Ecology

Wet and dry weight of aerial partsDry weight of aerial parts was significantly affected by drought stress and genotype treatments and their interactions (Table 2). With increasing drought stress the amount of dry weight of aerial parts in all genotypes was decreased. Dry functions in I2, I3, and I4 levels in H genotype (Lavandula gngustifolia cv. Hidecot) were 15.68%, 40.35% and 48.15%, respectively. In S genotype (Lavandula stricta) these amounts were 0.78%, 48.58% and 51.72%, respectively; and in M genotype (Lavandula angustifolia cv. Muneasted) they were 22.29%, 49.38% and 52.63%, respectively. Compared to the control group, the most reduction in dry weight of aerial parts was in M genotype. The highest amount of dry weight (11.40 g in plant) was observed in H genotype in drought stress of 90–100% of field capacity. The lowest amount of dry weight of aerial parts (3.07 g) was seen in S genotype in drought stress of 30–40% field capacity (Fig. 2).Table 2 Variance analysis of the effect of drought stress on enzymatic activity of antioxidant enzymes, and quantity of essential oil from different lavender genotypes.Full size tableFigure 2The effect of drought stress on dry weight of aerial parts in different lavender genotypes.Full size imageIn this study drought stress had a negative effect on biomass of lavender plants. This effect can be due to water shortage. Because drought stress cause reduction in swelling, total water potential in cell and withering, it also results in closing stomata, reduction in cell division, and cell enlargement47,48. Reduction in cell division and cell enlargement as a result of drought, reduce the leaf surface, photosynthesis and growth function of the plant. In other words, reduction in photosynthesis products, cause reduction in leaf’s surface; and reduction in transfer of assimilated materials to aerial part, as a result of drought, cause decrease in aerial yield of the plant49. In this regard, Abbaszadeh et al. (2020) reported that due to drought stress of 30% and 60% of field capacity, dry weight of aerial parts in Rosmarinus officinalis L. has decreased. While contrary to our results Rhizopoulou and Diamantoglou (1991) observed that dry weight of leaves from Marjoram plant (Origanum majorana) was increased with increased soil moisture deficiency; which can be due to differences in plant species and ecological conditions50,51.Proline content of leavesThe results of variance analysis showed that drought stress, genotype and their interactions have significantly affected proline content of leaves (Table 2). With increasing drought stress the proline content was increased. The highest amount of proline content (4.96 mg per g) was observed in H genotype in I4 drought level (30–40% of field capacity). While the lowest amount (1.08 mg per g) was observed in S genotype in irrigation of 90–100% of field capacity (Fig. 3). In each genotype separately, in I2 to I3 drought levels the amount of proline was equal, but in H and M genotypes with increasing drought stress, the amount of proline was increased, While in S genotype with increasing water deficit proline did not show a significant increase. This indicates that two genotypes (H and M) have a similar function for using these types of osmolyte to deal with this level of drought. Which this result may be exist another osmolite production as a resistance mechanism in S genotype52.Figure 3The effect of drought stress on proline content in different genotypes of lavender.Full size imageOne change that happens in biological and non-biological stresses is increasing the amount of osmolytes in plant. To prevent negative effects of drought stress, the plant increases the amount of its osmolytes including proline53. Proline is an amino acid which in addition to act as an osmolyte, plays an important role in maintaining and stabilizing membranes by adding membrane phospholipids and changing the hydrated layer around macromolecules. Proline is also recognized as a stabilizer for cellular homeostasis under stressful conditions. This is due to high ability of proline to stabilize sub-cellular structures such as proteins and cell membranes and its ability to eliminate free radicals54. In present study, increasing proline content in different genotypes of lavender as a result of drought, can be for the same reason. It is proved that in some plants, changes in amount of proline is related to their ability to tolerate and adapt with drought stress; so, the proline content can be used as an indicator to select drought-resistant plants. Hosseinpour et al. (2020), reported that in response to drought stress, accumulation of compatible metabolites such as proline can participate in water absorption. In accordance with our results, an increase in proline content in different genotypes of Calendula officinalis plant due to drought stress has been reported as well55,56. However, in some plant species, other osmolites are produced under biological stress, the most important of them is glycine betaine. So that it is probable that the relationship between glycine betaine accumulation and stress tolerance, such as drought stress, is species- or even genotype specific57. As a results, the S genotype likely produced glycine betaine under drought stress, obviously, completed studies are needed to confirm this hypothesis.Relative water content of leavesThe relative water content (RWC) of leaves was significantly affected by drought stress, genotype and their interaction (Table 2). The highest amount of RWC (87.43%) was observed in H genotype in no drought stress condition. The lowest RWC (19.60%) was observed in S genotype in 30–40% of field capacity (Fig. 4). The results of comparing average data showed that in highest level of drought stress RWC in H, S and M genotypes is 57.25%, 65.19% and 58.88%, respectively; which compared to the control group, it is decreased in all genotypes. This suggests higher resistance of H genotype to maintain RWC of leaves (Fig. 4). In all evaluated genotypes, with increasing drought, RWC was decreased.Figure 4The effect of drought stress on RWC of leaves in different lavender genotypes.Full size imageRWC is a suitable indicator for water stress in plants. Drought stress by reducing RWC and total water potential of cell, result in reduction in growth of plants. The osmoregulation mechanisms in drought-resistant plants, maintains high RWC in them. Reduction in RWC of leaves as a result of water deficiency stress, is due to reduction in amount of water in tissue, reduction in amount of water in soil, and the negative soil water potential58. Alinejad et al. (2020), reported that RWC of leaves in Datura stramonium L. plant was decreased due to drought, in a way that the highest amount of RWC (80.22%) was seen in 55% of field capacity, compared to 35% and 15% of field capacity59. Also Mohammadi et al. (2018) suggested that RWC of leaves in Thymu vulgari L. was decreased to 18.41%, after being exposed to drought60.Total phenolic and flavonoids contents in leavesDrought, genotype and their interaction had a significant effect on total phenolic content of leaves (Table 2). The results suggest that in different levels of drought, total phenolic content was different in lavender genotypes. In the highest level of drought, total phenolic content in H, S, and M genotypes was respectively increased 18.64%, 28.57% and 98.07% in comparison with the control group. The highest difference in total phenolic content compared to control group was observed in M genotype (Fig. 5).Figure 5The effect of total phenolic content of leaves in different genotypes of lavender.Full size imageTotal flavonoids content of leaves was significantly (p ≤ 0.01) affected by drought and genotype (Table 2). The results of comparing averages showed that the highest amount of total flavonoids (1.12 mg quercetin per g of fresh weight) was in H genotype, and the lowest amount (0.95 mg quercetin per g of fresh weight) was in M genotype (Table 3). Moreover, our results showed that drought level from I2 to I4 caused an increase of 12.74%, 14.61% and 15.38% in total flavonoid content of leaves, respectively. Which indicates an increase in flavonoid amount with increasing drought level (Table 3). Table 3 