Joint interactions of carbon and nitrogen metabolism dominated by bicarbonate and nitrogen in Orychophragmus violaceus and Brassica napus under simulated karst habitats

Karst habitats are uniquely characterized by high bicarbonate, high nitrate, and low ammonium, which are in-conducive to their growth and biodiversity. The occurrence of inorganic carbon and nitrogen in karst soil profoundly affects the carbon/nitrogen metabolism and adaptability of plants. However, there has been no final conclusion to the joint interactions of carbon and nitrogen metabolism in plants under karst habitats. In this study, we selected a karst-adaptable plant Orychophragmus violaceus (Ov), and a non-karst-adaptable plant Brassica napus (Bn) as experimental plants, and compared their joint effects of carbon and nitrogen metabolism under simulated karst habitats. It was found that the two species had different joint effects of carbon and nitrogen metabolisms. Bicarbonate and nitrate joint promoted photosynthetic activity and glucose metabolism, facilitating the carbon/nitrogen metabolism and growth of Ov, but their impacts on the carbon and nitrogen metabolism were insignificant in Bn. Bicarbonate and ammonium joint inhibited the photosynthesis and nitrogen metabolism, but promoted water use efficiency in Ov, leading to its enhance of growth reduction, ammonium toxicity alleviation, and drought resistance, while they inhibited the water use efficiency of Bn. In general, bicarbonate and nitrate/ammonium more significantly joint affected the carbon and nitrogen metabolism in Ov than Bn, which is vital for Ov to adapt to karst habitats.

unique strategies of inorganic carbon and nitrogen metabolism.
In addition to atmospheric carbon dioxide, dissolved inorganic carbon (DIC) existing in carbon dioxide (CO 2 ) and bicarbonate (HCO 3 − ) was demonstrated to be able to participate in the carbon assimilation. The pH value determines the carbon forms, and as the pH increase, DIC in water medium is mainly in the form of HCO 3 − , which are used by algae and plants, such as Orychophragmus violaceus [1], Chlamydomonas reinhardtii [6], Camptotheca acuminata [7], Potamogeton crispus and Potamogeton malaianus [8,9]. In a karst habitat, carbon dioxide and bicarbonate are alternatively used by plants and the latter can promote carbon assimilation and improve water availability as well as glucose metabolism [10]. Carbonic anhydrase (CA) catalyzes HCO 3 − to CO 2 and H 2 O, contributing to maintaining photosynthesis [1,2]. Furthermore, bicarbonate is involved in total inorganic carbon assimilation, and contributes 9.71% to the total inorganic carbon assimilation in Camptotheca acuminate under simulated karst drought [7]. It can also adjust the glucose metabolism in plants. Yao [10] indicated that 3 mM bicarbonate increased both the glycolytic pathway (EMP) and the pentose phosphate pathway (PPP) of Broussonetia papyrifera (Bp), resulting in the enhancement of the total glucose metabolism. Nitrate and ammonium are the primary inorganic nitrogen utilized by plants, and the former is the primary nitrogen source for most plants under aerobic soil conditions [11,12]. Typically, nitrate also plays an important role in physiological processes [13], but energy and NADPH/NADH reducing agents are required for plants to uptake it. Compared to ammonium, nitrate is theoretically more accessible for plants, but nitrate reduction requires 15 ATP, whereas ammonium assimilation requires only 5 ATP [14]. However, excessive ammonium leads to intracellular acidification and vein damage, which will result in the decline of photosynthetic products and plant growth [15]. In general, both nitrate nitrogen and ammonium nitrogen are necessary for the growth of most plants, and their distribution in soil determines the nitrogen utilization characteristics of plants [16][17][18]. Therefore, it is crucial to figure out the effects of nitrate and ammonium on the nitrogen utilization characteristics in plants. A karst habitat features nitrate-abundant and ammonium-rare soil, where karst-adaptable plants can better survive than non-karst-adaptable plants [1]. Lu [19] indicated that Ov would have higher nitrate utilization than Bn under simulated karst habitats. While these studies describe the utilization of inorganic carbon or nitrogen by plants in karst habitats, the symbiotic metabolism of these two elements remains unclear.
The carbon and nitrogen metabolisms of plants are closely coupled by energy and carbon skeletons. On the one hand, carbon assimilation affects nitrate reduction through the energy produced by photosynthesis, while the glycolytic pathway (EMP) supplies ATP to the nitrogen metabolism and recycles 75%of the cycle [20]. On the other hand, one ammonium ion released in mitochondria during serine biosynthesis from two glycines is re-assimilated in chloroplast by reduced ferridoxin. Meanwhile, nitrogen reduction not only provides enzymes and protein for photothesis, EMP and PPP, which are vital carbon skeletons of carbon metabolism, but also offers nitrogen to the composition of chlorophyll [17] as well as NAPDH to the pentose phosphate pathway (PPP) [18]. The carbon metabolism includes photosynthesis, EMP, PPP, and enzymes such as Ribulose bisphosphate carboxylase oxygenase (Rubisco) and sucrose synthetase (SS) [21][22][23]. In contrast, the nitrogen metabolism contains nitrate reduction and ammonium assimilation, which are obviously affected by nitrate reductase (NR) and glutamate synthase (GOGAT) [24]. The above indicators determine the carbon and nitrogen growth in plants regarding biomass and accumulation. Consequently, the above coupling relationships were determined to clarify the joint effects of carbon and nitrogen metabolisms in plants.
In a karst habitat, bicarbonate, nitrate, and ammonium have a profound influence on the inorganic carbon and nitrogen metabolisms [1,2], and therefore it is essential to clarify the conjugation of bicarbonate, nitrate, and ammonium to carbon and nitrogen metabolism in plants. However, the growth in plants is influenced by multiple factors, such as climate, rainfall, and biodiversity, making it difficult to quantify the conjugation of carbon and nitrogen metabolisms [3,19]. In this study, we simulated karst habitats in an artificial greenhouse, and then cultivated the Orychophragmus violaceus (Ov, karst-adaptable plant) and Brassica napus (Bn, non-karst-adaptable plant) to determine the joint effects of carbon and nitrogen metabolisms on different plant species. The followings are the main objectives of this study: (1) to compare the differential joint effects on the carbon and nitrogen metabolisms of bicarbonate and nitrate/ammonium in different plant species under the environment of a simulated karst habitat; (2) to determine the karst adapting mechanisms of carbon and nitrogen in karst-adaptable plants.

