Ectopic cambia in wisteria vines are associated with the expression of conserved KNOX genes

Domain: neuroscience
Published: 2026-01-30
Editorial outcome: minor_revision

Abstract
Secondary vascular growth is a conserved mechanism that gives rise to vascular tissues produced via a single vascular cambium. Molecular mechanisms underlying this process are characterized mainly in model species with typical vascular architectures, while the genetics underlying ecologically-important atypical vascular architectures remain unexplored. We used developmental anatomy, comparative transcriptomics, molecular evolutionary analyses, and heterologous gene expression to address this knowledge gap, investigating how multiple ectopic cambia (EC) form in the woody vine Japanese wisteria. Anatomical studies show EC in Japanese wisteria arise from cortical parenchyma, and cambium-specific transcriptome comparisons reveal that genes acting as regulators of typical cambium development in model species are likewise associated with EC development. Gene trees of KNOX genes suggest that duplication events may contribute to EC formation, including a Fabaceae-specific duplication of KNAT2/6, which is detected as being under positive selection. We also demonstrate that KNOX genes from Japanese wisteria show canonical KNOX-like activity in heterologous functional assays, although no vascular aberrations were observed. Overall, these findings provide the first insights into the genetics of EC formation in “natural variants”, advancing our understanding of the molecular mechanisms regulating vascular variants in seed plants.

Introduction
The vast majority of woody plants undergo a common type of secondary vascular growth that involves the production of a single meristematic niche called the vascular cambium, which is responsible for generating secondary xylem (wood) and secondary phloem (inner bark), thereby enabling the plants to expand in diameter1. With only a single vascular cambium, plants can live up to 5000 years (e.g., Bristlecone pines [Pinus longaeva D.K. Bailey] or build a sophisticated plumbing system to transport water upwards of 380 ft (e.g., Coastal Redwood [Sequoia sempervirens (D. Don) Endl.]2,3. However, some plants deviate from the canonical modality to create woody structures with unique growth patterns that result in novel anatomical formations, such as those found mainly within the stems of woody vines or “lianas.” Lianas reach the forest canopy by climbing over other plants rather than supporting their weight4. As they weave their way through dense forest understory canopy, the stems of lianas are frequently damaged either through the collapse of host trees or the regular wear and tear of twisting and twining5. The woody stems of many lianas exhibit “ectopic cambia.” Ectopic cambia is a convergently evolved vascular variant6 that may serve as a mechanism for lianas to repair their stems after injury while maintaining key vascular functions of transportation and support5. Ectopic cambia (EC) result from the development of multiple vascular cambia in atypical locations, after the first-formed and central vascular cambium is established7. In addition to their role in lianas, EC may also facilitate the success of self-supporting herbs, shrubs, and trees living in harsh conditions such as mangroves and arid environments7–9. Among lianas, EC have been identified in the stems of the temperate Wisteria vines10,11, potentially facilitating their success as invasive vines in the Eastern United States12. To date, all anatomical studies show that EC arise through three steps: (1) de-differentiation of living parenchyma cells, (2) cell division to give rise to a new cambium, and (3) production and differentiation of localized vascular tissues from the new cambium layer8,10,13. While the vast majority of the literature on the natural diversity of EC is purely anatomical and descriptive, transgenic phenotypes in model species suggest that ECs can emerge by simply manipulating the expression of conserved genes responsible for vascular development7. Recent investigations demonstrate that Arabidopsis thaliana (L.) Heynh and Populus spp. share a suite of genes in the regulatory pathway controlling the canonical model of secondary growth by a single vascular cambium1,14,15. Notably, transgenic lines that perturb the expression of conserved vascular genes produce “experimental mutants” with EC in stems and/or roots7. Various studies indicate that EC can appear as the result of the misexpression of members of the class III homeodomain-leucine zipper (HD-ZIPIII) transcription factor: for example, EC are developed in Poplar trees stems through overexpression of PRE, an ortholog of the gene REVOLUTA (REV)16, while EC have been shown to develop in A. thaliana through the overexpression of AtHB717 and repression of AtHB418. ECs can also be formed following overexpression of WOX4, a member of the WOX family19, and ECs were observed in double mutants following the overexpression of WOX4 and repression of PETALLOSS (PTL)20. Finally, regenerated cambium can be induced by girdling trees, allowing investigators to characterize how such cambia (here categorized as EC, given their de novo establishment) can function to stitch together a wounded vascular cambium21,22. KNOTTED1-like HOMEOBOX (KNOX) transcription factors––key regulators of vascular proliferation––often emerge as differentially expressed genes in cambium development studies17,18,20,23. Yet, their role is underexplored in the formation of EC development. Our current knowledge of EC formation arises from molecular manipulation or direct perturbation––either mechanical (i.e., girdling) or genetic (i.e. transgenics)––in model species that don’t naturally form EC. To understand how EC contribute to plant diversity in form and function, we must realize how EC are produced in systems in which they naturally occur (“natural variants”), particularly in lianas, where they have a key functional role in allowing plants to climb.

