AHL26, an AT-hook gene, negatively regulates hypocotyl growth and flowering time in Arabidopsis thaliana

AHL26, an AT-hook gene, negatively regulates hypocotyl growth and flowering time in Arabidopsis thaliana | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article AHL26, an AT-hook gene, negatively regulates hypocotyl growth and flowering time in Arabidopsis thaliana Shahbaz Ahmed, Anna Hulbert, Xin Xin, Michael Neff This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6264939/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 May, 2025 Read the published version in BMC Plant Biology → Version 1 posted 11 You are reading this latest preprint version Abstract Background The AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) gene family in Arabidopsis contains 29 members, which evolved into two phylogenetic clades. Genes from this family play a role in several biological processes, but most of the members' functions remain unknown. Results Here, we provide evidence that AHL26, a clade-a protein, negatively regulates hypocotyl growth and flowering time in Arabidopsis . Analysis of transgenic plants expressing an AHL26:AHL26:GUS translational fusion driven by 1.9 KB of the endogenous AHL26 promoter displayed GUS activity in the hypocotyl and apical meristem of light-grown seedlings. The overexpression of AHL26 resulted in the inhibition of hypocotyl growth and delayed flowering. However, the overexpression of a dominant-negative AHL26 with mutation in AT-hook motif, resulted in early flowering and longer hypocotyls than the WT and over-expression transgenic lines suggesting genetic redundancy between AHL26 and other AHL genes. Transcriptome analysis showed that the regulation of flowering time in AHL26 over-expression and dominant-negative mutants results from regulating flowering-related genes and pathways. Conclusion Our study highlights the significant role of AHL26 in hypocotyl growth and flowering time regulation. We further demonstrate that AHL26 regulates hypocotyl length in a light-dependent manner. Through transcriptomic analysis, we also show that the delayed flowering phenotype in our AHL26 over-expression plants is due to the negative regulation of flowering-promoting genes such as FT . Furthermore, transcriptome analysis provides insight into the biological processes and pathways through which AHL26 influences the control of flowering time. AT-hook AHL26 Flowering Dominant-negative mutation Arabidopsis thaliana RNA-Seq Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background The AHL gene family constitutes a crucial component of the regulatory machinery governing seedling and adult plant development (Street et al., 2008 ; Zhao et al., 2013 ). The evolutionary conservation of the AHL gene family underscores its fundamental importance in plant biology (Zhao et al., 2014 ). AHL proteins feature a conserved AT-hook motif at their N-terminal end and a plant and prokaryote conserved (PPC) domain, also known as the domain of unknown function #296 (DUF296), at their C-terminal end. The AT-hook, first described in the HIGH MOBILITY GROUP (HMG) family of non-histone chromosomal-associated proteins in mammals is a small motif that has a pattern centered around a glycine-arginine-proline (GRP) tripeptide (Aravind and Landsman, 1998). This sequence is necessary and sufficient to bind DNA. Mutations in this core sequence result in the loss of DNA binding properties of AHLs (Zhao et al., 2013 ).There are three types of AT-hooks present in the AHL gene family. Type-I is characterized by an additional module present at the C-terminal of the core GRP. They have a greater probability of glycine at the second position C-terminal to the GRP. Type-II is characterized by having a high probability of possessing lysine instead of glycine, two residues down from the GRP. Type-III is characterized by including features of both Type-I and Type-II AT-hooks (Aravind and Landsman, 1998). The PPC domain, approximately 120 amino acids in length, functions as an independent protein in bacteria, archaea, and some green algae (Fujimoto et al., 2004). In land plants, however, the PPC domain is fused with the AT-hook motif in the AHL family of proteins and is responsible for protein-protein interaction between AHLs and non-AHL proteins in the cell (Favero et al., 2016; Zhao et al., 2013 ). The PPC domains in plants can be categorized into Type-A and Type-B based on the conserved Gly-Arg-Phe-Glu-Ile-Leu motif. The conserved region in PPC domain is necessary for the protein-protein interaction of the AHL proteins. The AHL protein family in plants can be divided into two distinct clades (Clade A and Clade B) based on the type of AT-hook motif and the PPC domain (Zhao et al., 2014 ). The conserved characteristics of the AT-hook motif and PPC domain within AHL genes offer valuable insights into studying their gene function. However, the presence of numerous members of AHLs in plants presents a challenge for elucidating their precise functions through traditional gene knockout studies. In conventional gene knockout experiments, the deletion of a single AHL gene may not lead to significant observable phenotypic changes due to genetic redundancy present among the other family members (Street et al., 2008 ; Zhao et al., 2013 ). For instance, single-gene knockout mutants of AHL22 (ahl22-1), AHL29 (sob3-4) , and AHL27 (esc-8) exhibited no detectable phenotypic differences compared to the wild type (Street et al., 2008 ). However, when higher-order combinations of AHL genes were knocked out, such as in the quadruple mutant sob3-4 esc-8 ahl6 ahl22 , the resulting plants exhibited significantly more pronounced phenotypes compared to those with lower-order gene knockouts (Zhao et al., 2013 ). To address this limitation, the employment of dominant negative mutations has emerged as a valuable strategy, enabling researchers to overcome genetic redundancy and better unravel the functions of AHL genes in plants (Street et al., 2008 ; Tayengwa et al., 2020 ; Zhao et al., 2013 ). Dominant negative mutations are defined as mutation whose gene product adversely affect the wild-type gene product and/or interacting partners in the cell (Sheppard, 1994 ). In AHL proteins, these mutations are typically engineered to interfere with key functional domains, such as the AT-hook motif or the PPC domain. By selectively disrupting AHL-mediated transcriptional regulation through dominant negative mutations, our lab has uncovered the specific genes and pathways under AHL control, shedding light on their regulatory networks and molecular mechanisms First reported in AHL29 study, the gene containing a missense mutation in the R-G-R core of the AT-hook motif, resulted in suppression of gain-of-function phenotype (Street et al., 2008 ). Similarly, the dominant-negative mutation in AHL29 also showed more pronounced hypocotyl length compared to the quadruple mutant combination of sob3-4 , esc-8 , ahl6 , and ahl22 (Zhao et al., 2013 ). Dominant-negative mutation in the AHLs work by rendering these proteins incapable of executing their regulatory functions properly, leading to aberrant gene expression patterns and phenotypic abnormalities. (Jacques et al., 2022 ; Street et al., 2008 ; Tayengwa et al., 2020 ; Zhao et al., 2013 ). By combining dominant negative mutation studies with other genetic and molecular techniques, such as gene expression profiling, enables a comprehensive understanding of AHL gene function and their contributions to plant physiology. Our study used gain-of-function, and dominant-negative analysis to overcome the potential redundancy associated with the Clade-A gene AHL26 ( AT4g12050 ) in Arabidopsis thaliana . Transgenic plants overexpressing AHL26 produced shorter hypocotyls in a light-dependent manner with a delayed flowering phenotype under long-day, short-day, and continuous light conditions. In contrast, the dominant-negative mutation in AHL26 , which alters the second arginine in the conserved R-G-R core motif, disrupted its regulatory function, resulting in longer hypocotyls and an earlier flowering phenotype, highlighting the importance of the second arginine in the conserved R-G-R core motif for AHL26 function. Transcriptome analysis in transgenic plants over-expressing AHL26 further shows that the delayed flowering results from reduced cell division-related biological processes. Overall, the findings demonstrate that the AHL26 , like other members of AHL family, regulates hypocotyl development and flowering time in Arabidopsis thaliana . By identifying additional members with similar regulatory roles, our study adds to the growing evidence that AHL gene family members exhibit highly conserved functions, highlighting their importance in plant developmental processes. Methods Growth media conditions Different growth media were used for phenotypic analysis and to select transformants, as described by (Jacques et al., 2020 ). Briefly, selection media contained 0.5× Linsmaier and Skoog (LS) modified basal medium, 1.5% (w/v) sucrose, and 0.8% (w/v) Phytoblend (Caisson, Smithfield, UT) with appropriate antibiotics. Gellen gum (PhytoTechnology Laboratories, Inc.) was used as a solidifying agent in non-selection media along with 0.5× Linsmaier and Skoog (LS) modified basal medium and 1.5% (w/v) sucrose to ensure optimum plant growth. Selection of transformants All Arabidopsis thaliana plants used in this study are in Columbia (Col-0) background and are referred to as the wildtype (WT) in the manuscript. Transgenics were obtained through the floral-dip method (Clough and Bent, 1998 ). The putative T1 transgenic seeds were grown on selection media containing appropriate antibiotics. Transformants in the T2 generation were screened on selection media to identify transgenic lines containing single locus insertion based on chi-square analysis for a predicted 3 r :1 s segregation ratio. Multiple transgenic lines with single-locus insertions were grown further to select homozygous T3 lines. All the transformants were grown simultaneously to collect seeds to ensure uniform germination and seedling growth for phenotypic analysis. Seedlings germinated on non-selection media were used to record data. AHL26 overexpression A Gateway® Entry vector (pENTR223) containing the AHL26 (G23448) was obtained from the Arabidopsis Biological Resource Center (ABRC). The construct from the entry vector was LR cloned into the Gateway-compatible binary vector pED15 to over-express the AHL26 (AHL26OX) via the Cauliflower mosaic virus (CaMV) 35S constitutive promoter. WT plants were transformed with the resulting binary vectors carrying the AHL26OX (pAK8). Three homozygous T3 lines were used for AHL26OX transformants to carry out the phenotypic analysis. Generation of dominant-negative mutants The Q5® Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA) was used to carry out a target substitution at the 121–123 base site in AHL26 to substitute the wild-type codon AGA (arginine) to CAC (histidine). Non-overlapping primers (Table S1 ) were designed using the NEBaseChanger tool ( https://nebasechanger.neb.com/ ). AHL26OX carrying entry clones, as in the over-expression study, were used as templates in a PCR with substitution-specific primers to carry out the desired substitution. Newly generated constructs were sequenced to confirm the required substitution. Final constructs overexpressing mutated AHL26 (AHL26DN) were then cloned into Gateway-compatible pED15. Three independent transgenic lines each for AHL26DN (pAK17) were used for the phenotypic analysis. Hypocotyl Measurement After surface sterilization and placement on growth media, seeds were incubated for three days at 4°C followed by 4 hours of red-light treatment at 22°C to ensure uniform germination. Post cold and dark treatment, plates were subjected to 20 µmol m − 2 s − 1 of white light at 22°C for eight days in either long day (16 hours light and 8 hours dark) or short day (8 hours light and 16 hours dark) conditions (Zhao et al., 2013 ). Plates were wrapped with aluminum foil for dark-grown seedlings after the initial red-light treatment. After eight days, seedlings were transferred to transparencies and digitized at 800–1200 dots per inch (dpi) resolution using a flatbed scanner. The hypocotyls in the digitized images were measured using the NIH ImageJ software (Schneider et al., 2012 ) and analyzed using the ggplot package in R-studio. Flowering time For flowering time analysis, thirty-six seeds of multiple transgenic lines were directly sown in a pre-watered soil mix (Sunshine Mix4 [Aggregate] LA4; Green Island 28 Distributors Inc., Riverhead, NY). Trays were kept in the Conviron vernalization chamber ((Winnipeg, Manitoba, Canada) in darkness at 4 o C for four days to promote uniform germination. After cold treatment, trays were transferred to a growth chamber with 24-hour (FD), 16-hour (LD), and 8-hour (SD) day lengths at 21-22 o C, 60–70% humidity, and 200–225 µmol m − 2 s − 1 of light (Percival Intellus environmental controller, equipped with 4/8 fluorescent lamps and two Halco 9013 Frost T10FR25 25W Incandescent Bulbs). Seedlings were then thinned by clipping the hypocotyls with scissors to one seedling per pot. This approach was used to prevent damage to the roots of the remaining plant. Flowering time was measured by the number of rosette leaves and the number of days from emergence until the floral stem had grown 0.5cm. Data were analyzed in the ggplot package in RStudio. Generation of GUS constructs A 2963 bp region comprising the AHL26 promoter, 5’UTR, and coding sequence without the stop codon was PCR amplified using Gateway-compatible primers (Table S1 ). The resulting PCR product was purified, and the fragment was cloned into the Gateway-compatible entry vector pDONR221 using a BP reaction (Life Technologies, 2017 ). The entry vector with the fragment of interest was confirmed by sequencing. The construct from the entry vector was LR cloned into the Gateway-compatible destination vector pMDC163 (Curtis and Grossniklaus, 2003 ) via an LR reaction to generate an