Chung-Jui Tsai

In hybrid plants, phenotypic outcomes are governed by interactions between the two parental genomes. However, the mechanisms underlying the interplay of divergent regulatory networks from these genomes remain poorly understood. In this study, we compared gene-level and allele-specific expression patterns, as well as differentially enriched pathways between F₁ and complex backcross lines derived from a natural interspecific hybrid population of Populus fremontii S. Watson (Pf) and Populus angustifolia E. James (Pa). Metabolic differences between Pf and Pa, which exhibit low and high levels respectively of phenylpropanoid-derived condensed tannins, were leveraged. Using individualized transcriptome references, differential expression and clustering analyses revealed CBC-biased and F₁-biased expression for genes involved in phenylpropanoid metabolism and photosynthesis, respectively. Biased expression of these genes at the allele level was also observed in F1. At the whole-transcriptome level, Pa-biased genes predominated in F₁ hybrids, and Pa alleles displayed more conserved expression patterns than Pf alleles across examined samples. Further analyses indicated that allelic expression bias was significantly associated with parental origin, which could be driven by sequence variations in cis-regulatory elements and differences in Cytosine-phosphate-Guanine (CpG) island length. Our findings demonstrate strong parent-of-origin effects on divergent regulatory networks governing gene expression in poplar hybrids and provide clues for strategic parental selection tailored to specific metabolic pathways of interest.

Chromosome□scale genome engineering in plants typically relies on locus-specific recombination during meiosis to select desirable outcomes, limiting its application in species with long generation times. Here, we show that CRISPR targeting of abundant satellite DNA enables multiplexed chromosome restructuring in aspen, generating diverse large-scale structural variants in the first generation. These megabase rearrangements remain mitotically stable through clonal propagation and regeneration with no obvious growth defects.

Cytosolic ascorbate peroxidases (APXs) have been proposed to have bifunctional 4-coumarate 3-hydroxylase (C3H) activity, linking redox regulation to lignin biosynthesis in plants. Although this dual role has been shown in vitro, in vivo validation remains limited. Here, we used CRISPR/Cas9 gene editing to knock out cytosolic C3H/APX genes in Brachypodium distachyon and poplar (Populus tremula × Populus alba). In Brachypodium, BdC3H/APX1 catalyzed the ascorbate-dependent hydroxylation of 4-coumarate to caffeate in vitro. Loss of BdC3H/APX1 function led to reduced lignin content, altered monomer composition, elevated H2O2 levels, and impaired growth, while double monoallelic knockouts of BdC3H/APX1 combined with a biallelic BdC3H/APX2 mutation (Bdc3h/apx1&2) exhibited severe developmental defects. Exogenous caffeate and ferulate rescued the growth and lignin phenotype of the Bdc3h/apx1 knockout mutants, whereas catalase reduced H2O2 without restoring plant growth. Similarly, CRISPR/Cas9-mediated PtC3H/APX1 knockout in poplar resulted in stunted growth and altered lignin composition, while the double Ptc3h/apx1&2 mutants were unable to regenerate from tissue culture. These results provide in vivo evidence of C3H/APX bifunctionality, suggesting that perturbed lignin biosynthesis is the primary cause of the growth defects typically observed in C3H/APX-deficient plants.

SUMMARY: Caterpillars and beetles are among the most economically damaging defoliating insects, and their economic damage is predicted to increase in the coming decades. Hence the use of genetically derived resistance to supplement other pest control strategies is warranted. In soybean ( Glycine max (L.) Merr.), a major determinant for resistance is the quantitative trait locus, QTL‐M. Glyma07g14530 , the gene underlying QTL‐M, encodes a feeding‐inducible flavonol 3‐ O ‐glycosyltransferase (F3GlcT or UGT78D2) that glucosylates kaempferol, as well as quercetin, myricetin, and isorhamnetin. The resistant allele has a premature stop codon in it, thus preventing the glucosylation and sequestration of flavonols in the vacuole, leading to a concomitant accumulation of proanthocyanidins and manifestation of resistance. Expressing the dominant (susceptible) allele in resistant plants restores susceptibility, and silencing the susceptible allele results in resistance. The discovery and characterization of GmF3GlcT helps clarify the role of flavonoids in resistance to leaf‐chewing insects and facilitates the development of insect‐resistant cultivars that ultimately can lower production costs and reduce insecticide applications.

SUMMARY: Climate uncertainty is intensifying the need for greater plasticity in carbohydrate reserve utilization to support winter survival and spring growth in woody perennials. In poplar, the single‐copy SUT4 , which encodes a tonoplast‐localized sucrose transporter, and the SUT5 / SUT6 genome duplicates, which encode plasma membrane‐localized transporters, are expressed year‐round, with SUT4 showing the highest expression during cool seasons. Given its role in vacuolar sucrose efflux and winter‐predominant expression, SUT4 may play a key role in modulating seasonal carbohydrate dynamics. While SUT4‐ knockdown and knockout effects have been studied under greenhouse conditions, their impact under field conditions remains unexplored. Here, we report a field‐based study comparing CRISPR knockout mutants of winter‐expressed SUT4 and SUT5 / SUT6 in Populus tremula × alba . We show that sut4 , but not sut5/6 , mutants exhibited earlier autumn leaf senescence, delayed spring bud flush, reduced stem growth, and altered sugar partitioning in winter xylem and bark relative to controls. After 2 years in the field, all genotypes flowered before leaf flush in early spring; however, sut4 mutants produced sterile ovules despite developing normal‐looking catkins. Metabolic profiling revealed disrupted sucrose and raffinose dynamics in elongating sut4 catkins. This was accompanied by transcriptomic signatures of elevated stress and downregulation of proanthocyanidin biosynthesis and circadian clock genes. These findings highlight the critical role of SUT4 in coordinating sugar allocation, stress responses, and seasonal development in poplar.

SUMMARY: Multiplex CRISPR editing has emerged as a transformative platform for plant genome engineering, enabling the simultaneous targeting of multiple genes, regulatory elements, or chromosomal regions. This approach is effective for dissecting gene family functions, addressing genetic redundancy, engineering polygenic traits, and accelerating trait stacking and de novo domestication. Its applications now extend beyond standard gene knockouts to include epigenetic and transcriptional regulation, chromosomal engineering, and transgene‐free editing. These capabilities are advancing crop improvement not only in annual species but also in more complex systems such as polyploids, undomesticated wild relatives, and species with long generation times. At the same time, multiplex editing presents technical challenges, including complex construct design and the need for robust, scalable mutation detection. We discuss current toolkits and recent innovations in vector architecture, such as promoter and scaffold engineering, that streamline workflows and enhance editing efficiency. High‐throughput sequencing technologies, including long‐read platforms, are improving the resolution of complex editing outcomes such as structural rearrangements—often missed by standard genotyping—when targeting repetitive or tandemly spaced loci. To fully realize the potential of multiplex genome engineering, there is growing demand for user‐friendly, synthetic biology‐compatible, and scalable computational workflows for gRNA design, construct assembly, and mutation analysis. Experimentally validated inducible or tissue‐specific promoters are also highly desirable for achieving spatiotemporal control. As these tools continue to evolve, multiplex CRISPR editing is poised to become a foundational technology of next‐generation crop improvement to address challenges in agriculture, sustainability, and climate resilience.