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Mechanism of calcium lactate facilitating phytic acid degradation in soybean during germination Qianru Hui, Runqiang Yang, Chang Shen, Yulin Zhou, and Zhenxin Gu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01598 • Publication Date (Web): 20 Jun 2016 Downloaded from http://pubs.acs.org on June 28, 2016

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Journal of Agricultural and Food Chemistry

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Mechanism of calcium lactate facilitating phytic acid degradation in

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soybean during germination

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Qianru Hui, Runqiang Yang*, Chang Shen, Yulin Zhou, Zhenxin Gu*

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College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095,

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People's Republic of China

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ACS Paragon Plus Environment


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ABSTRACT: Calcium lactate facilitates the growth and phytic acid degradation of soybean sprouts,

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but the mechanism is unclear. In this study, calcium lactate (Ca) and calcium lactate plus lanthanum

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chloride (Ca+La) treated soybean sprouts to reveal the relevant mechanism. Results showed that

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phytic acid content decreased, the availability of phosphorus increased under Ca treatment. This

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must be due to the enhancement of enzymes activity related to phytic acid degradation. In addition,

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the energy metabolism was accelerated by Ca treatment. Energy status and energy

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metabolism-associated enzymes activity also increased. However, the trans-membrane transport of

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calcium was inhibited by La3+ and concentrated in intercellular space or between cell wall and cell

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membrane, thus Ca+La treatment showed reverse results compared with Ca treatment. Interestingly,

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the genes expression did not vary in accordance with their enzymes activity. These results

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demonstrated that calcium lactate increased phytic acid degradation via enhancing growth,

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phosphorus metabolism and energy metabolism.

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KEYWORDS: soybean sprout, calcium lactate, phytic acid, phosphorus metabolism, energy

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metabolism

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INTRODUCTION

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Soybean (Glycine max L.) products like soybean sprouts and soymilk are popular, because they

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are rich in various nutrients including proteins, polysaccharides, dietary fiber etc. Meanwhile,

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soybeans contain a great deal of phytic acid which is considered as an anti-nutritional factor. Phytic

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aicd can chelate metal cations (e.g., Ca2+, Mg2+, Zn2+) and affect their bioavailability. It is also

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easier to form complex with proteins.

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processing. Seeds normally accumulate phosphorus and store it in the form of phytic acid which

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represents 65%-85% of total phosphorus.

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metabolism which links to cell growth, energy status, inositol phosphate and phosphatidylinositol

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phosphate (PtdInsP) signaling and developmental pathways. 3 Furthermore, phytic acid degradation

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products lower inositol phosphates are known as a large family of signaling molecules, which

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participates in the phosphatidylinositol (PI) cycle, especially for inositol (1,4,5) trisphosphate

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(Ins(1,4,5)P3), it plays a role in intracellular secondary messenger and triggers the release of Ca2+

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from intracellular stores to activate a signaling cascade. 4 Meanwhile, lower inositol phosphates and

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their derivatives have health benefits in the protection against colon cancer, arteriosclerosis, neural

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tissue, and coronary heart diseases. 5

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1

Hence, phytic acid degradation is critical in soybean food

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Hence, phytic acid contributes to phosphorus

Ca2+ can regulate metabolism and accelerate physiological and biochemical reactions during 6

which enhance phytase and phosphatase activities,

7, 8

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seed germination,

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phytic aicd degradation. Regulation of cellular Ca2+ is an essential cell function which involves in

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pumps, secondary transporters, and ion channels.

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which would cause conformational changes of CaM and a series of physiological and biochemical

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reactions by the activation of downstream enzymes. It has been reported that CaCl2 could promote

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and thus facilitating

Ca2+ can also bind with calmodulin (CaM),



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growth and quality of soybean sprouts, 10 while lactic acid and thermal treatments could trigger the

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degradation of phytic acid and increase lower inositol phosphates content of barley.

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calcium lactate might facilitate plant growth and phytic acid degradation. In addition, calcium

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lactate is a good exogenous calcium supplement, due to a neutral taste, very high bioavailability,

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high solubility and high stability in solution. 12

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Therefore,

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Energy status is crucial for plant growth and development. Adenosine triphosphate (ATP) level

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could affect the rate of vital activities directly. Inositol phosphate pathway can provide materials for

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the synthesis of pyrophosphates or pyrophosphate derivatives, while pyrophosphates-inosisol

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phosphate is a high-energy phosphate source for ATP regeneration.

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(H+-ATPase) and Ca2+-Adenosine triphosphatase (Ca2+-ATPase) are ion channels inserted in plasma

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membrane to control H+ and Ca2+ transport, thus their activities could influence energy metabolism

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and Ca2+ function.

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enzymes in mitochondria respiration, their activities could affect energy synthesis and mitochondria

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structure. 14 Ca2+ enhancing phytic acid degradation might linked to energy metabolism. Therefore,

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it is of significance to research energy metabolism during investigating the effect of calcium lactate

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on plant growth and phytic acid metabolism.

13

3

H+-adenosine triphosphatase

Succinic dehydrogenase (SDH) and cytochrome c oxidase (CCO) are key

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However, no one has reported the effect of calcium lactate on phytic acid degradation and the

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internal mechanism. Therefore, this study investigated the activities and genes expression of phytic

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acid degradation-associated enzymes, Ca2+ distribution and energy metabolism to systematically

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reveal the mechanism that low concentration of calcium lactate could effectively degrade phytic

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acid and regulate vital activities.

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MATERIALS AND METHODS


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Plant materials and cultivation conditions. Soybean seeds (Glycine max L., cv. yunhe), were

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provided from Jiangsu Academy of Agricultural Sciences (Nanjing, China) and stored at -20 °C

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before experiments. One thousand seed weight was 161.6 g with a standard deviation of 0.8.

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Calcium lactate pentahydrate, Lanthanum (III) chloride heptahydrate were purchased from Sigma

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Chemical Co. (St Louis, MO, USA). All other reagents were of analytical grade.

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Two hundred of same-sized premium soybean seeds without deterioration were selected for

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every treatment, surface sterilized with 1% (v/v) sodium hypochlorite for 15 min, and then rinsed

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with distilled water 5 times. Soybean seeds were soaked in distilled water at 30 ºC for 6 h. Next, the

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soaked seeds were put into the automatic germination machines (BX801; Beixin Hardware

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Electrical Factory, Zhejiang, China), followed by a 4 days incubation in darkness (30 ºC) to

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germinate with 2 different cultivating solutions: (1) 0.27 mmol/L (mM) calcium lactate (Ca); (2)

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0.27 mM calcium lactate + 5 mM lanthanum chloride (Ca+La), distilled water was set as the control.

