Targeted protein degradation is an important and pervasive regulatory mechanism in plants, required for perception and response to the environment as well as developmental signaling. Despite the significance of this process, relatively few studies have assessed plant protein turnover in a quantitative fashion. Tandem fluorescent protein timers (tFTs) offer a powerful approach for the assessment of in vivo protein turnover in distinct subcellular compartments of single or multiple cells. A tFT is a fusion of two different fluorescent proteins with distinct fluorophore maturation kinetics, which enable protein age to be estimated from the ratio of fluorescence intensities of the two fluorescent proteins. Here, we used short-lived auxin signaling proteins and model N-end rule (N-recognin) pathway reporters to demonstrate the utility of tFTs for studying protein turnover in living plant cells of Arabidopsis (Arabidopsis thaliana) and Nicotiana benthamiana. We present transient expression of tFTs as an efficient screen for relative protein lifetime, useful for testing the effects of mutations and different genetic backgrounds on protein stability. This work demonstrates the potential for using stably expressed tFTs to study native protein dynamics with high temporal resolution in response to exogenous or endogenous stimuli.
The Arg/N-end rule pathway of ubiquitin-mediated proteolysis has multiple functions throughout plant development, notably in the transition from dormant seed to photoautotrophic seedling. PROTEOLYSIS6 (PRT6), an N-recognin E3 ligase of the Arg/N-end rule regulates the degradation of transcription factor substrates belonging to Group VII of the Ethylene Response Factor superfamily (ERFVIIs). It is not known whether ERFVIIs are associated with all known functions of the Arg/N-end rule, and the downstream pathways influenced by ERFVIIs are not fully defined. Here, we examined the relationship between PRT6 function, ERFVIIs and ABA signalling in Arabidopsis seedling establishment. Physiological analysis of seedlings revealed that N-end rule-regulated stabilisation of three of the five ERFVIIs, RAP2.12, RAP2.2 and RAP2.3, controls sugar sensitivity of seedling establishment and oil body breakdown following germination. ABA signalling components ABA INSENSITIVE (ABI)4 as well as ABI3 and ABI5 were found to enhance ABA sensitivity of germination and sugar sensitivity of establishment in a background containing stabilised ERFVIIs. However, N-end rule regulation of oil bodies was not dependent on canonical ABA signalling. We propose that the N-end rule serves to control multiple aspects of the seed to seedling transition by regulation of ERFVII activity, involving both ABA-dependent and independent signalling pathways.
Detection of potentially pathogenic microbes through recognition by plants and animals of both physical and chemical signals associated with the pathogens is vital for host well-being. Signal perception leads to the induction of a variety of responses that augment pre-existing, constitutive defences. The plant cell wall is a highly effective preformed barrier which becomes locally reinforced at the infection site through delivery of new wall material by the actin cytoskeleton. Although mechanical stimulation can produce a reaction, there is little understanding of the nature of physical factors capable of triggering plant defence. Neither the magnitude of forces nor the contact time required has been quantified. In the study reported here, mechanical stimulation with a tungsten microneedle has been used to quantify the response of Arabidopsis plants expressing an actin-binding protein tagged with green fluorescent protein (GFP) to reveal the organisation of the actin cytoskeleton. Using confocal microscopy, the response time for actin reorganisation in epidermal cells of Arabidopsis hypocotyls was shown to be 116 ± 49 s. Using nanoindentation and a diamond spherical tip indenter, the magnitude of the forces capable of triggering an actin response has been quantified. We show that Arabidopsis hypocotyl cells can detect a force as small as 4 μN applied for as short a time as 21.6 s to trigger reorganisation of the actin cytoskeleton. This force is an order of magnitude less than the potential invasive force determined for a range of fungal and oomycete plant pathogens. To our knowledge, this is the first quantification of the magnitude and duration of mechanical forces capable of stimulating a structural defence response in a plant cell.
BIG (also known as DOC1 and TIR3) is an 0.5 MDa protein that has been associated with multiple important functions in signalling and development through forward genetic screens in Arabidopsis thaliana. However, the biochemical function(s) of BIG are unknown. Here, we investigated whether BIG plays a role in the Arg/N-degron pathways, protein regulatory mechanisms in which substrate protein fate is influenced by the N-terminal (Nt) residue. In Arabidopsis, PROTEOLYSIS1 (PRT1) is an E3 ligase with specificity for aromatic amino acids, whereas PROTEOLYSIS6 (PRT6) targets basic N-terminal residues. We crossed a big loss-of-function allele toprt6andprt1mutants and examined the stability of protein substrates. Stability of model N-degron pathway substrates was enhanced inprt6-1 big-2andprt1-1 big-2relative to the respective single mutants. Abundance of the PRT6 physiological substrates, HYPOXIA RESPONSIVE ERF (HRE)2 and VERNALIZATION (VRN)2 was similarly increased in prt6 big double mutants, without increase in transcripts. Accordingly, hypoxia marker expression was enhanced inprt6 bigdouble mutants, in a manner requiring arginyltransferase activity and RAP-type ERFVII transcription factors. Transcriptomic analysis of roots not only demonstrated synergistically increased expression of a plethora of hypoxia responsive genes in the double mutant relative to prt6 but also revealed other roles for PRT6 and BIG, including regulation of suberin deposition through both ERFVII-dependent and independent mechanisms, respectively. Our results show that BIG acts together with PRT6 to regulate the hypoxia response and wider processes.
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