Carbonyl compounds are electrophiles and can react with nucleophilic groups on biomolecules to make a covalent bond 
. Typical reactions are Schiff base formation and Michael addition. Schiff base is formed between a carbonyl group and an amino group through dehydration (Figure 1
A). This reaction proceeds fast at pH 5, but slower at lower or higher pH conditions. The formed Schiff base can be hydrolyzed under a highly acidic condition. When a thiol group or an amino group reside nearby the Schiff base, the secondary reactions may proceed to form a complex structure 
, which may contribute to the formation of cross-link between proteins. Michael addition is the reaction of the electrophilic β carbon of an RCS molecule with the thiol sulfur, amino nitrogen, or the imidazole τ-nitrogen atom to form a covalent bond (Figure 1
B). They also react with the guanine base of nucleic acids, leading to a mutation 
. The Michael reaction with a thiol is reversible. The equilibrium constant of the reaction between GSH and an RCS differs greatly, depending on the type of RCS molecules 
Figure 1. Modification of amino acid residues on a protein with carbonyls. Panel A. Schiff base formation on an amino group with an aldehyde and possible secondary reactions. Panel B. Top, Michael addition of an RCS to a nucleophilic (-XH) group. X is for sulfur in Cys, ε-amino nitrogen in Lys and imidazole τ-nitrogen in His residues. From 2nd to bottom, Michael addition of a 4-hydroxy-(E)-2-alkenal to Cys, His and Lys residues. Primary adducts may undergo cyclization.
Michael addition of an RCS to a protein adds a carbonyl moiety on the protein (protein carbonylation), while Schiff base formation of RCS with a protein does not. Carbonyl moieties on a protein can also be formed by ROS directly at Trp, His, Tyr, Met and Cys residues 
. Protein carbonylation has been conventionally regarded as an indicator of protein oxidation in plants, but it should be noted that a considerable portion of the detected proteins as ‘oxidized proteins’ are modified with RCS 
The primary Michael adducts, or protein carbonyls, can proceed to secondary reactions with another nucleophilic group, to produce irreversible structural changes on the protein (Figure 1
B). The primary Michael adduct between a nucleophile and 4-hydroxyl-2-alkenals, e.g., HNE and HHE, can undergo cyclization. The cyclic secondary products may further react with another nucleophilic group 
. These reactions can lead to the formation of cross-links between proteins.
Effects of RCS modification on the protein function differs by the kinds of RCS and proteins. The RCS includes carbonyls with various structure; different carbon chain lengths, number of unsaturated bonds, and the extent of oxygenation 
. Hence, every RCS has different properties such as polarity, hydrophobicity/hydrophilicity, solubility, and volatility 
. This variety gives distinct RCS molecules different reactivity with proteins, implying that each species can bring distinct information via specific recognition by the targets and scavenging enzymes. For example, HNE and 4-oxo-(E
)-2-nonenal (ONE), two C9
-RCS differing only by the extent of oxidation at the 4th carbon, show a great difference in the reactivity with GSH; the second-order reaction constant of the former is 100-fold smaller than that of the latter 
. Interestingly, these two RCS react with the redox sensor protein mitoNEET differently, but in a manner not simply deduced from the reactivity discussed above; ONE is bound to Lys55 specifically, while HNE adds to Lys and His residues broadly on the protein 
. Several isozymes of glutathione transferase (GST) Tau class recognize acrolein and HNE as substrates, but there are ones that accept acrolein only 
. These examples suggest the difficulty in generalizing the mode of interaction between RCS and their target proteins.
Lists of proteins that are affected by RCS are available for animal proteins 
, but only a few plant proteins have been investigated, as described below. In general, when an RCS acts as a signal, the (putative) target protein probably will gain the function, so that modification of a small portion of the total population of the target might trigger the next signal. In case of damage, inactivation of an RCS target protein will affect a cellular process immediately if the activity of that protein limits the metabolic or signaling pathway involving it. Extensive modification of a protein will also facilitate cross-linking with other proteins, leading to aggregation unless the modified proteins are proteolytically removed (described below). Under severe and prolonged oxidative stress, larger populations of broader range of proteins will be inactivated, leading to extensive deterioration of cell functions.
