Background Phosphorus is one of the essential elements for plant growth and development, but available phosphorus (Pi) content in many soil types is low. As a fast-growing tree species for timber production, Chinese fir is in great demand of Pi, and the lack of Pi in soil restricts the increase of productivity of Chinese fir plantation. Root morphology and the synthesis and secretion of organic acids play an important role in the uptake of phosphorus, but the molecular mechanisms of Chinese fir root responses to Pi deficiency are largely unexplored. In this study, seedlings of Yang 061 clone were grown under three Pi supply levels (0, 5 and 10 mg·L-1 P) and morphological attributes, organic acid content and enzyme activity were measured. The transcriptome data of Chinese fir root system were obtained and the expression levels of phosphorus responsive genes and organic acid synthesis related genes on citric acid and glyoxylate cycle pathway were determined. Results We annotated 50,808 Unigenes from the transcriptome of Chinese fir roots. Among differentially expressed genes, seven genes of phosphate transporter family and 17 genes of purple acid phosphatase family were up-regulated by Pi deficiency, two proteins of SPX domain were up-regulated and one was down-regulated. The metabolic pathways of the citric acid and glyoxylate cycle pathway were mapped, and the expression characteristics of the related Unigenes under different phosphorus treatments were analyzed. The genes involved in malic acid and citric acid synthesis were up-regulated, and the activities of the related enzymes were significantly enhanced under long-term Pi stress. The contents of citric acid and malic acid in the roots of Chinese fir increased after 30 days of Pi deficiency. Conclusion Chinese fir roots showed increased expression of genes related with phosphorus starvation, citrate and malate synthesis genes, increased content of organic acids, and enhanced activities of related enzymes under Pi deficiency. The results provide a new insight for revealing the molecular mechanism of adaption to Pi deficiency and the pathway of organic acid synthesis in Chinese fir roots.

Chinese fir (Cunninghamia lanceolata) is an economical important timber species widely planted in southeastern Asia. Decline in yield and productivity during successive rotation is believed to be linked with abiotic stress, such as drought stress and nitrogen (N) and phosphorus (P) starvation. Molecular breeding could be an option to develop tolerant genotypes. For gene expression studies using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), stable reference genes are needed for normalization of gene expression under different experimental conditions. However, there is no internal reference genes identified for Chinese fir under abiotic stresses. Thus, nine internal reference genes based on transcriptome data were selected and analyzed in the root of Chinese fir under drought stress and N and P starvation. Data were analyzed using geNorm, NormFinder, and BestKeeper, to screen and identify the best reference genes. The results showed that the UBQ and GAPDH genes were the two most stable genes under drought stress and the Actin1 and GAPDH were the two most stable genes under P starvation. Further, it was discovered that the Actin1 and UBC were the two most stable genes under N starvation among nine candidate reference genes. The gene expression of drought stress induced expression protein 14-3-3-4, the P transporter gene ClPht1;3, and the nitrate transporter gene NRT1.1 were used to verify the stability of the selected reference genes under drought stress and P and N starvation, respectively, and the results revealed that the screened reference genes were sufficient to normalize expression of the target genes. In conclusion, the results demonstrate that the stability of reference genes was closely related to the external conditions and reference genes applied to the roots of Chinese fir under different abiotic stress treatments were different. Our data will facilitate further studies on stress ecology and gene function analysis in Chinese fir.

The study on the function and sequence of PHR1 (Phosphate Starvation Response gene 1) gene, which plays a central role in plant phosphorus (Pi) signal regulatory network, is of great significance to further study response mechanisms to Pi deficiency. In this work, the previously selected Pi-efficient Chinese fir clone M32 was used as research material to obtain the full-length sequence of ClPHR1 transcription factors in Chinese fir by RACE (Rapid Amplification of cDNA Ends) full-length cloning technique, and the structure, function and subcellular localization of ClPHR1 gene encoding protein were analyzed. The temporal and spatial expression characteristics of ClPHR1 transcription factors in Chinese fir under low Pi stress were also analyzed, and the overexpression of ClPHR1 gene in transgenic Arabidopsis thaliana was obtained to verify the function of ClPHR1 gene under low Pi stress. The results showed that the length of the ClPHR1 gene obtained by rapid amplification of cDNA ends technique was 1954 bp, of which 1512 bp was an open reading frame. ClPHR1 was predicted to be an unstable hydrophilic protein with only one possible transmembrane domain. The ClPHR1 gene had a highly conserved MYB-CC domain, which is similar to the PHR1 gene of other plants. Phylogenetic tree analysis showed that the sequence had high homology with PHR1 genes in the Prunus species. The ClPHR1 was expressed in all organs of Chinese fir, with the highest expression in the roots, followed by the leaves with the lowest expression in stems. ClPHR1 expression in roots was reduced dramatically at the beginning of Pi stress treatment and followed by an increase at 7days; in leaves, it increased dramatically at the beginning of Pi starvation treatment and showed a decreasing trend after 3 days; in stems, the expression level of ClPHR1 increased after 7 days of Pi stress treatment. The transient expression vector was introduced into plant cells, and it was found that ClPHR1 was located in the nucleus and was a MYB-CC transcription factor expressed in the cell nucleus. The ClPHR1 overexpression vector was constructed, and then introduced into Arabidopsis thaliana by agrobacterium infection inflorescence method. The expressions of Pi transporter genes, AtPHT1;1, AtPHT1;2, AtPHT1;8 and AtPHT1;9, was significantly higher in the overexpressing strain than that in the wild type strain. The results suggest that the ClPHR1 transcription factor could regulate the regulation of downstream Pi transporter gene and increase Pi utilization efficiency of the Chinese fir under Pi stress.

Background: Phosphorus is one of the essential elements for plant growth and development, but the content of plant available phosphorus (Pi) in many soil types is low. As a fast-growing timber species, Chinese fir is in great demand of Pi, and the lack of Pi in soil restricts the increase of productivity of Chinese fir plantation. Root morphology and the synthesis and secretion of organic acids play an important role in the uptake of phosphorus, but the molecular mechanisms of Chinese fir in response to Pi deficiency are largely unexplored. Results: In this study, seedlings of Yang 061 clone were grown under three Pi supply levels (0, 5 and 10 mg·L-1 P) and morphological attributes, organic acid content and enzyme activity were measured, the transcriptome data of Chinese fir root system were obtained and the expression levels of phosphorus responsive genes and organic acid synthesis related genes on citric acid and glyoxylate cycle pathway were determined. The results showed that there were 50,808 Unigenes annotated from the transcriptome of Chinese fir roots. Among differentially expressed genes, seven genes of phosphate transporter family and seventeen genes of purple acid phosphatase family were up-regulated by Pi deficiency, two proteins of SPX domain were up-regulated and one was down-regulated. The metabolic pathways of the citric acid and glyoxylate cycle pathway were mapped, and the expression characteristics of the related Unigenes under different phosphorus treatments were analyzed. The genes involved in malic acid and citric acid synthesis were up-regulated, and the activities of the related enzymes were significantly enhanced under long-term stress. The contents of citric acid and malic acid in the roots of Chinese fir increased after 30days of Pi deficiency. Conclusion: The Chinese fir roots showed increased expression of citrate and malate synthesis genes, increased content of organic acids and enhanced activities of related enzymes under Pi deficiency. The results provide a new insight for revealing the molecular mechanism of Pi deficiency and the pathway of organic acid synthesis in Chinese fir roots.