Large-Scale Phosphoproteomic Study of Arabidopsis Membrane Proteins Reveals Early Signaling Events in Response to Cold.

Md Mostafa Kamal,Kazuo Shinozaki,Yukio Kawamura,Fuminori Takahashi,Shinnosuke Ishikawa,Ko Suzuki,Taishi Umezawa,Matsuo Uemura,Masaharu Kamo

doi:10.3390/ijms21228631

Abstract

Cold stress is one of the major factors limiting global crop production. For survival at low temperatures, plants need to sense temperature changes in the surrounding environment. How plants sense and respond to the earliest drop in temperature is still not clearly understood. The plasma membrane and its adjacent extracellular and cytoplasmic sites are the first checkpoints for sensing temperature changes and the subsequent events, such as signal generation and solute transport. To understand how plants respond to early cold exposure, we used a mass spectrometry-based phosphoproteomic method to study the temporal changes in protein phosphorylation events in Arabidopsis membranes during 5 to 60 min of cold exposure. The results revealed that brief cold exposures led to rapid phosphorylation changes in the proteins involved in cellular ion homeostasis, solute and protein transport, cytoskeleton organization, vesical trafficking, protein modification, and signal transduction processes. The phosphorylation motif and kinase–substrate network analysis also revealed that multiple protein kinases, including RLKs, MAPKs, CDPKs, and their substrates, could be involved in early cold signaling. Taken together, our results provide a first look at the cold-responsive phosphoproteome changes of Arabidopsis membrane proteins that can be a significant resource to understand how plants respond to an early temperature drop.

Highlights

Throughout the Earth’s history, plants have evolved adaptive mechanisms against multiple abiotic and biotic stresses
We identified a total of 2795 unique phosphopeptides, which were phosphorylated in at least two biological replicates at all five time points, and these phosphopeptides constitute a total of 1360 proteins (Table S1A,B)
Because the objective of our study was to observe the phosphorylation-level changes in proteins potentially involved in the early cold response in the membrane fractions (MMFs), we performed an over-representation analysis on the cellular localization of the proteins and divided them into two groups based on significant enrichment (Fisher test, p < 0.05): (A) the protein group of interest, which includes proteins from the plasma membrane (PM), endoplasmic reticulum, Golgi body, vacuole, extracellular space, cell wall, apoplast, cytoskeleton, and plasmodesmata; and (B) the sub-portion of proteins from the cytosol, mitochondria, plastid, nucleus, and macromolecular complexes

Summary

Introduction

Throughout the Earth’s history, plants have evolved adaptive mechanisms against multiple abiotic and biotic stresses. Plants native to temperate climatic zones can take advantage of the colder autumn temperature and prepare themselves for the upcoming freezing conditions using an adaptive strategy called cold acclimation [4,5,6]. Perception of and response to a drop in temperature is essential for plants that originated in tropic and sub-tropic regions to deploy survival mechanisms and for those in temperate climatic zones to start the acclimation processes and prepare for upcoming freezing stress. Sensing early changes is an essential step for the plant to initiate the adaptive process and survive under cold temperatures. How plants respond to early temperature drops is necessary to develop cold-resistant/-tolerant plant varieties and secure crop supply in the global climate change era

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Conclusion