In this work, we seek to elucidate the developmental anatomy, transcriptomic profile, molecular evolution, and gene expression of molecular markers underlying cambium formation in a pair of vines in the Fabaceae family, the ectopic cambia-producing Japanese wisteria (Wisteria floribunda (Willd.) DC.) and the non-ectopic cambia-producing common bean (Phaseolus vulgaris L.). We confirm previous reports that ectopic cambia form within the cortex of Japanese wisteria in a haphazard fashion. To reveal the transcriptomic profile underlying EC formation, we leveraged tangential cryosectioning to isolate tissues of interest for RNA sequencing. Here, we report the differential expression of several conserved genes in species and cross-species analyses of cambium-specific samples, including known cambium markers and genes involved in epigenetic regulation. We identified KNOX genes as prospective candidates in EC development in Japanese wisteria, as multiple transcript clusters are differentially expressed in typical v. ectopic cambia contrasts. We explored the molecular evolution of KNOX genes across 45 species, spanning both gymnosperms and angiosperms, and used branch models to analyze the nonsynonymous to synonymous substitution ratio and detect positive selection in KNOX transcripts. Finally, we confirmed the conserved function of Japanese wisteria KNOX genes by heterologous expression in Arabidopsis thaliana, further supporting their role in plant development. Together, these results expand our understanding of both typical and atypical cambium development, while identifying promising contenders underpinning EC formation in woody plants.

Results
Common bean and Japanese wisteria exhibit typical cambium development; Japanese wisteria develops ectopic cambia during the later stages of stem growth

To characterize the origin of ectopic cambia (EC), we began by detailing the developmental anatomy of common bean and Japanese wisteria (Fig. 1a–j). Young stems of both species have the same organization of primary tissues, with vascular bundles arranged in a ring (Fig. 1b, f). In young stems (3rd to 5th internodes, see Fig. 1a, e), we find the initiation of the vascular cambium from the fascicular cambium within each bundle and the interfascicular cambium connecting the bundles (Fig. 1c, g). This continuous cambium gives rise to secondary xylem (wood) inward and secondary phloem (inner bark) outward (Fig. 1d, h). This stage marks the final step in the stem development of common bean (Fig. 1d), giving rise to the mature form; however, Japanese wisteria development continues with further elaborations (Fig. 1i) to reach maturity. In stems with at least 20 mm diameter, Japanese wisteria starts to produce additional cambia ectopically (Fig. 1j), with older stems displaying multiple layers of EC and its products, i.e., secondary xylem and secondary phloem (Fig. 1i). The first EC in Japanese wisteria arise from the cortex (Fig. 1j), where existing cortical cells undergo localized cell divisions, creating a mass of cells from which a new cambium will emerge to produce secondary xylem and phloem (Fig. 1j). These EC do not arise in a uniform concentric pattern but rather initiate asynchronously across the stem circumference forming discrete strands of secondary xylem and secondary phloem (Fig. 1i).

Transcriptomes of common bean and Japanese wisteria

Having isolated key developmental stages, we sought to identify which genes underlie the formation of EC. Transcriptomic profiles were made from the typical cambium of the common bean and Japanese wisteria and the EC of Japanese wisteria, with at least five biological replicates for each of the three tissues sampled. The tissues for transcriptomic analyses consisted of multiple sections of secondary xylem-vascular cambium-secondary phloem obtained through tangential cryosectioning (Fig. 2). Sequencing of 17 samples across cambium types and species yielded an average of 21,420,218 (128,521,310 total) and 23,468,348 (258,151,832 total) paired-end reads per sample for the common bean and Japanese wisteria, respectively. The transcriptome assemblies vary in BUSCO, mapping rate, and N50, but both assemblies yielded BUSCO scores and mapping rates over 80% with N50s greater than 1500 base pairs (bp) (Supplementary Table 1).

Differential cluster expression and gene ontology enrichment

Comparison across species. Using the transcript clusters for common bean (42,827) and Japanese wisteria (74,960), 10,498 one-to-one orthologous transcript clusters were identified and passed filtering (Expressed > 0.5 counts per million) (Supplementary Data 1). The number of differentially expressed clusters varied dramatically between contrasts (Table 1). The most differentially expressed (DE) transcript clusters were identified in the “Japanese wisteria v. Common bean” contrast, representing overall differences between the two species (>6000). This was followed by the number of DE transcripts when comparing the “Typical Cambia: Japanese wisteria v. Common bean” (>5000). The third highest number of DE transcripts was found in the “Ectopic Cambia v. Typical Cambia of Japanese wisteria and Common bean” contrast (>2000) (Table 1). Lastly, we compared the “Japanese wisteria: Ectopic v. Typical cambia” contrast, which surprisingly yielded no DE transcript clusters in this analysis (see further analyses “Comparisons within Japanese wisteria” below). In annotating the DE transcript clusters with Trinotate24 against the UniProt database for Fabales (UniProt Taxon ID 72025)25, we found transcript clusters related to DNA methylation (e.g., methyltransferases, DNA methylase), phytohormones (e.g., indole acetic acid, auxin response factors), cell cycle control (e.g., cyclins), cell differentiation (e.g., KNOX), and other cambial markers (e.g., PXY). The “Ectopic Cambia v. Typical Cambia of Japanese wisteria and Common bean” contrast is the only comparison that yielded enriched gene ontology terms based on identified DE clusters, and the GO terms are mostly related to RNA binding, splicing, and processing (Supplementary Table 2).