Plant materials
The experiments were carried out in an artificial greenhouse at the Institute of Geochemistry, Chinese Academy of Sciences (Guiyang, China) with a dimension of 10 (L) × 5 (W) × 4 (H) m. The light was provided by metal halide lamps (HPI-T400W/645, Philips, the Netherlands) while the temperature was controlled by air conditioning. Seedlings were then incubated in a photoperiod of 16/8 h under light/dark conditions, along with 500 ± 23 μmol m − 2 s − 1 of photosynthetic photon flux density. The greenhouse conditions were set as:temperature (day/night): 25/19 °C, constant light time of 12 h per day; relative humidity range: 55-60%. In this study, we selected Ov (karst-adaptable plant) and Bn (non-karst adaptable plant) as experimental plants and disinfected the seeds with 70% ethanol for 1 min with constant agitation. Additionally, the seeds were repeatedly rinsed 3-5 times and soaked for 6-8 h. The seeds were sown in 12-hole trays (size: 19 × 15 × 9.5 cm) and grown on a substrate (perlite: vermiculite = 1:3) with the modified Hoagland solution [2], which was changed every 3 days. After 28 days, the seedlings were transplanted and followed by 48 h without nitrogen. Finally, we chose 48 plants with uniform growth of Ov and Bn and randomly divided them into 16 groups (3 plants each group), so as to the following stress treatments.

Stress treatments
The continuous rainfall cycle in karst areas is usually 1-8 days, so karst drought (PEG6000, 10 g/L) was simulated for 8 days to cultivate Bn and Ov seedlings ( Table 1). The bicarbonate of wet soil is more than 10 mM under karst habitats. Therefore, 10 mM of NaHCO 3 was prepared to simulate the bicarbonate environment, and the pH was adjusted to 8.30 ± 0.05 with 1 mol/L KOH so that the bicarbonate exists in an alkaline environment [2,5]. Quantitatively, the culture solution was changed daily at 9:00 a.m., and the soil water content was maintained at 20-25%. Equal amounts of plants were assigned to the measurement of the photosynthesis, carbon and nitrogen metabolism enzymes, leaf carbon and nitrogen contents, and biomass.

The carbon and nitrogen metabolism enzymes
0.1 G of Ov and Bn fresh leaves (the 3rd or 4th euphylla of the seedling) were weighed and ground with liquid nitrogen. Then, the rubisco, SS, NR, GOGAT, PFK (A PFK ) and G6PDH activity (A G6PDH ) were tested with the bio enzyme kit (Sangon, Shanghai, China) The total glucose metabolism activity (EA ∑ ), EMP and PPP, GC and RC RUBP were calculated by the methods adopted by Yao and Wu [10].

Leaf carbon and nitrogen content
15-25 mg of the dried Ov and Bn leaves were wrapped in the tin foil, the leaf carbon and nitrogen contents were measured with the elemental analyzer (Elementar, Germany).

