Identification and Characterization of Secondary Wall-Associated NAC Genes and Their Involvement in Hormonal Responses in Tobacco (Nicotiana tabacum)

Secondary wall-associated NAC (SWN) genes are a subgroup of NAC (NAM, ATAF, and CUC) transcription factors (TF) that play a key role in regulating secondary cell wall biosynthesis in plants. However, this gene family has not been systematically characterized, and their potential roles in response to hormones are unknown in Nicotiana tabacum. In this study, a total of 40 SWN genes, of which 12 from Nicotiana tomentosiformis, 13 from Nicotiana sylvestris, and 15 from Nicotiana tabacum, were successfully identified. The 15 SWNs from Nicotiana tabacum were further classified into three groups, namely, vascular-related NAC domain genes (NtVNDs), NAC secondary wall thickening promoting factor genes (NtNSTs), and secondary wall-associated NAC domain genes (NtSNDs). The protein characteristic, gene structure, and chromosomal location of 15 NtSWNs (also named Nt1 to Nt15) were also analyzed. The NtVND and NtNST group genes had five conserved subdomains in their N-terminal regions and a motif (LP[Q/x] L[E/x] S[P/A]) in their diverged C- terminal regions. Some hormones, dark and low-temperature related cis-acting elements, were significantly enriched in the promoters of NtSWN genes. A comprehensive expression profile analysis revealed that Nt4 and Nt12 might play a role in vein development. Others might be important for stem development. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that in the NtNST group, genes such as Nt7, Nt8, and Nt13 were more sensitive than the genes in NtVND and NtSND groups under abiotic stress conditions. A transactivation assay further suggested that Nt7, Nt8, and Nt13 showed a significant transactivation activity. Overall, SWN genes were finally identified and characterized in diploid and tetraploid tobacco, revealing new insights into their evolution, variation, and homology relationships. Transcriptome, cis-acting element, qRT-PCR, and transactivation assay analysis indicated the roles in hormonal and stress responses, which provided further resources in molecular mechanism and genetic improvement.

Initial Characterization and Expression Pattern Analysis of Tobacco (Nicotiana Tabacum) HMGS Gene

3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS) is a member of condensing enzymes that catalyze a Claisen-like condensation reaction.The tobacco (nicotiana tabacum) HMGS gene was firstly characterized using the rapid amplification of cDNA ends methods based on one tobacco EST. The full-length tobacco HMGS gene mRNA was 1,773bp containing a 1389 bp open reading frame, which encodes a protein of 462 amino acids. Sequence analysis revealed that the HMGS of tobacco shares high homology with the HMGS of nicotiana tomentosiformis (96%), nicotiana attenuata (95%), Nicotiana sylvestris (95%), nicotiana benthamiana(94%), solanum lycopersicum(94%), solanum tuberosum(93%) and withania somnifera(93%). Results also showed that tobacco HMGS gene has a closer genetic relationship with the HMGS gene of withania somnifera. Tissue expression profile analysis revealed that the tobacco HMGS gene was highly expressed in flower, but moderately expressed in leaf and stem, and weakly expressed in root. Our experiment established the foundation for further research on this tobacco gene.

Isolation of the Histone Lysine Methyltransferase Gene PhSDG8 and Characterization of Function in Shoot Branching

Petunia (Petunia ×hybrida) is an important ornamental plant, and its branch development has become a hot research topic. In this study, PhSDG8, an ortholog of SET domain group 8 (SDG8), was cloned from the petunia cultivar Mitchell Diploid. It had an open reading frame (ORF) of 5070 bp and encoded 1689 amino acids, with Suppressor variegation 3–9, Enhancer of zeste, Trithorax (SET), Zinc finger-cysteine and tryptophan conserved (Zf-CW), associated with SET (AWS) and Post SET domains. The predicted amino acid sequence of PhSDG8 was most closely related to Nicotiana sylvestris ASHH2 (NsASHH2). Expression analysis revealed that PhSDG8 expressed highest in the stems and lowest in the axil. Subcellular localization analysis showed that PhSDG8 was localized in the nucleus. Overexpression of PhSDG8 reduced the branch number of Arabidopsis thaliana sdg8-2. The silencing of PhSDG8 resulted in an increase in the number of branches of petunia and significant upregulation of PhUGT74E2. These results suggested that PhSDG8 may be a candidate gene for regulating the branching of petunia.