AHL26::GUS expression binary vector. WT plants were transformed with binary vectors carrying AHL26::GUS (pAK27). Three homozygous T3 transgenic lines with single locus insertions were used to carry out histochemical staining as described by (Zhang et al., 2009 ). Transformants were subjected to continuous light and dark conditions for 8 and 12 days. The images of the histochemical-stained seedlings were recorded with Zeiss Stemi 508 microscope. A semi-quantitative PCR was performed on the cDNA of 8-day-old light and dark whole seedlings, hypocotyl plus roots, and adult flowering plant rosette leaves. RNA extraction, cDNA synthesis, and quantitative real-time RT-PCR RNA was extracted from the resettle leaves of the WT, overexpression, and dominant-negative transgenic lines grown in continuous light conditions after 17 days for AHL26 gene expression. Extraction was performed using Plant RNA mini kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. Extraction was treated with DNAses to degrade potential DNA contamination. Complimentary DNA (cDNA) was synthesized using iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA). Due to the increased homology of AHL26 with other genes from this family, alignment of the gene under study was done using an online tool ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ) with other genes to make the primers specific to AHL26 (Table S1 ). cDNA with a 10-fold dilution was used as a template to run the RT-qPCR using Bio-Rad’s SSO Advanced Universal SYBR Green Super Mix (Bio-Rad, Hercules, CA) on Bio-Rad’s CFX96 Touch Real-Time PCR Detection System. Melt curve analysis was done to eliminate the non-specific amplification. Data were normalized against the Actin primer used as an internal control. RNA seq library preparation RNA was isolated from 3 biological replicates of 17-day-old rosette leaves in the WT, AHL26OX , and AHL26DN grown in continuous light conditions using a Plant RNA mini kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. Messenger RNA was purified from the total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for the directional library or dTTP for the non-directional library. The library was checked with Qubit, real-time PCR for quantification, and a bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on Illumina platforms according to the effective library concentration and data amount. The clustering of the index-coded samples was performed according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina platform, and paired-end reads were generated. Data Analysis Quality control Raw data (raw reads) of fastq format were processed to obtain clean data (clean reads) by removing reads with adapter, reads containing ploy-N, and low-quality reads from raw data. At the same time, Q20, Q30, and GC content of the clean data were calculated. All the downstream analyses were based on clean data with high quality. Reads mapping to the reference genome Reference genome and gene model annotation files were downloaded from the genome website (ensemblplants_arabidopsis_thaliana_tair10_gca_000001735_1). The reference genome index was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. We selected Hisat2 as the mapping tool since it can generate a database of splice junctions based on the gene model annotation file and thus produce a better mapping result than other non-splice mapping tools. Quantification of gene expression level FeatureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. The FPKM of each gene was calculated based on the length of the gene, and the reads count was mapped to AHL26 . FPKM, the expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most used method for estimating gene expression levels. Differential expression analysis Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2R package (1.20.0). DESeq2 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using Benjamini and Hochberg's approach for controlling the false discovery rate. Genes with an adjusted P-value < = 0.05 found by DESeq2 were assigned as differentially expressed. GO and KEGG enrichment analysis of differentially expressed genes Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler R package, in which gene length bias was corrected. GO terms with corrected P-value less than 0.05 were considered significantly enriched by differential expressed genes. KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism, and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies ( http://www.genome.jp/kegg/ ). We used clusterProfiler R package to test the statistical enrichment of differential expression genes in KEGG pathways. Results The tissue-specific expression of AHL26 The tissue-specific expression pattern of AHL26 was analyzed under darkness and light-grown conditions by using a translational fusion with the β-glucuronidase (GUS) reporter which is under control of the endogenous AHL26 promoter. GUS expression in light-grown seedlings was concentrated in the apical meristem, hypocotyl, and leaves (Fig. 1 A). Expression in dark-grown seedlings, however, showed activity only in the apical meristem and leaves with no expression in the hypocotyl and weak or no expression in the roots (Fig. 1 B-C). GUS activity in 21-day-old plants grown under continuous light showed strong expression in the leaves with weak activity in the roots (Fig. 1 D). A semi-quantitative PCR analysis using light- and dark-grown seedlings confirmed in-situ expression patterns (Fig. S1 ). AHL26 plays a role in light-mediated hypocotyl growth To investigate the role of AHL26 in hypocotyl growth, we generated transgenic lines in Arabidopsis overexpressing AHL26 under the control of the CaMV 35S promoter (AHL26OX) . The resulting overexpression plants produced a short hypocotyl in both short- and long-day growth conditions compared to the wild type (WT) (Fig. 2 A). However, no significant effects on hypocotyl lengths were observed in seedlings grown in dark conditions (Fig. S2). Mutations in the core conserved region of the AT-hook motif in AHL genes have been shown to confer a dominant-negative phenotype (Street et al., 2008 ; Tayengwa et al., 2020 ; Zhao et al., 2013 ). Based on these studies, transgenic plants overexpressing a dominant-negative mutation in AHL26 (AHL26DN) were generated, where a conserved second arginine (R-G-R) in the AT-hook motifs necessary for DNA binding was replaced with histidine (R-G-H). The resulting overexpression of AHL26DN produced a short hypocotyl in both short- and long-day growth conditions compared to the WT (Fig. 2 B-D). Hypocotyl length for these AHL26DN overexpression plants conferred no significant differences when grown under dark conditions (Fig. S2). AHL26 plays a role in flowering time Transgenic Arabidopsis overexpressing AHL26 conferred a late-flowering phenotype compared to the WT, and plants overexpressing the dominant-negative mutation, irrespective of the day length conditions (Fig. 3 A). Along with delayed flowering, plants overexpressing AHL26 also displayed a distinct wrinkled leaf phenotype (S3). In contrast, transgenic Arabidopsis overexpressing AHL26 harboring a dominant-negative point mutation ( AHL26DN) displayed an early flowering phenotype compared to the WT, and over-expressing transgenics in all-day length conditions (Fig. 3 B). AHL26 Influences Flowering Pathways Through Extensive Transcriptomic Changes To investigate the molecular basis of flowering differences in AHL26 transgenic lines, the expression levels of AHL26 were first examined using reverse transcription-quantitative PCR (RT-qPCR). The results confirmed elevated AHL26 transcript levels in both AHL26OX and AHL26DN transgenic lines (Figure S3). Given that AHL proteins function as transcriptional regulators, RNA-seq analysis was subsequently performed to explore the broader transcriptomic impact in AHL26OX and AHL26DN transgenic lines. DESeq analysis was carried out on the transcriptome data to obtain differentially expressed genes (DEGs) between AHL26OX vs. the WT, AHL26DN vs. the WT, and AHL26OX vs. AHL26DN . A total of 1263 genes were differentially expressed in AHL26OX vs. the WT, with a higher number of downregulated genes (916) than upregulated genes (347) including flowering-related genes such as FLOWERING LOCUS T ( FT), Twin Sister of FT (TSF), CIRCADIAN CLOCK–ASSOCIATED1 (CCA1), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), and LATE ELONGATED HYPOCOTYL (LHY) , which were all downregulated (Fig. 4 A). In contrast, the number of DEGs in AHL26DN vs. the WT was significantly lower where only 138 genes were differentially expressed, with 55 upregulated to 83 downregulated genes including the upregulation of TSF , a key flowering promoter (Fig. 4 B). There were 462 DEGs present between AHL26DN vs. AHL26OX , with 243 upregulated to 219 downregulated genes. FT and TSF , which were repressed in the AHL26OX transgenic lines, accumulated more in the AHL26DN plants (Fig. 4 C). To further understand the similarity between AHL26DN and AHL26OX plants in relation to the upregulated and downregulated genes compared to the WT, venn diagrams were generated using upregulated and downregulated genes between AHL26OX vs. the WT and AHL26DN vs. the WT. Of the total genes repressed by AHL26OX and AHL26DN in relation to the WT, 91.4% (n = 882) were uniquely repressed by AHL26OX compared to only 5.1% (n = 49) by AHL26DN . There were only 34 genes that were both repressed by AHL26OX and AHL26DN (Fig. 4 D). Similarly, only eight genes were induced together by AHL26OX and AHL26DN . AHL26OX uniquely induced 339 genes to only 47 genes by AHL26DN when compared with the WT (Fig. 4 E). Gene Ontology-Based Functional Analysis of DEGs To understand the biological implications of the differentially expressed genes (DEGs), an enrichment analysis for Gene Ontology (GO) terms was performed across three groups. Figure 5 A shows the 30 most significant upregulated GO terms and the number of DEGs present in AHL26OX compared to the WT. The DEGs in this group were enriched to the biological process involved in flavonoid biosynthetic process, cold acclimation, sequence-specific double-stranded DNA binding, and AT-rich DNA binding. The downregulated biological process and molecular function categories in AHL26OX vs. the WT group included the mitotic cell cycle, cell division, cell cycle process, as well as cell cycle phase transition. (Fig. 5 B). In contrast to AHL26OX , molecular function GO terms in AHL26DN transgenics showed upregulation of the regulation of DNA binding transcription factor activity (Fig. 5 C). These upregulated GO terms included the well-known flower-inducing gene LHY . Other flowering-related biological processes upregulated in the AHL26DN transgenic lines were rhythmic and positive regulation of the developmental process. These included floral inducer genes such as FT , TSF , LHY , and CCA1 . Upregulation of these GO terms in AHL26DN transgenic lines are likely related to the early flowering phenotype compared to AHL26OX plants. The downregulated GO terms in AHL26DN vs. AHL26OX group included ribosome assembly, ribosome large subunit biogenesis, sequence-specific double-stranded DNA binding and AT-rich DNA binding (Fig. 5 D, Table S2). Regulatory Effects of AHL26 on Key Biological Pathways In the next step, KEGG pathway enrichment analysis was conducted to gain insight into the biological processes and pathways associated with the differentially expressed genes (DEGs) in AHL26 transgenic lines. Only three pathways were significantly upregulated in AHL26OX transgenic lines as overexpression of genes resulted in more downregulated than upregulated DEGs (Fig. 6 A). In contrast, multiple pathways were significantly downregulated in the AHL26OX compared to the WT. The downregulated DEGs in AHL26OX plants were enriched to the plant hormone signal transduction, photosynthesis-antenna proteins, and glucosinolate biosynthesis (Fig. 6 B). AHL26 dominant negative transgenic lines exhibited upregulation of KEGG-enriched pathways associated with circadian rhythm (Fig. 6 C), while pathways related to ribosome biogenesis were downregulated (Fig. 6 D). Discussion The role of AHL26 in seedling development The Arabidopsis hypocotyl, due to its simple anatomy, is an ideal phenotypic indicator for seedling development mutant studies (Boron and Vissenberg, 2014 ) and the basis for identification and genetic characterization of SUPPRESSOR OF PHYB #3 (SOB3)/AHL29 (Street et al., 2008 ). AHL26 is an intron-less gene that belongs to the same clade as AHL27 and AHL29 (Zhao et al., 2014 ). Our findings demonstrate that the overexpression of AHL26 results in a shorter hypocotyl than the WT when grown in long and short-day conditions (Fig. 2 A). However, no significant difference in hypocotyl lengths was present in seedlings grown in darkness, suggesting a light-dependent function for AHL26 (Fig. S2). This interpretation is further supported by the absence of AHL26-GUS protein expression in the hypocotyl region in dark-grown seedlings. The expression of AHL26-GUS in seedlings grown in dark conditions concentrates in apical meristems unlike light-grown seedlings, where expression can be seen in hypocotyl and leaves (Fig. 1 A-D, S1). We propose that the activity of AHL26 at the seedling stage in Arabidopsis is light-dependent and that the protein is either absent or present in low levels in the hypocotyls grown in the absence of light. Light-dependent hypocotyl activity was also observed in other clade-a AHL genes (Street et al., 2008 ). The AT-hook motif in AHL proteins is critical for their function as transcriptional regulators in Arabidopsis (Jacques et al., 2022 ). A conserved arginine residue within this motif is particularly important for their gene-over-expression gain-of-function phenotype. However, due to the presence of multiple closely related members within the AHL gene family, functional redundancy often masks phenotypic effects in single-gene knockout studies, making it difficult to determine the precise role of individual AHL genes. To address this challenge, our lab has developed