- Email: [email protected]
Dawn illumination prepares desert cyanobacteria for dehydration
Current Biology
Magazine various stressors including frequent hydration–dehydration cycles. Water is mainly provided as early-morning dew, followed by dehydration with rising temperatures and declining relative humidity. Earlier studies focused on community structure and cyanobacterial activities in various BSCs [1,2]. They identified genes present in dehydrationtolerant, but not -sensitive cyanobacteria [3], and suggested that abiotic conditions during natural dehydration (Figure 1A) are critical for the recovery upon rewetting. Inability of Leptolyngbya ohadii, which is abundant in the BSC examined here, to recover after rapid desiccation (Figure 1B) [4] suggested that the cells must prepare themselves toward forthcoming dehydration, but the nature of the signal involved was unknown. We show here that the rising dawn illumination, perceived by photo-sensors, serves
Dawn illumination prepares desert cyanobacteria for dehydration Nadav Oren1,2, Hagai Raanan1,2,3, Omer Murik1,2, Nir Keren2, and Aaron Kaplan2,4,* Desert biological soil crusts (BSC), among the harshest environments on Earth, are formed by the adhesion of soil particles to polysaccharides excreted mainly by filamentous cyanobacteria (see [1] and references therein). These species are the main primary producers in this habitat where they cope with A
Temperature [°C]
24 23 22 21 20 19 18 17 16
120
Ground Temp.
Water content
800 700
100 600 80
500
60
Light
400 300
Air Temp.
40
200 20
100
15 14
0
0 50
0
100
200
150
Relative transcript abundance (Δ log2)
Time, Minutes
C
Pheophorbide a oxygenase 2
• Natural dehydration • But darkness • With added FR • Blue light blocked
E
Glycosyl transferase 6 4
0
80%
60%
40%
20%
0% Natural
Darkness
+FR, 60 min
Trehalose synthase (TreS) 4
Continuous FR No blue light
F
-2 -4
8
60
-2
160
ABC bicarbonate transporter (cmpD)
Acyl-CoA dehydrogenase 2
-2 0
0 0
0
H
60
-2
160
I
Phytochrome (cph1)
-4 0
60
-6
160
Response regulator (rcp1)
J
2
6
6
6
0
4
4
4
-2
2
-4
2
2
0
-6
0
0
-2
-8
-2
-2
0
60
160
Dehydration time (min)
Fast
0
2
2
G
Relative transcript abundance (Δ log2)
D
Light intensity
25
Water content [% from initial]
26
as the signal inciting BSC-inhabiting cyanobacteria to prepare for forthcoming dehydration. L. ohadii filaments were exposed to simulated natural conditions from the morning of October 14th 2009, using our environmental chamber that enables accurate reproduction of BSC environment [4] (Supplemental Figure S1A). Samples were withdrawn at specific time points (Figure 1A), followed by RNA extraction and global transcript profiling (accession PRJNA391854). Four hours of dehydration led to up-regulation of 567 genes and down-regulation of 1597 (by more than 2-fold). Since BSCinhabiting organisms have not been used as genetic models, the functions of 3258 (43.5% of the 7487 L. ohadii genes [3]) are unknown. Nevertheless, a pronounced rise in transcript levels of genes involved in carbon metabolism, transport, osmolyte production, energy
B
900
140 27
O2 evolution (% from initial)
Correspondence
0
60
Dehydration time (min)
160
0
60
Dehydration time (min)
160
0
60
160
Orange carotenoid-binding protein (OCP)
0
60
160
Dehydration time (min)
Figure 1. Extent of revival and of gene expression following dehydration as affected by the illumination. (A) Time course of the changing water content, air and ground temperatures, and light intensity simulating the natural conditions on the morning of October 14th 2009. The sampling points for RNA analyses are indicated by the red circles. (B) The extent of recovery of the photosynthetic activity (used as a proxy for cell viability) upon re-wetting. The experimental procedure is described in Figure S1A. For the other treatments, L. ohadii filaments were kept under the natural conditions shown in (A) except for: Darkness, without illumination; FR 60 min, far-red light during the first hour; Continuous FR, far-red light throughout the dehydration protocol; No blue light, filter was used to remove the blueish part of the spectrum; Fast dehydration, the cells were dehydrated in less than 5 min. by dry air. (C–J) Examples of early- or late-responding genes up- or down-regulated by the dehydration protocol (examined by RT-qPCR). The selected dark-inhibited genes shown here also represent a combination of those affected by FR addition, removal of blue light or both, as well as various functions apparently essential for the ability to revive after dehydration. The complete gene profiling using RNAseq during natural dehydration is provided in accession PRJNA391854.
