R2R3 MYB transcription factor AtDIV1 regulates root growth through regulation of PIN5 expression in Arabidopsis

R2R3 MYB transcription factor AtDIV1 regulates root growth through regulation of PIN5 expression in Arabidopsis | 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 R2R3 MYB transcription factor AtDIV1 regulates root growth through regulation of PIN5 expression in Arabidopsis Liufeng Dai, Jiuting Xu, Chang Liu, Haoran Yin, Xiaojuan Wei, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7670153/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Key message The R2R3 MYB transcription factor AtDIV1 negatively regulates PIN5 expression to modulate primary root growth. As a crucial organ for nutrient uptake, the root plays a vital role in plant growth and development, a process regulated by multiple factors including phytohormones and transcriptional mechanisms. In this study, we identify AtDIV1, a R2R3-MYB transcription factor, as the key regulator of root development. Loss of function of AtDIV1 led to significantly shortened primary roots, accompanied by reductions in both cell number and cell length compared to the wild type. Pharmacological experiments demonstrated that exogenous IAA application partially rescued the root length defect in atdiv1 mutant, restoring cell number in both meristem and elongation zones. Notably, PIN5 expression was significantly upregulated in atdiv1 roots and the root developmental defects observed in atdiv1 mutants were fully rescued in atdiv1 pin5 double mutants. Collectively, our findings establish that AtDIV1 negatively regulates PIN5 expression to modulate primary root growth. Root development MYB PIN5 Figures Figure 1 Figure 2 Figure 3 Introduction Roots are crucial for plant growth and development as they drive nutrient and water uptake, provide structural support for the above-ground parts of plants, and engage with microbial communities in the soil (Ma et al., 2024 ). Primary roots are typically divided into meristematic, elongation and maturation zones where cells divide, elongate and differentiate, respectively (Somssich et al., 2016 ). These processes are regulated and determined by an array of factors, including different hormones and transcriptional regulators (Gouran and Brady, 2024 ; Motte et al., 2019 ; De Nittis et al., 2025 ). The hormone auxin is the key regulator of plant growth and development (Vanneste et al., 2025 ; Li et al., 2023 ). The distribution of auxin involves the coordinated interplay between long-distance and local polar transport. In brief, auxin is transported from above-ground tissues (such as the stem) to the roots via the vasculature, including the phloem parenchyma cells of the stele, primarily through non-polar diffusion or passive transport (Petrásek and Friml, 2009 ; Roychoudhry and Kepinski, 2022 ). Upon reaching the roots, auxin moves towards the root tip through polar transport in stele cells, particularly those surrounding the xylem. This process relies on the directional outward transport of auxin by PIN proteins, establishing an initial auxin gradient from the root base towards the root tip (Luschnig and Friml, 2024 ). These PINs include PIN3 and PIN7, which channel auxin towards the root apex region, including the quiescent center (QC) and root cap. From here, PIN2 drives apical transport of auxin through epidermal and outer cortical cells (Roychoudhry and Kepinski, 2022 ). In the small column cells of the root cap, PIN3, PIN4, and PIN7 distribute auxin from the lateral root cap outwards, while AUX/LAX proteins and PIN2 transport auxin from the lateral root cap to the epidermis (Hu et al. , 2021). Based on their critical functions in auxin transport, PINs play a crucial role in plant root growth and development. Indeed, the root length as well as root meristem size are reduced in pin1 and pin2 single mutants, pin3 , pin4 , and pin7 single mutants exhibit division defects in the QC and columella root cap, and the triple mutants pin1 pin2 pin7 , pin2 pin3 pin7 , pin2 pin4 pin7 and pin2pin3pin4 dramatically impair both root length and meristem size (Blilou et al., 2005 ). In contrast to these PINs that positively regulate root growth and development, overexpression of PIN5 leads to a reduced root length, while it increases in pin5 mutant (Mravec et al., 2009 ; Di Mambro et al., 2019 ). Compared with other PIN proteins, the regulatory mechanisms of PIN5 remain largely undetermined. The MYB transcription factor (TF) family is one of the largest in plants. MYBs are classified into four categories based on the number of amino acid repeats (R) in their DNA-binding domain, resulting in R1/2/3-MYB (1R-MYB), R2R3-MYB (2R-MYB), R1R2R3-MYB (3R-MYB), and 4R-MYB types (Martin and Paz-Ares, 1997 ; Li et al., 2019 ). These MYBs participate in the regulation of multiple plant developmental processes. For example, DRMY1, a 1R-MYB, regulates root tip growth and early development of vascular bundles (Kong et al., 2024 ). The R2R3-MYB is plant-specific and has more than 100 members in Arabidopsis thaliana (Li et al., 2019 ). This MYB category includes members that regulate plant growth, development, primary and secondary metabolism, cell differentiation, organelle biosynthesis and the response to biotic and abiotic stress (Zhang et al., 2025 ; Tominaga-Wada and Wada, 2014 ; Cao et al., 2020 ; Li et al., 2019 ; Frangedakis et al., 2024 ). In context of root development, AtMYB56 interacts with brassinosteroid (BR)-regulated TF BRI1‐EMS SUPPRESSOR 1 (BES1) to inhibit the expression of cell cycle genes, thereby ensuring low mitotic activity in the root meristem zone (Chen et al., 2022 ; Vilarrasa-Blasi et al., 2014 ). AtMYB59 binds to cis elements such as ERE (ATTTCAAA) to target the expression of cell cycle genes, thereby inhibiting root growth (Mu et al., 2009 ). Another R2R3-MYB, AtCDC5 promotes the development of the primary root by regulating the expression of the regulator of G2/M transition, AtCDKB1 gene (Lin et al., 2007 ; Chen et al., 2022 ). In the elongation zone, AtMYB11, AtMYB12, AtMYB30, AtMYB60 and AtMYB111 positively regulate root growth by affecting the content of auxin (IAA), through the influence of gibberellin (GA) signals (Huang et al., 2016 ; Sakaoka et al., 2018 ; Oh et al., 2011 ). In addition, AtMYB33, AtMYB65 and AtMYB101 are involved in the development of the primary root, where MYB65 accelerates the cell cycle to promote cell division in the root meristem (Xue et al., 2017 ). In this study, we identified the R2R3 MYB transcription factor AtDIV1 as a positive regulator of primary root growth. Gene expression, pharmacological and genetic analyses indicated that AtDIV1 regulates PIN5 expression to control primary root development. Results Seedlings with impaired AtDIV1 function display reduced root length To decipher molecular mechanisms of root growth and development, we screened a homozygous Arabidopsis T-DNA collection from Nottingham Arabidopsis Stock Centre (NASC) ( http://arabidopsis.info/ ) to identify potential functional genes involved in root development (Alonso et al., 2003 ). The root length of 7-days-old seedlings grown on 1/2 Murashige and Skoog (MS) medium were analyzed. One T-DNA insertion line, SALK_084867 with significantly shorter primary root length compared to WT were identified for further analysis. As the T-DNA insertion site located in the intron region of At5g58900 (Fig. 1 A), which was annotated as an R2R3-type MYB transcription factor AtDIV1, we named the T-DNA mutant atdiv1 . To further examine the root growth defect in atdiv1 , the primary root length was measured day by day from the 2nd day to the 9th day. The root growth became much slower in atdiv1 mutant starting from the 3rd day after germination (Fig. 1 A-B). To test whether the shorter root defect was caused by loss of function of AtDIV1 , we generated a fluorescently tagged AtDIV1 construct under its native promoter, pATDIV1::ATDIV1-GFP , and transformed it into atdiv1 mutant plants. Several transgenic plants were obtained, and the root growth defect was rescued in these complementation lines (Fig. 1 C-D). These data supported that AtDIV1 was the causal gene responsible for the short primary root phenotype observed in the atdiv1 mutants. The fluorescence of GFP was too weak to observe its localization, so we generated another construct, pATDIV1::ATDIV1-3×mNeonGreen , and transformed it into atdiv1 mutant plants. The expression of DIV1 and the root growth defect were rescued in these complementation lines (S1A, Fig. 1 C-D). Consistent with the notion that AtDIV1 is a transcription factor, we observed the mNeonGreen fluorescent signals in nucleus in the complemented lines (Fig. 1 E). AtDIV1 was highly expressed in roots, leaves and seedlings as analyzed via quantitative real-time PCR (S1B). However, no evident phenotype of above-ground tissues was observed in atdiv1 mutants compared to WT (S1C). We next investigated the major cause for the short primary roots in atdiv1 mutants. The lengths of the meristem and elongation zones were both significantly shorter in atdiv1 mutants than WT (Fig. 1 F-H). Detailed analysis of cell number and length revealed that the shorter meristem zone was primarily due to decreased cell number, as the meristem cell length was increased but the number of meristem cells was drastically decreased in the mutants, compared to WT (Fig. 1 I-J). Both the cell number and length in the elongation zone were significantly reduced in atdiv1 than WT (Fig. 1 K-L). Exogenous