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The concentration of calcium lactate has been optimaized in pre-experiment (Figure S1) and 5 mM

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lanthanum chloride was used according to the previous study.

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sprayed automatically once (for 2 minutes) every 1 hour. Soybean sprouts with different treatments

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were collected on the 0 day, the 2nd day and the 4th day respectively for further experiments.

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These cultured solutions were

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Determination of phytic acid, inorganic phosphorus, total phosphorus and phospholipid

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content. Phytic acid content was determined by the method of Ma et al. 16 with minor modifications.

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Five dried soybean sprouts were extracted by 30 mL of 100g/L Na2SO4 (dissolved in 1.2% (v/v)

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HCl) for 2 h at 30 °C, the supernatant was passed through an anion-exchange column filled with

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resin (AG1-X4 resin, 100–200 mesh; Alfa Aesar, Karlsruhe, Germany), 5 mL of collected eluate

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was mixed with 4 mL post-column reagent (0.03% FeCl3 solution + 0.3% sulfosalicylic acid). After



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centrifuged at 3,000×g for 10 min, the supernatant was measured in the absorbance at 500 nm.

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Phytic acid (Sigma, St. Louis, MO, USA) Standard curve used appropriate dilutions of 0.1 mg/mL.

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The results were expressed as micrograms per sprout.

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For inorganic phosphorus content determination, inorganic phosphorus was extracted by the 17

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method of Wilcox et al.

with some modifications. Five dried soybean sprouts were extracted by

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20 mL of 12.5% trichloroacetic acid (TCA) (w/v) containing 25 mM MgCl2 at room temperature

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with gentle shaking overnight. Then they were assayed as described by Chen et al. 18 The assay total

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volume (vol) was 4 mL, including 200 µL supernatant of extract, 1.8 mL ultrapure water (UPW)

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and 2 mL colorimetric reagent (1 vol 3 M H2SO4, 1 vol 0.02 M ammonium molybdate, 1 vol 10%

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(v/v) ascorbic acid and 2 vol UPW). Assays were incubated at 37 °C for 1 h, and the results were

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measured in the absorbance at 820 nm. The final results were expressed as milligrams per sprout.

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Total phosphorus content was determined by inductively coupled plasma optical emission

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spectroscopy (ICP-OES, Optima 2100 DV, Perkin Elmer, USA) following the method of Lasursen

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et al. 19 after microwave digestion with 5 soybean sprouts. The results were expressed as milligrams

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per sprout.

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Phospholipid content was evaluated using 2 g soybean sprouts oil. Petroleum ether (boiling

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point at 30 to 60 °C) can extract the oil in dried soybean sprouts powder, and then collected the oil

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after petroleum ether was evaporated by rotary evaporator. Phospholipid content was measured by

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the method of AOCS Ca 12-55. 20 The results were expressed as milligrams per gram oil.

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Determination of lower inositol phosphates content. IP3, tetraphosphate (IP4) and

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Pentaphosphate (IP5) were determined by high-performance liquid chromatography (HPLC)

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according to Kozlowska et al.

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with minor modifications. Five soybean sprouts samples were


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freeze-dried and ground to powder, followed by a stirring extraction with 20 mL 0.5 M HCl at

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20 °C for 4 h. The supernatant after centrifugation (3,000×g, 10 min) was filtered by

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anion-exchange column filled with resin (AG1-X4 resin, 100–200 mesh; Alfa Aesar, Karlsruhe,

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Germany), inositol phosphates were then washed by 2 mL of 2 M HCl. The eluate was evaporated

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to dryness by N2 blowing, and the residue was dissolved in mobile phase and analysed by HPLC

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(Agilent 1200, Agilent Technologies Co. Ltd., USA) equipped with a G1362A refractive index

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detector (RID). An eclipse XDB-C18 (5 µm particle size, 4.6×250 mm; Agilent Technologies Co.

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Ltd., USA) was used. Methanol, 0.05 M formic acid (1:1, v/v) and 1.3 mL per 100 mL of

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tetrabutylammonium hydroxide as mobile phase. The pH was adjusted to 4.3 by addition of 9 M

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sulphuric acid. The mobile phase was filtered through a 0.45 µm filter (Millipore Corp., Bedford,

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MA, USA). The flow rate was 1 mL/min and injection volume was 20 µL. A standard curve

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prepared with pure IP3, IP4 and IP5 (Sigma Chemical Co., St Louis, MO, USA). The final results

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were expressed as micrograms per sprout.

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Phytase, acid phosphatase, phosphatidylinositol-specific phospholipase C (PI-PLC)

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activity assay. Phytase activity was assayed according to the procedure described by Oomah et al.22

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with minor modifications. Every sample of 5 fresh soybean sprouts was ground with 30 mL ice cold

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buffer (10 mM Tris-HCl, pH 7.0). The crude homogenate was centrifuged at 10,000×g for 20 min,

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the supernatant was collected for enzyme activity measurement. Phytase activity was assayed in a

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solution containing 1mM sodium phytate and 1mM CaCl2 (dissolved in 200 mM sodium acetate

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buffer, pH 5.0), The assay mixture was incubated at 55 °C for 1 h and then measured by a detection

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solution containing 2.5 mM ammonium molybdate, 2 M H2SO4 and freshly prepared analytical

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reagent (AR) acetone (1:1:2, by vol). They were detected in the absorbance at 405 nm. One unit of



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phytase activity was defined as 1 µmol inorganic phosphorus release per minute, and the specific

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activity of phytase was expressed as units per sprout.

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Acid phosphatase activity was assayed according to the procedure described by Viveros et al. 23

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with some modifications. The extraction procedure was similar as phytase activity assay with 5

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fresh soybean sprouts. Each reaction mixture consisted of 0.5 mM p-nitrophenyl phosphate

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(dissolved in 50 mM sodium acetate buffer, pH 5.0) and crude enzyme sample. After 15 min of

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incubation at 30 °C, the reaction was stopped by the addition of 1 M NaOH necessary for color

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development. Acid phosphatase activity was measured at 400 nm by monitering the release of

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p-nitrophenol from p-nitrophenyl phosphate. One unit of acid phosphatase activity was defined as 1

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µmol p-nitrophenol liberated per minute, and the specific activity of acid phosphatase was

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expressed as units per sprout.

