Characterization of the Ubiquitin N-end Rule Pathway in Arabidopsis

The control of intracellular protein homeostasis is essential for the ability of plants to grow under different physiological conditions, as well as respond to various biotic or abiotic stresses. One of the ways that cells achieve this equilibrium is through the targeted proteolysis of proteins by the ubiquitin-proteasome system. A subset of this system, termed the N-end rule pathway, relates the in vivo longevity of a substrate protein to the nature of its N-terminal amino acid. Although the N-end rule pathway is known to regulate numerous physiological processes in plants relatively few substrates of the pathway have been identified to date. In this study experiments were conducted aimed at identifying N-end rule substrates in the model plant Arabidopsis thaliana. One group of candidate substrates is generated after their proteolytic cleavage by a bacterial effector protein. The transient expression of these candidate N-end rule substrates in tobacco coupled with pathogen inoculation and biochemical methods led to the identification of a group of protein fragments that are likely novel N-end rule substrates. Experiments were also conducted towards developing a molecular tagging tool with the aim of conducting a proteome-wide screen for N-end rule substrates. Additionally, experiments were carried out to characterize a component of the Arabidopsis N-end rule pathway by expressing this enzyme in the yeast S. cerevisiae under different conditions. This study will allow for a further understanding of the involvement of the N-end rule pathway in plant-pathogen interactions and provides several novel N-end rule substrates for future experiments aimed at dissecting the diverse functions of this pathway in plants

Ubiquitylation in plants: signaling hub for the integration of environmental signals

A fundamental question in biology is how organisms integrate the plethora of environmental cues that they perceive to trigger a co-ordinated response. The regulation of protein stability, which is largely mediated by the ubiquitin–proteasome system in eukaryotes, plays a pivotal role in these processes. Due to their sessile lifestyle and the need to respond rapidly to a multitude of environmental factors, plants are thought to be especially dependent on proteolysis to regulate cellular processes. In this review, we present the complexity of the ubiquitin system in plants, and discuss the relevance of the proteolytic and non-proteolytic roles of this system in the regulation and co-ordination of plant responses to environmental signals. We also discuss the role of the ubiquitin system as a key regulator of plant signaling pathways. We focus more specifically on the functions of E3 ligases as regulators of the jasmonic acid (JA), salicylic acid (SA), and ethylene hormone signaling pathways that play important roles to mount a co-ordinated response to multiple environmental stresses. We also provide examples of new players in this field that appear to integrate different cues and signaling pathway

Expression of KNUCKLES in the Stem Cell Domain Is Required for Its Function in the Control of Floral Meristem Activity in Arabidopsis

In the model plant Arabidopsis thaliana, the zinc-finger transcription factor KNUCKLES (KNU) plays an important role in the termination of floral meristem activity, a process that is crucial for preventing the overgrowth of flowers. The KNU gene is activated in floral meristems by the floral organ identity factor AGAMOUS (AG), and it has been shown that both AG and KNU act in floral meristem control by directly repressing the stem cell regulator WUSCHEL (WUS), which leads to a loss of stem cell activity. When we re-examined the expression pattern of KNU in floral meristems, we found that KNU is expressed throughout the center of floral meristems, which includes, but is considerably broader than the WUS expression domain. We therefore hypothesized that KNU may have additional functions in the control of floral meristem activity. To test this, we employed a gene perturbation approach and knocked down KNU activity at different times and in different domains of the floral meristem. In these experiments we found that early expression in the stem cell domain, which is characterized by the expression of the key meristem regulatory gene CLAVATA3 (CLV3), is crucial for the establishment of KNU expression. The results of additional genetic and molecular analyses suggest that KNU represses floral meristem activity to a large extent by acting on CLV3. Thus, KNU might need to suppress the expression of several meristem regulators to terminate floral meristem activity efficiently

Differential N-end rule degradation of RIN4/NOI fragments generated by the AvrRpt2 effector protease.