IAA treatment could partially rescue the root length defect in atdiv1 mutant Root growth is regulated by hormone auxin. To determine if the root growth defect in atdiv1 mutants was due to abnormal auxin homeostasis, different concentrations of exogenous IAA were applied to atdiv1 mutants. While 1 nM IAA had no significant effect on roots, application of 10 nM IAA significantly inhibited the primary root length in WT and the complementation lines, which was consistent with previous reports (Müssig et al., 2003 ). In contrast, 10 nM IAA significantly increased the root length in atdiv1 mutants (Fig. 2 A-B, S2 A). In accordance with these observations, the cell number of both meristem and elongation zones, as well as the cell length in meristem zone, were all partially rescued in atdiv1 mutants in the presence of IAA (Fig. 2 D-F). The cell length in the atdiv1 elongation zone was not affected by IAA treatment (Fig. 2 G). Similar experiments were performed with exogenous addition of synthetic IAA analogue 1-Naphthyl acetic acid, NAA, which entered cells via efflux carrier and free diffusion. Similar results were obtained compared with IAA treatment (Figure S2B-D). These results support that auxin can restore cell division and growth in atdiv1 mutants. PIN5 expression level was significantly increased in atdiv1 mutant The maintenance of auxin homeostasis is coordinately regulated by its metabolism, and polar transport mechanisms. Here, the PIN protein family, acting as an auxin efflux carrier, plays important functions, underpinning plant root growth and development. Previous studies have indicated that overexpression of PIN5 exhibited reduced primary root length, whereas the pin5 mutant displayed enhanced root growth (Di Mambro et al., 2019 ).,While mutants of other PINs result in reduced root length (Blilou et al., 2005 ; Mravec et al., 2009 ; Di Mambro et al., 2019 ). Elevated expression of PIN5 in atdiv1 was validated through qRT-PCR assay (Fig. 3 A). This result indicates that AtDIV1 might regulate the expression of PIN5 . Given the short root phenotype in atdiv1 mutants, we hypothesized that AtDIV1 regulated the growth of the primary root by inhibiting the transcription of PIN5 . To test this, we generated atdiv1 pin5 double mutants and found that the cell number and length in the meristem and elongation zones were significantly recovered compared to the atdiv1 single mutants (Fig. 3 D-H). Exogenous IAA (10 nM) significantly inhibited the primary root length of pin5 mutant and atdiv1 pin5 double mutants like WT (Fig. 3 B-C). Accordingly, the cell number of meristem zones were partially reduced in pin5 mutant and atdiv1 pin5 double mutants in the presence of IAA (Fig. 3 D-E). In contrast, the cell length of meristem zones in pin5 and atdiv1 pin5 mutant and the cell number of elongation zones were not affected by IAA treatment (Fig. 3 F-G). These data support that AtDIV1 governs root growth via transcriptional regulation of PIN5 . Discussion The polar distribution of auxin is crucial for its function. The proper expression and function of PIN transporters are essential for the regulation of auxin. Compared to other PIN proteins, PIN5 is an auxin transporter with specific ER localization, and its expression and functional mechanisms across various plant developmental stages, remains to be further elucidated. In this study, based on transcriptional, genetic and pharmacological analyses, we propose that AtDIV1 regulates the root growth through transcriptional repression of PIN5 . Auxin establishes and maintains its concentration gradient throughout the PIN protein-mediated polar transport. This gradient is crucial for the root growth and development. The majority of PINs localize to the plasma membrane, where they facilitate intercellular auxin transport. However, PIN5 predominantly resides in the endoplasmic reticulum (ER) and plays a key role in regulating intracellular auxin partitioning between the cytoplasm and the ER (Roychoudhry and Kepinski, 2022 ). PIN5 modulates free IAA levels by sequestering auxin in the ER, thereby promoting IAA conjugation into irreversible aspartate/glutamate derivatives (Ganguly et al., 2010 ), which is an important auxin inactivation process in plants. PIN5 overexpression in BY-2 cells led to an increase in IAA-Asp and IAA-Glu (non-reversible conjugates) and a decrease in free IAA levels (Ganguly et al., 2010 ). When PIN5 was expressed in yeast, the content of radio-labeled IAA and NAA decreased. Given that the mutation of AtDIV1 results in upregulation of PIN5 transcription, although total IAA levels remain largely unchanged in the atdiv1 mutant (unpubnished data), we propose that the subcellular IAA distribution between cytoplasm and ER, or the balance between conjugated and free auxin is altered in the atdiv1 mutants. Exogenous IAA supplementation to restore the shorter root defect in atdiv1 mutant might be via the regulation of the homeostatic balance between active and inactive IAA content. These unresolved aspects warrant further investigations. The regulation of PIN5 remains poorly understood compared to other PIN genes. In context of transcriptional PIN5 regulation, cytokinin regulates PIN5 through the ARR1 TF (Di Mambro et al., 2019 ) and CsMYB77 activates PIN5 expression, which leads to a decline in free IAA levels, and thus impaired auxin signaling, in the fruits of transgenic Hongkong kumquat lines (Zhang et al., 2024 ). There are also evidence that the function and cellular distribution of PIN5 is regulated via changes in some conserved acidic residues in AtNHX5 and AtNHX6, which are endosomal Na + , K + /H + antiporters (Fan et al., 2018 ). Our discovery that AtDIV1 acts as a negative regulator of PIN5 expression provides new insights into the regulatory network of PIN5 and thus auxin cellular distribution in plant cells. MATERIALS AND METHODS Plant materials and growth conditions The T-DNA insertion mutants atdiv1 (SALK_084867), pin5-3 (SALK_021738), were obtained from the ABRC. atdiv1 pin5 was obtained by crossing pin5 with atdiv1 . Arabidopsis seedlings were grown on vertical plates containing half-strength Murashige and Skoog (1/2 MS) media supplemented with 1% sucrose and 0.5% Gelzan (Sigma-Aldrich, G1910). The plants grow for different days at 22°C in 16h light/8h dark cycle. 7-day-old seedlings of Col-0 and atdiv1 were transferred to mixed soil and cultured in greenhouse for 8 weeks to record the phenotype of above-ground tissues. For IAA/NAA treatment, the surface sterilized seeds were germinated and grown on media supplemented with 1, 10 or 20 nM IAA (Sigma-Aldrich, I2886), 10, 30 or 50 nM NAA (Sigma-Aldrich, N0640) for 7 days. IAA and NAA was dissolved in 95% ethanol and diluted with ddH 2 O, the same dilution ratio of ethanol was added as control. Plasmid constructs and plant transformation To generate the ATDIV1-GFP fusion constructs, the nucleotide sequences containing the native promoter (2223 bp region before ATG) and genomic region were amplified with primer pATDIV1-F/ATDIV1-R. The sequence was then inserted into pCAMBIA1305-GFP vector between restriction sites of KpnI and BamHI via infusion cloning (Vazyme) to get pATDIV1:ATDIV1-GFP vector. Genomic sequences of ATDIV1 driven by the native promoter (2198 bp region before ATG) was inserted into pCAMBIA1300-3×mNeonGreen vector between restriction sites of SalI and XbaI via infusion cloning (Vazyme) to get pATDIV1:ATDIV1-3×mNeonGreen vector. All primers used for cloning are listed in Extended Data Table 1. The construct was sequence-verified and transformed to atdiv1 mutants via Agrobacterium tumefaciens strain GV3101. Transgenic plants were selected by antibiotic resistance and fluorescence intensity. RNA extraction and qPCR analysis Arabidopsis seedlings were grown on vertical plates containing half-strength Murashige and Skoog (1/2 MS) media for 7 days. Seedling roots were collected and weighed, then ground quickly into a powder in liquid nitrogen and transfer to a 1.5 mL sterile centrifuge tube. The RNAprep Pure Plant Kit (TIANGEN) was used for total RNA extraction from root. The cDNA was reversed translated from the total RNA by One-Step gDNA Removal and cDNA Synthesis Supermix (TRANSGEN). SYBR Green mix (TRANSGEN) was used for qPCR to analysis the expression levels of ATDIV1 and PIN5 using the ABI Quant Studio6 Real-Time PCR Systems. qPCR was run at 95°C for 10 s (denaturation), 60°C for 30 s (annealing and extension) for 40 cycles. The experiment was repeated at least three times with 4 technical replicates for each experiment. PI staining For PI staining, 7-days Arabidopsis seedlings were stained in 10 µg/mL PI for 1 minutes. Samples were observed under the confocal microscope (Zeiss LSM880) equipped with 20 X 0.8NA objective. The 561-nm laser were used for imaging. Image process was performed using the dark sectioning plugin in ImageJ (Cao et al., 2025 ). Fluorescent imaging and image analysis The subcellular localization of mNeonGreen-tagged proteins was obtained with a Zeiss LSM880 with 63 X 1.4NA oil immersion objective. Fluorescence intensity were measured using ImageJ software. Declarations Data availability Biological materials can be obtained upon request. Extended data and source data are provided with this paper. Acknowledgements We acknowledge the experimental technology center for life sciences, Beijing Normal University, and are grateful to Dr. Xiaoyan Zhang for technical support. This work was supported by grants from the National Natural