Comparing simple effects of genotype and drought stress on traits of lavender plant.Full size tableTotal phenolic content is related to stress-resistance, indirectly by helping cell protection, and directly as an antioxidant61. Phenolic compounds due to their reductive properties, act as a free radical remover62. Our findings are similar to those of a study on growth of Mentha piperita in drought stress54.Total antioxidant activityTotal antioxidant activity was significantly affected by drought stress and genotype (Table 2). With increasing drought, antioxidant activity in H and S genotypes was increased. The results of comparing average data showed that compared to the control group, in drought levels of I2, I3 and I4, antioxidant activity in H genotype was increased by 98.43%, 98.36% and 118.78%, respectively; and in S genotype this amounts were increased by 89.85%, 111.78%, and 131.90% respectively (Table 5). In M genotype the antioxidant activity has reached its highest amount (49.38 mg/g) in I3 level of drought, and then with increasing drought stress the antioxidant activity was decreased, in a way that in highest drought level it had the lowest antioxidant activity (23.18 mg/g). M genotype was used as control (Fig. 6). Our results indicate that in highest drought level, antioxidant activity of S genotype was more than others. Figure 6The effect of drought stress on antioxidant activity in different lavender genotypes.Full size imageAntioxidant enzymesEnzymatic activity of antioxidant enzymes in lavender leaves was significantly affected by genotype and drought stress (Table 2). Our results showed that the highest activity of SOD (304.75 μmol min−1 mg−1 protein) was observed in interaction of H genotype and I4 drought level, and the lowest activity of SOD (144.52 μmol min−1 mg−1 protein) was observed in S genotype with no drought (Fig. 7). Moreover, our observations showed that in I2 and I3 drought levels, the highest amount of SOD enzymatic activity was related to M genotype (Fig. 7). In the highest drought level, enzymatic activity of SOD was increased in H and S genotypes, and it decreased in M genotype.Figure 7The effect of drought stress on enzymatic activity of SOD, POX and CAT in different lavender genotypes.Full size imageEnzymatic activity of peroxidase (POX) enzyme was increased in all three genotypes, with increasing drought. In all drought levels, H genotype had the highest amount of POX activity, compared to other genotypes. There was no significant difference in POX activity in S and H genotypes. The results showed that the highest amount of POX activity (274.48 μmol min−1 mg−1 protein) was observed in interaction of H genotype and 30–40% field capacity, and the lowest amount (117.66 μmol min−1 mg−1 protein) was observed in interaction of S genotype and no drought condition (control) (Fig. 7).Catalase (CAT) enzyme was affected by drought, genotype and their interaction (Table 2). The results of catalase enzyme activity assessment showed that with increasing drought, catalase activity is different in H, M and S genotypes. The most different reaction in production of CAT was related to H genotype, which with increasing drought stress up to I3 level, the enzyme activity was increased. But regarding M and S genotypes, with increasing drought level, CAT activity was increased in both genotypes. In this study the highest amount of CAT (460.51 μmol min−1 mg−1 protein) was observed in interaction of S genotype with 30–40% of field capacity; and the lowest amount (157.06 μmol min−1 mg−1 protein) was observed in interaction of H genotype with 90–100% of field capacity (Fig. 7).No significant effect was observed for APX enzyme in interaction of genotype and drought (Table 2). The results of comparing average data, suggest that the highest amount of APX activity (284.96 μmol min−1 mg−1 protein) was observed in H genotype (Table 3). Also the results showed that I2, I3 and I4 drought level resulted in an increase in APX enzyme activity by 32.38%, 49.16%, and 65.53% respectively. This indicates that APX enzymatic activity increases with increasing drought level (Table 3).Using physiological and biochemical mechanisms to reduce effects of stress shows that to overcome drought, oxidative stress and to eliminate ROS, plants will increase the amount of antioxidant content55. One of major mechanisms to cope with oxidative stress in plants, is activation of antioxidant enzymes61. Findings of the present study indicates that different lavender genotypes showed partial resistance against drought. In this research, increased activity of antioxidant enzymes in lavender genotypes under drought condition, was considered as an important drought-resistance factor. Among all antioxidant enzymes, SOD can have a good response against drought stress. In a way that H, S, and M genotypes of lavender in the highest level of drought stress (I4), showed an increased amount of SOD, by 57.42%, 35.85% and 60.69% compared to normal conditions (Fig. 7).In this study, the minimum enzymatic changes were related to the POX enzyme and the highest enzymatic changes were related to the CAT enzyme. Moreover, it was observed that the highest amount of catalase enzymatic activity was in H genotype. In a way that in plants under drought stress CAT activity was increased up to I3 drought level; but, after this level with increasing drought (I4 drought level) CAT enzymatic activity was decreased. CAT and POX are among important plants enzymes which can protect plant cells against free radicals63. In this study, in drought period, enzymatic activity of CAT and POX was increased, this means that lavender genotypes, in the face of stress produce antioxidant enzymes to protect themselves. While in H genotype compared to other genotypes, in high drought stress, CAT activity was decreased which this response indicates the different function of this genotype in dealing with ROS. Enzymatic response to drought condition was different in various lavender genotypes. Generally, the negative effect of drought is shown by production of reactive oxygen species (ROS). Increased enzymatic activity of antioxidant enzymes, particularly CAT and POX can reduce the negative effects of drought64, 65. In this regard, increased activity of antioxidant enzymes in different genotypes of Calendula officinalis plant was reported to56.Malondialdehyde (MDA) contentReaction of different lavender genotypes under drought stress was different in terms of malondialdehyde (MDA) production and accumulation (Table 2). With increasing drought, MDA content was significantly increased in M and H genotypes. The highest amount of MDA in these genotypes was 14.34 and 9.50 nmolg − 1 FM respectively, which was observed in drought level of 30–40% of field capacity. This indicates a significant increase in MDA content with increasing drought (Fig. 8). While the process of production and accumulation of MDA in S genotype was different at various drought levels. For S genotype, in first level of drought (I2), MDA content was increased which showed the vulnerability of the cell membrane at this drought level. But with increasing drought, gradually, the S genotype plants adapted to the dry environment, which in this level cell membrane damage was not obvious. Then, increasing in drought stress resulted in increased MDA content. Generally, in I2 and I3 drought levels, lavender genotypes underwent varying degrees of damage, which in M and H genotypes followed by increasing enzymatic activity, and in S genotype it resulted in decreased enzymatic activity. But in the highest level of drought (I4), the cell membrane was seriously damaged and in all three genotypes and MDA content was significantly increased (Fig. 8).Figure 8The effect of drought stress on MDA content in different lavender genotypes.Full size imageMembrane lipid peroxidation due to the accumulation of active oxygen species leads to cell damage and death. In plants this lipid peroxidation happens under drought stress66. MDA is the final product of membrane peroxidation and membrane processes. Simultaneously with peroxidation, the MDA content increases significantly67. So the MDA content can be considered as an indicator of drought-resistance in plants. Among lavender