a dominant-negative mutant approach, which has been effective in overcoming both known and unknown genetic redundancy. This strategy involves the overexpression of a mutant version of the gene carrying a non-functional AT-hook motif, which interferes with the function of other AHL proteins that share similar binding properties (Street et al., 2008 ; Tayengwa et al., 2020 ; Zhao et al., 2013 ). To overcome the unknown genetic redundancy between AHL26 and other AHL and non- AHL genes, dominant negative mutants overexpressing AHL26 were generated with a non-functional AT-hook motif (AHL26DN). Plants overexpressing AHL26DN confer seedlings with significantly longer hypocotyls than the WT, and AHL26OX transgenic lines (Fig. 2 A-D). This increased hypocotyl length in the dominant-negative mutant plants suggests that additional proteins, possibly AHLs, are involved in the seedling growth in a light-dependent manner. However, the increase in hypocotyl length is not as dramatic as observed in the over-expression of sob3-6 (Street et al., 2008 ; Zhao et al., 2013 ), even though both genes harbor the same mutation in the AT-hook motif. Since AHLs tend to act as a transcriptional repressor (Favero et al., 2016; Jacques et al., 2022 ), another reason for the less severe hypocotyl phenotype in AHL26DN plants than sob3-6 might relate to the ability of SOB3 (AHL29) to repress different or additional genes than AHL26 . It is also possible that these phenotypic differences are due to levels of gene expression, protein accumulation/stability or experimental growth conditions. The role of AHL26 in flowering time Flowering in plants results from the expression of floral meristem identity genes, which transition cells in the shoot apical meristems from vegetative to floral (Krizek and Fletcher, 2005 ). The time and stage of floral formation is crucial for plants to continue their progeny and achieve maximum yield. Several AHLs have been demonstrated to play a role in controlling the onset of flowering in Arabidopsis (Ng et al., 2009 ; Tayengwa et al., 2020 ; Xiao et al., 2009 ; Yun et al., 2012 ; Zhao et al., 2013 ). Over-expression of AHL family genes in Arabidopsis has generally been associated with delayed flowering (Tayengwa et al., 2020 ; Xiao et al., 2009 ; Yun et al., 2012 ). Like other members of the AHL gene family, our gain-of-function study shows that the over-expression of AHL26 also confers a late flowering phenotype in Arabidopsis in short-day, long-day, and continuous light conditions (Fig. 3 A). As observed in the hypocotyl length analysis, AHL26 requires an intact AT-hook motif to impact the flowering time phenotype as over-expression of dominant-negative AHL26 (AHL26DN ) results in an earlier flowering phenotype than the WT, and over-expression plants. Similar results have also been reported for other clade-A AHLs in Arabidopsis (Tayengwa et al., 2020 ; Xiao et al., 2009 ). Transcriptome analysis supports the role of AHL26 in floral development To gain a deeper understanding of the specific effects of AHL26 on flowering time regulation, RNA-seq analysis was performed on AHL26OX and AHL26DN transgenic lines. By analyzing transcriptomes in these mutants, we aimed to uncover the impact of AHL26 on the expression of other flowering-related genes. Differential gene expression analysis reveals more downregulated genes in AHL26OX plants than upregulated ones (Fig. 4 A). Since most AHLs act as transcription factors due to their DNA binding ability, the more downregulated gene in AHL26OX transgenic lines suggests that AHL26 acts as a transcriptional repressor in controlling floral induction. To further understand the delayed flowering phenotype observed in AHL26 overexpression transgenic lines, the differentially expressed genes in AHL26OX plants were analyzed, focusing on key regulators of floral induction in Arabidopsis . For example, SOC1 , a well-established activator of floral development (Lee and Lee, 2010 ), exhibits reduced expression in AHL26OX plants, suggesting that AHL26 modulates flowering-related gene expression (Fig. 4 A). In continuous white light, the expression of FT and TSF is regulated by the circadian clock proteins LHY and CCA1 (Fujimoto et al., 2009). Our data indicate that the overexpression of AHL26 reduces the expression of FT and TSF by repressing the activity of LHY and CCA1 (Fig. 4 A). Additionally, FLC , a central repressor of flowering in the autonomous pathway, is significantly upregulated in AHL26OX transgenic lines, further contributing to the observed delay in flowering. In addition to the upregulation of flowering repressors, AHL26OX plants also exhibit increased transcript accumulation of several other AHL genes, including AHL19 , AHL2 , AHL1 , AHL15 , and AHL20 (Fig. 4 A). Similar to AHL26 , the overexpression of AHL20 and AHL15 has also been shown to delay flowering (Karami et al., 2020 ; Tayengwa et al., 2020 ), reinforcing the functional redundancy within the AHL gene family in regulating plant development. In contrast to AHL26 overexpressing plants, none of the negative regulators of flowering are among the upregulated differentially expressed genes (DEGs) in AHL26DN plants, resulting in an earlier flowering phenotype (Fig. 4 C). The substitution of the second arginine in the AHL26 AT-hook motif with histidine likely disrupts its regulatory function, leading to the loss of the late flowering phenotype in AHL26DN transgenic plants. This early flowering phenotype is associated with the upregulation of flowering-promoting genes such as LHY , CCA1 , FT , and TSF , along with the downregulation of FLC . These findings are consistent with previous dominant-negative studies (Lu et al., 2010 ; Tayengwa et al., 2020 ), further supporting the role of AHL26 in modulating flowering time through transcriptional regulation. Gene pathway analysis supports a transcriptional role of AHL26 on floral development The enrichment analysis of differentially expressed genes (DEGs) provides valuable insights into the biological pathways and functions significantly associated with AHL26 regulation. Comparing the active pathways between distinct groups reveals broader gene regulatory mechanisms beyond individual gene expression changes. Gene Ontology (GO) analysis categorizes genes based on biological processes, cellular components, and molecular functions, while Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations offer pathway-level insights into gene function. These analyses collectively enhance our understanding of AHL26 -mediated transcriptional regulation. One of the most striking phenotypes observed in AHL26 overexpression ( AHL26OX ) plants is delayed flowering, which is linked to slow leaf growth and reduced meristematic activity. The repression of meristem development and the positive regulation of developmental processes likely contributed to this phenotype. Additionally, a decrease in cell division processes may hinder the transition of vegetative meristems into floral meristems, further delaying flowering in AHL26OX plants (Fig. 5 A). The observed repression of these developmental processes aligns with the known transcriptional repressive nature of AHL26 . Our findings indicate that AHL26OX plants exhibit increased sequence-specific double-stranded DNA binding, AT-rich DNA binding, regulatory region nucleic acid binding, and transcription regulatory region DNA binding (Fig. 5 B). These molecular functions suggest that AHL26 exerts its effects by modulating key growth repressors. Consistent with our results, previous studies have also reported the transcriptional regulatory role of AHL proteins in developmental processes (Favero et al., 2020 ; Jacques et al., 2022 ). To further investigate the role of AHL26 in flowering regulation, AHL26DN plants, which carry a mutation in their AT-hook motif that disrupts DNA binding were examined. Unlike AHL26OX plants, AHL26DN transgenic lines did not exhibit upregulation of growth repressors, resulting in an early flowering phenotype (Fig. 4 C). A comparative GO analysis between AHL26OX and AHL26DN revealed a significant repression of sequence-specific double-stranded DNA binding in AHL26DN plants. Additionally, genes involved in AT-rich DNA binding were downregulated in AHL26DN plants compared to AHL26OX (Table S2). These findings suggest that the loss of AHL26 DNA binding activity prevents its ability to repress key flowering regulators, thereby accelerating the transition to reproductive growth. Interestingly, beyond transcriptional regulation, our GO analysis also revealed a significant downregulation of biological processes related to ribosome assembly and large ribosomal subunit biogenesis in AHL26DN plants. This suggests a potential role for AHL26 in regulating gene expression at the translational level, possibly influencing protein synthesis and overall cellular function. Given the observed downregulation of ribosomal biogenesis in AHL26DN plants, further investigations using techniques such as Translating Ribosome Affinity Purification followed by sequencing (TRAP-seq) could provide deeper insights into the role of AHL26 in modulating translational dynamics (Fig. 5 D). Overall, our findings highlight the critical role of AHL26 in repressing flowering by directly binding to DNA and modulating gene expression. The functional loss of DNA binding in AHL26DN plants further supports the importance of AHL26 as a transcriptional regulator of flowering time. Future studies integrating transcriptomic and translational analyses will be instrumental in unraveling the full spectrum of AHL26 -mediated gene regulation. Conclusion Our findings demonstrate that AHL26 plays a pleiotropic role in plant development, as its overexpression leads to a shorter hypocotyl and a delayed flowering phenotype. AHL26 exerts its regulatory function through a conserved AT-hook motif, as a missense mutation in this motif abolishes these phenotypic effects. Furthermore, AHL26 functions as a transcriptional repressor, modulating the expression of key growth repressors involved in floral meristem development and other essential developmental processes. These results highlight the critical role of AHL26 in regulating plant growth and flowering, providing a foundation for future studies on its broader regulatory network. Declarations Ethics approval and consent to participate Not applicable Consent for publications Not applicable Availability of data and materials The RNA-Seq dataset used in the study is available in the NCBI Sequence Read Archive (SRA) repository, accession number PRJNA1241335 under the following link: https://www.ncbi.nlm.nih.gov/sra/PRJNA1241335 The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by Authors’ contributions SA and MN designed the study. SA, AH, and XX performed the experiments. SA and MN wrote the manuscript. Acknowledgments We thank Dr. Cynthia Gleason for her support in providing access to her stereo microscope for GUS visualization. We also thank Drs. John Hadish, Ian Burke and Karen Sanguinet for their support References Boron, A.K., Vissenberg, K., 2014. The Arabidopsis thaliana hypocotyl, a model to identify and study control mechanisms of cellular expansion. Plant Cell Rep. 33, 697–706. https://doi.org/10.1007/s00299-014-1591-x Clough, S.J., Bent, A.F., 1998. Floral dip: a simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743. https://doi.org/10.1046/j.1365-313x.1998.00343.x Curtis, M.D., Grossniklaus, U., 2003. A Gateway Cloning Vector Set for High-Throughput Functional Analysis of Genes in Planta. Plant Physiol. 133, 462–469. https://doi.org/10.1104/pp.103.027979 Favero, D.S., Jacques, C.N., Iwase, A., Le, K.N., Zhao, J., Sugimoto, K., Neff, M.M., 2016. SUPPRESSOR OF PHYTOCHROME B4-#3 Represses Genes Associated with Auxin Signaling to Modulate Hypocotyl Growth. Plant Physiol. 171, 2701–2716. https://doi.org/10.1104/PP.16.00405 Favero, D.S., Kawamura, A., Shibata, M., Takebayashi, A., Jung, J.-H., Suzuki, T., Jaeger, K.E., Ishida, T., Iwase, A., Wigge, P.A., Neff, M.M., Sugimoto, K., 2020. AT-Hook Transcription Factors Restrict Petiole Growth by Antagonizing PIFs. Curr. Biol. 30, 1454-1466.e6. https://doi.org/10.1016/j.cub.2020.02.017 Fujimoto, S., Matsunaga, S., Yonemura, M., Uchiyama, S., Azuma, T., Fukui, K., n.d. Identification of a novel plant MAR DNA binding protein localized on chromosomal surfaces. Jacques, C.N., Favero, D.S., Kawamura, A., Suzuki, T., Sugimoto, K., Neff, M.M., 2022. SUPPRESSOR OF PHYTOCHROME B-4 #3 reduces the expression of PIF-activated genes and increases expression of growth repressors to regulate hypocotyl elongation in short days. BMC Plant Biol. 22, 399. https://doi.org/10.1186/s12870-022-03737-z Jacques, C.N., Hulbert, A.K., Westenskow, S., Neff, M.M., 2020. Production location of the gelling agent Phytagel has a significant impact on Arabidopsis thaliana seedling phenotypic analysis. PLOS ONE 15, e0228515. https://doi.org/10.1371/journal.pone.0228515 Karami, O., Rahimi, A., Khan, M., Bemer, M., Hazarika, R.R., Mak, P., Compier, M., van Noort, V., Offringa, R., 2020. A suppressor of axillary meristem maturation promotes longevity in flowering plants. Nat. Plants 6, 368–376. https://doi.org/10.1038/s41477-020-0637-z Krizek, B.A., Fletcher, J.C., 2005. Molecular mechanisms of flower development: an armchair guide. Nat. Rev. Genet. 6, 688–698. https://doi.org/10.1038/nrg1675 Lee, J., Lee, I., 2010. Regulation and function of SOC1, a flowering pathway integrator. J. Exp. Bot. 61, 2247–2254. https://doi.org/10.1093/jxb/erq098 Life Technologies, 2017. GATEWAY TM Cloning Technology. Lu, H., Zou, Y., Feng, N., 2010. Overexpression of AHL20 negatively regulates defenses in arabidopsis. J. Integr. Plant Biol. 52, 801–808. https://doi.org/10.1111/j.1744-7909.2010.00969.x Ng, K.-H., Yu, H., Ito, T., 2009. AGAMOUS Controls GIANT KILLER, a Multifunctional Chromatin Modifier in Reproductive Organ Patterning and Differentiation. PLoS Biol. 7, e1000251–e1000251. https://doi.org/10.1371/journal.pbio.1000251 Schneider, C.A., Rasband, W.S., Eliceiri, K.W., 2012. