Comparing photosynthetic characteristics of Isoetes sinensis Palmer under submerged and terrestrial conditions

Tao Yang; Xing Liu

doi:10.1038/srep17783

. 2015 Dec 4;5:17783. doi: 10.1038/srep17783

Comparing photosynthetic characteristics of Isoetes sinensis Palmer under submerged and terrestrial conditions

Tao Yang ¹, Xing Liu ^1,^a

PMCID: PMC4669503 PMID: 26634994

Abstract

Crassulacean acid metabolism (CAM) is widespread in terrestrial and aquatic species, plastic in response to environmental changes. Isoetes L. is one of the earliest basal vascular plants and CAM is popular in this genus. Isoetes sinensis Palmer is an amphibious species, alternating frequently between terrestrial and aquatic environments. Given this, we investigated and compared photosynthetic characteristics over a diurnal cycle under submerged condition (SC) and terrestrial condition (TC). The results suggest that I. sinensis possesses a stronger CAM capacity under SC. Compared with under TC, titratable acidity levels and organic acid concentrations were more enriched under SC, whereas soluble sugar or starch and protein levels were lower under SC. Transcript analyses for nine photosynthetic genes revealed that CAM-associated genes possessed high transcripts under SC, but C₃-related transcripts were highly expressed under TC. In addition, the enzyme activity measurements demonstrated that PEPC activity over a diurnal cycle was slightly higher under SC, whereas Rubisco activity during the daytime was greater under TC. This comprehensive study probably facilitates general understandings about the CAM photosynthetic characteristics of Isoetes in response to the environmental changes.

Crassulacean acid metabolism (CAM) photosynthesis is a key photosynthetic mode that C₃ (ribulose-1, 5-carboxlyase oxygenase: Rubisco) and C₄ (phosphoenolpyruvate carboxylase: PEPC) operate within a common cell but the two enzyme activities separate temporally¹. In the night, nocturnal CO₂ uptake from atmosphere or respiration is fixed by PEPC in the cytosol. A C₄ product, malic acid is transferred to central vacuole and catalyzed by malic acid dehydrogenase (MDH). Phosphoenolpyruvate carboxylase kinase (PPCK) is also associated with CO₂ uptake in the night. The electrochemical channel of H⁺-ATPase pump plays pivotal roles in the accumulation of malic acid in vacuole, which is activated by H⁺-translocation ATPase (V-ATPase)². During the following day, the generated CO₂ from decarboxylation of organic acids is fixed by Rubisco and further catalyzed by a multitude of enzymes in Calvin pathway, such as phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoribulokinase (PRK) and the others³,⁴. CAM photosynthesis is widespread in terrestrial and aquatic plants. It is present in approximately 16,800 species of 343 genera in 35 families⁵,⁶. CAM in aquatic vascular species has been investigated within the genera Isoetes L., Sagittaria, Crassula, Vallisneria, and Littorella⁷,⁸,⁹,¹⁰,¹¹. CAM for terrestrials is considered to respond to water-use efficiency in arid environments, whereas for aquatics, CAM is to improve competitive ability for carbon acquisition¹¹. In addition, CAM is a plastic process in response to the environmental changes, such as water, pH, temperature, CO₂, light level and ion concentrations¹²,¹³,¹⁴. Thus, expansive carbon fixation subcategories are observed, such as C₃, facultative CAM, obligate CAM, latent CAM, CAM idling, and rapid-cycling CAM cycles¹⁵,¹⁶. Considering the fact that CAM acts as a CO₂-concentration mechanism to reduce photorespiration and further elevate CO₂ availability in terrestrial and aquatic plants, yet, carbon assimilation rate in CAM cycle is lower than C₃ or C₄ photosynthesis, probably owning to the dynamics of carbon fixation over a diurnal cycle¹⁷,¹⁸. Moreover, compared with C₃, energetics and nitrogen use efficiency are not favorable choices for CAM photosynthesis¹⁸.

