Abstract Detail

Functional Genetics/Genomics

Tuma, Trevor T [1], McInnes, Holly [1], Pham, Hongduyen [1], Bewg, William P. [1], Tsai, Chung-Jui [2].

CRISPR knockout of sucrose transporter affects phenology and biomass accrual in field grown hybrid poplar.

Woody perennial species experience dynamic fluctuations between active growth and dormancy that are heavily influenced by carbohydrate allocation. In the stem of poplar, genes involved in carbohydrate biosynthesis, transport, and catabolism exhibit contrasting seasonal expression responses. Yet, robust studies investigating the genetic and molecular mechanisms responsible for seasonal fluctuations of oligosaccharides and cell wall polysaccharides in temperate trees have been limited. Sucrose transport proteins (SUTs) are thought to play pivotal roles in sucrose transport, seasonal carbohydrate mobilization, and overall plant growth rates. The poplar genome contains five members in the SUT gene family with contrasting seasonal expression patterns. Among them, the single-copy SUT4 and the SUT5/SUT6 paralogs all exhibit winter-biased expression, while the SUT1/SUT3 paralogs display summer-biased expression hinting at seasonal specific roles amongst the gene family members. Here, we investigated the relationship between seasonal expression patterns of SUTs and the interplay between carbohydrate allocation, phenology, and biomass accrual in a bioenergy crop under natural conditions through a multi-year field trial. We targeted the winter-biased SUT genes for CRISPR/Cas9-mediated mutagenesis to generate single SUT4, SUT5, SUT6, and double SUT5/6 knock-out (KO) mutants in the female Populus tremula x alba (INRA 717-1B4) hybrid clone. We confirmed biallelic frameshift mutations in multiple independent transgenic events using amplicon deep sequencing. We then released >200 potted SUT-KO mutants, Cas9 vector control, and wild-type trees at an APHIS-permitted field site. Phenotypic differences were striking, as both the timing of fall leaf senescence and spring bud flush were altered in the KO mutants. For example, in the fall, when roughly 90% of the SUT4-KO lines had already dropped leaves, the majority of the control trees (>90%) still retained green leaves. Furthermore, the timing of spring bud flush was delayed by SUT4 perturbation, as the SUT4-KO mutants flushed ~1.5 weeks later and a significantly lower proportion (<60%) of buds flushed at the time over 95% of the control trees had flushed. In addition, SUT4 perturbation altered multiple tree growth attributes. After the first growth season in the field, the mutants displayed a ~35% reduction in stem diameter growth and ~15% reduction in tree height. These growth phenotypes are striking considering that prior characterization of greenhouse grown SUT mutants display subtle, if any, differences in biomass and growth. Surprisingly, perturbation of SUT5 and SUT6 did not appear to substantially impact tree growth or seasonal phenology. These results highlight the power of CRISPR gene editing and necessity of field testing for uncovering complex relationships between carbohydrate allocation and mobilization across seasonal transitions in perennial tree species. In the future, we aim to further interrogate the phenotypes with RNA-sequencing, metabolite profiling, and cell wall composition analyses across seasonal timepoints to determine how SUTs impact tree growth.

1 - University of Georgia, Warnell School of Forestry & Natural Resources, Department of Plant Biology, & Department of Genetics, Davison Life Sciences, 120 E Green Street, Athens, GA, 30602, USA
2 - University of Georgia, Warnell School of Forestry & Natural Resources, Department of Plant Biology, & Department of Genetics, Davison Life Sciences, 120 E Green Street, GA, GA, 30602, USA

sucrose transporter

Presentation Type: Poster
Number: PCG003
Abstract ID:556
Candidate for Awards:None

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