BLAST search
Two groups of tobacco genes were examined for effects of RNAi knock-down on lateral shoot formation. The first 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 (T-genome) [22–24], both S- and T-alleles were identified 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 first group of the genes, the tobacco homologues, are also shown in Table 2. Among them, genes highly and specifically expressed in EA or VE tissue were NtLs and NtBl1. In all three samples, NtREV was highly expressed. The other homologues were either not specifically 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 sufficient for simultaneous knock-down of two alleles recommended by Parrish et al. [26]. In this process, it was possible to find 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 specifically.
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 five 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 five 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 identified 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 S- and T- genomes were combined by crossing the mutants, producing double recessive lines designated as SAS lines (Table 3). Because nonsense mutations were not identified in VE7 and VE12, these genes were not studied further.
The SAS lines, 15 plants each, were planted in an experimental field and were examined for lateral shoot development. Again, primary lateral shoots were not suppressed significantly 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 field 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 field 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 flue-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 significant differences were found as explained hereinafter. Coker319-ls-1 was earlier in days to flower, 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 confirmed 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 flowers of SAS-ls-1, SAS-ls-2, and Coker319-ls-1 plants were split (Fig. 2i) in greenhouses and in the field. In severe cases, pistils and stamens were completely exposed (Fig. 2j). Seed yield from these flowers 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 field trials (Supplemental Fig. S2). The seed yield from the flowers of Coker319-bl1-1 was 72% of that of 'Coker319' (Supplemental Fig. S1).