Cloning and characterization of the pepper CaPAO gene for defense responses to salt-induced leaf senescence.
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.
College of Horticulture, Northwest A&F University, Yangling, Shaanxi, P. R. China. yuanyi041ban@126.com.|College of Horticulture, Henan Agricultural University, Zhengzhou, Henan, P. R. China. yuanyi041ban@126.com.
Huai-Juan
HJ
Xiao
College of Horticulture, Henan Agricultural University, Zhengzhou, Henan, P. R. China. kekeliu1219@sina.com.
Ke-Ke
KK
Liu
College of Horticulture, Northwest A&F University, Yangling, Shaanxi, P. R. China. xndavid@nwsuaf.edu.cn.
Da-Wei
DW
Li
College of Horticulture, Northwest A&F University, Yangling, Shaanxi, P. R. China. mohhamedarisha@gmail.com.|Faculty of Agriculture, Zagazig University, Zagazig, Sharkia P. R. Egypt. mohhamedarisha@gmail.com.
Mohamed Hamed
MH
Arisha
Institute of Vegetables, Hangzhou Academy of Agricultural Sciences, Hangzhou, Zhejiang, P. R. China. kuni@21cn.com.
Wei-Guo
WG
Chai
College of Horticulture, Northwest A&F University, Yangling, Shaanxi, P. R. China. zhgong@nwsuaf.edu.cn.
Zhen-Hui
ZH
Gong
D000595:Amino Acid Sequence; D002212:Capsicum / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D018515:Plant Leaves / Q000502:physiology*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D055049:Salt Tolerance / Q000235:genetics*; D055051:Salt-Tolerant Plants / Q000235:genetics; D016415:Sequence Alignment; D014026:Nicotiana / Q000235:genetics / Q000502:physiology
D010940:Plant Proteins; D010105:Oxygenases; C112915:pheide a oxygenase