Written by/The West Wind Blows the Traveler
Editor/Zephyr
(Bermuda grass)
preface
Bermuda grass (Cynodon dactylon L.) is an important warm-season lawn grass variety with good creeping growth and development, laying the foundation for the rapid propagation of Bermuda grass plants through asexual clonal growth. However, the growth and development of Bermuda grass creeping is still poorly understood at the molecular level.
Our study will comprehensively analyze the acetylation and succinylation modifications of proteins in the Bermuda grass variety Yangjiang Creeping Creeping.
Liquid chromatography-tandem mass spectrometry was used to successfully identify 1914 lysine acetylation sites on 4657 proteins and 128 lysine succinylation sites on 226 proteins.
In addition, we found that 78 proteins and 81 lysine sites were simultaneously acetylated and succinylated. Functional enrichment analysis showed that acetylated proteins regulated different responses of carbohydrate metabolism and protein turnover, while succinylated proteins mainly regulated citrate cycles.
These findings partially explain the different growth disorders of Bermuda grass creeping stems treated with sodium butyrate and sodium malonate, which interfere with protein acetylation and succinylation, respectively.
In addition, there were 140 acetylated proteins and 42 succinylated proteins with similarly modified orthologs in other grass species.
Site-specific mutation-binding enzyme activity assays in the study showed that both conserved acetylation of catalase and succinylation of malate dehydrogenase inhibited their activity, further suggesting the important regulatory role of the two modifications.
The growth environment and reproductive characteristics of Bermuda grass
Bermuda grass (Cynodon dactylon L.) is a perennial warm-season lawn grass with great economic value, widely used in the cultivation of lawns in parks, golf courses, sports fields and home lawns in warm regions around the world.
Unlike domesticated cereal grasses such as rice, wheat, maize, sorghum and barley, Bermuda grass plants typically reproduce asexually by cloning regenerated seedlings from the Stallon node.
With this reproductive property, Bermuda grass can spread rapidly in open areas by constantly generating and regenerating creeping, laying the foundation for Bermuda grass's lawn-type varieties to form uniform and aesthetically pleasing lawns.
Over the past few years, high-throughput transcriptomics and proteomics studies have revealed the critical role of metabolic regulation in specialization and creeping growth in the growth of Bermuda grass creeping.
For example, comparative transcriptome analysis showed that lignin biosynthesis is impaired in creeping wild germplasm compared to upright wild germplasm. Comparative proteomics and enzymatic analysis showed that starch accumulation was highly active in creeping and glycolytic activity was more prominent in buds.
Compared to underground-grown stolons, aboveground-grown stolons have high levels of chlorophyll and high expression of enzymes involved in photosynthesis, suggesting that creeping stems may act as additional photosynthetic organs. However, the detailed molecular mechanisms that regulate the different metabolic responses of Bermuda grass are still poorly understood.
In the current study, we performed a global identification of protein lysine acetylation and succinylation modifications in fast-growing Bermuda grass creeping. A total of 1914 lysine acetylation sites on 4657 proteins and 128 lysine succinylation sites on 226 proteins were identified.
The characteristics and functions of the two PTMs in the creeping stems of fast-growing Bermuda grass were systematically analyzed, and the effects of the two PTMs on the activities of key enzymes were evaluated.
The results of this study not only provide the first comprehensive understanding of acetyl and succinin in turfgrass, but also expand the understanding of metabolic regulation in fast-growing plant organs.
Interference with protein lysine acetylation regulates the growth of Bermuda grass
Bermuda grass is a warm-season lawn grass with a variety of applications. Through rapid growth and constant differentiation of creeping growth, Bermuda grass plants can reproduce asexually.
Over the past few years, many exogenous small molecules have been found to affect the growth and development of Bermuda grass plants under normal and stress conditions.
Melatonin has also been found to alleviate H2O2-regulated plant growth, cell damage, and reactive oxygen species accumulation in Bermuda grass. In addition, both the protein phosphatase inhibitor NaF and the protein kinase inhibitor Astrosporine inhibit normal growth and starch accumulation in Bermuda grass.
In this study, it was also observed that the histone deacetylase inhibitor sodium butyrate and the succinate dehydrogenase inhibitor sodium malonate regulated the growth of Bermuda grass plants, especially in creeping animals.
Interestingly, both salts promoted creeping growth at low concentrations (10 mM) but inhibited creeping growth at high concentrations (50 mM), consistent with the toxic effect of high concentrations of malonic acid on Arabidopsis seedling growth.
In addition, sodium butyrate affects both soluble sugar and protein content, while sodium malonate has little effect on soluble protein content.
Since Bermuda grass cultivar, Yangjiang, is a salt-tolerant variety that maintains normal growth under 50 mM NaCl treatment, these results suggest that sodium butyrate and sodium malonate have different functions in regulating creeping growth in Bermuda grass, possibly by regulating acetylation and succinylation modifications of essential proteins, respectively.
Succinylation modification
We have performed acetyl and succinyl analysis experiments in different plant species. In these studies, as many as 3179 acetylated proteins were identified in paper mulberry, while the maximum and minimum amounts of succinylated proteins in rice and strawberries were reported to be 2593 and 116, respectively.
In this study, 1914 acetylated proteins and 128 succinylated proteins were successfully identified in fast-growing Bermuda grass creeping.
It is worth noting that the amount of succinylated protein is much lower than the amount of acetylated protein.