Biomass
The plants were dissected into three parts, roots, stems and leaves, then were dried at 108 °C for 30 min and 75 °C after cleaning. The samples were weighed by analytical balance (accuracy 0.0001 g) to obtain the biomass and plants and calculate the root/shoot ratio(R/S, %).

Statistical analysis
The experimental data were processed as follows: the images were drawn with Origin 9.0; ANOVA, Duncan and LSD in SPSS 25 were used to analyze the data; and the differences between the means were considered significant when the p-value was less than 0.05. The data were expressed as "mean ± standard deviation".

Photosynthesis
In Fig. 1, only nitrate promoted the photosynthesis of Bn, and Pn increased significantly at B0N91; while bicarbonate and nitrate/ammonium were not joint to promote the photosynthetic activity in Bn as they were insignificantly changed. Meanwhile, the Cond in Bn was promoted by the bicarbonate and nitrate joint and increased significantly at B10N91. The bicarbonate and nitrate joint promoted the photosynthesis in Ov, of which Pn, Cond and WUE significantly increased at B10N91, while it inhibited Tr in Ov, which was significantly reduced at B10N91. The bicarbonate and ammonium joint only significantly promoted WUE in Ov, but inhibited the photosynthesis of Ov, resulting in a significant reduction in Pn, Cond, Tr, and Ci of Ov at B10N19.

Carbon and nitrogen metabolizing enzymes
In Fig. 2, only nitrate significantly promoted the activities of carbon and nitrogen metabolism enzymes, and the activities of Rubisco, SS, NR and GOGAT in Bn increased significantly with the increase of nitrate. By contrast, bicarbonate and nitrate/ammonium were not jointd to affect the carbon and nitrogen metabolism enzyme activities, and the bicarbonate insignificantly changed the activities of Rubisco, SS, NRand GOGAT in Bn.
Bicarbonate and nitrate were not jointd to affect the carbon and nitrogen metabolism enzymatic activities in Ov, and the activities of Rubisco, SS, NR and GOGAT were insignificantly changed at B10N91. The bicarbonate and ammonium joint inhibited the nitrogen metabolism enzymatic activities of Ov, and the activities of NR and GOGAT were significantly reduced at B10N19.

Glucose metabolism
In Fig. 3, only nitrate significantly promoted the glucose metabolism in Bn. With the increase of nitrate, E EMP and the total glucose metabolic activity significantly increased, while bicarbonate and nitrate/ammonium were not joint to affect the glucose metabolism, and bicarbonate insignificantly changed E EMP , E PPP and the total glucose metabolic activity in Bn.
The bicarbonate and nitrate joint promoted the glucose metabolism in Ov, with the total glucose metabolic activity significantly increasing at B10N91. The bicarbonate and ammonium were not joint to affect the glucose metabolism, without significant changes in E EMP , E PPP, and the total glucose metabolic activity at B10N19 in Ov.

The growth capacity and the regeneration capacity of RUBP
As shown in Fig. 4, only nitrate significantly promoted the growth capacity (GC) and the regeneration capacity of RUBP (RC RUBP ) in Bn. With the increase of nitrate, GC and RC RUBP were promoted significantly, while bicarbonate and nitrate joint promoted the RC RUBP of Bn, which increased significantly at B10N91. However, bicarbonate and ammonium had no significant joint effect on GC and RC RUBP in Bn.
The bicarbonate and nitrate joint promoted GC in Ov, with a significant increase at B10N91, while the bicarbonate and ammonium joint inhibited it, with a significant decrease at B10N19.

Leaf carbon and nitrogen content in Bn and Ov
As shown in Fig. 5, only nitrate significantly promoted the leaf nitrogen content in Bn, which increased significantly at B0N91. In contrast, bicarbonate and nitrate/ ammonium had no significant joint effect on the leaf carbon and nitrogen content in Bn.
The bicarbonate and nitrate joint promoted the leaf carbon/nitrogen content in Ov, and the leaf carbon/ nitrogen content as well as C/N ratio increased significantly at B10N91, while bicarbonate and ammonium had no significant joint effects on the leaf carbon/nitrogen content in Ov, the leaf carbon /nitrogen content and C/N ratio did not change insignificantly at B10N19 in Ov. Table 2, the bicarbonate and nitrate joint promoted the roots, stems, leaves and total biomass in Bn and Ov, and they all increased significantly at B0N91. Compared to Bn, Ov had more significant total biomass. Bicarbonate and ammonium were not joint to affect the total biomass in either Bn or Ov, but the total biomass of B10N91 was higher than that of B0N19.

