Desiccation-induced oxidative stress causes metabolic flux in the terrestrial green alga Trentepohlia sp., suggesting extensive metabolic reprogramming for stress adaptation

doi:10.21203/rs.3.rs-8012098/v1

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Desiccation-induced oxidative stress causes metabolic flux in the terrestrial green alga Trentepohlia sp., suggesting extensive metabolic reprogramming for stress adaptation

https://doi.org/10.21203/rs.3.rs-8012098/v1

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In the present investigation, we report the impact of desiccation stress on the terrestrial green alga Trentepohlia sp. Stress imposition for a period of 7 d until the relative water content (RWC) of the algal thalli reached < 15%, resulted in a substantial change in morphological attributes, stress markers such as reactive oxygen species (ROS), oxidative stress markers, antioxidant metabolism, and metabolic responsesThe desiccation stress caused extensive changes in algal thalli, which were reflected in terms of loss of color and appearance of white patches as compared to the control. Microscopic studies revealed a considerable impact of desiccation stress on the algal filaments, which were manifested by bending and an increase in translucency due to the loss of cytoplasmic contents. Though desiccation stress could not impose any statistically significant impact on photosynthetic pigment levels, it caused a significant rise in ROS production and elevated the level of oxidative stress markers. With high ROS content and substantially high oxidative stress, the antioxidant metabolism was strongly affected in the alga during desiccation stress compared to the control. The metabolomic analysis revealed that desiccation causes a major metabolic shift in the alga, which could be attributed to the strategy for adaptation to desiccation through metabolic reprogramming. The results suggested that Trentepohlia is mostly susceptible to desiccation, but still manages to survive by sustaining a pool of some vital (signature) metabolites.

Desiccation

oxidative stress

ROS

metabolomics

Trentepohlia sp

Abiotic stresses are the major environmental constraints that affect almost all life forms on the planet. Among cryptogams, algae inhabit diverse habitats and are susceptible to various forms of abiotic stress. Although environmental stresses are natural, anthropogenic interferences have also significantly contributed to their recurring emergence. Furthermore, climate change has led to a significant shift in environmental dynamics, exacerbating these stresses and making them more severe and persistent. Life on this planet is dependent mainly on the availability of water. Water acts as a medium for all cellular activities. In the absence or scarcity of water, the cell loses its turgor and ceases to function normally. In recent times, global water scarcity has been increasing at an alarming rate, resulting in recurring droughts in many parts of the world. This largely occurs as a consequence of changes in the global precipitation pattern and the emergence of extreme climatic events.

Drought refers to a condition characterized by an extreme scarcity of available water, which affects the sustenance and survival of life (Takahashi et al., 2020). Desiccation is an extreme form of drought, where the cellular water content becomes significantly low and, in some cases, even attains a relative water content (RWC) of less than 10% (Gechev et al., 2012; Oliver et al., 2020). During desiccation, the plants experience extreme water loss, which exerts structural and mechanical constraints (Oliver et al., 2020). Desiccation in plants causes detachment of the cell wall from the plasma membrane, which alters the membrane permeability and risks the loss of cytoplasmic content outside the cellular environment (Walters et al., 2002; Oliver et al., 2020). Further, desiccation causes the loss of turgor, which substantially affects the movement of carbon and nitrogen in different metabolic pathways (Lawlor and Tezara, 2009; Krasensky and Jonak, 2012; Oliver et al., 2020). Desiccation stress has also been linked to the overproduction of reactive oxygen species (ROS), which are responsible for oxidative damage to cells and their components, and affect antioxidant metabolism, resulting in a lowering of the antioxidant pool (Shivaraj et al., 2021). A high concentration of ROS due to desiccation has also been directly implicated in poor plant growth and loss of metabolic function (Gechev and Petrov, 2020; Ravi et al., 2023).

Desiccation is considered lethal for most of plants, while scientific evidence suggests that the vast majority of lower plants, such as cyanobacteria, algae, lichens, bryophytes, and pteridophytes, can efficiently tolerate desiccation by modulating their physiological and metabolic functions (Holzinger and Karsten 2013; Gasulla et al. 2021; de Carvalho et al. 2019; Alejo-Jacuinde and Herrera-Estrella 2022). This unique attribute of these plants clearly indicates that desiccation tolerance is an early evolutionary phenomenon (Oliver et al., 2005, 2020; Proctor et al., 2007). In contrast to aquatic environments, the terrestrial green algae are more susceptible to desiccation owing to harsh and fluctuating climatic conditions that cause cellular dehydration. Water indeed is a key ecological requirement for survival for terrestrial algae growing in different habitats, ensuring the cellular integrity and physiological fitness (Karsten et al., 2010; Holzinger et. al., 2009; Karsten and Holzinger, 2012). The green alga Trentepohlia sp. primarily grows in terrestrial habitats, such as bare rock or concrete walls, that are subjected to varying water availability conditions; thus, it often exhibits an extreme form of desiccation tolerance (Rindi and Guiry, 2002). Studies have reported that the photosynthetic rates in the green alga Trentepohlia odorata are significantly inhibited due to low humidity and increased rate of desiccation (Ong et al., 1992). A relatively faster photosynthesis retrieval has been reported as a pronounced desiccation tolerance in green algae after a short rehydration period (Lüttge and Büdel, 2010).