R1056
Current Biology 27, R1037–R1059, October 9, 2017 © 2017 Elsevier Ltd.
Current Biology
Magazine dissipation and other cellular activities was observed. On the other hand, a declining transcript abundance for genes involved in light harvesting, photosynthetic metabolism, protein biosynthesis, cell division and other pathways was detected. The analysis unraveled clear distinctions between early- and late-responding genes. Supplemental Table S1 lists the 40 strongest differentially expressed genes verified by RT-qPCR and used in further analyses. A significant effect on transcript abundance was detected already at the 1h time point, suggesting that earlyresponding genes (and physiological performance) are affected by the rising illumination preceding the decline in water content (Figure 1A). To test this possibility, we repeated the dehydration protocol without illumination. Darkness significantly hampered the recovery after re-wetting (Figure 1B). It also blocked alterations in the transcript abundance of many genes strongly affected during natural dehydration (Figure 1C–J). Considering the very low illumination at dawn, and that L. ohadii filaments are located beneath the surface [2], earlymorning light likely serves as a signal rather than an energy source. Phytochromes sense and respond to red/far-red (FR) light in various organisms from bacteria to plants [5–9]. Addition of FR light (spectrum shown in Figure S1B) to the early-morning protocol (Figure 1A) completely blocked L. ohadii recovery after re-wetting, far more than darkness (Figure 1B). Removal of the FR light after 1h partly restored the extent of recovery, to the dark-treated level, suggesting that phytochromes are involved in both early- and late-responding processes. L. ohadii’s genome contains four phytochrome-encoding genes, cph1type, and all are transcribed. The transcript abundance (Table S1) is very high at time zero (92nd percentile of RNA reads) but declined thereafter (Figure 1H). Directly downstream of this gene is another strongly upregulated early gene encoding the response regulator Rcp1 (Figure 1I). In Synechocystis Rcp1 interacts with Cph1. Transcriptional profiling of some of the genes depicted in Table S1 distinguished those completely blocked by FR light (such as glycosyl transferase and acylCoA dehydrogenase, early- and lateresponding genes, respectively) from
those where FR reduced the response to dehydration (such as trehalose synthase, cph1 and others) and those blocked by darkness but unaffected by FR (such as the orange carotenoid protein, OCP). Interestingly, the glycosyl transferase and trehalose synthase types present in L. ohadii are among those specifically found in dehydration-tolerant but not in sensitive cyanobacteria [3]. In contrast, OCP, essential for energy dissipation [10], is ubiquitous in cyanobacteria. Use of a cut-off filter that removed the blue part of the incident light (Figure S1B) inhibited revival after re-wetting, although less so than darkness or FR (Figure 1B). This treatment blocked the expression of OCP and acyl-CoA dehydrogenase. In contrast, cmpD, involved in bicarbonate uptake and hence in dissipation of excess light energy, was upregulated, possibly compensating for the decline in OCP. Altogether, the selected examples among dark-inhibited genes (Figure 1C– J) cover the various possibilities — genes affected only by red/FR, or by cryptochrome, or by both. Our data show that BSC-inhabiting cyanobacteria must prepare themselves towards forthcoming dehydration, and that rising dawn illumination perceived by photo-sensors is critical for this process. The next challenges are to clarify how the twin responses to blue and red/FR light are orchestrated, and to identify the network of transcription factors and responding genes (particularly, but not solely, those exclusively present in cyanobacteria able to recover from dehydration [3]) that prepare the cells for desiccation. The experimental protocol used could have synchronized the circadian clock. However, it is not known whether a circadian rhythm is involved in the preparations towards dehydration. Further, since the cyanobacterial filaments are essentially desiccated most of the day, we doubt the circadian clock plays an important role in the BSC. The evolutionary origins of phytochrome and cryptochrome function in dehydration tolerance is also intriguing. We raise the possibility that the original genes were recruited from anoxygenic photosynthetic bacteria in ancient stromatolites, but in the absence of essential genomic information it is not possible to comprehensively investigate their phylogeny.
