Extracellular ATP sensing in living plant tissues with a genetically encoded, ratiometric fluorescent sensor.

Abstract

Adenosine 5′-triphosphate (ATP) is an important energy currency molecule for living organisms, which ensures the energy supply for biochemical reactions and the survival of organisms. Although ATP is produced within the intracellular spaces, animal, plant, and microbial cells can release ATP from the intracellular spaces into the extracellular matrix (Parish & Weibel, 1980; Boyum & Guidotti, 1997; Li et al., 2019). Different from the role of intracellular ATP as energy currency molecule, extracellular ATP (eATP) is considered to be a signaling molecule for regulating many physiological processes of cells (Colombo et al., 2018). The physiological functions of eATP in plants have been widely studied. It was reported that eATP can serve as an early signal of plant defense responses to pathogen infection and herbivore attack (Heil, 2009; Bouwmeester et al., 2011; Balagué et al., 2017; Jewell et al., 2022). Furthermore, much works have indicated that eATP is an important signal involved in the regulation of the response of plants to a variety of abiotic stresses, such as high salt, cold, and heavy metal (Sun et al., 2012; Deng et al., 2015; Hou et al., 2017). Otherwise, eATP is found to have ability to affect the plant growth, development, pollen germination, and root gravitropism (Tang et al., 2003; Reichler et al., 2009; Tanaka et al., 2010; Wu et al., 2018). One of the important aspects in the research on plant eATP is to detect the level of eATP in plants. Currently, the following techniques are mainly employed to detect the eATP level of plants: (1) the extracellular fluid of plants or the liquid medium that has immersed plant tissues is collected (Chivasa et al., 2010; Dark et al., 2011; Wu et al., 2012), and the ATP content in the extracellular fluid or the liquid medium is measured by luciferase–luciferin system that is based on the reaction: ATP + luciferase + luciferin → oxyluciferin + AMP + CO 2 + photon emission $$ \mathrm{ATP}+\mathrm{luciferase}+\mathrm{luciferin}\to \mathrm{oxyluciferin}+\mathrm{AMP}+{\mathrm{CO}}_2+\mathrm{photon}\ \mathrm{emission} $$ (Ramachandran et al., 2019); (2) due to the high molecular weight, luciferase applied exogenously cannot diffuse across the plasma membrane (PM) and thus only can contact the eATP of the plant, rather than intracellular ATP. After exogenous luciferase is applied, luciferin is supplemented to activate the reaction of ATP with luciferase and luciferin, and the production of photon emission from this reaction is proportional to the level of eATP (Kim et al., 2006; Chivasa et al., 2009; Myers Jr et al., 2022); (3) luciferase is ecto-expressed in the extracellular space of plants, and eATP level is measured by supplementing luciferin based on the same reaction as described in (1) (Clark et al., 2011). Based on these techniques, some important information about the effects of environmental factors on the plant eATP level or the dynamic changes of eATP level during plant development or growth were revealed (Tanaka et al., 2010, 2014). However, there still exist some obvious limitations or shortcoming in the current techniques on the detection of plant eATP level. For example, the ATP content in the extracellular fluid or the liquid medium that has immersed plant tissues may in reality reflect the amounts of the eATP that has been released in vitro, rather than the actual level of eATP in vivo. The luciferase that is applied exogenously or is ecto-expressed would hydrolyze ATP upon the measurement, which increases the difficulty of the continuous and real-time detection of plant eATP level. Recently, the sensors based on the gene-encoded fluorescent proteins have been developed as noninvasive tools with high spatial and temporal resolution for the ATP level measurement (Conley et al., 2017). The most known sensor for ATP detection is ATeam sensor, which is designed based on the Forster resonance energy transfer (FRET). The ATeam sensor consists of variants of cyan fluorescent protein (mseCFP) and yellow fluorescent protein (Venus), which are connected by the ε-subunit of ATP synthase from Bacillus sp. PS3 (Imamura et al., 2009; Kotera et al., 2010). When ATP binds to the ε-subunit of the ATP synthase from Bacillus sp. PS3, it induces a conformational change in the sensor structure modifying the relative orientation of the N- and C-terminal donor (mseCFP) and acceptor fluorophores (Venus), increasing FRET efficiency (Imamura et al., 2009). The FRET ratio (the FRET-derived