Tissue‐specific expression of barnase in tobacco delays axillary shoot development after topping (2021)

Shoot branching is an important agronomic trait that determines plant architecture and affects crop productivity (Shen et al., 2019). Molecular signals from the shoot apical meristem (SAM) create a hormonal environment that integrates with the expression of axillary bud-specific repressors such as BRANCHED1 (BRC1) to inhibit axillary shoot formation (Wang et al., 2019 and references therein). The signal is eliminated by topping (SAM removal), enabling the formation of new shoots (suckers) from axillary buds (Figure 1a). In tobacco (Nicotiana tabacum), topping is necessary to enhance leaf development/maturation before harvesting, but sucker growth after topping is undesirable because it reallocates resources to axillary buds, reducing yield and quality of the main leaves. Sucker growth can be inhibited by fatty alcohols, flumetralin or maleic hydrazide, but chemical control is time-consuming and expensive, and the chemicals may persist after leaf processing due to environmental variability (Bailey et al., 2019). Tobacco plants with delayed axillary bud initiation or shoot growth would therefore significantly improve harvest and/or product quality, as previously shown for other species (e.g., Groot et al., 1994). To analyse the transcriptomes of axillary meristems/buds from tobacco before and after topping, we grew plants in the greenhouse for 8 weeks and took seven samples (n = 3) including young leaf, SAM and axillary meristems/buds before and 2, 6, 24 and 72 h after topping, from plants with 8–10 fully expanded leaves. RNA was extracted for RNA-Seq analysis on an Illumina HiSeq 2000 device (100-bp single reads, at least 30 million reads per sample), and we identified 17 candidate genes that were deregulated in axillary buds post-topping (Figure 1b). Expression was validated by qPCR, and the six most promising genes were selected for further analysis. The corresponding promoters were analysed in the axillary meristem of a commercial dark tobacco before and after topping using a GUS reporter assay. Four of the promoters showed nonspecific activity, but gusA expression driven by promoters P#12.5kb and P#152.5kb was limited to the axillary meristem, with P#15 showing the more restricted spatial domain (Figure 1c). P#152.5kb::GUS activity was stable even 7 days post-topping, but GUS activity in the P#12.5kb::GUS transgenic lines declined shortly after topping. P#12.5kb sequence analysis revealed the presence of the sugar-repressible element TTATCCA (Tatematsu et al., 2005) at positions –2401 to –2407. Shortening P#12.5kb to 2.4 kb (P#12.4kb) did not change its axillary meristem specificity but prolonged its activity, so that GUS staining was still detected 10 days post-topping in P#12.4kb::GUS transgenic plants (Figure 1c). Gene#1 encoded a BRC1 homolog, and silencing enhanced sucker growth even before topping, as reported in other species, whereas strong overexpression driven by the constitutive CaMV35S promoter was lethal, allowing the regeneration of only one transgenic line with severely stunted growth (data not shown). Transgenic lines with weak gene#1 expression showed sucker development comparable to wild-type controls, and the expression of gene#1 driven by P#12.4kb only slightly reduced sucker growth (data not shown), probably reflecting endogenous regulation and/or a positive regulator of bud formation such as NtBRC2 (Ding et al., 2020). Gene#15 encoded a vicilin-like protein, and neither RNAi nor constitutive overexpression generated a notable phenotype (data not shown). To selectively inhibit axillary bud initiation and subsequent sucker growth, we expressed the cytotoxic ribonuclease barnase from the bacterium Bacillus amyloliquefaciens under the control of the axillary bud-specific promoters P#152.5kb and P#12.4kb in order to ablate the cells responsible for sucker formation. We initially generated 11 P#152.5kb::barnase transgenic plants, seven of which did not develop axillary bud primordia during vegetative growth. Next, we topped two lines (L10 and L11) and no axillary bud primordia were visible even 1 week post-topping (Figure 1d). These plants showed a normal phenotype, but axillary bud initiation was delayed by at least 3 weeks, resulting in fewer and shorter suckers with a weight reduction of 50%–79% even 4 weeks post-topping compared with wild-type controls (Figure 1d). Seeds from the remaining lines failed to germinate on selective medium, indicating that the P#152.5kb promoter (and thus barnase) is also active during seed formation/germination. We analysed the P#152.5kb::GUS transgenic lines again and confirmed weak GUS staining in the seeds (Figure 1d). Barnase expression driven by the P#12.4kb promoter also strongly inhibited sucker growth in T0 plants, and seven of an initial population of 21 plants produced no or only a few axillary bud primordia during vegetative growth (Figure 1e). Again, two lines (L55 and L57) were topped, revealing that sucker growth was delayed by at least 1 week. In contrast to the P#152.5kb::barnase plants, seeds from the P#12.4kb::barnase plants germinated, allowing comprehensive analysis of the T1 generation. Ten L7 and nine L23 plants were compared to 10 vector control plants, and again, no axillary bud primordia formed during vegetative growth in the transgenic plants (Figure 1e). After topping, axillary bud initiation in L7 plants was delayed by 2 weeks. Four weeks post-topping, all L7 plants formed fewer than eight, short suckers (vector control average ~16) with an average weight reduction of ~65% (Figure 1e). Axillary bud initiation was completely abolished in seven L23 plants even 4 weeks post-topping, and the other two plants produced fewer and shorter suckers with a reduced weight (Figure 1e). However, L23 plants were also smaller with thinner leaves than vector controls. Finally, we conducted a field study (Southern Piedmont AREC, Blackstone, Virginia) with 20 offspring each from three T1 parents of line L7. The T2 plants were cultivated from April to September 2019, and a comparative analysis of suckers 2 weeks post-topping revealed that 43% of the transgenic plants and the 10 wild-type controls formed >10 suckers (Figure 1f). However, 45% of the transgenic plants (27 of 60) formed ≤5 suckers and ~12% (7 of 60) formed 6–10 suckers, confirming the stability of the trait even under field conditions. In summary, we found that the axillary meristem-specific expression of barnase significantly delays and reduces sucker number, length and weight in tobacco after topping, adding to the body of knowledge on the control of axillary branching in plants. For tobacco field cultivation, our results may help to reduce the use of chemicals and the laborious work required for sucker control. The authors acknowledge Heike Hinte, Christiane Fischer, Thomas Schmelter, Michael Lahme and Andreas Wagner for technical assistance, and Dr. Richard M Twyman for manuscript editing. The authors are/were employees or contracted workers of Altria Client Services LLC, which funded the work. L.G., A.K., C.K., D.X., D.P. and G.N. designed the experiments L.G., J.M., F.B., M.J., Y.S., C.K. and J.Y. conducted the experiments. L.G., A.K., Y.S., C.K. and J.Y.: analysed the data. Y.S., C.K., D.X., J.Y., U.W. and J.S. contributed to reagents, materials and analysis tools. L.G., A.K. and G.N. wrote the manuscript. All authors revised the manuscript and approved the submitted version.