Isoetes is an ancient genus of heterosporous lycopsids with a unique position in plant evolution. Phylogenetic analysis shows that Isoetes is one of the earliest basal vascular plants, which can date back to the Devonian¹⁹. The genus has approximately 200 species characterized by a strongly reduced plant body²⁰. Since CAM in aquatic plants was first reported in Isoetes howellii Engelmann¹⁰, most species in this genus have shown similar CAM mode regardless of a few terrestrial species, including I. nuttallii A. Braun ex Engelm, I. butleri Engelm, I. stellenbossiensis A.V. Duthie, and I. durieui Bory⁹,¹⁰,¹¹,²¹. CAM in Isoetes is of great advantage to increase underwater photosynthesis at limited CO₂ concentrations and decrease photorespiration at apparently high O₂ concentrations²². In addition, typical isoetid growth form contributes to an alternative CO₂ source from sediments. Moreover, the achlorophyllous leaf bases play important roles in elevating underwater net photosynthesis and radial O₂ loss to the surrounding sediments²³,²⁴,²⁵. Since the Paleozoic Era, the species of Isoetes have frequently changed growth habitats from putative amphibious to terrestrial or lacustrine transitions¹¹. There is a notion that CAM pathway in modern Isoetes originates from seasonal wetlands and then spreads into oligotrophic lakes and terrestrial habitats¹¹. To date, the genus occupies a variety of niches, including oligotrophic softwater lakes, higher-altitude wetlands, seasonal pools and intermittent streams²⁶. Data in the published studies demonstrate that environmental changes have vital effects on the CAM activity in Isoetes¹⁷. It reveals that under terrestrial and semi-aquatic conditions, most species in this genus fail to produce functional stomata or loose CAM and further rely on C3 photosynthesis¹⁵,¹⁷,²⁷,²⁸,²⁹. But quite a few species still remain CAM as the leaves are exposed to air, such as I. karstenii, I. nuttallii and I. australis Williams¹¹,²². Furthermore, the shift from CAM to C3 mode is partially reversible, considering the fact that CAM cycle probably contributes to 40% total carbon acquisition under submerged condition (SC) but only less than 1% under terrestrial condition (TC)¹²,¹⁷.

Isoetes sinensis Palmer is an allotetraploid plant (2n = 4x = 44) distributed in East Asia³⁰,³¹. I. sinensis is categorized as critically endangered flora on the IUCN Red List due to water pollution or eutrophication, habit degradation or loss, and competitive exclusions³². Additionally, I. sinensis is a typical amphibious plant, growing in seasonal pools and intermittent streams. The plants alternate frequently between terrestrial and aquatic environments. Previous study indicates that I. sinensis shares the CAM activity under SC³³. But the photosynthetic characteristics are still unclear when exposed to TC. Moreover, little is known about the differences between the photosynthetic characteristics under TC and SC.

In this study, juvenile leaves of I. sinensis under TC and SC were harvested every three hours over a diurnal cycle, respectively. We used a series of experimental approaches, such as titratable acidity measurements, enzyme activity assays and quantitative real-time PCR (qPCR) analysis, to investigate the photosynthetic characteristics under TC and SC, and further compared the difference under SC with TC. Compared with under TC, the results revealed that I. sinensis possessed a stronger CAM capacity under SC. In addition, we identified nine photosynthetic genes and plotted a dynamic expression view based on qPCR analysis under both conditions. This comprehensive study probably facilitates general understandings about the photosynthetic characteristics of Isoetes in response to the changing environments and it might be instrumental for future investigations in Isoetes.

Results

Titratable acidity assay

Titratable acidity assay is considered to be one of the most popular diagnostic approaches for CAM activity¹¹,³⁴. In this study, titratable acidity was measured from the juvenile leaves which were harvested every three hours over a diurnal cycle under TC and SC, respectively. The acidity levels varied between 12.56 and 46.96 μmolequiv g⁻¹ FW under TC and between 22.44 and 102.95 μmolequiv g⁻¹ FW under SC (Fig. 1). Moreover, the acidity levels were higher under SC than TC and there was a statistically significant difference at 6, 12, 18, 0 and 3 h between the levels under SC and TC (Table 1). Under SC, the acidity levels decreased rapidly from 6 to 15 h during the daytime and increased markedly from 18 to 6 h in the night. Compared with under SC, the diurnal acidity fluctuation was lower under TC. Additionally, temperatures in water and air were recorded over a diurnal cycle, respectively. The temperature in water ranged from 14 to 23.1 °C and from 14.1 to 27.1 °C in air (Supplementary Figure S1, Table 1).

Juvenile and green leaves were harvested every three hours during the daytime (white) and night (black), under TC and SC, respectively. Each point represents the mean ± SE (n = 3).

Table 1. Diurnal fluctuation of the levels of temperature, titratable acidity, malate, citrate, starch, soluble sugar, soluble protein, PEPC activity, Rubisco activity and relative expression levels of nine photosynthetic genes under submerged (SC) and terrestrial conditions (TC).

