A heat stress transcription factor LlHSFA1 in lily and its relationship with LlHSFA2 was investigated, and its function in enhancing thermotolerance was confirmed by analyzing transgenic Arabidopsis thaliana overexpressed LlHSFA1. A large family of heat stress transcription factors that are involved in the heat stress response in plants can induce the expression of multiple genes related to thermotolerance including heat-shock proteins. In this study, a novel class A1 HSF named LlHSFA1 was isolated from leaves of lily (Lilium longiflorum cv. 'White Heaven') using the rapid amplification of cDNA ends technique. Analysis of the deduced amino acid sequence and construction of a phylogenetic tree showed that LlHSFA1 contained five critical domains and motifs and belonged to the A1 family of HSFs. Following the heat treatment of lily leaves, transcription of LlHSFA1 was induced to a varying extent, related to the time of measurement. The induced expression peak of LlHSFA1 occurred prior to that of LlHSFA2, during the early phase of heat stress. Following transient expression of LlHSFA1 in Nicotiana benthamiana, LlHSFA1 was found to be localized in both the nucleus and the cytoplasm. Analysis using bimolecular fluorescence complementation and a yeast two-hybrid assay demonstrated that LlHSFA1 could interact with LlHSFA2. Use of a yeast one-hybrid assay confirmed that LlHSFA1 had transcriptional activation activity. In transgenic Arabidopsis lines overexpressing LlHSFA1 under unstressed conditions, the expression of some putative target genes was up-regulated, in comparison with expression in wild-type plants, and furthermore, the thermotolerance of the transgenic lines was enhanced. Overall, LlHSFA1 was demonstrated to play an important role in the heat stress response of lily and to be a novel candidate gene for application in lily breeding, using genetic modification approaches.

The phytohormone abscisic acid (ABA) regulates plant development and is crucial for abiotic stress response. In this study, cold storage contributes to reducing endogenous ABA content, resulting in dormancy breaking of Gladiolus. The ABA inhibitor fluridone also promotes germination, suggesting that ABA is an important hormone that regulates corm dormancy. Here, we report the identification and functional characterization of the Gladiolus ABI5 homolog (GhABI5), which is a basic leucine zipper motif transcriptional factor (TF). GhABI5 is expressed in dormant vegetative organs (corm, cormel, and stolon) as well as in reproductive organs (stamen), and it is up-regulated by ABA or drought. Complementation analysis reveals that GhABI5 rescues the ABA insensitivity of abi5-3 during seed germination and induces the expression of downstream ABA response genes in Arabidopsis thaliana (EM1, EM6, and RD29B). Down-regulation of GhABI5 in dormant cormels via virus induced gene silence promotes sprouting and reduces the expression of downstream genes (GhLEA and GhRD29B). The results of this study reveal that GhABI5 regulates bud dormancy (vegetative organ) in Gladiolus in addition to its well-studied function in Arabidopsis seeds (reproductive organ).

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GhNPR1 shares similar functions as Arabidopsis NPR1 . Silencing of GhNPR1 in Gladiolus results in an enhanced susceptibility to Curvularia gladioli. We propose that GhNPR1 plays important roles in plant immunity. Gladiolus plants and corms are susceptible to various types of pathogens including fungi, bacteria and viruses. Understanding the innate defense mechanism in Gladiolus is a prerequisite for the development of new protection strategies. The non-expressor of pathogenesis-related gene 1 (NPR1) and bzip transcription factor TGA2 play a key role in regulating salicylic acid (SA)-mediated systemic acquired resistance (SAR). In this study, the homologous genes, GhNPR1 and GhTGA2, were isolated from Gladiolus and functionally characterized. Expression of GhNPR1 exhibited a 3.8-fold increase in Gladiolus leaves following salicylic acid treatment. A 1332 bp fragment of the GhNPR1 promoter from Gladiolus hybridus was identified. Inducibility of the GhNPR1 promoter by SA was demonstrated using transient expression assays in the leaves of Nicotiana benthamiana. The GhNPR1 protein is located in the nucleus and cytomembrane. GhNPR1 interacts with GhTGA2, as observed using the bimolecular fluorescence complementation system. Overexpression of GhNPR1 in an Arabidopsis npr1 mutant can restore its basal resistance to Pseudomonas syringae pv. tomato DC3000. Silencing of GhNPR1, using a tobacco rattle virus-based silencing vector, resulted in an enhanced susceptibility to Curvularia gladioli. In conclusion, these results suggest that GhNPR1 plays a pivotal role in the SA-dependent systemic acquired resistance in Gladiolus.

Understanding corm development in flower bulbs is of importance for securing the quality of cut flowers and propagation of commercial stocks. Gladiolus is one of the most popular bulb plants worldwide. Its corm development is characterized by starch accumulation. Previous research has shown that phytohormones (especially gibberellin (GA)) are involved in tuber development. However, the relationship between abscisic acid (ABA)/GA and starch during corm development remains unclear. To gain deeper insights into the biological process of corm development, we performed a detailed anatomical characterization of different stages of corm development and analyzed phytohormone levels. Our study showed that corm development is linked to hormones (ABA and GA) and carbohydrates (sucrose and starch). Exogenous hormone treatment and silencing of endogenous hormone biosynthesis genes indicated that ABA positively regulates corm development, while GA acts as an antagonist of ABA function. A sucrose synthase gene (GhSUS2) was shown to be involved in the antagonism between ABA and GA. GhSUS2 was upregulated by ABA and downregulated by GA. The increase in the transcript level of GhSUS2 coincided with the development of corm/cormels. Silencing of GhSUS2 repressed corm development and starch accumulation. In conclusion, we propose that GhSUS2, an essential enzyme in sucrose degradation, is differentially regulated by ABA and GA and controls corm development in Gladiolus.

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