@article{lu_yang_chiang_2011, title={Conservation and Diversity of MicroRNA-associated Copper-regulatory Networks in Populus trichocarpa}, volume={53}, ISSN={["1744-7909"]}, DOI={10.1111/j.1744-7909.2011.01080.x}, abstractNote={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.}, number={11}, journal={JOURNAL OF INTEGRATIVE PLANT BIOLOGY}, author={Lu, Shanfa and Yang, Chenmin and Chiang, Vincent L.}, year={2011}, month={Nov}, pages={879–891} }
 @article{shi_yang_lu_sederoff_chiang_2010, title={Specific down-regulation of PAL genes by artificial microRNAs in Populus trichocarpa}, volume={232}, ISSN={["0032-0935"]}, DOI={10.1007/s00425-010-1253-3}, number={6}, journal={PLANTA}, author={Shi, Rui and Yang, Chenmin and Lu, Shanfa and Sederoff, Ronald and Chiang, Vincent L.}, year={2010}, month={Nov}, pages={1281–1288} }
 @article{lu_sun_chiang_2009, title={Adenylation of plant miRNAs}, volume={37}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkp031}, abstractNote={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.}, number={6}, journal={NUCLEIC ACIDS RESEARCH}, author={Lu, Shanfa and Sun, Ying-Hsuan and Chiang, Vincent L.}, year={2009}, month={Apr}, pages={1878–1885} }
 @article{lu_li_yi_joshi_chiang_2008, title={Differential expression of three eucalyptus secondary cell wall-related cellulose synthase genes in response to tension stress}, volume={59}, ISSN={["1460-2431"]}, DOI={10.1093/jxb/erm350}, abstractNote={Trees constitute the majority of lignocellulosic biomass existing on our planet. Trees also serve as important feedstock materials for various industrial products. However, little is known about the regulatory mechanisms of cellulose synthase (CesA) genes of trees. Here, the cloning and characterization of three CesA genes (EgraCesA1, EgraCesA2, and EgraCesA3) from an economically important tree species, Eucalyptus grandis, are reported. All three genes were specifically expressed in xylem cells of eucalyptus undergoing secondary cell wall biosynthesis. The GUS gene, expressed under the control of the EgraCesA2 or EgraCesA3 promoter, was also localized in the secondary xylem in transgenic tobacco stems. However, the EgraCesA1 promoter alone or along with its 5'-UTR introns was insufficient to direct appropriate GUS expression. EgraCesA2 and EgraCesA3 gene expression was up-regulated in tension-stressed eucalyptus xylem cells. Accordingly, GUS expression directed by the EgraCesA2 or EgraCesA3 promoter was also up-regulated. EgraCesA1 had no such response. Thus, it is most unlikely that EgraCesA1 is a subunit of the EgraCesA2-EgraCesA3 complex. The presence of at least two types of cellulose biosynthesis machinery in wood formation is an important clue in deciphering the underpinnings of the perennial growth of trees in various environmental conditions. By analysing GUS gene expression directed by the EgraCesA3 promoter or its deletions, several negative and positive regulatory regions controlling gene expression in xylem or phloem were identified. Also a region which is likely to contain mechanical stress-responsive elements was deduced. These results will guide further studies on identifying cis-regulatory elements directing CesA gene transcription and wood formation regulatory networks.}, number={3}, journal={JOURNAL OF EXPERIMENTAL BOTANY}, author={Lu, Shanfa and Li, Laigeng and Yi, Xiaoping and Joshi, Chandrashekhar P. and Chiang, Vincent L.}, year={2008}, month={Feb}, pages={681–695} }
 @article{nakatsubo_li_hattori_lu_sakakibara_chiang_shimada_suzuki_umezawa_2008, title={Roles of 5-hydroxyconiferylaldehyde and caffeoyl CoA O-methyltransferases in monolignol biosynthesis in Carthamus tinctorius}, volume={41}, number={9-10}, journal={Cellulose Chemistry and Technology}, author={Nakatsubo, T. and Li, L. and Hattori, T. and Lu, S. and Sakakibara, N. and Chiang, V. L. and Shimada, M. and Suzuki, S. and Umezawa, T.}, year={2008}, pages={511–520} }
 @article{lu_sun_chiang_2008, title={Stress-responsive microRNAs in Populus}, volume={55}, ISSN={["1365-313X"]}, DOI={10.1111/j.1365-313X.2008.03497.x}, abstractNote={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.}, number={1}, journal={PLANT JOURNAL}, author={Lu, Shanfa and Sun, Ying-Hsuan and Chiang, Vincent L.}, year={2008}, month={Jul}, pages={131–151} }