genotypes, in the highest level of drought, MDA content in M genotype was significantly increased compared to others genotypes; whish suggests that M genotype is more vulnerable in comparison with the two other genotypes. An increase in MDA content under drought stress, was reported in Thymus species as well66.Quantity and quality of essential oilMutual interaction between drought stress and genotype had a significant effect on percentage and yield of essential oil in lavender plants (Table 2). Our findings suggested a different essential oil percentage for each genotype in various levels of drought stress. With increasing drought to I3 level, the essential oil percentage was increased in M and H genotypes, but after that with increasing drought to a higher level (I4), essential oil percentage in these genotypes was decreased. While in S genotype, increasing essential oil percentage totally had an upward trend (Fig. 9).Figure 9The effect of drought stress on essential oil percent in different lavender genotypes.Full size imageEvaluation of essential oil percentage in different levels of drought, showed that in I2 drought level, the highest amount of essential oil (0.81%) was observed in H genotype; and in I3 and I4 drought levels, the highest amounts of essential oil were 1.29% and 1.68% respectively, which were observed in S genotype. Moreover, our results suggest that the highest difference in essential oil percentage in the studied genotypes compared to the control, was related to S genotype (Fig. 9). Totally, the highest percentage of essential oil was observed in S genotype in I4 drought level. This shows the high capacity of this genotype to produce essential oil under drought stress.Essential oil yield was significantly affected by genotype and drought. The results showed that the essential oil yield in S genotype was different from the others. So that the highest yield of essential oil (0.055 g per plant) was observed in this genotype in I3 drought level. While in H and M genotypes the highest amounts were 0.068 g and 0.065 g respectively, which were gained in I2 drought level (Fig. 10). Results of comparing average data showed that the highest yield of essential oil at I2 and I3 levels was obtained with 151/85% and 122.22% difference compared to the control, respectively, and they gained from H genotype. This indicates the high potential of H genotype to maintain biomass and produce essential oil in drought stress. Also our results suggest that in the highest drought level (I4), the highest essential oil yield (0.046 g per plant) was observed in M genotype (Fig. 10).Figure 10The effect of drought stress on essential oil yield in different lavender genotypes.Full size imagePrincipal component analysis (PCA)PCA analysis was performed to identify susceptibility of genotypes in irrigation regimes. According to physiological traits in the PCA analysis (Fig. 11a, b), the first factor (PC1) explains about 90% of the total variance of variables, and the second factor (PC2) about 8%.Figure 11Principal component analysis (PCA) for genotypes (a) and physiological traits (b) based on water status calculated for physiological traits. (R 4.0.4 packages, https://rstudio.com/products/rstudio/).Full size imageThe results of PCA analysis of different irrigation regimes showed that in the first component, which shows 89.91% of changes, the best traits are antioxidant enzymes CAT, SOD, APX, while in the second component, with 8.10% changes, only the trait Catalase is the best trait. Also, in total, the first and second components, which show 98.01% of the changes, show CAT as the most effective trait (Fig. 11a).The results of PCA analysis in lavender genotypes showed that the first and second main components could explain 98.91% of the existing changes. So that the first main component with 91.13% and the second component with 7.78% had a share in the total variation. Therefore, using these two components and ignoring other components will only cause the loss of a small part of about 1.09% of the data changes (Fig. 11b). These two principal components include peroxidase, ascorbate peroxidase, and superoxide. Physiological responses of Lavandula genotypes (L. angustifolia cv. Hidcote, L. angustifolia cv. Munstead, and L. stricta) submitted to drought stress were evaluated through principal component analysis (PCA), and the results are illustrated in Fig. 11a. Lavandula stricta presents higher levels of CAT activity than L. angustifolia cv. Hidcote and L. angustifolia cv. Munstead. In addition, APX and CAT increase in stress-treated in 30–40% FC. This result shows that L. stricta exhibits the most affected physiological changes while trying to adjust to changes in the water status of the environment, under the imposed conditions and shows the highest resistance.The results of analysis of essentials oils from H, S and M genotypes is shown is Tables 4, 5 and 6. The trend of changes in essential oils composition is described in all three genotypes. By studying the mass spectra and the Kovats retention index, 23 compounds were identified in the H genotype’s essential oil (Table 4). The yield of H genotype essential oil from I1 to I4 drought levels was 99.89%, 82.78%, 81.09% and 82.85%, respectively. The main components of H genotype essential oil in I1 to I4 drought levels, include 1.8-Cineol compounds (5.94%, 7.73%, 4.24% and 3.50%), Linalool (23.20%, 16.30%, 11.90% and 10.57%), Camphor (3.41%, 4.65%, 2.32% and 2.87%), Borneol (4.89%, 3.34%, 3.65% and 3.01%), Bornyl formate (27.32%, 16.04%, 19.45% and 20.03%), Lavandulyl acetate (1.40%, 4.21%, 6 and 8.35%), Caryophyllene oxide (10.92%, 11.77%, 12.16% and 19.91%), α-Muurolene (4.38%, 3.20%, 1.20% and 0%) (Table 4). The results of grouping the essential oil compounds showed that the amount of hydrocarbon monoterpenes from I1 to I4 drought level were 12.88%, 8.86%, 8.53% and 6.06%, respectively. The amount of oxygen monoterpenes was 64.76%, 50.70%, 43.32% and 42.45%; and hydrocarbon sesquiterpene compounds were 13.12%, 11.45%, 13.03% and 13.96%. The amount of oxygen sesquiterpene compounds were 10.92%, 11.77%, 16.21%, and 19.91%; which shows that increasing drought level, result in decreasing monoterpene compounds, and increasing sesquiterpene compounds.Table 4 Chemical composition of essential oils extracted from Lavandula angustifolia cv. Hidcote plants under different irrigation regime.Full size tableTable 5 Chemical composition of essential oils extracted from Lavandula stricta plants under different irrigation regime.Full size tableTable 6 Chemical composition of essential oils extracted from Lavandula angustifolia cv. Munstead plants different irrigation regime.Full size tableHeat map for the essential oil profile in Lavandula angustifolia cv. Hidcote corresponding to the different irrigation regime The similar discrimination was also supported by the heatmap constructed for essential compounds. Accordingly, 22 rows and 4 columns were achieved. α- pinene, β-Pinene, δ-3-Carene, type of Cymene, 1,8-Cineol, Camphor and Linalool from the main compounds, peaked at control. Moreover, lavandulyl acetate, Myrtenyl acetate, caryophyllene oxide, camphene and γ-Cadinene revealed highest percentage at 30–40% FC, Some compounds, such as Camphor and Linalyl acetate, are at the levels of the intermediate irrigation regime (Fig. 12). It is remarkable that as the water limit increases, the amount of monoterpene compounds decreases and the amount of sesquiterpene compounds increases.Figure 12Heatmap for the essential oil profile in aerial parts of Lavandula angustifolia cv. Hidcote corresponding to irrigation regimes (CIMminer, https://discover.nci.nih.gov/cimminer/oneMatrix.do).Full size imageWith evaluation of the essential oil from S genotype, 18 compounds were identified (Table 5). The amount of essential oil in I1 to I4 drought levels was 99.41%, 98.48%, 99.53% and 99.93% respectively (Table 5). Among identified compounds in S genotype the followings were accounted for the highest amount of components in the essential oil in I1 to I4 levels respectively; Linalool (32.60%, 28.45%, 20.12% and 19.12%), decanal (10.26%, 15.21%, 18.56% and 19.27%), 1-Decanol (8.01%, 10.31%, 17.88% and 21.34%), Kessane (2.44%, 4.43%, 9.99% and 