acceptor emission : donor emission) of ATeam can reflect the level of ATP in cells (Ando et al., 2012; Lerchundi et al., 2020). The Arabidopsis (Arabidopsis thaliana) lines that have expressed ATeam in the cytoplasm, chloroplasts, and mitochondria have been constructed by De Col et al. (2017) and his colleagues, who reported the dynamics of intracellular ATP level through the measurement of changes in FRET ratio (De Col et al., 2017). In the present work, we set out to establish an Arabidopsis line for eATP sensing based on the extracellular expression of ATeam sensor. The CaMV 35S: -chitinase (chitinase signal sequence for extracellular targeting, Gao et al., 2004); ATeam1.03-nD/nA fusion constructs were cloned into pCAMBIA3301 vector and were used for the Agrobacterium-mediated transformation (Fig. 1a, Supporting Information Fig. S1; Methods S1). We generated Arabidopsis line expressing the ATeam1.03-nD/nA in the extracellular space and demonstrated that it can be used to monitor the eATP level in living plant tissues by noninvasive measurement of changes in eATP level in living plant tissues. We believe that such new sensor is useful to push beyond the current limitations in detection of plant eATP. For the generation of Arabidopsis line for eATP sensing, the ATeam1.03-nD/nA gene was fused with chitinase (chitinase signal sequence) for extracellular targeting and was expressed under the control of a CaMV 35S promoter. The Arabidopsis line expressing chitinase–ATeam1.03-nD/nA (i.e. the ex-ATeam line) has wild-type (WT)-like phenotype at the whole plant level (Fig. S2), ruling out that the expression of this fused gene might be deleterious for the plant. We performed fluorescence imaging of the ex-ATeam line by stereo-fluorescence microscopy. The result showed that the seedlings of the ex-ATeam line had obvious fluorescence in all of the organs, and a more detailed observation on the leaf of the ex-ATeam line showed that the trichomes and main vein presented stronger fluorescent signal than the other tissues of the leaf (Fig. 1b). Further analysis by confocal laser scanning microscopy with different excitation wavelengths demonstrated the co-expression of mseCFP and Venus in the ex-ATeam line (Fig. 1c), confirming that the ATeam1.03-nD/nA is composed of mseCFP and Venus. The subcellular localization of ATeam1.03-nD/nA in the ex-ATeam line was investigated, and the cyt-ATeam line (the Arabidopsis line expressing ATeam1.03-nD/nA in the cytosol; De Col et al., 2017) was used as the comparison. We performed confocal optical sections of root segments from the seedlings of the ex-ATeam and cyt-ATeam lines. The observation showed that the PM or cell wall of these two lines presented strong ATeam1.03-nD/nA signals (Fig. 1d). In order to get clearer pictures of localization of ATeam1.03-nD/nA in the ex-ATeam and cyt-ATeam lines, the roots of these two lines were subjected to cell plasmolysis by mannitol treatment. After the mannitol treatment, the ATeam1.03-nD/nA signals in the cyt-ATeam line were localized at the PM and in the cytoplasm (which was marked by the obvious signals from the transvacuolar strands), while no ATeam1.03-nD/nA signals were detected in the cell wall of the cyt-ATeam line (Fig. 2). By contrast, the plasmolyzed cells of the ex-ATeam line showed fluorescing PM and cell walls, while no ATeam1.03-nD/nA signals were detected from cytoplasm (Fig. 2). It has been well known that for living plant cells, DAPI (4′6-diamidino-2-phenylindole) cannot penetrate live cells, and thus, it can be used as extracellular staining (Liao et al., 2020). In the present work, the subcellular location of the ATeam1.03-nD/nA in the ex-ATeam line was further analyzed by its colocalization with DAPI. The results showed that in the either ex-ATeam or cyt-ATeam line, the ATeam1.03-nD/nA signals (green) colocalized with DAPI signals (blue) before plasmolysis (Fig. 3). After the ex-ATeam line was plasmolyzed, the ATeam1.03-nD/nA signals were still colocalized with DAPI signals, which were presented at PM and cell walls. By contrast, after the cyt-ATeam line was plasmolyzed, the DAPI signals were localized at PM and cell walls, while the ATeam1.03-nD/nA signals were localized at PM (Fig. 3). These observations indicate that ATeam1.03-nD/nA in the ex-ATeam line is localized at the extracellular space. In order to demonstrate whether the expressed ATeam1.03-nD/nA in the ex-ATeam line can act as a reliable fluorescent sensor for detecting the eATP level, we carried out some pharmacological treatments of the ex-ATeam line with ATP, apyrase, and antimycin