MicroRNAs (miRNAs) are small, noncoding RNAs that can play crucial regulatory roles in eukaryotes by targeting mRNAs for silencing. To test whether miRNAs play roles in the regulation of wood development in tree species, we isolated small RNAs from the developing xylem of Populus trichocarpa stems and cloned 22 miRNAs. They are the founding members of 21 miRNA gene families for 48 miRNA sequences, represented by 98 loci in the Populus genome. A majority of these miRNAs were predicted to target developmental-and stress/defense-related genes and possible functions associated with the biosynthesis of cell wall metabolites. Of the 21 P. trichocarpa miRNA families, 11 have sequence conservation in Arabidopsis thaliana but exhibited species-specific developmental expression patterns, suggesting that even conserved miRNAs may have different regulatory roles in different species. Most unexpectedly, the remaining 10 miRNAs, for which 17 predicted targets were experimentally validated in vivo, are absent from the Arabidopsis genome, suggesting possible roles in treespecific processes. In fact, the expression of a majority of the cloned miRNAs was upregulated or downregulated in woody stems in a manner consistent with tree-specific corrective growth against tension and compression stresses, two constant mechanical loads in trees. Our results show that plant miRNAs can be induced by mechanical stress and may function in one of the most critical defense systems for structural and mechanical fitness.

SummaryMicroRNAs (miRNAs), a group of small non-coding RNAs, have recently become the subject of intense study. They are a class of post-transcriptional negative regulators playing vital roles in plant development and growth. However, little is known about their regulatory roles in the responses of trees to the stressful environments incurred over their long-term growth. Here, we report the cloning of small RNAs from abiotic stressed tissues of Populus trichocarpa (Ptc) and the identification of 68 putative miRNA sequences that can be classified into 27 families based on sequence homology. Among them, nine families are novel, increasing the number of the known Ptc-miRNA families from 33 to 42. A total of 346 targets was predicted for the cloned PtcmiRNAs using penalty scores of £2.5 for mismatched patterns in the miRNA:mRNA duplexes as the criterion. Six of the selected targets were validated experimentally. The expression of a majority of the novel miRNAs was altered in response to cold, heat, salt, dehydration, and mechanical stresses. Microarray analysis of known Ptc-miRNAs identified 19 additional cold stress-responsive Ptc-miRNAs from 14 miRNA gene families. Interestingly, we found that individual miRNAs of a family responded differentially to stress, which suggests that the members of a family may have different functions. These results reveal possible roles for miRNAs in the regulatory networks associated with the long-term growth of tree species and provide useful information for developing trees with a greater level of stress resistance.

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...

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Terpenoids are the largest class of plant secondary metabolites and have attracted widespread interest. Salvia miltiorrhiza, belonging to the largest and most widely distributed genus in the mint family, is a model medicinal plant with great economic and medicinal value. Diterpenoid tanshinones are the major lipophilic bioactive components in S. miltiorrhiza. Systematic analysis of genes involved in terpenoid biosynthesis has not been reported to date. Searching the recently available working draft of the S. miltiorrhiza genome, 40 terpenoid biosynthesis-related genes were identified, of which 27 are novel. These genes are members of 19 families, which encode all of the enzymes involved in the biosynthesis of the universal isoprene precursor isopentenyl diphosphate and its isomer dimethylallyl diphosphate, and two enzymes associated with the biosynthesis of labdane-related diterpenoids. Through a systematic analysis, it was found that 20 of the 40 genes could be involved in tanshinone biosynthesis. Using a comprehensive approach, the intron/exon structures and expression patterns of all identified genes and their responses to methyl jasmonate treatment were analysed. The conserved domains and phylogenetic relationships among the deduced S. miltiorrhiza proteins and their homologues isolated from other plant species were revealed. It was discovered that some of the key enzymes, such as 1-deoxy-D-xylulose 5-phosphate synthase, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, hydroxymethylglutaryl-CoA reductase, and geranylgeranyl diphosphate synthase, are encoded by multiple gene members with different expression patterns and subcellular localizations, and both homomeric and heteromeric geranyl diphosphate synthases exist in S. miltiorrhiza. The results suggest the complexity of terpenoid biosynthesis and the existence of metabolic channels for diverse terpenoids in S. miltiorrhiza and provide useful information for improving tanshinone production through genetic engineering.