Abstract
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.
Similar content being viewed by others
References
Allen E, Xie Z, Gustafson AM, Carrington JC (2005) MicroRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121:207–221
Alvarez JP, Pekker I, Goldshmidt A, Blum E, Amsellem Z, Eshed Y (2006) Endogenous and synthetic microRNAs stimulate simultaneous, efficient, and localized regulation of multiple targets in diverse species. Plant Cell 18:1134–1151
Bartel DB (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297
Du J, Mansfield SD, Groover AT (2009) The Populus homeobox gene ARBORKNOX2 regulates cell differentiation during secondary growth. Plant J 60:1000–1014
Higuchi T (1997) Biochemistry and molecular biology of wood. Springer, New York, pp 131–181
Li L, Lu S, Chiang VL (2006) A genomic and molecular view of wood formation. Crit Rev Plant Sci 25:215–233
Li J, Brunner AM, Shevchenko O, Meilan R, Ma C, Skinner JS, Strauss SH (2008) Efficient and stable transgene suppression via RNAi in field-grown poplars. Transgenic Res 17:679–694
Llave C, Kasschau KD, Rector MA, Carrington JC (2002) Endogenous and silencing-associated small RNAs in plants. Plant Cell 14:1605–1619
Lu S, Sun YH, Shi R, Clark C, Li L, Chiang VL (2005) Novel and mechanical stress-responsive microRNAs in Populus trichocarpa that are absent from Arabidopsis. Plant Cell 17:2186–2203
Miki D, Itoh R, Shimamoto K (2005) RNA silencing of single and multiple members in a gene family of rice. Plant Physiol 138:1903–1913
Niu QW, Lin SS, Reyes JL, Chen KC, Wu HW, Yeh SD, Chua NH (2006) Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat Biotehcnol 24:1420–1428
Plomion C, Leprovost G, Stokes A (2001) Wood formation in trees. Plant Physiol 127:1513–1523
Ragauskas AJ, Williams CK, Davison BH, Britovsek G, Cairney J, Eckert CA, Frederick WJ Jr, Hallett JP, Leak DJ, Liotta CL et al (2006) The path forward for biofuels and biomaterials. Science 311:484–489
Rohde A, Morreel K, Ralph J, Goeminne G, Hostyn V, De Rycke R, Kushnir S, Van Doorsselaere J, Joseleau JP, Vuylsteke M et al (2004) Molecular phenotyping of the pal1 and pal2 mutants of Arabidopsis thaliana reveals far-reaching consequences on phenylpropanoid, amino acid, and carbohydrate metabolism. Plant Cell 16:2749–2771
Schlesinger WH, Lichter J (2001) Limited carbon storage in soil and litter of experimental forest plots under increased atmospheric CO2. Nature 411:466–469
Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18:1121–1133
Shi R, Chiang VL (2005) Facile means for quantifying microRNA expression by real-time PCR. Biotechniques 39:519–525
Shi R, Sun YH, Li Q, Heber S, Sederoff R, Chiang VL (2010) Towards a systems approach for lignin biosynthesis in Populus trichocarpa: transcript abundance and promoter sequence motifs of the monolignol biosynthetic genes. Plant Cell Physiol 51:144–163
Song J, Lu S, Chen ZZ, Lourenco R, Chiang VL (2006) Genetic transformation of Populus trichocarpa genotype Nisqually-1: a functional genomic tool for woody plants. Plant Cell Physiol 47:1582–1589
Suzuki S, Li L, Sun YH, Chiang VL (2006) The cellulose synthase gene superfamily and biochemical functions of xylem-specific cellulose synthase-like genes in Populus trichocarpa. Plant Physiol 142:1233–1245
Tuskan G, DiFazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U, Putnam N, Ralph S, Rombauts S, Salamov A et al (2006) The genome of black cottonwood, Populus trichocarpa (Torr& Gray). Science 313:1596–1604
Xu P, Zhang Y, Kang L, Roossinck MJ, Mysore KS (2006) Computational estimation and experimental verification of off-target silencing during posttranscriptional gene silencing in plants. Plant Physiol 142:429–440
Zeng Y, Wagner EJ, Cullen BR (2002) Both natural and designed microRNA can inhibit the expression of cognate mRNAs when expressed in human cells. Mol Cell 9:1327–1333
Acknowledgments
This work was supported by the Forest Biotechnology Industrial Research Consortium (FORBIRC) at North Carolina State University and The National Science Foundation, Plant Genome Research Program DBI-0922391.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Shi, R., Yang, C., Lu, S. et al. Specific down-regulation of PAL genes by artificial microRNAs in Populus trichocarpa . Planta 232, 1281–1288 (2010). https://doi.org/10.1007/s00425-010-1253-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00425-010-1253-3