Terrestrial	Hour (h)
Terrestrial	6	9	12	15	18	21	0	3
Temperature(°C)	14.23 ± 0.50	25.03 ± 0.26^a	25.10 ± 0.2^a	27.10 ± 0.21^a	23.03 ± 0.26^a	20.10 ± 0.32	14.13 ± 0.30	14.10 ± 0.21
Acidity(μmolequiv g−1 FW)	46.96 ± 1.98^a	31.75 ± 1.59	12.56 ± 1.8^a	17.09 ± 2.72	14.53 ± 2.2 5^a	26.90 ± 2.29	39.18 ± 4.39^a	30.13 ± 2.95^a
Malate(mg. g⁻¹ FW)	34.01 ± 1.79^a	26.18 ± 2.36^a	23.35 ± 1.71^a	23.30 ± 1.67^a	21.83 ± 1.50^a	30.97 ± 1.49^a	41.19 ± 2.57^a	49.68 ± 2.95^a
Citrate(mg. g⁻¹ FW)	35.30 ± 2.08	26.02 ± 2.33	27.30 ± 2.09^a	28.56 ± 1.73	27.48 ± 2.01	19.54 ± 2.37^a	27.09 ± 1.63	30.01 ± 1.6 3^a
Starch(%)	0.75 ± 0.01	0.80 ± 0.01^a	0.94 ± 0.03	0.86 ± 0.02	1.42 ± 0.06^a	1.03 ± 0.04	0.93 ± 0.02	1.25 ± 0.01^a
Soluble sugar(mg/g)	14.23 ± 0.38^a	68.61 ± 0.78^a	16.97 ± 0.28^a	53.35 ± 1.06^a	14.56 ± 0.25	50.28 ± 1.21^a	23.52 ± 0.25^a	38.26 ± 0.41^a
Soluble protein(μg/g)	2.91 ± 0.05^a	2.04 ± 0.04^a	1.30 ± 0.01^a	2.58 ± 0.02^a	2.29 ± 0.11	2.39 ± 0.0 4^a	1.85 ± 0.05	2.86 ± 0.20^a
PEPC activity(μmol mg⁻¹ protein min⁻¹)	3.66 ± 0.45	2.16 ± 0.18	2.08 ± 0.23^a	2.28 ± 0.27	2.11 ± 0.15	3.85 ± 0.09	4.69 ± 0.23	4.08 ± 0.18^a
Rubisco activity(μmol mg⁻¹ protein min⁻¹)	12.78 ± 0.67	19.69 ± 0.90	37.60 ± 1.43^a	25.64 ± 1.32	30.84 ± 1.19^a	12.72 ± 0.46	12.60 ± 0.86	10.01 ± 0.55^a
PEPC	0.16 ± 0.05^a	0.16 ± 0.04	0.09 ± 0.02	0.02 ± 0.01^a	0.14 ± 0.11^a	0.22 ± 0.05^a	0.31 ± 0.06^a	0.10 ± 0.0 8^a
PPCK	0.21 ± 0.01^a	0.37 ± 0.04	0.28 ± 0.02^a	0.07 ± 0.01^a	0.12 ± 0.07^a	0.66 ± 0.02	0.35 ± 0.19	0.29 ± 0.04
MDH	0.31 ± 0.05	0.54 ± 0.01	0.68 ± 0.00	0.14 ± 0.02^a	0.21 ± 0.01^a	0.46 ± 0.00	0.02 ± 0.10	0.15 ± 0.05
PPCK	5.28 ± 0.67^a	1.35 ± 0.20	10.31 ± 0.05^a	2.28 ± 0.34	8.35 ± 0.54	2.50 ± 0.90	2.12 ± 0.00	1.59 ± 0.30
Rubisco	1.00 ± 0.16^a	0.37 ± 0.06	0.56 ± 0.07^a	0.56 ± 0.05^a	0.47 ± 0.05	0.59 ± 0.07	0.58 ± 0.05	0.69 ± 0.06
GAPDH	1.72 ± 0.12^a	0.02 ± 0.04	0.40 ± 0.02	0.13 ± 0.01	0.21 ± 0.08	0.40 ± 0.01	0.24 ± 0.02	0.35 ± 0.05
GAPA	2.11 ± 0.01^a	0.03 ± 0.04	0.38 ± 0.01	0.16 ± 0.06	0.29 ± 0.02	0.41 ± 0.00	0.31 ± 0.04	0.30 ± 0.08
PGK	0.98 ± 0.03	1.01 ± 0.02	1.23 ± 0.03	8.00 ± 0.21^a	1.42 ± 0.03	3.09 ± 0.55^a	3.40 ± 0.08	0.87 ± 0.09
V-ATPase	0.25 ± 0.04^a	0.31 ± 0.02	0.50 ± 0.18	0.63 ± 0.03	0.47 ± 0.18	0.23 ± 0.04	0.11 ± 0.01	0.34 ± 0.05
