OsProT1 and OsProT3 Function to Mediate Proline- and γ-aminobutyric acid-specific Transport in Yeast and are Differentially Expressed in Rice (Oryza sativa L.)
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Proline (Pro) and γ-aminobutyric acid (GABA) play important roles in plant development and stress tolerance. However, the molecular components responsible for the transport of these molecules in rice remain largely unknown.
Here we identified OsProT1 and OsProT3 as functional transporters for Pro and GABA. Transient expression of eGFP-OsProTs in plant protoplasts revealed that both OsProT1 and OsProT3 are localized to the plasma membrane. Ectopic expression in a yeast mutant demonstrated that both OsProT1 and OsProT3 specifically mediate transport of Pro and GABA with affinity for Pro in the low affinity range. qRT-PCR analyses suggested that OsProT1 was preferentially expressed in leaf sheathes during vegetative growth, while OsProT3 exhibited relatively high expression levels in several tissues, including nodes, panicles and roots. Interestingly, both OsProT1 and OsProT3 were induced by cadmium stress in rice shoots.
Our results suggested that plasma membrane-localized OsProT1 and OsProT3 efficiently transport Pro and GABA when ectopically expressed in yeast and appear to be involved in various physiological processes, including adaption to cadmium stress in rice plants.
KeywordsOsProT1 OsProT3 Proline GABA Transporter Stress tolerance
Amino acid permease
Amino acid transporter
Aluminum-Activated Malate Transporter
1,1′- dioctadecyl- 3,3,3′,3′- tetramethylindocarbocyanine perchlorate
Enhanced green fluorescent protein
Quantitative real time polymerase chain reaction
Pro and GABA are key players in plant growth and resistance to stresses. Accumulation of Pro in pollen is crucial for pollen fertility and deficiency of Pro leads to morphological defects in leaves and inflorescences in Arabidopsis (Funck et al. 2012; Mattioli et al. 2012; Biancucci et al. 2015; Mattioli et al. 2018). In maize plants, Pro was also observed to contribute to final grain production (Spoljarevic et al. 2011). Moreover, Pro is proven to be a member of major compatible solutes which are highly soluble compounds with low molecular weight and are usually nontoxic even at high concentrations (Ashraf and Foolad 2007). It could provide osmotic effects for plants to cope with stresses including drought, salt, nutrient deficiency, heat, heavy metal toxicity and UV-B radiation (Szabados and Savoure 2010; Per et al. 2017). During stress, Pro content is dramatically elevated by enhanced synthesis and decreased degradation (Szabados and Savoure 2010; Kaur and Asthir 2015). Increased Pro synthesis is mainly attributed to upregulation of P5CS which is a key enzyme for Pro biosynthesis; thus enhancing P5CS expression by transgenic approach could effectively improve the ability of plant to tolerate stresses (Kishor et al. 1995; Igarashi et al. 2000).
GABA, a nonproteinogenic amino acid, rapidly accumulates in response to both abiotic and biotic stresses (Shelp et al. 2012; Bown and Shelp 2016). It has been shown that GABA functions in defense against insect herbivory and drought tolerance in Arabidopsis (Scholz et al. 2015; Bown and Shelp 2016; Mekonnen et al. 2016). Moreover, GABA can bind Aluminum-Activated Malate Transporter (ALMT) membrane channels and stimulate anion efflux or inhibit anion influx at the tonoplast or plasma membrane during drought-induced stomatal closure (Ramesh et al. 2015; Bown and Shelp 2016). In addition, GABA is considered as a pivotal amino acid in post-pollination fertilization (Biancucci et al. 2015).
The transport processes of Pro were considered to be important for altering Pro content inside plants, as no relation was observed between the accumulation and synthesis of Pro during grapevine berry maturation (Stines et al. 1999). Biosynthesis of Pro occurs mainly in the cytosol and chloroplasts (Szabados and Savoure 2010), but Pro can be detected in the xylem and phloem sap in several plant species (Weibull et al. 1990; Bialczyk et al. 2004; Lehmann et al. 2010), further confirming the cross-membrane and cross-tissue transport of Pro. Moreover, the long-distance transport of Pro in phloem is increased in response to water stress in alfalfa (Girousse et al. 1996), indicating that Pro transport is essential for plants under both normal and stress conditions. Similar observations were also obtained for GABA, as it can be released from mesophyll cells (Chung et al. 1992) and transported through vascular tissues (Shelp et al. 1999).
