Yue Zhang

Our previous studies have indicated that a partial NAC domain protein gene is strongly up-regulated by cold stress (4 °C) in tiger lily (Lilium lancifolium). In this study, we cloned the full-length of this NAC gene, LlNAC2, to further investigate the function of LlNAC2 in response to various abiotic stresses and the possible involvement in stress tolerance of the tiger lily plant. LlNAC2 was noticeably induced by cold, drought, salt stresses, and abscisic acid (ABA) treatment. Promoter analysis showed that various stress-related cis-acting regulatory elements were located in the promoter of LlNAC2; and the promoter was sufficient to enhance activity of GUS protein under cold, salt stresses and ABA treatment. DREB1 (dehydration-responsive binding protein1) from tiger lily (LlDREB1) was proved to be able to bind to the promoter of LlNAC2 by yeast one-hybrid (Y1H) assay. LlNAC2 was shown to physically interact with LlDREB1 and zinc finger-homeodomain ZFHD4 from the tiger lily (LlZFHD4) by bimolecular fluorescence complementation (BiFC) assay. Overexpressing LlNAC2 in Arabidopsis thaliana showed ABA hypersensitivity and enhanced tolerance to cold, drought, and salt stresses. These findings indicated LlNAC2 is involved in both DREB/CBF-COR and ABA signaling pathways to regulate stress tolerance of the tiger lily.

Background: The LA hybrid lily ‘Aladdin’ has both excellent traits of Longiflorum hybrids and Asiatic hybrids—such as big and vivid flower, strong stem, high self-propagation coefficient, and shorter low temperature time required to release bulb dormancy in contrast to Oriental hybrids. A genome-wide transcriptional analysis using transcriptome RNA-Seq was performed in order to explore whether there is a gibberellin floral induction pathway in the LA hybrid lily. Subsequently, gene co-expression network analysis was used to analyze the possible interactions of key candidate genes screened from transcriptome data. At the same time, a series of physiological, biochemical, and cultivation tests were carried out. Results: The content of five endogenous hormones changed sharply in the shoot apex during the treatment of 200 mg/L exogenous gibberellin and the ratio of ABA/GA₃ dropped and stayed at a lower level after 4 hours’ treatment from the higher levels initially, reaching a dynamic balance. In addition, the metabolism of carbohydrates in the bulbs increase during exogenous gibberellin treatment. A total of 124,041 unigenes were obtained by RNA-seq. With the transcriptome analysis, 48,927 unigenes and 48,725 unigenes respectively aligned to the NR database and the Uniprot database. 114,138 unigenes, 25,369 unigenes, and 19,704 unigenes respectively aligned to the COG, GO, and KEGG databases. 2148 differentially expression genes (DEGs) were selected with the indicators RPKM ≥ 0, FDR ≤ 0.05 and |log2(ratio)| ≥ 2. The number of the upregulated unigenes was significantly more than the number of the downregulated unigenes. Some MADS-box genes related to flowering transformation—such as AGL20, SOC1, and CO—were found to be upregulated. A large number of gibberellin biosynthesis related genes such as GA2ox, GA3ox, GA20ox, Cytochrome P450, CYP81, and gibberellin signal transduction genes such as DELLA, GASA, and GID1 were significantly differentially expressed. The plant hormones related genes such as NCED3 and sugar metabolism related genes such as α-amylase, sucrose synthase hexokinase, and so on were also found expressing differentially. In addition, stress resistance related genes such as LEA1, LEA2, LEA4, serine/threonine protein kinase, LRR receptor-like serine/threonine protein kinase, P34 kinase, histidine kinase 3 and epigenetic related genes in DNA methylation, histone methylation, acetylation, ubiquitination of ribose were also found. Particularly, a large number of transcription factors responsive to the exogenous gibberellin signal including WRKY40, WRKY33, WRKY27, WRKY21, WRKY7, MYB, AP2/EREBP, bHLH, NAC1, NAC2, and NAC11 were found to be specially expressing. 30 gene sequences were selected from a large number of differentially expressed candidate genes for qRT-PCR expression verification (0, 2, 4, 8, and 16 h) and compared with the transcriptome expression levels. Conclusions: 200mg/L exogenous GA₃ can successfully break the bulb’s dormancy of the LA hybrid lily and significantly accelerated the flowering process, indicating that gibberellin floral induction pathway is present in the LA lily ‘Aladdin’. With the GCNs analysis, two second messenger G protein-coupled receptor related genes that respond to gibberellin signals in the cell were discovered. The downstream transport proteins such as AMT, calcium transport ATPase, and plasma membrane ATPase were also discovered participating in GA signal transduction. Transcription factors including WRKY7, NAC2, NAC11, and CBF specially regulated phosphorylation and glycosylation during the ubiquitination degradation process of DELLA proteins. These transcription factors also activated in abscisic acid metabolism. A large number of transcription factors such as WRKY21, WRKY22, NAC1, AP2, EREB1, P450, and CYP81 that both regulate gibberellin signaling and low-temperature signals have also been found. Finally, the molecular mechanism of GA floral induction pathway in the LA hybrid lily ‘Aladdin’ was constructed.