Response To Chilling Stress In Pepper As Well As Characterization And Functional Analysis Of Chilling-related Genes

Low temperature is one of the most important abiotic factors limiting the growth,development and geographical distribution of plants. Pepper plant (Capsicum annuum L.)originates from tropical regions and is very sensitive to low temperature. The optimal growthtemperature for pepper plants ranges from21°C to27°C, and growth is retarded below12°Cand above30°C. As part of this effort, we are interested in investigating of plant defensemechanisms, in order to improve plant resistance to environmental stresses. In this study,‘P70’ is a typical pepper cultivar that is fairly tolerant of low temperatures. The pepper variety(cv.‘P70’) seedlings were pretreated with0.57mM abscisic acid (ABA) for72h and thensubjected to chilling stress. We investigated the effect of exogenous ABA on total antioxidantactivity and related genes expression in pepper seedlings subjected to chilling stress.Meanwhile, suppression subtractive hybridization analyzed genes regulated by application ofexogenous ABA in pepper plant leaves under chilling stress. Additionally, functionalidentification of several genes in pepper seedlings subjected to abiotic stress was done. Themain results of the study are as follows:(1) To elucidate how physiological and biochemical mechanisms of chilling stress areregulated by ABA pretreatment, the pepper seedlings were pretreated with0.57mM ABA for72h and then subjected to chilling stress at10°C/6°C (day/night). Chilling stress causedsevere necrotic lesions on the leaves and increased malondialdehyde and H2O2levels.Activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase(DHAR), glutathione reductase (GR), guaiacol peroxidase (POD), ascorbate peroxidase(APX), ascorbate (AsA), and glutathione (GSH) increased due to chilling stress during72h,while superoxide dismutase (SOD) and catalase (CAT) activities decreased during24h,suggesting that chilling stress activates the AsA-GSH cycle under CAT deactivation in pepperleaves. ABA pretreatment induced significant increases in the above-mentioned enzymeactivities and progressive decreases in AsA and GSH levels. On the other hand,ABA-pretreated seedlings under chilling stress increased SOD and POD activities andlowered concentrations of other antioxidants compared with untreated chilling-stressed plants.These seedlings showed concomitant decreases in foliage damage symptoms, and levels ofmalondialdehyde and H2O2. Induction of Mn-SOD and POD was observed in chilling-stressed plants treated with ABA. Overall, the results indicate that exogenous application of ABAincreases tolerance of plants to chilling-induced oxidative damage, mainly by enhancing SODand POD activities and related genes expression.(2) Application of exogenous ABA effectively alleviates the symptoms of chilling injury,such as wilting and formation of necrotic lesions on pepper leaves; however, the underlyingmolecular mechanism is not understood. The aim of this study was to identify genes that aredifferentially up-or downregulated in ABA-pretreated pepper seedlings incubated at6°C for48h, using a suppression subtractive hybridization (SSH) method. A total of235high-qualityESTs were isolated, clustered and assembled into a collection of73unigenes including18contigs and55singletons. A total of37unigenes (50.68%) showed similarities to genes withknown functions in the non-redundant database; the other36unigenes (49.32%) showed lowsimilarities or unknown functions. Gene ontology analysis revealed that the37unigenes couldbe classified into nine functional categories. The expression profiles of18selected genes wereanalyzed using quantitative RT-PCR; the expression levels of10of these genes were at leasttwo-fold higher in the ABA-pretreated seedlings under chilling stress than water-pretreated(control) plants under chilling stress. In contrast, the other8genes were downregulated inABA-pretreated seedlings under chilling stress, with expression levels that were one-third orless of the levels observed in control seedlings under chilling stress. These results suggest thatABA can positively and negatively regulate genes in pepper plants under chilling stress.