 @misc{lu_sun_amerson_chiang_2007, title={MicroRNAs in loblolly pine (Pinus taeda L.) and their association with fusiform rust gall development}, volume={51}, ISSN={["1365-313X"]}, DOI={10.1111/j.1365-313x.2007.03208.x}, abstractNote={MicroRNAs (miRNAs) are endogenous small RNAs that can have large-scale regulatory effects on development and on stress responses in plants. The endemic rust fungus Cronartium quercuum f. sp. fusiforme causes fusiform rust disease in pines, resulting in the development of spindle-shaped galls (cankers) on branches or stems. This disease is the most destructive disease of pines in the southern USA. To test whether miRNAs play roles in fusiform rust gall development, we cloned and identified 26 miRNAs from stem xylem of loblolly pine (Pinus taeda), which belong to four conserved and seven loblolly pine-specific miRNA families. Forty-three targets for nine of these 11 families were experimentally validated in vivo. Sequence analysis suggested that the target cleavage site may be determined not only by the miRNA sequence but also by the target sequence. Members of three loblolly pine-specific miRNA families target a large number of non-protein coding transcripts, and one of these families could also initiate secondary phased production from its target of a putative trans-acting short interfering RNA (ta-siRNA). Expression of 10 of these 11 miRNA families was significantly repressed in the galled stem. PCR-based transcript quantification showed complex expression patterns of these miRNAs and their targets in the galled tissues and in tissues surrounding the gall. We further predict 82 plant disease-related transcripts that may also response to miRNA regulation in pine. These results reveal a new genetic basis for host-pathogen interactions in the development of fusiform rust gall.}, number={6}, journal={PLANT JOURNAL}, author={Lu, Shanfa and Sun, Ying-Hsuan and Amerson, Henry and Chiang, Vincent L.}, year={2007}, month={Sep}, pages={1077–1098} }
 @misc{li_lu_chiang_2006, title={A genomic and molecular view of wood formation}, volume={25}, ISSN={["1549-7836"]}, DOI={10.1080/07352680600611519}, abstractNote={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.}, number={3}, journal={CRITICAL REVIEWS IN PLANT SCIENCES}, author={Li, Laigeng and Lu, Shanfa and Chiang, Vincent}, year={2006}, pages={215–233} }
 @article{lu_zhou_li_chiang_2006, title={Distinct roles of cinnamate 4-hydroxylase genes in Populus}, volume={47}, ISSN={["1471-9053"]}, DOI={10.1093/pcp/pcj063}, abstractNote={Cinnamate 4-hydroxylase (C4H) catalyzes the conversion of cinnamate into 4-hydroxy-cinnamate, a key reaction of the phenylpropanoid pathway which leads to the biosynthesis of several secondary metabolites. C4H genes exist as a multigene family in various plant species. In order to understand the roles of individual C4H members, four C4H cDNAs (PtreC4H) were isolated from Populus tremuloides and three C4H loci (PtriC4H) were identified in the P. trichocarpa genome. The ability of Populus C4H isoforms to convert trans-cinnamate into p-coumaric acid was verified by the examination of yeast recombinant PtreC4H proteins. Populus C4H genes were expressed in various tissues, including developing xylem, phloem and epidermis; however, the expression patterns of individual members were different from each other. Sequential analysis of C4H promoters showed that the differential expression of C4H genes was associated with cis-acting regulatory elements such as box L, box P and H box, suggesting that the divergent C4H isoforms played distinct roles in the production of secondary metabolites. The involvement of specific C4H isoforms in the biosynthesis of guaiacyl and syringyl monolignols is discussed.}, number={7}, journal={PLANT AND CELL PHYSIOLOGY}, author={Lu, Shanfa and Zhou, Yihua and Li, Laigeng and Chiang, Vincent L.}, year={2006}, month={Jul}, pages={905–914} }
 @article{song_lu_chen_lourenco_chiang_2006, title={Genetic transformation of Populus trichocarpa genotype Nisqually-1: A functional genomic tool for woody plants}, volume={47}, ISSN={["1471-9053"]}, DOI={10.1093/pcp/pcl018}, abstractNote={We report here the Agrobacterium-mediated genetic transformation of Nisqually-1, a Populus trichocarpa genotype whose genome was recently sequenced. Several systems were established. Internodal stem segments from vigorously growing greenhouse plants are the explants most amenable to transformation. For the most efficient system, approximately 40% of the stem segments infected with pBI121-containing Agrobacterium tumefaciens C58 produced transgenic calli, as confirmed by beta-glucuronidase (GUS) staining. The regeneration efficiency of independent transgenic plants was approximately 13%, as revealed by genomic Southern analysis. Some transgenic plants were produced in as little as 5 months after co-cultivation. This system may help to facilitate studies of gene functions in tree growth and development at a genome level.}, number={11}, journal={PLANT AND CELL PHYSIOLOGY}, author={Song, Jingyuan and Lu, Shanfa and Chen, Zenn-Zong and Lourenco, Rodrigo and Chiang, Vincent L.}, year={2006}, month={Nov}, pages={1582–1589} }