Plant proteins that are sensitive to or modified by RCS are listed. Addition of HNE to isolated mitochondria caused sensitive inactivation of lipoate enzymes such as H-subunit (SU) of glycine decarboxylase complex (GDC) 
and alternative oxidase 
. Addition of RCS, especially acrolein, to chloroplasts caused a rapid consumption of GSH, followed by the inactivation of phosphoribulokinase (PRK) preferentially, then fructose-1,6-bisphosphatase, glyceraldehyde-3-phosphate dehydrogenase, leading to the loss of the CO2
fixation ability 
. By a proteomic analysis of A. thaliana
cultured cells treated with oxidative agents such as H2
, menadione and antimycin A, subsets of inner membrane proteins and matrix proteins (totally 31 different proteins) were identified as HNE targets because they were modified by this RCS to greater extents under the stress 
. In heat-stressed spinach leaves, OEC33 protein in photosystem II (PSII) was modified with MDA and acrolein, while in A. thaliana
leaves the antenna LHCII protein was sensitively modified with MDA in heat stress 
. Mano et al. 
analyzed the RCS-modification of soluble proteins in leaves from salt stressed A. thaliana
plants. As detected with specific antibodies, protein modification with HNE, 4-hydroxy-(E
)-2-hexenal, acrolein, crotonaldehyde and malondialdehyde increased in leaves with the progress of the salt-stress treatment. In addition, the acrolein- and crotonaldehyde-modifications were increased significantly even under less severe stress conditions, in which there was no apparent tissue injury or the photoinhibition of PSII. The band pattern of Western blotting suggested these different RCS targeted a common set of proteins. With a quantitative proteomic analysis after immuno-affinity trapping of the HNE-modified proteins, 17 distinct proteins were identified as sensitive targets. Interestingly, these target proteins were distributed to various cellular compartments, i.e., cytoplasm, peroxisome, chloroplast, nucleus and even apoplast. Addition of acrolein to tobacco bright yellow-2 (BY-2) cultured cells or the cell extract activated caspase-1-like protease (C1LP) and caspase-3-like protease (C3LP) activities 
. Results of these experiments with different samples and treatments commonly suggest that certain subsets of proteins are sensitively modified with RCS under stress conditions. In other words, intracellular RCS do not necessarily react with broad range of proteins.
The fate of RCS-modified proteins has not been significantly investigated, both in animals and plants. In general, ‘damaged’ proteins should be immediately degraded before extensive modification leads to the formation of aggregated proteins, which may escape degradation and deposit in cells to cause detrimental effects. Mildly modified proteins are degraded by proteasomes 
. In mice, the 20S proteasome has been shown to be responsible for the elimination of most oxidized proteins 
. In A. thaliana
also, the 20S proteasome appears to be responsible for the degradation of oxidized proteins, while the 26S proteasome takes care of misfolded proteins 
. In mitochondria, ATP-dependent proteases such as Lon protease has been recognized as key proteases for the removal of oxidized proteins in mammals and yeast, but not in plants 
. These studies have investigated “oxidized” or “carbonylated” proteins collectively and have not distinguished ROS-mediated and RCS-mediated modification on proteins. There is a report that the degradation HNE-modified protein is catalyzed by cathepsin G in rat 
, but corresponding facts for plants are not available. It should be noted that the modification of proteins with certain kinds of RCS such as MDA are reversible 
. Thus, it is possible that a transient increase in the RCS concentration modifies the target protein(s) and afterwards the RCS is released from the protein, and then scavenged by certain enzymes (described below). In this case the target protein can be recruited to its physiological role although there has been no experimental data available for demonstrating such dynamics.