Comparison within Japanese wisteria. Given our focus on elucidating the genes related to EC, we further explored the “Japanese wisteria: Ectopic v. Typical cambia” contrast using differential expression for repeated measures (dream)26 to compare across cambium types with the individual as a random effect. This approach enabled us to isolate the most likely candidate genes associated with EC formation in Japanese wisteria. After filtering (Expressed > 0.5 counts per million in at least two samples), 46,225 transcript clusters were retained for differential transcript cluster expression analysis across the two Japanese wisteria cambium types using dream26, with 14 clusters (2 up and 12 down) found to be differentially expressed (Table 2). The differentially expressed clusters include transcripts related to DNA methylation, cell expansion (e.g., expansin-like B1), and cell differentiation (e.g., KNOX), all of which were down regulated in the EC of Japanese wisteria (Fig. 3). Only two transcript clusters are upregulated, one of which includes the epigenetic gene Methyltransf_7 (Table 2). Interestingly, we found five differentially expressed KNOX genes across the four contrasts (Table 3). KNOX clusters identified as two potential KNOX genes are linked to typical cambium development in Japanese wisteria and common bean (e.g., STM, KNATM), while EC formation is more directly associated with the expression of KNAT2/6 (Table 3, Supplementary Fig. 1). These observations make KNOX genes of particular interest, given their defined role in vascular cambium proliferation in model systems.

Identification, characterization, and phylogenetic analysis of KNOX genes analysis.

To investigate the relationship between KNOX genes and EC evolution, we began by testing the hypothesis that the diversification of KNOX genes has led to EC. To achieve this aim, we broadened our analyses to encompass all nine members of the KNOX gene family (based on the A. thaliana genome) across 45 seed plant species, including gymnosperms, monocots, and eudicots (Fig. 4; Supplementary Fig. 2). Our sampling included species with and without EC (Supplementary Data 2, 3). Although retrieved through the tBLASTx search, we removed eight species due to their poor alignment, which potentially resulted in an erroneous phylogenetic placement (Supplementary Fig. 3).

The final sequence alignment contained 466 sequences. The maximum likelihood phylogeny grouped KNOX genes into three main clades that correspond to defined classes: KNOX1, KNOX2, and KNATM (Fig. 4). In this phylogeny rooted with a representative of BEL1 genes, KNOX2 diverges first, with the subsequent divergence of KNOX1 and KNATM as sister lineages. The three KNOX classes can be further divided into major clades named according to their putative A. thaliana ortholog (Fig. 4; Supplementary Fig. 2). Class KNOX1 includes three main clades corresponding to STM, KNAT1, and KNAT2/6 (Clusters “W2” and “1332” are phylogenetically placed within a clade comprised of KNAT2 and 6 Arabidopsis reference sequences, highlighting their similarity to both genes. Hereafter, we use the notation “KNAT2/6” to refer to these genes). Class KNOX2 is subdivided into two clades corresponding to KNAT3/4/5 and KNAT7.

Using our phylogenetic approach, we determined the orthology of each of the differentially expressed KNOX transcript clusters (Fig. 4). All DE clusters belong to class KNOX1: Two of them group with A. thaliana STM ortholog, one of which is nested within a clade derived from a Fabaceae-specific gene duplication containing the soybean SBH1 reference; two transcript-clusters group with KNAT2/6, one being associated with each of the major clades resulting from gene duplication following the divergence of angiosperms (Supplementary Fig. 2); and one transcript-cluster groups within KNATM (Fig. 4; Supplementary Fig. 2).

The number of KNOX gene copies recovered from each taxon varies due to duplication events within major clades. We recovered duplication events in most lineages with EC including at the base of the angiosperms for KNAT2/6 (indicated by sequences of Amborella trichopoda Baill. in Fig. 4), eudicots for STM, KNAT2/6, and KNAT3/4/5) (indicated by sequences of Vitis vinifera L. in Fig. 4), as well as within the Fabaceae which included duplications for both KNOX1 and KNOX2 genes (indicated by sequences of Wisteria floribunda or Spatholobus suberectus Dunn in Fig. 4). Duplication events are also observed in lineages without EC, including the conifers for KNAT1 and the Poales (monocots) for KNAT1 and KNAT3/4/5 (indicated by sequences of Zea mays L. or Triticum dicoccoides (Asch. & Graebn.) Schweinf. in Fig. 4). We did not find an association between the number of KNOX genes and the presence/absence of EC across plants (Fig. 5). The A. thaliana genome contains nine KNOX, genes, but lineages with EC include both increases (e.g., Japanese wisteria with 16 copies and other species of Fabaceae with 17 copies) and decreases (e.g., Amaranthaceae species with five copies), including the possible absence of KNOX genes in the EC-producing gymnosperms Cycas panzhihuaensis L. Zhou & S.Y. Yang and Gnetum montanum Markgr. (Fig. 5). A Fabaceae-specific duplication in KNATM after the divergence of Cercis canadensis L. is apparent in this dataset (Fig. 5).

Selection analyses.

To test whether KNOX genes in lineages with EC are under positive selection (indicative of adaptive evolution), we compared PAML codon-based nested models (models 0 = one omega value for the tree, 1 = a unique omega per branch, 2 = foreground and background omega values)27. We tested these models in individual gene trees for members of the KNOX genes that were differentially expressed in the transcriptome analysis (i.e., STM, KNATM, and KNAT2/6). By comparing foreground (EC present) and background (EC absent or typical growth) branches (model 2), the only gene set that showed a significant difference was KNAT2/6 (p-value = 0.00406).