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PI-PLC activity was assayed by an assay kit (Maibo biotechnology Co. Ltd., Nanjing, China),

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assay procedure was according to corresponding protocol. Every sample of 5 fresh soybean sprouts

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was ground with 30 mL ice cold buffer (0.01 M phosphate buffered saline, pH 7.2). One unit of

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PI-PLC activity was defined as 1 µmol IP3 liberated per minute, and the specific activity of PI-PLC

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was expressed as units per sprout.

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ATP, Adenosine diphosphate (ADP), and Adenosine monophosphate (AMP) content and

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energy charge (EC) measurements. To analyze energy status, five fresh soybean sprouts were

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ground with 15 mL of 0.6 M perchloric acid. ATP, ADP and AMP content assays used HPLC

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(Agilent 1200, Agilent Technologies Co. Ltd., USA) equipped with a G1314B UV detector at 254

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nm, which was according to the method of Jin et al. 14 ATP, ADP, and AMP content was expressed

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as micrograms per sprout. EC was calculated using the following formula:


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ATP+0.5 × ADP ATP+ADP+AMP

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Energy metabolism enzymes assay. Five fresh soybean sprouts were extracted for enzymes

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assay. The activities of H+-ATPase, Ca2+-ATPase, SDH and CCO were measured using the method

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of Jin et al.

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µmol of phosphorus per minute under the assay condition. One unit of SDH activity was defined as

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an increase of 0.01 of absorbance at 600 nm per minute. One unit of CCO activity was defined as an

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increase of 0.1 of absorbance at 510 nm per minute. The specific activities of 4 energy metabolism

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enzymes were expressed as units per sprout.

EC=

14

One unit of H+-ATPase and Ca2+-ATPase activities were defined as the release of 1

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The detection for the subcellular location of calcium by transmission electron microscope

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(TEM). Calcium location employed antimonate precipitation technique, referring to the method of

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Slocum et al.

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into many 1 mm3 of cubes. Samples were soaked in a fixing agent containing 2% (w/v)

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paraformaldehyde and 1% (v/v) glutaraldehyde (dissolved in 2% (w/v) potassium pyroantimonate)

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for 8 hours to fix. Then they were cut into ultrathin slices (70 nm to 90 nm) by ultramicrotome

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(Leica UC-7, Leica Microsystems Co. Ltd., Germany). They were detected by TEM (Hitachi

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H-7650, Hitachi Group, Japan) and operated at accelerating voltage of 80 kilovolt.

24

with minor modifications. Five fresh soybean cotyledons and hypocotyls were cut

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Gene expression assay (QRT-PCR, Quantitative Real-Time PCR).Total RNA was extracted

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by an E.Z.N.A.TM Plant RNA Kit (Omega, Norcross, GA, USA; R6827-01) according to

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corresponding protocol. Five soybean sprouts were ground with liquid nitrogen, and 100 mg of

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ground sample was used to extract total RNA. First-strand cDNA was synthesized as described by

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yang et al. 25 The PCR amplification was performed using TaKaRa Ex-TaqTM polymerase for target

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genes and reference (Actin). For QRT-PCR analysis, the primers used in this study were shown in



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Table 1. Triplicate quantitative assays were performed on each cDNA with the SYBR®Premix Ex

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TaqTM (TAKARA: RR420A) by the ABI 7500 sequence detection system under manufacturer’s

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protocol (Applied Biosystems, Foster City, CA, USA). The PCR cycling conditions were as follows:

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1 cycle of 95 °C for 30 s, followed by 40 cycles of 95 °C for 3 s, 60 °C for 30 s.

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Statistical analysis. Each tested group in this study was replicated three times. The experiment

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was carried out for twice and got the similar results. The data were from one experiment and

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expressed as the mean ± SD of three replications. All statistical analyses were conducted using

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SPSS 16.0 (SPSS Inc., Chicago, IL, USA). The effects of each treatment and germination time were

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tested by two-way analysis of variance (ANOVA) and Duncan’s multiple range tests. Mean

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differences at p < 0.05 were considered significance.

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RESULTS

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Phytic acid, inorganic phosphorus, total phosphorus and phospholipid content. The

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degradation of phytic acid directly affects phosphorus metabolism in soybean sprouts. The changes

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of phytic acid content under Ca and Ca+La treatments during 4 days germination are shown in

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Figure 1A. Phytic acid degraded constantly under different treatments with germination time. Ca

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treatment had a positive effect on phytic acid degradation. Compared with the control, on the 4th day,

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phytic acid content decreased by 41.21% under Ca treatment while increased by 39.05% under

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Ca+La treatment (p < 0.05). Inorganic phosphorus content of different treatments on the 2nd and the

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4th day all increased significantly compared with those on 0 day (p < 0.05) and showed the converse

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trend compared with phytic acid content (Figure 1B). Total phosphorus content was almost stable at

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the same level (Figure1C). The changes of phospholipid content are shown in Figure 1D, its

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variation trend was similar to inorganic phosphorus content. Its content increased significantly


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under Ca treatment while decreased significantly under Ca+La treatment on both the 2nd and the 4th

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day compared with the control (p < 0.05). These results indicated that Ca treatment had a positive

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effect on the degradation of phytic acid and produced more inorganic phosphorus which contributed

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to the synthesis of other phosphorus-containing materials.

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Lower inositol phosphates content. Phytic acid can be converted into lower inositol

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phosphates, so the contents of lower inositol phosphates are very important for soybean sprouts

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growth. Under Ca treatment, the content of IP3 increased significantly on the 2nd and the 4th day

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germination compared with the control (p < 0.05). However, the content of Ca+La treatment was

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significantly lower than that of the control (p < 0.05) (Figure 2A). IP4 content varied without

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obvious trend, and almost remained stable (Figure 2B). IP5 content had a similar trend with IP3, but

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the content of each treatment on the 2nd day did not show the significant changes (p > 0.05) (Figure

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2C). The contents of IP3 and IP5 under Ca treatment on the 4th day were at the highest value

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compared with that on 0 day. The contents of IP3, IP4 and IP5 had the same trend under Ca+La

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treatment, and their contents on the 2nd day were all at the highest value.

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Phytase, acid phosphatase, PI-PLC activity and genes expression of relevant enzymes.

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Phytase activity, acid phosphatase activity and PI-PLC activity are shown in Figure 3A, 3B and 3C,

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respectively. The variation of phytase activity and acid phosphatase activity had a similar trend.

