Reduction of Lateral Shoots by RNA Interference and by Chemical Mutation of Genes Involved in Axillary Meristem Regulation and Field Trials of Mutant Lines in Nicotiana Tabacum L

Kaori Hamano (  kaori.hamano@jt.com ) Tobacco Institute Inc https://orcid.org/0000-0001-9381-6620 Seiki Sato Leaf Tobacco Research Center, Japan Tobacco Inc. Masao Arai Leaf Tobacco Research Center, Japan Tobacco Inc. Yuta Negishi Leaf Tobacco Research Center, Japan Tobacco Inc. Takashi Nakamura Leaf Tobacco Research Center, Japan Tobacco Inc. Tomoyuki Komatsu Leaf Tobacco Research Center, Japan Tobacco Inc. Tsuyoshi Naragino Leaf Tobacco Research Center, Japan Tobacco Inc. Shoichi Suzuki Leaf Tobacco Research Center, Japan Tobacco Inc.


Abstract
Background Lateral branches vigorously proliferate in tobacco after topping of in orescence portions of stems for maturation of leaves to be harvested. Therefore, tobacco varieties with reduced lateral shoots are highly desired by farmers.

Results
Genetic reduction of lateral shoots was attempted in tobacco. Two groups of genes were examined by RNA interference: homologues of the genes reported as involved in the formation of lateral shoots in other plants, and genes highly expressed in primordial stages of axillary buds. Although "primary" lateral shoots that grew after the plants were topped off when ower buds emerged were not much affected, "secondary" lateral shoots, which appeared from the abaxial sides of the bases of the primary lateral shoots, were suppressed signi cantly by knock-down of ve genes, NtLs, NtBl1, NtREV, VE7, and VE12. Chemical mutation of three of them, NtLs, NtBl1, and NtREV, similarly reduced secondary and "tertiary" lateral shoots but not primary ones. The mutation of NtLs and NtBl1 was backcrossed into an elite variety. The backcross lines were examined for agronomic characteristics in eld trials conducted in commercial tobacco production areas. The lines were satisfactory for leaf tobacco production overall and showed good potential as new tobacco varieties.