Evolutionary and Predictive Functional Insights into the Aquaporin Gene Family in the Allotetraploid Plant Nicotiana tabacum

Aquaporins (AQPs) are a class of integral membrane proteins that facilitate the membrane diffusion of water and other small solutes. Nicotiana tabacum is an important model plant, and its allotetraploid genome has recently been released, providing us with the opportunity to analyze the AQP gene family and its evolution. A total of 88 full-length AQP genes were identified in the N. tabacum genome, and the encoding proteins were assigned into five subfamilies: 34 plasma membrane intrinsic proteins (PIPs); 27 tonoplast intrinsic proteins (TIPs); 20 nodulin26-like intrinsic proteins (NIPs); 3 small basic intrinsic proteins (SIPs); 4 uncharacterized X intrinsic proteins (XIPs), including two splice variants. We also analyzed the genomes of two N. tabacum ancestors, Nicotiana tomentosiformis and Nicotiana sylvestris, and identified 49 AQP genes in each species. Functional prediction, based on the substrate specificity-determining positions (SDPs), revealed significant differences in substrate specificity among the AQP subfamilies. Analysis of the organ-specific AQP expression levels in the N. tabacum plant and RNA-seq data of N. tabacum bright yellow-2 suspension cells indicated that many AQPs are simultaneously expressed, but differentially, according to the organs or the cells. Altogether, these data constitute an important resource for future investigations of the molecular, evolutionary, and physiological functions of AQPs in N. tabacum.

Multi-Component Antioxidative System and Robust Carbohydrate Status, the Essence of Plant Arsenic Tolerance

Arsenic (As) contaminates the food chain and decreases agricultural production through impairing plants, particularly due to oxidative stress. To better understand the As tolerance mechanisms, two contrasting tobacco genotypes: As-sensitive Nicotiana sylvestris and As-tolerant N.tabacum, cv. ‘Wisconsin’ were analyzed. The most meaningful differences were found in the carbohydrate status, neglected so far in the As context. In the tolerant genotype, contrary to the sensitive one, net photosynthesis rates and saccharide levels were unaffected by As exposure. Importantly, the total antioxidant capacity was far stronger in the As-tolerant genotype, based on higher antioxidants levels (e.g., phenolics, ascorbate, glutathione) and activities and/or appropriate localizations of antioxidative enzymes, manifested as reverse root/shoot activities in the selected genotypes. Accordingly, malondialdehyde levels, a lipid peroxidation marker, increased only in sensitive tobacco, indicating efficient membrane protection in As-tolerant species. We bring new evidence of the orchestrated action of a broad spectrum of both antioxidant enzymes and molecules essential for As stress coping. For the first time, we propose robust carbohydrate metabolism based on undisturbed photosynthesis to be crucial not only for subsidizing C and energy for defense but also for participating in direct reactive oxygen species (ROS) quenching. The collected data and suggestions can serve as a basis for the selection of plant As phytoremediators or for targeted breeding of tolerant crops.

WOX9 functions antagonistic to STF and LAM1 to regulate leaf blade expansion in Medicago truncatula and Nicotiana sylvestris

Plant specific WOX family transcription factors are known to regulate embryogenesis, meristem maintenance and lateral organ development. Modern clade WOX genes function through a transcriptional repression mechanism, and the intermediate clade transcriptional activator WOX9 functions with the repressor WOX genes in embryogenesis and meristems maintenance, but the mechanism of this interaction is unclear. WOX1 homologues STF and LAM1 are required for leaf blade outgrowth in Medicago truncatula and Nicotiana Sylvestris, respectively. Here we show that WOX9 negatively regulates leaf blade outgrowth and functions antagonistically to STF and LAM1. While NsWOX9 ectopic expression enhances the lam1 mutant phenotype, and antisense expression partially rescues the lam1 mutant, both overexpression of NsWOX9 and knockout by CRISPR/Cas9 genome editing in N. sylvestris resulted in a range of severe leaf blade distortions, indicating that controlled negative regulation by NsWOX9 is required for proper blade development. Our results indicate that direct repression of WOX9 transcriptional activation activity by the transcriptional repressor STF/LAM1 is required for correct blade architecture and patterning in M. truncatula and N. sylvestris. These findings suggest that a balance between transcriptional activation and repression mechanisms by direct interaction of activator and repressor WOX genes may be required for cell proliferation and differentiation homeostasis, and could be an evolutionarily conserved mechanism for the development of complex and diverse morphology in higher plants.One sentence summaryWOX9 negatively regulates blade outgrowth antagonizing STF function but directly repressed by STF indicating WOX-mediated homeostasis in cell proliferation and differentiation during leaf morphogenesis.