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Phytase activity and acid phosphatase activity of Ca treatment increased significantly on both the

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2nd and the 4th day compared with the control, while they decreased significantly (p < 0.05) under

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Ca+La treatment. Under Ca treatment, phytase and acid phosphatase activities were much higher

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than those under other treatments. Ca treatment affected PI-PLC activity, and the activity on the 4th

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day was significantly higher than that on the 2nd day (p < 0.05). Moreover, PI-PLC activity on the



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4th day under Ca treatment was also significantly higher than that under Ca+La treatment and the

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control (p < 0.05).

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Phytase gene (PA) (Figure 4A), purple acid phosphatase gene (PAP) (Figure 4B) and histidine

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acid phosphatases gene (HAP) (Figure 4C) expression related to phytic acid degradation are

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examined. Interestingly, the variation trends of these genes expression were completely not in

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accordance with that of their enzyme activities. In terms of the relative expression of PA, PAP and

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HAP, Ca+La treatment resulted in the up-regulation compared with the control while Ca treatment

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caused an opposite result. For PI-PLC gene (PI-PLC), it up-regulated under each treatment with the

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germination time (Figure 4D). However, Ca treatment could not up-regulated PI-PLC significantly

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compared with the control and Ca+La treatment on the 4th day (p > 0.05).

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Changes in energy status. As shown in Figure 5, ATP content was obviously higher than ADP

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and AMP content under Ca and Ca+La treatments. ATP content under Ca treatment was

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significantly higher than that of Ca+La treatment during germination (p < 0.05) (Figure 5A). ATP

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content under Ca treatment was at the summit on the 4th day, whereas under Ca+La treatment and

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the control, the content declined after 2 days germination, so ATP contents of Ca+La treatment and

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the control were at the summit on the 2nd day. ADP content (Figure 5B) did not appear significant

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difference under both Ca and Ca+La treatments compared with the control (p > 0.05). AMP content

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is shown in Figure 5C, its content rose under Ca treatment and the control with the germination

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time, whereas AMP content increased on the 2nd day and then declined under Ca+La treatment (p
0.05). For 4 days germination, the activities of

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H+-ATPase and Ca2+-ATPase increased significantly (p < 0.05) under Ca treatment, while decreased

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significantly (p < 0.05) under Ca+La treatment in comparison with the control. The activities of

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H+-ATPase and Ca2+-ATPase under Ca treatment rose rapidly with the germination time, and

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H+-ATPase and Ca2+-ATPase activities on the 4th day increased by 72.73% and 71.43% compared

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with that on 0 day, respectively. The activities of H+-ATPase and Ca2+-ATPase under Ca+La

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treatment kept increasing for 2 days germination, but after 2 days, both H+-ATPase activity and

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Ca2+-ATPase activities were inhibited and kept stable. Both SDH (Figure 6C) and CCO (Figure 6D)

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activities were at a low level. SDH activity did not significantly change under different treatments

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(p > 0.05). CCO activity under Ca treatment was higher than that under Ca+La treatment on the 4th

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day (p < 0.05), and its activity changed with germination time.

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The variation trends of genes expression of these energy metabolism-associated enzymes were

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also not in accordance with energy metabolism-associated enzymes activities. Ca up-regulated

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H+-ATPase (Figure 7A) compared with the control while Ca+La down-regulated that. H+-ATPase

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expression of different treatments on the 2nd day all stood at the summit, then declined on the 4th day.



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Ca2+-ATPase expression up-regulated consecutively under Ca and Ca+La treatments with

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germination time (Figure 7B). Ca treatment on the 4th day up-regulated Ca2+-ATPase expression by

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4.08-times compared with the control, and by 51.04-times compared with that on the 0 day. SDH-1

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expression of each treatment rapidly up-regulated on the 2nd day, then they down-regulated (Figure

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7C). And SDH-1 expression of Ca treatment reached to the highest level compared with other

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treatments on the 2nd day. For SDH-2 expression (Figure 7D), it had the similar variation trend to

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SDH-1 before the 2nd day. Then SDH-1 expression of Ca+La treatment still up-regulated while other

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treatments down-regulated. CCO-1 expression (Figure 7E) and CCO-2 expression (Figure 7F)

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varied a little compared with other genes expression. CCO-1 expression and CCO-2 expression

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under Ca+La treatment were significantly up-regulated compared with other treatments (p < 0.05).

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Ca2+ and H+-ATPase are ion pumps, their variation trend is similar with their gene expression while

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SDH and CCO were not, which indicated the exogenous Ca2+ was not the main factor that affected

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their activities of these energy metabolism-associated enzymes.

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The subcellular location of calcium and total calcium content. Soybean subcellular location

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of calcium in cotyledons and hypocotyls detected by TEM is shown in Figure 8. It was found that

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the number of calcium precipitation points under Ca treatment was more than that under the control,

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and it could be seen calcium precipitation points in neither cotyledon nor hypocotyl under the

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control. In addition, it seemed that calcium could pass through cell membrane easily under Ca

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treatment, so proteins were surrounded by calcium precipitation points in cells of cotyledon in both

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the 2nd day's and the 4th day's cotyledon pictures. It was also found that the big vacuoles in

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hypocotyl pictures of both the 2nd day and the 4th day, with calcium on their surface or passing

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through their membranes. However, under Ca+La treatment, regardless of germination time and


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soybean tissues, it was obvious that calcium precipitation points concentrated on the middle area

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between cell walls and cell membranes or intercellular spaces. Moreover, more calcium

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precipitation points appeared under Ca treatment and under Ca+La treatment with germination time.

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DISCUSSION Calcium was extensively used for promoting plants growth.

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26

In this study, calcium lactate

294

with low concentration of 0.27 mM was firstly applied to soybean seeds during germination, which

295

had a positive effect on soybean growth and phytic acid degradation (Figure 1A). In addition,

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soybean sprouts had an extremely active growth status under Ca treatment (Figure S2 and Table S3).