There are limited studies that address the desiccation responses and tolerance in Trentepohlia sp. (Ong et al., 1992; Zhang et al., 2016, 2020; Ritchie and Heemboo, 2021). In Trentepohlia jolithus, desiccation stress resulted in a reversible down-regulation of PSII light absorption efficiency and activity, serving as a special adaptive mechanism for survival under terrestrial conditions (Zhang et al., 2016). This was attributed to a gradual decline in non-photochemical quenching (NPQ), a crucial photoprotective mechanism during dehydration (Zhang et al., 2020). Generally, environmental stress leads to significant cellular damage, resulting in either acclimation or cell death through apoptosis. As part of stress control and repair mechanisms, changes in fatty acid profiles, the accumulation of specific metabolites, or the manipulation of existing metabolite pools, membrane fluidity, chaperonin synthesis for rectifying denatured proteins, maintaining cell osmolality, and stable photosynthesis for energy balance are attributes that green algae tend to adopt. Terrestrial green algae are, therefore, evolutionarily advantaged to endure severe water stress capable of rejuvenating upon rehydration. With the advent of vast high-throughput analytical screening methods and advanced statistical tools for data analysis, novel traits in understanding plant responses to stresses have emerged. The physiological and biochemical datasets, accompanied by an inclusive metabolic analysis, provide a unique way to analyze the phenotypic response of the organism to the stresses (Obata and Fernie, 2012; Bueno and Lopes, 2020; Choudhury et al., 2021; Carrera et al., 2021). Herein, we report the response of a terrestrial green alga, Trentepohlia sp., to desiccation stress. Changes in stress markers such as ROS production, lipid peroxidation, and antioxidant defense metabolism in the alga under desiccation stress were evaluated. The metabolic disparity in the alga was probed using proton (¹H) nuclear magnetic resonance (NMR) and high-resolution liquid chromatography-mass spectrometry (HRLC-MS)-based studies.

Collection of samples and experimental setup

The experiment was designed to study the effect of desiccation stress on the terrestrial green alga Trentepohlia sp. The experimental design involves a stepwise increase in temperature and a drop in relative humidity (RH) (Fig. 1). Fresh samples of Trentepohlia sp. were collected during the month of May 2024 from tree bark inside the campus of Assam University, Silchar (24.6850°N and 92.7512°E), India, and brought to the laboratory in sterile plastic bags. The samples were cleaned of dust particles and any extraneous matter, then transferred to petri plates and allowed to acclimate for 48 hours inside a plant growth chamber at 30°C with 70% relative humidity (RH) and illumination of 40 µmol m^− 2 s^− 1 with a 12-hour alternate photoperiod. Before the onset of desiccation stress, petri plates containing the algal thalli were kept inside the laminar airflow chamber for 2 hours with a medium flow of air and then transferred to the growth chamber. The algal thalli were maintained inside the growth chamber at 30°C for 24h with 70% RH. The alga was then exposed to conditions with a linear decrease in RH from 70% to 50% over 24h at 35°C. At this stage, the algal thalli showed mild symptoms of stress. The RH declined from 50% to 30% over a 16-hour period at 40°C, during which the alga exhibited more visible stress symptoms. The alga was then grown under 30% RH for 7d, where the thalli exhibited significant stress symptoms such as loss of color, which was seen by the appearance of greyish-white patches (Fig. 1). The total time for onset of desiccation stress was found to be 40h. After the stress was imposed at 30% RH at 40°C, the relative water content (RWC) was measured on 1, 3, 5, and 7 days of stress. To determine the RWC, 0.5g algal thalli were immersed in 100 mL distilled water (dH₂O) at 30°C for 24h. The samples were removed, blotted, dried, weighed, and placed inside an oven at 80°C for 48h. The samples were weighed again to assess the dry weight. The RWC was calculated by using the formula:

$$\:RWC\:\left(\%\right)=\frac{Fwt-Dwt}{Twt-Dwt}\times\:100$$

where, F_wt = fresh biomass weight of the algal samples; D_wt = dry biomass weight of the algal samples;

T_wt = turgid weight of the algal samples

The RWC of ≤ 15% was considered the decisive desiccation-stressed condition of the algal thalli (Supplementary File S1), and algal filaments were collected for analysis. The results showed a reduction in RWC, suggesting that the effect of desiccation was distinct after 7d of stress imposition. At 7d, the RWC came down to almost 13%, indicating a severe water deficit condition. Thus, at this stage, the algal samples were considered desiccated and harvested for analysis.