Science Foundation of China (32100279 to T. W., 32400563 to L.D. and 32270350 and 32070194 to Y. Z.), the Fundamental Research Funds for the Central Universities (2243200007 to Y.Z.) and open funds of the State Key Laboratory of Plant Environmental Resilience (SKLPERKF2401). Author contributions T.W. and S.P. initiated the project. T.W., L.D. and J.X. designed the experiments; L.D. and J.X. performed the experiments; X.W. and H.Y. generated the atdiv1 and atdiv1 pin5 mutants; L.D. and J.X. analyzed the data; T.W., S.P., Y.Z., L.D. J.X. and C.L. wrote, reviewed and edited the manuscript with input from all of the authors. Competing interests The authors declare no competing interests. References Alonso, J.M., Stepanova, A.N., Leisse, T.J., et al. (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science (80-. ). , 301 , 653–7. Blilou, I., Xu, J., Wildwater, M., et al. (2005) The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots. Nature , 433 , 39–44. Cao, R., Li, Yaning, Zhou, Y., et al. (2025) Dark-based optical sectioning assists background removal in fluorescence microscopy. Nat. Methods , 22 , 1299–1310. Cao, Y., Li, K., Li, Y., Zhao, X. and Wang, L. (2020) MYB Transcription Factors as Regulators of Secondary Metabolism in Plants. Biology (Basel). , 9 . Chen, Z., Wu, Z., Dong, W., Liu, S., Tian, L., Li, J. and Du, H. (2022) MYB transcription factors becoming mainstream in plant roots. Int. J. Mol. Sci. , 23 . Fan, L., Zhao, L., Hu, W., et al. (2018) Na(+) ,K(+) /H(+) antiporters regulate the pH of endoplasmic reticulum and auxin-mediated development. Plant. Cell Environ. , 41 , 850–864. Frangedakis, E., Yelina, N.E., Billakurthi, K., Hua, L., Schreier, T., Dickinson, P.J., Tomaselli, M., Haseloff, J. and Hibberd, J.M. (2024) MYB-related transcription factors control chloroplast biogenesis. Cell , 187 , 4859-4876.e22. Ganguly, A., Lee, S.H., Cho, M., Lee, O.R., Yoo, H. and Cho, H.-T. (2010) Differential auxin-transporting activities of PIN-FORMED proteins in Arabidopsis root hair cells. Plant Physiol. , 153 , 1046–1061. Gouran, M. and Brady, S.M. (2024) The transcriptional integration of environmental cues with root cell type development. Plant Physiol. , 196 , 2150–2161. Hu, Yangjie, Omary, M., Hu, Yun, et al. (2021) Cell kinetics of auxin transport and activity in Arabidopsis root growth and skewing. Nat. Commun. , 12 , 1657. Huang, W., Khaldun, A.B.M., Chen, J., Zhang, C., Lv, H., Yuan, L. and Wang, Y. (2016) A R2R3-MYB transcription factor regulates the flavonol biosynthetic pathway in a traditional chinese medicinal plant, epimedium sagittatum. Front. Plant Sci. , 7 , 1089. Kong, S., Zhu, M., Scarpin, M.R., et al. (2024) DRMY1 promotes robust morphogenesis in Arabidopsis by sustaining the translation of cytokinin-signaling inhibitor proteins. Dev. Cell , 59 , 3141-3160.e7. Li, J., Han, G., Sun, C. and Sui, N. (2019) Research advances of MYB transcription factors in plant stress resistance and breeding. Plant Signal. Behav. , 14 , 1613131. Li, Y., Han, S. and Qi, Y. (2023) Advances in structure and function of auxin response factor in plants. J. Integr. Plant Biol. , 65 , 617–632. Lin, Z., Yin, K., Zhu, D., Chen, Z., Gu, H. and Qu, L.-J. (2007) AtCDC5 regulates the G2 to M transition of the cell cycle and is critical for the function of Arabidopsis shoot apical meristem. Cell Res. , 17 , 815–828. Luschnig, C. and Friml, J. (2024) Over 25 years of decrypting PIN-mediated plant development. Nat. Commun. , 15 , 9904. Ma, Y., Zhang, Y., Xu, J., Zhao, D., Guo, L., Liu, X. and Zhang, H. (2024) Recent advances in response to environmental signals during Arabidopsis root development. Plant Physiol. Biochem. , 215 , 109037. Mambro, R. Di, Svolacchia, N., Ioio, R. Dello, et al. (2019) The Lateral Root Cap Acts as an Auxin Sink that Controls Meristem Size. Curr. Biol. , 29 , 1199-1205.e4. Martin, C. and Paz-Ares, J. (1997) MYB transcription factors in plants. Trends Genet. , 13 , 67–73. Motte, H., Vanneste, S. and Beeckman, T. (2019) Molecular and Environmental Regulation of Root Development. Annu. Rev. Plant Biol. , 70 , 465–488. Mravec, J., Skůpa, P., Bailly, A., et al. (2009) Subcellular homeostasis of phytohormone auxin is mediated by the ER-localized PIN5 transporter. Nature , 459 , 1136–1140. Mu, R.-L., Cao, Y.-R., Liu, Y.-F., et al. (2009) An R2R3-type transcription factor gene AtMYB59 regulates root growth and cell cycle progression in Arabidopsis. Cell Res. , 19 , 1291–1304. Müssig, C., Shin, G.-H. and Altmann, T. (2003) Brassinosteroids promote root growth in Arabidopsis. Plant Physiol. , 133 , 1261–1271. Nittis, M. De, Vivo, M. De, Ioio, R. Dello and Sabatini, S. (2025) Hormonal regulation of primary root development. Cell Rep. , 44 , 115751. Oh, J.E., Kwon, Y., Kim, J.H., Noh, H., Hong, S.-W. and Lee, H. (2011) A dual role for MYB60 in stomatal regulation and root growth of Arabidopsis thaliana under drought stress. Plant Mol. Biol. , 77 , 91–103. Petrásek, J. and Friml, J. (2009) Auxin transport routes in plant development. Development , 136 , 2675–2688. Roychoudhry, S. and Kepinski, S. (2022) Auxin in root development. Cold Spring Harb. Perspect. Biol. , 14 . Sakaoka, S., Mabuchi, K., Morikami, A. and Tsukagoshi, H. (2018) MYB30 regulates root cell elongation under abscisic acid signaling. Commun. Integr. Biol. , 11 , e1526604. Somssich, M., Khan, G.A. and Persson, S. (2016) Cell Wall Heterogeneity in Root Development of Arabidopsis. Front. Plant Sci. , 7 , 1242. Tominaga-Wada, R. and Wada, T. (2014) Regulation of root hair cell differentiation by R3 MYB transcription factors in tomato and Arabidopsis. Front. Plant Sci. , 5 , 91. Vanneste, S., Pei, Y. and Friml, J. (2025) Mechanisms of auxin action in plant growth and development. Nat. Rev. Mol. Cell Biol. , 26 , 648–666. Vilarrasa-Blasi, J., González-García, M.-P., Frigola, D., et al. (2014) Regulation of plant stem cell quiescence by a brassinosteroid signaling module. Dev. Cell , 30 , 36–47. Xue, T., Liu, Z., Dai, X. and Xiang, F. (2017) Primary root growth in Arabidopsis thaliana is inhibited by the miR159 mediated repression of MYB33, MYB65 and MYB101. Plant Sci. , 262 , 182–189. Zhang, D., Zhou, H., Zhang, Yang, Zhao, Y., Zhang, Yiyi, Feng, X. and Lin, H. (2025) Diverse roles of MYB transcription factors in plants. J. Integr. Plant Biol. , 67 , 539–562. Zhang, L., Xu, Y., Li, Y., Zheng, S., Zhao, Z., Chen, M., Yang, H., Yi, H. and Wu, J. (2024) Transcription factor CsMYB77 negatively regulates fruit ripening and fruit size in citrus. Plant Physiol. , 194 , 867–883. Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7670153","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":528292515,"identity":"b876b619-993e-4e95-afc2-b90c3e941df8","order_by":0,"name":"Liufeng Dai","email":"","orcid":"","institution":"Beijing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Liufeng","middleName":"","lastName":"Dai","suffix":""},{"id":528292516,"identity":"509b009e-3c2c-4d3f-b0ea-dc0dadec16d8","order_by":1,"name":"Jiuting Xu","email":"","orcid":"","institution":"Beijing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Jiuting","middleName":"","lastName":"Xu","suffix":""},{"id":528292517,"identity":"bf8f5cf0-87cc-4e30-8d3a-32f3c5b686d9","order_by":2,"name":"Chang Liu","email":"","orcid":"","institution":"Beijing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Chang","middleName":"","lastName":"Liu","suffix":""},{"id":528292518,"identity":"a3cc2f6a-1239-48c6-a143-a23e82d8bc58","order_by":3,"name":"Haoran Yin","email":"","orcid":"","institution":"Beijing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Haoran","middleName":"","lastName":"Yin","suffix":""},{"id":528292519,"identity":"5db2edde-d8ff-4cb9-a28a-cc21e6c8d01f","order_by":4,"name":"Xiaojuan Wei","email":"","orcid":"","institution":"Beijing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xiaojuan","middleName":"","lastName":"Wei","suffix":""},{"id":528292520,"identity":"1a34ea92-66f4-4bcd-b5ef-963f4ba0b3fa","order_by":5,"name":"Yi Zhang","email":"","orcid":"","institution":"Beijing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Zhang","suffix":""},{"id":528292521,"identity":"732d79ff-d55f-4785-bc78-463fb27b5b26","order_by":6,"name":"Staffan Persson","email":"","orcid":"","institution":"University of Copenhagen Department of Plant Biology and Biotechnology: Kobenhavns Universitet Institut for Plantebiologi og Bioteknologi","correspondingAuthor":false,"prefix":"","firstName":"Staffan","middleName":"","lastName":"Persson","suffix":""},{"id":528292522,"identity":"d93e86e8-b187-4e9c-85ca-0a5c4560c8d0","order_by":7,"name":"Ting Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYNCCigNgSoIoxTxg8gzJWhjbSNFiL5H87OHXeXfyDA4wH7zNw2CXR9gWiTRzY9ltz4oNDrAlW/MwJBcT1iKdYCYtue1w4oYDPGbSPAwHEhsIa0n/Ji05B6SF/xuxWnLMJD82gG1hI1LL/Tdl0gzHniXOPMxmbDnHIJmwFvae49skf9TcSew73vzwxpsKO8JaQIAZHDnMIMKAGPVAwPiDSIWjYBSMglEwQgEA+3473lOqMogAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-1741-6657","institution":"Beijing Normal University","correspondingAuthor":true,"prefix":"","firstName":"Ting","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-09-22 08:25:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7670153/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7670153/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94440231,"identity":"62afbb09-e428-4733-870f-71a796908434","added_by":"auto","created_at":"2025-10-27 