The transporters responsible for Pro transport have been identified in some subfamilies of the amino acid transporter (AAT) family in Arabidopsis including the amino acid permease (AAP) family, the lysine-histidine transporter family and the Pro transporter (ProT) family. Meanwhile, some members of the ProT family and GABA transporters in Arabidopsis are also responsible for transporting GABA (Lehmann et al. 2010; Shelp et al. 2012). It is worth mentioning that ProTs frequently display specific transport activity for Pro, glycine betaine and GABA in different species including Arabidopsis, tomato, barley and the common bean (Schwacke et al. 1999; Grallath et al. 2005; Fujiwara et al. 2010; Lehmann et al. 2011; Chen et al. 2016).
Previous studies have identified 79 and 85 AAT genes in rice genomes (Lu et al. 2012; Zhao et al. 2012). However, the transport activities of the encoded proteins have rarely been examined. As such, the components responsible for Pro and GABA transport in rice remain unclear. As OsProTs are likely to participate in these transport processes and OsProT2 has been characterized as a functional Pro transporter through its expression in Xenopus oocytes (Igarashi et al. 2000), we identified and functionally characterized the other two OsProT members in order to uncover the details for Pro and GABA transport in rice. Our results demonstrated that plasma membrane-localized OsProT1 and OsProT3 are functional uptake transporters for Pro and GABA in yeast and might get involved in normal growth and stress tolerance in planta.
Phylogenetic Analysis and Prediction of Transmembrane Helices for OsProT1 and OsProT3
Membrane-spanning regions analyses using Phobius (http://phobius.sbc.su.se/) revealed that both OsProT1 and OsProT3 were predicted to contain 11 putative transmembrane regions (Fig. 1b). The regions between 9th and 10th transmembrane segments of OsProT1 and OsProT3 are slightly short.
OsProT1 and OsProT3 are Localized in the Plasma Membrane
OsProT1 and OsProT3 are Functional Transporters for pro and GABA in Yeast
OsProT1 and OsProT3 showed Differential Expression Patterns in Rice
The accumulation of plant compatible osmolytes, such as Pro and GABA, in response to stress is frequently observed, which is mainly attributed to a combination of increased synthesis and decreased degradation (Lehmann et al. 2010; Shelp et al. 2012; Kaur and Asthir 2015). Meanwhile, abundant evidence has proven that transport of these compatible osmolytes also plays important roles (Igarashi et al. 2000; Lehmann et al. 2010; Shelp et al. 2012). However, the transporters involved in transporting Pro and GABA are largely unknown.
A previous study reported that OsProT2 transports Pro when expressed in Xenopus oocytes (Igarashi et al. 2000). In this study, we characterized the other two OsProT members, OsProT1 and OsProT3, as functional transporters for Pro and GABA. AtProT1–3, HvProT2 and LeProTl were previously identified as efficient transporters for Pro, glycine betaine and GABA (Schwacke et al. 1999; Grallath et al. 2005; Fujiwara et al. 2010). Rice lacks the enzyme requirement for glycine betaine biosynthesis; thus, rice likely does not accumulate and transport glycine betaine (Rathinasabapathi et al. 1993). When expressed in yeast, OsProT1 and OsProT3 specifically mediated the transport of Pro and GABA (Figs. 3, 4 and 5). However, the affinity for Pro was different between the individual transporters (Fig. 5). OsProT3 showed a higher affinity for Pro than that of OsProT1, and they are both lower than that of the three AtProTs with Kmapp values of 0.427 ± 0.017 mM, 0.500 ± 0.005 mM, 0.999 ± 0.036 mM for Pro, respectively (Grallath et al. 2005).
OsProT1 and OsProT3 are both localized to the plasma membrane and function to uptake Pro and GABA from the apoplast (Fig. 2, 3 and 4). Further expression analyses indicated that OsProT1, OsProT3 and OsProT2 play different roles within rice plants. The high expression of OsProT1 in leaf sheath at the vegetative stage (Fig. 6a) suggests that OsProT1 might uptake Pro and GABA as nutrition for seeding growth. OsProT2 might be involved in Pro and GABA remobilization from flag leaf blade, as it shows much higher expression in flag leaf blade than in other tissues (Additional file 1: Figure S2). OsProT3 likely contributes to more physiological processes due to its high expression in more tissues at different stages (Fig. 6b). Consistently, ProTs in Arabidopsis also exhibit similar substrate specificity but different expression patterns (Grallath et al. 2005). AtProT1 is expressed mainly in phloem or phloem parenchyma throughout the whole plant, while AtProT2 is localized in the roots, and AtProT3 is expressed in the above-ground portions of plants (Grallath et al. 2005; Lehmann et al. 2011). In addition to ProTs, evidence indicates that AtAAP members also exhibit difference in tissue expression patterns which guarantees their unique functions in Arabidopsis (Tegeder and Rentsch 2010; Tegeder 2012; Tegeder and Masclaux-Daubresse 2018).