Changes in morphological attributes

To study any morphological changes in the algal filaments, microscopic observations were made after 7 days of desiccation stress and compared with control sets. The cells were studied at 400X magnification, and any visible changes were microphotographed using a light microscope (Leica, Germany).

Determination of chlorophyll content and stress markers

The pigment content in the alga Trentepohlia sp. was estimated by placing the algal thalli with 90% (v/v) acetone at 4°C overnight, and later homogenizing with 90% (v/v) acetone. The homogenate was centrifuged at 5,000g for 15 min, and the supernatant was collected. The supernatant was used to estimate chlorophyll a, b, and total chlorophyll by measuring the absorbance at 665, 645, and 630 nm using a UV-Visible spectrophotometer (Thermo Scientific, USA). The stress markers were measured in terms of the production of reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) and superoxide radical (O₂^Ÿ−), along with malondialdehyde (MDA) content as a marker of oxidative stress. The H₂O₂ and O₂^Ÿ− contents were determined as per the methods of Sagisaka (1976) and Elstner and Heupel (1976), respectively. The MDA content was determined as described by Heath and Packer (1968).

Antioxidant metabolism

To study the antioxidant metabolism in the alga under desiccation stress, we measured the activities of antioxidant enzymes, such as catalase (CAT) and superoxide dismutase (SOD), along with the levels of non-enzymatic antioxidants, including ascorbate (AsA) and glutathione (GSH). To extract antioxidant enzymes, 0.2g of algal samples was ground with liquid nitrogen and homogenized with 0.1M Na-phosphate buffer (pH 7.2). The homogenate was centrifuged at 16,000g for 20 min at 4°C. The supernatant was used for the assay and estimation of CAT (Aebi, 1984) and SOD (Beauchamp and Fridovich, 1971). To determine ascorbate (AsA) and glutathione (GSH) levels, 0.2g of algal samples was ground with liquid nitrogen and homogenized with 5% (w/v) sulfosalicylic acid. The homogenate was rested over ice for 5 minutes, and the supernatant extract was obtained through centrifugation at 12,000 g for 10 minutes at 4°C. AsA and GSH levels were determined according to the methods of Oser (1979) and Griffith (1980), respectively.

Metabolite extraction

For high-resolution liquid chromatography-mass spectrometry (HRLC-MS) analysis, metabolites were extracted by homogenizing 200mg of frozen algal samples with 5 mL of LC-grade methanol (Mhlongo et al., 2021). The algal homogenate was centrifuged at 6,000 g for 15 min at 4°C and dried in a desiccator under a nitrogen (N₂) gas flow. The dried samples were reconstituted with 1 mL of methanol and filtered through a 0.22 µm syringe filter. 250 µL of extract was transferred to LC-vials for analysis. The extraction of metabolites for ¹H-NMR analysis was carried out as per the method of Maravi et al. (2022). Briefly, 100mg of the algal sample was ground to a fine powder with liquid nitrogen and suspended in a 2 mL Eppendorf tube containing 1.5 mL of 80% (v/v) methanol. The samples were vortexed for 20 seconds and further extracted using ultrasonication. The homogenate was centrifuged at 12,000 g for 30 min at 4°C. The supernatant was collected in a fresh tube. The residual pellet was resuspended in 80% (v/v) methanol and extracted again. The supernatants obtained from the desiccated and control samples were dried in a vacuum for 3h under nitrogen (N₂) gas flow. The dried samples (extracts) were used for recording ¹H-NMR.

HRLC-MS and ¹H-NMR analysis

The HRLC-MS analysis used a 1290 HRLC (UHPLC)-MS system (Agilent Technologies, USA). The data acquisition mode was set to the lowest MS (m/z) range of 120 and the maximum MS (m/z) range of 1200, with a scan rate of 1.00 spectra per second (spectra sec^− 1). An amount of 3 µL of the sample was injected at a flow rate of 0.3 mL min^− 1, and the stop mode was set at 35 min. The solvent phase consisted of 100% ultrapure LC-grade water containing 0.1% (v/v) formic acid (solvent A) and 100% acetonitrile (solvent B). The flow of solvents A and B was adjusted (Table 1). The ¹H-NMR spectra were recorded using a solution of the extracted samples in deuterated dimethyl sulfoxide (DMSO-d6). The ¹H spectrum was obtained using a 400 MHz NMR spectrometer (Bruker, Switzerland) with 512 scans at 298 K using a 5 mm TX1 probe.