In plants, the protein RPM1-INTERACTING PROTEIN4 (RIN4) is a central regulator of both pattern-triggered immunity and effector-triggered immunity. RIN4 is targeted by several effectors, including the Pseudomonas syringae protease effector AvrRpt2. Cleavage of RIN4 by AvrRpt2 generates potentially unstable RIN4 fragments, whose degradation leads to the activation of the resistance protein RESISTANT TO P. SYRINGAE2. Hence, identifying the determinants of RIN4 degradation is key to understanding RESISTANT TO P. SYRINGAE2–mediated effector-triggered immunity, as well as virulence functions of AvrRpt2. In addition to RIN4, AvrRpt2 cleaves host proteins from the nitrate-induced (NOI) domain family. Although cleavage of NOI domain proteins by AvrRpt2 may contribute to pattern-triggered immunity regulation, the (in)stability of these proteolytic fragments and the determinants regulating their stability remain unexamined. Notably, a common feature of RIN4, and of many NOI domain protein fragments generated by AvrRpt2 cleavage, is the exposure of a new N-terminal residue that is destabilizing according to the N-end rule. Using antibodies raised against endogenous RIN4, we show that the destabilization of AvrRpt2-cleaved RIN4 fragments is independent of the N-end rule pathway (recently renamed the N-degron pathway). By contrast, several NOI domain protein fragments are genuine substrates of the N-degron pathway. The discovery of this set of substrates considerably expands the number of known proteins targeted for degradation by this ubiquitin-dependent pathway in plants. These results advance our current understanding of the role of AvrRpt2 in promoting bacterial virulence

Characterization of the Ubiquitin N-end Rule Pathway in Arabidopsis

The control of intracellular protein homeostasis is essential for the ability of plants to grow under different physiological conditions, as well as respond to various biotic or abiotic stresses. One of the ways that cells achieve this equilibrium is through the targeted proteolysis of proteins by the ubiquitin-proteasome system. A subset of this system, termed the N-end rule pathway, relates the in vivo longevity of a substrate protein to the nature of its N-terminal amino acid. Although the N-end rule pathway is known to regulate numerous physiological processes in plants relatively few substrates of the pathway have been identified to date. In this study experiments were conducted aimed at identifying N-end rule substrates in the model plant Arabidopsis thaliana. One group of candidate substrates is generated after their proteolytic cleavage by a bacterial effector protein. The transient expression of these candidate N-end rule substrates in tobacco coupled with pathogen inoculation and biochemical methods led to the identification of a group of protein fragments that are likely novel N-end rule substrates. Experiments were also conducted towards developing a molecular tagging tool with the aim of conducting a proteome-wide screen for N-end rule substrates. Additionally, experiments were carried out to characterize a component of the Arabidopsis N-end rule pathway by expressing this enzyme in the yeast S. cerevisiae under different conditions. This study will allow for a further understanding of the involvement of the N-end rule pathway in plant-pathogen interactions and provides several novel N-end rule substrates for future experiments aimed at dissecting the diverse functions of this pathway in plants

Characterization of the Ubiquitin N-end Rule Pathway in Arabidopsis

The control of intracellular protein homeostasis is essential for the ability of plants to grow under different physiological conditions, as well as respond to various biotic or abiotic stresses. One of the ways that cells achieve this equilibrium is through the targeted proteolysis of proteins by the ubiquitin-proteasome system. A subset of this system, termed the N-end rule pathway, relates the in vivo longevity of a substrate protein to the nature of its N-terminal amino acid. Although the N-end rule pathway is known to regulate numerous physiological processes in plants relatively few substrates of the pathway have been identified to date. In this study experiments were conducted aimed at identifying N-end rule substrates in the model plant Arabidopsis thaliana. One group of candidate substrates is generated after their proteolytic cleavage by a bacterial effector protein. The transient expression of these candidate N-end rule substrates in tobacco coupled with pathogen inoculation and biochemical methods led to the identification of a group of protein fragments that are likely novel N-end rule substrates. Experiments were also conducted towards developing a molecular tagging tool with the aim of conducting a proteome-wide screen for N-end rule substrates. Additionally, experiments were carried out to characterize a component of the Arabidopsis N-end rule pathway by expressing this enzyme in the yeast S. cerevisiae under different conditions. This study will allow for a further understanding of the involvement of the N-end rule pathway in plant-pathogen interactions and provides several novel N-end rule substrates for future experiments aimed at dissecting the diverse functions of this pathway in plants