Submerged
Temperature(°C)	14.10 ± 0.26	23.10 ± 0.21^a	23.03 ± 0.20^a	20.03 ± 0.15^a	20.03 ± 0.15^a	20.00 ± 0.00	14.07 ± 0.12	14.00 ± 0.12
Acidity(μmolequiv g−1 FW)	102.95 ± 1.50^a	36.79 ± 1.00	34.45 ± 0.59^a	22.44 ± 2.35	29.28 ± 1.29^a	39.06 ± 2.63	56.33 ± 3.08^a	75.77 ± 1.06^a
Malate(mg. g⁻¹ FW)	93.43 ± 3.99^a	84.17 ± 2.54^a	67.42 ± 1.68^a	62.52 ± 2.19^a	60.70 ± 2.20^a	72.70 ± 3.13^a	79.29 ± 3.28^a	180.93 ± 5.10^a
Citrate(mg. g⁻¹ FW)	40.94 ± 1.66	32.01 ± 1.76	52.44 ± 2.12^a	32.21 ± 1.27	31.04 ± 2.15	59.28 ± 2.74^a	29.44 ± 1.07	57.30 ± 2.26^a
Starch(%)	0.26 ± 0.03	0.27 ± 0.00^a	0.66 ± 0.03	0.78 ± 0.02	0.73 ± 0.02^a	0.61 ± 0.01	0.54 ± 0.00	0.22 ± 0.32^a
Soluble sugar(mg/g)	6.88 ± 0.12^a	26.43 ± 1.06^a	4.47 ± 0.6 4^a	8.34 ± 0.28^a	11.73 ± 1.52	12.50 ± 0.78^a	4.38 ± 0.15^a	22.20 ± 1.20^a
Soluble protein(μg/g)	1.48 ± 0.13^a	1.26 ± 0.09^a	1.87 ± 0.04^a	1.73 ± 0.00^a	2.14 ± 0.04	1.71 ± 0.12^a	2.04 ± 0.03	1.67 ± 0.04^a
PEPC activity(μmol mg⁻¹ protein min⁻¹)	4.69 ± 0.26	3.59 ± 0.18	3.43 ± 0.30^a	2.43 ± 0.31	2.19 ± 0.24	4.16 ± 0.29	4.83 ± 0.19	5.95 ± 0.26^a
Rubisco activity(μmol mg⁻¹ protein min⁻¹)	10.82 ± 0.55	14.95 ± 0.85	17.65 ± 1.19^a	20.72 ± 0.56	19.76 ± 1.71^a	18.75 ± 0.51	16.60 ± 0.82	18.66 ± 1.64^a
PEPC	1.00 ± 0.09^a	0.36 ± 0.02	0.17 ± 0.10	1.07 ± 0.06^a	1.51 ± 0.04^a	0.75 ± 0.08^a	1.58 ± 0.02^a	0.77 ± 0.03^a
PPCK	1.00 ± 0.09^a	0.73 ± 0.06	1.50 ± 0.05^a	0.75 ± 0.07^a	1.43 ± 0.11^a	0.90 ± 0.11	0.98 ± 0.08	0.84 ± 0.04
MDH	1.00 ± 0.01	0.53 ± 0.01	0.45 ± 0.00	3.00 ± 0.02^a	1.73 ± 0.01^a	1.08 ± 0.03	0.02 ± 0.03	0.90 ± 0.01
PPCK	1.00 ± 0.53^a	0.66 ± 0.14	2.60 ± 0.70^a	3.14 ± 0.55	5.90 ± 1.10	1.56 ± 0.08	1.12 ± 0.27	4.13 ± 0.35
Rubisco	1.00 ± 0.10^a	0.37 ± 0.04	0.13 ± 0.04^a	0.17 ± 0.02^a	0.35 ± 0.02	0.35 ± 0.05	0.70 ± 0.05	0.71 ± 0.03
GAPDH	1.00 ± 0.02^a	0.03 ± 0.04	0.001 ± 0.00	0.16 ± 0.02	0.06 ± 0.02	0.26 ± 0.03	0.07 ± 0.07	0.25 ± 0.04
GAPA	1.00 ± 0.03^a	0.03 ± 0.02	0.002 ± 0.11	0.20 ± 0.03	0.06 ± 0.03	0.26 ± 0.00	0.18 ± 0.18	0.20 ± 0.05
PGK	1.00 ± 0.03	0.82 ± 0.14	0.82 ± 0.04	2.90 ± 0.13^a	0.43 ± 0.10	15.58 ± 0.32^a	2.06 ± 0.07	1.05 ± 0.07
V-ATPase	1.00 ± 0.32^a	0.40 ± 0.05	0.65 ± 0.36	0.56 ± 0.04	0.13 ± 0.04	0.34 ± 0.01	0.62 ± 0.06	0.50 ± 0.09