 @article{li_cheng_lu_nakatsubo_umezawa_chiang_2005, title={Clarification of cinnamoyl co-enzyme a reductase catalysis in monolignol biosynthesis of aspen}, volume={46}, ISSN={["1471-9053"]}, DOI={10.1093/pcp/pci120}, abstractNote={Cinnamoyl co-enzyme A reductase (CCR), one of the key enzymes involved in the biosynthesis of monolignols, has been thought to catalyze the conversion of several cinnamoyl-CoA esters to their respective cinnamaldehydes. However, it is unclear which cinnamoyl-CoA ester is metabolized for monolignol biosynthesis. A xylem-specific CCR cDNA was cloned from aspen (Populus tremuloides) developing xylem tissue. The recombinant CCR protein was produced through an Escherichia coli expression system and purified to electrophoretic homogeneity. The biochemical properties of CCR were characterized through direct structural corroboration and quantitative analysis of the reaction products using a liquid chromatography-mass spectrometry system. The enzyme kinetics demonstrated that CCR selectively catalyzed the reduction of feruloyl-CoA from a mixture of five cinnamoyl CoA esters. Furthermore, feruloyl-CoA showed a strong competitive inhibition of the CCR catalysis of other cinnamoyl CoA esters. Importantly, when CCR was coupled with caffeoyl-CoA O-methyltransferase (CCoAOMT) to catalyze the substrate caffeoyl-CoA ester, coniferaldehyde was formed, suggesting that CCoAOMT and CCR are neighboring enzymes. However, the in vitro results also revealed that the reactions mediated by these two neighboring enzymes require different pH environments, indicating that compartmentalization is probably needed for CCR and CCoAOMT to function properly in vivo. Eight CCR homologous genes were identified in the P. trichocarpa genome and their expression profiling suggests that they may function differentially.}, number={7}, journal={PLANT AND CELL PHYSIOLOGY}, author={Li, LG and Cheng, XF and Lu, SF and Nakatsubo, T and Umezawa, T and Chiang, VL}, year={2005}, month={Jul}, pages={1073–1082} }
 @article{lu_sun_shi_clark_li_chiang_2005, title={Novel and mechanical stress-responsive microRNAs in Populus trichocarpa that are absent from Arabidopsis}, volume={17}, DOI={10.1105/tpc.105.033456}, abstractNote={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.}, number={8}, journal={Plant Cell}, author={Lu, S. F. and Sun, Y. H. and Shi, R. and Clark, C. and Li, L. G. and Chiang, V. L.}, year={2005}, pages={2186–2203} }
 @article{lu_shi_tsao_yi_li_chiang_2004, title={RNA silencing in plants by the expression of siRNA duplexes}, volume={32}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gnh170}, abstractNote={In animal cells, stable RNA silencing can be achieved by vector-based small interfering RNA (siRNA) expression system, in which Pol III RNA gene promoters are used to drive the expression of short hairpin RNA, however, this has not been demonstrated in plants. Whether Pol III RNA gene promoter is capable of driving siRNA expression in plants is unknown. Here, we report that RNA silencing was achieved in plants through stable expression of short hairpin RNA, which was driven by Pol III RNA gene promoters. Using glucuronidase (GUS) transformed tobacco as a model system, the results demonstrated that 21 nt RNA duplexes, targeting at different sites of GUS gene, were stably expressed under the control of either human H1 or Arabidopsis 7SL RNA gene promoter, and GUS gene was silenced in 80% of siRNA transgenics. The severity of silencing was correlated with the abundance of siRNA expression but independent of the target sites and uridine residue structures in siRNA hairpin transcripts. Thus, the specific expression of siRNA provides a new system for the study of siRNA silencing pathways and functional genomics in plants. Moreover, the effectiveness of the human H1 promoter in a plant background suggested a conserved mechanism underlying Pol III complex functionality.}, number={21}, journal={NUCLEIC ACIDS RESEARCH}, author={Lu, SF and Shi, R and Tsao, CC and Yi, XP and Li, LG and Chiang, VL}, year={2004} }