To detect whether some branches along KNAT2/6 lineages were under positive selection, we employed branch model 1 on the KNAT2/6 phylogenetic rooted tree, which showed nine branches with omega values greater than 1 (ω > 1), therefore, indicating positive selection in specific lineages (Fig. 6). Out of the nine branches, only two have EC, both of which are sequences differentially expressed from two transcript clusters in the gene expression analysis (Wisteria_floribunda_Cluster_W2, ω = 999; Wisteria_floribunda_Cluster_1332, ω = 958.234). Taxa under positive selection belonging to species with typical growth include Dioscorea rotundata (ω = 999), Populus trichocarpa (ω = 964.749 and 999), and Z. mays (ω = 1.29156), including some Fabaceae such as Phaseolus vulgaris (ω = 999) and Prosopis alba (1.27446) (Fig. 6). Despite statistical tests not supporting differences in synonymous v. nonsynonymous changes for the KNATM, results indicate only two branches with higher omega values, both of which are species with EC from the Fabaceae, including Phaseolus lunatus (ω = 1.53725) and W. floribunda (ω = 1.15616), representing the KNATM sequence from the DGE analysis (Cluster_4072) (Supplementary Fig. 4a). Only one branch belonging to P. trichocarpa (ω = 999) is under positive selection in the rooted STM tree (Supplementary Fig. 4b).

KNOX genes from Japanese Wisteria show canonical KNOX-like activity in heterologous functional assays.

Transcriptomic, phylogenetic, and selection analyses suggest that two KNAT2/6 orthologs (Cluster_W2 and Cluster_1332) are candidates to mediate EC formation. However, neither gene has been formally shown to possess KNOX activity. To directly test this, we turned to heterologous expression in Arabidopsis, as transgenesis has not yet been established in Japanese wisteria. Importantly, this approach has been used to demonstrate the functional conservation of KNOX genes from such evolutionarily distinct genera as Cardamine L., Liriodendron L., Solanum L., and Picea A. Dietr.28–31. Cluster_1332 was selected as a representative candidate, placed downstream of the strong UBIQUITIN10 promoter (pUBQ:W1332), and pUBQ:W1332 was transformed into wild type Arabidopsis. The overexpression of Cluster_1332 resulted in strong phenotypes in most primary transformants grown under both long-day (~30 days) and short-day (~90 days) conditions. For instance, transgenic plants were significantly smaller than their wildtype Col-0 counterparts in both experiments (Fig. 7), particularly in short-day plants (Supplementary Fig. 5). The leaves of transformants showed apparent developmental abnormalities, including reduced petiole lengths, rumpled leaf blades, and prolonged growth of their distal serrations (Fig. 7). Vascular aberrations were not observed (Supplementary Fig. 5). However, in short-day plants, transformants showed delayed bolting (~30 days after Col-0), which was characterized by delayed vascular development, including the absence of fibers in the secondary xylem at ~90 days post-germination (Supplementary Fig. 5).

Methods
Plants and stem sample collection
Wisteria floribunda (Japanese wisteria) plants were collected in two populations growing in Ithaca, NY, United States. We performed DNA barcoding using the chloroplast intergenic spacer gene “ndhJ-trnF” to confirm the identification of plants belonging to the species W. floribunda11. Phaseolus vulgaris (genotype L88-57; common bean) plants were grown at Cornell University Agricultural Experiment Station Guterman greenhouses (Ithaca, NY) under the following conditions: 14 h light, 75°F daytime temperature, 65°F nighttime temperature, and ambient humidity. Samples from these plants were used for developmental anatomy and RNAseq analyses (see “methods” below).

Histology and microscopy
Stem samples of wild plants of common bean and Japanese wisteria were hand-sectioned (with the aid of a razor blade) and processed to generate temporary slides or processed using embedding in polyethylene glycol, sectioning with rotary or sliding microtome, and staining with Safranin and Astra Blue to generate permanent slides60. Slides were imaged using a light microscope (Olympus BH2) equipped with a digital camera (AmScope MU1000) at New York University. Arabidopsis thaliana plants were either hand-sectioned or sectioned with a rotary microtome (ThermoScientific HM 355S), stained with Toluidine Blue O, and slides were imaged using a light microscope (Olympus BX51) equipped with a Raspberry Pi High Quality at Florida International University.

RNA sequencing and data processing
Sampling and RNA isolation. Developed stem samples were cut into small blocks containing cambium, secondary xylem, and secondary phloem, which were sectioned (~12 µm thick) using a cryostat at −20°C (tangential cryosectioning approach)23,61 to obtain the tissue for total RNA isolation with RNeasy Mini Kit (QIAGEN). Each sample used for tangential cryosectioning was roughly 6 × 2 × 4 mm. For RNA extraction, about 40–110 sections of each sample containing cambium, secondary xylem, and secondary phloem were used for each replicate (Fig. 2). Six biological replicates were used for the typical cambium of common bean and ectopic cambia (EC) of Japanese wisteria. Five replicates were used for the typical cambium of Japanese wisteria. The same adult plants used for comparative transcriptome analysis were also used for anatomical studies (see above). All sampling was performed at Cornell University.

RNA integrity and quantification. Total RNA was quantified using RNA Assay Kit in Qubit® 2.0 Fluorometer (Life Technologies, CA, USA), and RNA integrity was assessed using an AATI Fragment Analyzer (Agilent Technologies, CA, USA) at Cornell University. Purification, library preparation, and Illumina sequencing were performed by Novogene Co., Ltd (Davis, California). Quality control was performed on the RNA samples using Qubit® 2.0, and RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).