Table 1
Composition and flow of solvents used for the separation of metabolites using liquid chromatography (LC)
Solvent (mL)		Time (min)	Flow (mL min^− 1)	Pressure (bar)
Formic acid (0.1%)	Acetonitrile (100%)	Time (min)	Flow (mL min^− 1)	Pressure (bar)
95.0	5.0	1	0.30	1200.00
0.0	100.0	25
0.0	100.0	30
95.0	5.0	31
95.0	5.0	35

Data analysis

The ¹H-NMR spectrum was analyzed using MestReNova (Mestrelab Research, Spain). The spectral regions were bucketed from 0.1 to 8.5 ppm, and the peak intensity and area were calculated. The metabolite datasets obtained from ¹H-NMR and HRLC-MS spectral studies were analyzed using a web-based metabolomics platform, Metaboanalyst 6.0 (https://www.metaboanalyst.ca). The datasets were used to perform PCA biplot analysis, develop heat maps, fold-change analysis, and KEGG metabolite enrichment set analysis. The metabolites were identified from the ¹H-NMR spectrum by comparing the literature (Maravi et al. 2022; Macari et al. 2022; Ghosh et al. 2024) and Biological Magnetic Resonance Bank (https://bmrb.io).

Statistical analysis

The datasets obtained from biochemical analysis of pigments, stress markers, and antioxidant analysis were evaluated statistically to measure the significant difference among the treatment groups at p ≤ 0.001, 0.01, and 0.05. One-way analysis of variance (ANOVA) was performed, coupled with a post-hoc test, followed by a Tukey multiple comparison test using INSTAT (ver 3.0) and MS-Excel (Microsoft Inc.). The datasets presented are the mean of three replicates (n = 3).

Changes in morphological attributes

The effect on algal morphology during desiccation stress, as compared to the control, was assessed using a light microscope (Fig. 2). Irregularly branched filaments showing heterotrichous filaments having a prostrate basal system of filaments are observed from which numerous erect filaments emerge. The rust-red coloration of the alga owes its origin to the abundance of haematochrome, which often completely masks the chlorophyll. The algal cells were cylindrical with a length and diameter in the range 23–58 µm and 4–15 µm, respectively. The cell walls are frequently thickened, possibly to serve a protective role against various stress conditions, including desiccation. Chloroplast has a parietal band, usually breaking into irregularly shaped discs.

Upon desiccation, slight fading in pigmentation color was observed, possibly due to the scarcity of water in cells, particularly affecting chloroplast function and active photosynthesis. Minor constriction of the cell suggested loss of cell integrity due to a water-deficient condition, leading to disruption of energy transfer. Morphologically, there were no major alterations in cell shape, size, and color. The alga seems to be highly tolerant to desiccation, adapting well to the changing environment in terrestrial conditions. Slight shrinkage was observed in some cells of the desiccated alga, indicating a localized impact. However, cell integrity remains intact, implying high adaptability and dormant function of the alga even in extremely water-deficient conditions.

Changes in photosynthetic pigment content and stress markers

The filaments of Trentepohlia are often red-orange pigmented because of the presence of lipid droplets in cells containing β-carotenes. Such pigments probably play a stress-protective role in desiccation. In the alga Trentepohlia sp., the pigment composition was largely affected due to desiccation. A substantial decline in the chlorophyll a, b, and total chlorophyll contents was observed in the alga due to desiccation stress (Fig. 3A). The difference was observed to be relatively constant for all the pigments measured. However, a marked effect of desiccation was observed on the total chlorophyll content, indicating perturbation of photosynthetic efficiency that affects plant biomass and energy metabolism. The stress markers in the alga, in response to desiccation stress, were determined as overproduction of ROS (H₂O₂ and O₂^•−) and malondialdehyde (MDA) content, which serves as an indicator of lipid peroxidation. A significant increase in H₂O₂ content upon desiccation stress was observed (Fig. 3B). The increase in H₂O₂ level was almost 1.3-fold higher during desiccation stress with respect to the control. Significant increase (p ≤ 0.001) in O₂^Ÿ− levels was also observed in the alga due to desiccation stress, which was almost 3-fold higher as compared to the control. Associated with the rise in ROS overproduction, the MDA content was significantly (p ≤ 0.05) increased in the alga as a result of desiccation stress. The MDA content was almost 3.2-fold higher in the desiccated alga as compared to the control. This indicated that the species, Trentepohlia, suffered cellular ROS overproduction, and the onset of oxidative stress affected the cellular metabolism and function.

Changes in antioxidant metabolism

To study the antioxidant metabolism in Trentepholia sp. under desiccation stress, we determined the activities of antioxidant enzymes such as CAT and SOD (Fig. 4A) along with the concentration of non-enzymatic antioxidants such as AsA and GSH (Fig. 4B). In the desiccated alga, a significant (p ≤ 0.001) increase in CAT activity was observed with respect to the control. In addition to enhanced CAT activity, a significant increase in SOD activity was also observed in the alga subjected to desiccation stress. The CAT and SOD activities were found to be approximately 2.2- and 3-fold higher, respectively, in the desiccated alga. The concentration level of non-enzymatic antioxidants such as AsA and GSH showed a 2-fold increase (p ≤ 0.01), while GSH levels showed almost 3.5-fold increase under desiccated conditions (p ≤ 0.05). This pattern of antioxidant metabolism reflects that the alga possesses a robust antioxidant defense metabolism to quench ROS and protect the cells from oxidative damage. Thus, desiccation stress caused a significant inhibition of antioxidant defense metabolism, resulting in the overproduction of ROS and a loss of cellular redox homeostasis.

