ClNAC100 Is a NAC Transcription Factor of Chinese Fir in Response to Phosphate Starvation.

Phosphate (Pi) deficiency is one of the most limiting factors for Chinese fir growth and production. Moreover, continuous cultivation of Chinese fir for multiple generations led to the reduction of soil nutrients, which hindered the yield of Chinese fir in southern China. Although NAC (NAM, ATAF, and CUC) transcription factors (TFs) play critical roles in plant development and abiotic stress resistance, it is still unclear how they regulate the response of Chinese fir to phosphate (Pi) starvation. Based on Pi-deficient transcriptome data of Chinses fir root, we identified a NAC transcription factor with increased expression under Pi deficiency, which was obtained by PCR and named ClNAC100. RT-qPCR confirmed that the expression of ClNAC100 in the root of Chinese fir was induced by phosphate deficiency and showed a dynamic change with time. It was positively regulated by ABA and negatively regulated by JA, and ClNAC100 was highly expressed in the roots and leaves of Chinese fir. Transcriptional activation assay confirmed that ClNAC100 was a transcriptional activator. The promoter of ClNAC100 was obtained by genome walking, which was predicted to contain a large number of stress, hormone, and growth-related cis-elements. Tobacco infection was used to verify the activity of the promoter, and the core promoter was located between -1519 bp and -589 bp. We identified 18 proteins bound to the ClNAC100 promoter and 5 ClNAC100 interacting proteins by yeast one-hybrid and yeast two-hybrid, respectively. We speculated that AHL and TIFY family transcription factors, calmodulin, and E3 ubiquitin ligase in these proteins might be important phosphorus-related proteins. These results provide a basis for the further study of the regulatory mechanism and pathways of ClNAC100 under Pi starvation.

Transcriptome Level Analysis of Genes of Exogenous Ethylene Applied under Phosphorus Stress in Chinese Fir.

Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) is a widely grown gymnosperm in China. Phosphorus (P) is an indispensable nutrient for the growth of Chinese fir. Inorganic phosphate (Pi) deficiency exists in soils of many Chinese fir planting area regions, and the trees themselves have limited efficiency in utilizing P from the soil. Ethylene is important in regulation responses to nutrient deficiencies. However, little is known about how ethylene signals participate in Pi stress in Chinese fir. A total of six different treatments were performed to reveal the transcript levels of Chinese fir under Pi, ethephon (an ethylene-releasing compound), and CoCl2 (cobalt chloride, an ethylene biosynthesis inhibitor) treatments. We assembled a full-length reference transcriptome containing 22,243 unigenes as a reference for UMI RNA-seq (Digital RNA-seq). There were 586 Differentially Expressed Genes (DEGs) in the Pi starvation (NP) group, while DEGs from additional ethephon or CoCl2 in NP were 708 and 292, respectively. Among the DEGs in each treatment, there were 83 TFs in these treatment groups. MYB (v-myb avian myeloblastosis viral oncogene homolog) family was the most abundant transcription factors (TFs). Three ERF (Ethylene response factor) family genes were identified when only ethylene content was imposed as a variable. Enrichment analysis indicated that the ascorbate and aldarate metabolism pathway plays a key role in resistance to Pi deficiency. This study provides insights for further elucidating the regulatory mechanism of Pi deficiency in Chinese fir.