Mechanism Analysis Of Response To Heat Stress Of LIHSFA1and Its Downstream LIHSP70from Lily (Lilium Longiflorum)

As one of the most important ornamental plants worldwide, lilies are well adapted to cool and humid environments at temperatures of around16～24℃; however, lilies grown in most parts of China frequently suffer from high temperatures in the summer, which result in diminished cut-flower quality and in degeneration of the bulb, and which consequently restrict the year-round production of lilies as cut flowers. Improvement of thermotolerance in Lily is therefore a key approach to resolving this problem. Under the high temperature condition, the expression level of heat stress-responsive genes are enhanced, of which heat stress transcription factors (HSFs) and heat shock proteins (HSPs) play a main and positive role in the thermotolerant stress. Research on heat-responsive mechanism that HSFs-HSPs are involved in has been a basic research on revealing thermotolerance of plants. In the present study, LIHSFA1and LIHSP70were isolated from leaves of Lilium longiforum ’White Heaven’ that shows higher thermotolerance. We further analyzed their biochemical characteristics, biological functions and relationship responsive to heat stress. The results are shown as below.1. LlHSFA1belongs to the HSFA1subgroup members. A novel class A1HSF named LlHSFA1in full length with1,587bp ORF encoding a protein of528amino acid residues was isolated from leaves of Lilium longiforum ’White Heaven’ using the rapid amplification of cDNA ends technique. The deduced amino acid sequence of LlHSFA1contained five typical domains and motifs of HSF, and showed a considerably greater degree of similarity to and a closest genetic relationship with OsHSFAla.2. LlHSFA1was induced by high temperature and other stresses. The expression level of LlHSFA1was tested using quantitative real-time PCR. Under the normal conditions, LlHSFA1was expressed constitutively; however, under the continues heat stress at42℃, LlHSFA1expression in leaves showed an alternating saw-tooth pattern of rise and fall. A peak of expression (about4.3-fold greater than the control level) was observed at2h after heat treatment. Moreover, LlHSFA1was induced intensely by mannitol and NaCl.3. LlHSFA1is a nuclear-and cytoplasmic-localized protein, and possesses transcriptional activation activity. Following transient expression of LlHSFA1in onion(Allium cepa)and tobacco (Nicotiana benthamiana) epidermal cells, LlHSFA1was found to be localized in both the nucleus and the cytoplasm. Use of a yeast one-hybrid assay confirmed that LlHSFA1had transcriptional activation activity.4. LlHSFA1may be an upstream factor of LlHSFA2, and both of them can interact with each other. During the early phase of heat stress, the induction of expression of LlHSFA1occurred prior to that of LIHSFA2. We therefore conjectured that LlHSFA1might lie up-stream of LIHSFA2. Analysis using bimolecular fluorescence complementation and a yeast two-hybrid assay demonstrated that LlHSFA1could interact with LlHSFA2.5. LlHSFA1-overexpressive Arabidopsis shows improvement of thermotolerance in transgenic plants. The function of LlHSFA1in heat tolerance was further investigated through ectopic overexpression of LlHSFA1in Arabidopsis. Three representative lines with higher and different expression levels were selected for following experiments. Under heat stress conditions, overexpressing LIHSFA1in Arabidopsis enhances obviously thermotolerance in transgenic plants.6. LlHSP70belongs to a HSP70protein of cytoplasm subtype. A heat shock protein70gene named LlHSP70whose ORF was a1,938bp encoding a stabilized protein of645amino acid residues was cloned from leaves of lily (L. longiforum cv.’White Heaven’). The amino acid sequence analysis indicated that the LlHSP70protein contained three highly conserved signatures of HSP70family of eucaryon and a characteristic motif of EEVD at the C-terminal. A phylogenetic tree showed that LlHSP70was matched best with TaHSP70of Triticum aestivum.1. LlHSP70was induced by high temperature and might be a downstream gene of LlHSFA1in the pathway of heat stress signal transduction. Transient expression analysis of LIHSP70in onion(Allium cepa)epidermal cells indicated that the GFP fluorescence of LlHSP70-GFP was located in both the nucleus and the cytoplasm. A yeast two-hybrid assay confirmed that LlHSP70could interact with LlHSFA1.8. Overexpressing LIHSP70in Arabidopsis enhances thermotolerance in transgenic plants. The biological role of LIHSP70was further analyzed by overexpressing LIHSP70in Arabidopsis. Three transgenic lines with higher and different expression levels of LlHSP70were selected for following investigation. After heat stress, three lines showed higher survival rate compared to wild type plants, which suggested that overexpression of LlHSP70in Arabidopsis improved the thermotolerance of transgenic plants.Taken together, LlHSFA1and LlHSP70are both the important components in the heat signaling transduction in lily, regulating the thermotolerance of lily. The present work provides new insights into the mechanism of heat stress transcription factor and heat shock protein in regulation of lily thermotolerance.