In the ever-evolving landscape of agricultural research, the spotlight shines brightly on the dynamic interplay between plants and their environment. As terrestrial organisms, plants contend with an array of stressors, from extreme temperatures to drought and, significantly, high salinity. The latter, in particular, disrupts the natural distribution of crops, leading to substantial yield losses in agricultural ecosystems.
A crucial revelation in plant science is the intricate web of responses orchestrated by multiple genes and metabolic pathways when faced with abiotic stress. This complexity poses a challenge, as enhancing plant resilience to external stressors requires a holistic understanding that goes beyond the regulation of individual genes or proteins.
Enter proteomics, a powerful tool in unraveling the molecular intricacies of plant stress responses. By delving into the world of proteins and their dynamic expressions, proteomics provides a comprehensive view of how plants navigate the challenges posed by high salinity and other stressors. Specifically, in the context of salinity stress, proteomic analyses shed light on the differential abundance of proteins, unveiling key players in the plant's adaptive strategies.
Proteomic techniques, such as nano liquid chromatography-tandem mass spectrometry (nano LC-MS/MS), offer a lens into the intricate dance of proteins within plant cells. This analytical approach allows researchers to identify and quantify glycoproteins, among other protein modifications, providing insights into how these molecular players contribute to salt stress tolerance.
As demonstrated in recent studies on Arabidopsis thaliana, a model organism in plant research, glycoproteomic analysis uncovered 97 proteins with significant abundance changes in response to salt stress. These findings not only enhance our understanding of the molecular mechanisms at play but also offer potential targets for fortifying plant resilience in the face of salinity-induced challenges.
Case. Unraveling Arabidopsis' Response to Salt Stress Through Glycoproteomic Analysis [1]
As sessile organisms, terrestrial plants often face various stresses and adverse conditions, such as extreme temperatures, drought, and high salinity. In agricultural production, high salinity affects the natural distribution of crops in the ecosystem, causing significant yield losses. Numerous studies indicate that multiple genes and/or metabolic pathways synergistically contribute to the plant's response to abiotic stress, making it challenging to significantly enhance plant resistance to external stress by regulating a single effective gene or protein.
In plants, N-glycosylation participates in various biological processes, including ER quality control of steroid hormone receptors, gametophyte recognition, subcellular transport, plant innate immunity, and stomatal development. This regulation is primarily achieved by modulating the stability, function, or subcellular localization of substrate proteins. Additionally, research has revealed that a failure in complex N-glycan biosynthesis leads to salt sensitivity in Arabidopsis seedlings, yet the underlying molecular mechanisms remain poorly understood.
This study utilized nano liquid chromatography-tandem mass spectrometry (nano LC-MS/MS) technology for label-free proteomics analysis, comparing the protein profile differences between the wild type (WT) and two salt-resistant mutants. A total of 727 glycosylation sites corresponding to 371 glycoproteins were identified from 632 peptides. Most N-glycosylation sites exhibited typical NXS/T consensus sequences. The number of identified N-glycoproteins varied among samples. In the control group, 311 glycoproteins were identified in mns1 mns2, 300 in the cgl1-3 mutant, and only 203 in WT. In WT, the reduced abundance of N-glycoproteins could be attributed to the lower enrichment of glycoproteins carrying complex N-glycans compared to mutants with high mannose-type N-glycans.
However, in the presence of high salinity, the number of detected glycoproteins slightly increased in WT (from 203 to 244). Conversely, a decrease was observed in mns1mns2 (from 311 to 297) and cgl1-3 (from 300 to 263; Figure 1). One possibility is that the abundance of glycoproteins responding to salt stress is regulated by attached N-glycans. The immature state of N-glycans renders salt stress-related glycoproteins unstable, consistent with previous research highlighting the crucial role of mature N-glycans in plant salt stress tolerance.
Figure 1. Overview of Identified Glycopeptides and Proteins
The study further analyzed the biological functions of differentially abundant glycoproteins (DAG) under salt stress through GO annotation and KEGG metabolic pathways. More reduced glycoproteins were observed in both mutants, particularly in cgl1-3 (Figure 2 A–C). Notably, compared to WT, hydrolases were enriched in both mns1 mns2 and cgl1-3 mutants under salt stress (Figure 2 D–F). Under salt stress, the abundance of STT3A (OST subunit), purple acid phosphatase 2 (PAP2), and globulin-10 (GLP10) involved in root growth and carbon metabolism changed in WT and both ER glucosyltransferase mutants. STT3A increased in WT but decreased in mns1 mns2 and cgl1-3; PAP2 increased in WT and mns1 mns2 but decreased in cgl1-3, while GLP10 increased in all three samples. Further molecular function analysis revealed enriched hydrolase activity in mns1 mns2 and cgl1-3 compared to WT (Figure 2 E,G). Biological processes of proteins identified in WT were mostly stress-related, while DAG from mutants were associated with carbohydrate metabolism processes (Figure 3 G). These findings suggest that salt-responsive glycoproteins in mns1 mns2 and cgl1-3 mutants fail to respond properly to stress, possibly due to incorrect N-glycan modifications leading to abnormal subcellular localization.
Figure 2. GO Annotation of Differentially Abundant Glycoproteins under Salt Stress
Mounting evidence suggests that the adaptive response of plants to salt stress involves the participation of mature N-glycans on relevant proteins. However, little is known about the salt-responsive glycoproteins involved in this process. This study identified salt-responsive glycoproteins in wild-type (WT) Arabidopsis thaliana and two N-glycan maturation-defective mutants, mns1 mns2, and cgl1. Through quantitative analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a total of 97 proteins with abundance changes > 1.5 were identified in response to salt stress. This indicates the crucial role of N-glycans in regulating stress response protein levels and lists several novel glycoproteins responsible for Arabidopsis salt tolerance.
Reference
- Liu, Chuanfa, et al. "Comparative label-free quantitative proteomics analysis reveals the essential roles of N-glycans in salt tolerance by modulating protein abundance in Arabidopsis." Frontiers in Plant Science 12 (2021): 646425.
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