Dehydration-induced responses of primary photosynthetic processes and spectral reflectance indices in Antarctic Nostoc commune

In this study, we investigated the relationship between
relative water content (RWC) of N. commune colonies recorded
during gradual dehydration and (i) normalized difference
vegetation index (NDVI), (ii) photochemical reflectance index
(PRI), and (iii) primary photochemical processes of
photosynthesis, effective quantum yield of photosynthetic
processes (PhiPSII) in photosystem II particular. PRI increased
from -0.05 to 0.02 with RWC decrease from 100% (full hydration)
to 0% (dry state). NDVI showed somewhat curvilinear
relationship with desiccation with minimum value of 0.25 found
at 10% RWC. Negative effect of suprasaturation of N. commune
colony with water on effective quantum yield (PhiPSII) was
found at RWC range 80-100%. At the RWC range, PhiPSII reached
only 50 % of maximum found at RWC of 30%. In general,
desiccation-response curve of showed polyphasic character with
three main phases (phase I – constant PhiPSII values, phase II
– an increase with desiccation at RWC 80-30%, and phase III –
sigmoidal decrease with desiccation at RWC 0-30%).


Introduction
Nostoc commune is frequently found in terrestrial and semi-aquatic Antarctic habitats. It is reported from maritime Antarctica (Victoria et al. 2013) as well as from continental Antarctica (e.g. Novis et Smissen 2006). Generally, the ability of N. commune to fix atmospheric nitrogen and, to resist desiccation may explain its dominance in many terrestrial habitats in Antarctica. In Antarctic ecosystems, such as margins of freshwater terrestrial lakes, temporal ponds, streams and seepages, rich in melt water during austral summer Nostoc commune typically forms colonies. Among Nostoc species forming sheetlike colonies, N. commune, and closely related N. flagelliforme are reported (Sand-Jensen 2014). At James Ross Island, Antarctica, N. commune colonies are quite abundant in shallow streams and seepages (Skácelová et al. 2013, Komárek et al. 2015. N. commune colonies form inactive crusts when dry, but rapidly changes to gelatinous structures when wet (e.g. Novis et al. 2007). Major part of colonies are composed of the extracellular matrix -up to 60% of dry mass is reported (Hill et al. 1997). Matrix forms upper and lower surface of the colony as well as an intracolonial matter filling the space in between individual filaments of N. commune cells. The matrix is formed primarily of several polysaccharides (Rossi et Philippis 2015) that have a high viscosity and molecular weight (Sand-Jensen 2014). In general, N. commune matrix contains also monosaccharides, uronic acid, deoxy-sugars, pyruvate, acetate and peptides (Pereira et al. 2009). Physico-chemical properties of extracellular matrix may vary according to number and arrangement of exopolysaccharides. However, quantitative data on polysacharides in extracellular matrix are still insuffient for functional analysis of N. commune during wet-dry cycles. However, 1-4-linked xylogalactoglucan backbone with D-ribofuranose and 3-0-[(R)-1-carboxyethyl]-D-glucuronic acidpendant groups are reported from desiccation tolerant N. commune .
N. commune is typical by a high tolerance to a great variety of environmental stressors including desiccation (Satoh et al. 2002, Tamaru et al. 2005. The species is considered extremely tolerant to long-term desiccation, some biomolecules may stay active even when rewetted after hundred years in dry state (Shirkey et al. 2000). During desiccation, several photoprotective mechanisms are activated in Nostoc. Among them, zeaxanthin, but also nostoxanthin and caloxanthin, and ketocarotenoids echinenone were detected in Nostoc sp. (Schagerl et Müller 2006) as well as carotenoids (Potts et al. 1987) and beta carotene that represents a strong photoprotective quenchers.
Photosynthetic processes in N. commune collonies from polar regions have been investigated by a variety of chlorophyll fluorescence techniques in response to light and temperature (Kosugi et al. 2010), UV-B radiation (Estêvāo 2015).
In this paper, we focused on changes in primary photosynthetic processes monitored by chlorophyll fluorescence parameters during desiccation in N. commune colony. We hypothesized that, similarly to cyanolichens (Lange et al. 1996), water suprasaturation effect resulting in inhibition of photosynthetic processes could be detected at high thallus hydration. We, therefore, also hypothesized that optimum hydration of N. commune colony would be less than full hydration, i.e. under 100% of relative water content. Simultaneously, we focused on changes in spectral reflectance indices NDVI and PRI. Similarly to the study of Yamano et al. (2006) who studied soil crusts. The letter one is associated with activation of xanthophyll-cycle pigments and studied in higher plants frequently. In lichens and cryptogamic polar autotrophs forming microbiological crusts, number of studies is limited. Recently, an interest in spectral reflectance properties of polar soil crust has been increased (see e.g. Walker et al. 2012), as well as ecophysiological characteristics, such as e.g. water holding capacity. The same trend is apparent also in the research of temperate zone ecosystems (Gypser et al. 2016). However, knowledge on physiological background of PRI changes in response to hydration status is still insufficient polar autotrophs.

