RESUMO
Nutrient starvation-induced autophagy is a conserved process in eukaryotes. Plants defective in autophagy show hypersensitivity to carbon and nitrogen limitation. However, the role of autophagy in plant phosphate (Pi) starvation response is relatively less explored. Among the core autophagy-related (ATG) genes, ATG8 encodes a ubiquitin-like protein involved in autophagosome formation and selective cargo recruitment. The Arabidopsis thaliana ATG8 genes, AtATG8f and AtATG8h, are notably induced in roots under low Pi. In this study, we show that such upregulation correlates with their promoter activities and can be suppressed in the phosphate response 1 (phr1) mutant. Yeast one-hybrid analysis failed to attest the binding of the AtPHR1 transcription factor to the promoter regions of AtATG8f and AtATG8h. Dual luciferase reporter assays in Arabidopsis mesophyll protoplasts also indicated that AtPHR1 could not transactivate the expression of both genes. Loss of AtATG8f and AtATG8h leads to decreased root microsomal-enriched ATG8 but increased ATG8 lipidation. Moreover, atg8f/atg8h mutants exhibit reduced autophagic flux estimated by the vacuolar degradation of ATG8 in the Pi-limited root but maintain normal cellular Pi homeostasis with reduced number of lateral roots. While the expression patterns of AtATG8f and AtATG8h overlap in the root stele, AtATG8f is more strongly expressed in the root apex and root hair and remarkably at sites where lateral root primordia develop. We hypothesize that Pi starvation-induction of AtATG8f and AtATG8h may not directly contribute to Pi recycling but rely on a second wave of transcriptional activation triggered by PHR1 that fine-tunes cell type-specific autophagic activity.
RESUMO
Autophagy in plants is regulated by diverse signaling cascades in response to environmental changes. Fine-tuning of its activity is critical for the maintenance of cellular homeostasis under basal and stressed conditions. In this study, we compared the Arabidopsis autophagy-related (ATG) system transcriptionally under inorganic phosphate (Pi) deficiency versus nitrogen deficiency and showed that most ATG genes are only moderately upregulated by Pi starvation, with relatively stronger induction of AtATG8f and AtATG8h among the AtATG8 family. We found that Pi shortage increased the formation of GFP-ATG8f-labeled autophagic structures and the autophagic flux in the differential zone of the Arabidopsis root. However, the proteolytic cleavage of GFP-ATG8f and the vacuolar degradation of endogenous ATG8 proteins indicated that Pi limitation does not drastically alter the autophagic flux in the whole roots, implying a cell type-dependent regulation of autophagic activities. At the organismal level, the Arabidopsis atg mutants exhibited decreased shoot Pi concentrations and smaller meristem sizes under Pi sufficiency. Under Pi limitation, these mutants showed enhanced Pi uptake and impaired root cell division and expansion. Despite a reduced steady-state level of several PHOSPHATE TRANSPORTER 1s (PHT1s) in the atg root, cycloheximide treatment analysis suggested that the protein stability of PHT1;1/2/3 is comparable in the Pi-replete wild type and atg5-1. By contrast, the degradation of PHT1;1/2/3 is enhanced in the Pi-deplete atg5-1. Our findings reveal that both basal autophagy and Pi starvation-induced autophagy are required for the maintenance of Pi homeostasis and may modulate the expression of PHT1s through different mechanisms.