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675. https://doi.org/10.1038/nmeth.2089 Sheppard, D., 1994. Dominant negative mutants: tools for the study of protein function in vitro and in vivo. Am. J. Respir. Cell Mol. Biol. 11, 1–6. https://doi.org/10.1051/acarologia/20132076 Street, I.H., Shah, P.K., Smith, A.M., Avery, N., Neff, M.M., 2008. The AT-hook-containing proteins SOB3/AHL29 and ESC/AHL27 are negative modulators of hypocotyl growth in Arabidopsis. Plant J. 54, 1–14. https://doi.org/10.1111/j.1365-313X.2007.03393.x Tayengwa, R., Sharma-Koirala, P., Pierce, C.F., Werner, B.E., Neff, M.M., 2020. Overexpression of AtAHL20 causes delayed flowering in Arabidopsis via repression of FT expression (preprint). In Review. https://doi.org/10.21203/rs.3.rs-38575/v1 Xiao, C., Chen, F., Yu, X., Lin, C., Fu, Y.-F., 2009. Over-expression of an AT-hook gene, AHL22, delays flowering and inhibits the elongation of the hypocotyl in Arabidopsis thaliana. Plant Mol. Biol. 71, 39–50. https://doi.org/10.1007/s11103-009-9507-9 Yun, J., Kim, Y.-S., Jung, J.-H., Seo, J., Park, C.-M., 2012. The AT-hook Motif-containing Protein AHL22 Regulates Flowering Initiation by Modifying FLOWERING LOCUS T Chromatin in Arabidopsis * □ S. https://doi.org/10.1074/jbc.M111.318477 Zhang, J., Vankova, R., Malbeck, J., Dobrev, P.I., Xu, Y., Chong, K., Neff, M.M., 2009. AtSOFL1 and AtSOFL2 Act Redundantly as Positive Modulators of the Endogenous Content of Specific Cytokinins in Arabidopsis. PLOS ONE 4, e8236. https://doi.org/10.1371/journal.pone.0008236 Zhao, J., Favero, D.S., Peng, H., Neff, M.M., 2013. Arabidopsis thaliana AHL family modulates hypocotyl growth redundantly by interacting with each other via the PPC/DUF296 domain. Proc. Natl. Acad. Sci. U. S. A. 110, E4688-97. https://doi.org/10.1073/pnas.1219277110 Zhao, J., Favero, D.S., Qiu, J., Roalson, E.H., Neff, M.M., 2014. Insights into the evolution and diversification of the AT-hook Motif Nuclear Localized gene family in land plants. https://doi.org/10.1186/s12870-014-0266-7 Additional Declarations No competing interests reported. Supplementary Files suppdataarabidopsisbmcplant32525.docx Cite Share Download PDF Status: Published Journal Publication published 30 May, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 07 May, 2025 Reviews received at journal 05 May, 2025 Reviewers agreed at journal 29 Apr, 2025 Reviews received at journal 10 Apr, 2025 Reviewers agreed at journal 04 Apr, 2025 Reviewers agreed at journal 31 Mar, 2025 Reviewers invited by journal 31 Mar, 2025 Editor assigned by journal 26 Mar, 2025 Editor invited by journal 26 Mar, 2025 Submission checks completed at journal 25 Mar, 2025 First submitted to journal 25 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6264939","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":441437995,"identity":"49f81c3b-f347-43d2-84ef-68b9e584851d","order_by":0,"name":"Shahbaz Ahmed","email":"","orcid":"","institution":"Washington State University","correspondingAuthor":false,"prefix":"","firstName":"Shahbaz","middleName":"","lastName":"Ahmed","suffix":""},{"id":441437996,"identity":"73c2d282-8600-4698-8cfe-c4d9c56ffdd4","order_by":1,"name":"Anna Hulbert","email":"","orcid":"","institution":"Washington State University","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Hulbert","suffix":""},{"id":441437997,"identity":"37cf7b34-9d29-4b83-84fa-aeb3c2f6fb44","order_by":2,"name":"Xin Xin","email":"","orcid":"","institution":"Washington State University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Xin","suffix":""},{"id":441437998,"identity":"d8da0328-6720-4919-944d-17d9fa8ee3d0","order_by":3,"name":"Michael Neff","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAoUlEQVRIiWNgGAWjYLCCChsbCIOHaC1n0tJI13KYBC26084efHAg4Xzi/BkJjA/ethGhxex2XrLBgYTbiRtuJDAbziVOS46Z9Mcft3M3SCSwSfMSqcX8x4GEc7lAh7H/JlaLGcOBhAO5DTcS2JiJ1WIscSAhuX7DmYfNknPOEafF8MOBBDtj+fbkgx/elBGhBQkwNpCmfhSMglEwCkYBbgAAkmI8Ft0C9LsAAAAASUVORK5CYII=","orcid":"","institution":"Washington State University","correspondingAuthor":true,"prefix":"","firstName":"Michael","middleName":"","lastName":"Neff","suffix":""}],"badges":[],"createdAt":"2025-03-20 00:08:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6264939/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6264939/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-06764-8","type":"published","date":"2025-05-30T15:57:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81071324,"identity":"d94588e8-585d-4d9f-a071-851e09751ba8","added_by":"auto","created_at":"2025-04-22 01:42:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":910923,"visible":true,"origin":"","legend":"\u003cp\u003eTissue-specific expression pattern of AHL26. GUS activity in AHL26:AHL26-GUS expressing plants: (A) 12-day-old seedlings grown under 20 μmol m\u003csup\u003e−2\u003c/sup\u003e s\u003csup\u003e−1\u003c/sup\u003e of continuous white light, (B) 8-day-old seedlings grown under dark, (C) 12-day-old seedlings grown under dark conditions, and (D) 21-day-old plant grown under 200-225 μmol m\u003csup\u003e−2\u003c/sup\u003e s\u003csup\u003e−1\u003c/sup\u003e of continuous white light.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6264939/v1/c92e2a915ec9b2ffddd17e82.png"},{"id":81070766,"identity":"e0a189ad-a10c-4072-b5cf-14731212f692","added_by":"auto","created_at":"2025-04-22 01:34:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":624443,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of hypocotyl lengths of 8-day-old seedlings grown in 20 μmol m\u003csup\u003e−2\u003c/sup\u003e s\u003csup\u003e−1\u003c/sup\u003e of while light under short-day (SD) and long (LD) conditions. (A) Quantitative analysis of three independent AHL26 overexpression transgenic lines under SD and LD conditions compared to the WT. (B) Quantitative analysis of hypocotyl length of three independent AHL26 dominant-negative transgenic lines under SD and LD conditions. (C-D) A comparison of hypocotyl phenotypes between the WT, AHL26OX, and AHL26DN under SD (C) and LD (D) conditions. n ³ 20. The error bars represent the standard error of the mean (SEM). Different letters indicate statistical significance (ANOVA; P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6264939/v1/2eb1e4ae2b175a51dd1ae1b5.png"},{"id":81070768,"identity":"e5cdae24-9a56-4aa2-9a5f-4539a46a3f6e","added_by":"auto","created_at":"2025-04-22 01:34:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":631640,"visible":true,"origin":"","legend":"\u003cp\u003eQuantitative analysis of flowering time in AHL26transgenics lines under full-day (FD), long (LD), and short-day (SD) conditions. (A) Quantitative analysis of flowering time in two independent AHL26overexpression transgenic lines under long-day, short-day, and full-day light conditions compared to the WT. (B) Quantitative analysis of flowering time in two independent AHL26 dominant-negative transgenic lines under long-day, short-day, and full-day light conditions compared to the WT. The error bar denotes SEM. Different letters indicate statistical significance (ANOVA; P \u0026lt; 0.05). n=36\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6264939/v1/7cff64d2e1d0dc73b6c4707c.png"},{"id":81071326,"identity":"6e681864-4128-4d83-a13b-40cad4acbb8e","added_by":"auto","created_at":"2025-04-22 01:42:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":718166,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eAHL26OX\u003c/em\u003e and \u003cem\u003eAHL26DN\u003c/em\u003emutants result in differential gene expression patterns. Volcano plots of differentially expressed genes among different groups, padj\u0026lt;0.05 and |log2FoldChange|\u0026gt;1. The x-axis represents the log2FoldChange, while the y-axis represents the statistical significance of each gene. (A) Volcano plot of DEGs comparing \u003cem\u003eAHL26OX\u003c/em\u003e vs. the WT, (B) Volcano plot of DEGs between \u003cem\u003eAHL26DN\u003c/em\u003evs. the WT group. (C) Volcano plot comparing DEGs between \u003cem\u003eAHL26DN vs. AHL26OX\u003c/em\u003e. (D) Venn diagram comparing the downregulated genes by \u003cem\u003eAHL26OX \u003c/em\u003eand\u003cem\u003eAHL26DN\u003c/em\u003e with respect to the WT. (E) Venn diagram representing the genes commonly upregulated by both \u003cem\u003eAHL26OX \u003c/em\u003eand\u003cem\u003e AHL26DN\u003c/em\u003e with respect to the WT.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6264939/v1/c42041d7d12cf58fb3cf2c08.png"},{"id":81070770,"identity":"b4f2d771-9feb-44f3-b7de-90eb51160cae","added_by":"auto","created_at":"2025-04-22 01:34:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1224025,"visible":true,"origin":"","legend":"\u003cp\u003eSignificantly enriched (qadj \u0026lt; 0.05) GO terms among DEGs. (A) upregulated GO terms in AHL26OX vs. the WT group. (B) downregulated GO terms in AHL26OX vs. the WT group. (C) upregulated GO terms in AHL26DN vs. AHL26OX. (D) downregulated GO terms in AHL26DN vs. AHL26OX. The color and size of the dots represent the range of q-value and the number of DEGs mapped.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6264939/v1/afef389edeac022991fba88c.png"},{"id":81071384,"identity":"200aeec8-a3fa-4175-8edf-7ca77dec95a3","added_by":"auto","created_at":"2025-04-22 01:50:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":313841,"visible":true,"origin":"","legend":"\u003cp\u003eSignificantly enriched (qadj \u0026lt; 0.05) KEGG pathways among DEGs. (A) upregulated KEGG pathways in AHL26OX vs. the WT group. (B) downregulated KEGG pathways in AHL26OX vs. the WT group. (C) upregulated KEGG pathways in AHL26DNvs. AHL26OX. (D) downregulated KEGG pathways in AHL26DN vs. AHL26OX. The color and size of the dots represent the range of q-value and the number of DEGs mapped.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6264939/v1/7f0eacff819f869e08265834.png"},{"id":83782808,"identity":"fe38d205-ab0e-42fc-8ac6-ee7e97c2ff1b","added_by":"auto","created_at":"2025-06-02 16:06:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5508569,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6264939/v1/e84c7243-6ac4-44c2-a587-232553a52752.pdf"},{"id":81071325,"identity":"6255a66b-d3d4-4522-96e0-aa75ac3858b8","added_by":"auto","created_at":"2025-04-22 01:42:01","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1679380,"visible":true,"origin":"","legend":"","description":"","filename":"suppdataarabidopsisbmcplant32525.docx","url":"https://assets-eu.researchsquare.com/files/rs-6264939/v1/7124f35d36c5a49401d6eae2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"AHL26, an AT-hook gene, negatively regulates hypocotyl growth and flowering time in Arabidopsis thaliana","fulltext":[{"header":"Background","content":"\u003cp\u003eThe \u003cem\u003eAHL\u003c/em\u003e gene family constitutes a crucial component of the regulatory machinery governing seedling and adult plant development (Street et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The evolutionary conservation of the \u003cem\u003eAHL\u003c/em\u003e gene family underscores its fundamental importance in plant biology (Zhao et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). AHL proteins feature a conserved AT-hook motif at their N-terminal end and a plant and prokaryote conserved (PPC) domain, also known as the domain of unknown function #296 (DUF296), at their C-terminal end.\u003c/p\u003e \u003cp\u003eThe AT-hook, first described in the HIGH MOBILITY GROUP (HMG) family of non-histone chromosomal-associated proteins in mammals is a small motif that has a pattern centered around a glycine-arginine-proline (GRP) tripeptide (Aravind and Landsman, 1998). This sequence is necessary and sufficient to bind DNA. Mutations in this core sequence result in the loss of DNA binding properties of AHLs (Zhao et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).There are three types of AT-hooks present in the \u003cem\u003eAHL\u003c/em\u003e gene family. Type-I is characterized by an additional module present at the C-terminal of the core GRP. They have a greater probability of glycine at the second position C-terminal to the GRP. Type-II is characterized by having a high probability of possessing lysine instead of glycine, two residues down from the GRP. Type-III is characterized by including features of both Type-I and Type-II AT-hooks (Aravind and Landsman, 1998).