ABSTRACT Genetic transformation is a powerful tool in the realm of plant sciences, enabling the study of endogenous and heterologous genes and conferring novel traits to transformed plant species, notably in crop species of agricultural importance. The ability to genetically transform plants has historically been contingent upon the development of tissue culture methods for the species of interest, a significant technical limitation due to the recalcitrance of many plant species in tissue culture. Recent advances in molecular techniques and biotechnological resources have facilitated the development of methods that enable the genetic transformation of plants without the need for tissue culture. In this study, we have developed a method to transform the model tree system Populus using Agrobacterium rhizogenes carrying the RUBY visual marker gene as well as methods to regenerate whole transgenic plants, all with a tissue culture‐free protocol. This method greatly simplifies the genetic transformation procedure for the poplar model system and could potentially be applied to other tree species that lack developed tissue culture protocols.
ABSTRACT JASMONATE ZIM‐DOMAIN (JAZ) proteins act as central repressors of jasmonate signaling, modulating plant development, stress resilience, and secondary metabolism. Despite the economic and ecological importance of the aromatic tree Cinnamomum burmannii , its JAZ family has not been systematically explored. We performed a genome‐wide survey and identified 12 CbJAZ genes, all harboring conserved TIFY and Jas domains and unevenly distributed across seven chromosomes. Phylogenetic reconstruction assigned them to four clades, each exhibiting conserved exon–intron structures and motif compositions. Segmental duplication events primarily drove the expansion of this family. Promoter cis ‐element analysis revealed a rich repertoire of hormone‐, light‐, and stress‐responsive motifs. Tissue‐specific expression profiling showed that most CbJAZ transcripts accumulate preferentially in flowers and fruits. Upon exogenous methyl jasmonate (MeJA) treatment, the majority of CbJAZ genes were significantly induced, whereas abscisic acid (ABA) and drought stress generally suppressed their expression. Notably, CbJAZ8 and CbJAZ9 emerged as robust responders to all three treatments, and their encoded proteins localized to the nucleus, implicating them as candidate genes for future investigation of JA, ABA, and drought signaling crosstalk and potential regulators of essential oil biosynthesis. This first comprehensive characterization of the CbJAZ family not only furnishes a valuable genetic resource but also pinpoints CbJAZ8 and CbJAZ9 as prime candidates for future investigations into JA‐mediated stress adaptation and essential oil regulation in this economically relevant tree species.
ABSTRACT Base editing (BE) technologies enable precise genome modification but often generate undesired editing outcomes, including indels, bystander edits, and editing outside the optimal activity windows. Here, we report a modular BE strategy, termed Free‐BE, where the cytidine deaminase and Cas9 nickase are delivered as separate components rather than as a single fusion protein. In the moss Physcomitrium patens , Free‐BE showed lower editing efficiency than the conventional fused system (Fused‐BE). However, it generated fewer indels, a higher proportion of single‐nucleotide edits, and reduced out‐of‐window editing. Free‐BE also altered the spectrum of base substitutions, producing a higher proportion of C‐to‐G and C‐to‐A transversions than Fused‐BE in P. patens . No mutations were detected at four predicted off‐target loci examined in P. patens . We further validated Free‐BE in crop systems. In a transient maize protoplast reporter assay, Free‐BE achieved targeted base conversions, whereas stable transformation of wheat demonstrated successful editing of endogenous loci, although with lower efficiency than Fused‐BE. In wheat, Free‐BE similarly reduced multicytidine editing and by‐products. Together, these results establish Free‐BE as a functional modular BE strategy that broadens the plant genome‐editing toolbox and may be advantageous for applications requiring greater control over editing outcomes, despite lower overall editing efficiency.
Chalcone isomerase (CHI) catalyzes the cyclization of 3-ring scaffolds of flavonoids, a class of plant-based natural products important for nutrition and disease prevention. A persistent question has been whether the enzyme uses dynamics to facilitate conformational rearrangements of substrates within the active site. To help resolve this question, CHI was crystallized with phloretin, a flexible substrate analogue that cannot undergo cyclization. The crystal structure possesses eight protein molecules per asymmetric unit, revealing different active site conformations that accommodate different bound conformers of phloretin. Together, the structural snapshots depict a series of coordinated, dynamic chemical interactions that lower barriers to substrate rearrangements approaching bond formation. Differential scanning fluorimetry combined with mutational analysis and enzyme kinetics further confirm that phloretin binds to the enzyme active site and that it acts as a competitive inhibitor of CHI. Together these findings answer outstanding questions about the flexibility and dynamics of CHI catalysis, information that may be useful for future biosynthetic design and enzyme engineering goals. Overall, this work supports a catalytic model in which the CHI enzyme operates as a dynamic ensemble of structures necessary to facilitate catalytic substrate rearrangements. ### Competing Interest Statement The authors have declared no competing interest. * CHI : chalcone isomerase CHI-I : type-1 chalcone isomerase CHI-2 : type-2 chalcone isomerase Mt CHI-I : Medicago truncatula type-1 CHI DSF : differential scanning fluorimetry App. Kd : apparent equilibrium dissociation constant ΔTmmax : maximum thermal shift RMS : root mean square National Institutes of Health, T34GM136467
ABSTRACT Plant vasculature is essential for the transport of water, nutrients, and signaling molecules across organs, while also providing critical mechanical support for growth and development. Disruptions in vascular bundle formation can therefore lead to severe physiological and developmental defects. In maize, ethyl methanesulfonate (EMS)‐induced dominant nonallelic Wilty mutants exhibit a pronounced wilting phenotype even under well‐watered conditions, indicating underlying defects in vascular function. In this study, we characterized the Wi3 mutant, identified as ZmDrought‐Overly‐Sensitive1/DOS1, and compared it with the previously described Wi2 mutant to uncover shared mechanisms underlying their phenotypes. We provide evidence, by bulk segregant resequencing linkage disequilibrium of SNPs adjacent to the causal Wilty SNPs in respective ß‐ and α‐tubulin genes, for the personal communication from Gerry Neuffer that Wi2/ß‐tub6 provenance is from ACR‐related stock, whereas Wi3/α‐tub4 allele is from Mo17, not B73 as claimed by the authors who cloned Dos1. Histochemical staining and Fourier‐transform infrared (FTIR) spectroscopy of vascular bundles in Wi3 indicated apparent alterations in cellulose and lignin content consistent with those observed in Wi2. Transcriptome analysis of shoot meristems further indicated that similar sets of genes and pathways are differentially expressed in both mutants, suggesting convergence on common biological pathways. Using bulk‐segregant whole‐genome resequencing, we identified alpha‐tubulin4 (TUA4) as the causal gene in Wi3 (ZmDOS1), harboring a C‐to‐T substitution within the N‐terminal GTPase‐binding domain. This mutation results in a glutamic acid196‐to‐lysine substitution. Given that α‐ and β‐tubulin subunits heterodimerize, and in many plants and animal mutant alleles are dominant‐negative gains‐of‐function, we infer Wi2, Wi3, and likely Wi4, based on very similar FTIR biophysical difference spectra, may act as effectors of vascular bundle cell wall deposition, potentially involving vesicle trafficking as recently shown for asymmetric cell divisions in maize stomatal development. Together, these findings highlight the functional interdependence of tubulin subunits and provide a plausible mechanistic framework for the striking biophysical, transcriptomic, and phenotypic similarities observed between Wi2, Wi3/ZmDOS1, and Wi4 mutants.