Metabolic analysis

To study the desiccation stress-induced metabolic disparity in the alga Trentepohlia sp., HRLC-MS and ¹H-NMR-based metabolite profiles were obtained from aqueous methanolic extracts of the alga. The HRLC-MS analysis of methanolic extracts of Trentepohlia sp. under desiccation and control conditions displayed a set of twenty-eight (28) metabolites identified in both positive (+ ve) and negative (-ve) ionization modes (Supplementary File S2 and S3). The ¹H-NMR spectroscopy of methanolic extracts exhibited twenty-five (25) identified metabolites along with some unidentified moieties (Supplementary File S4 and S5). These metabolites primarily belong to amino acids, organic acids, carbohydrates, and secondary metabolites. These metabolites are known to possess unique stress-responsive functions, which are anticipated to be important in revealing desiccation tolerance traits in the alga. To study the metabolic disparity in the alga under desiccated and control conditions, PCA and loading plots were generated from the identified metabolite profile using HRLC-MS (Fig. 5A-B) and ¹H-NMR (Fig. 5C-D) analysis. As shown in Fig. 5A, a clear difference in metabolite abundance is observed, which clearly separates the desiccated conditions from the control along PC1. As PC1 explains 98.1% of the variance, it clearly indicates that a strong metabolic shift occurred in the alga due to desiccation. Both control and desiccated conditions were clustered opposite to each other in the PCA plot. The loading plot (Fig. 5B) suggested accumulation of specific metabolites in the alga under desiccation stress, which are present on the positive side of the PC1. These include arginine, glyceroglycolipids, carboxyspermidine, maltose, sphingolipid (CerP), 2-acyl-sn-glycero-3-phosphoethanolamine (LysoPE), and tyrosyl-glutamate. Among these metabolites, ornaline, β-carotene, oxalosuccinic acid, citrate, and cercosporin fell on the negative side of the PC1, indicating that the metabolite abundance was higher in the control condition compared to the desiccated ones. The PCA and loading plot indicated that during desiccation, diverse metabolic pathways are strongly affected, which are mostly associated with stress adaptive and signalling functions.

The PCA plots derived from the metabolic profiles obtained from ^¹H-NMR analysis reflected a clear and strong separation between control and desiccated conditions (Fig. 5C). PC1 accounted for 100% variance, distinctly suggesting a significant metabolic shift among the treatments. Thus, the alga showed a robust and one-way metabolic shift in response to desiccation. The loading plot (Fig. 5D) showed that metabolites such as sugars (sucrose, trehalose, and fructose) and amino acids (alanine, valine, threonine, and cysteine), along with the TCA cycle intermediate (citrate), were clustered on the positive side of PC1. The metabolites such as GABA, linoleic acid, choline, maltose, and arabinose, as well as amino acids (isoleucine, leucine, glycine, and asparagine), sugar alcohol (myo-inositol), and the metabolites associated with energy metabolism (pyruvate and succinate) were abundant in the control, as they are on the negative side of PC1. Both HRLC-MS and ¹H-NMR-based metabolic analysis reflected substantial metabolic shift in the alga due to imposition of desiccation stress.

