Effects of Zn Deficiency and Bicarbonate on the Growth and Photosynthetic Characteristics of Four Plant Species

Calcareous soils are characterized by low nutrient contents, high bicarbonate (HCO3−) content, and high alkalinity. The effects of HCO3− addition under zinc-sufficient (+Zn) and zinc-deficient (−Zn) conditions on the growth and photosynthetic characteristics of seedlings of two Moraceae species (Broussonetia papyrifera and Morus alba) and two Brassicaceae species (Orychophragmus violaceus and Brassica napus) were investigated. These four species were hydroponically grown in nutrient solution with 0 mM Zn (−Zn) or 0.02 mM Zn (+Zn) and 0 mM or 10 mM HCO3−. The photosynthetic response to HCO3− treatment, Zn deficiency, or both varied according to plant species. Of the four species, Broussonetia papyrifera showed the best adaptability to Zn deficiency for both the 0 mM and 10 mM HCO3− treatments due to its strong growth and minimal inhibition of photosynthesis and photosystem II (PS II). Brassica napus was sensitive to Zn deficiency, HCO3− treatment, or both as evidenced by the considerable inhibition of photosynthesis and high PS II activity. The results indicated different responses of various plant species to Zn deficiency and excess HCO3−. Broussonetia papyrifera was shown to have potential as a pioneer species in karst regions.


Introduction
Bicarbonate (HCO 3 − ) is the product for the catalysis of carbon dioxide (CO 2 ) hydration by carbonic anhydrase (CA). It can be used as an inorganic carbon source to supplement CO 2 in leaf cells [1]. Additionally, HCO 3 − is an essential constituent of the water-oxidizing complex of photosystem II (PS II). This complex is stabilized by HCO 3 − by binding to other components of PS II and influences the molecular processes associated with the electron acceptor and electron donor sides of PS II [1,2]. Finally, HCO 3 − supplies CO 2 and H 2 O through the actions of CA in photosynthetic oxygen evolution under environmental stress [3]. However, excess HCO 3 − is harmful for crop growth due to the inhibition of protein synthesis and respiration and decreased nutrient absorption [4]. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Zinc (Zn) is an essential microelement for plant growth in all kinds of soils. It influences many biological processes, including carbohydrate metabolism, cell proliferation and phosphorus-Zn interactions [5][6][7]. Zn also serves as an integral component of some enzyme structures, such as CA, alcohol dehydrogenase, and glutamate dehydrogenase [7]. Therefore, Zn deficiency causes the rapid inhibition of plant growth and development, which results in increased reactive oxygen species (ROS) due to photo-oxidative damage and consequently decreased net photosynthesis and photosynthetic electron transport [8].
Excess HCO 3 − or Zn deficiency inhibits photosynthesis and PS II, which influences photosynthetic and chlorophyll (Chl) fluorescence parameters [9][10][11]. HCO 3 − , which is considered the key factor that influences Fe deficiency chlorosis and Zn deficiency in many plant species [12], is the major anion found in calcareous soils in karst regions. However, few studies have shown how plant growth and photosynthetic physiology react to the dual impact of Zn stress and excess HCO 3 − . The response to excess HCO 3 − in different rice genotypes has indicated that Zn-efficient rice cultivars can sustain root growth in the presence of high HCO 3 − when grown in soils with low Zn availability, whereas root growth in Zn-inefficient genotypes is severely inhibited [13,14]. Karst regions are characterized by calcareous soils with a low bioavailability of plant nutrients (e.g., phosphorus, Zn, and iron), high calcium content (in the form of calcium carbonate), and high alkalinity (pH 7.5 to 8.5) [15]. Zn deficiency in plants is particularly associated with calcareous soils in karst regions, where the HCO 3 − concentration in surface runoff water is approximately 5 mM [16]. HCO 3 − is considered an important factor for inhibiting plant growth in calcareous soil, especially in rice and wheat [17]. To grow in these challenging regions, plants must adapt and overcome the prevalent nutrient deficiency in these soils. In a previous study, nine calcifuge and nine acidifuge plants exuded different organic acids from their roots when grown in calcareous soils [18]. Wu