Material and Methods
The colonies of Nostoc commune were collected in James Ross Island, Antarctica, from the long-term research plot located close to Czech Antarctic station J. G. Mendel (63° 48′ 02″ S, 57° 52′ 57″ E). Sampling plot is typical by patchy moss cover (more than 50% of total plot area) with several lichen species (Barták et al. 2015b). The plot is wet at least for some part of austral summer season. It is supplied by melt water form two temporal streams originating in neighbouring annual snow field. Austral summer microclimate is typical by air temperature ranging from -10°C (monthly mean of September) to 6°C (monthly mean of February) -measured at 30 cm above surface (Láska et al. 2011). Austral summer surface temperature (absolute daily minimum, maximum) ranges from -7.1 to 12.0°C (Barták et Váczi 2014), and close-to-ground relative air humidity is found within the range of 80-100% (Láska et al. 2011). Dehydration response curves of PRI and NDVI were measured at J. G. Mendel station laboratory, while chlorophyll fluorescence parameters were measured after transfer to Brno, Czech Republic.

Dehydration and spectral indices measurements
Measurements of spectral reflectance indices and photosynthetic measurements were done in samples desiccating from fully hydrated to fully dry state in a laboratory (Mendel station). During the desiccation, room temperature was kept constant (18°C, 40% RH) and lichen thalli were left in open Petri dishes to desiccate naturally desiccation. Relative water content (RWC) was evaluated before each single individual PRI, NDVI measurements using a gravimetric method. Samples were weighted on an analytical RADWAG scale (XA 60/220/X), and RWC calculated according to the equation:

Chlorophyll fluorescence
Repetitive measurements of effective quantum yield ( PSII ) of photosystem II in Nostoc sp. started from fully wet state and ended in dry state. Samples were allowed to desiccate at room temperature (t=22°C, EEL laboratory, Brno, Czech Republic) and low irradiance (20 mol m -2 s -1 ). During gradual slow-rate desiccation (according to sample size 7-14 h from fully wet to dry state), actual fresh mass (weight) F M was evaluated (Mettler AS100, Germany) as well as several chlorophyll fluorescence parameters using a PAM-2000 fluorometer (H.Walz, Germany): variable fluorescence at steady state (F S ). The measurements were taken simultaneously so that the response of particular chlorophyll fluorescence parameters (see below) to RWC (Eqn. 1) could be evaluated. Light-adapted chlorophyll fluorescence comprised signals (F S , F M´) and parameters ( PSII , qN). They were measured each 20 min. until dry state was reached.

Results
In N. commune, NDVI showed a curvilinear relationship to RWC with desiccation (see Fig. 1). Maximum values were found in the range of 40-70% RWC. Minimum NDVI of 0.12 (individual measure-ment) was found at low RWC (0%). PRI values exhibited an increase with desiccation (within a range from -0.02 to 0.01) with maximum found at RWC of 10%. Dehydration-response curve of  PSII had polyphasic character with 3 main phases (Fig. 2). At RWC range 80-100% RWC (phase I),  PSII was more or less constant (0.15). With gradual dehydration (phase II) from RWC of 80 to 30%, in-creased and showed a maximum of 0.27 at RWC of 30%. Then, with further dehydration (phase III, RWC 0-30%), N. commune colony showed a S-shaped decline in  PSII with 0 found at RWC of about 5% RWC. Phase I with limited  PSII could be related to reduction of the diffusive flux of dissolved inorganic carbon to the colony thanks to boundary layer thickness (Sand-Jensen 2014). Variable fluorescence at steady state (F S ) declined with desiccation in a polyphasic manner (see Fig. 3). It showed, however, constantly high values (about 0.730 mV) at RWC range of 79-100%, and, at final phase of dehydration (RWC 0-17%), constantly low values (0.160 mV). Non-photochemical quenching (qN) showed constant value at the RWC range (79-100%), followed by an increase towards maximum (RWC 17-79% corresponding to phase II in  PSII ). Then, maxium qN of about 0.94 was maintained during following desiccation to fully desiccated state (from 17 to 0% RWC). Non-photochemical quenching (qN) of absorbed light energy showed constant value of about 0.7 at full hydration, then increased with following dehydration (RWC range of 79-100%), and reached its maximum at RWC of 17% indicating that capacity of protective mechanisms in photosynthetic apparatus was fully exploited at the RWC. Fig. 3. Dehydration -response curves of steady-state chlorophyll fluorescence (F S , left), and nonphotochemical quenching (qN, right). Decrease in relative water content (RWC ) from full hydration (RWC=100%) to desiccated state (RWC=0%) leads to a polyphasic decrease in F S , and an increase in qN.

