Variation in floral form of CRISPR knock-outs of the poplar homologs of LEAFY and AGAMOUS after FT heat-induced early flowering

Plant migration and gene flow from genetically modified or exotic trees to nearby lands or by crossing with wild relatives is a major public and regulatory concern.Many genetic strategies exist to mitigate potential gene flow;however,the long delay in onset of flow-ering is a severe constraint to research progress.We used heat-induced FT overexpression to speed assessment of the expected floral phenotypes after CRISPR knockout of poplar homologs of the key floral genes,LEAFY and AGAMOUS.We selected events with previously characterized CRISPR-Cas9 induced biallelic changes then re-transformed them with the Arabidopsis thaliana FLOWERING LOCUS T(AtFT)gene under control of either a strong constitutive promoter or a heat-inducible promoter.We successfully obtained flowering in both a male and female clone of poplar,observing a wide range of inflorescence and floral forms among flowers,ramets,and insertion events.Overall,flowers obtained from the selected LFY and AG targeted events were consistent with what would be predicted for loss-of-function of these genes.LFY-targeted events showed small catkins with leaf-like organs,AG-targeted events had nested floral organs consistent with reduction in floral determinacy and absence of well-formed carpels or anthers.These findings demonstrate the great developmental plasticity of Populus flowers during genetically accelerated flowering,which may be of horticultural value.They also provide an informative early view of floral phenotypes and apparent sterility from knockouts of both these gene targets.

GWAS identifies candidate genes controlling adventitious rooting in Populus trichocarpa

Adventitious rooting(AR)is critical to the propagation,breeding,and genetic engineering of trees.The capacity for plants to undergo this process is highly heritable and of a polygenic nature;however,the basis of its genetic variation is largely uncharacterized.To identify genetic regulators of AR,we performed a genome-wide association study(GWAS)using 1148 genotypes of Populus trichocarpa.GWASs are often limited by the abilities of researchers to collect precise phenotype data on a high-throughput scale;to help overcome this limitation,we developed a computer vision system to measure an array of traits related to adventitious root development in poplar,including temporal measures of lateral and basal root length and area.GWAS was performed using multiple methods and significance thresholds to handle non-normal phenotype statistics and to gain statistical power.These analyses yielded a total of 277 unique associations,suggesting that genes that control rooting include regulators of hormone signaling,cell division and structure,reactive oxygen species signaling,and other processes with known roles in root development.Numerous genes with uncharacterized functions and/or cryptic roles were also identified.These candidates provide targets for functional analysis,including physiological and epistatic analyses,to better characterize the complex polygenic regulation of AR.

Overexpression of SHORT VEGETATIVE PHASE-LIKE (SVL) in Populus delays onset and reduces abundance of flowering in field-grown trees

The spread of transgenes and exotic germplasm from planted crops into wild or feral species is a difficult problem for public and regulatory acceptance of genetically engineered plants,particularly for wind-pollinated trees such as poplar.We report that overexpression of a poplar homolog of the floral repressor SHORT VEGETATIVE PHASE-LIKE(SVL),a homolog of the Arabidopsis MADS-box repressor SHORT VEGETATIVE PHASE(SVP),delayed the onset of flowering several years in three genotypes of field-grown transgenic poplars.Higher expression of SVL correlated with a delay in flowering onset and lower floral abundance,and did not cause morphologically obvious or statistically significant effects on leaf characteristics,tree form,or stem volume.Overexpression effects on reproductive and vegetative phenology in spring was modest and genotype-specific.Our results suggest that use of SVL and related floral repressors can be useful tools to enable a high level of containment for vegetatively propagated short-rotation woody energy or pulp crops.

Genetic down-regulation of gibberellin results in semi-dwarf poplar but few non-target effects on chemical resistance and tolerance to defoliation

Aims Plant stature can be strongly modified via regulation of endog-enous levels and signalling of the plant hormone gibberellin(GA).Down-regulation of GA can produce semi-dwarf tree varieties with improved qualities such as reduced susceptibility to wind damage,enhanced root growth and more compact cultivation.However,these modifications may have unintended,non-target consequences for defence against herbivores,via either of two mechanisms:(i)reduced biomass production may cause trade-offs with chemical resistance traits,as predicted by the growth-differentiation balance hypothesis,and(ii)altered biomass allocation to either roots or pho-tosynthetic tissues may affect regrowth potential and thus tolerance to defoliation.Methods We studied GA down-regulated(GE)and non-transgenic wild-type hybrid poplar(Populus alba×P.tremula)in an outdoor,above-ground common garden and defoliated half of all replicate trees to simulate defoliation.We then quantified the independent and inter-active effects of genotype and defoliation on growth and chemical resistance-related traits,including phenolic glycosides(PGs),con-densed tannin and nitrogen.We also calculated tolerance to defo-liation as the differential in relative growth between undefoliated and defoliated trees.Important Findings Our results indicate that two of the four GA down-regulated geno-types had significantly reduced stem height,basal diameter,vol-ume(d2h),total biomass and increased allocation to leaves relative to the wild type.One of those two genotypes also had reduced allocation to roots.One and sometimes both of these same two genotypes also had at least 20%lower levels of condensed tannins and PGs and similar increases in lignin and nitrogen.Tolerance,as calculated by the differential in relative growth between unde-foliated and defoliated trees,was similar among all experimental genotypes.However,two GE genotypes flushed fewer leaves in response to defoliation relative to the wild type.Our results indi-cate that GA down-regulation strongly alters biomass production and allocation in poplar but does not necessarily compromise the ability of these trees to tolerate damage.However,some of the modifications we observed do have the potential to alter non-target resistance traits over time,and warrant further research,especially under plantation conditions.

Reconfiguring Plant Metabolism for Biodegradable Plastic Production

For decades,plants have been the subject of genetic engineering to synthesize novel,value-added compounds.Polyhydroxyalkanoates(PHAs),a large class of biodegradable biopolymers naturally synthesized in eubacteria,are among the novel products that have been introduced to make use of plant acetyl-CoA metabolic pathways.It was hoped that renewable PHA production would help address environmental issues associated with the accumulation of nondegradable plastic wastes.However,after three decades of effort synthesizing PHAs,and in particular the simplest form polyhydroxybutyrate(PHB),and seeking to improve their production in plants,it has proven very difficult to reach a commercially profitable rate in a normally growing plant.This seems to be due to the growth defects associated with PHA production and accumulation in plant cells.Here,we review major breakthroughs that have been made in plant-based PHA synthesis using traditional genetic engineering approaches and discuss challenges that have been encountered.Then,from the point of view of plant synthetic biology,we provide perspectives on reprograming plant acetyl-CoA pathways for PHA production,with the goal of maximizing PHA yield while minimizing growth inhibition.Specifically,we suggest genetic elements that can be considered in genetic circuit design,approaches for nuclear genome and plastome modification,and the use of multiomics and mathematical modeling in understanding and restructuring plant metabolic pathways.