\u003c/p\u003e \u003cp\u003eThe PPC domain, approximately 120 amino acids in length, functions as an independent protein in bacteria, archaea, and some green algae (Fujimoto et al., 2004). In land plants, however, the PPC domain is fused with the AT-hook motif in the AHL family of proteins and is responsible for protein-protein interaction between AHLs and non-AHL proteins in the cell (Favero et al., 2016; Zhao et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The PPC domains in plants can be categorized into Type-A and Type-B based on the conserved Gly-Arg-Phe-Glu-Ile-Leu motif. The conserved region in PPC domain is necessary for the protein-protein interaction of the AHL proteins. The AHL protein family in plants can be divided into two distinct clades (Clade A and Clade B) based on the type of AT-hook motif and the PPC domain (Zhao et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe conserved characteristics of the AT-hook motif and PPC domain within \u003cem\u003eAHL\u003c/em\u003e genes offer valuable insights into studying their gene function. However, the presence of numerous members of \u003cem\u003eAHLs\u003c/em\u003e in plants presents a challenge for elucidating their precise functions through traditional gene knockout studies. In conventional gene knockout experiments, the deletion of a single \u003cem\u003eAHL\u003c/em\u003e gene may not lead to significant observable phenotypic changes due to genetic redundancy present among the other family members (Street et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). For instance, single-gene knockout mutants of \u003cem\u003eAHL22 (ahl22-1), AHL29 (sob3-4)\u003c/em\u003e, and \u003cem\u003eAHL27 (esc-8)\u003c/em\u003e exhibited no detectable phenotypic differences compared to the wild type (Street et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). However, when higher-order combinations of \u003cem\u003eAHL\u003c/em\u003e genes were knocked out, such as in the quadruple mutant \u003cem\u003esob3-4 esc-8 ahl6 ahl22\u003c/em\u003e, the resulting plants exhibited significantly more pronounced phenotypes compared to those with lower-order gene knockouts (Zhao et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). To address this limitation, the employment of dominant negative mutations has emerged as a valuable strategy, enabling researchers to overcome genetic redundancy and better unravel the functions of \u003cem\u003eAHL\u003c/em\u003e genes in plants (Street et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Tayengwa et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDominant negative mutations are defined as mutation whose gene product adversely affect the wild-type gene product and/or interacting partners in the cell (Sheppard, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). In AHL proteins, these mutations are typically engineered to interfere with key functional domains, such as the AT-hook motif or the PPC domain. By selectively disrupting AHL-mediated transcriptional regulation through dominant negative mutations, our lab has uncovered the specific genes and pathways under AHL control, shedding light on their regulatory networks and molecular mechanisms First reported in \u003cem\u003eAHL29\u003c/em\u003e study, the gene containing a missense mutation in the R-G-R core of the AT-hook motif, resulted in suppression of gain-of-function phenotype (Street et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Similarly, the dominant-negative mutation in \u003cem\u003eAHL29\u003c/em\u003e also showed more pronounced hypocotyl length compared to the quadruple mutant combination of \u003cem\u003esob3-4\u003c/em\u003e, \u003cem\u003eesc-8\u003c/em\u003e, \u003cem\u003eahl6\u003c/em\u003e, and \u003cem\u003eahl22\u003c/em\u003e (Zhao et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Dominant-negative mutation in the \u003cem\u003eAHLs\u003c/em\u003e work by rendering these proteins incapable of executing their regulatory functions properly, leading to aberrant gene expression patterns and phenotypic abnormalities. (Jacques et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Street et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Tayengwa et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). By combining dominant negative mutation studies with other genetic and molecular techniques, such as gene expression profiling, enables a comprehensive understanding of \u003cem\u003eAHL\u003c/em\u003e gene function and their contributions to plant physiology.\u003c/p\u003e \u003cp\u003eOur study used gain-of-function, and dominant-negative analysis to overcome the potential redundancy associated with the Clade-A gene \u003cem\u003eAHL26\u003c/em\u003e (\u003cem\u003eAT4g12050\u003c/em\u003e) in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Transgenic plants overexpressing \u003cem\u003eAHL26\u003c/em\u003e produced shorter hypocotyls in a light-dependent manner with a delayed flowering phenotype under long-day, short-day, and continuous light conditions. In contrast, the dominant-negative mutation in \u003cem\u003eAHL26\u003c/em\u003e, which alters the second arginine in the conserved R-G-R core motif, disrupted its regulatory function, resulting in longer hypocotyls and an earlier flowering phenotype, highlighting the importance of the second arginine in the conserved R-G-R core motif for \u003cem\u003eAHL26\u003c/em\u003e function. Transcriptome analysis in transgenic plants over-expressing \u003cem\u003eAHL26\u003c/em\u003e further shows that the delayed flowering results from reduced cell division-related biological processes. Overall, the findings demonstrate that the \u003cem\u003eAHL26\u003c/em\u003e, like other members of \u003cem\u003eAHL\u003c/em\u003e family, regulates hypocotyl development and flowering time in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. By identifying additional members with similar regulatory roles, our study adds to the growing evidence that \u003cem\u003eAHL\u003c/em\u003e gene family members exhibit highly conserved functions, highlighting their importance in plant developmental processes.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGrowth media conditions\u003c/h2\u003e \u003cp\u003eDifferent growth media were used for phenotypic analysis and to select transformants, as described by (Jacques et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Briefly, selection media contained 0.5\u0026times; Linsmaier and Skoog (LS) modified basal medium, 1.5% (w/v) sucrose, and 0.8% (w/v) Phytoblend (Caisson, Smithfield, UT) with appropriate antibiotics. Gellen gum (PhytoTechnology Laboratories, Inc.) was used as a solidifying agent in non-selection media along with 0.5\u0026times; Linsmaier and Skoog (LS) modified basal medium and 1.5% (w/v) sucrose to ensure optimum plant growth.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSelection of transformants\u003c/h3\u003e\n\u003cp\u003eAll \u003cem\u003eArabidopsis thaliana\u003c/em\u003e plants used in this study are in Columbia (Col-0) background and are referred to as the wildtype (WT) in the manuscript. Transgenics were obtained through the floral-dip method (Clough and Bent, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). The putative T1 transgenic seeds were grown on selection media containing appropriate antibiotics. Transformants in the T2 generation were screened on selection media to identify transgenic lines containing single locus insertion based on chi-square analysis for a predicted 3\u003csup\u003er\u003c/sup\u003e:1\u003csup\u003es\u003c/sup\u003e segregation ratio. Multiple transgenic lines with single-locus insertions were grown further to select homozygous T3 lines.\u003c/p\u003e \u003cp\u003eAll the transformants were grown simultaneously to collect seeds to ensure uniform germination and seedling growth for phenotypic analysis. Seedlings germinated on non-selection media were used to record data.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAHL26\u003c/b\u003e \u003cb\u003eoverexpression\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA Gateway\u0026reg; Entry vector (pENTR223) containing the \u003cem\u003eAHL26\u003c/em\u003e (G23448) was obtained from the Arabidopsis Biological Resource Center (ABRC). The construct from the entry vector was LR cloned into the Gateway-compatible binary vector pED15 to over-express the \u003cem\u003eAHL26 (AHL26OX)\u003c/em\u003e via the Cauliflower mosaic virus (CaMV) 35S constitutive promoter. WT plants were transformed with the resulting binary vectors carrying the \u003cem\u003eAHL26OX\u003c/em\u003e (pAK8). Three homozygous T3 lines were used for \u003cem\u003eAHL26OX\u003c/em\u003e transformants to carry out the phenotypic analysis.\u003c/p\u003e\n\u003ch3\u003eGeneration of dominant-negative mutants\u003c/h3\u003e\n\u003cp\u003eThe Q5\u0026reg; Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA) was used to carry out a target substitution at the 121\u0026ndash;123 base site in \u003cem\u003eAHL26\u003c/em\u003e to substitute the wild-type codon AGA (arginine) to CAC (histidine). Non-overlapping primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were designed using the NEBaseChanger tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://nebasechanger.neb.com/\u003c/span\u003e\u003cspan address=\"https://nebasechanger.neb.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). \u003cem\u003eAHL26OX\u003c/em\u003e carrying entry clones, as in the over-expression study, were used as templates in a PCR with substitution-specific primers to carry out the desired substitution. Newly generated constructs were sequenced to confirm the required substitution. Final constructs overexpressing mutated \u003cem\u003eAHL26 (AHL26DN)\u003c/em\u003e were then cloned into Gateway-compatible pED15. Three independent transgenic lines each for AHL26DN (pAK17) were used for the phenotypic analysis.\u003c/p\u003e\n\u003ch3\u003eHypocotyl Measurement\u003c/h3\u003e\n\u003cp\u003eAfter surface sterilization and placement on growth media, seeds were incubated for three days at 4\u0026deg;C followed by 4 hours of red-light treatment at 22\u0026deg;C to ensure uniform germination. Post cold and dark treatment, plates were subjected to 20 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of white light at 22\u0026deg;C for eight days in either long day (16 hours light and 8 hours dark) or short day (8 hours light and 16 hours dark) conditions (Zhao et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Plates were wrapped with aluminum foil for dark-grown seedlings after the initial red-light treatment. After eight days, seedlings were transferred to transparencies and digitized at 800\u0026ndash;1200 dots per inch (dpi) resolution using a flatbed scanner. The hypocotyls in the digitized images were measured using the NIH ImageJ software (Schneider et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and analyzed using the ggplot package in R-studio.\u003c/p\u003e\n\u003ch3\u003eFlowering time\u003c/h3\u003e\n\u003cp\u003eFor flowering time analysis, thirty-six seeds of multiple transgenic lines were directly sown in a pre-watered soil mix (Sunshine Mix4 [Aggregate] LA4; Green Island 28 Distributors Inc., Riverhead, NY). Trays were kept in the Conviron vernalization chamber ((Winnipeg, Manitoba, Canada) in darkness at 4\u003csup\u003eo\u003c/sup\u003eC for four days to promote uniform germination. After cold treatment, trays were transferred to a growth chamber with 24-hour (FD), 16-hour (LD), and 8-hour (SD) day lengths at 21-22\u003csup\u003eo\u003c/sup\u003eC, 60\u0026ndash;70% humidity, and 200\u0026ndash;225 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of light (Percival Intellus environmental controller, equipped with 4/8 fluorescent lamps and two Halco 9013 Frost T10FR25 25W Incandescent Bulbs). Seedlings were then thinned by clipping the hypocotyls with scissors to one seedling per pot. This approach was used to prevent damage to the roots of the remaining plant. Flowering time was measured by the number of rosette leaves and the number of days from emergence until the floral stem had grown 0.5cm. Data were analyzed in the ggplot package in RStudio.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of GUS constructs\u003c/h2\u003e \u003cp\u003eA 2963 bp region comprising the \u003cem\u003eAHL26\u003c/em\u003e promoter, 5\u0026rsquo;UTR, and coding sequence without the stop codon was PCR amplified using Gateway-compatible primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The resulting PCR product was purified, and the fragment was cloned into the Gateway-compatible entry vector pDONR221 using a BP reaction (Life Technologies, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The entry vector with the fragment of interest was confirmed by sequencing. The construct from the entry vector was LR cloned into the Gateway-compatible destination vector pMDC163 (Curtis and Grossniklaus, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) via an LR reaction to generate an \u003cem\u003eAHL26::GUS\u003c/em\u003e expression binary vector. WT plants were transformed with binary vectors carrying \u003cem\u003eAHL26::GUS\u003c/em\u003e (pAK27). Three homozygous T3 transgenic lines with single locus insertions were used to carry out histochemical staining as described by (Zhang et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Transformants were subjected to continuous light and dark conditions for 8 and 12 days. The images of the histochemical-stained seedlings were recorded with Zeiss Stemi 508 microscope. A semi-quantitative PCR was performed on the cDNA of 8-day-old light and dark whole seedlings, hypocotyl plus roots, and adult flowering plant rosette leaves.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA extraction, cDNA synthesis, and quantitative real-time RT-PCR\u003c/h3\u003e\n\u003cp\u003eRNA was extracted from the resettle leaves of the WT, overexpression, and dominant-negative transgenic lines grown in continuous light conditions after 17 days for \u003cem\u003eAHL26\u003c/em\u003e gene expression. Extraction was performed using Plant RNA mini kit (Qiagen, Valencia, CA) following the manufacturer\u0026rsquo;s protocol. Extraction was treated with DNAses to degrade potential DNA contamination. Complimentary DNA (cDNA) was synthesized using iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA). Due to the increased homology of \u003cem\u003eAHL26\u003c/em\u003e with other genes from this family, alignment of the gene under study was done using an online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/Tools/msa/clustalo/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/Tools/msa/clustalo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with other genes to make the primers specific to \u003cem\u003eAHL26\u003c/em\u003e (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). cDNA with a 10-fold dilution was used as a template to run the RT-qPCR using Bio-Rad\u0026rsquo;s SSO Advanced Universal SYBR Green Super Mix (Bio-Rad, Hercules, CA) on Bio-Rad\u0026rsquo;s CFX96 Touch Real-Time PCR Detection System. Melt curve analysis was done to eliminate the non-specific amplification. Data were normalized against the Actin primer used as an internal control.\u003c/p\u003e\n\u003ch3\u003eRNA seq library preparation\u003c/h3\u003e\n\u003cp\u003eRNA was isolated from 3 biological replicates of 17-day-old rosette leaves in the WT, \u003cem\u003eAHL26OX\u003c/em\u003e, and \u003cem\u003eAHL26DN\u003c/em\u003e grown in continuous light conditions using a Plant RNA mini kit (Qiagen, Valencia, CA) following the manufacturer\u0026rsquo;s protocol. Messenger RNA was purified from the total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for the directional library or dTTP for the non-directional library. The library was checked with Qubit, real-time PCR for quantification, and a bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on Illumina platforms according to the effective library concentration and data amount. The clustering of the index-coded samples was performed according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina platform, and paired-end reads were generated.