Library preparation for transcriptome sequencing. RNA library for poly(A)-dependent RNA-seq was prepared using NEBNext Ultra II RNA Library Prep Kit for Illumina (New England BioLabs). The libraries were subjected to 150 bp paired-end sequencing using NovaSeq 6000 with the v1.5 S4 reagent kit. The library was checked with Qubit and real-time PCR for quantification and a bioanalyzer for size distribution detection.

Transcriptome assembly and annotation. Raw paired-end reads were trimmed for adapter sequences using an overlap stringency of three, a Phred score cutoff of twenty, and a minimum sequence length of 50 base pairs (bp) using Trim Galore! version 0.6.662. De novo transcriptome assemblies were developed for P. vulgaris and W. floribunda. For each trimmed dataset, the range of potential kmer values was estimated using Kmergenie version 1.701663. De novo assemblies were generated following part of the TransPi pipeline that uses a multi-assembler approach, followed by merging assemblies, reducing complexity, and annotation64 (see Supplementary Methods 2 for additional details). Within the TransPi pipeline, the UniProt database for Fabales (UniProt Taxon ID 72025)25 was used for annotation (Supplementary Methods 2).

Differential expression and gene ontology enrichment. Trimmed reads were subset to 20 million reads, mapped to their respective transcriptomes with Hisat2 version 2.2.165, including all multiple alignments, and the mate-pair information of the resulting SAM files was fixed using Picard Tools version 2.27.5 FixMateInformation before being converted to BAM files with SAMtools version 1.1466. Corset version 1.0767 was used to group the mapped transcripts into transcript clusters and estimate read counts for each cluster separately for both transcriptomes. Each cluster was annotated using the Trinotate annotation of the longest sequence within the cluster. Clusters were retained if at least two samples in each species had a count of 0.5 or more per million. For the cross-species comparison, Orthofinder2 version 2.5.268 was used with our protein annotations from TransDecoder, and the proteomes of Vigna unguiculata (UniProt Taxon ID: 3917), Cajanus cajan (UniProt Taxon ID: 3821), Lotus japonicus69, Medicago truncatula70 and Mucuna pruriens (UniProt Taxon ID: 157652) from UniProt25 and Phytozome71 to identify orthologs between P. vulgaris and W. floribunda. We identified one-to-one orthologs at the transcript cluster level by removing any clusters with more than one ortholog group and any ortholog groups that contained sequences from more than one cluster. Differential cluster expression was tested using limma72 and voom73 comparing between species regardless of cambium type, between cambium types regardless of species, between the typical cambium of each species, and between the ectopic and typical cambium of Japanese wisteria (see Supplementary Methods 3 for additional details). Gene ontology enrichment was tested using the differentially expressed transcript clusters for each using GOseq74. To better leverage the repeated sampling within an individual, Japanese wisteria clusters were tested separately from the cross-species comparison using differential expression for repeated measures (dream)26 with voom73 and variancePartition75 to compare across cambium types with individual as a random effect. Gene ontology enrichment for differentially expressed clusters was implemented in GOseq. For comparison with the cross-species analysis, ortholog groups were subsequently mapped back to the differentially expressed clusters identified by Dream. For all statistical analyses, p-values were adjusted for false discovery rates using a Benjamini-Hochberg procedure using two-sided tests.

Phylogenetic and selection analyses. To investigate the role of KNOX genes in EC formation across seed plants, we performed a phylogenetic analysis and selection analysis for this gene family. Candidate genes were identified from available genomes and transcriptomes using a BLAST pipeline that was previously used for investigating gene evolution across angiosperms76. Coding sequences (cds) were downloaded from Genbank or TreeGenes for 39 species, and raw RNA-seq files were downloaded from the SRA for six species (Supplementary Data 2.). In total, 45 species were included in the analysis, 11 containing EC. Raw sequencing files were cleaned with fastp version 0.23.377 and assembled with Trinity version 2.14.077,78 with the following flags: --max_memory 200G --no_normalize_reads --min_contig_length 250 --trimmomatic. Reference sequences of candidate genes from A. thaliana were downloaded from Genbank, and a tBLASTx search was performed using blast version 2.1379. A maximum of 30 hits per taxon were retained if the matching length was 10 bp and an e-value of 0.0001. A fasta file containing all retrieved sequences was generated and checked for duplicates using the sRNAtoolbox80. Twenty-one sequences from different KNOX genes and taxa were downloaded from Genbank and included as additional references to identify specific clades (Supplementary Data 3). A species tree to depict this sampling (Fig. 4) was constructed using the R package V.PhyloMaker2 with scenario 3 for taxa binding81. Finally, the sequences of five transcript clusters identified as KNOX genes were included in the phylogenetic analysis. The resulting fasta file for phylogenetic reconstruction contained 466 sequences (Supplementary Methods 4, Supplementary Data 8 for additional details). These contigs were first aligned with MAFFT version 7.5282 with the following options: --auto --adjustdirectionaccurately --op 3 --leavegappyregion. The sequences were then realigned with the codon-aware aligner MACSE version 2.0783 and the alignSequences command. The resulting alignment was cleaned up with trimAL version 1.4.184 with the -gappyout option before converting premature stop codons to NNN while retaining the final stop codons with the exportAlignment command in MACSE, which were cleaned up with trimAL version 1.4.184. See Supplementary Dataset 9, 10. for aligned and unaligned sequences. Tree building was performed repeatedly to exclude poorly aligned and gappy contigs. Trees were generated with RaxML version 8.2.12 with the GTRGAMMA model of molecular evolution and 100 bootstrap replicates85.

