Establishment of the rapid and accurate detection method for acid invertase activity from red pitaya

【Objective】Sucrose, glucose and fructose are major and important types of soluble sugar in high plants. Acid invertase (AIN) containing vacuole invertase (VIN) and cell wall invertase (CWIN), degrades sucrose into glucose and fructose irreversibly in vacuoles and extracellular space, respectively. Sucrose metabolism mediated by AINs plays vital roles in plant growth and development, yield and quality formation, biotic and abiotic stress resistance. The enzyme activity determination of AINs is a prerequisite for studying their physiological functions. Currently, yeast complementation assay offers advantages such as simple and no need for instruments, making it the most commonly used approach for AINs activity detection. However, yeast complementation method has defects: (1) judge qualitatively only, and misjudge when vector control grows weakly; (2) time- consuming, usually needs 48- 96 hours; (3) low efficiency. Aiming to limitations of yeast complementation assay, this study established a rapid and accurate method for detecting AIN activities by yeast extracellular acidification in vivo. 【Methods】Using 0.006% Bromocresol purple (BCP) as the acidification indicator, color changes under a series of different pH values were investigated. Then, absorption curves of BCP solutions under the wavelength range of 350-650 nm were scanned by microplate spectrophotometer (Multiskan GO, Thermo Fisher, USA). Under different pH values, the absorbance value A432 and A488 at the wavelength of 432 nm and 488 nm were obtained, respectively. Then, R432/488 values (A432/A488) at different pH values were generated. The linear fitting between pH and R432/488 values was conducted by Excel 2007 software, and the linear function was obtained for the convertion of R432/488 to pH value. Using exogenous invertase enzyme to supplement sucrose degradation defect of invertase deficient baker's yeast (Saccharomyces cerevisiae) SEY2102 strain, color and pH changes with BCP as indicator were investigated, which was used to explore the feasibility of detecting INV enzyme activity based on extracellular acidification. Using BCP as the indicator, extracellular color and pH value changes of yeast expressing red pitaya AIN gene HpVIN1 under control of expression vector pDR196 was investigated with 2% glucose and 2% sucrose as the sole carbon source, respectively. The influence of culture time (0, 2, 4 and 6 h), yeast OD600 value (0, 0.3, 0.6 and 1.2) and sucrose concentration (0%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1.0% and 2.0% ) on pH changes was studied. Yeast single clones expressing red pitaya AIN genes HpVIN1, HpVIN4 or HpCWIN6 after screened by the solid medium, were selected for culturing by using 2% sucrose as the sole carbon source. Meanwhile, the extracellular acidification was detected, which was used for simplifying the procedure and testing the possibility of high throughput detection.【Results】At the pH range of 4.00-8.00, BCP solution color changed from yellow to purple as the pH value increase. There were two obvious light absorption peaks at λ=432 nm and λ=589 nm, respectively, and A488 value was extremely stable at λ=488 nm. Under the pH range of 4.50-8.00, R432/488 showed negative linear correlation with pH value. The equation between R432/488 and pH value was: R432/488= (-0.718 7)×pH+6.043 6, R2 =0.988 3. Under glucose as carbon source, culture solution color of yeast mutant (adding exogenous INV enzyme or not) changed from purple to yellow and pH value decreased by 1.90±0.01 and 1.94± 0.02, revealing that yeast cells could utilize glucose for inducing extracellular acidification. Using sucrose as carbon source, culture solution color of yeast mutant kept purple, and pH value decreased only by 0.28±0.04, which indicated that the yeast mutant could not degrade sucrose into glucose and fructose. Then, after adding exogenous INV enzyme for yeast mutant, solution color changed from purple to yellow, and its pH value decreased by 1.79±0.03, suggesting that the feasibility of sucrose degrade activity of INV enzyme indicated by extracellular acidification. Under glucose as carbon source, solution color of yeast expressing HpVIN1 or vector control pDR196 both turned yellow after 12 h, and pH values decreased by 2.03±0.02. When using sucrose as carbon source, the color of vector control remained purple and its pH value decreased only by 0.03 ± 0.01 after 24 h; the solution color of yeast expressing HpVIN1 turned yellow after 24 h, and its pH value decreased by 1.93±0.01. After fixed the culture time and sucrose concentration, the pH value decreased almost linearly with the yeast OD600 value increase. Under fixed the sucrose concentration and yeast OD600 value, the pH value also decreased linearly with the culture time extension. When the yeast OD600 value and incubation time are fixed, within the sucrose concentration range of 0- 0.05% , the pH value decreases rapidly as sucrose concentration increases; whereas within the range of 0.5%-2.0%, the pH value decreases gradually with increasing sucrose concentration. The main detection conditions were as follows: the yeast OD600 was 1.0, final sucrose concentration was 0.5%-2.0% and culture time was 24 h. The extracellular acidification of yeast single clones expressing HpVIN1, HpVIN4 or HpCWIN6 from solid medium were directly selected to be cultured and detected at the same time. After incubation of 24 h, the solution color of yeast expressing HpVIN1 and HpVIN4 both changed from purple to yellow, whereas HpCWIN6 and pDR196 both remained purple. The pH value of yeast expressing HpVIN1 and HpVIN4 decreased by 1.93±0.02 and 1.40±0.40, which were significantly larger than that of vector control (0.02±0.01). The pH value of yeast expressing HpCWIN6 decreased only by 0.13±0.25, which was not significantly different from vector control. In conclusion, based on extracellular acidification tests of yeast expression, it proved that enzyme activity results of HpVIN1, HpVIN4 and HpCWIN6 coincided with the previous yeast complementation detection.【Conclusion】Based on the extracellular acidification principle, this study establishes a method for detecting AIN enzyme activity in living yeast cells. Compared to the current used yeast complementation assay, it has obvious advantages: qualitative and quantitative combination, high accuracy, timesaving, simplifying steps and high detection efficiency, which can be used for rapid detection of plant AIN enzyme activity or high throughput screening of AIN mutant libraries.