Conclusion
Suppression of the ve genes reduced only secondary and tertiary lateral shoots in tobacco although similar approaches reduced all branches in other plant species. Nevertheless, the mutant lines might greatly relieve farmers because secondary and tertiary lateral shoots are especially cumbersome because they emerge when farmers are burdened by the labor-intensive leaf harvest. Background Development of lateral shoots, a fundamental process in plants, is regulated in a unique way in every species. In crop species, the harvest quality and productivity are affected directly by how lateral shoot formation is controlled. Therefore, genes involved in axillary meristem development have been studied extensively in various plant species. Mutation in Lateral suppressor genes Ls in tomato [1] and LAS in Arabidopsis [2] inhibit axillary shoot formation during the vegetative phase. Mutation in an orthologue, Monocolum (MOC1), in rice suppressed tillering and decreased rachis branches and spikelets [3]. Mutation in Blind gene strongly suppressed axillary meristem formation in tomato [4,5]. Three Regulator of Axillary Meristems (RAX) genes, RAX1, RAX2, and RAX3, were identi ed in Arabidopsis as homologous of Blind. The triple recessive mutants almost completely inhibited axillary shoot formation [6,7]. Blind and RAX genes code for R2R3 Myb transcription factors, which play a central role in the initiation of axillary meristems during the vegetative phase. Blind also controls the axillary meristem initiation in the reproductive phase [5,6]. Decapitation of primary shoots did not stimulate outgrowth of axillary shoots in a las mutant or in a rax1 mutant in Arabidopsis [2,7]. LAX1 gene in rice and BA1 gene in maize carried bHLH domains and were involved in branching of in orescence and vegetative shoots [8,9]. ROX gene is an orthologue of LAX1 and BA1 in Arabidopsis. It is reportedly involved in lateral shoot formation in the early vegetative stage [10].
Mutation in some genes regulating axillary meristem formation affects the development of main shoot apices. Mutation in the Revolute (REV) gene in Arabidopsis remarkably reduced outgrowth of both rosette and cauline leaves and reduced stimulation of axillary shoots by decapitation of primary shoots [11,12]. In addition, rev mutation sometimes arrested development of the primary shoot apical meristems at an early stage. Cup-Shaped Cotyledon (CUC) genes CUC1, CUC2, and CUC3, [13][14][15] and Lateral Organ Fusion (LOF) gene [16] regulate both organ separation and axillary meristem formation in Arabidopsis. Hairy Meristem (HAM) genes in Petunia [17] and pepper [18] and their homologue, Lost Meristem (LOM) gene, in Arabidopsis [19] play important roles in shoot apical meristem and axillary meristem development. Consequently, ham mutants in Petunia and pepper and lom1-lom2-lom3 triple mutant in Arabidopsis exhibited premature cession of shoot apex and arrest of axillary shoot development. Mutation in Far-Red Elongated Hypocotyl (FHY3) gene in Arabidopsis inhibited axillary bud outgrowth. Also, rev-fhy3 double mutation more drastically repressed axillary bud formation than the single recessive mutations [20].
Because tobacco is cultivated for harvesting of the leaves, when plants start to ower, apical portions of stems, mostly in orescence, are topped off so that leaves well develop and mature for processing. Although tobacco generally exhibits strong apical dominance, this topping releases lateral buds from dormancy. Lateral shoots, which are often called "suckers" in tobacco, start vigorous development. Tobacco farmers must remove these "primary" lateral shoots soon, but then "secondary" lateral shoots grow immediately from the abaxial sides of the bases of the primary lateral shoots. In this manner, lateral shoots emerge sequentially during the cultivation period up to "tertiary" ones. The lateral shoots must be removed manually or suppressed by chemicals, "suckercides", to maintain the leaf harvest quality and quantity. Control of lateral shoots is a labor-intensive and cost-intensive process. Secondary and tertiary lateral shoots are especially cumbersome because, when they emerge, farmers are burdened by another labor-intensive task: leaf harvest. Therefore, tobacco varieties with fewer lateral shoots, especially with fewer secondary and tertiary ones, are highly desired. Nevertheless, breeding including conventional and biotechnological approaches has not remarkably reduced lateral shoots to date.
Little is known about tobacco genes involved in the regulation of axillary shoot development and their functions. However, it is possible that homologues of the genes in other plants mentioned above are present and function similarly in tobacco. For example, a tobacco homologue (GenBank EU935981) of Ls was cloned [21], although its function was not studied. In this study, RNA interference (RNAi) of the homologues and a number of other genes preferentially expressed in the axillary meristems was attempted in tobacco. Then, chemically induced mutations were identi ed for the genes, which showed reduction of lateral shoots in the RNAi screening. Mutant lines were characterized in greenhouse and eld trials.

BLAST search
Two groups of tobacco genes were examined for effects of RNAi knock-down on lateral shoot formation. The rst group included homologues of the genes reported as involved in lateral shoot formation in other plants. BLAST search of GenBank and the in-house tobacco cDNA database was conducted for the tobacco homologues (Table 1). Because tobacco is an amphidiploid plant that inherited its genome from N. sylvestris (S-genome) and N. tomentosiformis (Tgenome) [22][23][24], both S-and T-alleles were identi ed for each homologue. NtBl and NtCUC genes were numbered, respectively in descending order of homology with Blind and a tomato homologue (GeneBank HM210879) of the CUC genes.

