Main conclusion Provide an evolutionary and an empirical molecular genetic foundation of the Sus gene family in tobacco and will be beneficial for further investigations of Sus gene functions Sucrose synthase (Sus) has been well characterized as the key enzyme participating in sucrose metabolism, and the gene family encoding different Sus isozymes has been cloned and characterized in several plant species. However, scant information about this gene family is available to date in tobacco. Here, we identified 14, 6, and 7 Sus genes in the genomes of Nicotiana tabacum, N. sylvestris and N. tomentosiformis, respectively. These tobacco Sus family members shared high levels of similarity in their nucleotide and amino acid sequences. Phylogenetic analysis revealed distinct evolutionary paths for the tobacco Sus genes. Sus1–4, Sus5, and Sus6–7 originated from three Sus precursors, respectively, which were generated by duplication before the split of monocots and eudicots. There were two additional duplications, before and after the differentiation of the Solanaceae, which separately gave rise to Sus3/4 and Sus1/2. Gene exon/intron structure analysis showed that the tobacco Sus genes contain varying numbers of conserved introns, resulting from intron loss under different selection pressures during the course of evolution. The expression patterns of the NtSus genes differed from each other in various tobacco tissues. Transcripts of Ntab0259170 and Ntab0259180 were detected in leaves at all tested developmental stages, suggesting that these two genes play a predominant role in sucrose metabolism during leaf development. Expression of Ntab0288750 and Ntab0234340 were conspicuously induced by low temperature and virus treatment, indicating that these two isozymes are important in meeting the increased glycolytic demand that occurs during abiotic stress. Our results provide an evolutionary and an empirical molecular genetic foundation of the Sus gene family in tobacco, and will be beneficial for further investigations of Sus gene functions in the processes of tobacco leaf development and tobacco resistance to environmental stresses.

Abscisic acid (ABA) is an important phytohormone for plant growth, development and responding to stresses such as drought, salinity, and pathogen infection. Pyrabactin Resistance 1 (PYR1)/PYR1-Like (PYL)/Regulatory Component of ABA Receptor (RCAR) (hereafter referred to as PYLs) has been identified as the ABA receptors. The PYL family members have been well studied in many plants. However, the members of PYL family have not been systematically identified at genome level in cultivated tobacco (Nicotiana tabacum) and its two ancestors. In this study, the phylogenic relationships, chromosomal distribution, gene structures, conserved motifs/regions, and expression profiles of NtPYLs were analyzed. We identified 29, 11, 16 PYLs in the genomes of allotetraploid N. tabacum, and its two diploid ancestors N. tomentosiformis and N. sylvestris, respectively. The phylogenetic analysis revealed that NtPYLs can be divided into three subfamilies, and each NtPYL has one counterpart in N. sylvestris or N. tomentosiformis. Based on microarray analysis of NtPYL transcripts, four NtPYLs (from subfamily II, III), and five NtPYLs (from subfamily I) are highlighted as potential candidates for further functional characterization in N. tabacum seed development, response to ABA, and germination, and resistance to abiotic stresses, respectively. Interestingly, the expression profiles of members in the same NtPYL subfamily showed somehow similar patterns in tissues at different developmental stages and in leaves of seedlings under drought stress, suggesting particular NtPYLs might have multiple functions in both plant development and drought stress response. NtPYLs are highlighted for important functions in seed development, germination and response to ABA, and particular in drought tolerance. This work will not only shed light on the PYL family in tobacco, but also provides some valuable information for functional characterization of ABA receptors in N. tabacum.

The HD-ZIP Ⅳ transcription factors have been identified and functional characterized in many plant species. However, no tobacco HD-ZIP IV gene has been isolated, and it is not yet known whether HD-ZIP IV genes are involved in controlling flavonols accumulation in plants. Here, we cloned a HD ZIP gene named NtHDG2 from Nicotiana tabacum, which belongs to the class IV of HD-ZIP family, and the NtHDG2-GFP fusion protein is localized to the nucleus. We further observed that the flavonols contents in the NtHDG2 overexpression leaves increase to 1.9-4.5 folds of that in WT plants, but in the NtHDG2-RNAi plants the flavonols contents reduce to 20.9%-52.7% of that in WT plants. The transcriptions of one regulatory gene NtMYB12, and three structural genes (NtPAL, NtF3'H, NtF3GT), contributing to flavonols biosynthesis, were significantly induced by NtHDG2. However, the transcription level of NtNAC002, a flavonols biosynthesis repressor, was also significantly up-regulated in NtHDG2-overexpression lines, but significantly down-regulated in the RNAi lines, indicating that HDG2 regulates the synthesis of flavonols as a complex regulatory network. Moreover, ectopic expression of NtHDG2 gene promoted the transcription of several AP2/ERF genes, including NtERF1-5, NtERF109, NtDREB1, and NtCIPK11, which participate in regulating root development and resistance to abiotic stresses. Our findings reveal the new function of HD-ZIP IV transcription factors in flavonoids biosynthesis, and indicate that HD-ZIP IV members may play an important role in plant resistance to abiotic stress. The NtHDG2 gene provides a promising target for genetically manipulating to increase the amounts of flavonols in tobacco leaves.