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eQuality control\u003c/h2\u003e \u003cp\u003eRaw data (raw reads) of fastq format were processed to obtain clean data (clean reads) by removing reads with adapter, reads containing ploy-N, and low-quality reads from raw data. At the same time, Q20, Q30, and GC content of the clean data were calculated. All the downstream analyses were based on clean data with high quality.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eReads mapping to the reference genome\u003c/h2\u003e \u003cp\u003eReference genome and gene model annotation files were downloaded from the genome website (ensemblplants_arabidopsis_thaliana_tair10_gca_000001735_1). The reference genome index was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. We selected Hisat2 as the mapping tool since it can generate a database of splice junctions based on the gene model annotation file and thus produce a better mapping result than other non-splice mapping tools.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of gene expression level\u003c/h2\u003e \u003cp\u003eFeatureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. The FPKM of each gene was calculated based on the length of the gene, and the reads count was mapped to \u003cem\u003eAHL26\u003c/em\u003e. FPKM, the expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most used method for estimating gene expression levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDifferential expression analysis\u003c/h2\u003e \u003cp\u003eDifferential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2R package (1.20.0). DESeq2 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using Benjamini and Hochberg's approach for controlling the false discovery rate. Genes with an adjusted P-value\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.05 found by DESeq2 were assigned as differentially expressed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGO and KEGG enrichment analysis of differentially expressed genes\u003c/h2\u003e \u003cp\u003eGene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler R package, in which gene length bias was corrected. GO terms with corrected P-value less than 0.05 were considered significantly enriched by differential expressed genes. KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism, and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.genome.jp/kegg/\u003c/span\u003e\u003cspan address=\"http://www.genome.jp/kegg/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). We used clusterProfiler R package to test the statistical enrichment of differential expression genes in KEGG pathways.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThe tissue-specific expression of AHL26\u003c/h2\u003e \u003cp\u003eThe tissue-specific expression pattern of AHL26 was analyzed under darkness and light-grown conditions by using a translational fusion with the \u003cem\u003eβ-glucuronidase (GUS)\u003c/em\u003e reporter which is under control of the endogenous \u003cem\u003eAHL26\u003c/em\u003e promoter. GUS expression in light-grown seedlings was concentrated in the apical meristem, hypocotyl, and leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Expression in dark-grown seedlings, however, showed activity only in the apical meristem and leaves with no expression in the hypocotyl and weak or no expression in the roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C). GUS activity in 21-day-old plants grown under continuous light showed strong expression in the leaves with weak activity in the roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). A semi-quantitative PCR analysis using light- and dark-grown seedlings confirmed \u003cem\u003ein-situ\u003c/em\u003e expression patterns (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAHL26\u003c/b\u003e \u003cb\u003eplays a role in light-mediated hypocotyl growth\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the role of \u003cem\u003eAHL26\u003c/em\u003e in hypocotyl growth, we generated transgenic lines in \u003cem\u003eArabidopsis\u003c/em\u003e overexpressing \u003cem\u003eAHL26\u003c/em\u003e under the control of the CaMV 35S promoter \u003cem\u003e(AHL26OX)\u003c/em\u003e. The resulting overexpression plants produced a short hypocotyl in both short- and long-day growth conditions compared to the wild type (WT) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). However, no significant effects on hypocotyl lengths were observed in seedlings grown in dark conditions (Fig. S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMutations in the core conserved region of the AT-hook motif in \u003cem\u003eAHL\u003c/em\u003e genes have been shown to confer a dominant-negative phenotype (Street et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Tayengwa et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Based on these studies, transgenic plants overexpressing a dominant-negative mutation in \u003cem\u003eAHL26 (AHL26DN)\u003c/em\u003e were generated, where a conserved second arginine (R-G-R) in the AT-hook motifs necessary for DNA binding was replaced with histidine (R-G-H). The resulting overexpression of \u003cem\u003eAHL26DN\u003c/em\u003e produced a short hypocotyl in both short- and long-day growth conditions compared to the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D). Hypocotyl length for these \u003cem\u003eAHL26DN\u003c/em\u003e overexpression plants conferred no significant differences when grown under dark conditions (Fig. S2).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAHL26\u003c/b\u003e \u003cb\u003eplays a role in flowering time\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTransgenic \u003cem\u003eArabidopsis\u003c/em\u003e overexpressing \u003cem\u003eAHL26\u003c/em\u003e conferred a late-flowering phenotype compared to the WT, and plants overexpressing the dominant-negative mutation, irrespective of the day length conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Along with delayed flowering, plants overexpressing \u003cem\u003eAHL26\u003c/em\u003e also displayed a distinct wrinkled leaf phenotype (S3). In contrast, transgenic \u003cem\u003eArabidopsis\u003c/em\u003e overexpressing \u003cem\u003eAHL26\u003c/em\u003e harboring a dominant-negative point mutation (\u003cem\u003eAHL26DN)\u003c/em\u003e displayed an early flowering phenotype compared to the WT, and over-expressing transgenics in all-day length conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAHL26\u003c/b\u003e \u003cb\u003eInfluences Flowering Pathways Through Extensive Transcriptomic Changes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the molecular basis of flowering differences in \u003cem\u003eAHL26\u003c/em\u003e transgenic lines, the expression levels of \u003cem\u003eAHL26\u003c/em\u003e were first examined using reverse transcription-quantitative PCR (RT-qPCR). The results confirmed elevated \u003cem\u003eAHL26\u003c/em\u003e transcript levels in both \u003cem\u003eAHL26OX\u003c/em\u003e and \u003cem\u003eAHL26DN\u003c/em\u003e transgenic lines (Figure S3).\u003c/p\u003e \u003cp\u003eGiven that AHL proteins function as transcriptional regulators, RNA-seq analysis was subsequently performed to explore the broader transcriptomic impact in \u003cem\u003eAHL26OX\u003c/em\u003e and \u003cem\u003eAHL26DN\u003c/em\u003e transgenic lines. DESeq analysis was carried out on the transcriptome data to obtain differentially expressed genes (DEGs) between \u003cem\u003eAHL26OX\u003c/em\u003e vs. the WT, \u003cem\u003eAHL26DN\u003c/em\u003e vs. the WT, and \u003cem\u003eAHL26OX\u003c/em\u003e vs. \u003cem\u003eAHL26DN\u003c/em\u003e. A total of 1263 genes were differentially expressed in \u003cem\u003eAHL26OX\u003c/em\u003e vs. the WT, with a higher number of downregulated genes (916) than upregulated genes (347) including flowering-related genes such as \u003cem\u003eFLOWERING LOCUS T\u003c/em\u003e (\u003cem\u003eFT), Twin Sister of FT (TSF), CIRCADIAN CLOCK\u0026ndash;ASSOCIATED1 (CCA1), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), and LATE ELONGATED HYPOCOTYL (LHY)\u003c/em\u003e, which were all downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In contrast, the number of DEGs in \u003cem\u003eAHL26DN\u003c/em\u003e vs. the WT was significantly lower where only 138 genes were differentially expressed, with 55 upregulated to 83 downregulated genes including the upregulation of \u003cem\u003eTSF\u003c/em\u003e, a key flowering promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). There were 462 DEGs present between \u003cem\u003eAHL26DN\u003c/em\u003e vs. \u003cem\u003eAHL26OX\u003c/em\u003e, with 243 upregulated to 219 downregulated genes. \u003cem\u003eFT\u003c/em\u003e and \u003cem\u003eTSF\u003c/em\u003e, which were repressed in the \u003cem\u003eAHL26OX\u003c/em\u003e transgenic lines, accumulated more in the \u003cem\u003eAHL26DN\u003c/em\u003e plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further understand the similarity between \u003cem\u003eAHL26DN\u003c/em\u003e and \u003cem\u003eAHL26OX\u003c/em\u003e plants in relation to the upregulated and downregulated genes compared to the WT, venn diagrams were generated using upregulated and downregulated genes between \u003cem\u003eAHL26OX\u003c/em\u003e vs. the WT and \u003cem\u003eAHL26DN\u003c/em\u003e vs. the WT. Of the total genes repressed by \u003cem\u003eAHL26OX\u003c/em\u003e and \u003cem\u003eAHL26DN\u003c/em\u003e in relation to the WT, 91.4% (n\u0026thinsp;=\u0026thinsp;882) were uniquely repressed by \u003cem\u003eAHL26OX\u003c/em\u003e compared to only 5.1% (n\u0026thinsp;=\u0026thinsp;49) by \u003cem\u003eAHL26DN\u003c/em\u003e. There were only 34 genes that were both repressed by \u003cem\u003eAHL26OX\u003c/em\u003e and \u003cem\u003eAHL26DN\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Similarly, only eight genes were induced together by \u003cem\u003eAHL26OX\u003c/em\u003e and \u003cem\u003eAHL26DN\u003c/em\u003e. \u003cem\u003eAHL26OX\u003c/em\u003e uniquely induced 339 genes to only 47 genes by \u003cem\u003eAHL26DN\u003c/em\u003e when compared with the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eGene Ontology-Based Functional Analysis of DEGs\u003c/h2\u003e \u003cp\u003eTo understand the biological implications of the differentially expressed genes (DEGs), an enrichment analysis for Gene Ontology (GO) terms was performed across three groups. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA shows the 30 most significant upregulated GO terms and the number of DEGs present in \u003cem\u003eAHL26OX\u003c/em\u003e compared to the WT. The DEGs in this group were enriched to the biological process involved in flavonoid biosynthetic process, cold acclimation, sequence-specific double-stranded DNA binding, and AT-rich DNA binding. The downregulated biological process and molecular function categories in \u003cem\u003eAHL26OX\u003c/em\u003e vs. the WT group included the mitotic cell cycle, cell division, cell cycle process, as well as cell cycle phase transition. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast to \u003cem\u003eAHL26OX\u003c/em\u003e, molecular function GO terms in \u003cem\u003eAHL26DN\u003c/em\u003e transgenics showed upregulation of the regulation of DNA binding transcription factor activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). These upregulated GO terms included the well-known flower-inducing gene \u003cem\u003eLHY\u003c/em\u003e. Other flowering-related biological processes upregulated in the \u003cem\u003eAHL26DN\u003c/em\u003e transgenic lines were rhythmic and positive regulation of the developmental process. These included floral inducer genes such as \u003cem\u003eFT\u003c/em\u003e, \u003cem\u003eTSF\u003c/em\u003e, \u003cem\u003eLHY\u003c/em\u003e, and \u003cem\u003eCCA1\u003c/em\u003e. Upregulation of these GO terms in \u003cem\u003eAHL26DN\u003c/em\u003e transgenic lines are likely related to the early flowering phenotype compared to \u003cem\u003eAHL26OX\u003c/em\u003e plants. The downregulated GO terms in \u003cem\u003eAHL26DN\u003c/em\u003e vs. \u003cem\u003eAHL26OX\u003c/em\u003e group included ribosome assembly, ribosome large subunit biogenesis, sequence-specific double-stranded DNA binding and AT-rich DNA binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, Table S2).