Molecular Dissection Of Arabidopsis F-box-containing Gene AtPP2-B11 In Regulation Of Salt Stress Tolerance

Soil salinity has an important effect on plant growth, plant development, and crop productivity. To survive, plants have evolved diverse and elaborate mechanisms to protect themselves from salt stress through a series of physiological and morphological changes, such as the Salt Overly Sensitive(SOS) regulatory pathway, the mitogen-activated protein kinase(MAPK) cascade, the different categories of ion channels and the biosynthesis of products that can alleviate salt stress responses including antioxidants, chaperones, and late embryogenesis abundant(LEA) proteins.The F-box-containing protein family is large, functionally diverse and distributed in all eukaryotes which play important roles in protein degradation. Protein degradation is an important posttranslational regulatory process in cell cycle transitions, developmental programs and responses to environmental signals. Most proteins are degraded via ubiquitin/26 S proteasome(Ub/26S) pathway, which is the major proteolytic route in eukaryotes. The Ub/26 S pathway catalyses the covalent attachment of polyubiquitin chains to substrate proteins. The ubiquitinated substrates are recognised and degraded by the 26 S proteasome, but Ub moieties are recycled. Three enzymes participate in the ubiquitin-mediated protein degradation, including Ub-activating enzyme(E1), Ub-conjugating enzyme(E2) and Ub-ligase(E3). Among these three enzymes, E3 s are the most diverse proteins in the Ub-mediated protein degradation pathway to confer substrate selectivity for an extensive range of substrates. F-box protein is an important component in E3 s and is a key regulatory protein in many cellular processes, including hormone response, photomorphogenesis, floral development, senescence and various signal transduction pathways. Now, the regulation information of F-box genes and abiotic stress is becoming the hotspot.In Arabidopsis, the F-box-containing gene AtPP2-B11 plays an important role in response to salt stress. Its expression pattern and function is analyzed in detail.(1) AtPP2-Bl1 belongs to F-box-containing protein family in Arabidopsis. The expression parttern of AtPP2-B11 in tussiue was constitutive, but the intensity was different: the expressions in rosette leaf and flower were relative higher than other tussiues. qRT-PCR and Western blot analysis proved that AtPP2-B11 was induced by salt stress.(2) Transgenic plants overexpressing AtPP2-B11 exhibited increased salt stress tolerance at seedling and adult growth stages. However, the RNA interference lines showed more sensitive to salt stress than wild type plants.(3) In order to further investigate the regulation mechanism of AtPP2-B11 involved in salt stress resistance, iTRAQ analysis was used to explore the change of proteome, and 4,311 proteins were quantitated, covering a wide range of metabolic and signaling pathways. Proteins that were up-regulated upon high salinity were enriched in the GO categories “Response to stimulus” and “Antioxidant activity”, thereby demonstrating that AtPP2-B11 may be involved in salt tolerance by regulating the stress-responsive proteins and redox status in Arabidopsis.(4) Based on the results of iTRAQ analysis, we focused on the twofold up-regulated proteins. Interestingly, plants overexpressing AtPP2-B11 remarkably increased the expressions of protein family of Annexins compared to the wild type plants under salt stress conditions. Annexins are capable of forming a Ca2+-permeable conductance in an oxidized membrane simulating ROS signaling caused by abiotic stress, promoting Ca2+ influx. The results showed that the mRNA and protein levels of AnnAt1 were induced in AtPP2-B11 overexpressing plants under salt stress conditions compared with those in wild type plants, indicating that AtPP2-B11 functioned as a signaling mediator between ROS and salt stress response.(5) AtPP2-B11 overexpressing lines exhibited significent lower ROS levels. Adding 600μM GSH or 300μM DTT to 1/2MS medium containing 200 mM NaCl could rescue the salinity hypersensitivity of the seedlings.(6) AtPP2-B11 could maintain Na+ homeostasis under salt stress conditions. AtPP2-B11 functioned in the salt stress response at least partially by indirectly regulating the transcription of genes, such as NHX1 and SOS3, to maintain Na+ homeostasis. The high levels of NHX1 could transport the remaining Na+ to vacuoles, and SOS3 contributed to transport the excess Na+ out of cells in order to avoid Na+ toxicity in cells.(7) To determine whether AtPP2-B11 actually functioned as an F-box protein, the interactions between At PP2-B11 and ASK proteins were examined by yeast two-hybrid analysis. Results showed that AtPP2-B11 selectively interacted with ASK7, 18, and 19 proteins, respectively, functioning as an E3 ligase.(8) Yeast two-hybrid and bimolecular fluorescence complementation(Bi FC) assay proved that AtPP2-B11 could interact with At LEA14 in plants. AtPP2-B11 did not influence the protein level of AtLEA14, thus that AtPP2-B11 could not degrade At LEA14 under salt stress conditions. Whereas, AtLEA14 could stabilize the protein level of AtPP2-B11, and the stabilization of AtPP2-B11 proteins by At LEA14 promoted AtPP2-B11 functioning better.(9) AtLEA14 was obviously induced by salt stress at both mRNA and protein levels. Overexpressing AtLEA14 in Arabidopsis and yeast led to considerable increased tolerance to salt stress. Moreover, AtLEA14 functioned in the salt stress response by indirectly regulating transcript accumulation of salt-inducible genes, such as COR15 a, KIN1, RD29 B and ERD10.