SWEET1 sugar transporter paralogs in hybrid poplar: Functional analysis and phenotypic characterization of CRISPR/Cas9 knockout lines

Sugar transporters of the SWEET family play a crucial role in carbon allocation in plants and enable the transport of sugars between different tissues in all plant organs. In trees, this process is particularly important for the supply of root systems and symbiotic partners such as ectomycorrhizae. The SWEET1 gene family in Populus tremula x alba comprises seven highly similar paralogs that have arisen due to the complex ge-nome structure of this hybrid species. However, the functional significance and evolu-tionary specialization of these paralogs was largely unknown.
The aim of this work was to characterize the functional role of SWEET1 paralogs by CRISPR/Cas9-mediated knockout and to investigate their importance for ectomycor-rhizal symbiosis. For this purpose, a multiplex CRISPR/Cas9 system was developed that targets all seven paralogs simultaneously. Evolutionary analyses using a haplotype-resolved genome enabled a systematic nomenclature and identification of gene clusters on chromosome 2 and 5. Transgenic lines were generated by agrobacteria-mediated transformation and characterized at the molecular, physiological and morphological level.
The results showed that SWEET1 paralogs are functionally specialized: While complete knockout of all paralogs resulted in severe growth defects, chimeric lines that had lost only chromosome 2 paralogs exhibited normal development. Complete knockout lines accumulated only about half the biomass of wild-type controls and showed altered carbon allocation with reduced leaf mass density and shifted biomass distribution in favor of leaves. Grafting experiments provided additional evidence for a disturbance of source-sink dynamics. Contrary to expectation, both knockout lines formed structurally normal ectomycorrhizas with Pisolithus microcarpus, suggesting that SWEET1 transporters are not required for the basic establishment of the symbiosis.
This study demonstrates for the functional specialization of gene duplicates in a complex hybrid genome and establishes methodological approaches for the analysis of gene families in tree hybrids. The results expand our understanding of carbon allocation in trees and have implications for forest management and breeding.