An assay for assessing 1-aminocyclopropane-1-carboxylate malonyl (MACC) transferase (AMT) activity and its regulation by ethylene

IF 4.2 2区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Plant Science Pub Date : 2025-01-30 DOI:10.1016/j.plantsci.2025.112401

J. Pattyn , M. Geerts Danau , D. De Ruysscher , S. Roden , T. Snoekx , J. Masschelein , J. Vaughan-Hirsch , B. Van de Poel

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引用次数: 0

Abstract

Background

N-malonyl 1-aminocyclopropane-1-carboxylic acid (MACC) is a major conjugate of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and may therefore play an important role in regulating ethylene production, as well as ethylene-independent ACC signalling. While the enzyme responsible for this derivatization, ACC malonyltransferase (AMT), has been studied in the past, its identity remains unknown. Methods to assay AMT activity are not well established, and no standardized assay has been described.

Results

We optimized an AMT activity assay and investigated the biological implications of AMT. This assay can be divided into three parts: total protein extraction, in vitro AMT activity assay, and MACC detection. For these three parts, different parameters were optimized and combined into an integrated and robust protocol. We used gas chromatography for the indirect detection of MACC, which was compared to a direct LC-MS approach, indicating that the GC-based method is a good alternative readily available to most labs studying ethylene. Next, we used this in vitro AMT activity assay to study the biological function of MACC formation. We observed an ontogenetic, tissue-specific and an ethylene-mediated feedback effect on AMT activity in tomato and Arabidopsis. The feedback of ethylene on AMT activity seems to be important to regulate ethylene production levels.

Conclusions

The optimized and robust AMT activity assay presented here will enable other plant researchers to investigate the biochemistry of the ethylene biosynthesis pathway through ACC conjugation into MACC. Our AMT activity method was deployed both in tomato and Arabidopsis, and revealed that AMT activity is tightly controlled by ethylene itself in a tissue-specific way.

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来源期刊

Plant Science 生物-生化与分子生物学

CiteScore

9.10

自引率

1.90%

发文量

322

审稿时长

33 days

期刊介绍： Plant Science will publish in the minimum of time, research manuscripts as well as commissioned reviews and commentaries recommended by its referees in all areas of experimental plant biology with emphasis in the broad areas of genomics, proteomics, biochemistry (including enzymology), physiology, cell biology, development, genetics, functional plant breeding, systems biology and the interaction of plants with the environment. Manuscripts for full consideration should be written concisely and essentially as a final report. The main criterion for publication is that the manuscript must contain original and significant insights that lead to a better understanding of fundamental plant biology. Papers centering on plant cell culture should be of interest to a wide audience and methods employed result in a substantial improvement over existing established techniques and approaches. Methods papers are welcome only when the technique(s) described is novel or provides a major advancement of established protocols.