Rnaseq Study
Genes that were not studied in other plants might also be involved in the regulation or development of axillary meristems and shoots. Consequently, as the second group, genes that are highly expressed in primordial stages of axillary buds were sought by RNA sequencing. As presented in Fig. 1, tissues were sampled from zones of axillary meristems in the early stage (EA) and the very early stage (VE) and control zones of tobacco plants by laser micro-dissection.
RNA was prepared from the tissues and was analyzed using a next generation sequencer: 454 GS FLX. Approximately 900,000 reads, about 400 bp in length on average, were obtained from each tissue sample and were assembled into 38,569 unique genes. The second group of the genes was chosen according to the criteria, (a) read count of a gene in EA or VE was at least 10 times higher than that in the control, (b) the assembled gene was longer than 200 bp, and (c) the gene was of a transcription factor or unknown protein in the annotation. We selected 11 genes highly expressed in EA tissue, EA1-EA11, and 13 genes highly expressed in VE tissue, VE1-VE13 ( Table 2).
The counts of the RNA reads assigned to the rst group of the genes, the tobacco homologues, are also shown in Table 2. Among them, genes highly and speci cally expressed in EA or VE tissue were NtLs and NtBl1. In all three samples, NtREV was highly expressed. The other homologues were either not speci cally expressed in EA and VE tissues or were not detectable at all. In the literature, LAS and RAX were highly expressed in the axil of leaf primordia [2,6,7]. Also, REV was expressed not only in the early stage of lateral shoot meristem but also in the center of the SAM in inverted-cup-shaped population of the cell [12]. Consequently, at least some of the homologues, NtLs, NtBl1, and NtREV, were similar to their counterparts in other species in the expression patterns.
It is noteworthy that the sequence analysis revealed that VE7, which carried a bHLH domain, was a homologue of ROX gene, which was published after the BLAST s BLAST search described above [10]. In addition, VE12, which carried a NAC domain and which lacked a recognition site of miR164, turned out to be a homologue of CUC3 gene. ROX [10] and its maize orthologue BA1 [9] were expressed at the adaxial boundary of leaf primordia. Expression of CUC3 was detected at the boundaries between leaf primordia and the shoot meristem [25]. Consequently, VE7 and VE12 were similar to the homologues also in the expression patterns. After all, the RNAseq study well supplemented the homologue search.

RNAi knock-down
A total of 36 trigger sequences, 430 bp in length on average, were designed for RNA interference of the 12 homologues and the 24 genes from RNAseq screening (Supplemental Table S1). For each target, the S-allele and T-allele were compared. A region of high homology was chosen as the trigger so that both alleles would be knocked down effectively by a single trigger sequence. The average homology between the trigger regions of the S-and T-alleles was 95.4% identity, which was higher than the level of homology su cient for simultaneous knock-down of two alleles recommended by Parrish et al. [26]. In this process, it was possible to nd less conserved regions, less than 70% identity, among NtBl genes and among NtCUC genes. Therefore, it was likely that a trigger was able to knock down the S-T pair of the target gene speci cally.
The RNAi genes were introduced into tobacco 'Petit Havana SR-1'. The T1 progeny of three single locus transformants for each construct were grown in a greenhouse and were examined for the target gene expression. The target genes were well suppressed ( Table 2) except for VE3 transformants, in which the target gene was expressed higher than the null segregants, for reasons that remain unknown. Results showed that all transgenic plants produced as many and as much primary lateral shoots as the null segregants (Table 2). Therefore, the RNAi of no target suppressed primary lateral shoots. However, the secondary lateral shoots were well suppressed in number and/or weight in the transformants of ve constructs: NtLs, NtBl1, NtREV, VE7, and VE12. For example, although a secondary lateral bud was visible at the base of the abaxial side of a primary lateral shoot of a wild type plant (Fig. 2a), there was none at the position of an RNAi knock-down plant for NTLs (Fig. 2b). After removal of the secondary lateral shoots, no more lateral shoot formation was observed during greenhouse testing. Therefore, these ve genes were studied further. However, the transformants of EA5 and VE6 produced more secondary lateral shoots than the null segregants.