The difference in metabolite abundance in Trentepholia sp., heat map analysis, and partial least square discriminant analysis (PLS-DA), such as variable score plots (VIP), based on metabolic profiles identified using HRLC-MS (Fig. 6A-B) and ¹H-NMR (Fig. 7A-B) analysis. As shown in Fig. 6A, the metabolic profiles represented a substantial divergence in the alga under desiccated conditions, compared to the control. Furthermore, based on VIP scores, we identified several metabolites in the alga that distinguish between desiccated conditions and control. Among these metabolites that showed VIP scores greater than one (VIP score > 1.0) were considered significant in separating the treatment groups (Fig. 6B). Metabolites that are considered the most significant discriminating factor in separating the desiccated and control groups include carboxyspermidine, arginine, morroniside, and tyrosyl-glutamate, which showed the maximum VIP scores of between 2.5 and 1.8. Moderate contribution in the separation of the treatment group was observed in the case of 2-pyrolidine-5-carboxyllic acid, harderoporphyrin, pyrensylfate, and β-carotene with VIP score greater than one (VIP score > 1.0). The heat map generated with the metabolites identified in the alga under desiccated and control conditions using ¹H-NMR analysis also showed major disparity in metabolite abundance, reflecting a substantial metabolic flux (Fig. 7A). The analysis of VIP score plot (Fig. 7B) showed citrate and myo-inositol to be the most significant contributors (VIP score > 1.6) in the separation of control and desiccated conditions. Other metabolites that contributed moderately (VIP score > 1.0) towards separating the two treatment groups include sugars (arabinose and maltose), amino acids (threonine, valine, glycine, and isoleucine), and other metabolites such as acetate and ascorbate. This suggests that desiccation has a significant impact on metabolic function, particularly indicating a major metabolic shift that may contribute to substantial adaptation and survival during stress. The significant difference in metabolite expression, based on fold change (FC) and statistical significance (p-value), was analyzed using volcano plots to explain the effect of desiccation stress on Trentepohlia sp. in relation to the control (Fig. 8A-B and Supplementary Files S6 and S7). The metabolites profiled using HRLC-MS (Fig. 8A and Supplementary File S6) showed the most significant upregulation of tyrosyl-glutamate, followed by morroniside, arginine, carboxyspermidine, 2-acyl-sn-glycero-3-phosphoethanolamine (LysoPE), and gingerglycolipid. These metabolites exhibited a positive fold change compared to the control. In response to desiccation, significant downregulation of oxalosuccinic acid, 1,3,6-tri-O-galloylglucose, 3-oxo-12,18,28-ursadienic acid, pyrenylsulfate, auriculoside, lucidenic acid, and 3-indolepropinoic acid was observed in the alga. The volcano plot derived from the metabolites profiled with ¹H-NMR analysis (Fig. 8B and Supplementary File S7) also showed significant metabolic flux in the alga in response to desiccation. Significant upregulation in organic acids (acetate and citrate), sugar moieties (sucrose and trehalose), and amino acids (cysteine, alanine, threonine, and valine) was observed in the alga during desiccation stress compared to the control. Metabolites that were most significantly downregulated in response to desiccation in the alga include GABA and linoleic acid, followed by amino acids (glycine, proline, and isoleucine), sugars (arabinose and maltose), sugar alcohol (myo-inositol), lactate, and the metabolites of energy metabolism such as pyruvate and succinate. Metabolites such as choline and leucine showed no substantial change in either of the conditions in the alga. This clearly indicates that desiccation stress creates a substantial metabolic flux in the alga, critically affecting its physiological state and cellular homeostasis.

Metabolic pathway analysis

Based on pathway enrichment analysis and identified metabolites with HRLC-MS, we developed the pathway interaction map in Trentepohlia sp. under desiccation stress (Fig. 9). The pathway enrichment analysis showed that desiccation stress resulted in significant and moderate enrichment of arginine biosynthesis and glutathione metabolism pathway, while the pathway associated with glycerophospholipid metabolism has substantially low but relevant enrichment. The higher enrichment of arginine biosynthesis, followed by glutathione metabolism and glycerophospholipid biosynthesis pathways, reflected their central role in metabolic reprogramming in the alga during desiccation. Integrating the metabolic pathways, arginine biosynthesis fuels the synthesis of polyamines, as evidenced by the abundant level of carboxyspermidine in the alga under desiccated conditions. The carboxyspermidine is further converted to polyamines such as spermidine, which have major stress-responsive functions. Besides, glutamate may also be converted to proline, and further glutamate, along with cysteine is forms glutathione. The component metabolites of glycerophospholipids, such as LysoPE (20:2(11Z.14Z/0:0), can be attributed to lipid metabolism, while the phosphorylated ceramide (CerP(d18:1/12:0) functions in stress signaling mechanisms. The breakdown of starch sugar moieties is converted into intermediate metabolites of the glycolytic cycle, fueling the metabolic components of the TCA cycle. The pyruvate can also be converted to form lysine, which can be converted to alkaloids such as ormosanine.

The pathway enrichment analysis of metabolites identified in the alga during desiccation stress using ¹H-NMR analysis (Fig. 10) showed significant enrichment of metabolic pathways for sulfur metabolism, starch and sucrose metabolism, glyoxylate and dicarboxylate metabolism, and pantothenate and CoA biosynthesis. Additionally, moderate to low enrichment of pathways such as the citrate (TCA) cycle, amino acid (alanine, aspartate, and glutamate) metabolism, pyruvate metabolism, and the Calvin cycle for carbon fixation was also observed in the alga during desiccation stress. The metabolic map revealed a central link among the pathways in the alga, enabling it to survive during desiccation. Starch, for example, is converted to glucose, which can be further converted to trehalose, a molecule with a vital role in stress regulation and signaling. Glucose can be phosphorylated to form glucose-6-phosphate or can be converted into sucrose, which fuels the triose phosphate pool and balances the energy requirements of the cells. The 3-phosphoglycerate (3-PGA), which is presumed to be formed by the carbon fixation pathways, generates pyruvate, a key metabolite of energy metabolism. The metabolism of amino acids such as alanine, aspartate, and glutamate is also significantly important in maintaining the cellular metabolite pool, as they act as precursor for several essential metabolites. Alanine combines with pantoate to form pantothenate, which ultimately forms CoA, an important cofactor in energy metabolism. In the alga, higher levels of alanine metabolism may also contribute to the pyruvate pool, which is decarboxylated to form acetyl CoA and enters the citrate (TCA) cycle. This was evident as TCA intermediates, such as citrate, were observed to be high in the alga under desiccated conditions. Glutamate metabolism generates GABA, which, via the GABA shunt, is converted to succinate, a key intermediate in the TCA cycle.

