Huai-Juan Xiao, Ke-Ke Liu, Da-Wei Li, Mohamed Hamed Arisha³, Wei-Guo Chai and Zhen-Hui Gong

BMC Biotechnology October 2015, 15:100 doi:10.1186/s12896-015-0213-1

http://www.biomedcentral.com/1472-6750/15/100

Background

Pheophorbide a oxygenase (PAO) is an important enzyme in the chlorophyll catabolism pathway and is involved in leaf senescence. It opens the porphyrin macrocycle of pheophorbide a and finally forms the primary fluorescent chlorophyll catabolite. Previous studies have demonstrated the function of PAO during cell death. However, the characterizaton of PAO during leaf senescence induced by environmental factors is not well understood.

Methods

Homology-based cloning and RACE techniques were used to obtain the full-length cDNA of the CaPAO gene. CaPAO expression was determined by quantitative real-time PCR. Function of CaPAO gene were studied using virus-induced gene silencing and transgenic techniques with tobacco plants (Nicotiana tabacum).

Results

A novel PAO gene CaPAO was isolated from pepper (Capsicum annuum L.). The full-length CaPAO cDNA is comprised of 1838 bp, containing an open reading frame of 1614 bp, and encodes a 537 amino acid protein. This deduced protein belongs to the Rieske-type iron-sulfur superfamily, containing a conserved Rieske cluster. CaPAO expression, as determined by quantitative real-time PCR, was higher in leaves than roots, stems and flowers. It was upregulated by abscisic acid, methyl jasmonate and salicylic acid. Moreover, CaPAO was significantly induced by high salinity and osmotic stress treatments and also was regulated by Phytophthora capsici. The virus-induced gene silencing technique was used to silence the CaPAO gene in pepper plants. After 3 days of high salt treatment, the chlorophyll breakdown of CaPAO-silenced pepper plants was retarded. RD29A promoter-inducible expression vector was constructed and transferred into tobacco plant. After 7 days of salt treatment, the leaves of transgenic plants were severely turned into yellow, the lower leaves showed necrotic symptom and chlorophyll content was significantly lower than that in the control plants.

Conclusions

The expression of CaPAO gene was induced in natural senescence and various stresses. The CaPAO gene may be related to defense responses to various stresses and play an important role in salt-induced leaf senescence.

Fig. 1. Multiple sequence alignment of the CaPAO protein and PAO proteins from other plants using DNAMAN software. The arrow indicates the cleavage site of the chloroplast peptide, and the single line indicates the Rieske iron-binding motif. The dashed line indicates the mononuclear iron-binding motif, and a conserved CxxC motif is indicated by a double line. Besides CaPAO, other amino acid sequences included in this alignment were S. lycopersicum lethal leaf spot 1-like protein (NP_001234535), N. tabacum PAO1 (ABY19384.1), N. tabacum PAO2 (ABY19385.1), V. vinifera PAO (ACO56118.1), R. communis PAO (XP_002523735.1), B. napus PAO (ABD60317.1), and A. thaliana PAO (AEE77964.1). Shaded regions show identical amino acid residues among all species.