For further investigation of specific candidate genes, a fasta file containing the nucleotide sequences of specific KNOX genes identified in the previous tree was aligned with MAFFT version 7.52, realigned with the codon-aware aligner MACSE version 2.07, and then cleaned with trimAL version 1.4.1. The inferred phylogeny and cleaned alignment were input into PAML version 10.627 for codeml analysis to explore signatures of selection. Both model = 0 (one omega value for the entire alignment), model = 1 (omega value for each branch), and model = 2 (foreground and background omega values) were conducted on both the rooted and unrooted trees. Branch-specific omega values were plotted on the phylogeny with the R package ggtree version 3.6.286.

Molecular cloning and plant transformation. A genomic fragment of Cluster_1332 (KNAT2/6) from the start codon to the stop codon was PCR amplified from Japanese wisteria genomic DNA as a 4936 bp band using W1332FpUBQ10OL and W1332Rpcr8OL primers. A 4812 bp fragment comprised of pUBQ10 and the pCR/GW/TOPO vector was amplified from an existing template using pCR8 F W1332 OL and pUBQ10pCR8RW1332OL primers. Fragments were assembled using Gibson assembly (NEBuilder® HiFi DNA Assembly Master Mix Cat. E2621L) and sequence validated using Plasmidsaurus. The resulting pUBQ10:W1332 cassette was then cloned into a modified pB7GW binary vector (VIB Ghent) using Gateway LR technology (Gateway™ LR Clonase™ II Enzyme mix Cat. 11791020) and transformed into the Col-0 ecotype of Arabidopsis thaliana using standard Agrobacterium-mediated floral dip protocols. Primary transformants were grown at 22°C under long (16-h light, 8-h dark) and short (8-h light, 16-h dark) day conditions on soil for 4 weeks (~30 days) or 12 weeks (~90 days) post-germination, respectively. Plants were grown and imaged at the University of Pennsylvania. Cloning primers are listed in Supplementary Data 11.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The paper by Cunha Neto et al describes the identification of transcripts that are differentially expressed in ectopic cambia, with a view to understanding how this enigmatic property of woody vines is regulated. The authors identify members of the KNOX family as being preferentially expressed in these structures and show that they are under positive selection. The manuscript is engaging and exceptionally well-written, describing the background and experimental programme in a series of clear logic steps, leading to a balanced discussion. However, there are two pieces of data that I believe would further strengthen the authors arguments:\nThe first is spatial gene expression data. This could come in the form of RNA-in situ hybridization (challenging in woody species), but probably a graphical representation of the authors existing RNA-seq data, showing section number on the x-axis (including a representation of position along the radial axis) and relative expression on the Y is enough to assist the reader in visualising expression along the radial axis of the plant.\nThe second is in planta evidence of W. floribunda genes of interest regulating cambium, e.g. over-expression or cross-complementation in e.g. Arabidopsis.", "reviewer_2": "In this manuscript, the authors try to characterize the regulatory network underlying ectopic cambia forms in Japanese wisteria. The results are interesting and may be interested by the related readers. However, the current version is descriptative and lack of functional validation. Several suggestions could be useful for improving your manuscript.\n1.What factors are essential for determining EC formaton in plants? What are the differences between EC and vascular camibum? Comparation of common genes derived from the transcriptomic data in two species may provide some clues.\n2.Funcitional analysis of key KNOX genes associated with EC formation in planta is necessary.\n3.It is unclear how to sample EC for transcriptome analysis.\n4.What KNOXs are specific for the regulation of vascular cambium while what KNOXs are for EC? Along this logic, the authors should extract the useful information from the phylogenetic tree of KNOX genes."}, "author_response": "Reviewer #1 (Remarks to the Author):\nThe paper by Cunha Neto et al describes the identification of transcripts that are differentially expressed\nin ectopic cambia, with a view to understanding how this enigmatic property of woody vines is regulated.\nThe authors identify members of the KNOX family as being preferentially expressed in these structures\nand show that they are under positive selection. The manuscript is engaging and exceptionally well-\nwritten, describing the background and experimental programme in a series of clear logic steps, leading to\na balanced discussion. However, there are two pieces of data that I believe would further strengthen the\nauthors arguments:\nWe appreciate the positive feedback! We address each specific comment below.\nThe first is spatial gene expression data. This could come in the form of RNA-in situ hybridization\n(challenging in woody species), but probably a graphical representation of the authors existing RNA-seq\ndata, showing section number on the x-axis (including a representation of position along the radial axis)\nand relative expression on the Y is enough to assist the reader in visualising expression along the radial\naxis of the plant.\nWe appreciate the suggestions. Current Fig. 2 is a new graphical element to accommodate\nthis comment.\nThe second is in planta evidence of W. floribunda genes of interest regulating cambium, e.g. over-\nexpression or cross-complementation in e.g. Arabidopsis.\nThank you for the suggestion. We are pleased to present an essay demonstrating the\nheterologous expression of Japanese wisteria KNOX genes in Arabidopsis. We show that the\nKNAT2/6 genes have a conserved role, as indicated by the phenotypes in our overexpression\nstudy. Due to this change, we have added a new figure (Fig. 7), and the primers are listed in\nSupplementary Table 8. The methods, results, and discussion of the paper were revised to reflect\nthese updates.