A Novel Function for Arabidopsis CYCLASE1 in Programmed Cell Death Revealed by Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Analysis of Extracellular Matrix Proteins

Programmed cell death is essential for plant development and stress adaptation. A detailed understanding of the signal transduction pathways that regulate plant programmed cell death requires identification of the underpinning protein networks. Here, we have used a protagonist and antagonist of programmed cell death triggered by fumonisin B1 as probes to identify key cell death regulatory proteins in Arabidopsis. Our hypothesis was that changes in the abundance of cell death-regulatory proteins induced by the protagonist should be blocked or attenuated by concurrent treatment with the antagonist. We focused on proteins present in the mobile phase of the extracellular matrix on the basis that they are important for cell–cell communications during growth and stress-adaptive responses. Salicylic acid, a plant hormone that promotes programmed cell death, and exogenous ATP, which can block fumonisin B1-induced cell death, were used to treat Arabidopsis cell suspension cultures prior to isobaric-tagged relative and absolute quantitation analysis of secreted proteins. A total of 33 proteins, whose response to salicylic acid was suppressed by ATP, were identified as putative cell death-regulatory proteins. Among these was CYCLASE1, which was selected for further analysis using reverse genetics. Plants in which CYCLASE1 gene expression was knocked out by insertion of a transfer-DNA sequence manifested dramatically increased cell death when exposed to fumonisin B1 or a bacterial pathogen that triggers the defensive hypersensitive cell death. Although pathogen inoculation altered CYCLASE1 gene expression, multiplication of bacterial pathogens was indistinguishable between wild type and CYCLASE1 knockout plants. However, remarkably severe chlorosis symptoms developed on gene knockout plants in response to inoculation with either a virulent bacterial pathogen or a disabled mutant that is incapable of causing disease in wild type plants. These results show that CYCLASE1, which had no known function hitherto, is a negative regulator of cell death and regulates pathogen-induced symptom development in Arabidopsis.Programmed cell death (pcd)¹ is a genetically controlled dismantling of cells, which is indispensable for plant development and stress-adaptive responses. In development, pcd is invoked to facilitate xylem tracheary element differentiation, to remodel leaf shape, and to delete ephemeral cells and organs such as embryonic suspensor cells (1–3). In response to drought stress, pcd is used to break root apical meristem dominance in order to remodel root system architecture as an adaptive response to water deficit (4). Additionally, a specialized form of pcd known as the hypersensitive response kills plant cells at the epicenter of attack by certain pathogens, which activate the effector-triggered immune response (5, 6). A detailed understanding of the signal transduction pathways that trigger, propagate, and terminate plant pcd requires identification of the key components of the underlying protein networks. Our group has been using Arabidopsis cell death induced by fumonisin B1 (FB1) as an experimental system to study plant pcd and identify the key regulatory proteins (6).FB1, a mycotoxin that triggers cell death in both animal and plant cells (8, 9), disrupts sphingolipid biosynthesis via inhibition of ceramide synthase (10). Several proteins directly involved in sphingolipid biosynthesis and metabolism have been shown to regulate FB1-induced plant pcd because of their influence on levels of metabolic intermediates, such as long chain bases (LCBs), which act as second messengers of plant cell death. For example, activity of serine palmitoyltransferase, the enzyme catalyzing the first rate-limiting step in sphingolipid biosynthesis, strongly controls Arabidopsis sensitivity to FB1 (11). Serine palmitoyltransferase has two subunits – LCB1 and LCB2. Resistance to FB1-induced death is manifested in Arabidopsis loss-of-function mutants of LCB1 (12) and LCB2a (13) genes. Overexpression of endogenous Arabidopsis 56 amino acid polypeptides that interact with and stimulate serine palmitoyltransferase activity increases sensitivity to FB1, whereas RNA interference lines have reduced sensitivity to the mycotoxin (11).Although exogenous ceramide can suppress FB1-induced death in animal cells (14), it fails to block cell death in Arabidopsis (15), indicating that other factors work in concert with ceramide depletion in pcd induction in Arabidopsis. Identification of these factors is essential to the understanding of general pcd regulation in plants, given that Arabidopsis responses to FB1 share common features with the pathogen-induced hypersensitive response (15). Clues that may lead to mechanistic details of pcd could arise from focusing on known regulatory signals that control FB1-mediated responses. FB1-induced cell death is regulated by extracellular ATP (eATP) (16) and the plant defense hormone, salicylic acid (SA) (17). NahG transgenic plants, which degrade SA, are resistant to FB1 as are pad4–1 mutants, which have an impaired SA amplification mechanism (17). Mutants that constitutively accumulate greater amounts of SA, cpr1 and cpr6, manifest increased susceptibility to FB1 (17). Thus, SA functions as a positive regulator of FB1-induced pcd. In contrast, eATP is a negative regulator of FB1-triggered pcd in Arabidopsis. Accordingly, FB1 activates eATP depletion prior to onset of death and addition of exogenous ATP to FB1-treated Arabidopsis cell suspension cultures blocks pcd (16). This suggests that SA- and eATP-mediated signaling converge onto the signal transduction cascade activated by FB1 to promote or inhibit pcd, respectively.We have developed an experimental system, which harnesses the effects of exogenous ATP and SA on FB1-induced death, to identify important proteins that regulate Arabidopsis pcd. It utilizes Arabidopsis cell suspension cultures treated with these compounds and proteomic analyses restricted to the mobile phase of the extracellular matrix. The extracellular matrix proteome consists of cell surface proteins fully or partially embedded in the plasma membrane, proteins immobilized in the cell wall, and soluble mobile proteins in the apoplastic fluid – the mobile phase. The rationale for this is predicated on the hypothesis that cells constantly communicate with their neighbors by releasing and sensing signal molecules in the mobile phase (18). Arabidopsis has more than 600 plasma membrane receptor kinases (19) and ∼400 G-protein-coupled receptors (20, 21), which sense extracellular signals at the cell surface and activate a cytoplasmic response. We hypothesize that upon receiving an exogenous chemical, cell–cell signaling is activated either by directly binding the chemical if it has a cell surface receptor, or by modulating signal regulatory proteins in the mobile phase to reset the communication and transmit new signals. Therefore, in this study, we used ATP and SA treatments to identify pcd regulatory proteins in the mobile phase of the Arabidopsis extracellular matrix. We provide a novel extracellular matrix putative cell death regulatory protein network and present evidence validating the role of CYCLASE1 in FB1- and pathogen-induced pcd and the control of disease symptoms.