Establishment and Application of a High-throughput Screening Method for Salicylic Acid Metabolic Mutants in Rice

doi:10.11983/CBB24148

Abstract

Abstract: INTRODUCTION Salicylic acid (SA) plays an important role in the plant immune system. The quantitative analysis of SA in plants is fundamental to studying SA metabolism and its biological functions. Although high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC/MS) are widely used for SA determination, their low throughput limits their suitability for large-scale analysis. However, the SA biosynthetic pathway in rice is not well understood, highlighting the need for efficient methods to screen SA-related mutants and elucidate SA metabolic pathways. RATIONALECurrent methods for measuring endogenous SA levels, such as HPLC and LC/MS, involve labor-intensive sample preparation, making them unsuitable for high-throughput analysis. While a lux gene-based SA biosensor has been successfully used in tobacco and Arabidopsis, a reliable and efficient method for SA detection in rice remains unavailable. To address this problem, we optimized sample processing and operational workflows to enable high- throughput SA quantification in rice plants.RESULTS We developed a streamlined, high-throughput method for SA quantification in rice, eliminating time-consuming steps such as sample weighing, tissue grinding, and centrifugation. This approach significantly simplifies the process while maintaining efficiency and accuracy. We validated the method’s feasibility using published rice SA metabolic mutants. We then applied it to screen a Cobalt-60 induced rice mutant library, identifying mutants with altered SA metabolism. Endogenous SA levels in these mutants were confirmed using HPLC. The results demonstrate the method’s effectiveness in screening SA-related metabolic mutants, providing a valuable tool for studying SA metabolism and its roles in rice and other crops. The method was validated using known SA genetic materials. SA content-altered mutants were successfully isolated for further research.CONCLUSION This study establishes a rapid and cost-effective method for measuring SA content in rice tissues using the SA biosensor Acinetobacter sp. ADPWH_lux. Given the pivotal role of SA in plant defense, our method adopts streamlined sampling process, requiring only leaf clipping and boiling in LB medium, and dramatically reduces the time and effort associated with tissue collection and processing. This high-throughput approach is well-suited for large-scale screening of greenhouse-grown or hydroponic plants, providing a powerful platform for advancing research on SA metabolism and its biological functions in crops.
Modified manipulating process for high-throughput determination of salicylic acid (SA) content using SA biosensor Acinetobacter sp. ADPWH_lux strain in rice

Key words: salicylic acid assay, biosensor, high throughput method, salicylic acid metabolism mutant, rice

Can Ye, Linbo Yao, Ying Jin, Rong Gao, Qi Tan, Xuying Li, Yanjun Zhang, Xifeng Chen, Bojun Ma, Wei Zhang, Kewei Zhang. Establishment and Application of a High-throughput Screening Method for Salicylic Acid Metabolic Mutants in Rice[J]. Chinese Bulletin of Botany, 2025, 60(4): 586-596.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB24148

https://www.chinbullbotany.com/EN/Y2025/V60/I4/586

Figures/Tables 6

Figure 1 Standard curve of luminescence of Acinetobacter sp. ADPWH_lux strain Equation: Y=235621X-77045, Coefficient of determination: R2=0.9723. Data are means ± SD (n=3).

Figure 2 Modified manipulating process for high-throughput determination of salicylic acid (SA) content using SA biosensor Acinetobacter sp. ADPWH_lux strain in rice (adapted from Marek et al., 2010) (1) Rice was hydroponic for about 3 weeks in 96-well PCR plates (The bottom of tube was cut) (As controls, 3 wild type,1 oss5hDM/NIP and S5H1-OE plants were added to each plate); (2) Took two leaves of 5 cm (about 0.015 g) from each rice seedling; (3) Kneaded the leaf into a 2 mL 96-well plate with 600 μL LB, and the positions of the 96-well PCR plate and the 96-well 2 mL plate were one-to-one corresponding; (4) The 2 mL 96-well plate was bathed in water at 95°C for 30 minutes (the lid of the 96-well plate should be re-closed every 5 minutes, because high temperature would make the lid open); (5) After the leaf extract liquid from the 2 mL 96-well plate was cooled to room temperature, 50 μL of the extract liquid was pipetted into the black enzyme-labeled plate that had been added with 50 μL biosensor in advance, and the position of the enzyme-labeled plate was one-to-one corresponding to the 96-well 2 mL plate (two plates were repeated for each rice seedling in each experiment); (6) Incubation at 37°C in a incubator for 1.5 hours in the dark; (7) The relative luminescence value was measured with an enzyme-linked immunodetector (each enzyme plate needs to be measured twice).

Figure 3 The salicylic acid (SA) luminescence (A) and SA content (B) of wild type (NIP), oss5hDM/NIP and S5H1-OE Rice plants were cultured in greenhouse, and the culture conditions were the same. Data are means ± SD (n=3). The statistical significances were calculated by t-test, * P<0.05; **P<0.01

Figure 4 The salicylic acid (SA) luminescence of all rice on the 96-well PCR plate (A) and SA luminescence in the selected candidate mutants (B) Data are means ± SD (n=3). Statistical significance was calculated by t-test, * P<0.05; ** P<0.01; *** P<0.001

Table 1 The salicylic acid (SA) concentration corresponding to the SA luminescence of mutant in the standard curve and estimated SA content in rice leaves

No.	Sample name	Luminescence	SA concentration in extracting solution (nmol·mL^-1)	Estimated SA content in rice leaves (nmol·g^-1)
1	NIP	240906.67±33164.52	1.35±0.14	53.98±5.63
1	41-4A	373098.67±23587.55**	1.91±0.10*	76.42±4.00*
2	NIP	251577.33±48090.31	1.39±0.20	55.79±8.16
2	133-3H	7778.33±1563.11***	0.36±0.01***	13.21±0.27***
3	NIP	253330.67±50076.11	1.40±0.21	56.09±8.50
3	134-6D	21748.67±2827.30**	0.42±0.01**	16.77±0.48**
4	NIP	232799.67±29917.16	1.32±0.13	52.60±5.08
4	144-5B	44528±4907.68***	0.52±0.02***	20.64±0.83***
5	NIP	232799.67±29917.16	1.32±0.13	52.60±5.08
5	144-5F	72798.67±7982.44***	0.64±0.03***	25.44±1.36***

Figure 5 Histogram of salicylic acid (SA) content in wild type (NIP) and 5 SA rice mutant plants FW: Fresh weight. Data are means ± SD (n=3). The statistical significance was calculated by t-test, **P<0.01; ***P<0.001

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