11.50%), Hexadecane (1.26%, 5.77%, 6.10% and 11.9%), 2-methyl-1-hexadecanol (11.1%, 9.32%, 8.15% and 2.37%) and Hexahydrofarnesyl acetone (6.8%, 6.34%, 3.78% and 1.26%) (Table 5). The most obvious point was the high percentage of Linalool, decanal and 1-Decanol in the S genotype. With increasing drought, Linalool compounds were decreased and decanal and 1-Decanol compounds were increased. The grouping of essential oil components also showed that among the 18 compounds identified, the following were the highest in I1 to I4 drought levels, respectively; 3 hydrocarbon monoterpenes with total of (5.34%, 5.44%, 4.57% and 4.34%), 6 oxygen monoterpenes with total of (60.49%, 61.03%, 59.57% and 60.45%), 3 hydrogen sesquiterpenes with total of (5.69%, 10.27%, 11.85% and 15.24%) and 6 oxygen sesquiterpenes with total of (27.89%, 28.09%, 29.44% and 18.32%). With increasing drought, the amounts of hydrocarbon monoterpenes and oxygen sesquiterpenes were decreased; while the amount of hydrocarbon sesquiterpenes was increased. Also the highest amount of oxygen monoterpenes, by 61.03%, was seen in I2 drought level.Heat map for the essential oil profile in Lavandula stricta corresponding to the different irrigation regime The parallel discrimination was also supported by the heatmap constructed for essential compounds. Accordingly, 18 rows and 4 columns were achieved. α- pinene, Amyl isovalerate, Citronellol, β-Ionone and Linalool from the main compounds, peaked at control. Moreover, α-Thujene, decanal, 1-Decanol, Sesquiphellandrene, Kessane and Hexadecane revealed highest percentage at 30–40% FC (Fig. 13). These results confirm the results obtained from the Lavandula angustifolia cv. Hidcote so that as the water limit increases, the amount of monoterpene compounds decreases and the amount of Sesquiterpene compounds increases.Figure 13Heatmap for the essential oil profile in aerial parts of Lavandula stricta corresponding to irrigation regimes (CIMminer, https://discover.nci.nih.gov/cimminer/oneMatrix.do).Full size imageEssential oil yield in M genotype from I1 to I4 drought levels was obtained 99.90%, 98.38%, 93.08% and 87.04% (Table 6). As it is shown in Table 6, analysis of the essential oil from M genotype included 27 compounds which its major part was consisted of Camphor (16.82%, 16.32%, 17.11% and 18.30%), Borneol (44.96%, 42.80%, 37.54% and 30.99%) and Caryophyllene oxide (14.68%, 15.21%, 15.90% and 17.21%) from I1 to I4 drought levels, respectively. comparison of essential oil components (Table 6) showed that from 27 identified compounds in M genotype, the followings were the most prevalent from I1 to I4 levels respectively, including hydrocarbon monoterpene with total of (17.82%, 17.45%, 13.91% and 9.96%), 12 total oxygen monoterpene compounds with total of (65.95%, 62.05%, 56.96% and 50.42%), 4 hydrocarbon sesquiterpenes with total of (1.58%, 23.23%, 5.42% and 8.09%) and 2 oxygen sesquiterpenes with total of (14.91%, 15.65%, 16.79% and 18.37%). The highest drought level resulted in 31.76% and 17.23% increase in Camphor and Caryophyllene oxide. It also caused 31.07% decrease in Borneol compared to the control (Table 6). Totally, with increasing drought level, monoterpene compounds were decreased and sesquiterpene compounds were increased in lavender genotypes.The major components of essential oil were different in various lavender genotypes in the highest level of drought (I4). In this study in H genotype, the compounds Linalool, Bornyl formate and Caryophyllene oxide; in S genotype the compounds Linalool, decanal, 1-Decanol, Kessane and Hexadecane; and in M genotype the compounds Camphor, Borneol and Caryophyllene oxide, were the most prevalent components of essential oil. In this study, Borneol compound was not observed in S genotype. regarding the fact that essential oil extraction was performed on flowering branches in all three genotypes, and they were studied under similar drought conditions; and also comparing the results of this study with finding of other studies shows that the difference in types and percentage of essential oil’s components can be due to the effect of genetic differences; and to some extent, environmental factors on essential oil in different genotypes.A total comparison of essential oil analysis results for different lavender genotypes under drought stress showed that oxygen monoterpenes are the most prevalent components of the essential oil, which will decrease with increasing drought level. Sarker et al. (2012) reported that the essential oil of lavender (Lavandula angustifolia) contains high amounts of linalool and linalool acetate, along with scares amount of other monoterpenes68. A study by Hassan et al. (2014) showed that the compounds carvacrol, phenol-2-amino-4, 6-bis, trans-2-caren-4-ol, and n-hexadecanoic acid are the main constituents of Lavandula stricta plants which were collected from the Shaza Mountains in southern Saudi Arabia69. Total results from essential oil analysis in this study showed that Linalool was the main ingredient of essential oils in H and S genotypes. This compound is an oxygen monoterpene with a density of 0.85 and a pleasant smell, and is the main component of the essential oil from lavender plant. While in M genotype, Borneol was the main component of the essential oil, which is a circular monoterpene compound with density of Mohammadnejad ganji et al. (2017) suggested that the difference in natural quality of the essential oil from lavender plants is related to intrinsic factors (genetic or heredity capabilities and maturity), and external factors including sunlight, water, heat, pressure, latitude, and soil which affect plant growth and essential oil production70.Heat map for the essential oil profile in Lavandula angustifolia cv. Munstead corresponding to the different irrigation regime The parallel discrimination was also supported by the heatmap constructed for essential compounds. Accordingly, 18 rows and 4 columns were achieved. α- pinene, Tricycle, Camphene, Thuja-2,4(10)-diene, δ-3-Carene, ρ-Cymene, Borneol and limonene from the main compounds, peaked at control. Moreover, Camphor, α-Santalene, γ-Cadinene, δ-Cadinene, Caryophyllene oxide, α-Muurolene and Ledene oxide-(II) revealed highest percentage at 30–40% FC (Fig. 14). The results showed that the composition of the compounds was similar to the previous two genotypes and the water limit increases, the amount of monoterpene compounds decreases and the amount of Sesquiterpene compounds increases.Figure 14Heatmap for the essential oil profile in aerial parts of Lavandula angustifolia cv. Munstead corresponding to irrigation regimes (CIMminer, https://discover.nci.nih.gov/cimminer/oneMatrix.do).Full size imageEssential oils are generally in the group of terpenoids and The structure of terpenoids consists of two main precursors, isopentenyl pyrophosphate (IPP) and its isomer, dimethylallyl pyrophosphate (DMAPP). These compounds are synthesized via the cytosolic pathway of mevalonic acid (MVA) or plasticity of methylerythritol phosphate (MEP)71. The MVA pathway is primarily responsible for the synthesis of Sesquiterpenoids and triterpenoids, while the MEP pathway is used for the biosynthesis of monoterpenoids, diterpenoids and tetraterpenoids72. Monoterpenes and Sesquiterpenes are the main constituents of essential oils that play a role in aroma, flavor, photosynthetic pigments and antioxidant activities73.In drought conditions, the amount of these isoprenes does not decrease in relation to the mediators of the MEP pathway and in contrast sometimes increases. Therefore, sesquiterpene compounds increase in drought conditions because most of these compounds are synthesized through the MVA pathway74. Another reason for the decrease in MEP path flux is the location of this path, which has a significant impact in drought conditions. In this case, plastids are not able to provide the required IPP of this path, so most monoterpene compounds are reduced75.Also, since the quality of the essential oil is due to the presence of linalool and linalyl acetate76. According to the results obtained from heatmaps related to essential oils, three genotypes are identified, the highest amount of linalool amount in S genotype was remained under