Avena phytochrome a overexpressed in transgenic tobacco seedlings differentially affects red/far-red reversible and very-low-fluence responses (cotyledon unfolding) during de-etiolation

Etiolated seedlings of tobacco (Nicotiana tabacum L.) were exposed to single light pulses predicted to establish different proportions of phytochrome in its far-red absorbing form (Pfr/P). The angle between the cotyledons was compared in wild-type and transgenic seedling overexpressing Avena phytochrome A over the range of both very low-fluence responses (VLFR) and low-fluence responses (LFR). The unfolding of the cotyledons increased linearly for 24 h after the light pulse. At this time the Pfr/P-response curve showed two linear segments. The segment below a calculated Pfr/P = 3% (i.e. VLFR) was steeper than the segment above 3% (i.e. LFR). In the VLFR range the slope was almost threefold higher in transgenic than wild-type seedlings. However, in the LFR range the difference was less than 50%. From these data we propose that Avena phytochrome A makes a higher contribution to VLFR than LFR in etiolated tobacco seedlings.Abbreviations FR far-red light - LFR low-fluence response - Pfr/P proportion of phytochrome (P) in its FR-absorbing form (Pfr) - R red light - VLFR very low-fluence response Financial support was provided by the University of Buenos Aires and Fundación Antorchas (Argentina) to J.J.C., CONICET (Argentina) to R.A.S. and the U.S. Department of Energy (DE-FG02-88ER13968) to R.D.V.     

Ubiquitylome analysis reveals a central role for the ubiquitin-proteasome system in plant innate immunity

Protein ubiquitylation profoundly expands proteome functionality and diversifies cellular signaling processes, with recent studies providing ample evidence for its importance to plant immunity. To gain a proteome-wide appreciation of ubiquitylome dynamics during immune recognition, we employed a two-step affinity enrichment protocol based on a 6His-tagged ubiquitin (Ub) variant coupled with high sensitivity mass spectrometry to identify Arabidopsis proteins rapidly ubiquitylated upon plant perception of the microbe-associated molecular pattern (MAMP) peptide flg22. The catalog from 2-week-old seedlings treated for 30 min with flg22 contained 690 conjugates, 64 Ub footprints, and all seven types of Ub linkages, and included previously uncharacterized conjugates of immune components. In vivo ubiquitylation assays confirmed modification of several candidates upon immune elicitation, and revealed distinct modification patterns and dynamics for key immune components, including poly- and monoubiquitylation, as well as induced or reduced levels of ubiquitylation. Gene ontology and network analyses of the collection also uncovered rapid modification of the Ub-proteasome system itself, suggesting a critical auto-regulatory loop necessary for an effective MAMP-triggered immune response and subsequent disease resistance. Included targets were UBIQUITIN-CONJUGATING ENZYME 13 (UBC13) and proteasome component REGULATORY PARTICLE NON-ATPASE SUBUNIT 8b (RPN8b), whose subsequent biochemical and genetic analyses implied negative roles in immune elicitation. Collectively, our proteomic analyses further strengthened the connection between ubiquitylation and flg22-based immune signaling, identified components and pathways regulating plant immunity, and increased the database of ubiquitylated substrates in plants.

Proteome-wide catalogs of ubiquitylated proteins reveal a rapid engagement of the ubiquitin–proteasome system in Arabidopsis innate immunity.