Chemically induced mutants
The EMS mutant library of tobacco 'Tsukuba 1' was screened for nonsense mutations of NtLS, NtBl1, NtREV, VE7 and VE12. As depicted in Fig. 3, the following mutations were identi ed and mapped: two each in NtLs-S and NtBl1-S and one each in NtLs-T, NtBl1-T, NtRev-S, and NtRev-T. Also, mutations in Sand T-genomes were combined by crossing the mutants, producing double recessive lines designated as SAS lines (Table 3). Because nonsense mutations were not identi ed in VE7 and VE12, these genes were not studied further.
The SAS lines, 15 plants each, were planted in an experimental eld and were examined for lateral shoot development. Again, primary lateral shoots were not suppressed signi cantly in the mutant lines (Table 4). Then, the number and weight of secondary lateral shoots were well suppressed. Those of tertiary ones were suppressed nearly completely. Therefore, the observation made in the RNAi lines in a greenhouse was faithfully reproduced in the EMS mutants in the eld trial. It is particularly interesting that the position of the primary lateral shoots was shifted upward in the SAS-ls and SAS-bl1 mutant lines ( Fig. 2d and 2f) in the eld trial and also in a greenhouse (data not shown). No such shift was observed in the RNAi lines in the greenhouse (Fig. 2b). The positions of the secondary and tertiary lateral shoots in the mutant lines and the RNAi lines were unchanged. Only the position of the primary lateral shoots was affected, for reasons that remain unclear.
Field trials in commercial production areas Field trials in commercial production areas were conducted to examine agronomic characteristics of the mutant lines. The ls-1 and bl1-1 mutations were introduced into a ue-cured tobacco variety, 'Coker319', widely grown in Japan by backcross breeding ( Table 3). Because of space limitations, the rev mutation was not tested.
Results of the trials of the backcross lines are presented in Table 5 for Coker319-ls-1 at four locations and Coker319-bl1-1 at two locations. A plot of 10 plants was replicated twice for each line at all locations. Overall, the mutant lines did not differ much from the original 'Coker319' in leaf yield or the other measurements, although small but statistically signi cant differences were found as explained hereinafter. Coker319-ls-1 was earlier in days to ower, higher in plant height, larger in leaf length, larger in leaf width, with darker leaf color than 'Coker319' at one or more locations. Coker319-bl1-1 had lower plant height, more numerous leaves, smaller leaf width at 1/2 from the top, and lighter leaf color than 'Coker319' at one or two locations. Therefore, no problems were found in the mutant lines as raw materials. The continuation of the trials was decided. In addition, reduction of lateral shoot developments, especially of secondary and tertiary lateral shoots, was observed. The labor necessary for removal of lateral shoots of Coker319-ls-1 was lower by 52% than that of 'Coker319' in a preliminary survey (Supplemental Table S2).

Other characteristics of the mutants
During the course of the study, some other phenotypic alterations were noticed. Leaves of 4-5-week-old Coker319-ls-1 plants occasionally turned yellow in nursery boxes (Fig. 2h). It was then con rmed that the same phenotype was observed in a growth chamber when 4-5-week-old Coker319-ls-1 plants were grown at 15 °C, although they were normal at 28 °C. Results show that the Coker319-ls-1 leaf color was cold-sensitive. Some plants were probably exposed to low temperatures in the nursery boxes. However, yellowed plants recovered quickly and showed no growth delay.
Petals of most of the owers of SAS-ls-1, SAS-ls-2, and Coker319-ls-1 plants were split (Fig. 2i) in greenhouses and in the eld. In severe cases, pistils and stamens were completely exposed (Fig. 2j). Seed yield from these owers decreased considerably, by 35% and 55% of the wild type lines, respectively, by natural and hand pollination (Supplemental Fig. S1).
Flowers of Coker319-bl1-1 plants were 73% fewer than those of the wild type lines in the eld trials (Supplemental Fig. S2). The seed yield from the owers of Coker319-bl1-1 was 72% of that of 'Coker319' (Supplemental Fig. S1).

