The drnf1 Gene from the Drought-Adapted Cyanobacterium Nostoc flagelliforme Improved Salt Tolerance in Transgenic Synechocystis and Arabidopsis Plant

G C A T T A C G G C A T genes Article The drnf1 Gene from the Drought-Adapted Cyanobacterium Nostoc flagelliforme Improved Salt Tolerance in Transgenic Synechocystis and Arabidopsis Plant Lijuan Cui 1,† , Yinghui Liu 1,† , Yiwen Yang 1 , Shuifeng Ye 2 , Hongyi Luo 1 , Baosheng Qiu 1, * and Xiang Gao 1, * ID 1 2 * † Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan 430079, China; (L.C.); (Y.L.); (Y.Y.); (H.L.) Shanghai Agrobiological Gene Center, Shanghai 201106, China; Correspondence: (B.Q.); (X.G.) These authors have contributed equally to this work. Received: 11 August 2018; Accepted: 28 August 2018; Published: 4 September 2018



 Abstract: Environmental abiotic stresses are limiting factors for less tolerant organisms, including soil plants. Abiotic stress tolerance-associated genes from prokaryotic organisms are supposed to have a bright prospect for transgenic application. The drought-adapted cyanobacterium Nostoc flagelliforme is arising as a valuable prokaryotic biotic resource for gene excavation. In this study, we evaluated the salt-tolerant function and application potential of a candidate gene drnf1 from N. flagelliforme, which contains a P-loop NTPase (nucleoside-triphosphatase) domain, through heterologous expression in two model organisms Synechocystis sp. PCC 6803 and Arabidopsis thaliana. It was found that DRNF1 could confer significant salt tolerance in both transgenic organisms. In salt-stressed transgenic Synechocystis, DRNF1 could enhance the respiration rate; slow-down the accumulation of exopolysaccharides; up-regulate the expression of salt tolerance-related genes at a higher level, such as those related to glucosylglycerol synthesis, Na+ /H+ antiport, and sugar metabolism; and maintain a better K+ /Na+ homeostasis, as compared to the wild-type strain. These results imply that DRNF1 could facilitate salt tolerance by affecting the respiration metabolism and indirectly regulating the expression of important salt-tolerant genes. Arabidopsis was employed to evaluate the salt tolerance-conferring potential of DRNF1 in plants. The results show that it could enhance the seed germination and shoot growth of transgenic plants under saline conditions. In general, a novel prokaryotic salt-tolerant gene from N. flagelliforme was identified and characterized in this study, enriching the candidate gene pool for genetic engineering in plants. Keywords: terrestrial cyanobacteria; Nostoc flagelliforme; abiotic stress; transgenic study; salt-tolerant genes 1. Introduction Environmental abiotic stresses, such as drought, salinity, and high and low temperatures, are detrimental factors limiting the growth of less tolerant organisms, including soil plants. Generating transgenic plants with abiotic stress tolerance-associated genes is a promising way to alleviate growth inhibition and crop loss [1,2]. Three major groups of genes are reported to be associated with abiotic stress responses: those involving signaling cascades and transcriptional regulation; those involving the protection of membranes and proteins; those involving water and ion uptake and transport [2,3]. Excavation of new gene resources and elucidation of their physiological or cellular roles Genes 2018, 9, 441; doi:10.3390/genes9090441 www.mdpi.com/journal/genes Genes 2018, 9, 441 2 of 14 in stress response or tolerance is still a critical issue in current plant biology. Angiosperm resurrection plants and halophytes have come to be considered excellent potential sources for exploring these genes in recent years [4,5]. However, it also deserves to be noted that only a few prokaryotic genes are commercially applied in transgenic crops, although numerous genes associated with plant stress resistance have been identified and characterized [6]. This so-called trans-boundary expression (transfer of prokaryotic genes into plants) is thus highly appreciated by some experts. Cyanobacteria are cosmopolitan prokaryotic microorganisms that can be found from temperate to extreme habitats. In xeric regions, much of the success of cyanobacterial species is related to their ability to withstand long-term desiccation and frequent dehydration-rehydration cycles [7]. Desiccation-tolerant cells implement structural, physiological, and molecular mechanisms to survive severe water deficit [8,9]. At the same time, the periodic loss of water from cells leads to alterations of intracellular ion composition and concentration, imposing potential ion toxicity [10]. Thus, there is some overlap in the adaptation of cyanobacteria to desiccation stress and salt stress. Nostoc flagelliforme, a filamentous nitrogen-fixing cyanobacterium, is distributed in arid or semi-arid steppes of the west and northwestern parts of China [11]. Its habitats are characterized by high evaporation rates, substantial temperature differences, and intense solar radiation [11]. This species may serve as a valuable prokaryotic biotic resource for gene excavation [6]. However, so far only limited abiotic stress-responsive genes or proteins have been isolated or identified in it [12–16]. Thus, excavation of new abiotic stress-tolerant genes from N. flagelliforme is still needed, for expanding the candidate gene pool for transgenic use. In our previous study, we isolated some osmotic stress-responsive complementary DNA (cDNA) fragments, and identified three of the predicted genes, named as drought resistant genes of Nostoc flagelliforme (drnf ) 3, 5, and 9, respectively [14,16]. In the preliminary test via the transgenic Escherichia coli strain, another candidate cDNA fragment was found to be most efficient in conferring salt tolerance, named as drnf1; its full gene sequence (NCBI accession no. JX417708.1) was obtained according to the reported partial genome sequences [17]. The deduced DRNF1 protein harbors a conservative P-loop NTPase (nucleoside-triphosphatase) domain and a helix-turn-helix domain (CD-search, NCBI). The family of P-loop NTPases participates in various cellular processes, including translation, transcription, replication and repair, intracellular trafficking, membrane transport, and activation of various metabolites [18,19]. Several P-loop NTPases were reported to be involved in stress response and regulation in bacteria or plants, including SufC [20], Fap7 [21], SKD1 [22], and Hsp100/ClpB [23]. Recently, a rice P-loop NTPase YchF1 was reported to function as a negative regulator in abiotic stress and plant-defense responses [24]. As a potential new P-loop NTPase, the biological function (most possibly in salt tolerance) of DRNF1 awaits further investigation. In this study, we mainly investigated its salt-tolerant roles by over-expressing the drnf1 gene in the model cyanobacterium Synechocystis sp. PCC 6803 and then evaluated its transgenic application potential in plants by over-expressing it in the model plant Arabidopsis (...truncated)