WRKY transcription factors (TFs) are of great importance in plant responses to different abiotic stresses. However, research on their roles in the regulation of thermotolerance remains limited. Here, we investigated the function of LlWRKY39 in the thermotolerance of lily (Lilium longiflorum ‘white heaven’). According to multiple alignment analyses, LlWRKY39 is in the WRKY IId subclass and contains a potential calmodulin (CaM)-binding domain. Further analysis has shown that LlCaM3 interacts with LlWRKY39 by binding to its CaM-binding domain, and this interaction depends on Ca2+. LlWRKY39 was induced by heat stress (HS), and the LlWRKY39-GFP fusion protein was detected in the nucleus. The thermotolerance of lily and Arabidopsis was increased with the ectopic overexpression of LlWRKY39. The expression of heat-related genes AtHSFA1, AtHSFA2, AtMBF1c, AtGolS1, AtDREB2A, AtWRKY39, and AtHSP101 was significantly elevated in transgenic Arabidopsis lines, which might have promoted an increase in thermotolerance. Then, the promoter of LlMBF1c was isolated from lily, and LlWRKY39 was found to bind to the conserved W-box element in its promoter to activate its activity, suggesting that LlWRKY39 is an upstream regulator of LlMBF1c. In addition, a dual-luciferase reporter assay showed that via protein interaction, LlCaM3 negatively affected LlWRKY39 in the transcriptional activation of LlMBF1c, which might be an important feedback regulation pathway to balance the LlWRKY39-mediated heat stress response (HSR). Collectively, these results imply that LlWRKY39 might participate in the HSR as an important regulator through Ca2+-CaM and multiprotein bridging factor pathways.

Chinese medicinal herbs have a similar appearance and are easily confused, complicating identification via traditional methods. This study provided a scientific approach, based on DNA barcoding, to accurately and rapidly identify Anoectochilus roxburghii and its adulterants. This technology complements traditional methods of identification of medicinal herbs. A comparison of the DNA barcodes matK, psbA-trnH and ITS2 was performed to verify that the ITS2 sequence is an effective marker for rapidly and accurately identifying A. roxburghii and its closely related species. Genomic DNA extracted from A. roxburghii and its adulterants were used as templates and the ITS2 sequence was amplified using PCR amplification and sequencing. Species identification was conducted using BLAST1 and neighbor-joining trees. The 12 samples were successfully classified into four species based on the ITS2 sequence. The ITS2 sequence length of A. roxburghii was 253 bp. The average intra-specific genetic distance of A. roxburghii was 0.0021, markedly lower than the inter-specific genetic distance between A. roxburghii and its adulterants (0.0380). Our findings illustrate that ITS2 sequence can accurately and efficiently distinguish A. roxburghii and its adulterants. In addition, the results provided reference for molecular identification of other Chinese herbal medicine.

The NTL (NAC with transmembrane motif 1-like) transcription factors with a conserved transmembrane motif (TM) are members of the NAC family and are important in plant development and response to stress. However, the knowledge of their regulatory pathways is scarce, especially in heat stress (HS). Here, a novel lily (Lilium longiflorum) NTL, LlNAC014, was cloned and identified that increased thermotolerance. High temperature repressed LlNAC014 expression but activated its protein. LlNAC014 contained a typical TM at its far C-terminal and was normally located on membranes, but with HS, it entered the nucleus as a transcription factor. LlNAC014 had a transactivation domain at its C-terminus, and its active form of removing TM, LlNAC014ΔC, could function as a trans-activator in both yeast and plant cells. LlNAC014ΔC overexpression in lily and Arabidopsis increased thermotolerance and also caused growth defects; but silencing LlNAC014 in lily decreased thermotolerance. LlNAC014ΔC could constitutively activate heat stress response by inducing the expression of heat-responsive genes that were dependent or not on the HSF (heat stress transcription factor) pathway. Further analysis showed LlNAC014 was a direct regulator of the DREB2-HSFA3 module and bound to the CTT(N7)AAG element in the promoters of LlHSFA3A, LlHSFA3B, and LlDREB2B to activate their expressions. Thus, LlNAC014 increased thermotolerance by sensing high temperature and translocating to the nucleus to activate the DREB2-HSFA3 module.