Photosynthetic parameter measurements
Photosynthetic parameters, such as net photosynthetic rate (P N ), transpiration (E), stomatal conductance (g s ), and intercellular CO 2 concentration (Ci), were measured using a portable LI-6400XT photosynthesis system (LI-COR Inc., Lincoln, NE, USA). The fourth-youngest fully expanded leaf from the top was used for measurement between 9:00 and 11:00 a.m. The photosynthetic active radiation, temperature, and CO 2 concentration during measurement collection were 600 μmol m −2 s −1 , 25˚C, and 380 μmol mol −1 , respectively. The water use efficiency (WUE) was calculated as P N /E.

Chl fluorescence measurements
Chl fluorescence parameters were measured using an IMAGING-PAM Chl fluorometer (Heinz Walz GmbH, Effeltrich, Germany) that applied the same array of blue light-emitting diodes (peak wavelength, 470 nm) for fluorescence excitation, actinic illumination, and saturating light pulses. Plants were dark-adapted for 30 min prior to measurement using the upper middle fully expanded leaves [23]. The minimum Chl fluorescence (Fo) was determined using a measuring beam, whereas the maximum Chl fluorescence (Fm) was measured during an 800-ms exposure to a saturating light intensity (6000 μmol m −2 s −1 ). The Fm 0 (maximal fluorescence yield of a light-adapted leaf) and steady-state Chl fluorescence (Ft) were determined. The maximum quantum yield of PS II (Fv/Fm) was calculated as (Fm − Fo)/Fm. The effective PS II quantum yield (FPS II) was calculated as (Fm 0 − Ft)/Fm 0 . Therefore, the relative photosynthetic electron transport rate (ETR) was calculated as FPS II × PPFD × 0.5 × 0.84.

Chl content and biomass measurements
The Chl content was determined for the 3 rd and 6 th fully expanded leaves (in two leaf-age stages) counted from the top of the plants (three measurements per leaf) using SPAD-502 readings (Konica Minolta Sensing Inc., Osaka, Japan). Each measurement was repeated five times. Leaf, stem, and root samples of plants from each of the four treatments were dried at 105˚C for 30 min and then weighed at 70˚C to obtain their dry weight (DW).

Zinc concentration measurement in roots, stems and leaves
The dried samples of roots, stems and leaves were digested with HNO 3 -HClO 4 , and the Zn concentrations in the plants were determined using a TAS-990 hydride-flame atomic absorption spectrometer (Persee Inc., Beijing, China).

Determination of the variation in growth and photosynthetic characteristics
To compare the plant response to Zn stress and HCO 3 − treatment, variations in plant biomass, photosynthetic parameters, Chl fluorescence parameters, and Chl content were calculated according to Eqs (1)(2)(3)(4).
where G is the plant biomass, photosynthetic parameters, Chl fluorescence parameters, Chl content, or Zn concentration of organs under Zn stress and/or HCO 3 − treatment; A 1 is the influence of HCO 3 − treatment under +Zn conditions, and the control is the +Zn0 treatment (Eq (1)); A 2 is the influence of HCO 3 − treatment under −Zn conditions, and the control is the −Zn0 treatment (Eq (2)); A 3 is the influence of Zn deficiency under no HCO 3 − conditions, and the control is the +Zn0 treatment (Eq (3)); and A 4 is the influence of Zn deficiency under HCO 3 − treatment, and the control is the +Zn10 treatment (Eq (4)).

