Using Chlorophyll Fluorescence to Test for Desiccation Tolerance Among Green Algae
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From River to Desert: Using Chlorophyll Fluorescence to Test for Desiccation Tolerance Among Green Algae Hannah L. Gershone 1844 Blanchard Campus Center Mount Holyoke College 50 College Street, South Hadley MA 01075 Mentors: Dr. Zoe Cardon and Dr. Elena Peredo The Ecosystems Center 7 MBL Street Woods Hole, MA 02543 Semester in Environmental Science December 18, 2016 Gershone 1 Abstract Desert crusts are composed of a mix of organisms, including green algae, and provide many invaluable ecosystem functions. Desert crusts are a growing importance for ecosystem restoration. Understanding characteristics such as desiccation tolerance among crust organisms is critical for improving our restoration methods. I examined and compared the desiccation tolerance of three very closely related species of green algae from the genus Scenedesmus: Scenedesmus deserticola, Scenedesmus bajacalifornicus, and Scenedesmus obliquus. Scenedesmus deserticola was originally isolated from desert microbiotic crust. Scenedesmus bajacalifornicus was originally isolated from desert microbiotic crust, however, these cultures were lost. I used a Scenedesmus bajacalifornicus isolate from brackish water in a tidal river in Spain. Lastly, Scenedesmus obliquus is a freshwater isolate. To test for desiccation tolerance, I grew all of my algae under common garden conditions in liquid media. I then dried the algae slowly in the dark. After the algae were desiccated, I recorded fluorescence and humidity data during three cycles of desiccation and rehydration. I used the fluorescence data as an indicator of photosynthetic activity in the green algae. I found that S. obliquus most likely died during desiccation and/or rehydration. During the fluorescence assays, S. obliquus’ photosynthetic quantum yield of PSII was essentially zero. S. deserticola and S. bajacalifornicus had relatively high photosynthetic quantum yield of PSII values during all three hydration and desiccation cycles. However, S. bajacalifornicus had the highest average maximum photosynthetic quantum yield of PSII for all three cycles. I ultimately found S. obliquus had extremely low desiccation tolerance while S. deserticola and S. bajacalifornicus had high desiccation tolerance. Because S. bajacalifornicus had the highest average maximum photosynthetic quantum yield of PSII for all three cycles, it was the most desiccation tolerant. Key Words Scenedesmus deserticola, Scenedesmus bajacalifornicus, Scenedesmus obliquus, green algae, desiccation, fluorescence, photosynthetic quantum yield of PSII, microbiotic crust, restoration Gershone 2 Introduction Unappreciated for their physical beauty, microbiotic crusts are key components in desert ecosystems. Organisms combined with their by-products form crusts on top of the soil, creating a buffer against soil disturbance and erosion (Johnston 1997; USGS 2016). Crusts are invaluable to desert ecosystems; they can be the main carbon and nitrogen fixers in the ecosystem, store and filter water, and increase seed germination (Johnston 1997; USGS 2016). Large portions of crusts are composed of cyanobacteria, but a mix of diatoms, bacteria, rhizoid communities, fungi, and in particular, green algae are also found in crusts (Johnston 1997; Cardon et al. 2008; USGS 2016). I examined and compared the desiccation tolerance of three very closely related species of green algae from the genus Scenedesmus: Scenedesmus deserticola, Scenedesmus bajacalifornicus, and Scenedesmus obliquus. Two of these species made independent leaps to land (Cardon et al. 2008) and were isolated from desert crusts (Lewis and Flechtner 2004). One of the green algae species I studied that transitioned to land was Scenedesmus deserticola. S. deserticola (SNI-2) was originally isolated from desert microbiotic crust in San Nicolas Island, California (Lewis and Flechtner 2004). The second green algae species I examined, which also made an independent leap to land, was Scenedesmus bajacalifornicus. S. bajacalifornicus was first isolated from desert microbiotic crust in Baja, California and sequenced by Lewis and Flechtner (2004). However, the S. bajacalifornicus cultures isolated by Lewis and Flechtner (2004) were lost in Zoe Cardon and Elena Peredo’s lab. Instead, I tested another S. bajacalifornicus isolate (BEA 747B). BEA 747B was recently identified as S. bajacalifornicus, or a very similar species, based on a comparison of its 18S rRNA gene sequences to those of the S. bajacalifornicus desert isolates published by Lewis and Flechtner (2004). The S. bajacalifornicus isolate I tested is from brackish water in a tidal river in Spain (Nervión River Estuary-Station 3, Vizcaya, Banco Español de Algas 2016). The last green