Discussion
A polyphasic curve of  PSII to RWC relationship reflects several limitations of photosynthetic processes in N. commune colony. Initial phase of desiccation (WP range 100 to 80%), at which  PSII values are constant but far below their maximum, is attributed to photosynthesis exploiting carbon available inside Nostoc cells thanks to carbon concentrating mechanisms exclusively. At this RWC range no atmospheric carbon contribute to photosynthetic processes thank to a physical barrier of water-saturated colony for CO 2 diffusion. Such phenomenon is called suprasaturation (i.e. the photosynthetizing poikilohydric organism contains more water than necessary to saturate photosynthesis) and well described for lichens (see e.g. Lange et al. 1996Lange et al. , 2001. In the following phase (RWC from 80 to 30%), the atmospheric CO 2 diffusion into the colony is enhanced and an increased  PSII can be explained as a coaction of increased CO 2 intake and fixation by the colony (when considering an equilibrium between photochemical and biochemical processes). The fall of  PSII in the last RWC range (WP decreasing from 30 to 0%) is associated dehydration-decline in photosynthesis in Nostoc cells.
Our results showing a steep  PSII decline at RWC below 20% suggest that even a partially hydrated Nostoc commune colony is capable to maintain high rates of photosynthetic processes in desiccating colonies. This conclusion might be supported by the data presented by Kvíderová et al. (2011), who reported unchanged F V /F M and  PSII for at least 6 h from the start of desiccation. Similar rate is reported for rehydration, Gupta et Kashyap (1995) showed that water uptake by desiccated thalli of N. commune occurring in Schirmacher Oasis (Antarctica) took about 5 hours for the thalli to absorb water to a saturating level. In the field, however, desiccation rate and gradual loss of photosynthetic activity is even slower than reported by Kvíderová et al. (2011) because of contact of the colonies with wet surfaces such as e.g. soil, mosses. Such contact allows to slow down desiccation and prolong the time of photosynthetic activity. In the field, size of N. commune colony may play a role since a faster desiccation in small-area colonies is reported (Gao et Ai 2004). It has been demonstrated earlier that Nostoc colonies decrease their mean diameter from 150-250 µm to 50-100 µm during desiccation from wet to dry state (Belnap et Gardner 1993). Apart of colony size, a 3-D complexity of colony may affect the rate of desiccation as well. Typically, surface to volume ratio and morphological structures (Esseen et al. 2015) may affect water holding capacity and the rate of desiccation.
Decrease of F S and an increase in qN is a general phenomenon found in desiccating poikilohydric autotrophs. Such phenomenon is a mechanism providing effective protection of photosynthetic apparatus. For terrestrial cyanobacteria, numerous pigments, carotenoids in particular (Vincent et al. 1993, Lakatos et al. 2001, and other quenchers, such as e.g. inhibition of state 2 to state 1 transition (Trnková et Barták 2016, accepted), are involved into the protection during desiccation, which, together with fast reactivation of photosynthetic pigments (Abed et al. 2014) and lipids resynthesis after wetting (Taranto et. al. 1993), represent a strategy to cope with repeated drought stress. Specifically for N. commune, these mechanisms make the species extremely desiccation and photoinhibition tolerant (Fukuda et al. 2008).
Optimum RWC at which maximum NDVI was reached ranged 40-60%. The RWC range is well comparable to the values reported in our earlier studies (similarly to Barták et al. (2015a), Trnková et Barták 2016, accepted). NDVI index might be affected by phycocynine since spectral reflectances between 610 and 650 nm are included into PC index (see e.g. Kutser et al. 2006). PRI exhibited negative values at RWC range 40-100%. It means that wellhydrated Nostoc sp. colony has lower reflection at 570 than 531 nm. Possible explanation is an interaction with absorption spectra of phycobilisomes (phycoerythrin, phycocyanin) that overlap at the wavelength about 570 nm (Singh et al. 2015). Similarly, recent study of Li et al. (2015) indicated a decrease in reflectance 570 nm in cyanobacterial community and attributed the decrease to absorption by phycoerythrin.