The major impact of climate change is the recurrent emergence of prolonged periods of dryness. Such an environmental situation has become a common occurrence in many parts of the world, adversely affecting diverse life forms on the planet. Adaptation to such environmental conditions is not a common occurrence among the majority of plant species. During the course of evolution, lower plant groups such as algae have acquired a substantial degree of adaptability to adverse environmental conditions, enabling them to occupy diverse and extreme habitats. Besides, algae are considered important primary producers of both terrestrial and aquatic ecosystems (Bar-On et al., 2018; Becker et al., 2020). Thus, it was interesting to elucidate the physiological and cellular responses of lower plants, such as algae, to adverse climatic conditions, including desiccation stress. In the present study, we observed an interesting response of the terrestrial green alga Trentepohlia sp. to desiccation stress. The imposition of desiccation stress for a period of 7 d resulted in a substantial increase in ROS production, leading to significant oxidative damage and decline in antioxidant metabolism, followed by a strong metabolic flux. Furthermore, desiccation also caused a minimum level of decline in photosynthetic pigments in response to desiccation, although it was not statistically significant. The ROS are considered as inevitable entities of aerobic metabolism, whose production increases under stress conditions (Choudhury et al., 2013; Wang et al., 2024; Xu et al., 2024). During desiccation or water-deficient conditions, ROS production intensifies several-fold, directly affecting physiological functions and altering cellular redox homeostasis (Verma et al. 2019). In Trentepohlia, the imposition of desiccation stress resulted in a substantial increase in H2O2 and O2•• levels, thereby increasing the levels of stress markers, such as MDA. This response could be attributed directly to the inactivation of antioxidant metabolism in the alga. Previous studies on the effect of desiccation stress in the rhodophycean alga Gracilaria corticate and other seaweed species have shown significantly high accumulation of ROS, which caused an extensive increase in lipid peroxidation rate (Kumar et al., 2011, 2014; Taenzer et al., 2024). In the microalga Desmodesmus subspicatus, desiccation or drought-like conditions caused overproduction of ROS, which resulted in a decline of antioxidant metabolism, leading to oxidative stress (Javed et al. 2024).

The metabolomics of desiccation stress responses in the green alga Trentepohlia sp. revealed substantial disparity in metabolite abundance compared to the control. Comprehensive metabolomic analysis, such as multivariate analysis, PLS-DA, volcano plot, and pathway enrichment analysis, revealed that desiccation causes substantial metabolite flux in the alga, which could be attributed to a major metabolic shift, possibly aiming towards the (metabolic) adaptation to sustain the desiccation. The desiccation stress caused the enrichment of several metabolic pathways in the alga, which could be linked to maintaining a specific metabolite pool during desiccation. Arginine biosynthesis was significantly enriched in response to desiccation. It is an important amino acid that is directly linked to polyamine biosynthesis (Winter et al. 2015). A higher level of carboxyspermidine, a precursor for the synthesis of polyamine spermidine, was observed in the alga during desiccation stress. It suggests that arginine biosynthesis may increase the polyamine pool, which in turn serves as an important metabolite in regulating stress responses and adaptation (He et al. 2022). Spermidine is an important metabolite that provides protection against stress and fuels the biosynthesis of spermine, GABA, and TCA cycle metabolites, such as succinate, as well as components of glutathione metabolism (Killiny and Nehela, 2020). Other metabolites that were abundant in the alga during desiccation stress include -pyrolidine-5-carboxylic acid (pyroglutamic acid), morroniside, and tyrosyl-glutamate, all of which have a major role in stress signaling and adaptation (Shen et al., 2021; Song et al., 2024). In the presence of 5-oxoprolinase, pyroglutamic acid can be converted into L-glutamic acid, which could be further converted to GABA by the glutamic acid (Kumar and Bachhawat, 2012; Lee et al., 2019). Tyrosine-glutamate, a dipeptide whose function is not clearly known in plants in response to abiotic stresses, including desiccation. Morroniside is known as a bioactive compound whose function in regulating plant stress responses is not yet understood. However, its use as a potential antioxidant compound has been very well demonstrated in reducing ROS formation, MDA production, restoring mitochondrial function, and regulating microRNA (miRNA) in animal or human cells (Ma et al., 2022; Hwangbo et al., 2024). Thus, it could be presumed that a higher abundance of morronoside possibly functions as a significant antioxidant that controls ROS overproduction in the alga during desiccation stress. In addition, higher levels of 2-acyl-sn-glycero-3-phosphoethanolamine (LysoPE) and phosphorylated ceramide (CerP) suggest their potential role in lipid biosynthesis and stress signaling (Michaelson et al., 2016). Glutamate, which acts as a precursor for arginine that also forms proline, or, along with the amino acid cysteine, it synthesizes important antioxidant metabolite glutathione that helps to maintain the cellular redox homeostasis (Hasanuzzaman et al. 2017).