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The reason might be, firstly, calcium is an essential element for germination to meet the basic need

298

(e.g., component forms cell wall). Secondly, calcium functions as a second messenger to regulate

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the intracellular reactions. Thirdly, intracellular Ca2+ controls a number of important processes and

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enzymes activity, which associated with seed germination. 6

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In fact, phytic acid degradation also closely linked with soybean sprouts growth status. Plants

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growth and development need supplies of essential elements and materials. Especially for phytic

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acid located in cotyledon is a storage vault of phosphorus, inositol skeletons and some other

304

minerals, it degraded rapidly into small molecules to supply substances and energy for soybean

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sprouts growth. Ca treatment could directly lift intracellular Ca2+ level (Figure 8 and S5), which

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accelerated more physiological and biochemical reactions as well as enzymes activity in soybean

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sprouts, including phytase and acid phosphatase, their activities were enhanced (Figure 3), which

308

resulted in the degradation of phytic acid. In addition, growth hormones content increased under Ca

309

treatment (Figure S4), which indicated that Ca might affect secondary metabolism about hormones.

310

27

As a result, growth-promoting hormones enhanced the growth and development of soybean



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sprouts, then further facilitating the degradation of phytic acid. Thus, Ca treatment notably

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accelerated the process of phosphorus and phytic acid metabolism. Phosphorus is important for cells

313

growth, signaling transduction, energy metabolism, gene transcription and translation, etc. It is a

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component of key molecules such as nucleic acids, phospholipids, and ATP, etc.

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acid degraded, inorganic phosphorus, phospholipid and lower inositol phosphates (IP3, IP4, IP5)

316

content increased (Figure 1 and 2), these materials were metabolic intermediate products existed in

317

phytic acid and phosphorus cycle. Soybean sprouts utilized phytic acid to meet basic needs of

318

phosphorus, and metabolic intermediate products of phytic acid provides raw materials for

319

phosphoinositide (PI) pathway. In eukaryotes, phosphoinositides are essential metabolites as labile

320

messengers regulating cellular physiology while traveling within and between cells.

321

steps couple with Ca2+ in PI pathway and Ins(1,4,5)P3 metabolism. Ins(1,4,5)P3 involved in

322

signaling is generated via the hydrolysis of PtdInsP2 by PI-PLC. In plants, all PLCs are most likely

323

to relate to the mammalian PI-PLC ζ type and regulated by Ca2+. Thus under Ca treatment, PI-PLC

324

activity increased, its gene expression also up-regulated. With more Ins(1,4,5)P3 was produced,

325

Ins(1,4,5)P3 could finally function on regulating Ca2+ release in return. 4 These results indicated that

326

calcium lactate resulted in the synthesis of growth hormones, facilitating the growth of soybean

327

sprouts. Therefore, the availability of phytic acid phosphorus increased, and the degradation of

328

phytic acid further accelerated.

28

Once phytic

9

Their key

329

Phytic acid degradation is also associated with energy metabolism. The most apparent evidence

330

is the variation of respiratory rate which is related to ATP, ADP and AMP level. Soybean sprouts

331

could not synthesize organic compounds by photosynthesis in darkness, they could only consume

332

nutrients by respiration to provide all energy for vital activities, thus respiratory rate, ATP content,


Page 17 of 33


333

and EC increased under calcium lactate (Figure S4 and 5), which meant their energy status

334

enhanced. Ca2+-ATPases mediate efflux of Ca2+ from cytoplasm, Ca2+ channels mediate influx of

335

Ca2+. In plants, Ca2+ transport in isolated membrane vesicles has relied on the P-type Ca2+-ATPase,

336

which is stimulated by CaM and acidic phospholipids, transports one Ca2+ per ATP hydrolyzed, and

337

shows increased levels of the phosphoenzyme in the presence of La3+. 9 The plasma membrane

338

H+-ATPase is an electrogenic pump that directly couples ATP hydrolysis with the vectorial transport

339

of H+ out of the cell and thus creating pH and electrical potential differences across the plasma

340

membrane. SDH is a key enzyme in mitochondria of eucaryote cells, and it is the only enzyme that

341

participates in both the tricarboxylic acid cycle and the electron transport chain. SDH might contain

342

CaM subunit, thus its activity was regulated by Ca2+-CaM.

343

transport chain, which plays a critical role in energy metabolism.

344

enhanced the synthesis of energy and the activities of energy metabolism-associated enzymes,

345

improving the transformation efficiency of ATP, ADP and AMP. Finally, these effects could supply

346

the synthesis of substances containing phosphorus with raw materials and energy, thereby reducing

347

the content of phytic acid in soybean sprouts.

29

CCO is the last enzyme of electron 30

The effects of calcium lactate

348

Interestingly, Ca treatment down-regulated the expression of PA, PAP and HAP (Figure 4). The

349

similar phenomenon was also found in gamma-aminobutyric acid (GABA) pathway of soybean

350

sprouts. 31 The most possible explanation was that Ca treatment could directly lift intracellular Ca2+

351

level (Figure 8 and S5), which accelerated enzyme activities in soybean sprouts, thus, they did not

352

need to up-regulate genes expression to translate more proteins. Gene expression of Ca2+-ATPase

353

and H+-ATPase up-regulated by Ca treatment while SDH and CCO did not show a similar trend.

354

The reason might be that Ca2+-ATPase and H+-ATPase control ion transport across the plasma



Page 18 of 33

355

membrane, thus their gene expressions were up-regulated in order to transport more ions.

356

Meanwhile, due to SDH and CCO are multi-subunit enzymes, the variation of SDH-1, SDH-2,

357

CCO-1 and CCO-2 expression might not represent whole enzymes. In addition, the expression of

358

CaM1, CaM3 and CaM4 up-regulated under Ca treatment (Figure S6), which means Ca2+-CaM

359

complex might have positive effect on some enzymes activity researched in this study. This

360

complex could regulate enzymes activity downstream after CaM combined with Ca2+.

361

LaCl3 negatively affected Ca2+ function and thus inhibited the activities of phytase, phosphatase,

362

PI-PLC and energy metabolism-associated enzymes (Figure 3 and 6). Therefore, in order to

363

alleviate the stress, the genes expression of soybean sprouts up-regulated.

364

9

However,

Germination is one of the most effective biological methods to degrade phytic acid,

32

Ca

365

treatment plus germination had a more positive effect on soybean sprouts growth and phytic acid

366

degradation. However, Ca+La treatment is negative for the growth of soybean sprouts, the reason is

367

that LaCl3 is a Ca2+ channel blocker in cytoplasmic membrane.