SUPPLEMENTAL INFORMATION Supplemental information includes a description of the experimental methods, one figure and one table, and can be found with this article online at http://dx.doi. org/10.1016/j.cub.2017.08.027. ACKNOWLEDGMENTS This study was supported by the USIsrael Binational Agricultural Research and Development fund (BARD). REFERENCES 1. Weber, B., Büdel, B., and Belnap, J. (2016). Biological soil crusts: An organizing principle in drylands, Volume http://dx.doi. org/10.1016/10.1007/978-3-319-30214-0, (Springer). 2. Raanan, H., Felde, V.J.M.N.L., Peth, S., Drahorad, S., Ionescu, D., Eshkol, G., Treves, H., Felix-Henningsen, P., Berkowicz, S.M., and Keren, N. (2016). The 3D structure and cyanobacterial activity in a desert biological soil crust. Environ. Microbiol. 18, 372–383. 3. Murik, O., Oren, N., Shotland, Y., Raanan, H., Treves, H., Kedem, I., Keren, N., Hagemann, M., Pade, N., and Kaplan, A. (2017). What distinguishes cyanobacteria able to revive after desiccation from those that cannot: The genome aspect. Environ. Microbiol. 19, 535–550. 4. Raanan, H., Oren, N., Treves, H., Berkowicz, S.M., Hagemann, M., Padec, N., Keren, N., and Kaplan, A. (2016). Simulated soil crust conditions in a chamber system provide new insights on cyanobacterial acclimation to desiccation Environ. Microbiol. 18, 414–426. 5. Falciatore, A., and Bowler, C. (2005). The evolution and function of blue and red light photoreceptors. Curr. Top. Dev. Biol. 68, 317–350. 6. Schmitz, O., Katayama, M., Williams, S.B., Kondo, T., and Golden, S.S. (2000). CikA, a bacteriophytochrome that resets the cyanobacterial circadian clock. Science 289, 765–768. 7. Wiltbank, L.B., and Kehoe, D.M. (2016). Two cyanobacterial photoreceptors regulate photosynthetic light harvesting by sensing teal, green, yellow, and red light. mBio 7. 8. Yeh, K.-C., Wu, S.-H., Murphy, J.T., and Lagarias, J.C. (1997). A cyanobacterial phytochrome two-component light sensory system. Science 277, 1505–1508. 9. Fiedler, B., Borner, T., and Wilde, A. (2005). Phototaxis in the cyanobacterium Synechocystis sp PCC 6803: Role of different photoreceptors. Photochem. Photobiol. 81, 1481–1488. 10. Leverenz, R.L., Sutter, M., Wilson, A., Gupta, S., Thurotte, A., Bourcier de Carbon, C., Petzold, C.J., Ralston, C., Perreau, F., and Kirilovsky, D. (2015). A 12 Å carotenoid translocation in a photoswitch associated with cyanobacterial photoprotection. Science 348, 1463–1466.