algae species I studied was S. obliquus (UTEX 393), which is a freshwater isolate (AlgaeBase 2016 B). Desiccation tolerance is critical in desert microbiotic crusts (Cardon et al. 2008). Desert crusts must survive in areas exposed to harsh sunlight where water availability is extremely low (Rosentreter and Belnap 2003). S. obliquus, the freshwater isolate, has very little desiccation tolerance (Cardon et al., unpublished data). On the other end of the spectrum is Scenedesmus deserticola, the desert microbiotic crust isolate, which is highly desiccation tolerant (Cardon et Gershone 3 al., unpublished data). Finally, Scenedesmus bajacalifornicus’ desiccation tolerance was unknown before my study. Both S. deserticola (desert) and S. bajacalifornicus (tidal river) undergo cycles of rehydration and desiccation in their respective ecosystems (Rosentreter and Belnap 2003). Desiccated algae undergo an additional stress; they’re still absorbing light. When an alga absorbs light energy, the energy first excites chlorophyll molecules in the antenna complexes (Amarnath et al. 2015). When an alga is hydrated, absorbed excitation energy from the antenna complexes is transferred to the plant’s photosynthesis reaction center where electrons flow into a ‘z scheme,’ eventually creating ATP and NADPH (Amarnath et al. 2015). A hydrated alga gives off huge chlorophyll fluorescence spikes, during very strong pulses of light that saturate photosynthesis, suggesting the alga’s photosynthetic machinery is active and functional (Veerman et al. 2007). However, when an alga is exposed to sustained high light intensity and/or begins to desiccate, the alga activates safety mechanisms (Gray et al. 2007; Veerman et al. 2007; Lunch et al. 2013). These safety mechanisms’ activities contribute to non-photochemical quenching, or NPQ (Maxwell and Johnson 2000; Veerman et al. 2007). When activated during desiccation, the safety mechanisms prevent light energy from being transferred to PSII (Veerman et al. 2007). We know these mechanisms are at work when we detect quenching of fluorescence from PSII at room temperature during saturating pulses of light (Veerman et al. 2007). If this dissipation didn’t occur, the photosynthetic machinery would be damaged in desert conditions! I hypothesized S. bajacalifornicus had a desiccation tolerance between S. deserticola and S. obliquus, but would be on the higher end of the desiccation range. This is because S. bajacalifornicus undergoes similar osmotic stress in a tidal river system as it does in a desert. S. bajacalifornicus must be able to handle salty conditions and long periods without water in both ecosystems. To test my desiccation hypothesis, I measured photosynthetic activity by collecting chlorophyll fluorescence data during cycles of desiccation and rehydration of the algae. Methods Algal Species and Culturing Methods: I grew cultures of all three green algae species: Scenedesmus obliquus, Scenedesmus deserticola, and Scenedesmus bajacalifornicus. Gershone 4 Scenedesmus obliquus (UTEX 393): In the class Chlorophyceae (AlgaeBase 2016 B). S. obliquus was isolated from freshwater (AlgaeBase 2016 B) and obtained from the Culture Collection of Algae at the University of Texas at Austin (2016). They divide in groups of four and are shaped like crescents (personal observations). It has very little desiccation tolerance (Cardon et al., unpublished data). Scenedesmus deserticola (SNI-2): In the class Chlorophyceae (Algaebase 2016 A). Originally isolated from desert soil surface in San Nicolas Island, California 33.2ºN latitude, 119.2º W longitude (Algaebase 2016 A; Lewis and Flechtner 2004) and sequenced by Lewis and Flechtner (2004). The cells are shaped like crescent moons, lemons, and/or bananas (Algaebase 2016 A; personal observations). S. deserticola is highly desiccation tolerant (Cardon et al., unpublished data). Scenedesmus bajacalifornicus (BEA 747B): In the class Chlorophyceae (Banco Español de Algas 2016). It was collected from Vizcaya, Spain from the estuary of the Nervión River (43.36ºN latitude, -3.04º W longitude) by Aitor González and isolated by V. Cruz Alamo (Banco Español de Algas 2016). Its 18S rRNA gene sequence identified it as S. bajacalifornicus, based off of Lewis and Flechtner’s sequencing (2004). They are very round and shaped like bubbles (personal observations). Its desiccation tolerance is unknown. I grew all three species of green algae under common garden conditions in a liquid culture mixture one half modified Bold Basal Medium (Stein 1973) and one half Woods Hole Medium (Stein 1973) as described in Lunch et al. (2013). I grew each species in a 250 mL Erlenmeyer flask. I filled every flask with 100 mL of the BBM and Woods Hole medium mixture and added 50 mL of already actively growing cultures of the respective algae. I grew the algae for one