulose content and fiber length increased, whereas the lignin content decreased, suggesting that PsnSuSy2 plays a significant role in cleaving sucrose into UDP-glucose and fructose to facilitate cellulose biosynthesis and that promotion of cellulose biosynthesis suppresses lignin biosynthesis. Additionally, the noticeable increase in the lodging resistance in transgenic tobacco stem suggested that the cell wall characteristics were altered by PsnSuSy2 overexpression. Scanning electron microscopy was performed to study the cell wall morphology of stem, and surprisingly, we found that the secondary cell wall was significantly thicker in transgenic tobacco. However, the thickened secondary cell wall did not negatively affect the height of the plants because the PsnSuSy2- overexpressing lines grew taller than the wildtype plants. This systematic analysis demonstrated that PsnSuSy2 plays an important role in cleaving sucrose coupled with cellulose biosynthesis in wood tissue.

Identification of genes required for cellulose synthesis by regression analysis of public microarray data sets.

Coexpression patterns of gene expression across many microarray data sets may reveal networks of genes involved in linked processes. To identify factors involved in cellulose biosynthesis, we used a regression method to analyze 408 publicly available Affymetrix Arabidopsis microarrays. Expression of genes previously implicated in cellulose synthesis, as well as several uncharacterized genes, was highly coregulated with expression of cellulose synthase (CESA) genes. Four candidate genes, which were coexpressed with CESA genes implicated in secondary cell wall synthesis, were investigated by mutant analysis. Two mutants exhibited irregular xylem phenotypes similar to those observed in mutants with defects in secondary cellulose synthesis and were designated irx8 and irx13. Thus, the general approach developed here is useful for identification of elements of multicomponent processes.