368

shown (Figure 8), Ca2+ precipitation points increased compared with the control and they

369

distributed throughout the cell especially in Ca2+ storage organelles (e.g., vacuole) under Ca

370

treatment. But under Ca+La treatment, La3+ blocked Ca2+ transport across plasma membrane, thus

371

Ca2+ were detained in intercellular space or between cell wall and cell membrane, and could not

372

enter in cells. The growth of soybean sprouts and phytic acid degradation were inhibited. As a result,

373

the activity of phytase, acid phosphatase, PI-PLC and energy metabolism-associated enzymes

374

decreased, the content of lower inositol phosphates also decreased. La3+ inhibited the function of

375

Ca2+, for La3+ blocked the trans-membrane function of Ca2+, which indicated that the influx and

376

efflux across plasma membranes of Ca2+ was crucial for phytic acid degradation of soybean sprouts.

33


Therefore, as the TEM images

Page 19 of 33


377

In conclusion, the addition of calcium lactate significantly improved the transfer and absorption

378

of calcium in soybean sprouts. And therefore, on the one hand, calcium lactate resulted in the

379

synthesis of growth hormones, facilitating the growth of soybean sprouts. Meanwhile, the activities

380

of phytase and phosphatase were also improved, thereby improving the availability of phytic acid

381

phosphrous and promoting the degradation of phytic acid. On the other hand, the effects of calcium

382

lactate enhanced the synthesis of energy and the activities of energy metabolism-associated

383

enzymes, improving the transformation efficiency of ATP, ADP and AMP, so that soybean sprouts

384

could make full use of phytic acid phosphorus. Finally, these effects could supply the synthesis of

385

substances containing phosphorus with raw materials and energy, thereby reducing the content of

386

phytic acid in soybean sprouts. In addition, the influx and efflux across plasma membranes of Ca2+

387

was crucial for phytic acid degradation of soybean sprouts (Figure S7).

388

AUTHOR INFORMATION

389

Corresponding Authors

390

Tel/Fax: + 86-25-84396293.

391

E-mail: [email protected] (Runqiang Yang); [email protected] (Zhenxin Gu).

392

Funding source

393

The authors appreciate the finance supported by the National Natural Science Foundation of

394

China (No. 31471596).

395

Notes

396

The authors declare no competing financial interest.

397

ASSOCIATED CONTENT

398

Supporting Information Description:



399

Figure S1. Effect of different calcium lactate concentrations on phytic acid content of soybean

400

sprouts for 4 days germination.

401

Figure S2. Image of the effect of Ca and Ca+La treatment on soybean sprouts growth status.

402

Figure S3. Changes in respiratory rate under Ca and Ca+La treatment.

403

Figure S4. Changes in auxin (IAA, A), gibberellin acid (GA, B), brassinolide (BR, C), cytokinin

404

(CTK, D) content under Ca and Ca+La treatment.

405

Figure S5. Changes in total calcium content under Ca and Ca+La treatment.

406

Figure S6. Changes in relative gene expression of calmodulin1 (CaM1, A), calmodulin2 (CaM2, B),

407

calmodulin3 (CaM3, C), calmodulin4 (CaM4, D), calmodulin5 (CaM5, E) under Ca and Ca+La

408

treatment.

409

Figure S7. Schematic draw of pathway for the discussion of this paper.

410

Table S1. The primers used for QRT-PCR.

411

Table S2. Effect of Ca and Ca+La on length and water content of soybean sprouts.

412

REFERENCES

413

1.

Thavarajah, P.; Thavarajah, D.; Vandenberg, A. Low phytic acid lentils (Lens culinaris L.): a

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9044-9049.

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and phytic acid. J. Plant Physiol. 2001, 158, 489-497.

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Raboy, V.; Young, K. A.; Dorsch, J. A.; Cook, A. Genetics and breeding of seed phosphorus

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Dorsch, J. A.; Cook, A.; Young, K. A.; Anderson, J. M.; Bauman, A. T.; Volkmann, C. J.;

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Murthy, P. P. N.; Raboy, V. Seed phosphorus and inositol phosphate phenotype of barley low

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Munnik, T., Ed.; Springer Berlin Heidelberg: Berlin, Heidelberg, 2010; pp 145-160.

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Im, Y. J.; Phillippy, B. Q. Perera, I. Y., InsP3 in Plant Cells. In Lipid Signaling in Plants,

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fermentation: a 31P NMR study. J. Agric. Food Chem. 2004, 52, 6300-6305.

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Roux, S. J. Calcium-regulated metabolism in seed germination. In Recent Advances in the

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Development and Germination of Seeds, Taylorson, R. B., Ed.; Springer US: Boston, MA,

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Bush, D. S. Calcium regulation in plant cells and its role in signaling. Annu. Rev. Plant Biol. 1995, 46, 95-122.

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11. Metzler-Zebeli, B. U.; Deckardt, K.; Schollenberger, M.; Rodehutscord, M.; Zebeli, Q. Lactic

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acid and thermal treatments trigger the hydrolysis of myo-inositol hexakisphosphate and

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modify the abundance of lower myo-inositol phosphates in barley (Hordeum vulgare L.). Plos

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12. Pathomrungsiyounggul, P.; Grandison, A. S.; Lewis, M. J. Effect of calcium carbonate,

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calcium citrate, tricalcium phosphate, calcium gluconate and calcium lactate on some



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physicochemical properties of soymilk. Int. J. Food Sci. Technol. 2010, 45, 2234-2240. 13. Sanders, D.; Pelloux, J.; Brownlee, C.; Harper, J. F. Calcium at the Crossroads of Signaling. The Plant Cell 2002, 14, S401-S417.

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14. Jin, P.; Zhang, Y.; Shan, T.; Huang, Y.; Xu, J.; Zheng, Y. Low-Temperature Conditioning

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Alleviates Chilling Injury in Loquat Fruit and Regulates Glycine Betaine Content and Energy

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Status. J. Agric. Food Chem. 2015, 63, 3654-3659.

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the protective effect of exogenous calcium on the germinating soybean response to salt stress.

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16. Ma, G.; Jin, Y.; Piao, J.; Kok, F.; Guusje, B.; Jacobsen, E. Phytate, calcium, iron, and zinc

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2005, 53, 10285-10290.

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17. Wilcox, J. R.; Premachandra, G. S.; Young, K. A.; Raboy, V. Isolation of high seed inorganic P, low-phytate soybean mutants. Crop Sci. 2000, 40, 1601-1605. 18. Chen, P. S.; Toribara, T. Y. Warner, H., Microdetermination of Phosphorus. Anal. Chem. 1956, 28, 1756-1758.

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19. Laursen, K. H.; Schjoerring, J. K.; Olesen, J. E.; Askegaard, M.; Halekoh, U.; Husted, S.