Regulatory interplay between LEAFY, APETALA1/CAULIFLOWER and TERMINAL FLOWER1: New insights into an old relationship

The gene regulatory network comprised of LEAFY (LFY), APETALA1 (AP1), the AP1 paralog CAULIFLOWER (CAL), and TERMINAL FLOWER1 (TFL1) is a major determinant of the flowering process in Arabidopsis thaliana. TFL1 activity in the shoot apical meristem provides inflorescence identity while the transcription factors LFY and AP1/CAL confer floral identity to emerging floral primordia. It has been thought that LFY and AP1/CAL control the onset of flowering in part by repressing TFL1 expression in flowers. However, in the June issue of Plant Physiology, we reported that LFY and AP1 act antagonistically in the regulation of several key flowering regulators, including TFL1. Specifically, TFL1 transcription was suppressed by AP1 but promoted by LFY. Here, we present additional evidence for the role of LFY as an activator of TFL1 and propose that this regulatory activity is pivotal for the indeterminate growth of the SAM during the reproductive phase of development

Transcription Factor Interplay between LEAFY and APETALA1/CAULIFLOWER during Floral Initiation

The transcription factors LEAFY (LFY) and APETALA1 (AP1), together with the AP1 paralog CAULIFLOWER (CAL), control the onset of flower development in a partially redundant manner. This redundancy is thought to be mediated, at least in part, through the regulation of a shared set of target genes. However, whether these genes are independently or cooperatively regulated by LFY and AP1/CAL is currently unknown. To better understand the regulatory relationship between LFY and AP1/ CAL and to obtain deeper insights into the control of floral initiation, we monitored the activity of LFY in the absence of AP1/ CAL function. We found that the regulation of several known LFY target genes is unaffected by AP1/CAL perturbation, while others appear to require AP1/CAL activity. Furthermore, we obtained evidence that LFY and AP1/CAL control the expression of some genes in an antagonistic manner. Notably, these include key regulators of floral initiation such as TERMINAL FLOWER1 (TFL1), which had been previously reported to be directly repressed by both LFY and AP1. We show here that TFL1 expression is suppressed by AP1 but promoted by LFY. We further demonstrate that LFY has an inhibitory effect on flower formation in the absence of AP1/CAL activity. We propose that LFY and AP1/CAL act as part of an incoherent feed-forward loop, a network motif where two interconnected pathways or transcription factors act in opposite directions on a target gene, to control the establishment of a stable developmental program for the formation of flowers

The N-end rule pathway regulates pathogen responses in plants

To efficiently counteract pathogens, plants rely on a complex set of immune responses that are tightly regulated to allow the timely activation, appropriate duration and adequate amplitude of defense programs. The coordination of the plant immune response is known to require the activity of the ubiquitin/proteasome system, which controls the stability of proteins in eukaryotes. Here, we demonstrate that the N-end rule pathway, a subset of the ubiquitin/proteasome system, regulates the defense against a wide range of bacterial and fungal pathogens in the model plant Arabidopsis thaliana. We show that this pathway positively regulates the biosynthesis of plant-defense metabolites such as glucosinolates, as well as the biosynthesis and response to the phytohormone jasmonic acid, which plays a key role in plant immunity. Our results also suggest that the arginylation branch of the N-end rule pathway regulates the timing and amplitude of the defense program against the model pathogen Pseudomonas syringae AvrRpm1