Upon exposure to salt, plants often accumulate Pro (Ueda et al. 2001; Mansour and Ali 2017; Per et al. 2017). However, gene expression data revealed that salinity hardly affects expression levels of OsProTs (Fig. 7e-h, Additional file 1: Figure S3c and d). Consistent with our results, OsProT2 was not induced by salt treatment in a previous study (Igarashi et al. 2000). Surprisingly, Cd stress induced the expression of OsProT1 and OsProT3 in shoots (Fig. 7e and f). The accumulation of Pro frequently occurs in response to heavy metal stress (Sharma and Dietz 2006; Amna et al. 2015), and high constitutive Pro content was detected in metal-tolerant plants (Sharma and Dietz 2006). Given that the transport of Pro might participate in Cd stress tolerance, as the expression of OsProT1 and OsProT3 in shoots were induced by Cd, additional investigation into Pro transport process may improve plant tolerance to Cd.
In the present study, two plasma membrane-localized OsProT members, OsProT1 and OsProT3, were characterized as functional molecular components for transporting Pro and GABA. When ectopically expressed in yeast, OsProT1 and OsProT3 specifically mediated the uptake of Pro and GABA, and both exhibited low affinity for transporting Pro. OsProT1 and OsProT3 might function in differentially physiological processes, including stress tolerance, based on their differential expression patterns. Our work identified new molecular components for controlling the transport of Pro and GABA, and provides new clues to improve stress tolerance in rice by manipulating these transporters.
Materials and methods
Plant material and growth conditions
Uniformly germinated seeds (Oryza sativa L. var. Nipponbare) were cultivated in 96-well plates with removed bottoms (Li et al. 2015) and were then grown hydroponically in Yoshida solution (Adjust pH to 5.8, refreshed every 2 d) at 28 °C, approximately 60% humidity and a 16-h-light/8-h-dark photoperiod. 14-day-old seedlings were further treated for the analysis of gene expression. Yoshida solution without NH4NO3 or containing 10 mM NH4NO3 was used for nitrogen-starvation or high-nitrogen experiments, respectively. Yoshida solution with 50 μM CdCl2 or 100 mM NaCl was used for abiotic stress treatments.
To determine gene expression at the reproductive stage, 3-week-old rice seedlings were transplanted into flooded soil in pots with two seedlings per hill. Soil properties were pH =6.66, organic matter = 48.51 g·kg− 1, total N = 2.2 g·kg− 1, total P = 0.8 g·kg− 1, total K = 6.32 g·kg− 1, available N = 123.24 mg·kg− 1, available P = 26.04 mg·kg− 1, and available K = 118.19 mg·kg− 1. Fertilizers used were urea for N at dose of 34.5 g·m− 2, single superphosphate for P at dose of 50 g·m− 2, and potassium chloride for K at doses of 20 g·m− 2. Different tissues at reproductive stage were collected for further RNA extraction.
Radial tree construction and multiple alignments of OsProTs
OsProT protein sequences were obtained (http://rice.plantbiology.msu.edu/) according to accession numbers LOC_Os01g68050.1 for OsProT1, LOC_Os03g44230.1 for OsProT2, and LOC_Os07g01090.1 for OsProT3 and were then aligned using CLUST W with default parameters. The homologous sequences in other species were obtained via blasting on the NCBI database using OsProT sequences as references. The phylogenetic tree of ProT proteins was constructed by NGPhylogeny.fr (Lemoine et al. 2019) with 1000 bootstrap replications using the default parameters after removing redundant sequences and selecting representative splice forms of the same gene.