1
These authors contributed equally to this study. 2Department of Plant and Environmental Sciences, the Hebrew University of Jerusalem, Jerusalem 9190401, Israel. 3Present address: Institute of Earth, Ocean and Atmospheric Sciences, Rutgers University 71 Dudley Road, New Brunswick, NJ 08901, USA. 4 Lead contact. *E-mail: [email protected]
Current Biology 27, R1037–R1059, October 9, 2017 R1057
Magazine various stressors including frequent hydration–dehydration cycles. Water is mainly provided as early-morning dew, followed by dehydration with rising temperatures and declining relative humidity. Earlier studies focused on community structure and cyanobacterial activities in various BSCs [1,2]. They identified genes present in dehydrationtolerant, but not -sensitive cyanobacteria [3], and suggested that abiotic conditions during natural dehydration (Figure 1A) are critical for the recovery upon rewetting. Inability of Leptolyngbya ohadii, which is abundant in the BSC examined here, to recover after rapid desiccation (Figure 1B) [4] suggested that the cells must prepare themselves toward forthcoming dehydration, but the nature of the signal involved was unknown. We show here that the rising dawn illumination, perceived by photo-sensors, serves
Dawn illumination prepares desert cyanobacteria for dehydration Nadav Oren1,2, Hagai Raanan1,2,3, Omer Murik1,2, Nir Keren2, and Aaron Kaplan2,4,* Desert biological soil crusts (BSC), among the harshest environments on Earth, are formed by the adhesion of soil particles to polysaccharides excreted mainly by filamentous cyanobacteria (see [1] and references therein). These species are the main primary producers in this habitat where they cope with A
Temperature [°C]
24 23 22 21 20 19 18 17 16
120
Ground Temp.
Water content
800 700
100 600 80
500
60
Light
400 300
Air Temp.
40
200 20
100
15 14
0
0 50
0
100
200
150
Relative transcript abundance (Δ log2)
Time, Minutes
C
Pheophorbide a oxygenase 2
• Natural dehydration • But darkness • With added FR • Blue light blocked
E
Glycosyl transferase 6 4
0
80%
60%
40%
20%
0% Natural
Darkness
+FR, 60 min
Trehalose synthase (TreS) 4
Continuous FR No blue light
F
-2 -4
8
60
-2
160
ABC bicarbonate transporter (cmpD)
Acyl-CoA dehydrogenase 2
-2 0
0 0
0
H
60
-2
160
I
Phytochrome (cph1)
-4 0
60
-6
160
Response regulator (rcp1)
J
2
6
6
6
0
4
4
4
-2
2
-4
2
2
0
-6
0
0
-2
-8
-2
-2
0
60
160
Dehydration time (min)
Fast
0
2
2
G
Relative transcript abundance (Δ log2)
D
Light intensity
25
Water content [% from initial]
26
as the signal inciting BSC-inhabiting cyanobacteria to prepare for forthcoming dehydration. L. ohadii filaments were exposed to simulated natural conditions from the morning of October 14th 2009, using our environmental chamber that enables accurate reproduction of BSC environment [4] (Supplemental Figure S1A). Samples were withdrawn at specific time points (Figure 1A), followed by RNA extraction and global transcript profiling (accession PRJNA391854). Four hours of dehydration led to up-regulation of 567 genes and down-regulation of 1597 (by more than 2-fold). Since BSCinhabiting organisms have not been used as genetic models, the functions of 3258 (43.5% of the 7487 L. ohadii genes [3]) are unknown. Nevertheless, a pronounced rise in transcript levels of genes involved in carbon metabolism, transport, osmolyte production, energy
B
900
140 27
O2 evolution (% from initial)
Correspondence
0
60
Dehydration time (min)
160
0
60
Dehydration time (min)
160
0
60
160
Orange carotenoid-binding protein (OCP)
0
60
160
Dehydration time (min)
Figure 1. Extent of revival and of gene expression following dehydration as affected by the illumination. (A) Time course of the changing water content, air and ground temperatures, and light intensity simulating the natural conditions on the morning of October 14th 2009. The sampling points for RNA analyses are indicated by the red circles. (B) The extent of recovery of the photosynthetic activity (used as a proxy for cell viability) upon re-wetting. The experimental procedure is described in Figure S1A. For the other treatments, L. ohadii filaments were kept under the natural conditions shown in (A) except for: Darkness, without illumination; FR 60 min, far-red light during the first hour; Continuous FR, far-red light throughout the dehydration protocol; No blue light, filter was used to remove the blueish part of the spectrum; Fast dehydration, the cells were dehydrated in less than 5 min. by dry air. (C–J) Examples of early- or late-responding genes up- or down-regulated by the dehydration protocol (examined by RT-qPCR). The selected dark-inhibited genes shown here also represent a combination of those affected by FR addition, removal of blue light or both, as well as various functions apparently essential for the ability to revive after dehydration. The complete gene profiling using RNAseq during natural dehydration is provided in accession PRJNA391854.
