Inhibition of Lateral Shoot Formation by RNA Interference and Chemically Induced Mutations to Genes Expressed in the Axillary Meristem of Nicotiana Tabacum L

Hamano et al. BMC Plant Biol (2021) 21:236 https://doi.org/10.1186/s12870-021-03008-3 RESEARCH ARTICLE Open Access Inhibition of lateral shoot formation by RNA interference and chemically induced mutations to genes expressed in the axillary meristem of Nicotiana tabacum L. Kaori Hamano* , Seiki Sato, Masao Arai, Yuta Negishi, Takashi Nakamura, Tomoyuki Komatsu, Tsuyoshi Naragino and Shoichi Suzuki Abstract Background: Lateral branches vigorously proliferate in tobacco after the topping of the inforescence portions of stems for the maturation of the leaves to be harvested. Therefore, tobacco varieties with inhibited lateral shoot formation are highly desired by tobacco farmers. Results: Genetic inhibition of lateral shoot formation was attempted in tobacco. Two groups of genes were examined by RNA interference. The frst group comprised homologs of the genes mediating lateral shoot formation in other plants, whereas the second group included genes highly expressed in axillary bud primordial stages. Although “primary” lateral shoots that grew after the plants were topped of when fower buds emerged were unafected, the growth of “secondary” lateral shoots, which were detected on the abaxial side of the primary lateral shoot base, was signifcantly suppressed in the knock-down lines of NtLs, NtBl1, NtREV, VE7, and VE12. Chemically induced mutations to NtLs, NtBl1, and NtREV similarly inhibited the development of secondary and “tertiary” lateral shoots, but not primary lateral shoots. The mutations to NtLs and NtBl1 were incorporated into an elite variety by backcrossing. The agronomic characteristics of the backcross lines were examined in feld trials conducted in commercial tobacco production regions. The lines were generally suitable for tobacco leaf production and may be useful as new tobacco varieties. Conclusion: The suppressed expression of NtLs, NtBl1, NtREV, VE7, or VE12 inhibited the development of only the secondary and tertiary lateral shoots in tobacco. The mutant lines may beneft tobacco farmers by minimizing the work required to remove secondary and tertiary lateral shoots that emerge when farmers are harvesting leaves, which is a labor-intensive process. Keywords: Chemically induced mutation, Lateral shoot inhibition, RNAi, RNA-seq, Sucker, Tobacco Background how lateral shoot formation is controlled. Genes involved in Lateral shoot development, which is a fundamental process axillary meristem development have been studied in vari- in plants, is uniquely regulated in all species. In crop spe- ous plant species. Mutations in Lateral suppressor genes cies, harvest quality and productivity are directly afected by in tomato (Ls) [1] and Arabidopsis (LAS) [2] inhibit axillary shoot formation during the vegetative phase. A mutation in a rice ortholog, Monoculm 1 (MOC1), results in suppressed *Correspondence: [email protected] tillering and a decrease in the number of rachis branches and Leaf Tobacco Research Center, Japan Tobacco Inc., 1900 Idei, Oyama, spikelets [3]. A mutation in the Blind gene strongly suppresses Tochigi 323-0808, Japan © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. 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. Hamano et al. BMC Plant Biol (2021) 21:236 Page 2 of 12 axillary meristem formation in tomato [4, 5]. Tree Regula- cultivation period (up to “tertiary” lateral shoots). Tese tor of Axillary Meristems (RAX) genes, RAX1, RAX2, and lateral shoots must be removed manually or suppressed RAX3, were identifed in Arabidopsis as homologs of Blind. by chemicals (i.e., “suckercides”) to enable plants to pro- Triple recessive mutants exhibit almost completely inhibited duce many high-quality leaves. However, this control axillary shoot formation [6, 7]. Blind and RAX genes encode of lateral shoots is a labor-intensive and costly process. R2R3 MYB transcription factors, which are critical for ini- Removing the secondary and tertiary lateral shoots is tiating axillary meristem development during the vegeta- especially inconvenient because the timing of their emer- tive phase. Previous studies revealed that Blind also controls gence coincides with when farmers must harvest the axillary meristem initiation in the reproductive phase [5, 