Abstract：Polyamines which play an important role in plant growth and development are aliphatic nitrogen-containing bases with low molecular weight and biological activity. Polyamine uptake transporters (PUT) mediate the intracellular and intercellular transport of amino acids, polyamines, paraquat and thiamine. Studies showed that Arabidopsis thaliana polyamine transporter 3 (AtPUT3) mutant atput3 has the ability to resist 0.1 μmol/L paraquat. In order to study the role of rice (Oryza sativa) polyamine transporters (OsPUT) in plant growth and development under paraquat stress, 6 polyamine transporters were identified in the genome of rice by bioinformatics methods. The results showed that OsPUT1 and AtPUT3 proteins which have similar molecular weights and structures were membrane proteins with 12 transmembrane structures and amino acid transport domains, and the N-terminal and C-terminal were on the cytoplasmic side. The overexpression vector pFGC1300-35S-OsPUT1 was constructed by double restriction endonuclease digestion and sequencing, and then transformed into Arabidopsis thaliana atput3 mutant. In the whole life cycle, under short-day condition, the phenotypic differences were not obvious among wild-type (WT), mutant atput3 and overexpression (OE) lines, and there was no significant difference between the length of bolting time and the number of rosette leaves during bolting. There was no significant difference among the 3 germination rate when seeds germinated on 1/2 MS medium (0 μmol/L PQ) for 48 h. However, the seed germination rate of WT and OE lines was significantly lower than that of atput 3 (P<0.05) when seeds germinated on 1/2 MS medium (0.1 μmol/L PQ) for 5 d. After 10 d of seedling growth on 1/2 MS medium (0.1 μmol/L PQ), the phenotype of OE lines returned to WT state. Compared with atput3, the length of taproot of WT and OE lines decreased significantly (P<0.05). It shows that the function of OsPUT1 gene restores the phenotype of atput3 to WT, which proved that rice OsPUT1 protein and Arabidopsis AtPUT3 protein had similar functions. This study provides a reference for further exploring the function of OsPUT1 gene, and provides some enlightenment for further exploring the resistance of rice to PQ.
[1] Ahmed S, Ariyaratne M, Patel J, et al.2017. Altered expression of polyamine transporters reveals a role for spermidine in the timing of flowering and other developmental response pathways[J]. Plant Science, 258: 146-155. [2] Begam R A, Good A G.2017. The Arabidopsis paraquat resistant1 mutant accumulates leucine upon dark treatment[J]. Botany, 95(7): 751-761. [3] Begam R A, Entremont J D, Good A.2020. The Arabidopsis L-type amino acid transporter 5(LAT5/PUT5) is expressed in the phloem and alters seed nitrogen content when knocked out[J]. Plants, (11): 1519. [4] Benavides M P, Gallego S M, Comba M E, et al.2000. Relationship between polyamines and paraquat toxicity in sunflower leaf discs[J]. Plant Growth Regulation, 31(3): 215-224. [5] Childs AC, Mehta DJ, Gerner EW.2003. Polyamine-dependent gene expression[J]. Cellular And Molecular Life Sciences, 60(7): 1394-1406. [6] Clough SJ, Bent AF.1998. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J]. Plant Journal, 16: 735-743. [7] Dong S, Hu H, Wang Y,et al.2016. A pqr2 mutant encodes a defective polyamine transporter and is negatively affected by ABA for paraquat resistance in Arabidopsis thaliana[J]. Journal of Plant Research, 129(5): 899-907. [8] Fujita M, Fujita Y, Iuchi S, et al.2012. Natural variation in a polyamine transporter determines paraquat tolerance in Arabidopsis[J]. Proceedings of the National Academy of Sciences of the USA, 109(16): 6343-6347. [9] Hart JJ, Ditomaso JM, Linscott DL, et al.1992. Transport interactions between paraquat and polyamines in roots of intact maize seedlings[J]. Plant Physiology, 99(4): 1400-1405. [10] Igarashi K, Kashiwagi K.2010. Modulation of cellular function by polyamines[J]. International Journal of Biochemistry and Cell Biology, 42(1): 39-51. [11] Kusano T, Berberich T, Tateda C, et al.2008. Polyamines: Essential factors for growth and survival[J]. Planta, 228(3): 367-381. [12] Li J, Mu J, Bai J, et al.2013. PARAQUAT RESISTANT1,a golgi-localized putative transporter protein, is involved in intracellular transport of paraquat1[J]. Plant Physiology, 162(1): 470-483. [13] Martinis J, Gas-Pascual E, Szydlowski N, et al.2016. Long-distance transport of thiamine (vitamin B1) is concomitant with that of polyamines[J]. Plant Physiology, 171(1): 542-553. [14] Mattoo A K, Sobolev A P, Neelam A, et al.2006. Nuclear magnetic resonance spectroscopy-based metabolite profiling of transgenic tomato fruit engineered to accumulate spermidine and spermine reveals enhanced anabolic and nitrogen-carbon interactions[J]. Plant Physiology, 142(4): 1759-1770. [15] Mehta R A, Cassol T, Li N, et al.2002. Engineered polyamine accumulation in tomato enhances phytonutrient content, juice quality, and vine life[J]. Nature Biotechnology, 20(6): 613-618. [16] Mulangi V, Chibucos M C, Phuntumart V, et al.2012. Kinetic and phylogenetic analysis of plant polyamine uptake transporters[J]. Planta, 236(4): 1261-1273. [17] Okumoto S, Pilot G.2011. Amino acid export in plants: A missing link in nitrogen cycling[J]. Molecular Plant, 4(3): 453-463. [18] Sagor G H M, Berberich T, Kojima S, et al.2016. Spermine modulates the expression of two probable polyamine transporter genes and determines growth responses to cadaverine in Arabidopsis[J]. Plant Cell Reports, 35(6): 1247-1257. [19] Seiler N, Raul F.2005. Polyamines and apoptosis[J]. Journal of Cellular and Molecular Medicine, 9(3): 623. [20] Shen Y, Ruan Q, Chai H, et al.2016. The Arabidopsis polyamine transporter LHR1/PUT3 modulates heat responsive gene expression by enhancing mRNA stability[J]. Plant Journal for Cell and Molecular Biology, 88(6): 1006. [21] Soar C J, Preston C, Karotam J, et al.2004. Polyamines can inhibit paraquat toxicity and translocation in the broadleaf weed Arctotheca calendula[J]. Pesticide Biochemistry and Physiology, 80(2): 94-105. [22] Wipf D, Ludewig U, Tegeder M, et al.2002. Conservation of amino acid transporters in fungi, plants and animals[J]. Trends in Biochemical Sciences, 27(3): 139-147.
