Functional Characterization Of PtrA/NINV, An Alkaline/Neutral Invertase Gene, From Poncirus Trifoliata In Stress Tolerance

Poncirus trifoliata(L.) Raf is a widely involved rootstock in citriculture, due to its tolerance to freezing temperatures lower than –20 °C after fully cold acclimation. However, its molecular mechanisms under freezing stress remained to be investigated. Due to the difficulties of traditional citrus breeding programs, the exploitation of modern biotechnological tools has become the approach for producing stress-tolerant citrus cultivars. However, availability of candidate genes and understanding the molecular effects of the inserted transgene at the whole plant level is a prerequisite for developing stresstolerant citrus. Previously, our lab screened out an alkaline/neutral invertase EST, an enzyme that degrades sucrose permanently into fructose and glucose, from P. trifoliata using suppression subtractive hybridization(SSH) c DNA library.In this research, as we do not know the function of this gene, we purposed, therefore, to test whether this gene, nominated as Ptr A/NINV, can confer enhanced abiotic stress tolerance. To confirm this idea, the full-length ORF sequence and the promoter region of Ptr A/NINV were isolated from trifoliate orange, identified and bioinformatics analyzed. We also determined the subcellular targeting of Ptr A/NINV. Moreover, the transgenic tobacco plants overexpressing Ptr A/NINV were generated to illustrate whether Ptr A/NINV contributes to enhancing abiotic stress tolerance. The obtained results are following: 1. The sequence analysis of Ptr A/NINV revealed that it is encoding a protein of 678 amino acid residues with a theoretical p I of 6.59. We found that the molecular weight of this protein was 76.4 k Da. The phylogenetic study of Ptr A/NINV in parallel with 57 putative A/N-INV proteins from various higher plants and cyanobacteria exhibited that Ptr A/NINV is clustered into a group, which its members localized in mitochondria and have oxidative stress tolerance. MEME analysis of Ptr A/NINV protein sequence with other genes investigated that all tested sequences shared in nine conserved motifs(motif 1-9) that construct GH100 conserved domain. Multiple sequence alignment demonstrated that Ptr A/NINV has a 58-81% sequence similarity to the other tested sequences.2. The Ptr A/NINV promoter region of 1709 bp was isolated from trifoliate orange. The sequence analysis showed the presence of several cis–acting regulatory binding sites that involved in abiotic stress or light response. That was partially supported by the induction of the expression level of Ptr A/NINV in trifoliate orange under abiotic stress and by the circadian expression pattern of Ptr A/NINV in trifoliate orange under the normal condition at time course. 3. The prediction analysis of subcellular localization exhibited the possible targeting of Ptr A/NINV protein into both chloroplast and mitochondria, which we further experimentally verified by microscopy determination. 4. Overexpression of Ptr A/NINV resulted in rendering tolerance to multiple abiotic stresses, including cold, high salinity, and drought. That was concurrently emphasized by better plant performance and associated physiological indices, such as lower levels of reactive oxygen species(ROS), less oxidative damage, decreased water loss rate, and higher photosynthesis efficiency compared to wild-type(WT) plant. The transgenic plants displayed more activity of A/N-INV and higher Glucose and fructose levels under the normal and stress conditions.In Conclusion, we demonstrated that Ptr A/NINV gene from P. trifoliata is an essential gene implicates in sucrose catabolism. We found that the Ptr A/NINV gene fulfills a positive function in conferring tolerance against abiotic stress, which is attributed to the enhancement of osmotic homoeostasis, ROS scavenging, and photosynthesis efficiency. Collectively, Ptr A/NINV has great potential to be used in transgenic breeding for stress tolerance improvement of citrus cultivars.