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Juvenile and green leaves were harvested every three hours during the daytime (from 6 to 18 h) and night (from 18 to 6 h) under TC and SC, respectively. Letters indicate statistically significant differences (ANOVA and Tukey post hoc test; P < 0.01) between the levels under TC and SC. Numbers indicate the mean ± SE (n = 3).

Organic acid determination

Organic acid is another key indicator to estimate the CAM capacity¹¹,³⁴. To further investigate the CAM capacity under both conditions, we measured malate and citrate concentrations using high-performance liquid chromatograph (HPLC) analysis. The malate concentration varied between 60.70 and 180.93 mg. g⁻¹ FW under SC and between 21.83 and 49.68 mg. g⁻¹ FW under TC (Fig. 2). Compared with under TC, a markedly higher malate level was observed under SC over a diurnal cycle (Table 1). Moreover, a low amplitude during the daytime was present under both conditions, whereas in the night, the concentration obviously increased from 0 to 3 h under SC and only a slightly increased extent was occurred under TC. Similarily, the citrate concentration was higher under SC than TC and a statistically significant difference was present at 12, 21 and 3 h by comparing the concentrations under SC with TC (Fig. 2, Table 1). The citrate concentration varied between 29.44 and 52.44 mg. g⁻¹ FW under SC and between 19.54 and 35.30 mg. g⁻¹ FW under TC (Fig. 2).

Juvenile and green leaves were harvested every three hours during the daytime (white) and night (black), under TC and SC, respectively. Then the malate and citrate concentrations were measured using high-performance liquid chromatograph. Each point represents the mean ± SE (n = 3).

Starch and soluble sugar measurements

Starch and soluble sugar are considered to be vital products in the process of photosynthesis¹⁸,³⁵. We examined the accumulation levels of soluble sugar and starch over a diurnal change under SC and TC, respectively. The result showed that the starch and soluble sugar concentrations over a diurnal cycle were consistently higher under TC than SC (Fig. 3). The starch levels in the leave tissues varied between 0.75% and 1.42% under TC and between 0.22% and 0.78% under SC. Moreover, a statistically significant difference was present at 9, 18 and 3 h by comparing the starch levels under SC with TC (Table 1). Furthermore, there was an elevated amplitude under both conditions during the daytime from 6 to 15 h, whereas a reduced fluctuation was observed throughout the night under SC and from 18 to 0 h under TC. Additionally, there was a large diurnal amplitude for the soluble sugar levels under TC, ranging from 14.23 to 68.61 mg/g (Fig. 3, Table 1).

Soluble protein and enzyme activity

Growth under TC and SC had a statistically significant effect on the soluble protein at most of the times (Supplementary Figure 2, Table 1). Totally, the soluble protein levels were higher under TC than SC, ignoring the levels at 12 h.

PEPC and Rubisco enzymes play important roles in CAM photosynthesis by operating temporary function separately within a common cell⁵,¹⁶. In this study, we conducted enzyme activity assay to investigate the activities of PEPC and Rubisco during a diurnal cycle under TC and SC, respectively. The PEPC activity on a protein basis varied between 2.08 and 4.69 μmol mg⁻¹ protein min⁻¹ under TC and between 2.19 and 5.95 μmol mg⁻¹ protein min⁻¹ under SC (Fig. 4). The activity under SC was slightly higher than under TC over a diurnal cycle and there was a statistically significant difference at 3, 9 and 12 h by comparing the activity under SC with TC (Table 1). Moreover, the PEPC activity consistently decreased during the daytime from 6 to 18 h and increased in the night from 18 to 0 h under both conditions, whereas the activity was decreased from 0 to 3 h under TC. Otherwise, the Rubisco activity varied between 10.01 and 37.60 μmol mg⁻¹ protein min⁻¹ under TC and between 10.82 and 20.72 μmol mg⁻¹ protein min⁻¹ under SC (Fig. 4). Compared with under SC, the Rubisco activity under TC was higher from 6 to 18 h during the daytime and lower from 21 to 3 h in the night. Furthermore, there was a statistically significant difference at 12, 18 and 3 h by comparing the activity under TC with SC (Table 1).

