Characterisation of the

Background: Mentha canadensis L. has important economic value for the production of essential oils, which are synthesised, secreted and stored in peltate glandular trichomes. As a typical multicellular secretory trichome, glandular trichomes are important biological factories for the synthesis of some specialised metabolites. However, little is known about the molecular mechanism of glandular trichome development in M. canadensis. Results: In this study, the R2R3-MYB transcription factor gene McMIXTA was isolated to investigate its function in glandular trichome development. Bioinformatics analysis indicated that McMIXTA belonged to the subgroup 9 R2R3MYB, with a R2R3 DNA-binding domain and conserved subgroup 9 motifs. A subcellular localisation assay indicated that McMIXTA was localised in the nucleus. Transactivation analysis indicated that McMIXTA was a positive regulator, with transactivation regions located between positions N253 and N307. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that McMIXTA formed a complex with McHD-Zip3, a trichome developmentrelated HD-ZIP IV transcription factor. Overexpression of McMIXTA in Mentha × piperita L. caused an increase in peltate glandular trichomes density of approximately 25% on the leaf abaxial surface. Conclusions: Our results demonstrated that the subgroup 9 R2R3-MYB transcription factor McMIXTA has a positive effect on regulating peltate glandular trichome development and the MIXTA/HD-ZIP IV complexes might be conserved regulators for glandular trichome initiation. These results provide useful information for revealing the regulatory mechanism of multicellular glandular trichome development. Keyword: Mentha canadensis L. ; Glandular trichome; McMIXTA; McHD-Zip3 © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Background Trichomes are appendages derived from cells of the aerial epidermis, and are found in numerous plant species worldwide [1]. Trichomes vary in size, morphology, cellular composition, and function, and can be categorised into unicellular and multicellular or secretory and non-secretory types according to their cell number and secretory ability [2, 3]. For instance, Arabidopsis thaliana trichomes are unicellular and non-secretory [4]. Secretory or glandular trichomes synthesise or secrete large amounts of specialised metabolites that enhance plant fitness and may also be beneficial for humans [5, 6]. For example, glandular trichomes of Artemisia annua secrete the sesquiterpene metabolite artemisinin, which is an effective anti-malarial drug [7]. Open Access *Correspondence: liangcy618@cnbg.net Xiwu Qi and Zequn Chen are contributed equally to this work. 1 Jiangsu Key Laboratory for the Research and Utilization of Plant Resources, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China Full list of author information is available at the end of the article Page 2 of 11 Qi et al. BMC Plant Biology (2022) 22:219 The amounts of metabolites secreted by glandular trichomes are generally correlated to their density [7, 8]. Increased glandular trichome density is considered to be an effective strategy to increase the accumulation of specialised metabolites [7]. Therefore, the genetic networks of glandular trichome development provide an important basis for genetic engineering of glandular trichome. In A. thaliana, more than 70 genes regulating trichome development have been identified and characterised, revealing a complex regulatory network for trichome [9–14]. In contrast to the unicellular trichomes of Arabidopsis, the regulatory networks of multicellular glandular trichome development in other species remain poorly understood [15]. To date, only a few genes regulating multicellular glandular trichome development have been identified, which mainly encode some transcription factors, including MYB, HD-ZIP, C2H2, bHLH and so on [15]. The MIXTA gene, which was first characterised in Antirrhinum majus, encodes one of the most important regulators of glandular trichomes and promotes trichome initiation [16–18]. Homologous genes have also been identified in A. annua and Solanum lycopersicum, which have been shown to have similar functions in promoting glandular trichome initiation [19, 20]. In addition to MIXTA genes, several HD-ZIP IV transcription factors have also been shown to participate in regulating glandular trichome development [15]. For example, AaHD1 and AaHD8 in A. annua can promote the development of glandular trichome [21, 22]. In S. lycopersicum, SlWOOLLY and SlCD2 have been shown to participate in regulation of glandular trichome initiation [23–25]. Further, there is evidence that the glandular trichome-related MIXTA and HD-ZIP transcription factors may function through interaction. In A. annua, AaMIXTA1 and AaHD8 show similar functions in promoting glandular trichome initiation, and they interact to form complexes. The interaction of AaMIXTA1 and AaHD8 produces a much greater effect than their additive effects [22]. In S. lycopersicum, SlMIXTA-like and SlCD2 may also interact to form a co-regulation network [15]. Mentha canadensis L. (Lamiaceae) can produce a large amount of aromatic essential oils, which are widely used in food additives, cosmetics and medical industries [26]. The main components of essential oils are monoterpenoids, which are synthesised, secreted, and stored in glandular trichomes [27, 28]. The biosynthetic pathway of monoterpenoids in essential oils has been extensively studied in the genus Mentha [29–31]. However, the genetic mechanism of the involvement of glandular trichomes in essential oil biosynthesis has rarely been explored. The surface of M. canadensis bears three types of trichomes: non-glandular, capitate glandular, and peltate glandular trichomes; among these, peltate glandular trichomes are the centre of essential oil biosynthesis [32]. In our previous study, we demonstrated that McHDZip3, a HD-Zip IV transcription factor of M. canadensis, participated in promoting glandular trichome development [33]. However, the regulation mechanism of glandular trichome development in M. canadensis requires further study. Therefore, in this study, we cloned the MIXTA homologue gene McMIXTA from M. canadensis and characterised its regulatory role in glandular trichome development. We further examined whether McMIXTA interacted with the HD-ZIP IV transcription factor McHD-Zip3 to form a complex. The results of this study will contribute to elucidating the molecular mechanism of multicellular glandular trichome development in M. canadensis.