Plant biomass and Chl content
Under +Zn conditions, the biomass of all four plant species increased with the presence of HCO 3 − (+Zn10 treatment) (Fig 1A and 1B). The addition of HCO 3 − inhibited the Chl content in all four plant species (Fig 1C). Compared to those under +Zn0 treatments, Ma and Bp showed maximum and minimum increases in aboveground (underground) biomass of 43.16% (37.96%) and 37.55% (18.90%), respectively. As observed in  (Fig 1). Under HCO 3 − -treatment conditions, Zn deficiency had a significant inhibitory effect on biomass, which had the greatest inhibitory effect on the aboveground biomass of Ma and the underground biomass of Bn (Fig 1A and 1B). Compared to those in the +Zn10 treatment, the decreases in Ma in the aboveground biomass and Bn in the underground biomass were 58.81% and 60.43%, respectively. The Chl content decreased much more in the two Brassicaceae plants than in the two Moraceae species. Bp showed a small increase in Chl content (Fig 1).

Chl fluorescence and photosynthetic parameters
Under +Zn conditions, HCO 3 − addition increased Fo and decreased P N , g s , Fv/Fm, and ETR.  (Figs 2 and 3).

Zn concentration in plant organs
As shown in Table 1, the Zn concentration in four plant organs significantly decreased with Zn deficiency and excess HCO 3 − . The Zn concentration in the organs of two Moraceae plants was significantly higher than that in the two Brassicaceae plants.
Under +Zn conditions, the Zn concentration of organs from all four plant species decreased in the presence of HCO 3 − (+Zn10 treatment), except for that of the roots and stems of Ov.
There was a significantly greater decrease in Zn concentration in the organs of the two Moraceae plants than in those of the two Brassicaceae plants. , and the Zn concentration decrease in the underground parts (roots) in all four species was greater than 45%, particularly in the roots of Ma, where the Zn concentration decreased by as much as 55.65%. The Zn concentration decrease in the aboveground parts (leaves and stems) of all four species was greater than 55%, particularly in the leaves and stems of Bn, where the Zn concentration decreased by as much as 69.71% and 66.61%, respectively (Table 1).