14:23:44","extension":"xml","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9510,"visible":true,"origin":"","legend":"","description":"","filename":"pcrePCRED2501109.xml","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/aa86b7967f5c73883ebe9ecd.xml"},{"id":94440093,"identity":"ec905110-cfe6-4823-a4c1-0f5d6720e0fb","added_by":"auto","created_at":"2025-10-27 14:23:27","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":937,"visible":true,"origin":"","legend":"","description":"","filename":"PCRED250110919680.go.xml","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/34d40dd8f9fd11b04a541fa8.xml"},{"id":94439843,"identity":"d11eff73-09ad-402a-832c-1aec90f26c75","added_by":"auto","created_at":"2025-10-27 14:23:10","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":803,"visible":true,"origin":"","legend":"","description":"","filename":"PCRED2501109Import.xml","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/d132bce0efa6d64890f813a2.xml"},{"id":94439971,"identity":"f121935c-0cc4-4255-a927-824ff9683c04","added_by":"auto","created_at":"2025-10-27 14:23:18","extension":"xml","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":84985,"visible":true,"origin":"","legend":"","description":"","filename":"PCRED25011090enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/eeba9407695c2c55af5384d6.xml"},{"id":94440092,"identity":"a9c48196-33b5-425b-bc45-1d6115aad140","added_by":"auto","created_at":"2025-10-27 14:23:26","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":673972,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/16743f1ab86dce78e5c74fda.jpeg"},{"id":94440232,"identity":"6507e1b9-25b4-4dd1-a854-e4751dada34d","added_by":"auto","created_at":"2025-10-27 14:23:44","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":298220,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/f900e53cf4bb3a891536d4c2.jpeg"},{"id":94439294,"identity":"fce60577-afa5-4ced-8794-551c549846d9","added_by":"auto","created_at":"2025-10-27 14:22:34","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":744800,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/427e77d39bee5a7e0de15007.jpeg"},{"id":94439171,"identity":"e2dda0be-3ba8-43d7-942d-6630cc022173","added_by":"auto","created_at":"2025-10-27 14:22:25","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":220398,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/d35aad81259af6c8df6a954d.jpeg"},{"id":94439591,"identity":"ce10e680-cd80-4b67-ac66-936b21e26d80","added_by":"auto","created_at":"2025-10-27 14:22:56","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":857902,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/2a164c41d75c39da68b64a0f.jpeg"},{"id":94439778,"identity":"91f77817-6e0d-4cc8-a28d-f1c6d8ed9b5c","added_by":"auto","created_at":"2025-10-27 14:23:07","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":100228,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/8048bc6715407a5de6f3eb4f.png"},{"id":94489769,"identity":"158a38d6-ec1e-4de2-a4ad-9869d88291f5","added_by":"auto","created_at":"2025-10-27 17:05:55","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":42908,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/591f9f4ced145863abf5336b.png"},{"id":94439620,"identity":"f69c367c-97c5-40ec-8aaa-59d75039bc9e","added_by":"auto","created_at":"2025-10-27 14:22:59","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":101141,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/c56192ba11522f5bb1b6e949.png"},{"id":94440088,"identity":"8a8e101e-0116-4b0b-9125-5787e6d55c1a","added_by":"auto","created_at":"2025-10-27 14:23:26","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":29615,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/b3d50bb9019151350658b880.png"},{"id":94439621,"identity":"5a3b9dbf-6da0-4d2e-af21-b12bca005ade","added_by":"auto","created_at":"2025-10-27 14:22:59","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127512,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/5e555e3ea5600eb862a4d52f.png"},{"id":94439182,"identity":"7483333a-ffd3-40b6-a1cb-ffae2ad84211","added_by":"auto","created_at":"2025-10-27 14:22:25","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":83398,"visible":true,"origin":"","legend":"","description":"","filename":"PCRED25011090structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/b1a485f2e459b4c8801e56a4.xml"},{"id":94439974,"identity":"a627fe7d-706e-42c1-9b3f-1763499d4289","added_by":"auto","created_at":"2025-10-27 14:23:19","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":91573,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/88fe92400e5e983a1e1f60aa.html"},{"id":94439579,"identity":"ea25d02d-7bb8-4df1-be2f-093b57d36128","added_by":"auto","created_at":"2025-10-27 14:22:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":358348,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSeedlings with impaired AtDIV1 function display reduced root length\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) T-DNA insertion site in the \u003cem\u003eATDIV1\u003c/em\u003e gene. Boxes indicate coding regions; black thick line indicate intron. Black arrows indicate primers used for qRT-PCR.\u003c/p\u003e\n\u003cp\u003e(B) Root length of Col-0 and \u003cem\u003eatdiv1 \u003c/em\u003eseedlings at 2-9 days after seed germination. n ≥ 38 seedlings for each genotype.\u003c/p\u003e\n\u003cp\u003e(C) 7-day-old Col-0, \u003cem\u003eatdiv1\u003c/em\u003e and the complementation lines seedlings grown on half MS media. Scale bar, 10 mm.\u003c/p\u003e\n\u003cp\u003e(D) Root length of (C). n ≥ 40 seedlings for each genotype.\u003c/p\u003e\n\u003cp\u003e(E) Representative images of 7-day-old roots expressing ATDIV1 fused with 3×mNeonGreen driven by the native promoter in \u003cem\u003eatdiv1\u003c/em\u003e mutant background. Scale bar, 10 µm.\u003c/p\u003e\n\u003cp\u003e(F) Representative root images of 7-day-old Col-0, \u003cem\u003eatdiv1\u003c/em\u003e and the complementation lines treated with 10 μM PI for 1 min. Arrowheads indicate the junction of the meristematic zone and the elongation zone. Arrow indicates the junction of the elongation zone and the maturation zone. Scale bar, 50 µm.\u003c/p\u003e\n\u003cp\u003e(G-H) Meristem zone (G) and elongation zone (H) length of 7-day-old Col-0, \u003cem\u003eatdiv1\u003c/em\u003e and the complementation lines. n = 20 seedlings for each genotype.\u003c/p\u003e\n\u003cp\u003e(I-J) Quantification of cortex cell number (n = 10) (I) and cell length (n ≥ 150) (J) of meristem zone.\u003c/p\u003e\n\u003cp\u003e(K-L) Quantification of cortex cell number (n = 10) (K) and cell length (n ≥ 45) (L) of elongation zone.\u003c/p\u003e\n\u003cp\u003eValues are mean ± SD; *P \u0026lt; 0.05, ***P \u0026lt; 0.001, ns, not significant; two-tailed Student’s t-test in (B, D, G-L).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/8d8a29045af2005b6df08426.png"},{"id":94439262,"identity":"f1868570-bd81-4ac8-9e9b-7c2719a5ae6e","added_by":"auto","created_at":"2025-10-27 14:22:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":347078,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExogenous IAA treatment could partially rescue the root length defect in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eatdiv1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) 7-day-old seedlings of Col-0, \u003cem\u003eatdiv1\u003c/em\u003e and the complementation lines grown on half MS media (Upper Panel), on half MS media supplemented with 10 nM IAA (Lower Panel). Scale bar, 10 mm.\u003c/p\u003e\n\u003cp\u003e(B) Quantification of root length of (A). n ≥ 25 seedlings for each genotype and treatment.\u003c/p\u003e\n\u003cp\u003e(C) Representative images of PI-stainied roots of 7-day-old seedlings in (A). Arrowheads indicate the junction of the meristematic zone and the elongation zone. Arrows indicate the junction of the elongation zone and the maturation zone. Scale bar, 50 µm.\u003c/p\u003e\n\u003cp\u003e(D-E) Quantification of cortex cell number (n = 10) (D) and cell length (n ≥ 138) (E) of root meristem zone.\u003c/p\u003e\n\u003cp\u003e(F-G) Quantification of cortex cell number (n = 10) (F) and cell length (n ≥ 45) (G) of root elongation zone. Groups not sharing a common letter were statistically significantly different, P value \u0026lt; 0.05, One-way ANOVA in (B), (D), (E), (F), (G).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/8c45f643395a7ad324246c90.png"},{"id":94439625,"identity":"c3f5c660-bc15-4d88-9571-6afae1a8478c","added_by":"auto","created_at":"2025-10-27 14:22:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":410221,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePIN5 expression level was significantly increased in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eatdiv1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) qRT-PCR of \u003cem\u003ePIN5\u003c/em\u003ein 7-day-old roots of Col-0 and \u003cem\u003eatdiv1\u003c/em\u003e. ACTIN2 was used as the internal control.\u003c/p\u003e\n\u003cp\u003e(B) 7-day-old Col-0, \u003cem\u003eatdiv1, pin5\u003c/em\u003e and \u003cem\u003eatdiv1 pin5 \u003c/em\u003edouble mutant\u003cem\u003e \u003c/em\u003eseedlings grown on half MS media (Upper Panel), on half MS media supplemented with 10 nM IAA (Lower Panel). Scale bar, 10 mm.\u003c/p\u003e\n\u003cp\u003e(C) Quantification of root length of (B). n ≥ 46 seedlings for each genotype.\u003c/p\u003e\n\u003cp\u003e(D) Representative images of 7-day-old roots of Col-0, \u003cem\u003eatdiv1, pin5, atdiv1 pin5\u003c/em\u003eseedlings treated with 10 μM PI for 1 min. Arrowheads indicate the junction of the meristematic zone and the elongation zone. Arrow indicates the junction of the elongation zone and the maturation zone. Scale bar, 50 µm.