Discussion
Genetic reduction of lateral shoots was attempted in tobacco. Two groups of tobacco genes were examined for effects of RNAi knock-down on lateral shoot formation. One source of the candidate genes was homologues of the genes reported as involved in the formation of lateral shoots in other plants. Such genes have been studied most extensively in Arabidopsis. The following eight genes or gene families were identi ed: LAS [2], RAX family [6,7], ROX [10], REV [11,12], CUC family [13][14][15], LOF [16], LOM [19], and FHY3 [20]. In tobacco, the BLAST search found homologues of seven of them, excluding ROX, which was reported after the BLAST search was performed. Then VE7, a homologue of ROX, was found from another source, genes highly expressed in primordial stages of axillary buds in tobacco in the RNA seq analysis. A homologue of CUC3, VE12, was also found from the second source. Therefore, tobacco and Arabidopsis apparently shared similar sets of the genes.
However, some characteristics of the knock-down and mutant tobacco were not reported in other plants. Primary lateral shoots were not suppressed, whereas secondary and tertiary ones were well suppressed in tobacco. In addition, the positions of primary lateral shoots in the tobacco mutants of NtLs and NtBl1 shifted upward. The mechanisms for differences between the plant species remain unclear, but the presence of the difference is not surprising because diverse ways of regulating lateral branching must be a key factor for a wide variety of plant shapes.
The other tobacco homologues were not effective in the knock-down study. The expression of these genes was either not detected in or was not speci c to the primordial stages of axillary buds. They were probably not functional counterparts of the homologues of the other plants.
The second source of the candidate genes examined were genes that were highly expressed in the primordial stages of axillary buds. Quite a few, 24, genes were chosen from the RNAseq analysis. They were highly expressed in and speci c to EA or VE tissues. However, except for VE7 and VE12 described above, knock-down of no gene caused signi cant reduction of lateral shoots. These genes might not be playing crucial regulatory roles in lateral shoot development. Alternatively, their functions might be redundant in tobacco.
Two mutant tobacco lines under the genetic background of variety 'Coker319' were examined in eld trials at commercial tobacco production sites. A general conclusion is that these lines were satisfactory for leaf tobacco production and that trials are worth repeating. Regarding the reduction of secondary and tertiary lateral shoots, the labor required for removal of lateral shoots in a preliminary survey was observed. Although cold sensitivity at the seedling stage and some abnormalities of owers that decreased seed yield were observed in the mutants of NtLs, these characteristics were not regarded as di culties likely to affect leaf tobacco production practices.
Therefore, the 'Coker319' derivatives showed good potential as new tobacco varieties. They can relieve farmers from the di cult labor necessary for control of secondary and tertiary lateral shoots. In addition, breeding of other lines with mutations in NtLs or NtBl1, the combination of these mutations, and further studies of NtLs, NtBl1, NtREV, VE7, and VE12 genes are among important future tasks.

Conclusions
Development of tobacco lines with reduced lateral shoots, which is strongly desired by tobacco farmers, was attempted. Indeed, RNA interference of ve genes and the chemical mutation of three of them signi cantly suppressed lateral shoot development in tobacco in a greenhouse and in eld tests conducted at an experimental site. Two mutant lines were evaluated for agronomic performance in eld trials conducted at commercial production sites. Although primary lateral shoots did not decrease in the lines, the reduction of secondary and tertiary lateral shoots, which are especially cumbersome in tobacco production, was an important step forward.

BLAST analysis
Query genes for BLAST search of NCBI BLAST database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and a database of tobacco cDNA sequences accumulated by a number of in-house studies are shown in Table 1.