6]. leaves, which is another labor-intensive task. Terefore, Te decapitation of primary shoots does not stimulate the tobacco varieties with fewer lateral shoots, especially outgrowth of axillary shoots in the Arabidopsis las mutant secondary and tertiary ones, are highly desired. Unfor- or rax1 mutant [2, 7]. Te LAX1 gene in rice and the BA1 tunately, breeding involving conventional and biotech- gene in maize encode bHLH domains and are involved in the nological approaches has not resulted in varieties with branching of inforescence and vegetative shoots [8, 9]. Te substantially fewer lateral shoots. ROX gene in Arabidopsis is an ortholog of LAX1 and BA1. It Te tobacco genes involved in the regulation of axil- is reportedly involved in lateral shoot formation in the early lary shoot development and their functions remain rela- vegetative stage [10]. tively uncharacterized. Nevertheless, it is possible that Mutations in some genes regulating axillary meristem homologs of the above-mentioned genes in other plants formation afect the development of the main shoot api- are present and function similarly in tobacco. For exam- ces. For example, a mutation in the Revoluta (REV) gene ple, a tobacco homolog (GenBank EU935981) of Ls has in Arabidopsis substantially decreases the outgrowth of been cloned [21], but its function was not investigated. rosette and cauline leaves and inhibits the stimulation In this study, the expression of the homologs of these of axillary shoots following the decapitation of primary genes as well as a number of other genes preferentially shoots [11, 12]. Additionally, the rev mutation sometimes expressed in the axillary meristems was knocked down leads to the arrested development of the primary shoot in tobacco by RNA interference (RNAi). Additionally, the apical meristem (SAM) at an early stage. Te Cup-Shaped chemically induced mutations to selected genes inhibited Cotyledon (CUC ) genes CUC1, CUC2, and CUC3 [13– lateral shoot formation. Mutant lines were characterized 15] and the Lateral Organ Fusion (LOF) gene [16] regu- in greenhouse and feld trials. late organ separation and axillary meristem formation in Arabidopsis. Hairy Meristem (HAM) genes in petunia Results [17] and pepper [18] and their homolog in Arabidopsis, BLAST search Lost Meristem (LOM) [19], are important for SAM and Two groups of tobacco genes were examined to assess the axillary meristem development. Consequently, petunia efects of RNAi knock-down on lateral shoot formation. and pepper ham mutants and the Arabidopsis lom1- Te frst group included homologs of the genes involved lom2-lom3 triple mutant exhibit premature termination in lateral shoot formation in other plants. A BLAST of the shoot apex and arrested axillary shoot develop- search of the GenBank database and an in-house tobacco ment. A mutation in the Far-Red Elongated Hypocotyl cDNA database was conducted to identify tobacco (FHY3) gene in Arabidopsis leads to inhibited axillary homologs (Table 1). Because tobacco is an amphidip- bud outgrowth. Additionally, the rev-fhy3 double muta- loid species that inherited its genome from Nicotiana tion represses axillary bud formation considerably more sylvestris (S-genome) and Nicotiana tomentosiformis than the single recessive mutations [20]. (T-genome) [22–24], both S-genes and T-genes were Because tobacco is cultivated for its leaves, when plants identifed for each homolog. Te NtBl and NtCUC genes start to fower, the apical portions of stems (mainly were numbered respectively in descending order of inforescences) are cut of (i.e., topping) to enhance leaf homology with Blind and Goblet (GenBank HM210879), development and maturation prior to harvest. Although a tomato homolog of the CUC genes. tobacco plants generally exhibit strong apical dominance, topping releases lateral buds from dormancy. Te result- RNA sequencing (RNA‑seq) analysis ing lateral shoots, which are often called “suckers” in Genes that were not studied in other plants might also tobacco, undergo vigorous development. Tobacco farm- be involved in the regulation or development of axil- ers must remove these “primary” lateral shoots soon after lary meristems and shoots. Tus, as the second group, they develop, but “secondary” lateral shoots grow imme- genes that are highly expressed in axillary bud primordial diately from

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