\u003c/p\u003e\n\u003cp\u003e(E-F) Quantification of cortex cell number (n = 10) (E) and cell length (n ≥ 160) (F) of meristem zone.\u003c/p\u003e\n\u003cp\u003e(G-H) Quantification of cortex cell number (n = 10) (G) and cell length (n ≥ 45) (H) of elongation zone. Groups not sharing a common letter were statistically significantly different, P value \u0026lt; 0.05, One-way ANOVA in (C), (E-H).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/f0aa3a70d6d92b20b12ab8e3.png"},{"id":100546018,"identity":"83745ad8-c69f-47c1-832b-c1671bf39f67","added_by":"auto","created_at":"2026-01-19 07:33:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2346554,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/c6a61042-b475-40dd-9109-ce87106dc5c2.pdf"},{"id":94439266,"identity":"4909caa1-3f94-4975-beb9-ad8675ac151e","added_by":"auto","created_at":"2025-10-27 14:22:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":536254,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7670153/v1/da97b11460db09ee60dc47e4.docx"}],"financialInterests":"","formattedTitle":"R2R3 MYB transcription factor AtDIV1 regulates root growth through regulation of PIN5 expression in Arabidopsis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRoots are crucial for plant growth and development as they drive nutrient and water uptake, provide structural support for the above-ground parts of plants, and engage with microbial communities in the soil (Ma et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Primary roots are typically divided into meristematic, elongation and maturation zones where cells divide, elongate and differentiate, respectively (Somssich et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These processes are regulated and determined by an array of factors, including different hormones and transcriptional regulators (Gouran and Brady, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Motte et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; De Nittis et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe hormone auxin is the key regulator of plant growth and development (Vanneste et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The distribution of auxin involves the coordinated interplay between long-distance and local polar transport. In brief, auxin is transported from above-ground tissues (such as the stem) to the roots via the vasculature, including the phloem parenchyma cells of the stele, primarily through non-polar diffusion or passive transport (Petr\u0026aacute;sek and Friml, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Roychoudhry and Kepinski, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Upon reaching the roots, auxin moves towards the root tip through polar transport in stele cells, particularly those surrounding the xylem. This process relies on the directional outward transport of auxin by PIN proteins, establishing an initial auxin gradient from the root base towards the root tip (Luschnig and Friml, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These PINs include PIN3 and PIN7, which channel auxin towards the root apex region, including the quiescent center (QC) and root cap. From here, PIN2 drives apical transport of auxin through epidermal and outer cortical cells (Roychoudhry and Kepinski, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the small column cells of the root cap, PIN3, PIN4, and PIN7 distribute auxin from the lateral root cap outwards, while AUX/LAX proteins and PIN2 transport auxin from the lateral root cap to the epidermis (Hu \u003cem\u003eet al.\u003c/em\u003e, 2021). Based on their critical functions in auxin transport, PINs play a crucial role in plant root growth and development. Indeed, the root length as well as root meristem size are reduced in \u003cem\u003epin1\u003c/em\u003e and \u003cem\u003epin2\u003c/em\u003e single mutants, \u003cem\u003epin3\u003c/em\u003e, \u003cem\u003epin4\u003c/em\u003e, and \u003cem\u003epin7\u003c/em\u003e single mutants exhibit division defects in the QC and columella root cap, and the triple mutants \u003cem\u003epin1 pin2 pin7\u003c/em\u003e, \u003cem\u003epin2 pin3 pin7\u003c/em\u003e, \u003cem\u003epin2 pin4 pin7\u003c/em\u003e and \u003cem\u003epin2pin3pin4\u003c/em\u003e dramatically impair both root length and meristem size (Blilou et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In contrast to these PINs that positively regulate root growth and development, overexpression of \u003cem\u003ePIN5\u003c/em\u003e leads to a reduced root length, while it increases in \u003cem\u003epin5\u003c/em\u003e mutant (Mravec et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Di Mambro et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Compared with other PIN proteins, the regulatory mechanisms of PIN5 remain largely undetermined.\u003c/p\u003e\u003cp\u003eThe MYB transcription factor (TF) family is one of the largest in plants. MYBs are classified into four categories based on the number of amino acid repeats (R) in their DNA-binding domain, resulting in R1/2/3-MYB (1R-MYB), R2R3-MYB (2R-MYB), R1R2R3-MYB (3R-MYB), and 4R-MYB types (Martin and Paz-Ares, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These MYBs participate in the regulation of multiple plant developmental processes. For example, DRMY1, a 1R-MYB, regulates root tip growth and early development of vascular bundles (Kong et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The R2R3-MYB is plant-specific and has more than 100 members in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Li et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This MYB category includes members that regulate plant growth, development, primary and secondary metabolism, cell differentiation, organelle biosynthesis and the response to biotic and abiotic stress (Zhang et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Tominaga-Wada and Wada, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Cao et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Frangedakis et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In context of root development, AtMYB56 interacts with brassinosteroid (BR)-regulated TF BRI1‐EMS SUPPRESSOR 1 (BES1) to inhibit the expression of cell cycle genes, thereby ensuring low mitotic activity in the root meristem zone (Chen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Vilarrasa-Blasi et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). AtMYB59 binds to cis elements such as ERE (ATTTCAAA) to target the expression of cell cycle genes, thereby inhibiting root growth (Mu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Another R2R3-MYB, AtCDC5 promotes the development of the primary root by regulating the expression of the regulator of G2/M transition, \u003cem\u003eAtCDKB1\u003c/em\u003e gene (Lin et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the elongation zone, AtMYB11, AtMYB12, AtMYB30, AtMYB60 and AtMYB111 positively regulate root growth by affecting the content of auxin (IAA), through the influence of gibberellin (GA) signals (Huang et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sakaoka et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Oh et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In addition, AtMYB33, AtMYB65 and AtMYB101 are involved in the development of the primary root, where MYB65 accelerates the cell cycle to promote cell division in the root meristem (Xue et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, we identified the R2R3 MYB transcription factor AtDIV1 as a positive regulator of primary root growth. Gene expression, pharmacological and genetic analyses indicated that AtDIV1 regulates \u003cem\u003ePIN5\u003c/em\u003e expression to control primary root development.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSeedlings with impaired AtDIV1 function display reduced root length\u003c/h2\u003e\u003cp\u003eTo decipher molecular mechanisms of root growth and development, we screened a homozygous Arabidopsis T-DNA collection from Nottingham Arabidopsis Stock Centre (NASC) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://arabidopsis.info/\u003c/span\u003e\u003cspan address=\"http://arabidopsis.info/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to identify potential functional genes involved in root development (Alonso et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The root length of 7-days-old seedlings grown on 1/2 Murashige and Skoog (MS) medium were analyzed. One T-DNA insertion line, SALK_084867 with significantly shorter primary root length compared to WT were identified for further analysis. As the T-DNA insertion site located in the intron region of At5g58900 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), which was annotated as an R2R3-type MYB transcription factor AtDIV1, we named the T-DNA mutant \u003cem\u003eatdiv1\u003c/em\u003e. To further examine the root growth defect in \u003cem\u003eatdiv1\u003c/em\u003e, the primary root length was measured day by day from the 2nd day to the 9th day. The root growth became much slower in \u003cem\u003eatdiv1\u003c/em\u003e mutant starting from the 3rd day after germination (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). To test whether the shorter root defect was caused by loss of function of \u003cem\u003eAtDIV1\u003c/em\u003e, we generated a fluorescently tagged AtDIV1 construct under its native promoter, \u003cem\u003epATDIV1::ATDIV1-GFP\u003c/em\u003e, and