Although the immunoaffinity enrichment efficiency of the two PTMs may be different, resulting in differences between the two identification results, considering that the amount of succinylated protein identified in paper mulberry trees is also much lower than that of acetylated proteins, the uneven distribution of the two PTMs inherently in Bermuda grass creeping stems cannot be ruled out.
The mean acetylation and succinylation sites for each protein in Bermuda grass creeping were 21.2 (43/4657) and 1914.1, respectively. These figures are comparable to the results for other plant species.
For example, the average acetylation site for each protein in soybean leaves was 128.2 (05/3148), while the average succinylation site for each protein in Oliver alder was 16.1 (79/653).
Notably, in Bermuda grass creeping, two proteins were identified as having more than 28 succinylation sites. This result further supports the suspicion that protein lysine acetylation and succinylation may be present in Bermuda grass creeping at different frequencies, as multiple succinylated peptides of a single protein are rarely obtained from inefficient immunoaffinity enrichment processes.
In addition, amino acid composition analysis showed that tyrosine, glutamate, and lysine were often distributed around the acetylated lysine site, and isoleucine, phenylalanine, and glutamate were often distributed around the bermuda grass creeping ketone succinylated lysine site.
Similar amino acid compositions have been found in other large-scale acetylation and succinylation identification studies. For example, tyrosine and lysine residues are frequently detected around acetylated lysine sites in P. asperata, while glutamate is frequently detected around succinylated lysine sites in C. cathayensis and D. officinale.
Together, these results suggest that there may be a conserved mechanism in different plant species to add and remove acetylation and succinylation modifications of specific proteins.
Given that protein acyltransferases and succinyltransferases remain unidentified in plants, biased non-enzymatic modification of lysine residues surrounded by specific amino acids may be a possible mechanism.
Identification of lysine acetylated and succinylated proteins in Bermuda grass creeping
Although large-scale acetyl and succinyl studies have been successfully performed in several plant species, the detailed regulatory functions of specific acetylation and succinylation modifications have been characterized in only a few plants.
For example, acetylation at K407 and K425 increases the enzymatic activity of long-chain acyl-CoA synthase in Pseudomonas trigonumas.
The acetylation of 172 lysine sites decreased the oxaloacetic acid reduction activity of MDH protein in Arabidopsis, while acetylation of MDH protein at K32 increased its activity in Arabidopsis.
Succinylation of K55 reduces the enzymatic activity of CAT protein in rice, while succinylation of K241 or K10 increases the enzymatic activity of glutathione S-transferase protein in rice.
In this study, we expressed and purified wt and acetylated/deacetylated-mimicking CAT proteins and wt and succinylating/desuccinylation-mimicking MDH proteins. Enzymatic assays showed that both acetylation of CAT at K332 and succinylation of MDH at K7 reduced their enzymatic activity.
Considering that these results were obtained from purified proteins in vitro, the decrease in enzymatic activity of the acetylated mimicking CAT protein and the succinylation-mimicking MDH protein may be caused only by changes in protein charge, while deacetylation/desuccinylation mimicking mutations have little effect on enzyme activity because the charge of both proteins remains constant.
Interestingly, both modification sites are preserved in the fast-growing organs of three types of grasses. Previous studies have shown that Arabidopsis mutant plants lacking mitochondrial MDH protein exhibit small, slow-growing, and impaired growth phenotypes after seed germination.
Arabidopsis catalase triple mutants also exhibit severe redox disorders and growth defects, implying that these two enzymes play an important role in rapid organ growth.
The results of this study further show that the activities of the two enzymes can be regulated by conserved acetylation and succinylation, respectively, which provides new ideas for the dynamic metabolic regulation in plant growth and development.
Our analysis successfully identified thousands of lysine acetylation modifications and hundreds of lysine succinylation modifications in the rapidly growing creeping vegetation of Bermuda grass, however, many acetylated proteins and succinylated proteins that may have important functions may not be identified because of their low abundance or poor reactivity with affinity antibodies.
On the other hand, many studies have shown that both acetylation and succinylation modifications exist in very low stoichiometry.
In addition, experimental evidence further suggests that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is acetylated in Arabidopsis thaliana with a very low stoichiometry and cannot meaningfully affect its enzymatic activity in vivo, and a modest increase in acetylation levels does not significantly alter its maximum activity.
Considering that purified acetylated/deacetylated mimicking CAT protein and succinylating/desuccinylation-mimicking MDH protein can only simulate 100% and 0% stoichiometry of both proteins by acetylation and succinylation modifications, the actual effect of the two modifications on enzyme activity in vivo may be weaker than the results of in vitro enzymatic assays.
In the future, we will not only further explore unknown acetylation and succinylation modifications using state-of-the-art mass spectrometry technology and high-affinity antibodies.
Also use isotope-labeled peptide standards to determine the stoichiometry of modifications of interest to obtain their actual biological function.
conclusion
In summary, acetylated proteins with 4657 lysine acetylation sites and 226 succinylated proteins with 128 lysine succinylation sites were first identified in Bermuda grass creeping in 1914.
Acetylated and succinylated proteins are located in different cellular compartments, forming complex networks of interactions and participating in different cellular processes.
Conservative acetylation of catalase and succinylation of malate dehydrogenase further reveal reduced enzyme activity in vitro, providing preliminary evidence for the regulation of acetylation and succinylation-mediated growth of Bermuda grass Storon at the biochemical level.
In the future, using existing genomic resources and genetic transformation techniques, the detailed physiological functions of specific acetylation and succinylation modifications in Bermuda grass plants can be elucidated.