mind- (I2) till severe-drought (I4) condition. This indicates more compatibility with maintaining the desired quality of drought conditions in this plant than the other two commercial genotypes. And then the H genotype is in the second stage due to the presence of important compounds.Comparing the grouping created in the heat maps related to the essential oil of 3 genotypes, it is clear that the two genotypes S and H were divided into two groups I1, I2 and I3, I4 in the genotype. But in the genotype M, the results were divided into I4 and I3 groups I2 were divided into genotypes. This can be due to differences in the resistance mechanism of plants in different genotypes, so in genotypes S and H of the plant through increasing sesquiterpene compounds showed resistance to drought stress, while in genotype M increased resistance to drought levels through higher monoterpene compounds. Another conclusion that can be drawn from these heat maps is that in genotypes S and H, the rate of drought resistance in the first and second levels of drought with the third and fourth levels has shown more changes in the type of essential oil compounds, while in the third genotype (M) these changes in the last level drought has been most evident.At a glance, it seems Genotype S has a different mechanism in reducing the negative effects of drought compared to genotypes M and H, So that, among the enzymatic and non-enzymatic mechanisms, it tends to use the enzymatic pathway more. In association with the production of “proline”, drought stress index osmolyte, genotype S has a different trend from genotypes H and M and this osmolyte in this plant has a lower production flux compared to other genotypes. Also, due to the fact that the production of soluble sugars in this plant has been moderate compared to other genotypes, it is expected this genotype replace proline with another osmolyte or uses an enzymatic mechanism to deal with drought, as the results of antioxidant enzyme “catalase” related to genotype S had the highest value with a significant difference under drought stress, while, in the H and M genotypes, the SOD enzyme was responsive to drought.On the other hand, the high resistance of genotype S can be attributed to the greater activation of the pathway of essential oil compounds. Because by examining the constituents of the essential oil (monoterpene and sesquiterpene), it can be concluded that genotype H and then M at high drought levels still retain the ability to produce monoterpene compounds, while in genotype S with increasing drought, the amount of semi-heavy compounds (sesquiterpene) has increased significantly (Fig. 15), this can confirm the existence of a different resistance mechanism in the S genotype. Because some structural compounds of the membrane, such as sterols, are made from the mevalonic acid (MVA) pathway of acetyl coenzyme A origin. For this reason it seems that S genotype by setting up terpenoid pathways involved in the production of steroids another solution to drought is by preserving its plasma membrane. Steroids are derivatives of triterpenes that, along with phospholipids, are major components of plasma membranes70. Also, the study of MDA content as the final product of membrane lipid peroxidation in genotypes at the fourth level of drought (the most severe drought) showed the M genotype is most sensitive to drought. In this way, the two genotypes S and H have almost equal MDA content, so that it can be said that with a small difference from genotype S, genotype H has less composition.Figure 15The amount of monoterpenes and sesquiterpene compounds in different genotypes under irrigation regimes.Full size imageContinuous production of isoprene under drought conditions shows that despite the reduction in the synthesis of osmolyte and relative increasing of MDA (with very little difference from genotype H) that occurs under these conditions, the function of this pathway is essential for the S genotype. Isoprene has long been used to protect plants from drought, high temperatures and oxidative stress are recommended77. Of course, it was showed which is possible with increasing drought, sufficient isoprene is not produced to counteract and launch defense pathways and instead used as a general signal to increase drought tolerance78,79.Reasons such as further activation of terpenoid skeletal pathways towards the production of semi-heavy (sesquiterpene) compounds, production of steroids via the MVA pathway could be a reason for lower susceptibility of S genotype and high resistance of this genotype through these mechanisms compared to other genotypes. In contrast, on the one hand, H genotype using proline production, soluble sugar levels and decreased MDA in response to stress caused by drought and on the other hand, the ability to produce substances important monoterpenes, such as Linalool and Linalyl acetate, with the aim of using medicine and aromatherapy76, It (H genotype) can be considered as a cultivar with high commercial value and significant resistance to M genotype. More

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Overview of the FluxNet methodThe FluxNet approach is based on a mechanistic model, which includes multiple species/types of phytoplankton, bacteria, dissolved and particulate organic matter (DOM, POM), inorganic nutrients, micronutrients and inhibitors (see Table 1). For phytoplankton—bacteria carbon flux, which is the focus here, phytoplankton produce organic carbon by exudation and death. For exudation, living phytoplankton produce total DOM at constant and photosynthesis-proportional rates (ke, ef), with a composition defined by an exudation fraction (Fe) for each DOM species. These parameters vary by phytoplankton type. For example, for green algae (gre), the constant exudation rate is kegre and the fraction of glucose-containing HMW DOM (gl2) is Fegre,gl2. For one phytoplankton type the total DOM production varies in time with the photosynthesis rate, but the composition is constant. Phytoplankton die by a general death function and inhibition. The death function is time-variable (a bell-shaped function with a maximum at a specific time of year) and does not differentiate between various death mechanisms like zooplankton grazing or viral lysis, but presumably it represents mostly grazing in this case. Upon death, the phytoplankton biomass is converted to POM and DOM, where e.g., the content of chrysolaminarin (chr) for the diatom Rhizosolenia styliformis (rst) is defined by a composition fraction (Fxrst,chr). POM dissolves to DOM at a first-order rate. Bacteria consume DOM using Monod-level kinetics, where e.g. the affinity for Polaribacter (pol) for chrysolaminarin is defined by a half-saturation constant (Kshpol,chr).Table 1 Model components.Full size tableThe novel aspect is the upscaling to hundreds of state variables and thousands of parameters, which is accompanied by several conceptual and practical modeling challenges. To balance mass and account for the action of unobserved components, cryptic or hypothetical species are included [17], like DOM types d01-d15, which may represent e.g., threonine [18]. To simulate a diverse community with a smaller number of drivers (“paradox of the plankton”) and control chaos, interaction via micronutrients and inhibitors, as well as dormancy is included [19,20,21,22]. Parameters are optimized/calibrated to minimize the discrepancy between the model and observations. Which parameters are optimized and the corresponding ranges is based on available information (complete model equations and parameters are in Table S1–S25). For example, the constant DOM production rate (ke) is optimized for all phytoplankton, with a range adopted from a previous modeling study [23]. For rst (Rhizosolenia styliformis), the exudation fractions for most DOM components, like the cryptic species d01 (Ferst,d01), are optimized. Others, like glucose-containing HMW DOM (Ferst,gl2), are fixed based on literature (Table S14). The optimization is challenging because of the many components, nonlinear interactions, and resulting local optima in the objective function. We developed an optimization routine customized for microbial ecosystems with a number of key features.First, the method mimics natural