transformed it into \u003cem\u003eatdiv1\u003c/em\u003e mutant plants. Several transgenic plants were obtained, and the root growth defect was rescued in these complementation lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). These data supported that \u003cem\u003eAtDIV1\u003c/em\u003e was the causal gene responsible for the short primary root phenotype observed in the \u003cem\u003eatdiv1\u003c/em\u003e mutants. The fluorescence of GFP was too weak to observe its localization, so we generated another construct, \u003cem\u003epATDIV1::ATDIV1-3\u0026times;mNeonGreen\u003c/em\u003e, and transformed it into \u003cem\u003eatdiv1\u003c/em\u003e mutant plants. The expression of DIV1 and the root growth defect were rescued in these complementation lines (S1A, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). Consistent with the notion that AtDIV1 is a transcription factor, we observed the mNeonGreen fluorescent signals in nucleus in the complemented lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). \u003cem\u003eAtDIV1\u003c/em\u003e was highly expressed in roots, leaves and seedlings as analyzed via quantitative real-time PCR (S1B). However, no evident phenotype of above-ground tissues was observed in \u003cem\u003eatdiv1\u003c/em\u003e mutants compared to WT (S1C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next investigated the major cause for the short primary roots in \u003cem\u003eatdiv1\u003c/em\u003e mutants. The lengths of the meristem and elongation zones were both significantly shorter in \u003cem\u003eatdiv1\u003c/em\u003e mutants than WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-H). Detailed analysis of cell number and length revealed that the shorter meristem zone was primarily due to decreased cell number, as the meristem cell length was increased but the number of meristem cells was drastically decreased in the mutants, compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-J). Both the cell number and length in the elongation zone were significantly reduced in \u003cem\u003eatdiv1\u003c/em\u003e than WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK-L).\u003c/p\u003e\u003cp\u003e\u003cb\u003eExogenous IAA treatment could partially rescue the root length defect in\u003c/b\u003e \u003cb\u003eatdiv1\u003c/b\u003e \u003cb\u003emutant\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRoot growth is regulated by hormone auxin. To determine if the root growth defect in \u003cem\u003eatdiv1\u003c/em\u003e mutants was due to abnormal auxin homeostasis, different concentrations of exogenous IAA were applied to \u003cem\u003eatdiv1\u003c/em\u003e mutants. While 1 nM IAA had no significant effect on roots, application of 10 nM IAA significantly inhibited the primary root length in WT and the complementation lines, which was consistent with previous reports (M\u0026uuml;ssig et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In contrast, 10 nM IAA significantly increased the root length in \u003cem\u003eatdiv1\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). In accordance with these observations, the cell number of both meristem and elongation zones, as well as the cell length in meristem zone, were all partially rescued in \u003cem\u003eatdiv1\u003c/em\u003e mutants in the presence of IAA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-F). The cell length in the \u003cem\u003eatdiv1\u003c/em\u003e elongation zone was not affected by IAA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Similar experiments were performed with exogenous addition of synthetic IAA analogue 1-Naphthyl acetic acid, NAA, which entered cells via efflux carrier and free diffusion. Similar results were obtained compared with IAA treatment (Figure S2B-D). These results support that auxin can restore cell division and growth in \u003cem\u003eatdiv1\u003c/em\u003e mutants.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePIN5\u003c/b\u003e \u003cb\u003eexpression level was significantly increased in\u003c/b\u003e \u003cb\u003eatdiv1\u003c/b\u003e \u003cb\u003emutant\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe maintenance of auxin homeostasis is coordinately regulated by its metabolism, and polar transport mechanisms. Here, the PIN protein family, acting as an auxin efflux carrier, plays important functions, underpinning plant root growth and development. Previous studies have indicated that overexpression of \u003cem\u003ePIN5\u003c/em\u003e exhibited reduced primary root length, whereas the \u003cem\u003epin5\u003c/em\u003e mutant displayed enhanced root growth (Di Mambro et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).,While mutants of other PINs result in reduced root length (Blilou et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Mravec et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Di Mambro et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Elevated expression of \u003cem\u003ePIN5\u003c/em\u003e in \u003cem\u003eatdiv1\u003c/em\u003e was validated through qRT-PCR assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). This result indicates that AtDIV1 might regulate the expression of \u003cem\u003ePIN5\u003c/em\u003e. Given the short root phenotype in \u003cem\u003eatdiv1\u003c/em\u003e mutants, we hypothesized that AtDIV1 regulated the growth of the primary root by inhibiting the transcription of \u003cem\u003ePIN5\u003c/em\u003e. To test this, we generated \u003cem\u003eatdiv1 pin5\u003c/em\u003e double mutants and found that the cell number and length in the meristem and elongation zones were significantly recovered compared to the \u003cem\u003eatdiv1\u003c/em\u003e single mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-H). Exogenous IAA (10 nM) significantly inhibited the primary root length of \u003cem\u003epin5\u003c/em\u003e mutant and \u003cem\u003eatdiv1 pin5\u003c/em\u003e double mutants like WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C). Accordingly, the cell number of meristem zones were partially reduced in \u003cem\u003epin5\u003c/em\u003e mutant and \u003cem\u003eatdiv1 pin5\u003c/em\u003e double mutants in the presence of IAA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E). In contrast, the cell length of meristem zones in \u003cem\u003epin5\u003c/em\u003e and \u003cem\u003eatdiv1 pin5\u003c/em\u003e mutant and the cell number of elongation zones were not affected by IAA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-G). These data support that AtDIV1 governs root growth via transcriptional regulation of \u003cem\u003ePIN5\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe polar distribution of auxin is crucial for its function. The proper expression and function of PIN transporters are essential for the regulation of auxin. Compared to other PIN proteins, PIN5 is an auxin transporter with specific ER localization, and its expression and functional mechanisms across various plant developmental stages, remains to be further elucidated. In this study, based on transcriptional, genetic and pharmacological analyses, we propose that AtDIV1 regulates the root growth through transcriptional repression of \u003cem\u003ePIN5\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eAuxin establishes and maintains its concentration gradient throughout the PIN protein-mediated polar transport. This gradient is crucial for the root growth and development. The majority of PINs localize to the plasma membrane, where they facilitate intercellular auxin transport. However, PIN5 predominantly resides in the endoplasmic reticulum (ER) and plays a key role in regulating intracellular auxin partitioning between the cytoplasm and the ER (Roychoudhry and Kepinski, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). PIN5 modulates free IAA levels by sequestering auxin in the ER, thereby promoting IAA conjugation into irreversible aspartate/glutamate derivatives (Ganguly et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), which is an important auxin inactivation process in plants. \u003cem\u003ePIN5\u003c/em\u003e overexpression in BY-2 cells led to an increase in IAA-Asp and IAA-Glu (non-reversible conjugates) and a decrease in free IAA levels (Ganguly et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). When \u003cem\u003ePIN5\u003c/em\u003e was expressed in yeast, the content of radio-labeled IAA and NAA decreased. Given that the mutation of \u003cem\u003eAtDIV1\u003c/em\u003e results in upregulation of \u003cem\u003ePIN5\u003c/em\u003e transcription, although total IAA levels remain largely unchanged in the \u003cem\u003eatdiv1\u003c/em\u003e mutant (unpubnished data), we propose that the subcellular IAA distribution between cytoplasm and ER, or the balance between conjugated and free auxin is altered in the \u003cem\u003eatdiv1\u003c/em\u003e mutants. Exogenous IAA supplementation to restore the shorter root defect in \u003cem\u003eatdiv1\u003c/em\u003e mutant might be via the regulation of the homeostatic balance between active and inactive IAA content. These unresolved aspects warrant further investigations.