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

Abstract

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 PtrMIR397a in transgenic P. trichocarpa. In each of all nine transgenic lines tested, 17 PtrLACs were down-regulated as analyzed by RNAseq. 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.L ignin, an abundant biological polymer affecting the ecology of the terrestrial biosphere, is vital for the integrity of plant cell walls, the strength of stems, and resistance against pests and pathogens (1). Lignin is also a major barrier in the pulping and biomass-to-ethanol processes (2-4). For extracting cellulose (pulping) or for enzymatic degradation of cellulose for bioethanol, harsh chemical or physical treatments are used to reduce interactions with lignin or other cell wall components (2-4). Reducing lignin content or altering lignin structure to reduce its recalcitrance are major goals for more efficient processing.Lignin is polymerized primarily from three monolignol precursors, p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol (1, 5). Over five decades, efforts have been made to understand the biosynthesis of the primary monolignols and to modify the quantity or composition of lignin. The polymerization of monolignols into a lignin polymer has long been thought to occur through oxidative polymerization catalyzed by either laccases or peroxidases (6). The mechanisms and specificity of the roles of the oxidative enzymes in lignin polymerization have been controversial (7).Laccases (EC. 1.10.3.2) are multicopper oxidoreductases. Plant laccase was the first enzyme sh...

SND1 Transcription Factor-Directed Quantitative Functional Hierarchical Genetic Regulatory Network in Wood Formation in Populus trichocarpa

“…These two sequencerelated Ptr-MYBs share eight common targets, of which five are laccase genes (Ptr-LAC14, Ptr-LAC15, Ptr-LAC40, Ptr-LAC41, and Ptr-LAC49). Using transgenic P. trichocarpa overexpressing a microRNA (miRNA), ptr-miR397a, we recently demonstrated that this miRNA and many TF genes, including Ptr-MYB021, regulate directly 13 laccase (including Ptr-LAC14, Ptr-LAC15, Ptr-LAC40, Ptr-LAC41, and Ptr-LAC49) and four peroxidase (Ptr-PO) genes in a transcriptional regulatory network controlling lignin content during wood formation (Lu et al, 2013). These miRNA transgenic results strongly supported the accuracy of developed top-down algorithm (Figure 4) for predicting the direct TF-DNA relationships.…”

“…Step II (Figure 4), which we developed previously (Lu et al, 2013), uses a partial correlation-based graphical Gaussian model (GGM) (Whittaker, 1990;Edwards, 2000) for a vigorous assessment of direct interactions between Ptr-SND1-B1 and responsive genes to infer Ptr-SND1-B1's direct targets. SDX protoplasts were isolated using the dipping method and transfected with Ptr-SND1-B1 to result in 178 DEGs based on RNA-seq analysis.…”

“…The five Ptr-LACs and Arabidopsis LAC4 are homologs and were shown to control lignin quantity in P. trichocarpa (Lu et al, 2013) and Arabidopsis (Berthet et al, 2011), respectively. At-LAC4 is a predicted direct target of At-MYB46 based on the MYB46-responsive cis-acting binding site (RKTWGGTR) in the At-LAC4 gene promoter.…”

“…The frequency of raw counts was determined and normalized as described (Lu et al, 2013). DEGs were identified using edgeR (Robinson et al, 2010) by comparing the relative transcript abundance for each gene between the Ptr-SND1-B1 and the sGFP (control) transfection at each incubation time point (7, 12, and 25 h).…”

“…Briefly, the Ptr-SND1-B1 cDNA sequence was amplified from P. trichocarpa xylem cDNA with primers Ptr-SND1-B1-1F/R (see Supplemental Data Set 6 online) and then inserted into pBI121 using BamHI-SacI sites. The construct was sequence confirmed and mobilized into Agrobacterium tumefaciens strain C58 to transform P. trichocarpa as described (Song et al, 2006;Lu et al, 2013). qRT-PCR was conducted as described above to quantify the transcript abundance of Ptr-SND1-B1 and the selected direct targets of Ptr-SND1-B1 (Figure 9).…”