JA/ethylene and NaWRKY6/3 regulated Alternaria resistance depends on ethylene response factor 1B-like in wild tobacco.

Biosynthesis of the phytoalexins scopoletin and scopolin in Nicotiana species is regulated by upstream signals including jasmonate (JA), ethylene (ET) and NaWRKY3 in response to the necrotrophic fungus Alternaria alternata, which causes brown spot disease. However, how these signals are coordinated to regulate these phytoalexins remains unknown. By analyzing RNA sequencing data and RNA interference, we identified NaERF1B-like (NaERF1B-L) as a key player in Nicotiana attenuata during A. alternata infection by regulating the transcripts of Feruloyl-CoA 6'-hydroxylase 1 (NaF6'H1), encoding a key enzyme for scopoletin biosynthesis, and NaVS1-like (NaVS1-L), a putative biosynthetic gene of the phytoalexin solavetivone. We further demonstrated that the synergistic induction of these two genes by JA and ET signaling is mediated by NaERF1B-L. Additionally, we found that the two closely related proteins NaWRKY6 and NaWRKY3 physically interact to enhance NaERF1B-L expression by directly binding and activating the NaERF1B-L promoter. Collectively, our current results demonstrate that NaERF1B-L plays a positive role in resistance to A. alternata by modulating phytoalexins biosynthesis through the integration of JA/ET and NaWRKY6/3 signaling. Our findings reveal a fine-tuned transcriptional regulatory hierarchy mediated by NaERF1B-L for brown spot disease resistance in wild tobacco.

Heterologous Expression of Platycodon grandiflorus PgF3'5'H Modifies Flower Color Pigmentation in Tobacco.

Flavonoid-3',5'-hydroxylase (F3'5'H) is the key enzyme for the biosynthesis of delphinidin-based anthocyanins, which are generally required for purple or blue flowers. Previously, we isolated a full-length cDNA of PgF3'5'H from Platycodon grandiflorus, which shared the highest homology with Campanula medium F3'5'H. In this study, PgF3'5'H was subcloned into a plant over-expression vector and transformed into tobacco via Agrobacterium tumefaciens to investigate its catalytic function. Positive transgenic tobacco T0 plants were obtained by hygromycin resistance screening and PCR detection. PgF3'5'H showed a higher expression level in all PgF3'5'H transgenic tobacco plants than in control plants. Under the drive of the cauliflower mosaic virus (CaMV) 35S promoter, the over-expressed PgF3'5'H produced dihydromyricetin (DHM) and some new anthocyanin pigments (including delphinidin, petunidin, peonidin, and malvidin derivatives), and increased dihydrokaempferol (DHK), taxifolin, tridactyl, cyanidin derivatives, and pelargonidin derivatives in PgF3'5'H transgenic tobacco plants by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis, resulting in a dramatic color alteration from light pink to magenta. These results indicate that PgF3'5'H products have F3'5'H enzyme activity. In addition, PgF3'5'H transfer alters flavonoid pigment synthesis and accumulation in tobacco. Thus, PgF3'5'H may be considered a candidate gene for gene engineering to enhance anthocyanin accumulation and the molecular breeding project for blue flowers.

Transcriptome Analysis of Key Genes Involved in Color Variation between Blue and White Flowers of Iris bulleyana.

Iris bulleyana Dykes (Southwest iris) is an extensively distributed Iridaceae species with blue or white flowers. Hereby, we performed a systematic study, employing metabolomics and transcriptomics to uncover the subtle color differentiation from blue to white in Southwest iris. Fresh flower buds from both cultivars were subjected to flavonoid/anthocyanin and carotenoid-targeted metabolomics along with transcriptomic sequencing. Among 297 flavonoids, 24 anthocyanins were identified, and 13 showed a strong down-accumulation pattern in the white flowers compared to the blue flowers. Significant downregulation of 3GT and 5GT genes involved in the glycosylation of anthocyanins was predicted to hinder the accumulation of anthocyanins, resulting in white coloration. Besides, no significant altered accumulation of carotenoids and expression of their biosynthetic genes was observed between the two cultivars. Our study systematically addressed the color differentiation in I. bulleyana flowers, which can aid future breeding programs.

Phylogenetic Analysis of Wild Species and the Maternal Origin of Cultivars in the Genus Lilium Using 114 Plastid Genomes.

Lilies are one of the most important ornamental flowers worldwide with approximately 100 wild species and numerous cultivars, but the phylogenetic relationships among wild species and their contributions to these cultivars are poorly resolved. We collected the major Lilium species and cultivars and assembled their plastome sequences. Our phylogenetic reconstruction using 114 plastid genomes, including 70 wild species representing all sections and 42 cultivars representing six hybrid divisions and two outgroups, uncovered well-supported genetic relationships within Lilium. The wild species were separated into two distinct groups (groups A and B) associated with geographical distribution, which further diversified into eight different clades that were phylogenetically well supported. Additional support was provided by the distributions of indels and single-nucleotide variants, which were consistent with the topology. The species of sections Archelirion, Sinomartagon III, and Leucolirion 6a and 6b were the maternal donors for Oriental hybrids, Asiatic hybrids, Trumpet hybrids, and Longiflorum hybrids, respectively. The maternal donors of the OT hybrids originated from the two sections Archelirion and Leucolirion 6a, and LA hybrids were derived from the two sections Leucolirion 6b and Sinomartagon. Our study provides an important basis for clarifying the infrageneric classification and the maternal origin of cultivars in Lilium.

Integrating physiological and metabolites analysis to identify ethylene involvement in petal senescence in Tulipa gesneriana.

Flower senescence is classified into ethylene-dependent and ethylene-independent manners and determines the flower longevity which is valuable for ornamental plants. However, the manner of petal senescence in tulip is still less defined. In this study, we characterized the physiological indexes in the process of petal senescence, as well as metabolic and ethylene responses in tulip cultivar 'American Dream', and further identified the role of ethylene biosynthesis genes TgACS by transgenic and transient assays. Primary metabolites profiling revealed that sugars, amino acids and organic acids preferentially accumulated in senescent petals. Additionally, senescence-associated genes were identified and significantly up-regulated, coupled with increased ROS contents, rapid water loss and accelerated cell membrane breakdown. Moreover, ethylene production was stimulated as evidenced by increasing in ACS activity and ethylene biosynthesis-related genes expression. Exogenous treatment of cutting flowers with 1-MCP or ethephon resulted in delayed or enhanced petal senescence, respectively. Transient down-regulation of TgACS by VIGS assay in tulip petals delayed senescence, while over-expressed TgACS1 in tobacco promoted leaf senescence. Taken together, this study provides evidences to certify ethylene roles and TgACS functions during flower senescence in tulip.