AUTHOR=Yu Jing , Lei Bo , Zhao Huina , Wang Bing , Kakar Kaleem U. , Guo Yushuang , Zhang Xiaolian , Jia Mengao , Yang Hui , Zhao Degang 

TITLE=Cloning, characterization and functional analysis of NtMYB306a gene reveals its role in wax alkane biosynthesis of tobacco trichomes and stress tolerance

JOURNAL=Frontiers in Plant Science

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.1005811

DOI=10.3389/fpls.2022.1005811

ISSN=1664-462X

ABSTRACT=<p>Trichomes are specialized hair-like organs found on epidermal cells of many terrestrial plants, which protect plant from excessive transpiration and numerous abiotic and biotic stresses. However, the genetic basis and underlying mechanisms are largely unknown in <italic>Nicotiana tabacum</italic> (common tobacco), an established model system for genetic engineering and plant breeding. In present study, we identified, cloned and characterized an unknown function transcription factor <italic>NtMYB306a</italic> from tobacco cultivar K326 trichomes. Results obtained from sequence phylogenetic tree analysis showed that <italic>NtMYB306a</italic>-encoded protein belonged to S1 subgroup of the plants’ R2R3-MYB transcription factors (TFs). Observation of the green fluorescent signals from NtMYB306a-GFP fusion protein construct exhibited that NtMYB306a was localized in nucleus. In yeast transactivation assays, the transformed yeast containing pGBKT7-NtMYB306a construct was able to grow on SD/-Trp-Ade+X-α-gal selection media, signifying that NtMYB306a exhibits transcriptional activation activity. Results from qRT-PCR, <italic>in-situ</italic> hybridization and GUS staining of transgenic tobacco plants revealed that <italic>NtMYB306a</italic> is primarily expressed in tobacco trichomes, especially tall glandular trichomes (TGTs) and short glandular trichomes (SGTs). RNA sequencing (RNA-seq) and qRT-PCR analysis of the <italic>NtMYB306a</italic>-overexpressing transgenic tobacco line revealed that <italic>NtMYB306a</italic> activated the expression of a set of key target genes which were associated with wax alkane biosynthesis. Gas Chromatography<italic>–</italic>Mass Spectrometry (GC-MS) exhibited that the total alkane contents and the contents of <italic>n</italic>-C28, <italic>n</italic>-C29, <italic>n</italic>-C31, and <italic>ai</italic>-C31 alkanes in leaf exudates of <italic>NtMYB306a</italic>-OE lines (OE-3, OE-13, and OE-20) were significantly greater when compared to WT. Besides, the promoter region of <italic>NtMYB306a</italic> contained numerous stress-responsive <italic>cis</italic>-acting elements, and their differential expression towards salicylic acid and cold stress treatments reflected their roles in signal transduction and cold-stress tolerance. Together, these results suggest that <italic>NtMYB306a</italic> is necessarily a positive regulator of alkane metabolism in tobacco trichomes that does not affect the number and morphology of tobacco trichomes, and that it can be used as a candidate gene for improving stress resistance and the quality of tobacco.</p>