Because lignin limits the use of wood for fiber, chemical, and energy production, strategies for its downregulation are of considerable interest. We have produced transgenic aspen (Populus tremuloides Michx.) trees in which expression of a lignin biosynthetic pathway gene Pt4CL1 encoding 4-coumarate:coenzyme A ligase (4CL) has been downregulated by antisense inhibition. Trees with suppressed Pt4CL1 expression exhibited up to a 45% reduction of lignin, but this was compensated for by a 15% increase in cellulose. As a result, the total lignin-cellulose mass remained essentially unchanged. Leaf, root, and stem growth were substantially enhanced, and structural integrity was maintained both at the cellular and whole-plant levels in the transgenic lines. Our results indicate that lignin and cellulose deposition could be regulated in a compensatory fashion, which may contribute to metabolic flexibility and a growth advantage to sustain the long-term structural integrity of woody perennials.

Summary• Salicin-based phenolic glycosides, hydroxycinnamate derivatives and flavonoidderived condensed tannins comprise up to one-third of Populus leaf dry mass. Genes regulating the abundance and chemical diversity of these substances have not been comprehensively analysed in tree species exhibiting this metabolically demanding level of phenolic metabolism.• Here, shikimate-phenylpropanoid pathway genes thought to give rise to these phenolic products were annotated from the Populus genome, their expression assessed by semiquantitative or quantitative reverse transcription polymerase chain reaction (PCR), and metabolic evidence for function presented.• Unlike Arabidopsis , Populus leaves accumulate an array of hydroxycinnamoylquinate esters, which is consistent with broadened function of the expanded hydroxycinnamoyl-CoA transferase gene family. Greater flavonoid pathway diversity is also represented, and flavonoid gene families are larger. Consistent with expanded pathway function, most of these genes were upregulated during wound-stimulated condensed tannin synthesis in leaves.• The suite of Populus genes regulating phenylpropanoid product accumulation should have important application in managing phenolic carbon pools in relation to climate change and global carbon cycling.

CRISPR (clustered regularly interspaced short palindromic repeats), first discovered as an immune system of prokaryotes, has become a powerful tool for genome editing in eukaryotes (Gaj et al., 2013). Co-expression of the CRISPR-associated endonuclease (Cas9) with a chimeric guide-RNA (gRNA) targeting a GN 19 NGG motif results in a double-strand DNA break near NGG, the protospacer adjacent motif (PAM) (Jinek et al., 2012). Processing by the endogenous DNA repair machinery generates small indels that, when located within a coding sequence, can disrupt the reading frame and render the gene nonfunctional. The CRISPR/ Cas9 system has been successfully applied in several herbaceous systems (Belhaj et al., 2013;Harrison et al., 2014). Here we report its application in the woody perennial Populus using the 4-coumarate:CoA ligase (4CL) gene family as a case study. We achieved 100% mutational efficiency for two 4CL genes targeted, with every transformant examined carrying biallelic modifications. The CRISPR/Cas9 system is highly sensitive to single nucleotide polymorphisms (SNPs), as cleavage for a third 4CL gene was abolished due to SNPs in the target sequence. For outcrossing species with a highly heterozygous genome, gRNA design must take into account the frequent occurrence of SNPs to achieve efficient genome editing.Two 4CL genes, 4CL1 and 4CL2, associated with lignin and flavonoid biosynthesis, respectively (Hu et al., 1998;Harding et al., 2002), were targeted for CRISPR/Cas9 editing. The Populus tremula 9 alba clone 717-1B4 (717) routinely used for transformation is divergent from the genome-sequenced Populus trichocarpa (Hamzeh & Dayanandan, 2004). Therefore, the 4CL1 and 4CL2 gRNAs designed from the reference genome were interrogated with in-house 717 RNA-Seq data to ensure the absence of SNPs which could limit Cas9 efficiency (Supporting Information Fig. S1). A third gRNA designed for 4CL5, a genome duplicate of 4CL1, was also included. The corresponding 717 sequence harbors one SNP in each allele near/within the PAM, both of which are expected to abolish targeting by the 4CL5-gRNA (Fig. S1). All three gRNA target sites are located within the first exon (Fig. S1a).

SUMMARYPlasma membrane, proton-coupled Group II sucrose symporters (SUT) mediate apoplastic phloem loading and sucrose efflux from source leaves in Arabidopsis and agricultural crop species that have been studied to date. We now report that the most abundantly expressed SUT isoform in Populus tremula · alba, PtaSUT4, is a tonoplast (Group IV) symporter. PtaSUT4 transcripts were readily detected in conducting as well as mesophyll cells in stems and source leaves. In comparison, Group II orthologs PtaSUT1 and PtaSUT3 were very weakly expressed in leaves. Both Group II and Group IV SUT genes were expressed in secondary stem xylem of Populus. Transgenic poplars with RNAi-suppressed PtaSUT4 exhibited increased leaf-to-stem biomass ratios, elevated sucrose content in source leaves and stems, and altered phenylpropanoid metabolism. Transcript abundance of several carbohydrate-active enzymes and phenylalanine ammonia-lyases was also altered in transgenic source leaves. Nitrogen-limitation led to a down-regulation of vacuolar invertases in all plants, which resulted in an augmentation of sucrose pooling and hexose depletion in source leaves and secondary xylem of the transgenic plants. These results are consistent with a major role for PtaSUT4 in orchestrating the intracellular partitioning, and consequently, the efflux of sucrose from source leaves and the utilization of sucrose by lateral and terminal sinks. Our findings also support the idea that PtaSUT4 modulates sucrose efflux and utilization in concert with plant N-status.