\u003c/p\u003e \u003cp\u003e \u003cb\u003eRegulatory Effects of\u003c/b\u003e \u003cb\u003eAHL26\u003c/b\u003e \u003cb\u003eon Key Biological Pathways\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the next step, KEGG pathway enrichment analysis was conducted to gain insight into the biological processes and pathways associated with the differentially expressed genes (DEGs) in \u003cem\u003eAHL26\u003c/em\u003e transgenic lines. Only three pathways were significantly upregulated in \u003cem\u003eAHL26OX\u003c/em\u003e transgenic lines as overexpression of genes resulted in more downregulated than upregulated DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In contrast, multiple pathways were significantly downregulated in the \u003cem\u003eAHL26OX\u003c/em\u003e compared to the WT. The downregulated DEGs in \u003cem\u003eAHL26OX\u003c/em\u003e plants were enriched to the plant hormone signal transduction, photosynthesis-antenna proteins, and glucosinolate biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). \u003cem\u003eAHL26\u003c/em\u003e dominant negative transgenic lines exhibited upregulation of KEGG-enriched pathways associated with circadian rhythm (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), while pathways related to ribosome biogenesis were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cb\u003eThe role of\u003c/b\u003e \u003cb\u003eAHL26\u003c/b\u003e \u003cb\u003ein seedling development\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eArabidopsis\u003c/em\u003e hypocotyl, due to its simple anatomy, is an ideal phenotypic indicator for seedling development mutant studies (Boron and Vissenberg, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and the basis for identification and genetic characterization of \u003cem\u003eSUPPRESSOR OF PHYB #3 (SOB3)/AHL29\u003c/em\u003e (Street et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). \u003cem\u003eAHL26\u003c/em\u003e is an intron-less gene that belongs to the same clade as \u003cem\u003eAHL27\u003c/em\u003e and \u003cem\u003eAHL29\u003c/em\u003e (Zhao et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Our findings demonstrate that the overexpression of \u003cem\u003eAHL26\u003c/em\u003e results in a shorter hypocotyl than the WT when grown in long and short-day conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). However, no significant difference in hypocotyl lengths was present in seedlings grown in darkness, suggesting a light-dependent function for \u003cem\u003eAHL26\u003c/em\u003e (Fig. S2). This interpretation is further supported by the absence of AHL26-GUS protein expression in the hypocotyl region in dark-grown seedlings. The expression of AHL26-GUS in seedlings grown in dark conditions concentrates in apical meristems unlike light-grown seedlings, where expression can be seen in hypocotyl and leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-D, S1). We propose that the activity of AHL26 at the seedling stage in \u003cem\u003eArabidopsis\u003c/em\u003e is light-dependent and that the protein is either absent or present in low levels in the hypocotyls grown in the absence of light. Light-dependent hypocotyl activity was also observed in other clade-a \u003cem\u003eAHL\u003c/em\u003e genes (Street et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe AT-hook motif in AHL proteins is critical for their function as transcriptional regulators in \u003cem\u003eArabidopsis\u003c/em\u003e (Jacques et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A conserved arginine residue within this motif is particularly important for their gene-over-expression gain-of-function phenotype. However, due to the presence of multiple closely related members within the \u003cem\u003eAHL\u003c/em\u003e gene family, functional redundancy often masks phenotypic effects in single-gene knockout studies, making it difficult to determine the precise role of individual \u003cem\u003eAHL\u003c/em\u003e genes. To address this challenge, our lab has developed a dominant-negative mutant approach, which has been effective in overcoming both known and unknown genetic redundancy. This strategy involves the overexpression of a mutant version of the gene carrying a non-functional AT-hook motif, which interferes with the function of other AHL proteins that share similar binding properties (Street et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Tayengwa et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). To overcome the unknown genetic redundancy between \u003cem\u003eAHL26\u003c/em\u003e and other \u003cem\u003eAHL\u003c/em\u003e and non-\u003cem\u003eAHL\u003c/em\u003e genes, dominant negative mutants overexpressing \u003cem\u003eAHL26\u003c/em\u003e were generated with a non-functional AT-hook motif \u003cem\u003e(AHL26DN).\u003c/em\u003e\u003c/p\u003e \u003cp\u003ePlants overexpressing \u003cem\u003eAHL26DN\u003c/em\u003e confer seedlings with significantly longer hypocotyls than the WT, and \u003cem\u003eAHL26OX\u003c/em\u003e transgenic lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). This increased hypocotyl length in the dominant-negative mutant plants suggests that additional proteins, possibly AHLs, are involved in the seedling growth in a light-dependent manner. However, the increase in hypocotyl length is not as dramatic as observed in the over-expression of \u003cem\u003esob3-6\u003c/em\u003e (Street et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), even though both genes harbor the same mutation in the AT-hook motif. Since AHLs tend to act as a transcriptional repressor (Favero et al., 2016; Jacques et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), another reason for the less severe hypocotyl phenotype in \u003cem\u003eAHL26DN\u003c/em\u003e plants than \u003cem\u003esob3-6\u003c/em\u003e might relate to the ability of \u003cem\u003eSOB3 (AHL29)\u003c/em\u003e to repress different or additional genes than \u003cem\u003eAHL26\u003c/em\u003e. It is also possible that these phenotypic differences are due to levels of gene expression, protein accumulation/stability or experimental growth conditions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe role of\u003c/b\u003e \u003cb\u003eAHL26\u003c/b\u003e \u003cb\u003ein flowering time\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFlowering in plants results from the expression of floral meristem identity genes, which transition cells in the shoot apical meristems from vegetative to floral (Krizek and Fletcher, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The time and stage of floral formation is crucial for plants to continue their progeny and achieve maximum yield. Several \u003cem\u003eAHLs\u003c/em\u003e have been demonstrated to play a role in controlling the onset of flowering in \u003cem\u003eArabidopsis\u003c/em\u003e (Ng et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Tayengwa et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Xiao et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Yun et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Over-expression of \u003cem\u003eAHL\u003c/em\u003e family genes in \u003cem\u003eArabidopsis\u003c/em\u003e has generally been associated with delayed flowering (Tayengwa et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Xiao et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Yun et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Like other members of the \u003cem\u003eAHL\u003c/em\u003e gene family, our gain-of-function study shows that the over-expression of \u003cem\u003eAHL26\u003c/em\u003e also confers a late flowering phenotype in \u003cem\u003eArabidopsis\u003c/em\u003e in short-day, long-day, and continuous light conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). As observed in the hypocotyl length analysis, AHL26 requires an intact AT-hook motif to impact the flowering time phenotype as over-expression of dominant-negative \u003cem\u003eAHL26 (AHL26DN\u003c/em\u003e) results in an earlier flowering phenotype than the WT, and over-expression plants. Similar results have also been reported for other clade-A AHLs in \u003cem\u003eArabidopsis\u003c/em\u003e (Tayengwa et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Xiao et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTranscriptome analysis supports the role of\u003c/b\u003e \u003cb\u003eAHL26\u003c/b\u003e \u003cb\u003ein floral development\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo gain a deeper understanding of the specific effects of \u003cem\u003eAHL26\u003c/em\u003e on flowering time regulation, RNA-seq analysis was performed on \u003cem\u003eAHL26OX\u003c/em\u003e and \u003cem\u003eAHL26DN\u003c/em\u003e transgenic lines. By analyzing transcriptomes in these mutants, we aimed to uncover the impact of \u003cem\u003eAHL26\u003c/em\u003e on the expression of other flowering-related genes. Differential gene expression analysis reveals more downregulated genes in \u003cem\u003eAHL26OX\u003c/em\u003e plants than upregulated ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Since most AHLs act as transcription factors due to their DNA binding ability, the more downregulated gene in \u003cem\u003eAHL26OX\u003c/em\u003e transgenic lines suggests that \u003cem\u003eAHL26\u003c/em\u003e acts as a transcriptional repressor in controlling floral induction.\u003c/p\u003e \u003cp\u003eTo further understand the delayed flowering phenotype observed in \u003cem\u003eAHL26\u003c/em\u003e overexpression transgenic lines, the differentially expressed genes in \u003cem\u003eAHL26OX\u003c/em\u003e plants were analyzed, focusing on key regulators of floral induction in \u003cem\u003eArabidopsis\u003c/em\u003e. For example, \u003cem\u003eSOC1\u003c/em\u003e, a well-established activator of floral development (Lee and Lee, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), exhibits reduced expression in \u003cem\u003eAHL26OX\u003c/em\u003e plants, suggesting that \u003cem\u003eAHL26\u003c/em\u003e modulates flowering-related gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In continuous white light, the expression of \u003cem\u003eFT\u003c/em\u003e and \u003cem\u003eTSF\u003c/em\u003e is regulated by the circadian clock proteins LHY and CCA1 (Fujimoto et al., 2009). Our data indicate that the overexpression of \u003cem\u003eAHL26\u003c/em\u003e reduces the expression of \u003cem\u003eFT\u003c/em\u003e and \u003cem\u003eTSF\u003c/em\u003e by repressing the activity of \u003cem\u003eLHY\u003c/em\u003e and \u003cem\u003eCCA1\u003c/em\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Additionally, \u003cem\u003eFLC\u003c/em\u003e, a central repressor of flowering in the autonomous pathway, is significantly upregulated in \u003cem\u003eAHL26OX\u003c/em\u003e transgenic lines, further contributing to the observed delay in flowering. In addition to the upregulation of flowering repressors, \u003cem\u003eAHL26OX\u003c/em\u003e plants also exhibit increased transcript accumulation of several other \u003cem\u003eAHL\u003c/em\u003e genes, including \u003cem\u003eAHL19\u003c/em\u003e, \u003cem\u003eAHL2\u003c/em\u003e, \u003cem\u003eAHL1\u003c/em\u003e, \u003cem\u003eAHL15\u003c/em\u003e, and \u003cem\u003eAHL20\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eSimilar to \u003cem\u003eAHL26\u003c/em\u003e, the overexpression of \u003cem\u003eAHL20\u003c/em\u003e and \u003cem\u003eAHL15\u003c/em\u003e has also been shown to delay flowering (Karami et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tayengwa et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), reinforcing the functional redundancy within the \u003cem\u003eAHL\u003c/em\u003e gene family in regulating plant development. In contrast to \u003cem\u003eAHL26\u003c/em\u003e overexpressing plants, none of the negative regulators of flowering are among the upregulated differentially expressed genes (DEGs) in \u003cem\u003eAHL26DN\u003c/em\u003e plants, resulting in an earlier flowering phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The substitution of the second arginine in the \u003cem\u003eAHL26\u003c/em\u003e AT-hook motif with histidine likely disrupts its regulatory function, leading to the loss of the late flowering phenotype in \u003cem\u003eAHL26DN\u003c/em\u003e transgenic plants. This early flowering phenotype is associated with the upregulation of flowering-promoting genes such as \u003cem\u003eLHY\u003c/em\u003e, \u003cem\u003eCCA1\u003c/em\u003e, \u003cem\u003eFT\u003c/em\u003e, and \u003cem\u003eTSF\u003c/em\u003e, along with the downregulation of \u003cem\u003eFLC\u003c/em\u003e. These findings are consistent with previous dominant-negative studies (Lu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Tayengwa et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), further supporting the role of \u003cem\u003eAHL26\u003c/em\u003e in modulating flowering time through transcriptional regulation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGene pathway analysis supports a transcriptional role of\u003c/b\u003e \u003cb\u003eAHL26\u003c/b\u003e \u003cb\u003eon floral development\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe enrichment analysis of differentially expressed genes (DEGs) provides valuable insights into the biological pathways and functions significantly associated with \u003cem\u003eAHL26\u003c/em\u003e regulation. Comparing the active pathways between distinct groups reveals broader gene regulatory mechanisms beyond individual gene expression changes. Gene Ontology (GO) analysis categorizes genes based on biological processes, cellular components, and molecular functions, while Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations offer pathway-level insights into gene function. These analyses collectively enhance our understanding of \u003cem\u003eAHL26\u003c/em\u003e-mediated transcriptional regulation.\u003c/p\u003e \u003cp\u003eOne of the most striking phenotypes observed in \u003cem\u003eAHL26\u003c/em\u003e overexpression (\u003cem\u003eAHL26OX\u003c/em\u003e) plants is delayed flowering, which is linked to slow leaf growth and reduced meristematic activity. The repression of meristem development and the positive regulation of developmental processes likely contributed to this phenotype. Additionally, a decrease in cell division processes may hinder the transition of vegetative meristems into floral meristems, further delaying flowering in \u003cem\u003eAHL26OX\u003c/em\u003e plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The observed repression of these developmental processes aligns with the known transcriptional repressive nature of \u003cem\u003eAHL26\u003c/em\u003e. Our findings indicate that \u003cem\u003eAHL26OX\u003c/em\u003e plants exhibit increased sequence-specific double-stranded DNA binding, AT-rich DNA binding, regulatory region nucleic acid binding, and transcription regulatory region DNA binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These molecular functions suggest that \u003cem\u003eAHL26\u003c/em\u003e exerts its effects by modulating key growth repressors. Consistent with our results, previous studies have also reported the transcriptional regulatory role of AHL proteins in developmental processes (Favero et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Jacques et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo further investigate the role of \u003cem\u003eAHL26\u003c/em\u003e in flowering regulation, \u003cem\u003eAHL26DN\u003c/em\u003e plants, which carry a mutation in their AT-hook motif that disrupts DNA binding were examined. Unlike \u003cem\u003eAHL26OX\u003c/em\u003e plants, \u003cem\u003eAHL26DN\u003c/em\u003e transgenic lines did not exhibit upregulation of growth repressors, resulting in an early flowering phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). A comparative GO analysis between \u003cem\u003eAHL26OX\u003c/em\u003e and \u003cem\u003eAHL26DN\u003c/em\u003e revealed a significant repression of sequence-specific double-stranded DNA binding in \u003cem\u003eAHL26DN\u003c/em\u003e plants. Additionally, genes involved in AT-rich DNA binding were downregulated in \u003cem\u003eAHL26DN\u003c/em\u003e plants compared to \u003cem\u003eAHL26OX\u003c/em\u003e (Table S2). These findings suggest that the loss of AHL26 DNA binding activity prevents its ability to repress key flowering regulators, thereby accelerating the transition to reproductive growth.