Functional Analysis of OsProTs in 22△10α
Coding sequences of OsProT1 and OsProT3 were amplified (primers are listed in Additional file 1: Table S1) from cDNA template of Nipponbare followed by cloning into the p426GPD vector with the BamH1 and EcoR1 restriction sites, leading to the final p426GPD-OsProT1 and p426GPD-OsProT3 constructs, respectively. p426GPD-OsProT1, p426GPD-OsProT3 and the empty vector p426GPD were separately transformed into mutant yeast 22△10α cells as previously described (Elble 1992). Functional complementation assays were performed as described (Fischer et al. 2002; Besnard et al. 2016) with minor modifications. Yeast cells were grown to log phase using SD medium containing 3 mM (NH4)2SO4. Then, cells were collected, rewashed, diluted to an OD600 of 0.8 in sterile water, and spotted onto SD plates containing indicated L-amino acid as the sole nitrogen source at dose of 3 mM. The cells were then grown for about 4 d before being photographed. For 15N-Pro uptake assays, 1.5 mM 15N labeled L-Pro (99 atom %) was added to the culture after yeast cells had reached log phase, and the cells were collected at 0 min, 15 min and 30 min. Then, the cells were immediately washed three times by ultrapure water and dried at 80 °C for 24 h. 15N retained in the cells was measured as previously described (He et al. 2017) using a continuous- flow isotope ratio mass spectrometer coupled to a carbon nitrogen elemental analyzer.
Determination of Kmapp for Pro by Yeast Uptake Assay
Analyses of Kmapp were performed as described previously (Hsu et al. 1993; Hirner et al. 2006; Wang and Tsay 2011) with minor modifications. Yeast cells expressing OsProT1 or OsProT3 were cultured with SD medium containing 3 mM (NH4)2SO4 until the OD600 reached 0.8. Then, the cells were cultured with SD medium containing different concentrations of 15N-Pro ranging from 0.25 mM to 40 mM for 10 min. The collection of cells and determination of 15N contents were performed as described above. The curves and the Kmapp values were obtained by fitting to the Michaelis-Menten equation in the SigmaPlot 12.0 program.
Subcellular Localization Analysis of OsProT1 and OsProT3
The amplified fragment of OsProT1 and OsProT3 (primers used for amplifying are listed in Additional file 1: Table S1) coding sequences were cloned in frame with eGFP into pA7-eGFP vector using Xba1 and BamH1, resulting in eGFP-OsProT1 and eGFP-OsProT3 constructs, respectively, driven by the 35S promoter. Fusion constructions were then transiently expressed in Arabidopsis protoplasts as described previously (Yoo et al. 2007). Transformed protoplasts were incubated in the dark at 22 °C overnight, and eGFP images were subsequently obtained using confocal microscopy (Olympus-FV1000) (Peng et al. 2017). The fluorescent dye Dil was used at a concentration of 10 μM to indicate the plasma membrane (Wang et al. 2019).
Quantitative RT-PCR (Reverse Transcription Polymerase Chain Reaction)
RNA was extracted using TRIzol reagent (Invitrogen, 15,596,026) as previously described (Meng et al. 2016). First-strand cDNA was synthesized using cDNA Synthesis Kit (Vazyme, R212), and quantitative RT–PCR was performed using SYBR Green with a StepOnePlus instrument (Applied Biosystems). OsACTIN1 (LOC_Os03g50885.1) was selected as internal reference gene. Primers used for assays are all listed in Additional file 1: Table S1. The different samples used for the qPCR are biological replicates, and relative expression levels are the expression of OsProTs normalized to that of OsACTIN1 using formula Ratio = 2 (Ct_OsActin − Ct_OsProT).
We are grateful to Dr. Guillaume Pilot (Virginia Tech) and Dr. Nan Guo (Nanjing Agricultural University) for providing the yeast mutant 22△10α, and to Dr. Ren-Jie Tang (Shanghai Institutes for Biological Sciences) for providing p426GPD vector.
S M, JHZ and NHY designed the research. S M, JHL, ZJX, JX, JSP and GBZ performed the experiments. S M, JHL and JZ analyzed the data. S M, JMG, JSP, GBZ and ZXY wrote the paper. All authors read and approved the final manuscript.
This work was supported by the National Key Research and Development Program of China (Grant 2017YFD0301502), the Hunan Provincial Natural Science Foundation of China (Grants 2018JJ3230, 2018JJ3235, 2018JJ2173), the National Natural Science Foundation of China (Grant 31700212), the Fund for Excellent Talents in Crop Discipline of Hunan Agricultural University (Grants YXQN2018–5) and open project of National Key Laboratory of Plant Molecular Genetics.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
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