\u003c/p\u003e\u003cp\u003eThe regulation of \u003cem\u003ePIN5\u003c/em\u003e remains poorly understood compared to other PIN genes. In context of transcriptional PIN5 regulation, cytokinin regulates \u003cem\u003ePIN5\u003c/em\u003e through the ARR1 TF (Di Mambro et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and CsMYB77 activates \u003cem\u003ePIN5\u003c/em\u003e expression, which leads to a decline in free IAA levels, and thus impaired auxin signaling, in the fruits of transgenic Hongkong kumquat lines (Zhang et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). There are also evidence that the function and cellular distribution of PIN5 is regulated via changes in some conserved acidic residues in AtNHX5 and AtNHX6, which are endosomal Na\u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e/H\u003csup\u003e+\u003c/sup\u003e antiporters (Fan et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Our discovery that AtDIV1 acts as a negative regulator of \u003cem\u003ePIN5\u003c/em\u003e expression provides new insights into the regulatory network of \u003cem\u003ePIN5\u003c/em\u003e and thus auxin cellular distribution in plant cells.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003ePlant materials and growth conditions\u003c/h2\u003e\u003cp\u003eThe T-DNA insertion mutants \u003cem\u003eatdiv1\u003c/em\u003e (SALK_084867), \u003cem\u003epin5-3\u003c/em\u003e (SALK_021738), were obtained from the ABRC. \u003cem\u003eatdiv1 pin5\u003c/em\u003e was obtained by crossing \u003cem\u003epin5\u003c/em\u003e with \u003cem\u003eatdiv1\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eArabidopsis seedlings were grown on vertical plates containing half-strength Murashige and Skoog (1/2 MS) media supplemented with 1% sucrose and 0.5% Gelzan (Sigma-Aldrich, G1910). The plants grow for different days at 22\u0026deg;C in 16h light/8h dark cycle. 7-day-old seedlings of Col-0 and \u003cem\u003eatdiv1\u003c/em\u003e were transferred to mixed soil and cultured in greenhouse for 8 weeks to record the phenotype of above-ground tissues.\u003c/p\u003e\u003cp\u003eFor IAA/NAA treatment, the surface sterilized seeds were germinated and grown on media supplemented with 1, 10 or 20 nM IAA (Sigma-Aldrich, I2886), 10, 30 or 50 nM NAA (Sigma-Aldrich, N0640) for 7 days. IAA and NAA was dissolved in 95% ethanol and diluted with ddH\u003csub\u003e2\u003c/sub\u003eO, the same dilution ratio of ethanol was added as control.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePlasmid constructs and plant transformation\u003c/h3\u003e\n\u003cp\u003eTo generate the ATDIV1-GFP fusion constructs, the nucleotide sequences containing the native promoter (2223 bp region before ATG) and genomic region were amplified with primer pATDIV1-F/ATDIV1-R. The sequence was then inserted into pCAMBIA1305-GFP vector between restriction sites of KpnI and BamHI via infusion cloning (Vazyme) to get pATDIV1:ATDIV1-GFP vector. Genomic sequences of ATDIV1 driven by the native promoter (2198 bp region before ATG) was inserted into pCAMBIA1300-3\u0026times;mNeonGreen vector between restriction sites of SalI and XbaI via infusion cloning (Vazyme) to get pATDIV1:ATDIV1-3\u0026times;mNeonGreen vector. All primers used for cloning are listed in Extended Data Table\u0026nbsp;1. The construct was sequence-verified and transformed to \u003cem\u003eatdiv1\u003c/em\u003e mutants via Agrobacterium tumefaciens strain GV3101. Transgenic plants were selected by antibiotic resistance and fluorescence intensity.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eRNA extraction and qPCR analysis\u003c/h2\u003e\u003cp\u003eArabidopsis seedlings were grown on vertical plates containing half-strength Murashige and Skoog (1/2 MS) media for 7 days. Seedling roots were collected and weighed, then ground quickly into a powder in liquid nitrogen and transfer to a 1.5 mL sterile centrifuge tube. The RNAprep Pure Plant Kit (TIANGEN) was used for total RNA extraction from root. The cDNA was reversed translated from the total RNA by One-Step gDNA Removal and cDNA Synthesis Supermix (TRANSGEN). SYBR Green mix (TRANSGEN) was used for qPCR to analysis the expression levels of ATDIV1 and PIN5 using the ABI Quant Studio6 Real-Time PCR Systems. qPCR was run at 95\u0026deg;C for 10 s (denaturation), 60\u0026deg;C for 30 s (annealing and extension) for 40 cycles. The experiment was repeated at least three times with 4 technical replicates for each experiment.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePI staining\u003c/h3\u003e\n\u003cp\u003eFor PI staining, 7-days Arabidopsis seedlings were stained in 10 \u0026micro;g/mL PI for 1 minutes. Samples were observed under the confocal microscope (Zeiss LSM880) equipped with 20 X 0.8NA objective. The 561-nm laser were used for imaging. Image process was performed using the dark sectioning plugin in ImageJ (Cao et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eFluorescent imaging and image analysis\u003c/h3\u003e\n\u003cp\u003eThe subcellular localization of mNeonGreen-tagged proteins was obtained with a Zeiss LSM880 with 63 X 1.4NA oil immersion objective. Fluorescence intensity were measured using ImageJ software.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBiological materials can be obtained upon request. Extended data and source data are provided with this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the experimental technology center for life sciences, Beijing Normal University, and are grateful to Dr. Xiaoyan Zhang for technical support. This work was supported by grants from the National Natural Science Foundation of China (32100279 to T. W., 32400563 to L.D. and 32270350 and 32070194 to Y. Z.), the Fundamental Research Funds for the Central Universities (2243200007 to Y.Z.) and open funds of the State Key Laboratory of Plant Environmental Resilience (SKLPERKF2401).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT.W. and S.P. initiated the project. T.W., L.D. and J.X. designed the experiments; L.D. and J.X. performed the experiments; X.W. and H.Y. generated the \u003cem\u003eatdiv1\u003c/em\u003e and \u003cem\u003eatdiv1 pin5\u003c/em\u003e mutants; L.D. and J.X. analyzed the data; T.W., S.P., Y.Z., L.D. J.X. and C.L. wrote, reviewed and edited the manuscript with input from all of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u003cstrong\u003eAlonso, J.M., Stepanova, A.N., Leisse, T.J., et al.\u003c/strong\u003e (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. \u003cem\u003eScience (80-. ).\u003c/em\u003e, \u003cstrong\u003e301\u003c/strong\u003e, 653\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eBlilou, I., Xu, J., Wildwater, M., et al.\u003c/strong\u003e (2005) The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots. \u003cem\u003eNature\u003c/em\u003e, \u003cstrong\u003e433\u003c/strong\u003e, 39\u0026ndash;44.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eCao, R., Li, Yaning, Zhou, Y., et al.\u003c/strong\u003e (2025) Dark-based optical sectioning assists background removal in fluorescence microscopy. \u003cem\u003eNat. Methods\u003c/em\u003e, \u003cstrong\u003e22\u003c/strong\u003e, 1299\u0026ndash;1310.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eCao, Y., Li, K., Li, Y., Zhao, X. and Wang, L.\u003c/strong\u003e (2020) MYB Transcription Factors as Regulators of Secondary Metabolism in Plants. \u003cem\u003eBiology (Basel).\u003c/em\u003e, \u003cstrong\u003e9\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eChen, Z., Wu, Z., Dong, W., Liu, S., Tian, L., Li, J. and Du, H.\u003c/strong\u003e (2022) MYB transcription factors becoming mainstream in plant roots. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e, \u003cstrong\u003e23\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eFan, L., Zhao, L., Hu, W., et al.\u003c/strong\u003e (2018) Na(+) ,K(+) /H(+) antiporters regulate the pH of endoplasmic reticulum and auxin-mediated development. \u003cem\u003ePlant. Cell Environ.\u003c/em\u003e, \u003cstrong\u003e41\u003c/strong\u003e, 850\u0026ndash;864.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eFrangedakis, E., Yelina, N.E., Billakurthi, K., Hua, L., Schreier, T., Dickinson, P.J., Tomaselli, M., Haseloff, J. and Hibberd, J.M.\u003c/strong\u003e (2024) MYB-related transcription factors control chloroplast biogenesis. \u003cem\u003eCell\u003c/em\u003e, \u003cstrong\u003e187\u003c/strong\u003e, 4859-4876.e22.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eGanguly, A., Lee, S.H., Cho, M., Lee, O.R., Yoo, H. and Cho, H.-T.\u003c/strong\u003e (2010) Differential auxin-transporting activities of PIN-FORMED proteins in Arabidopsis root hair cells. \u003cem\u003ePlant Physiol.\u003c/em\u003e, \u003cstrong\u003e153\u003c/strong\u003e, 1046\u0026ndash;1061.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eGouran, M. and Brady, S.M.\u003c/strong\u003e (2024) The transcriptional integration of environmental cues with root cell type development. \u003cem\u003ePlant Physiol.\u003c/em\u003e, \u003cstrong\u003e196\u003c/strong\u003e, 2150\u0026ndash;2161.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eHu, Yangjie, Omary, M., Hu, Yun, et al.\u003c/strong\u003e (2021) Cell kinetics of auxin transport and activity in Arabidopsis root growth and skewing. \u003cem\u003eNat. Commun.\u003c/em\u003e, \u003cstrong\u003e12\u003c/strong\u003e, 1657.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eHuang, W., Khaldun, A.B.M., Chen, J., Zhang, C., Lv, H., Yuan, L. and Wang, Y.\u003c/strong\u003e (2016) A R2R3-MYB transcription factor regulates the flavonol biosynthetic pathway in a traditional chinese medicinal plant, epimedium sagittatum. \u003cem\u003eFront. Plant Sci.\u003c/em\u003e, \u003cstrong\u003e7\u003c/strong\u003e, 1089.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eKong, S., Zhu, M., Scarpin, M.R., et al.