Cloning And Functional Analysis Of Leaf Senescence-related Genes In Capsicum Annuum L.

Pepper (Capsicum annuum L.), widely cultivated in the world, is an important vegetablecrop with high economic value. Recently, growth of pepper is seriously affected by variousstress factors for the deterioration of the global environmental conditions, which lead to thepremature of the plant. Leaf is an important organ of the plant for photosynthesis. Prematureof the pepper leaves lead to decrease of photoperiod, and reducing of the nutrient materialstransporting from leaf to fruit, which finally affect the yield and quality of pepper fruits.However, the report on premature of pepper leaf is little. This study discussed the relationshipbetween leaf senescence regulated by exogenous ABA and antioxidases, chlorophylldegradation-related genes in pepper. Three leaf senescence-related genes, includingCaCP(cysteine proteinase), CaPAO(pheophorbide a oxygenase) and CaRCCR(redchlorophyll catabolite reductase), were cloned. Expression patterns of the three genes undersignaling molecules, biotic and abiotic stresses were performed. Meanwhile, function ofCaCP and CaPAO genes were studied using virus-induced gene silencing (VIGS) andtransgenic techniques with tobacco plants (Nicotiana tabacum L.). The main results of thestudy are as follows:1. The detached leaves were treated under dark condition after pretreatment withexogenious ABA. The results showed that dark treatment led to chlorophyll breakdown andyellowing of detached leaves. Moreover, ABA pretreatment could accelerate degradation ofchlorophyll and made the leaves yellowing earlier. During dark treatment for4d, MDAcontent increased, SOD and CAT activities decreased, and POD acitivity increased.Compared to the dark treatment, ABA pretreatment could decrease SOD activity, while PODincrease activity from2d to the end of treatment. ABA could strongly evoke higher levels ofthree genes encoding chlorophyll degradation metabolism-related enzyme, CaPPH, CaPAOand CaRCCR, and two senscence-related genes, CaCP and CaSGR. In addition, thetranscription levels of these genes reach to maximum after ABA treatment for3d, whichwas significantly different from dark treatment. All results indicated that exogenous ABA,which decreased SOD activity, increased POD activity, and strongly upregulated expressionof CaPPH, CaPAO and CaRCCR, could accelerate the senescence progress of detached leaves of pepper.2. Three leaf senescence-related genes of pepper, CaCP (KC176710), CaPAO(KC176709) and CaRCCR (KC176711), were cloned. CaCP gene was located onchromosome2. The full-length CaCP cDNA comprised of1316bp, contained1044nucleotides in open reading frame (ORF), and encodes a347amino acid protein. CaCPbelonged to papain-like superfamily. CaPAO gene, full length of which was1838bp,containing an ORF of1614bp and encoding537amino acids, was located on chromosome11.CaPAO belonged to Rieske-type iron–sulfur superfamily. The full length CaRCCR cDNAwas comprised of1173bp, containing an ORF of945bp and encodes314amino acids,CaRCCR gene was located on chromosome4. CaRCCR belonged to ferredoxin-dependentbilin reductase superfamily.3. CaCP located to the vacuole of plant cells.The expression level of CaCP in leavesand flowers was significantly higher than that in roots. Moreover, the transcriptions ofCaCPwere induced upon during leaf senescence. CaCP expression was upregulated by planthormones of ABA, MeJA and SA. CaCP was also significantly induced by abiotic and bioticstress treatments, including high salinity, mannitol and Phytophthora capsici infection. AVIGS technique was used to silence the CaCP in pepper plants, the leaf discs from thesilenced pepper plants were exposed to NaCl or mannitol solutions. The leaf discs of theempty vector control plants were more severely bleached after3d of treatment, compared tothose of CaCP-silenced plants under the same concentration. Meanwhile, chlorophyllbreakdowm and the accumulation of lipid peroxidation in CaCP-silenced plants were lowerthan that in the empty vector control plants. PVBG2307-CaMV35S-CaCP overexpressedvector was constructed and transferred into tobacco. The transgenic plants were treated withsalt and osmotic stress. The results showed that chlorophyll breakdown and the accumulationof lipid peroxidation of the transgenic plants were faster than that of the control plants after3d of treatment. The results were opposite to that observed in the gene silenced plants. Allresults demonstrated that CaCP, which was a leaf senescence-related gene, involved innatural senescence and salt-and osmotic-induced leaf senescence in pepper plants.4. CaPAO transcripts were detected in roots, stems, leaves flowers and fruits, and thehighest expression was found in leaves. CaPAO transcript was detectable in young leaves andfully expanded leaves, and significantly upregulated in yellowing senescent leaves. It wasupregulated by ABA, MeJA and SA in different extents. Moreover, CaPAO was significantlyinduced by high salinity and drought stress treatments and also regulated by Phytophthoracapsici infetion. A VIGS technique was used to silence the CaCP gene in pepper plants. After3d of ABA or high salt treatment, the chlorophyll breakdown of CaPAO-silenced pepper plants was retarded. PVBG2307-PRD29A-CaPAO vector was constructed and transfferedinto tobacco. After7d of ABA or salt treatment, the leaves of transgenic plants were severelyyellow, the lower leaves showed necrotic symptom, and chlorophyll content was significantlylower than that in the control plants. All of the aboved results indicated that CaPAO geneacted as a positive regulator to participate in natural senescence and ABA-and salt-inducedleaf senescence in pepper plants.5. CaRCCR transcripts were detected in roots, stems, leaves flowers and fruits, and thehighest expression was found in leaves. CaRCCR transcript was increased slightly inyellowing senescent leaves, but the transcript levels of CaRCCR had no significant differencein fully expanded leaves and senescent leaves, compared to young leaves. The expressionlevel of CaRCCR in pepper leaves was upregulated to some extent after treatment withphytohormone, including ABA, MeJA and SA. Similarly, the environmental stress factors,including high salt, mannitol and Phytophthora capsici infection could also induceupregulation of CaRCCR.