speciation, where a coarse-grained model is gradually de-lumped to a finer resolution, a strategy also used in manual model development [13, 24, 25]. This is illustrated in Fig. 1, which shows how the model starts with just one component in each ecological compartment (Fig. 1E). This model is optimized until a threshold is reached, and then all species are de-lumped/split into two, followed by another round of optimization and so on. During the course of the optimization, with time or model runs, the number of components and parameters increase, and the total error generally decreases, although there can be a transient increase when new species are introduced (Fig. 1A, B). This way the optimization routine works with a smaller model on average and computational effort can be directed to a smaller set of parameters corresponding to newly introduced species, and the performance increases (Fig. 1C).Fig. 1: FluxNet inference method illustration.A Number components and optimized parameters. B Error for entire model (Total) and selected individual observations (rst = R. styliformis, pol = Polaribacter, lam = particulate chrysolaminarin). Best of 128 replicate runs. C Diversification of chrysolaminarin uptake affinity (max. heterotrophy rate/half-saturation constant). D Method performance with and without de-lumping. E Network corresponding to different de-lump levels. See Table 1 for component names and abbreviations.Full size imageAt each de-lumping level, the new species generally inherits the parameter values (i.e., the genome [26]) from the old species. Subsequent optimization then diversifies the population. This is illustrated in Fig. 1C, which shows the uptake affinity of all bacteria species for chr. However, different parameter values can also be specified for the new species, and then they are adopted and overwrite those inherited from the old species. This is used, for example, to assign species-specific cell sizes or prevent species from taking up a substrate. In Fig. 1C, those species that are not capable of assimilating chr, like rei (Reinekea), have an affinity equal to 0. The method thus allows for natural and automated expansion of the model to very large scale, yet provides a way to constrain/curate it based on available information.Second, the routine includes multi-parameter optimization (Nelder-Mead simplex method) on selected subsets of dependent parameters, like those involved in the production and consumption of chrysolaminarin (chr) or directly affecting the photosynthesis of the diatom R. styliformis (rst). Dependence between parameters, like max. photosynthesis rate and nutrient half-saturation constant, are explicitly considered. Also, Monte Carlo scans are performed on selected parameter sets at various points in the process.Application to Helgoland time seriesThe FluxNet method is applied to a four-year time series at Helgoland [27], including near-daily observations of 15 phytoplankton and 38 heterotrophic bacteria types (e.g., species, strains) and various bulk and auxiliary parameters (e.g., Chlorophyll a, DAPI, temperature, nitrate+nitrite, ammonium, phosphate, light extinction) (Tables S19 and S20). Data from more focused studies characterizing DOM and POM are also included [28, 29] (Table S21).In addition to the time-series data, the model is informed by literature information. Model parameters, incl. general properties like phytoplankton exudation fraction or bacteria growth efficiency, are constrained based on past models and data. Also, constraints are implemented for parameters controlling composition, exudation and utilization for the specific components included in the model. Those were based on a literature meta-analysis, where we searched primarily for studies with strains from Helgoland, but included strains from other locations if necessary. These constraints include, for example, for the phytoplankton storage polysaccharide chrysolaminarin, the typical content (~30% for diatoms, none for dinoflagellates) and ability of bacteria to assimilate it (yes for Polaribacter, no for Roseobacters and Reinekea) (Tables S4 and S11). Imposing constraints from the literature generally results in a worse agreement with the observations, but also increased realism of the model. Removing the constraints of phytoplankton composition (Table S4) significantly improves the agreement with observations, but also predicts substantial glycogen content of diatoms (e.g., Fxmhe,gly+ply = 0.19). Removing uptake constraints by bacteria (Table S11) reduces the error, but not significantly, suggesting that there is enough flexibility of the model to reproduce the observations even with this constraint. However, that model also includes features that disagree with literature, like substantial uptake of chr by s11 (Kshs11,chr = 25 L/mmolC/d).Carbon fluxes through and within in the ecosystemThe final model includes 210 components and their behavior and interaction are described by a total of 8200 calibrated parameters of 50 different parameter types (e.g., the composition of each of the 53 microbes is described by 76 fractions Fx, or 4000 total parameters) (Fig. 1), and it constitutes a mass-balancing, mechanistically-constrained, quantitative representation of the ecosystem. It reproduces many of the observed patterns of summary parameters like Chlorophyll a (chl), total bacteria (dap), particulate chrysolaminarin (lam), various high-molecular weight (HMW) DOM compounds, as well as absolute concentrations of individual phytoplankton and bacteria species (Fig. 2A–C). Only subset of the hundreds of model components is shown in Fig. 2B, C, which were selected based on (a) importance (e.g., rst is the dominant OM producer in 2009), (b) availability of data (e.g., chrysolaminarin, [29]) and (c) illustration of co-blooming (panel B) and succession (panel C). All model-data comparisons are presented in the SI (Fig. S1). The model under-predicts total DOM (doc), probably because a large fraction of observed DOM is more refractory allochthonous material, which is not considered in the model.Fig. 2: FluxNet model results and comparison to data.A All model types lumped. Phytoplankton (chl, μgChla/L), POM (poc, incl. microbes, μmolC/L ×0.1*), DOM (doc, μmolC/L ×0.1*), bacteria (dap, 1e6/mL ×3*). Gray shading are spring blooms, defined as the first time of the year the phytoplankton exceeds 3 µgChla/L plus 28 days. B Selected types for 2009 spring bloom. Rhizosolenia styliformis (rst, centric diatom, 1e6/L ×1.2*), Thalassiosira nordenskioeldii (tno, centric diatom, 1e6/L ×0.05*), particulate chrysolaminarin (lam = phr + phytoplankton content, μmolC/L ×0.002*), dissolved chrysolaminarin (chr, μmolC/L ×0.002*, no data available), Polaribacter (pol, DAPI × CARD-FISH, 1e6/mL ×0.1*), NS3a marine group (ns3, 1e6/mL ×0.2*). C Selected types for 2010 spring bloom. Mediopyxis helysia (mhe, centric diatom, 1e6/L), Thalassiosira nordenskioeldii (tno, centric diatom, 1e6/L), glucose-containing HMW DOM (glc, μmolC/L ×0.01*), arabinose-containing HMW DOM (ara, μmolC/L ×3*), Reinekea (rei, 1e6/mL ×5*), Alteromonas (alt, 1e6/mL ×1.5*). Lines are model and symbols are data [27,28,29]. *Individual concentration series scaled to illustrate dynamics. See Fig. S1 for all model-data comparisons. Upside-down triangles mark various bloom stages for networks in (D) and Fig. 4A. D Inferred carbon flux network. Nodes are components. Size indicates in/outflux (μmolC/L/d), color varied randomly within each ecological compartment. Lines are fluxes. Thickness is proportional to log flux (μmolC/L/d), colored based on the source node, lines below a threshold distance are colored gray to highlight most important fluxes. Italic numbers are total fluxes (μmolC/L/d). Flux cut off is 0.01%. See Table 1 for component names and abbreviations. See Movie S1.Full size imageIt is important to understand that the model was calibrated to these observations, so this is not a prediction per se. The main information produced by this analysis (emergent property) are the mass fluxes. Predicted ecosystem-level fluxes can be compared to independent estimates, which were not used as input here. For the period 2009–2012, the gross primary production rate in the model is 28 (±1.2 standard