A (AA), respectively. And, the plate reader-based measurement, which was developed by De Col et al. (2017), was employed to record the fluorescence of Venus and mseCFP, and the ratio of Venus : CFP (i.e. the FRET ratio) was calculated from the intensities of the fluorescence emission form Venus and mseCFP. Because ATP has high charge, ATP applied exogenously cannot freely diffuse across the PM and thus can increase the eATP level (Tanaka et al., 2010). In the present work, the exogenous 0.001–10 mM ATP was applied to the ex-ATeam line, and the effects of exogenous ATP on the values of FRET ratio of the ex-ATeam line were monitored. The result showed that the values of FRET ratio of the ex-ATeam line continuously increased with the increase in concentration of exogenous ATP from 0.1 to 2.5 mM (Fig. 4a). Higher concentration of exogenous ATP (5–10 mM) did not further increased the values of FRET ratio, implying that the ATeam1.03-nD/nA sensors in the ex-ATeam line could be saturated with such higher concentration of exogenous ATP at such conditions. We also noted that 0.001 and 0.01 mM ATP did not significantly affect the values of FRET ratio. This is not surprising. In fact, many natural components as the barriers, such as the cuticle or waxiness, can prevent polar compounds entering into the plants (DiTomaso, 1999). Thus, the sensitivity of the FRET ratio of the ex-ATeam line to exogenous ATP is considerably lower than that to the physiological level of eATP with the same concentration. Apyrase is an ATP-degrading enzyme. Because of its high molecular weight, apyrase, when applied exogenously, cannot diffuse across the PM. Thus, exogenous addition of apyrase on plants can effectively reduce the eATP level (Chivasa et al., 2005, 2009, 2010). Furthermore, the ex-ATeam line was exogenously treated with apyrase. The results showed that the values of FRET ratio of the ex-ATeam line were significantly decreased by the apyrase, while the application of denatured apyrase did not affect the values of FRET ratio of the ex-ATeam line (Fig. 4b), indicating that the FRET ratio of the ex-ATeam line is responsive to the reduction in the eATP level by the apyrase. It is well known that AA can inhibit mitochondrial electron transport at complex III and thus reduce the intracellular ATP content (Zhang et al., 2001). In the previous work, treatment of the cyt-ATeam line with AA triggered a rapid and pronounced decrease in FRET ratio, confirming that the sensitiveness of the intracellular ATP level to AA (De Col et al., 2017). In the present work, we compared the effects of AA on the FRET ratio of the cyt-ATeam line and ex-ATeam line. Consistent with the observation by De Col et al. (2017), treatment of the cyt-ATeam line with AA caused significant decrease in FRET ratios (Fig. 4c). By contrast, the seedlings of the ex-ATeam line after the same treatment did not present significant decline of the FRET ratio (Fig. 4d). This observation suggests that the ATeam1.03-nD/nA expressed in the ex-ATeam line did not respond to the changes of intracellular ATP content. Thus, combined this observation with the results from the effects of exogenous ATP and apyrase, the changes in the values of FRET ratio of the ex-ATeam line is specific to the eATP. We also further evaluated whether the ATeam1.03-nD/nA expressed in the ex-ATeam line can act as a reliable fluorescent sensor for detecting the eATP level under physiological situation. The seedlings of the ex-ATeam line were exposed to two different stress condition, either wounding or NaCl stress, which has been known to effectively enhance the eATP level of plants (Kim et al., 2009; Myers Jr et al., 2022). Consistent with previous studies, our results showed that wounding or NaCl stress significantly increased the FRET ratio of the seedlings of the ex-ATeam line (Fig. 5), demonstrating that the ex-ATeam line has utility in detecting the physiological changes of eATP level. Otherwise, one could assume that the ATeam1.03-nD/nA expressed in the ex-ATeam line would compete with the eATP receptors to bind eATP and thus attenuate the eATP-regulated physiological responses, when the ex-ATeam line was also used to test the eATP-regulated responses. Previous work by Choi et al. (2014) confirmed that the expression of WRKY domain transcription factor 40 (WRKY40; AT1G80840) and calcium-dependent protein kinase 28 (CPK28; AT5G66210) are up-regulated by eATP. Thus, we compared the expression of these two eATP-responsive marker genes between the WT and ex-ATeam lines with or without the treatment with exogenous ATP as described by