\n###############\nReviewer #2 (Remarks to the Author):\nIn this manuscript, the authors try to characterize the regulatory network underlying ectopic cambia forms\nin Japanese wisteria. The results are interesting and may be interested by the related readers. However,\nthe current version is descriptative and lack of functional validation. Several suggestions could be useful\nfor improving your manuscript.\nWe appreciate the feedback. Below, we address each of the suggestions.\n1.What factors are essential for determining EC formaton in plants? What are the differences between EC\nand vascular camibum? Comparation of common genes derived from the transcriptomic data in two\nspecies may provide some clues.\nThe manuscript (and its references) provides a substantial amount of information on how\nEC forms in terms of developmental anatomy. This topic has been investigated in several papers,\nshowing that there is a common developmental series leading to the formation of EC; hence, we\nturned to the question: What is the molecular regulation of this EC formation? Our paper presents\nthe first exploration of candidate genes involved in EC formation. From our transcriptomes, we\nwere able to compare differentially expressed genes across species (Japanese wisteria v.\nCommon bean) and cambium types (both across species and within Japanese wisteria). These\ncomparisons provide a solid basis for identifying genes associated with cambium formation in\nJapanese wisteria, with a focus on one key regulator: the KNOX genes. We welcome the reviewer's\nsuggestions on alternatives for comparisons among the candidate genes. Additionally, in this\nrevised version, we have included functional analyses to validate the role of KNOX genes (see\nbelow), which represents a major step towards elucidating the genetic basis of these vascular\nvariants.\n2.Funcitional analysis of key KNOX genes associated with EC formation in planta is necessary.\nThat was a very important suggestion, also recommended by Reviewer 1. Thank you! As\nmentioned earlier, we successfully included the first experimental essay to explore the function of\nJapanese wisteria KNOX genes through heterologous expression in Arabidopsis. These results\nare presented in the new section “KNOX genes from Japanese Wisteria show canonical KNOX-like\nactivity in heterologous functional assays”.\n3.It is unclear how to sample EC for transcriptome analysis.\nThanks for your comment. Indeed, we missed the chance to clearly explain how the samples were\ncollected. To fix this, we added a new figure (Fig. 2) showing a 3D view of the tangential\ncryosectioning approach and a high-magnification cross-section of the EC cambium of Japanese\nwisteria, illustrating the tissue amount collected for each sample. These updates greatly improve\nthe description of our methodology and how we obtained these important results. We truly\nappreciate the suggestion!\n4.What KNOXs are specific for the regulation of vascular cambium while what KNOXs are for EC? Along\nthis logic, the authors should extract the useful information from the phylogenetic tree of KNOX genes.\nWe appreciate this comment. We amended the results to provide a more direct description of the\nKNOX proteins that are potentially more closely associated with the development of each cambium\ntype in our species. To that end, we examined the results already presented in Table 5, which\ndescribes the transcript clusters that were differentially expressed in each contrast, thus providing\na clue about which proteins are associated with typical v. EC. These changes are noted in lines 177-\n180."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The resubmitted manuscript by Cunha Neto et al successfully addresses the issues raised in the first round of review. As such, no further revisions are necessary. Congratulations on an interesting paper!", "reviewer_2": "Currently, the genetic mechanism underlying ecological atypical vascular architectures in plants remains unclear. This manuscript illustrated an association of ectopic cambia in the woody vine Japanese wisteria with KNOX gene expression. Undoubtly, their finding is meaningful. In the resubmited manuscript,the authors adderssed most of my concerns except for functional validation. Although they ectopically expressed wisteria vine Cluster_1332, the ortholog of KNAT2/6, in Arabidopsis, and found that the transgenic plants displayed developmental abnormalities in petioles and leaves. Unfortunately, these phenotypic alterations are not related with ectopic cambia (EC), the topic of this manuscript. It is suggested that the authors might observe the effect of wisteria vine Cluster_1332 overexpression on EC formation in the poplar stem. This should be more informative."}, "author_response": "REVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nThe resubmitted manuscript by Cunha Neto et al successfully addresses the issues raised in the first\nround of review. As such, no further revisions are necessary. Congratulations on an interesting paper!\nThanks for the positive response.\nReviewer #1 (Remarks on code availability):\nI have looked through the folders. The necessary files are present, accompanied by readme documents\nexplaining the experimental steps.\nThanks for checking that.