deviation) mmolC/m2/d. Uncertainty of fluxes and parameters are based on top 5% of 128 replicate runs, as in [23]. This flux compares well to a regional estimate of 29 (26–33) mmolC/m2/d for the Transitional East Region of the North Sea for the same period [30]. At the end of March, the bacterial production rate in the model is 0.32 (±0.041), 0.14 (±0.017), 0.20 (±0.025) and 0.45 (±0.057) μmolC/L/d for the 4 years, respectively. This is consistent with measurements of 0.20 μmolC/L/d in 1992 ~30 km from Helgoland [31].These comparisons provide confidence in other aggregate fluxes predicted by the model. The C, N and P fluxes to the sediment bed, via settling of phytoplankton and POM, are 5.8 (±0.91) mmolC/m2/d, 0.87 (±0.14) mmolN/m2/d and 0.054 (±0.0085) mmolP/m2/d, which constitute 20%, 16% and 18% of the input via photosynthesis (C) or external input (N, P) (see Fig. S2). External “new” input of N is 0.66 μmolN/L/d, which is 6.0 time higher than the 0.11 (±0.023) μmolN/L/d released or “recycled” by bacteria.The resulting flux network includes quantitative carbon fluxes between all components at each time point, like 28 days into the 2009 spring bloom (Fig. 2D, Dataset S1 list all fluxes). The dominant source of organic matter is rst at 0.36 (±0.19) μmolC/L/d, 30% of which is dissolved and particulate chrysolaminarin (chr + phr). These instantaneous fluxes exhibit a higher uncertainty than the integrated fluxes discussed in the previous paragraph, which can be explained by small timing differences (Table S26). The DOM is consumed by a diverse consortium of bacteria, mostly Polaribacter (pol) at 0.46 (±0.22) μmolC/L/d, 35% of which is chr. chr has a through-flux of 0.25 (±0.049) μmolC/L/d and a turnover time of 8.8 (±2.0) days. In the model, phytoplankton and bacteria interact via DOM, but the carbon flux can be traced and used to quantify phytoplankton – bacteria associations. Here, the carbon flux via all DOM types from rst to pol is 0.27 (±0.20) μmolC/L/d, 58% of carbon to pol, making this the second-strongest (after ns3) microbial linkage in the system at this time. This who produces/consumes how much of what when information is the main output of the FluxNet method, and it is critical for moving our understanding of microbial ecosystem functioning beyond bulk parameters like respiration and photosynthesis rates towards a higher resolution.Whereas the 2009 spring bloom illustrates co-blooming of phytoplankton and bacteria, the 2010 bloom shows succession of phytoplankton, DOM and bacteria. Several factors control this pattern in the model. Reinekea (rei) is negative for chrysolaminarin (chr) based on literature (Table S11), but is predicted to have a relatively high affinity for other glucose-containing DOM (gl2) (khrei / Kshrei,gl2 = 63 (±22) L/mmolC/d). A substantial fraction of gl2 is produced relatively early by phytoplankton exudation, and it is the primary substrate for rei at bloom stage 14 days. Alteromonas (alt) is predicted to have a low affinity for gl2 (khalt / Kshalt,gl2 = 0.015 (±0.0097) L/mmolC/d), but it is positive for chr based on literature and predicted to have a high affinity (khalt / Kshalt,chr = 52 (±4.7) L/mmolC/d). Chr is a death (i.e. grazing) product of phytoplankton and produced relatively later in the bloom, and it is the primary substrate for alt at this time. The substrate spectra of bacteria emerge in the analysis, within literature constraints, and can be considered a prediction testable with modern experimental techniques [6].Oligotrophic and copiotrophic carbon processingThe network includes concentrations and fluxes for each bacteria type, and a natural question is to what extend they are correlated. There is increasing awareness that high abundance may not necessarily mean high importance and vice versa, including the over-proportional role of rare species in biogeochemical cycles [32]. In the model, there is a strong correlation between concentration and carbon flux of bacteria, but for the same concentration there is also about an order of magnitude variation in flux (Fig. 3). The spread reflects differences in growth rates during the bloom periods. Some species, like the oligotroph SAR11 (s11), have consistently lower flux and others, like the copiotroph Polaribacter (pol), have consistently higher flux. There are also some, like the cryptic alphaproteobacteria (alx), that go in different directions in different years.Fig. 3: Correlation between spring bloom abundance and importance.Concentration and carbon flux for all model bacteria types during spring bloom periods (see Fig. 2 caption for definition). Lines: All(dashed)/Olig.(thick)/Copi.(thin), log Flux = –0.93/–1.03/–0.81 + 0.93/1.00/0.94 × log Conc., R2 = 0.88/0.92/0.92.Full size imageIt is important to realize that, in dynamic systems, microbial interactions and the corresponding networks are not static [3, 33]. The dynamics of the entire Helgoland flux network over the four-year period is illustrated in an animation, which shows the production of DOM and POM during and after phytoplankton blooms and later blooming of bacteria (Movie S1). These features are also evident in the phytoplankton – DOM – bacteria interactions at two selected time points during the 2009 spring bloom (Fig. 4A, B). At the onset of the bloom, the oligotroph SAR11 (s11) consumes the most DOM, primarily the cryptic species d08, which comes mostly from grazing death of green algae (gre) and exudation by rst. After 28 days the copiotroph pol dominates, which consumes primarily chr, a death product of mostly rst. SAR11 continues to be a major carbon processor in the early parts of the bloom, which was unexpected, because it is an inferior competitor at this time (growth rate s11 = 0.051 vs. pol = 0.15 1/d, bloom average), but can be explained by the higher biomass concentration (s11 = 0.68 vs. pol = 0.13 μmolC/L, bloom start). The flux is proportional to concentration and growth rate, and neither measure alone is a good proxy for the importance of a species [4]. Across all four years, oligotrophic bacteria, defined based on below-average growth rates (literature classifications are often ambiguous), dominate carbon processing for the first 18 days, generally past the phytoplankton peak (Fig. 4C).Fig. 4: Carbon processing during the course of blooms.A Phytoplankton—DOM—bacteria carbon flux network for the start and +28 days of 2009 spring bloom. See Fig. 2 legend. Flux cut off is 0.3%. B Cell concentrations, growth rate and relative carbon processing for s11 and pol for 2009 spring bloom. C Fraction of DOM processed by oligotrophic bacteria and exudate fraction in DOM pool for all blooms. Oligotrophs are defined based on literature as shown in Table S23 or based on below-average growth rates (kg). For the later, the oligotrophic fraction or weight given for type i, is based on fOLIi = kgAVEn / (kgAVEn + kgin), n = 5. kg is the net growth rate calculated from biomass change, plus dilution rate.Full size imageThe use of d08 by s11 and chr by pol in 2009 suggests are more general pattern, i.e., use of exudation products earlier by oligotrophs and death (i.e., grazing) products later by copiotrophs. Across all years, the fraction of DOM produced by exudation decreases during the course of the bloom (Fig. 4C), a common feature of phytoplankton blooms [33]. This is reflected in the diet of these bacterial groups, i.e., for oligotrophs (vs. copiotrophs), exudates make up a higher portion of the diet (27 vs. 18%), and they have a higher affinity for exudates (39 vs. 35 L/mmolC/d), which is also consistent with experimental evidence from another system [7].After the model was developed, while this paper was in peer review, metaproteomic data for the Helgoland Island spring bloom in 2016 were published that suggest that algal storage compounds (e.g., chrysolaminarin) are used throughout the bloom, whereas cell wall-related compounds (e.g., fucose-containing) are used at later bloom stages [34]. Our model also predicts an increase in the consumption of cell-wall vs. storage compounds at later bloom stages (Fig. 5), which validates our outcomes, although a direct comparison is not possible because of the different time.Fig. 5: Consumption of cell wall vs. storage compounds during the course of