Shanfa Lu

Novel and Mechanical Stress–Responsive MicroRNAs in Populus trichocarpa That Are Absent from Arabidopsis

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 tree-specific 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.

Stress-responsive microRNAs in Populus

MicroRNAs (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 Ptc-miRNAs 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.

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.

Adenylation of plant miRNAs

The modification or degradation of RNAs including miRNAs may play vital roles in regulating RNA functions. The polyadenylation- and exosome-mediated RNA decay is involved in the degradation of plant RNAs including the primary miRNA processing intermediates. However, plant miRNA levels are not affected by exosome depletion. Here, we report the cloning of a large number of 5′ and/or 3′ truncated versions of the known miRNAs from various tissues of Populus trichocarpa (black cottonwood). It suggests that plant miRNAs may be degraded through either 5′ to 3′ or 3′ to 5′ exonucleolytic digestion. We also show that a significant portion of the isolated miRNAs contains, at the 3′-end, one or a few post-transcriptionally added adenylic acid residues, which are distinct in length from the polyadenylate tail added to other plant RNAs for exosome-mediated degradation. Using an in vitro miRNA degradation system, where synthesized miRNA oligos were degraded in extracts of P. trichocarpa cells, we revealed that the adenylated miRNAs were degraded slower than others without adenylation. It indicates that addition of adenylic acid residues on the 3′-end plays a negative role in miRNA degradation. Our results provide new information for understanding the mechanism of miRNA degradation.

A Genomic and Molecular View of Wood Formation

Wood formation is a process derived from plant secondary growth. Different from primary growth, plant secondary growth is derived from cambium meristem cells in the vascular and cork cambia and leads to the girth increase of the plant trunk. In the secondary growth process, plants convert most of photosynthesized products into various biopolymers for use in the formation of woody tissues. This article summarizes the new developments of genomic and genetic characterization of wood formation in herbaceous model plant and tree plant systems. Genomic studies have categorized a collection of the genes for which expression is associated with secondary growth. During wood formation, the expression of many genes is regulated in a stage-specific manner. The function of many genes involved in wood biosyntheses and xylem differentiation has been characterized. Although great progress has been achieved in the molecular and genomic understanding of plant secondary growth in recent years, the profound genetic mechanisms underlying this plant development remain to be investigated. Completion of the first tree genome sequence (Populus genome) provides a valuable genomic resource for characterization of plant secondary growth.

Conservation and diversity of microRNA-associated copper-regulatory networks in Populus trichocarpa.

Plants develop important regulatory networks to adapt to the frequently-changing availability of copper (Cu). However, little is known about miRNA-associated Cu-regulatory networks in plant species other than Arabidopsis. Here, we report that Cu-responsive miRNAs in Populus trichocarpa (Torr. & Gray) include not only conserved miR397, miR398 and miR408, but also Populus-specific miR1444, suggesting the conservation and diversity of Cu-responsive miRNAs in plants. Copper-associated suppression of mature miRNAs is in company with the up-regulation of their target genes encoding Cu-containing proteins in Populus. The targets include miR397-targeted PtLAC5, PtLAC6 and PtLAC110a, miR398-targeted PtCSD1, PtCSD2a and PtCSD2b, miR408-targeted PtPCL1, PtPCL2, PtPCL3 and PtLAC4, and miR1444-targeted PtPPO3 and PtPPO6. Consistently, P. trichocarpa miR408 promoter-directed GUS gene expression is down-regulated by Cu in transgenic tobacco plants. Cu-response elements (CuREs) are found in the promoters of Cu-responsive miRNA genes. We identified 34 SQUAMOSA-promoter binding protein-like (SPL) genes, of which 17 are full-length PtSPL proteins or partial sequences with at least 300 amino acids. Phylogenetic analysis indicates that PtSPL3 and PtSPL4 are CuRE-binding proteins controlling Cu-responsive gene expression. Cu appears to be not involved in the regulation of these transcription factors because neither PtSPL3 nor PtSPL4 is Cu-regulated and no CuRE exists in their promoters.

Specific down-regulation of PAL genes by artificial microRNAs in Populus trichocarpa

Artificial microRNAs (amiRNAs) are similar to microRNAs (miRNAs) in that they are able to reduce the abundance of specific transcripts in plants by RNA-Induced Silencing Complex (RISC)-mediated cleavage and degradation, but differ in that they are designed for specific targets. The long generation times of forest trees have limited the discovery of mutations by conventional genetics. AmiRNAs can create gene-specific transcript reduction in transgenic trees in a single generation and may have broad application for functional genomics of trees. In this paper, we describe the specific down-regulation of multiple genes in the phenylalanine ammonia-lyase (PAL) gene family of Populus trichocarpa using amiRNA sequences incorporated in a P. trichocarpa miRNA-producing precursor, ptc-MIR408. Two different amiRNA constructs were designed to specifically down-regulate two different subsets of PAL genes, revealing differential regulation within the gene family. Down-regulation of subset A (PAL2, PAL4 and PAL5) by amiRNA-palA led to an increase in transcript abundance of subset B (PAL1 and PAL3). The reciprocal effect was not observed.

Genetic modification of wood quality for second-generation biofuel production

How the abundant tree biomass resources can be efficiently used for future biofuel production has attracted a great deal of interest and discussion in the past few years. Capable technologies are expected to be developed to realize the production of biofuel from wood biomass. A significant effort is put into the field of modifying wood properties of trees and simplifying the process of biomass-to-ethanol conversion, which includes mainly genetic engineering of lignin, cellulose and hemicellulose of woods. Current research in this field has achieved some promising results and opened up new opportunities to utilize wood biomass efficiently. This review will discuss the main developments in genetic modification of lignin, cellulose and hemicellulose biosynthesis in trees as well as other potential genetic technology of biofuel production from wood biomass.