\nReviewer #2 (Remarks to the Author):\nCurrently, the genetic mechanism underlying ecological atypical vascular architectures in plants remains\nunclear. This manuscript illustrated an association of ectopic cambia in the woody vine Japanese wisteria\nwith KNOX gene expression. Undoubtly, their finding is meaningful. In the resubmited manuscript,the\nauthors adderssed most of my concerns except for functional validation. Although they ectopically\nexpressed wisteria vine Cluster_1332, the ortholog of KNAT2/6, in Arabidopsis, and found that the\ntransgenic plants displayed developmental abnormalities in petioles and leaves. Unfortunately, these\nphenotypic alterations are not related with ectopic cambia (EC), the topic of this manuscript. It is\nsuggested that the authors might observe the effect of wisteria vine Cluster_1332 overexpression on EC\nformation in the poplar stem. This should be more informative.\nWe thank the reviewer for their\nthoughtful and positive feedback on our\nfindings. Both Arabidopsis and poplar do not\nnaturally exhibit ectopic cambia; both\npresent secondary vascular growth;\ntherefore, they are equally suitable for the\nheterologous overexpression of KNAT2/6 for\nfunctional validation. We opted to use\nArabidopsis given the efficient\ntransformation time and in-house expertise.\nIn our first revisions, we demonstrated that\nthe transgenic plants displayed typical KNOX\nphenotypes, indicating homology between\nthe Japanese wisteria and Arabidopsis\ncopies. In the current revision, as suggested by the reviewer, we investigated whether the\nArabidopsis transgenics in fact displayed vascular defects in the inflorescence stem, leaves, and\nroots in both long- (~30 days) and short-day plants (~90 days). We did not observe significant\nchanges to the vascular anatomy; notably, ectopic cambia were absent. A new figure is included\nin the supplemental materials (Supplementary Fig. S5) (see image on the side). The absence of\nectopic cambia in Arabidopsis transgenics indicates that misexpression of KNAT2/6 alone is\ninsufficient to induce this phenotype. In this work, we were careful not to overstate that KNAT2/6\nalone is the sole contributor; rather, it is clearly part of the regulatory network that produces\nectopic cambia in Wisteria.\nThe corresponding text has been edited accordingly as follows:\nAbstract (page 1):\nBefore:\n“We also demonstrate that KNOX genes from Japanese wisteria show canonical KNOX-like activity in\nheterologous functional assays.”\nNow:\n“We also demonstrate that Japanese wisteria KNOX genes exhibit typical KNOX-like activity in\nheterologous functional assays, although no vascular aberrations were observed.”\nResults (page 7):\nBefore:\n“The resulting overexpression of Cluster_1332 conditioned strong phenotypes in the majority of primary\ntransformants. For instance, transgenic plants were significantly smaller than their wildtype Col-0\ncounterparts (Fig. 7). In addition, their leaves showed apparent developmental abnormalities, including\nreduced petiole lengths, rumpled leaf blades, and prolonged growth of their distal serrations (Fig. 7).”\nNow:\n“The overexpression of Cluster_1332 resulted in strong phenotypes in most primary transformants grown\nunder both long-day (~30 days) and short-day (~90 days) conditions. For instance, transgenic plants were\nsignificantly smaller than their wildtype Col-0 counterparts in both experiments (Fig. 7), particularly in\nshort-day plants (Supplementary Fig. 5). The leaves of transformants showed apparent developmental\nabnormalities, including reduced petiole lengths, rumpled leaf blades, and prolonged growth of their distal\nserrations (Fig. 7). Vascular aberrations were not observed (Supplementary Fig. 5). However, in short-day\nplants, transformants showed delayed bolting (~30 days after Col-0), which was characterized by delayed\nvascular development, including the absence of fibers in the secondary xylem at ~90 days post-\ngermination (Supplementary Fig. 5).”\nDiscussion (p. 9)\nBefore:\n“Our findings that the transcript-cluster identified as KNAT2/6 is downregulated in the “Japanese wisteria:\nEctopic v. Typical cambia” contrast concur with the downregulation of other KNOX proteins in A. thaliana\nmutants with cambia formed ectopically20 (Supplementary Table 2). However, just as the mechanism by\nwhich KNAT6 functions in vascular development remains largely unclear in model species37,43,45,47,\nadditional studies will be required to clarify how KNAT2/6 plays a role in EC development, as well as the\ninvolvement of other KNOX proteins (e.g., STM), which is also downregulated in the contrast comparing\n“Ectopic Cambia v. Typical Cambia of Japanese wisteria and Common bean”.”\nNow:\n“Our findings that the transcript-cluster identified as KNAT2/6 is downregulated in the “Japanese wisteria:\nEctopic v. Typical cambia” contrast concur with the downregulation of other KNOX proteins in A. thaliana\nmutants with cambia formed ectopically20 (Supplementary Table 2). Complementary, our heterologous\nfunctional assay using the Japanese wisteria KNAT2/6 ortholog resulted in shorter Arabidopsis plants with\nrumpled leaves and delayed development, but without vascular abnormalities. Therefore, just as the\nmechanism by which KNAT6 functions in vascular development remains largely unclear in model\nspecies37,43,45,47, additional studies will be required to clarify how KNAT2/6 plays a role in EC\ndevelopment, as well as the involvement of other KNOX proteins (e.g., STM), which is also downregulated\nin the contrast comparing “Ectopic Cambia v. Typical Cambia of Japanese wisteria and Common bean”.”\nMethods (page 13)\nBefore:\n“Primary transformants were grown at 22 oC under long day conditions (16-hr light, 8-hr dark) on soil for\n17- or 23-day post-germination before imaging. Cloning primers are listed in Supplementary Table 7.”\nNow\n“Primary transformants were grown at 22 oC under long (16-hr light, 8-hr dark) and short (8-hr light, 16-hr\ndark) day conditions on soil for 4 weeks (~30 days) or 12 weeks (~90 days) post-germination,\nrespectively. Plants were grown and imaged at the University of Pennsylvania. Cloning primers are listed\nin Supplementary Table 7.”"}]

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