RNA sequencing (RNAseq)
Apical tissues of shoots were collected from seedlings of tobacco 'Petit Havana SR-1' at 29-37 days after germination and were xed in an ice-cold 3:1 solution of ethanol and acetic acid. Protocols for preparation of para n-embedded sections and laser-microdissection were described by Takahashi et al. [27].
Total RNA was extracted using a PicoPure TM RNA isolation Kit (Thermo Fisher Scienti c Inc.) and was examined using an Agilent 2100 Bioanalyzer and an RNA 6000 Pico kit (Agilent Technologies Inc.). Also, cDNA was synthesized using an oligo-dT primer with the T7 promoter sequence; then antisense RNA was transcribed by T7 RNA polymerase. Again, cDNA was synthesized from the antisense RNA using random primers. The secondary cDNA was sequenced by 454 GS FLX. The obtained reads were assembled by Newbler ver. 2.6.

Cloning of genes
After coding sequence information was obtained from databases (GenBank and the in-house cDNA database), 5′ or 3′-RACE was performed with a SMARTer RACE cDNA Ampli cation Kit (Clontech) to obtain full-length cDNA clones of the coding sequences. RNAs were extracted from a dormant axillary bud, shoot apex, ower bud, or root (Magtraton®; Precision System Science Co., Ltd.). Ampli ed fragments were sequenced with a capillary sequencer (3730xl DNA Analyzer, Thermo Fisher Scienti c Inc.) using a BigDye®v3.1 Cycle Sequencing Kit (Thermo Fisher Scienti c Inc.). DNA manipulations in this study were performed according to standard procedures [28].

RNAi
Trigger sequences were ampli ed using the primers listed in Supplemental Table S1 from the cloned cDNAs or RNA isolated from dormant axillary buds, shoot apices, ower buds or roots of tobacco 'Petit Havana SR-1' with Magtraton® technology. The ampli ed fragments were cloned into pENTR™⁄D-TOPO vector (Takara Bio) and were then introduced into a modi ed pHellsgate12 binary vector [29], which had an expression cassette of a green uorescence gene driven by the CaMV 35S promoter. The binary vectors were introduced into Agrobacterium tumefaciens strain LBA4404. Tobacco 'Petit Havana SR-1' was transformed with the resultant strains. Transgene locus number was determined by segregation ratios of the green uorescence in the T1 generation. Expression level of the target genes was measured using quantitative PCR. RNAs were prepared with Magtration® technology from leaves, roots or aboveground tissues excluding leaves. The cDNAs were synthesized using PrimeScript RT reagent kit with gDNA Eraser (Takara Bio). Real-time PCR was conducted from the cDNAs using StepOnePlus (Thermo Fisher Scienti c Inc.) and TaqMan Fast Advanced Master Mix (Thermo Fisher Scienti c Inc.). Primers and probes are presented in Supplemental Table S3. Elongation factor-1α (AF120093) was used as a reference gene.

Chemically induced mutants
A library of tobacco 'Tsukuba 1' mutated with ethyl methanesulfonate (EMS) constructed by Tajima et al. [30] was screened using the protocol described by Takakura et al. [31] with primers listed in Supplemental Table S4.

Plant cultivation
Tobacco plants were grown in soil pots (12 cm) in a greenhouse under natural day length at 25 °C or in a growth chamber under the cycle of 12 h light at 25 °C and 12 h dark at 18 °C, in an experimental eld at the Leaf Tobacco Research Center of Japan Tobacco Inc., or in elds in commercial tobacco production areas in southwest Japan, according to the respective standard cultivation practice. When ower buds emerged, the plants were topped off. The lateral shoots were removed and measured weekly for ve weeks in the growth chamber or for eight weeks in the eld after the topping. Leaf color was scored using a Leaf Color Chart 2019A for tobacco (Fujihira Industry, Supplemental Table S5). Funding Japan Tobacco Inc., with which all authors are a liated, is the sole funder of the research.

Availability of data and materials
The datasets generated or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate Not applicable.

Con ict of interest
All authors are a liated with Japan Tobacco Inc., a funder of this study.