blooms.Total consumption (all bacteria) in µmolC/L/d of cell wall compounds divided by storage compounds. Cell wall compounds = man (mannan) + glo (glucoromannan) + fcs (FCSP). Storage compounds = chr (chrysolaminarin) + gly (glycogen) + sta (starch). Averages for all four years.Full size imagePhytoplankton functional similarity decouples them from bacteriaAn important question is to what extent the patterns recur from year to year [27]. We compare networks of phytoplankton producers, DOM exchanged and bacteria consumers, as well as phytoplankton – bacteria interactions quantified in absolute (μmolC/L/d moving between phytoplankton X to bacteria Y) and relative (% of carbon for bacteria Y supplied by phytoplankton X) terms (Fig. 6A). All networks show significant similarity so there is recurrence from year to year. The recurrence is higher for DOM than phytoplankton, suggesting that different phytoplankton produce similar DOM, which is expected considering similar composition (e.g., chr in diatoms). There are no phytoplankton producers that recur in the top quartile every year, but chr and others are in the top quartile of DOM exchanged (produced and consumed) every year. The recurrence is lower for bacteria consumers suggesting factors beyond DOM shape the bacteria community.Fig. 6: Recurring patterns and comparison of FluxNet and LSA methods.A Similarity of networks for spring blooms. Error bars are 95% confidence limits. Bray-Curtis similarity was calculated as 1 – Bray-Curtis dissimilarity. Text on top of symbols lists components that recur in the top quartile every year, listed in order of average rank. B Carbon flux networks for top recurring bacterial consumer, top four DOM sources and top coupled phytoplankton. (C&D) LSA network (showing top 15% of significant local similarity scores) and sample time series.Full size imageAn important question is how specific interactions are and how tightly networks are interconnected [35, 36], which depends on the mechanisms of interaction and will affect the recurrence. Consistent with the relatively low recurrence of phytoplankton producers, phytoplankton—bacteria coupling shows relatively low recurrence, i.e. low specificity. The primary substrate for the consumer pol is mostly chr and gl2, although it does change from year to year with varying DOM, consistent with the known assimilation capabilities of pol (Polaribacter) [37] (Fig. 6B). However, the primary associated phytoplankton for pol is different each year, although it is always a diatom. The de-coupling of phytoplankton production and bacteria consumption was also concluded from the lower recurrence of phytoplankton and higher recurrence of bacteria abundance in the same dataset [27]. It suggests that carbon processing is resilient to changes in phytoplankton, which may arise from factors like species invasion or climate change.The above discussion focused on one-way/commensal (phytoplankton  > DOM  > bacteria) interactions, but the network also includes specific two-way/mutualistic phytoplankton-bacteria interactions. Phaeocystis (pha) has the highest exudation fraction and Bacteroidetes nvi the highest affinity for DOM d04, whereas nvi has the highest exudation fraction and pha the highest requirement for micronutrient m15. Such mutualism is observed in other systems and the interactions predicted here can be tested experimentally [20]. Alternatively, experimentally-observed interactions could be used as input to the method, as constraints.Robustness of the analysisTo understand the effect of some of the choices made in the model structure we repeated the analysis with added or removed components or processes. Models without micronutrients or inhibitors produce significantly worse fit to the data (Fig. 7A), highlighting the need for a two-way interaction between phytoplankton and bacteria to maintain diversity. Models with more micronutrients or inhibitors are similar to the basecase. Together, these results provide some justification for the complexity (i.e., number of parameters) in the basecase model. The analysis including osmotrophy (aka absorbotrophy, i.e., phytoplankton can perform heterotrophy) produces a better fit to the observations, but that model was not adopted as basecase, because the osmotrophy process is poorly constrained and includes some probably unrealistic features/fluxes, like significant exudation and uptake of the same substance by one phytoplankton species. Importantly, excluding the runs with worse fit to the observations, the main conclusions (as shown in Figs. 4C and 5A) are the same, confirming that the results are reproducible and robust to some of the choices made in model structure.Fig. 7: Reproducibility of main results.A Total error for runs with different models. “w” or “b” indicates performance is significantly worse (open bars, think lines) or better than basecase, p  ns9 interaction ranks in the top 1% for LSA and FluxNet (relative interaction). However, the lack of mechanistic constraints is evident. One of the strongest links for the 2009 spring bloom (rank 13%) is between the diatom Chaetoceros debilis (cde) and Roseobacters (ros) (Fig. 6C, D). The shifted peaks line up nicely, but the bacteria biomass is higher than that of the phytoplankton and genome analysis suggests ros do not assimilate chrysolaminarin [37], which is a major death product of diatoms. Considering this, growth yield and other competing consumers, it is unlikely that cde is a major source of carbon to ros.Summary and outlookModern observational tools are generating high-resolution descriptions of the components of microbial ecosystems, and an ongoing grand challenge is to use these data to understand how systems function. Our method predicts dynamic mass fluxes between marine phytoplankton and bacteria, which provides insights into the functioning of the ecosystem. Specifically, it showed that there is a strong correlation between concentration and flux of bacteria during blooms, but oligotrophs are relatively less important than copiotrophs. However, due to their higher biomass, they are major carbon processors during early phases of blooms, well past the peak. Oligotrophs grow preferentially on exudation products, which are more abundant earlier in the bloom. Also, our results suggest that phytoplankton are functionally similar in terms of what organic carbon species they produce, and that this decouples them from bacteria.FluxNet is an inference method for microbial time series data that serves the same general purpose as existing empirical inference methods, like LSA [38]. In general, both approaches have strengths and weaknesses (see Introduction) and may complement each other. A main advantage of FluxNet is that it produces quantitative concentrations and fluxes, and associated conclusions (e.g., preferential use of exudates by oligotrophs). Also, it is constrained by mass balance and additional information from the literature (i.e., beyond the time series data), which make the results more realistic.The existing FluxNet code can readily be applied at a higher resolution (microdiversity), explicit representation of other ecosystem components, like viruses and zooplankton, and more processes, like photoheterotrophy and mixotrophy. It may also be applied to understand other microbial ecosystems, like the human gut or wastewater treatment plants. For an inference method it is important to be applicable to various types of observations, including modern environmental -omics observations, like transcript, protein and metabolite levels, and the present model will have to evolve in this direction [39]. The present model includes a relatively simple representation of the various processes, and the current biological understanding supports increasing the mechanistic realism (and complexity). For example, the present version assumes constant composition of DOM produced by phytoplankton, but observations show that it changes with physiology and interaction with bacteria [18, 40]. Also, the model assumes simple first-order dissolution of POM to DOM and direct utilization by bacteria, whereas break-down of especially polysaccharides is often mediated by extracellular enzymes [41]. More

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