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Multielemental fingerprinting as a tool for authentication of organic wheat, barley, faba bean,

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and potato. J. Agric. Food Chem. 2011, 59, 4385-4396.

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20. AOCS. Official Methods and Recommended Practices of the American Oil Chemists’ Society; AOCS Press: Champaign, IL, USA, 2009; Method Ca 12-55. 21. Kozlowska, H.; Honke, J.; Sadowska, J.; Frias, J.; Vidal-Valverde, C. Natural fermentation of


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lentils: influence of time, concentration and temperature on the kinetics of hydrolysis of

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inositol phosphates. J. Sci. Food Agric. 1996, 71, 367-375.

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22. Oomah, B. D.; Luc, G.; Leprelle, C.; Drover, J. C.; Harrison, J. E.; Olson, M. Phenolics, phytic

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acid, and phytase in Canadian-grown low-tannin faba bean (Vicia faba L.) genotypes. J. Agric.

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Food Chem. 2011, 59, 3763-3771.

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23. Viveros, A.; Centeno, C.; Brenes, A.; Canales, R.; Lozano, A. Phytase and acid phosphatase activities in plant feedstuffs. J. Agric. Food Chem. 2000, 48, 4009-4013.

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24. Slocum, R. D.; Roux, S. J. An improved method for the subcellular localization of calcium

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using a modification of the antimonate precipitation technique. J. Histochem. Cytochem. 1982,

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30, 617-629.

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25. Yang, R.; Hui, Q.; Gu, Z. Effects of ABA and CaCl2 on GABA accumulation in fava bean germinating under hypoxia-NaCl stress. Biosci., Biotechnol,. Biochem. 2015, 1-7. 26. Hepler, P. K. Calcium: a central regulator of plant growth and development. Plant Cell 2005, 17, 2142-2155.

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27. Sembdner, G.; Gross, D.; Liebisch, H.-W.; Schneider, G. Biosynthesis and Metabolism of

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Plant Hormones. In Hormonal Regulation of Development I: Molecular Aspects of Plant

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Hormones, MacMillan, J., Ed.; Springer Berlin Heidelberg: Berlin, Heidelberg, 1980; pp

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281-444.

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28. Schachtman, D. P.; Reid, R. J.; Ayling, S. M. Phosphorus uptake by plants: from soil to cell. Plant Physiol. 1998, 116, 447-453. 29. Patel, A.; Ting, I. P. Relationship between respiration and CAM-cycling in Peperomia camptotricha. Plant Physiol. 1987, 84, 640-642.



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30. Millar, A.; Atkin, O.; Lambers, H.; Wiskich, J.; Day, D., A critique of the use of inhibitors to

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estimate partitioning of electrons between mitochondrial respiratory pathways in plants.

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Physiol. Plant. 1995, 95, 523-532.

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31. Yin, Y.; Yang, R.; Guo, Q.; Gu, Z. NaCl stress and supplemental CaCl2 regulating GABA metabolism pathways in germinating soybean. Eur. Food Res. Technol. 2014, 238, 781-788.

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32. El-Adawy, T. Nutritional composition and antinutritional factors of chickpeas (Cicer arietinum

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L.) undergoing different cooking methods and germination. Plant Foods Hum. Nutr. 2002, 57,

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83-97.

495 496

33. Bhattacharjee, S., Calcium-dependent signaling pathway in the heat-induced oxidative injury in Amaranthus lividus. Biol. Plant. 2008, 52, 137-140.

497

Figure captions

498

Figure 1. Changes in phytic acid (A), inorganic phosphorus (B), total phosphorus (C) and

499

phospholipid (D) content under Ca and Ca+La treatment.

500

Figure 2. Changes in IP3 content (A), IP4 content (B) and IP5 content (C) under Ca and Ca+La

501

treatment.

502

Figure 3. Changes in Phytase activity (A), acid phosphatase activity (B) and PI-PLC activity (C)

503

under Ca and Ca+La treatment.

504

Figure 4. Changes in relative expression of PA (A), PAP (B), HAP (C) and PI-PLC (D) under Ca

505

and Ca+La treatment.

506

Figure 5. Changes in ATP content (A), ADP content (B), AMP content (C) and EC (D) under Ca

507

and Ca+La treatment.

508

Figure 6. Changes in H+-ATPase activity (A), Ca2+-ATPase activity (B), SDH activity (C), and


Page 24 of 33

Page 25 of 33


CCO activity (D) under Ca and Ca+La treatment.

510

Figure 7. Changes in relative gene expression of H+-ATPase (A), Ca2+-ATPase (B), SDH subunit-1

511

(C), SDH subunit-2 (D), CCO subunit-1 (E) and CCO subunit-2 (F) under Ca and Ca+La treatment.

512

Figure 8. Subcellular location of calcium in soybean cotyledons and hypocotyls detecting by TEM

513

under Ca and Ca+La treatment.

514

Figures

0.5

(A)

d0

d4

d2

(B)

a a

a a 2

a

a

a

a a

b b

b b

c b

1

0.4

a b

3

0.3

b b a

c c

a

a

b a

b

c

a c b c a

c a

0.1

Total phosphorus content (mg/sprout)

0

0.0

1.2

12

(C) 1.0

a a b b b ab

a a aaaa

a a b b c b

(D)

a

a 10

a b

0.8

8

0.6

6 a

0.4 0.2

a

b b c

b

c a

4 b b a c

a c

0.0

2 0

Control

515

0.2

b

Phospholipid content (mg/ g oil)

Phytic acid content (mg/sprout)

4

Inorganic phosphorus content (mg/sprout)

509

Ca

Ca+La

Treatment

Control

Ca

Ca+La

Treatment

516

Figure 1. Changes in phytic acid (A), inorganic phosphorus (B), total phosphorus (C) and

517

phospholipid (D) content under Ca and Ca+La treatment. Error bars show the standard deviation.

518

The different letters showed the significant differences under treatments (p < 0.05). The significant

519

differences with germination time for 0 day (d 0), 2 days (d 2) and 4 days (d 4) are shown by

520

superscript letters (p < 0.05).



20 IP3 content (µg/sprout)

(A)

d0 d2 d4

15 a b

10

5

0 20

IP4 content (µg/sprout)

a a

15

b c a

a

b

b

c a

a

a a ba b c

a

a b c cb

(B) a a c b c b

a c a ab

10

5

0 20 IP5 content (µg/sprout)

(C) 15

10

a bb ca a

a

c

b a

a

a a c a cb a

5

0 Control

521

Ca

Ca+La

Treatment

522

Figure 2. Changes in IP3 content (A), IP4 content (B) and IP5 content (C) under Ca and Ca+La

523

treatment. Error bars show the standard deviation. The different letters showed the significant

524

differences under treatments (p < 0.05). The significant differences with germination time for 0 day

525

(d 0), 2 days (d 2) and 4 days (d 4) are shown by superscript letters (p < 0.05).