Transcript determinations of photosynthetic genes using RNA-sequencing technology

We performed RNA-sequencing to determine photosynthetic genes in Isoetes. The sequencing datasets have been deposited in the National Center of Biotechnology Information Short Read Archive database with the accession numbers SRR1648119 under SC and SRR1646513 under TC, respectively. Out of the datasets, we totally identified nine putative homologous genes related to photosynthesis which play important roles in the process of CAM metabolism. In terms of the nine homologous genes, there were three genes related to CAM mode, including PEPC, PPCK and MDH; five associated with C₃ mode, including Rubisco, GADPH, glyceraldehyde-3-phosphate dehydrogenase (NADP+) (GAPA), PRK and PGK; and V-ATPase associated with malic acid accumulation via the electrochemical channel of tonoplast.

Transcript accumulations of photosynthetic genes using qPCR

We further detected the transcript accumulations by using qPCR analysis from the harvested materials under TC and SC, respectively. Totally, the results demonstrated that the PEPC, PPCK and MDH transcripts over a diurnal cycle were consistently higher under SC than TC (Fig. 5). For the PEPC transcript, the difference between the expression levels under TC and SC was statistically significant from 15 to 6 h (Table 1). For the PPCK and MDH transcripts, the diurnal expression fluctuation was larger under SC than TC, whereas the transcript difference under both conditions was statistically significant at 6, 12, 15 and 18 h for the PPCK transcript, and at 15 and 18 h for the MDH transcript (Table 1).

Juvenile and green leaves were harvested every three hours during the daytime (white) and night (black), under TC and SC, respectively. In total, nine photosynthetic genes were identified, including *PEPC*, *PPCK* and *MDH* for CAM mode; *Rubisco*, *GADPH*, *GAPA*, *PRK* and *PGK* for C₃ mode; and *V-ATPase* associated with malate accumulation via the electrochemical channel of tonoplast. The plots were conducted using the ggplot2 module in R program.

The accumulations of five transcripts related to C₃ mode, including Rubisco, GADPH, GAPA, PGK and PRK genes, were evaluated under both conditions (Fig. 5). For the Rubisco gene, the transcript under SC was gradually decreasing during the daytime (from 6 to 15 h) and increasing in the night (from 18 to 3 h). Nonetheless, there was a slight increase under TC in the night and an obvious decrease from 6 to 9 h during the daytime (Table 1). In addition, the diurnal amplitude for GAPDH and GAPA transcripts resembled each other under both conditions. And the expression levels were significantly decreased from 6 to 9 h under both conditions (Table 1). Furthermore, there was a statistically significant difference at 15 and 21 h by comparing the PGK transcript under TC with SC (Table 1). Moreover, a large amplitude for the PRK transcript was occurred under both conditions and there was a statistically significant difference at 6 and 12 h by comparing the transcript under TC with SC (Table 1). In terms of the V-ATPase gene, the transcript under TC was increasing from 6 to 15 h and decreasing from 15 to 0 h, whereas the transcript shared a large diurnal fluctuation under SC (Fig. 5, Table 1).