The metabolite profile, as determined by 1H-NMR-based analysis, showed significant involvement in various vital metabolic pathways. The pathway enrichment analysis showed high to moderate enrichment of the pathways was observed in the alga during desiccation stress, which include starch and sucrose metabolism, sulfur metabolism, pantothenate and CoA biosynthesis, glyoxylate and dicarboxylate metabolism, amino acid (alanine, asparagine, and glutamate) metabolism, selenocompound metabolism, and carbon fixation by the Calvin cycle. Other metabolic pathways, including pyruvate metabolism, have significantly low enrichment in the alga subjected to desiccation stress. Sulfur metabolism can be metabolically interconnected to glutathione and pyruvate biosynthesis. The pyruvate pool in the alga during desiccation stress was also presumed to be fueled by glycolytic intermediates formed from the breakdown of glucose molecules, formed from starch metabolism, or even indirectly from threonine. Additionally, cellular activation of glucose also produces trehalose, a crucial stress-responsive metabolite. Thus, the response of Trentepohlia to desiccation was characterized by the exclusive balancing of energy needs, as evident from the glyoxylate shunt, which bypasses the energy-expensive steps of the TCA cycle. The biosynthesis of amino acids, such as cysteine, requires sulfide, which is generated through sulfur metabolism. Cysteine can be converted to an important antioxidant metabolite, glutathione, which helps to maintain cellular redox homeostasis during stress. Furthermore, desiccation led to glutamate metabolism, which may potentially form GABA. In plants, GABA accumulation is typically increased to maintain ionic balance, and it also functions as a strong antioxidant (Guo et al., 2023). Alternatively, GABA may also be converted to a TCA cycle intermediate, succinate, via the GABA shunt (Ravasz et al., 2017). Thus, based on the comprehensive analysis of stress markers, antioxidant metabolism, and metabolic disparity in the alga Trentepohlia sp. under desiccation stress compared to the control, complex biological responses could be inferred that involve major metabolic reprogramming.

In this study, the use of metabolomics has unfolded novel traits and relevant biomarkers in the terrestrial green alga, Trentepohlia sp., in response to desiccation. The metabolic profiles studied using high-throughput analytical platforms have revealed substantial metabolic reprogramming in the alga in response to desiccation stress, which constitutes an adaptation strategy to tolerate desiccation. While the alga is quite desiccation-sensitive, it exhibits remarkable competence in subtly modulating its metabolic machinery by accumulating specific metabolites and regulating others to survive harsh water-stress conditions.

Acknowledgments We express sincere thanks to SAIF, Indian Institute of Technology, Bombay (IITB), Mumbai, for the HRLC-MS facility. Authors are thankful to Transzend Scientific, Bangalore, for providing the NMR facility.

Author contribution Conceptualization: Jayahree Rout, Shuvasish Choudhury; Methodology: Shuvasish Choudhury, Jayashree Rout; Formal analysis and Investigation: Parul Sharma; Writing- original draft preparation: Parul Sharma, Shuvasish Choudhury; Writing- review and editing: Jayashree Rout, Shuvasish Choudhury; Resources- Jayashree Rout, Shuvasish Choudhury; Supervision- Jayashree Rout, Shuvasish Choudhury

Funding: We declare that no funding was received from any agency for this research work.

Data availability: No existing data were used for the study. All the data are included in the manuscript and as Supplementary Data Files

Conflict of Interest We declare that there is no conflict of interest.

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Desiccation-induced oxidative stress causes metabolic flux in the terrestrial green alga Trentepohlia sp., suggesting extensive metabolic reprogramming for stress adaptation

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Collection of samples and experimental setup

Changes in morphological attributes

Determination of chlorophyll content and stress markers

Antioxidant metabolism

Metabolite extraction

HRLC-MS and ¹H-NMR analysis

Data analysis

Statistical analysis

Results

Changes in morphological attributes

Changes in photosynthetic pigment content and stress markers

Changes in antioxidant metabolism

Metabolic analysis

Metabolic pathway analysis

Discussion

Conclusion

Declarations

References

Supplementary Files

Status:

Version 1

Desiccation-induced oxidative stress causes metabolic flux in the terrestrial green alga Trentepohlia sp., suggesting extensive metabolic reprogramming for stress adaptation

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Collection of samples and experimental setup

Changes in morphological attributes

Determination of chlorophyll content and stress markers

Antioxidant metabolism

Metabolite extraction

HRLC-MS and 1H-NMR analysis

Data analysis

Statistical analysis

Results

Changes in morphological attributes

Changes in photosynthetic pigment content and stress markers

Changes in antioxidant metabolism

Metabolic analysis

Metabolic pathway analysis

Discussion

Conclusion

Declarations

References

Supplementary Files

Status:

Version 1

HRLC-MS and ¹H-NMR analysis