\u003c/strong\u003e (2024) DRMY1 promotes robust morphogenesis in Arabidopsis by sustaining the translation of cytokinin-signaling inhibitor proteins. \u003cem\u003eDev. Cell\u003c/em\u003e, \u003cstrong\u003e59\u003c/strong\u003e, 3141-3160.e7.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eLi, J., Han, G., Sun, C. and Sui, N.\u003c/strong\u003e (2019) Research advances of MYB transcription factors in plant stress resistance and breeding. \u003cem\u003ePlant Signal. Behav.\u003c/em\u003e, \u003cstrong\u003e14\u003c/strong\u003e, 1613131.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eLi, Y., Han, S. and Qi, Y.\u003c/strong\u003e (2023) Advances in structure and function of auxin response factor in plants. \u003cem\u003eJ. Integr. Plant Biol.\u003c/em\u003e, \u003cstrong\u003e65\u003c/strong\u003e, 617\u0026ndash;632.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eLin, Z., Yin, K., Zhu, D., Chen, Z., Gu, H. and Qu, L.-J.\u003c/strong\u003e (2007) AtCDC5 regulates the G2 to M transition of the cell cycle and is critical for the function of Arabidopsis shoot apical meristem. \u003cem\u003eCell Res.\u003c/em\u003e, \u003cstrong\u003e17\u003c/strong\u003e, 815\u0026ndash;828.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eLuschnig, C. and Friml, J.\u003c/strong\u003e (2024) Over 25 years of decrypting PIN-mediated plant development. \u003cem\u003eNat. Commun.\u003c/em\u003e, \u003cstrong\u003e15\u003c/strong\u003e, 9904.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eMa, Y., Zhang, Y., Xu, J., Zhao, D., Guo, L., Liu, X. and Zhang, H.\u003c/strong\u003e (2024) Recent advances in response to environmental signals during Arabidopsis root development. \u003cem\u003ePlant Physiol. Biochem.\u003c/em\u003e, \u003cstrong\u003e215\u003c/strong\u003e, 109037.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eMambro, R. Di, Svolacchia, N., Ioio, R. Dello, et al.\u003c/strong\u003e (2019) The Lateral Root Cap Acts as an Auxin Sink that Controls Meristem Size. \u003cem\u003eCurr. Biol.\u003c/em\u003e, \u003cstrong\u003e29\u003c/strong\u003e, 1199-1205.e4.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eMartin, C. and Paz-Ares, J.\u003c/strong\u003e (1997) MYB transcription factors in plants. \u003cem\u003eTrends Genet.\u003c/em\u003e, \u003cstrong\u003e13\u003c/strong\u003e, 67\u0026ndash;73.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eMotte, H., Vanneste, S. and Beeckman, T.\u003c/strong\u003e (2019) Molecular and Environmental Regulation of Root Development. \u003cem\u003eAnnu. Rev. Plant Biol.\u003c/em\u003e, \u003cstrong\u003e70\u003c/strong\u003e, 465\u0026ndash;488.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eMravec, J., Skůpa, P., Bailly, A., et al.\u003c/strong\u003e (2009) Subcellular homeostasis of phytohormone auxin is mediated by the ER-localized PIN5 transporter. \u003cem\u003eNature\u003c/em\u003e, \u003cstrong\u003e459\u003c/strong\u003e, 1136\u0026ndash;1140.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eMu, R.-L., Cao, Y.-R., Liu, Y.-F., et al.\u003c/strong\u003e (2009) An R2R3-type transcription factor gene AtMYB59 regulates root growth and cell cycle progression in Arabidopsis. \u003cem\u003eCell Res.\u003c/em\u003e, \u003cstrong\u003e19\u003c/strong\u003e, 1291\u0026ndash;1304.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eM\u0026uuml;ssig, C., Shin, G.-H. and Altmann, T.\u003c/strong\u003e (2003) Brassinosteroids promote root growth in Arabidopsis. \u003cem\u003ePlant Physiol.\u003c/em\u003e, \u003cstrong\u003e133\u003c/strong\u003e, 1261\u0026ndash;1271.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eNittis, M. De, Vivo, M. De, Ioio, R. Dello and Sabatini, S.\u003c/strong\u003e (2025) Hormonal regulation of primary root development. \u003cem\u003eCell Rep.\u003c/em\u003e, \u003cstrong\u003e44\u003c/strong\u003e, 115751.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eOh, J.E., Kwon, Y., Kim, J.H., Noh, H., Hong, S.-W. and Lee, H.\u003c/strong\u003e (2011) A dual role for MYB60 in stomatal regulation and root growth of Arabidopsis thaliana under drought stress. \u003cem\u003ePlant Mol. Biol.\u003c/em\u003e, \u003cstrong\u003e77\u003c/strong\u003e, 91\u0026ndash;103.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003ePetr\u0026aacute;sek, J. and Friml, J.\u003c/strong\u003e (2009) Auxin transport routes in plant development. \u003cem\u003eDevelopment\u003c/em\u003e, \u003cstrong\u003e136\u003c/strong\u003e, 2675\u0026ndash;2688.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eRoychoudhry, S. and Kepinski, S.\u003c/strong\u003e (2022) Auxin in root development. \u003cem\u003eCold Spring Harb. Perspect. Biol.\u003c/em\u003e, \u003cstrong\u003e14\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eSakaoka, S., Mabuchi, K., Morikami, A. and Tsukagoshi, H.\u003c/strong\u003e (2018) MYB30 regulates root cell elongation under abscisic acid signaling. \u003cem\u003eCommun. Integr. Biol.\u003c/em\u003e, \u003cstrong\u003e11\u003c/strong\u003e, e1526604.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eSomssich, M., Khan, G.A. and Persson, S.\u003c/strong\u003e (2016) Cell Wall Heterogeneity in Root Development of Arabidopsis. \u003cem\u003eFront. Plant Sci.\u003c/em\u003e, \u003cstrong\u003e7\u003c/strong\u003e, 1242.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eTominaga-Wada, R. and Wada, T.\u003c/strong\u003e (2014) Regulation of root hair cell differentiation by R3 MYB transcription factors in tomato and Arabidopsis. \u003cem\u003eFront. Plant Sci.\u003c/em\u003e, \u003cstrong\u003e5\u003c/strong\u003e, 91.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eVanneste, S., Pei, Y. and Friml, J.\u003c/strong\u003e (2025) Mechanisms of auxin action in plant growth and development. \u003cem\u003eNat. Rev. Mol. Cell Biol.\u003c/em\u003e, \u003cstrong\u003e26\u003c/strong\u003e, 648\u0026ndash;666.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eVilarrasa-Blasi, J., Gonz\u0026aacute;lez-Garc\u0026iacute;a, M.-P., Frigola, D., et al.\u003c/strong\u003e (2014) Regulation of plant stem cell quiescence by a brassinosteroid signaling module. \u003cem\u003eDev. Cell\u003c/em\u003e, \u003cstrong\u003e30\u003c/strong\u003e, 36\u0026ndash;47.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eXue, T., Liu, Z., Dai, X. and Xiang, F.\u003c/strong\u003e (2017) Primary root growth in Arabidopsis thaliana is inhibited by the miR159 mediated repression of MYB33, MYB65 and MYB101. \u003cem\u003ePlant Sci.\u003c/em\u003e, \u003cstrong\u003e262\u003c/strong\u003e, 182\u0026ndash;189.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eZhang, D., Zhou, H., Zhang, Yang, Zhao, Y., Zhang, Yiyi, Feng, X. and Lin, H.\u003c/strong\u003e (2025) Diverse roles of MYB transcription factors in plants. \u003cem\u003eJ. Integr. Plant Biol.\u003c/em\u003e, \u003cstrong\u003e67\u003c/strong\u003e, 539\u0026ndash;562.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eZhang, L., Xu, Y., Li, Y., Zheng, S., Zhao, Z., Chen, M., Yang, H., Yi, H. and Wu, J.\u003c/strong\u003e (2024) Transcription factor CsMYB77 negatively regulates fruit ripening and fruit size in citrus. \u003cem\u003ePlant Physiol.\u003c/em\u003e, \u003cstrong\u003e194\u003c/strong\u003e, 867\u0026ndash;883.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Root development, MYB, PIN5","lastPublishedDoi":"10.21203/rs.3.rs-7670153/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7670153/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eKey message\u003c/p\u003e\n\u003cp\u003eThe R2R3 MYB transcription factor AtDIV1 negatively regulates \u003cem\u003ePIN5\u003c/em\u003e expression to modulate primary root growth.\u003c/p\u003e\n\u003cp\u003eAs a crucial organ for nutrient uptake, the root plays a vital role in plant growth and development, a process regulated by multiple factors including phytohormones and transcriptional mechanisms. In this study, we identify AtDIV1, a R2R3-MYB transcription factor, as the key regulator of root development. Loss of function of \u003cem\u003eAtDIV1\u003c/em\u003e led to significantly shortened primary roots, accompanied by reductions in both cell number and cell length compared to the wild type. Pharmacological experiments demonstrated that exogenous IAA application partially rescued the root length defect in \u003cem\u003eatdiv1\u003c/em\u003e mutant, restoring cell number in both meristem and elongation zones. Notably, \u003cem\u003ePIN5\u003c/em\u003e expression was significantly upregulated in \u003cem\u003eatdiv1\u003c/em\u003e roots and the root developmental defects observed in \u003cem\u003eatdiv1\u003c/em\u003e mutants were fully rescued in \u003cem\u003eatdiv1\u003c/em\u003e \u003cem\u003epin5 \u003c/em\u003edouble mutants. \u0026nbsp;Collectively, our findings establish that AtDIV1 negatively regulates \u003cem\u003ePIN5 \u003c/em\u003eexpression to modulate primary root growth.\u003c/p\u003e","manuscriptTitle":"R2R3 MYB transcription factor AtDIV1 regulates root growth through regulation of PIN5 expression in Arabidopsis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-27 10:45:24","doi":"10.21203/rs.3.rs-7670153/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"088d7f81-c955-41da-a314-69a0cda393fc","owner":[],"postedDate":"October 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-13T15:36:41+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-27 10:45:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7670153","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7670153","identity":"rs-7670153","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}