Discussion

CAM shares a significant carbon-concentrating mechanism for aquatic plants in submerged environments and it is plastic in response to the environmental changes¹¹,¹⁴,¹⁷,²⁸. In this study, I. sinensis was used to elucidate the photosynthetic characteristics under SC and TC. Previous researches demonstrate that I. sinensis exhibits appreciable CAM photosynthesis under SC³³. However, dynamic characteristics for the CAM photosynthesis in I. sinensis are poorly elucidated. In this article, we gradually investigated the photosynthetic characteristics during a diurnal cycle and further compared the characteristics under SC with TC. The results showed that titratable acidity levels and organic acid concentrations during a diurnal cycle were higher under SC than TC, indicating that I. sinensis possessed a stronger CAM capacity under SC than TC. When exposed to aerial environments, most species in Isoetes alternate the photosynthetic pathway from CAM to C₃¹¹,¹⁵. Yet, there are still quite a few species remaining CAM as the leaves are exposed to air, such as I. karstenii, I. nuttallii and I. australis¹¹,²². Although the mechanism for CAM loss or reservation in Isoetes is still unknown, a widely acceptable opinion suggests that switching between CAM and C3 is to respond to the elevated CO₂ in air, whereas the fixed CAM is to be a conservative strategy, considering that switching back and forth between C₃ and CAM is of little benefit¹¹,¹⁵,²². Titratable acidity and organic acid assays are popular diagnostic tests to detect the CAM activity⁵,¹⁶. Under TC, the total titratable acidity levels and malate concentrations were slightly higher in the night than the daytime, indicating that I. sinensis under TC shared a reduced CAM activity. Statistical data show that the titratable acidity levels from substantial CAM plants are variable from 5 to 300 μmolequiv g−1 FW¹⁰,¹¹,¹²,³⁶. Moreover, previous research declares that the acidity levels for I. sinensis under SC vary between 10.47 and 11.06 μmolequiv g−1 FW in the late afternoon and between 105.71 and 111.57 μmolequiv g−1 FW in the early morning³³. In this article, our results revealed that the acidity levels under SC ranged from 22.44 to 29.28 μmolequiv g−1 FW in the late afternoon (from 15 to 18 h) and from 75.77 to 102.95 μmolequiv g−1 FW in the early morning (from 3 to 6 h). The results are understandable that CAM activity is variable depending on seasons and environments, such as pH, temperature, CO₂, light level and ion concentrations¹³,¹⁴,²⁸. Compared with under SC, the acidity levels under TC varied between 14.53 and 17.09 μmolequiv g−1 FW in the late afternoon and between 30.13 and 46.96 μmolequiv g−1 FW in the early morning, further revealing a decreased CAM activity under TC. Additionally, the malate concentration was decreased from 3 to 6 h in the early morning, speculating that I. sinensis probably brought forward to make preparations to satisfy the requirements in the following day. Citric acid is another carbon-fixed product in the night and occupies 10–20% dark-fixed components based on steady-state ¹⁴C labeling studies¹⁰,¹¹. In this study, the citrate concentration showed a large diurnal amplitude under both conditions, suggesting that citric acid probably is not a suitable indicator to reflect the CAM activity in Isoetes¹¹,²². But the malate and citrate concentrations consistently present obviously diurnal fluctuations in the typical CAM plant Mesembryanthemum crystallinum¹⁵,³⁷.

Starch is an important product in the photosynthetic pathway and it is stored in leaves during the daytime and broken out in the following night¹⁸. Starch plays a pivotal role in serving as a carbon overflow to allow photosynthetic process faster and acting as a continuous carbon supply at night³⁵,³⁸. For the CAM plants, starch has an added role in providing the three-carbon CO₂ acceptor molecule phosphoenolpyruvate in glycolysis pathway³⁹. Moreover, starch metabolism is plastic to adapt to the environmental changes in terrestrial and aquatic plants¹⁸,⁴⁰,⁴¹. In this study, the starch and soluble sugar levels were higher under TC than SC. Furthermore, the starch levels during the daytime were increasing under both conditions. In the night, the amplitude of starch level was low under SC, whereas a large amplitude was observed under TC (Fig. 3). Although starch can provide the substrate for malic acid synthesis, the starch accumulation in leaves is generally not completely in accordance with the malate and acidity levels¹⁸. The fact further demonstrates that starch is transferred to the corm or translated into phosphoenolpyruvate at proper times¹¹,¹⁸.

Nocturnal CO₂ in the cytosol is captured by PEPC enzyme in the night¹⁶. However, the PEPC activity probably is not limited in the dark, considering the fact that the enzyme can enhance Calvin activity during the daytime¹¹. The PEPC activity over a diurnal cycle was slightly higher under SC than TC and there was a statistically significant difference at 9,12 and 3 h by comparing the activity under TC with SC (Table 1). Moreover, the PEPC activity consistently decreased during the daytime and increased from 18 to 0 h under both conditions (Fig. 4). In addition, for the PEPC gene, the transcript was more enriched under SC than TC and there was a statistically significant difference at most of the times by comparing the expression levels under TC and SC (Fig. 5). Furthermore, the total PEPC expression levels were higher in the night than the daytime under both conditions. The discrepancy between mRNA levels and protein abundance is considered that changes in mRNA stability and translational efficiency are likely to regulate the genes’ expression in response to the environmental changes⁴²,⁴³. Moreover, PEPC probably has some paralogous members recruited in the family to fulfill the increased carbon flux demand for CAM photosynthesis⁴⁴.