R1056
Current Biology 27, R1037–R1059, October 9, 2017 © 2017 Elsevier Ltd.
Current Biology
Magazine dissipation and other cellular activities was observed. On the other hand, a declining transcript abundance for genes involved in light harvesting, photosynthetic metabolism, protein biosynthesis, cell division and other pathways was detected. The analysis unraveled clear distinctions between early- and late-responding genes. Supplemental Table S1 lists the 40 strongest differentially expressed genes verified by RT-qPCR and used in further analyses. A significant effect on transcript abundance was detected already at the 1h time point, suggesting that earlyresponding genes (and physiological performance) are affected by the rising illumination preceding the decline in water content (Figure 1A). To test this possibility, we repeated the dehydration protocol without illumination. Darkness significantly hampered the recovery after re-wetting (Figure 1B). It also blocked alterations in the transcript abundance of many genes strongly affected during natural dehydration (Figure 1C–J). Considering the very low illumination at dawn, and that L. ohadii filaments are located beneath the surface [2], earlymorning light likely serves as a signal rather than an energy source. Phytochromes sense and respond to red/far-red (FR) light in various organisms from bacteria to plants [5–9]. Addition of FR light (spectrum shown in Figure S1B) to the early-morning protocol (Figure 1A) completely blocked L. ohadii recovery after re-wetting, far more than darkness (Figure 1B). Removal of the FR light after 1h partly restored the extent of recovery, to the dark-treated level, suggesting that phytochromes are involved in both early- and late-responding processes. L. ohadii’s genome contains four phytochrome-encoding genes, cph1type, and all are transcribed. The transcript abundance (Table S1) is very high at time zero (92nd percentile of RNA reads) but declined thereafter (Figure 1H). Directly downstream of this gene is another strongly upregulated early gene encoding the response regulator Rcp1 (Figure 1I). In Synechocystis Rcp1 interacts with Cph1. Transcriptional profiling of some of the genes depicted in Table S1 distinguished those completely blocked by FR light (such as glycosyl transferase and acylCoA dehydrogenase, early- and lateresponding genes, respectively) from
those where FR reduced the response to dehydration (such as trehalose synthase, cph1 and others) and those blocked by darkness but unaffected by FR (such as the orange carotenoid protein, OCP). Interestingly, the glycosyl transferase and trehalose synthase types present in L. ohadii are among those specifically found in dehydration-tolerant but not in sensitive cyanobacteria [3]. In contrast, OCP, essential for energy dissipation [10], is ubiquitous in cyanobacteria. Use of a cut-off filter that removed the blue part of the incident light (Figure S1B) inhibited revival after re-wetting, although less so than darkness or FR (Figure 1B). This treatment blocked the expression of OCP and acyl-CoA dehydrogenase. In contrast, cmpD, involved in bicarbonate uptake and hence in dissipation of excess light energy, was upregulated, possibly compensating for the decline in OCP. Altogether, the selected examples among dark-inhibited genes (Figure 1C– J) cover the various possibilities — genes affected only by red/FR, or by cryptochrome, or by both. Our data show that BSC-inhabiting cyanobacteria must prepare themselves towards forthcoming dehydration, and that rising dawn illumination perceived by photo-sensors is critical for this process. The next challenges are to clarify how the twin responses to blue and red/FR light are orchestrated, and to identify the network of transcription factors and responding genes (particularly, but not solely, those exclusively present in cyanobacteria able to recover from dehydration [3]) that prepare the cells for desiccation. The experimental protocol used could have synchronized the circadian clock. However, it is not known whether a circadian rhythm is involved in the preparations towards dehydration. Further, since the cyanobacterial filaments are essentially desiccated most of the day, we doubt the circadian clock plays an important role in the BSC. The evolutionary origins of phytochrome and cryptochrome function in dehydration tolerance is also intriguing. We raise the possibility that the original genes were recruited from anoxygenic photosynthetic bacteria in ancient stromatolites, but in the absence of essential genomic information it is not possible to comprehensively investigate their phylogeny.