Choi et al. (2014). The result showed that there was no significant difference in the expression level of WRKY40 and CPK28 between the WT and ex-ATeam lines (Fig. S5). Nonetheless, since we cannot test all of the eATP-regulated responses in the ex-ATeam line, other eATP-regulated responses that one researcher want to study in the ex-ATeam line are still needed to be evaluated. As introduced above, the physiological roles of eATP in plants have been extensively studied in the last decades, and some reporter for detecting the plant eATP have been developed. Kim et al. (2006) fused a cellulose-binding domain (CBD) with luciferase. This CBD-luciferase reporter was expressed in Escherichia coli and was applied to the roots of the plant after being purified. A subsequent work by Clark et al. (2011) established a transgenic Arabidopsis line for eATP detection, in which a signal peptide from the Brassica oleracea pollen coat protein was incorporated at the N-terminus of the luciferase to target luciferase for extracellular secretion. Besides using luciferase, Vanegas et al. (2015) described a self-referencing electrochemical biosensor for the measurement of eATP in living organisms based on the glycerol kinase and glycerol-3-phosphate oxidase. Although these methods are applicable to the assay of eATP of plants, some methodologic limitations still exist. When luciferase is employed, which is either ecto-expressed or exogenously applied, luciferin solution has to been supplied for the assay of eATP. When electrochemical biosensor is employed, the plant or its tissues has to be immersed in liquid phase microenvironment. These could cause some unexpected effects on real level of plant eATP, especially during long-term measurement. The genetically encoded FRET-based eATP sensor can be used for the noninvasive measurement of eATP level in plants without the requirements for reagents and special microenvironment, and thus helps to overcome such these limitations, although development of such transgenic line in other plant species could be still a challenge. Our understanding of the spatial and temporal changes in eATP levels during plant development and responses to stresses may benefit from such eATP sensor. In any case, we believe that this sensor for plant eATP will become a useful tool for the further studies of plant eATP. We thank Prof. Markus Schwarzländer (University of Münster) for the kind gift of the pH2GW7_cATeam1.03nD/nA plasmid and seeds of the Arabidopsis line expressing ATeam1.03-nD/nA in the cytosol. This work was supported by the National Natural Science Foundation of China (nos. 31870246 and 22164018), the Major Project of Science and Technology Plan of Gansu Province (no. 22ZD1NA001), the Special Fund for Guiding Scientific and Technological Innovation Development of Gansu Province (no. 2019ZX-05), the Open Fund of New Rural Development Institute of Northwest Normal University, the Key Research and Development Project of Gansu Province (no. 22YF7NA119), the Gansu Provincial Department of Education: Excellent Graduate ‘Innovation Star’ project (no. 2021CXZX-214), the Graduate Research Funding Project of Northwest Normal University (nos. 2021KYZZ01037 and 2022KYZZ-B061), and the Outstanding Doctoral Dissertation Cultivation Funding Project of Northwest Normal University. None declared. HF conceived the project and designed the experiments. ZS, YZ, XW, HP, LJ, KS, JZ, and JD performed the experiments and the data analysis. HF and ZS wrote the manuscript. All authors read and approved the manuscript. The data generated and/or analyzed during this study are available from the corresponding author on reasonable request. Fig. S1 Map of the vector that was used for the expression of chitinase-ATeam1.03-nD/nA fusion constructs. Fig. S2 Phenotyping of the seedlings of wild-type and ex-ATeam lines. Fig. S3 Dynamics of fluorescence emission intensity of Venus and CFP in the wells that contained the seedlings of ex-ATeam line after treatment with exogenous adenosine 5′-triphosphate or apyrase. Fig. S4 Dynamics of fluorescence emission intensity of Venus and CFP in representative individual wells that contained the seedlings of cyt-ATeam or ex-ATeam line after treatment with antimycin A or solvent alone. Fig. S5 Adenosine 5′-triphosphate-induced expressions of WRKY domain transcription factor 40 (WRKY40) and calcium-dependent protein kinase 28 (CPK28) between the wild-type and ex-ATeam seedlings. Methods S1 Description of the materials and methods used in this work. Please note: Wiley is not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.