\u003c/p\u003e \u003cp\u003eInterestingly, beyond transcriptional regulation, our GO analysis also revealed a significant downregulation of biological processes related to ribosome assembly and large ribosomal subunit biogenesis in \u003cem\u003eAHL26DN\u003c/em\u003e plants. This suggests a potential role for AHL26 in regulating gene expression at the translational level, possibly influencing protein synthesis and overall cellular function. Given the observed downregulation of ribosomal biogenesis in \u003cem\u003eAHL26DN\u003c/em\u003e plants, further investigations using techniques such as Translating Ribosome Affinity Purification followed by sequencing (TRAP-seq) could provide deeper insights into the role of AHL26 in modulating translational dynamics (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eOverall, our findings highlight the critical role of AHL26 in repressing flowering by directly binding to DNA and modulating gene expression. The functional loss of DNA binding in \u003cem\u003eAHL26DN\u003c/em\u003e plants further supports the importance of AHL26 as a transcriptional regulator of flowering time. Future studies integrating transcriptomic and translational analyses will be instrumental in unraveling the full spectrum of \u003cem\u003eAHL26\u003c/em\u003e-mediated gene regulation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur findings demonstrate that \u003cem\u003eAHL26\u003c/em\u003e plays a pleiotropic role in plant development, as its overexpression leads to a shorter hypocotyl and a delayed flowering phenotype. AHL26 exerts its regulatory function through a conserved AT-hook motif, as a missense mutation in this motif abolishes these phenotypic effects. Furthermore, AHL26 functions as a transcriptional repressor, modulating the expression of key growth repressors involved in floral meristem development and other essential developmental processes. These results highlight the critical role of \u003cem\u003eAHL26\u003c/em\u003e in regulating plant growth and flowering, providing a foundation for future studies on its broader regulatory network.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publications\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA-Seq dataset used in the study is available in the NCBI Sequence Read Archive (SRA) repository, accession number PRJNA1241335 under the following link: https://www.ncbi.nlm.nih.gov/sra/PRJNA1241335\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSA and MN designed the study.\u003c/p\u003e\n\u003cp\u003eSA, AH, and XX performed the experiments.\u003c/p\u003e\n\u003cp\u003eSA and MN wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Cynthia Gleason for her support in providing access to her stereo microscope for GUS visualization. We also thank Drs. John Hadish, Ian Burke and Karen Sanguinet for their support\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBoron, A.K., Vissenberg, K., 2014. The Arabidopsis thaliana hypocotyl, a model to identify and study control mechanisms of cellular expansion. Plant Cell Rep. 33, 697\u0026ndash;706. https://doi.org/10.1007/s00299-014-1591-x\u003c/li\u003e\n \u003cli\u003eClough, S.J., Bent, A.F., 1998. Floral dip: a simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana. Plant J. 16, 735\u0026ndash;743. https://doi.org/10.1046/j.1365-313x.1998.00343.x\u003c/li\u003e\n \u003cli\u003eCurtis, M.D., Grossniklaus, U., 2003. A Gateway Cloning Vector Set for High-Throughput Functional Analysis of Genes in Planta. Plant Physiol. 133, 462\u0026ndash;469. https://doi.org/10.1104/pp.103.027979\u003c/li\u003e\n \u003cli\u003eFavero, D.S., Jacques, C.N., Iwase, A., Le, K.N., Zhao, J., Sugimoto, K., Neff, M.M., 2016. SUPPRESSOR OF PHYTOCHROME B4-#3 Represses Genes Associated with Auxin Signaling to Modulate Hypocotyl Growth. Plant Physiol. 171, 2701\u0026ndash;2716. https://doi.org/10.1104/PP.16.00405\u003c/li\u003e\n \u003cli\u003eFavero, D.S., Kawamura, A., Shibata, M., Takebayashi, A., Jung, J.-H., Suzuki, T., Jaeger, K.E., Ishida, T., Iwase, A., Wigge, P.A., Neff, M.M., Sugimoto, K., 2020. AT-Hook Transcription Factors Restrict Petiole Growth by Antagonizing PIFs. Curr. Biol. 30, 1454-1466.e6. https://doi.org/10.1016/j.cub.2020.02.017\u003c/li\u003e\n \u003cli\u003eFujimoto, S., Matsunaga, S., Yonemura, M., Uchiyama, S., Azuma, T., Fukui, K., n.d. Identification of a novel plant MAR DNA binding protein localized on chromosomal surfaces.\u003c/li\u003e\n \u003cli\u003eJacques, C.N., Favero, D.S., Kawamura, A., Suzuki, T., Sugimoto, K., Neff, M.M., 2022. SUPPRESSOR OF PHYTOCHROME B-4\u0026nbsp;#3 reduces the expression of PIF-activated genes and increases expression of growth repressors to regulate hypocotyl elongation in short days. BMC Plant Biol. 22, 399. https://doi.org/10.1186/s12870-022-03737-z\u003c/li\u003e\n \u003cli\u003eJacques, C.N., Hulbert, A.K., Westenskow, S., Neff, M.M., 2020. Production location of the gelling agent Phytagel has a significant impact on Arabidopsis thaliana seedling phenotypic analysis. PLOS ONE 15, e0228515. https://doi.org/10.1371/journal.pone.0228515\u003c/li\u003e\n \u003cli\u003eKarami, O., Rahimi, A., Khan, M., Bemer, M., Hazarika, R.R., Mak, P., Compier, M., van Noort, V., Offringa, R., 2020. A suppressor of axillary meristem maturation promotes longevity in flowering plants. Nat. Plants 6, 368\u0026ndash;376. https://doi.org/10.1038/s41477-020-0637-z\u003c/li\u003e\n \u003cli\u003eKrizek, B.A., Fletcher, J.C., 2005. Molecular mechanisms of flower development: an armchair guide. Nat. Rev. Genet. 6, 688\u0026ndash;698. https://doi.org/10.1038/nrg1675\u003c/li\u003e\n \u003cli\u003eLee, J., Lee, I., 2010. Regulation and function of SOC1, a flowering pathway integrator. J. Exp. Bot. 61, 2247\u0026ndash;2254. https://doi.org/10.1093/jxb/erq098\u003c/li\u003e\n \u003cli\u003eLife Technologies, 2017.\u0026nbsp;GATEWAY\u003csup\u003eTM\u003c/sup\u003e Cloning Technology.\u003c/li\u003e\n \u003cli\u003eLu, H., Zou, Y., Feng, N., 2010. Overexpression of AHL20 negatively regulates defenses in arabidopsis. J. Integr. Plant Biol. 52, 801\u0026ndash;808. https://doi.org/10.1111/j.1744-7909.2010.00969.x\u003c/li\u003e\n \u003cli\u003eNg, K.-H., Yu, H., Ito, T., 2009. AGAMOUS Controls GIANT KILLER, a Multifunctional Chromatin Modifier in Reproductive Organ Patterning and Differentiation. PLoS Biol. 7, e1000251\u0026ndash;e1000251. https://doi.org/10.1371/journal.pbio.1000251\u003c/li\u003e\n \u003cli\u003eSchneider, C.A., Rasband, W.S., Eliceiri, K.W., 2012.\u0026nbsp;NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671\u0026ndash;675. https://doi.org/10.1038/nmeth.2089\u003c/li\u003e\n \u003cli\u003eSheppard, D., 1994. Dominant negative mutants: tools for the study of protein function in vitro and in vivo. Am. J. Respir. Cell Mol. Biol. 11, 1\u0026ndash;6. https://doi.org/10.1051/acarologia/20132076\u003c/li\u003e\n \u003cli\u003eStreet, I.H., Shah, P.K., Smith, A.M., Avery, N., Neff, M.M., 2008. The AT-hook-containing proteins SOB3/AHL29 and ESC/AHL27 are negative modulators of hypocotyl growth in Arabidopsis. Plant J. 54, 1\u0026ndash;14. https://doi.org/10.1111/j.1365-313X.2007.03393.x\u003c/li\u003e\n \u003cli\u003eTayengwa, R., Sharma-Koirala, P., Pierce, C.F., Werner, B.E., Neff, M.M., 2020. Overexpression of AtAHL20 causes delayed flowering in Arabidopsis via repression of FT expression (preprint). In Review. https://doi.org/10.21203/rs.3.rs-38575/v1\u003c/li\u003e\n \u003cli\u003eXiao, C., Chen, F., Yu, X., Lin, C., Fu, Y.-F., 2009. Over-expression of an AT-hook gene, AHL22, delays flowering and inhibits the elongation of the hypocotyl in Arabidopsis thaliana. Plant Mol. Biol. 71, 39\u0026ndash;50. https://doi.org/10.1007/s11103-009-9507-9\u003c/li\u003e\n \u003cli\u003eYun, J., Kim, Y.-S., Jung, J.-H., Seo, J., Park, C.-M., 2012. The AT-hook Motif-containing Protein AHL22 Regulates Flowering Initiation by Modifying FLOWERING LOCUS T Chromatin in Arabidopsis * □ S. https://doi.org/10.1074/jbc.M111.318477\u003c/li\u003e\n \u003cli\u003eZhang, J., Vankova, R., Malbeck, J., Dobrev, P.I., Xu, Y., Chong, K., Neff, M.M., 2009. AtSOFL1 and AtSOFL2 Act Redundantly as Positive Modulators of the Endogenous Content of Specific Cytokinins in Arabidopsis. PLOS ONE 4, e8236. https://doi.org/10.1371/journal.pone.0008236\u003c/li\u003e\n \u003cli\u003eZhao, J., Favero, D.S., Peng, H., Neff, M.M., 2013. Arabidopsis thaliana AHL family modulates hypocotyl growth redundantly by interacting with each other via the PPC/DUF296 domain. Proc. Natl. Acad. Sci. U. S. A. 110, E4688-97. https://doi.org/10.1073/pnas.1219277110\u003c/li\u003e\n \u003cli\u003eZhao, J., Favero, D.S., Qiu, J., Roalson, E.H., Neff, M.M., 2014. Insights into the evolution and diversification of the AT-hook Motif Nuclear Localized gene family in land plants. https://doi.org/10.1186/s12870-014-0266-7\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"AT-hook, AHL26, Flowering, Dominant-negative mutation, Arabidopsis thaliana, RNA-Seq","lastPublishedDoi":"10.21203/rs.3.rs-6264939/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6264939/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eAT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL)\u003c/em\u003e gene family in \u003cem\u003eArabidopsis\u003c/em\u003e contains 29 members, which evolved into two phylogenetic clades. Genes from this family play a role in several biological processes, but most of the members' functions remain unknown.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHere, we provide evidence that AHL26, a clade-a protein, negatively regulates hypocotyl growth and flowering time in \u003cem\u003eArabidopsis\u003c/em\u003e. Analysis of transgenic plants expressing an \u003cem\u003eAHL26:AHL26:GUS\u003c/em\u003e translational fusion driven by 1.9 KB of the endogenous \u003cem\u003eAHL26\u003c/em\u003e promoter displayed GUS activity in the hypocotyl and apical meristem of light-grown seedlings. The overexpression of \u003cem\u003eAHL26\u003c/em\u003e resulted in the inhibition of hypocotyl growth and delayed flowering. However, the overexpression of a dominant-negative \u003cem\u003eAHL26\u003c/em\u003e with mutation in AT-hook motif, resulted in early flowering and longer hypocotyls than the WT and over-expression transgenic lines suggesting genetic redundancy between \u003cem\u003eAHL26\u003c/em\u003e and other \u003cem\u003eAHL\u003c/em\u003e genes. Transcriptome analysis showed that the regulation of flowering time in \u003cem\u003eAHL26\u003c/em\u003e over-expression and dominant-negative mutants results from regulating flowering-related genes and pathways.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur study highlights the significant role of \u003cem\u003eAHL26\u003c/em\u003e in hypocotyl growth and flowering time regulation. We further demonstrate that \u003cem\u003eAHL26\u003c/em\u003e regulates hypocotyl length in a light-dependent manner. Through transcriptomic analysis, we also show that the delayed flowering phenotype in our \u003cem\u003eAHL26\u003c/em\u003e over-expression plants is due to the negative regulation of flowering-promoting genes such as \u003cem\u003eFT\u003c/em\u003e. Furthermore, transcriptome analysis provides insight into the biological processes and pathways through which \u003cem\u003eAHL26\u003c/em\u003e influences the control of flowering time.\u003c/p\u003e","manuscriptTitle":"AHL26, an AT-hook gene, negatively regulates hypocotyl growth and flowering time in Arabidopsis thaliana","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-22 01:33:56","doi":"10.21203/rs.3.rs-6264939/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-07T07:20:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-05T14:26:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"74454691570310496249681684883358207047","date":"2025-04-29T13:56:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-10T09:19:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"233870217067702013163117779552879445845","date":"2025-04-04T10:23:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"127529907667072290917686734853076139768","date":"2025-04-01T03:41:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-01T03:19:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-27T02:51:36+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-26T06:40:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-25T20:42:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-03-25T20:41:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"01f19323-efe1-48ca-82d0-cf5fbc095357","owner":[],"postedDate":"April 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-02T15:59:37+00:00","versionOfRecord":{"articleIdentity":"rs-6264939","link":"https://doi.org/10.1186/s12870-025-06764-8","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-05-30 15:57:09","publishedOnDateReadable":"May 30th, 2025"},"versionCreatedAt":"2025-04-22 01:33:56","video":"","vorDoi":"10.1186/s12870-025-06764-8","vorDoiUrl":"https://doi.org/10.1186/s12870-025-06764-8","workflowStages":[]},"version":"v1","identity":"rs-6264939","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6264939","identity":"rs-6264939","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}