Rubisco enzyme not only plays critical roles in photosynthetic pathway but also it is functional in photorespiration metabolism. In this study, the total Rubisco activity during the daytime was higher under TC than SC and the activity under both conditions presented a statistically significant difference at 12 and 18 h (Table 1). An apparent midday depression for photosynthesis and high light intensity presumably contribute to the decreasing Rubisco activity from 12 to 15 h under TC. However, for the Rubisco transcript, the mRNA levels were greater under TC than SC at most of the times and there was a statistically significant difference at 6, 12 and 15 h by comparing the expression levels under TC with SC. Nonetheless, the Rubisco transcript was decreasing during the daytime and increasing in the night under SC. When exposed to under TC, the plants suffered greater photorespiration and light intensity than under SC, which partially have effect on the discrepancy between transcripts and protein activity. Furthermore, post-transcription modification, temporal translation, mRNA unstability, low-translation efficiency and other Rubisco homologues consistently contribute to the temporal difference between mRNA levels and protein activity¹⁶,²³,⁴². Compared the total enzyme activity of Rubisco with PEPC, the Rubisco activity was from 2.24 to 9.2-fold higher than PEPC activity under SC and from 2.52 to 18.60-fold under TC (Supplementary Figure S3). Nevertheless, previous studies reveal that the Rubisco activities in I. howellii, I. lacustrisis and I. orcuttii are from 3.4 to 7.2-fold higher than PEPC activity under SC and from 30.7 to 32.0- fold changes under TC¹¹. The dynamic enzyme activities in this study are reliable with the diurnal changes, further suggesting that enzyme activity is affected by species diversity and environmental factors¹¹. Isoetes can absorb additional CO₂ from sediments in order to enhance carbon pool¹¹. More than 95% CO₂ is captured from sediments when CO₂ levels in water are decreased, whereas the root uptake probably drops into less than 50% as increased CO₂ levels in water¹¹. PEPC has a high CO₂ affinity under SC, probably resulting from the fact that vacuole occupies a limited storage capacity for malic acid¹¹. Additionally, amphibious species in Isoetes do not have functional stomata in submerged environments³⁴. The behaviors of stomata with the environmental changes in I. sinensis should be further investigated in the future.

CAM photosynthesis is controlled not only by environmental factors but also by genes associated with the regulations of transcription, post–transcription and post–translation¹³. To date, CAM research is mainly limited to biochemistry and physiological aspects. Molecular resources are very limited in public database. High-throughput sequencing approach becomes a perfect alternative for non-model plants to produce multiple sequencing data at a relatively low cost⁴⁵,⁴⁶,⁴⁷,⁴⁸. Furthermore, RNA-sequencing technology has become popular to discover novel genes and explore different expression profiles under various conditions⁴⁶,⁴⁷. In this study, we used RNA-sequencing to excavate photosynthetic genes in I. sinensis. In total, nine homologous genes were identified, including PEPC, PPCK, MDH, Rubisco, GADPH, GAPA, PRK, PGK and V-ATPase. Furthermore, we applied qPCR to describe expression profiles from the harvested materials under TC and SC, respectively. In general, the C₃-related transcripts were enriched under TC, including the genes Rubisco, PGK, GAPDH and GAPA, whereas CAM-related transcripts were abundant under SC, including PEPC, PPCK and MDH transcripts. We further plotted dynamic diagrams to compare the expression patterns under TC with SC (Supplementary Figure S4 and S5). Compared with under SC, the transcripts showed a larger fluctuation under TC, probably indicating that the plants attempt to respond to the external stresses in terrestrial environments with high light intensity, limited water and CO₂. In summary, investigation for the photosynthetic genes might be a breakthrough improvement to explore CAM pathway in Isoetes and it probably provides a novel insight into the study of CAM photosynthesis.

Methods

Plant materials and growth conditions

I. sinensis was collected from Xinan River with a fluctuated water level in Zhejiang Province, China (29°28′N; 119°14′E) and then cultivated in a greenhouse in Wuhan University. Initially, all experimental materials were maintained in submerged cultures with five plants in one growth chamber (30 × 40 × 50 cm), containing soil and 60 L tap water. We randomly chose a part of experimental materials to still keep the plants under SC with the water about 4 cm above the tops of leaves. The others were transferred to TC with leaves and shoots fully in air for an additional month. During the experimental period, weeds or snails were removed every day to maintain the experimental materials as a monoculture. Temperatures in water and air were recorded every three hours during a diurnal cycle. Juvenile and green leaves under SC and TC were collected over a diurnal cycle, respectively, including at 9, 12 15 and 18 h during the daytime and at 21, 0, 3 and 6 h in the night. All the materials were harvested in triplicate. Then the materials were washed in distilled water, immediately frozen into liquid nitrogen and stored at −80 °C, respectively.