Joint interactions of bicarbonate and nitrate Joint promotions on bicarbonate and nitrate in Bn and Ov
In this study, we found that nitrate enhanced the carbon and nitrogen metabolisms in both Ov and Bn. This is because nitrate promoted nitrate reduction and facilitated enzyme formation, then enhanced the Rubisco, SS, NR and GOGAT activities that promoted the carbon and nitrogen metabolisms in plants [25,26].
The joint promotion of bicarbonate and nitrate in Bn and Ov was different, and the bicarbonate and nitrate joint promoted the photosynthesis, glucose metabolism and growth only in Ov. Bicarbonate provided protons for nitrate reduction, which promoted the formation of NADPH and RC RUBP , resulting in the enhancement of the photosynthesis and biomass in plants [27]. However, the promotion in Bn was offset by the adverse effect of bicarbonate [28,29]. Compared to Bn, Ov was more capable of utilizing bicarbonate. On the one hand, it decreased the toxicity of bicarbonate [1]. On the other hand, the carbonic anhydrase of Ov was increased, resulting in more HCO 3 − catalyzed into H 2 O and CO 2 to alleviate the stomatal closure and water use efficiency under karst drought, thus promoting the recovery of photosynthesis [1,2]. Finally, the total glucose metabolism and biomass in Ov were promoted (Fig. 1).

Joint inhibitions on bicarbonate and nitrate in Bn and Ov
In this study, we found that the bicarbonate and nitrate joint only inhibited Tr of Ov. Compared to Bn, Ov was more capable of utilizing bicarbonate under karst drought [1,2]. Dominated to CA, which excellently catalyzed HCO 3 − to CO 2 and H 2 O [4,5], thus increasing the water use efficiency, and reducing Tr in Ov (Fig. 1).

Joint interactions of bicarbonate and ammonium Joint promotions on bicarbonate and ammonium in Bn and Ov
In this study, the bicarbonate and ammonium joint only promoted Ci in Bn and WUE in Ov. Compared to Ov, Bn had a greater demand for ammonium [30,31], which made nitrogen accumulation increase dramatically, leading the photosynthesis enhance [32]. Therefore, the demand for CO 2 and Ci increased in Bn. Meanwhile, Ov had lower demand for ammonium and therefore the nitrogen assimilation decreased, leading to the decrease of photosynthesis and leaf area [33]. Consequently, the transpiration decreased and the water use efficiency significantly enhanced in Ov.

Joint inhibitions on bicarbonate and ammonium in Bn and Ov
In this study, the bicarbonate and ammonium joint inhibited both the photosynthesis and the nitrogen metabolism in Ov. Previous studies have shown that bicarbonate can provide electrons to balance the cell potential imbalance caused by extra ammonium in the alkaline environment, thus reducing intracellular acidification [34,35]. Therefore, the Bn enhanced its leaf biomass by using more ammonium, which significantly promoted Tr, resulting in the decrease of WUE in Bn. Compared to Bn, the Ov consumed a lower amount of ammonium, which decreased the nitrogen accumulation, resulting in the decline of the C and N metabolism enzymes, photosynthesis, NR., GOGAT, Pn and GC in Ov (Figs. 1 and 2).

Joint interactions of C and N in karst-adaptable plants
The Bn and Ov have different carbon/nitrogen coupling mechanisms under karst habitats (Tables 3 and 4), and the latter is more adaptable to high bicarbonate, high nitrate and high pH [1,2,5]. In this study, we found that the bicarbonate and nitrate joint promoted the photosynthesis, glucose metabolism and water use efficiency in Ov, enabling it to adapt well to the drought, high bicarbonate, abundant nitrate and bare aluminium habitats. At the same time, the bicarbonate and ammonium joint inhibited the carbon/ nitrogen metabolism and growth of Ov, but promoted the water use efficiency, helping Ov alleviate the poison of ammonium to resistant the karst habitats (Fig. 6a). Additionally, in this study, bicarbonate and nitrate/ammonium did not clearly joint affected the carbon/nitrogen metabolism in Bn (Fig. 6b), attributing to its weak karst adaptions. Hence, the C and N joint interactions are vital physiological mechanisms of karst adaptations in Ov.

Conclusions
The bicarbonate and nitrate/ammonium jointly affected the carbon and nitrogen metabolisms, of which there were significant differences between a karst-adaptable plant (Ov) and a non-karst-adaptable plant (Bn). The Ov was more adaptable to karst habitats as bicarbonate and nitrate joint promoted photosynthesis and glucose metabolism in Ov, enhancing the carbon and nitrogen metabolism and growth, while they didn't significantly affect the carbon and nitrogen metabolism in Bn.