(3) Based on differential genes identified by SSH technique, in accordance with thetobacco rattle virus (TRV)-based VIGS technique, five genes were isolated using ahomology-based candidate gene method and designated as CaNAC2, CaMBF, CaF-box,CaMADS-box, CaDHN, respectively. They contained complete open reading frames (ORF)and GenBank numbers were JX402928, JX402927, JX402925, JX402926, JZ198814,respectively.In this report, pepper CaNAC2belonging to NAC2subfamily, was predicted to encode aprotein of409amino acid residues. The deduced CaNAC2protein was localized in thenucleus of onion epidemical cells and proven to have transactivation and DNA-bindingactivities in yeast. The expression of CaNAC2was largely higher in roots and seeds than thatin other tissues. CaNAC2transcript in pepper leaves was induced by sodium chloride (NaCl)and ABA, indicating that this gene was involved in ABA-mediated salt signaling. However,the expression of CaNAC2was downregulated by mannitol and salicylic acid (SA). Heavymetal Hg treatment had no obvious effect on CaNAC2expression.To further examine the effect of loss-of-function of the CaNAC2gene on abiotic stresstolerance, we silenced the CaNAC2gene in pepper plants using a VIGS technique. Loss-of-function of CaNAC2in pepper plants led to increased susceptibility to chilling stress.Following chilling stress pretreated with ABA or water, the thiobarbituric acid reactivesubstances (TBARS) content of the CaNAC2-silenced leaves was significantly higher thanthat of the empty vector control plants. The combination of ABA with chilling stress led to anincreased level of H2O2in the CaNAC2-silenced leaves. However, H2O2content inCaNAC2-silenced seedlings subjected to chilling stress was very low, indicating that H2O2isinvolved in signaling transduction. Meanwhile, compared with the empty vector controlplants, the leaf discs of gene-silenced plants were more severely green under salinity stress.However, no phenotypic differences were observed between leaf discs from empty vectorcontrol and CaNAC2-silenced plants following treatment with300mM mannitol.(4) Pepper CaMBF contained an open reading frame (ORF) of420bp. The deducedCaMBF protein consisted of140amino acids with a calculated molecular weight of15.3kDaand an isoelectric point (pI) of9.86. The expression level of CaMBF in flowers and seeds washigher and was not easily detected in roots. CaMBF transcript in pepper seedlings wasupregulated by cold stress and downregulated in response to salinity, osmotic stress, heavymetal Hg and SA. Meanwhile, the expression level of CaMBF in CaMBF-overexpressingArabidopsis was downregulated in response to salinity, cold stress and ABA. Transgenicplants were larger than non-transgenic plants (control), especially the length and width ofrosette leaves were70%and60%larger than control. To study the tolerance ofCaMBF-expressing plants to cold stress, we subjected2-week-old wild-type and transgenicseedlings to4°C and observed visual damage symptoms. Transgenic plants were moresusceptibility than wild-type plants to cold stress. Furthermore, overexpression of CaMBF inArabidopsis negatively regulated some defense transcripts in response to cold stress such asRD22, ERD15, RD29A, PDF1.2and PR2.(5) Pepper CaF-box belonging to an ERF branch of F-box superfamily, encoded apolypeptide of638amino acids. CaF-box was mainly expressed in stems and seeds. ABA, SAand cold treatments induced CaF-box transcript; the expression of CaF-box was down-regulated in response to mannitol and heavy metal Hg treatments. To further examine theeffect of loss-of-function of the CaF-box gene on abiotic stress tolerance, we silenced theCaF-box gene in pepper plants using a VIGS technique. The measurement of TBARS andelectrolyte leakage levels revealed stronger lipid peroxidation and cell death in theCaF-box-silenced plants than in the empty vector control plants, suggesting thatloss-of-function of CaF-box significantly compromised cold stress tolerance in pepper plants.When treated with300mM NaCl and300mM mannitol, the leaf discs fromCaF-box-silenced leaves were more severely bleached, indicating that loss-of-function of CaF-box in pepper plants showed early senescence in response to salinity and osmotic stress.(6) Pepper CaMADS-box contained an ORF of726bp. The deduced CaMADS-boxprotein consisted of241amino acids and belonged to an SEP1/AGL2-like subfamily. Theexpression level of CaMADS-box in flowers was higher and was not easily detected in roots,leaves and seeds. ABA and heavy metal Hg treatments had no obvious effect onCaMADS-box expression; CaMADS-box transcript gradually decreased during12h of SAtreatment and upregulated by salinity and osmotic stress. Pepper CaDHN, belonging toSK2-type dehydrins, contained an ORF of660bp. Pepper CaDHN was expressed mainly inflowers and fruits. CaDHN transcript in pepper seedlings was differentially upregulated bycold, salinity, osmotic stress and SA and downregulated in response to heavy metal Hgtreatment.