Page 26 of 33

Page 27 of 33


0.030

PI-PLC activity (U/sprout)

Acid phosphatase activity (U/sprout)

Phytase activity (U/sprout)

(A) 0.025

a

a

d0 d2 d4

a b

0.020

a

0.015

b

b b

0.010 0.005

a c b cb a

c a

c a

0.000 50

(B)

a

a

40 30 20

a

10 a

b

a b

c

b a

c a

0 0.035 0.030 0.025

a a

(C) b ab a ab b

a

aa bb c

b ba a

a a cbbc

Ca

Ca+La

0.020 0.015 0.010 0.005 0.000 Control

526

Treatment

527

Figure 3. Changes in Phytase activity (A), acid phosphatase activity (B) and PI-PLC activity (C)

528

under Ca and Ca+La treatment. Error bars show the standard deviation. The different letters showed

529

the significant differences under treatments (p < 0.05). The significant differences with germination

530

time for 0 day (d 0), 2 days (d 2) and 4 days (d 4) are shown by superscript letters (p < 0.05).



Page 28 of 33

2.0

2.0

a

a b

b b c

ba aa a

a

c a

a

b c c b

a

a

b b bc

a a b a c a

a a

b c c

0.5

0.0

Relative expression of HAP

1.5

4

(C) a

a

(D)

a a

a a

a

1.0

b b b b

a

a

a a

a

3

a a

a a a a a b a

b b

a a

a a

b a a

2

1

b c

0.0

0 Control

531

1.0

0.5

0.0

0.5

1.5

Relative expression of PAP

d0 d2 d4

1.5

1.0

(B)

Relative expression of PI-PLC

Relative expression of PA

(A)

Ca

Ca+La

Treatment

Control

Ca

Ca+La

Treatment

532

Figure 4. Changes in relative expression of PA (A), PAP (B), HAP (C) and PI-PLC (D) under Ca

533

and Ca+La treatment. Error bars show the standard deviation. The different letters showed the

534

significant differences under treatments (p < 0.05). The significant differences with germination

535



Page 29 of 33


100

30

80

a ab a b

d0 a a a a

d4

d2

60 40

a

b

a

b

a

a b b c c

(B)

a

a

ab a a a

b

a a

a

a

a

b

a a a

a a

20

15

5 0

20

1.0

(C)

(D)a

15

a

a a a a b

a b

b

10 a

b

c

b

a

aa aa a

a a

a

b

c

c a

a

a a a a a c

0.8 0.6

b

EC

AMP content (µg/sprout)

20

10

0

c

0.4

5

0.2

0

0.0 Control

536

25

ADP content (µg/sprout)

ATP content (µg/sprout)

(A)

Ca

Ca+La

Treatment

Control

Ca

Ca+La

Treatment

537

Figure 5. Changes in ATP content (A), ADP content (B), AMP content (C) and EC (D) under Ca

538

and Ca+La treatment. Error bars show the standard deviation. The different letters showed the

539

significant differences under treatments (p < 0.05). The significant differences with germination

540




Page 30 of 33

d0 d2 d4

0.012 0.009

b a

0.006

a

a

a

(B)

a

b a a a b

b

c a

a

a

b

a

b

a a

a b b

c

a

a 0.015 0.012

b a a ca

a

b

a

0.006

b

0.003

0.003

0.000

0.000

0.06

(C) SDH activity (U/sprout)

0.009

a

a

(D)

a ab

0.05

a

a ab

0.20

a

0.15 0.04

a

0.03 0.02

Ca2+-ATPase activity (U/sprout)

0.018

(A)

a

aa

0.01

a

a a a

a a a a b

a a

a a

a

a

a a

b

a

b

a

0.10

a

a

0.05

a

0.00

CCO activity (U/ sprout)

H+-ATPase activity (U/sprout)

0.015

0.00 Control

Ca

Ca+La

Treatment

541

Control

Ca

Ca+La

Treatment

542

Figure 6. Changes in H+-ATPase activity (A), Ca2+-ATPase activity (B), SDH activity (C), and

543

CCO activity (D) under Ca and Ca+La treatment. Error bars show the standard deviation. The

544

different letters showed the significant differences under treatments (p < 0.05). The significant

545

differences with germination time for 0 day (d 0), 2 days (d 2) 4 days (d 4) are shown by superscript

546

letters (p < 0.05).


80

(A)

(B) d0 d2 d4

30

20

a

a

a

a

a b a b

a

60

b 40 b b

a a b b

10 c a

b a

a a

a

c a

c

b

a a b b

b

b a

b a

Relative expression of SDH-1

0 15

(C) 10

a b

a

a b

a a

b b

a

a

a b

a

a

60

b

a b 40

a c

5 20 b a

c a

b a

a

a a

a

b b 0.5

b

a

b

c

(F)

b a b b c b

a

0 4

a a

(E) 1.0

a

c

c a

c c

a

a b

a b c

c a

b

a a

a b a

b

3

2 c a

0.0

1

0 Control

547

0 80

a

0 1.5 Relative expression of CCO-1

a a

(D) a

20

Relative expression of SDH-2

Relative expression of H+-ATPase

40

Relative expression of Ca2+-ATPase


Relative expression of CCO-2

Page 31 of 33

Ca

Ca+La

Treatment

Control

Ca

Ca+La

Treatment

548

Figure 7. Changes in relative gene expression of H+-ATPase (A), Ca2+-ATPase (B), SDH subunit-1

549

(C), SDH subunit-2 (D), CCO subunit-1 (E) and CCO subunit-2 (F) under Ca and Ca+La treatment.

550

Error bars show the standard deviation. The different letters showed the significant differences

551

under treatments (p < 0.05). The significant differences with germination time for 0 day (d 0), 2

552

days (d 2) and 4 days (d 4) are shown by superscript letters (p < 0.05).



553 554

Figure 8. Subcellular location of calcium in soybean cotyledons and hypocotyls detecting by TEM

555

under Ca and Ca+La treatment. The scale unit is 2 µm.

556 557 558 559 560 561 562 563 564 565 566


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