Response of plant growth to excess HCO 3 − and/or Zn deficiency
As shown in Fig 4, excess HCO 3 − influenced plant growth; excess HCO 3 − results in an intracellular ion charge imbalance through passive diffusion, thus directly inhibiting plant growth [2]. It also inhibited plant growth indirectly through increased pH, which led to a decrease in available nutrient elements, such as Zn, iron, and copper, and then inhibited plant growth via two pathways. In the first pathway, excess HCO 3 − initially appeared in all plants as chlorosis related to nutrient element deficiency [24], a considerable decrease of photosynthetic capability was associated with the lower Zn contents [25], followed by the inhibition of PS II activity and decreased photosynthetic parameters, such as P N and WUE, and Chl fluorescence parameters, such as Fo, Fv/Fm, and ETR. In the second pathway, excess HCO 3 − decreased the Zn concen-  Zn deficiency decreased the Zn concentration in plant tissue and then inhibited the activity of CA, a Zn-containing enzyme that catalyzes the reversible reaction between CO 2 hydration and HCO 3 − dehydration (Fig 4). Low CA activity resulted in a slight conversion of HCO 3 − into CO 2 and H 2 O under Zn deficiency [27,28]. As such, Zn deficiency inhibited the plant HCO 3 − -use capacity and PS II activity due to the presence of excess HCO 3 − . Meanwhile, Zn deficiency inhibited other Zn-containing enzymes, such as alcohol dehydrogenase and glutamate dehydrogenase, thus inhibiting plant growth [29]. . In addition, the activity of CA in Bp was about 2 times higher than that in Ma on either an sunny days or a cloudy days in Karst soils [19]. Bp had a significantly greater CA activity, at least five times greater than that in Ma under 10 mM bicarbonate treatments in hydroponically culture [3]. High concentrations of bicarbonate decreased the photosynthetic assimilation of inorganic carbon in Bp and Ma in none bicarbonate treaments [3]. Therefore, the growth of Bp increased under these conditions. Bp had a greater HCO 3 − -use capacity than did Ma [3,26]. Although there was a decrease in P N in Bp, the Zn concentration was greater than that in Ma, and the growth of Bp was stimulated because of the considerable HCO 3 − -use capacity under excess HCO 3 − conditions. The conclusion is identical with our previous research conclusions in the field soil cultivations a higher CA activity of Bp supplied both water and CO 2 for the photosynthesis of mesophyll cells [3,19]. However, we illuminated Zn deficiency or excess bicarbonate, or both as the crucial factors influenced bicarbonate-use capacity, the photosynthetic response to excess HCO 3 − or Zn  , the Ai of biomass, P N (WUE, g s ) and ETR (Fv/Fm, Chl content) were more than 20%, 0 and 0, respectively, when the criterion index was # # # # #; 5~20%, -15~0%, and -10~0%, respectively, when the criterion index was # # # #; -15~5%, -30~-15%, and -20~-10%, respectively, when the criterion index was # # #; -30~-15%, -30~-45%, and -30~-20%, respectively, when the criterion index was # #; and less than -30%, -45% and -30%, respectively, when the criterion index was #. Under Zn deficiency, the Ai of biomass, P N (WUE, ETR, Fv/Fm) and Chl content were more than -10%, -15% and 0 when the criterion index was # # # # #; -20~-10%, -25~-15%, and -10~0, respectively, when the criterion index was # # # #; -30~-20%, -35~-25%, and -20~-10%, respectively, when the criterion index was # # #; -40~-30%, -45~-35%, and -30~-20%, respectively, when the criterion index was # #; and -40%, -45% and -30%, respectively, when the criterion index was #. -use capacity and was also influenced by the photosynthetic rate and inorganic carbon-use capacity [30]. The smallest decrease in the WUE in Bp indicated that Bp had a greater capacity for HCO 3 − use than did the other three species, and the Zn concentration in the organs of Bp was the highest among the four species. Thus, Bp showed the smallest decrease in growth, and the organ activity of Bp was higher than that in the other three species. Although the P N of Bn slightly decreased, the PS II ETR, g s and Zn availability in Bn organs were severely inhibited. Bn also did not adapt well to excess HCO 3 − under Zn deficiency. As a result, growth, particularly root growth, was inhibited. Thus, Bp showed the greatest capacity of all four plant species to resist excess HCO 3 − under Zn deficiency ( Table 2). Few substrates compensate for photosynthesis under the dual influence of Zn deficiency and HCO 3 − treatment. Therefore, the interaction of Zn deficiency and HCO 3 − treatment (−Zn10 treatment) severely inhibited growth, photosynthesis, Zn accumulation in plant organs and electron transport. The photosynthesis of Bp was least affected, and the Zn concentration in the Bp organs was the highest, indicating that Bp might have a greater capacity for inorganic carbon use and resistance to Zn deficiency than the other species that were tested. Bp also grew the most rapidly. Bn had the greatest inhibition in photosynthesis and PS II reaction center activity, indicating that it had the weakest resistance to Zn deficiency and HCO 3 − addition (−Zn10 treatment) ( Table 2).

Conclusions
Four plant species showed different photosynthetic responses to Zn deficiency, HCO 3 − treatment, or both. Bp showed the greatest adaptability to HCO 3 − treatment, Zn deficiency, or both, which involved the greatest HCO 3 − -use capacity. Bn was sensitive to Zn deficiency, HCO 3 − treatment, or both, due to the great inhibition of photosynthesis and PS II reaction center activity. In summary, the plants had different adaptive modes in response to Zn deficiency, HCO 3 − treatment, or both. According to this research and the previous studies we suggested that Bp has the potential to be a pioneer species for ecological restoration in environments with Zn deficiency and excess HCO 3 -, such as karst regions.
Supporting Information S1 Table. The original data of plant biomass, Zn concentration, chlorophyll contents and chlorophyll fluorescence and photosynthetic parameters of two plants. (XLS)