SUPPLEMENTAL INFORMATION Supplemental information includes a description of the experimental methods, one figure and one table, and can be found with this article online at http://dx.doi. org/10.1016/j.cub.2017.08.027. ACKNOWLEDGMENTS This study was supported by the USIsrael Binational Agricultural Research and Development fund (BARD). REFERENCES 1. Weber, B., Büdel, B., and Belnap, J. (2016). Biological soil crusts: An organizing principle in drylands, Volume http://dx.doi. org/10.1016/10.1007/978-3-319-30214-0, (Springer). 2. Raanan, H., Felde, V.J.M.N.L., Peth, S., Drahorad, S., Ionescu, D., Eshkol, G., Treves, H., Felix-Henningsen, P., Berkowicz, S.M., and Keren, N. (2016). The 3D structure and cyanobacterial activity in a desert biological soil crust. Environ. Microbiol. 18, 372–383. 3. Murik, O., Oren, N., Shotland, Y., Raanan, H., Treves, H., Kedem, I., Keren, N., Hagemann, M., Pade, N., and Kaplan, A. (2017). What distinguishes cyanobacteria able to revive after desiccation from those that cannot: The genome aspect. Environ. Microbiol. 19, 535–550. 4. Raanan, H., Oren, N., Treves, H., Berkowicz, S.M., Hagemann, M., Padec, N., Keren, N., and Kaplan, A. (2016). Simulated soil crust conditions in a chamber system provide new insights on cyanobacterial acclimation to desiccation Environ. Microbiol. 18, 414–426. 5. Falciatore, A., and Bowler, C. (2005). The evolution and function of blue and red light photoreceptors. Curr. Top. Dev. Biol. 68, 317–350. 6. Schmitz, O., Katayama, M., Williams, S.B., Kondo, T., and Golden, S.S. (2000). CikA, a bacteriophytochrome that resets the cyanobacterial circadian clock. Science 289, 765–768. 7. Wiltbank, L.B., and Kehoe, D.M. (2016). Two cyanobacterial photoreceptors regulate photosynthetic light harvesting by sensing teal, green, yellow, and red light. mBio 7. 8. Yeh, K.-C., Wu, S.-H., Murphy, J.T., and Lagarias, J.C. (1997). A cyanobacterial phytochrome two-component light sensory system. Science 277, 1505–1508. 9. Fiedler, B., Borner, T., and Wilde, A. (2005). Phototaxis in the cyanobacterium Synechocystis sp PCC 6803: Role of different photoreceptors. Photochem. Photobiol. 81, 1481–1488. 10. Leverenz, R.L., Sutter, M., Wilson, A., Gupta, S., Thurotte, A., Bourcier de Carbon, C., Petzold, C.J., Ralston, C., Perreau, F., and Kirilovsky, D. (2015). A 12 Å carotenoid translocation in a photoswitch associated with cyanobacterial photoprotection. Science 348, 1463–1466.
1
These authors contributed equally to this study. 2Department of Plant and Environmental Sciences, the Hebrew University of Jerusalem, Jerusalem 9190401, Israel. 3Present address: Institute of Earth, Ocean and Atmospheric Sciences, Rutgers University 71 Dudley Road, New Brunswick, NJ 08901, USA. 4 Lead contact. *E-mail: [email protected]
Current Biology 27, R1037–R1059, October 9, 2017 R1057