Functional characterization of cell wall damage response genes in Arabidopsis thaliana

Abstract

Plant cell walls form the first barrier during exposure to biotic and abiotic stresses and are constantly remodelled during developmental and growth processes. Thus, it is conceivable that a dedicated monitoring system perceives changes in cell wall integrity and initiates a coordinated set of responses. Currently, there is only limited knowledge on how this system functions. Recent findings have suggested that cell wall damage (CWD) caused by chemical or genetic inhibition of cellulose biosynthesis and pathogen attack triggers a canonical set of overlapping responses. These include ectopic deposition of lignin, increases in production of jasmonic (JA) and salicylic acid (SA), ethylene, reactive oxygen species (ROS), callose deposition, transcriptional activation of stress response mechanisms and changes in cell wall composition and structure. Isoxaben is a chemical inhibitor of cellulose biosynthesis that induces CWD. Microarray-based gene expression profiling has previously identified several genes whose expression is transiently induced in Arabidopsis thaliana seedlings exposed to isoxaben. In the work presented here, the function of two of these genes, ATL2 and AT5G43450, was characterized. To achieve this, loss of function knock-out lines for each gene were used in phenotypical analyses based on the isoxaben-induced responses. Additionally, molecular tools were developed to study the global expression pattern and sub-cellular localization of ATL2 and AT5G43450, and complementation constructs for atl2 and at5g43450 were generated as well. ATL2 was found to be involved in regulation of JA biosynthesis and lignin deposition in response to cellulose biosynthesis inhibition. Global gene expression pattern analysis using promoter-reporter constructs showed that ATL2 is expressed in most of the plant tissues in seedlings and adult plants. AT5G43450 was suggested to be involved in inhibition of cell elongation upon isoxaben addition. at5g43450 seedlings exhibited reduced sensitivity to inhibition of cell elongation caused by isoxaben. In accordance with this observation, at5g43450 lines expressing the full-length genomic AT5G43450 exhibited wild-type levels of cell elongation inhibition. AT5G43450 was found to localize to the nucleus and cytosol, as determined by using an AT5G43450::CITRINE translational fusion protein under the control of the constitutive promoter 35S. Furthermore, by using a cell cycle marker line containing a CYCB1;1::GUS reporter, isoxaben was shown to inhibit cell cycle progression 6 h after the start of the treatment. Interestingly, providing osmotic support suppressed the isoxaben effect, as CYCB1;1::GUS activity levels were not changed under these conditions. Similarly, the addition of MeJA counteracted the isoxaben-induced cell cycle inhibition as well. Moreover, ATL2 and AT5G43450 were found to be involved in cell cycle regulation since atl2 and at5g43450 containing the same reporter construct exhibited stronger activity levels of CYCB1;1::GUS compared to the control. In particular, ATL2 was found to be involved in the MeJA modulation of cell cycle progression since CYCB1;1::GUS activity levels substantially increased in atl2 after addition of MeJA. Together, these results show that ATL2 and AT5G43450 are involved in the isoxaben-induced CWD response and are negative regulators of cell cycle progression.