Lack of FTSH4 Protease Affects Protein Carbonylation, Mitochondrial Morphology, and Phospholipid Content in Mitochondria of Arabidopsis: New Insights into a Complex Interplay.

FTSH4 is one of the inner membrane-embedded ATP-dependent metalloproteases in mitochondria of Arabidopsis (Arabidopsis thaliana). In mutants impaired to express FTSH4, carbonylated proteins accumulated and leaf morphology was altered when grown under a short-day photoperiod, at 22°C, and a long-day photoperiod, at 30°C. To provide better insight into the function of FTSH4, we compared the mitochondrial proteomes and oxyproteomes of two ftsh4 mutants and wild-type plants grown under conditions inducing the phenotypic alterations. Numerous proteins from various submitochondrial compartments were observed to be carbonylated in the ftsh4 mutants, indicating a widespread oxidative stress. One of the reasons for the accumulation of carbonylated proteins in ftsh4 was the limited ATP-dependent proteolytic capacity of ftsh4 mitochondria, arising from insufficient ATP amount, probably as a result of an impaired oxidative phosphorylation (OXPHOS), especially complex V. In ftsh4, we further observed giant, spherical mitochondria coexisting among normal ones. Both effects, the increased number of abnormal mitochondria and the decreased stability/activity of the OXPHOS complexes, were probably caused by the lower amount of the mitochondrial membrane phospholipid cardiolipin. We postulate that the reduced cardiolipin content in ftsh4 mitochondria leads to perturbations within the OXPHOS complexes, generating more reactive oxygen species and less ATP, and to the deregulation of mitochondrial dynamics, causing in consequence the accumulation of oxidative damage.

The mitochondrial protease AtFTSH4 safeguards Arabidopsis shoot apical meristem function.

The shoot apical meristem (SAM) ensures continuous plant growth and organogenesis. In LD 30 °C, plants lacking AtFTSH4, an ATP-dependent mitochondrial protease that counteracts accumulation of internal oxidative stress, exhibit a puzzling phenotype of premature SAM termination. We aimed to elucidate the underlying cellular and molecular processes that link AtFTSH4 with SAM arrest. We studied AtFTSH4 expression, internal oxidative stress accumulation, and SAM morphology. Directly in the SAM we analysed H2O2 accumulation, mitochondria behaviour, and identity of stem cells using WUS/CLV3 expression. AtFTSH4 was expressed in proliferating tissues, particularly during the reproductive phase. In the mutant, SAM, in which internal oxidative stress accumulates predominantly at 30 °C, lost its meristematic fate. This process was progressive and stage-specific. Premature meristem termination was associated with an expansion in SAM area, where mitochondria lost their functionality. All these effects destabilised the identity of the stem cells. SAM termination in ftsh4 mutants is caused both by internal oxidative stress accumulation with time/age and by the tissue-specific role of AtFTSH4 around the flowering transition. Maintaining mitochondria functionality within the SAM, dependent on AtFTSH4, is vital to preserving stem cell activity throughout development.

Genomic screens identify a new phytobacterial microbe-associated molecular pattern and the cognate Arabidopsis receptor-like kinase that mediates its immune elicitation.

BACKGROUND: The recognition of microbe-associated molecular patterns during infection is central to the mounting of an effective immune response. In spite of their importance, it remains difficult to identify these molecules and the host receptors required for their perception, ultimately limiting our understanding of the role of these molecules in the evolution of host-pathogen relationships. RESULTS: We employ a comparative genomics screen to identify six new immune eliciting peptides from the phytopathogenic bacterium Pseudomonas syringae. We then perform a reverse genetic screen to identify Arabidopsis thaliana leucine-rich repeat receptor-like kinases required for the recognition of these elicitors. We test the six elicitors on 187 receptor-like kinase knock-down insertion lines using a high-throughput peroxidase-based immune assay and identify multiple lines that show decreased immune responses to specific peptides. From this primary screen data, we focused on the interaction between the xup25 peptide from a bacterial xanthine/uracil permease and the Arabidopsis receptor-like kinase xanthine/uracil permease sensing 1; a family XII protein closely related to two well-characterized receptor-like kinases. We show that xup25 treatment increases pathogenesis-related gene induction, callose deposition, seedling growth inhibition, and resistance to virulent bacteria, all in a xanthine/uracil permease sensing 1-dependent manner. Finally, we show that this kinase-like receptor can bind the xup25 peptide directly. These results identify xup25 as a P. syringae microbe-associated molecular pattern and xanthine/uracil permease sensing 1 as a receptor-like kinase that detects the xup25 epitope to activate immune responses. CONCLUSIONS: The present study demonstrates an efficient method to identify immune elicitors and the plant receptors responsible for their perception. Further exploration of these molecules will increase our understanding of plant-pathogen interactions and the basis for host specificity.

NbCSPR underlies age-dependent immune responses to bacterial cold shock protein in Nicotiana benthamiana.

Plants use receptor kinases (RKs) and receptor-like proteins (RLPs) as pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs) that are typical of whole classes of microbes. After ligand perception, many leucine-rich repeat (LRR)-containing PRRs interact with the LRR-RK BRI1-ASSOCIATED KINASE 1 (BAK1). BAK1 is thus expected to interact with unknown PRRs. Here, we used BAK1 as molecular bait to identify a previously unknown LRR-RLP required for the recognition of the csp22 peptide derived from bacterial cold shock protein. We established a method to identify proteins that interact with BAK1 only after csp22 treatment. BAK1 was expressed transiently in Nicotiana benthamiana and immunopurified after treatment with csp22. BAK1-associated proteins were identified by mass spectrometry. We identified several proteins including known BAK1 interactors and a previously uncharacterized LRR-RLP that we termed RECEPTOR-LIKE PROTEIN REQUIRED FOR CSP22 RESPONSIVENESS (NbCSPR). This RLP associates with BAK1 upon csp22 treatment, and NbCSPR-silenced plants are impaired in csp22-induced defense responses. NbCSPR confers resistance to bacteria in an age-dependent and flagellin-induced manner. As such, it limits bacterial growth and Agrobacterium-mediated transformation of flowering N. benthamiana plants. Transgenic expression of NbCSPR into Arabidopsis thaliana conferred responsiveness to csp22 and antibacterial resistance. Our method may be used to identify LRR-type RKs and RLPs required for PAMP perception/responsiveness, even when the active purified PAMP has not been defined.

Organ-specific regulation of growth-defense tradeoffs by plants.

Plants grow while also defending themselves against phylogenetically unrelated pathogens. Because defense and growth are both costly programs, a plant's success in colonizing resource-scarce environments requires tradeoffs between the two. Here, we summarize efforts aimed at understanding how plants use iterative tradeoffs to modulate differential organ growth when defenses are elicited. First, we focus on shoots to illustrate how light, in conjunction with the growth hormone gibberellin (GA) and the defense hormone jasmonic acid (JA), act to finely regulate defense and growth programs in this organ. Second, we expand on the regulation of growth-defense trade-offs in the root, a less well-studied topic despite the critical role of this organ in acquiring resources in an environment deeply entrenched with disparate populations of microbes.

Mitochondrial ATP-dependent proteases in protection against accumulation of carbonylated proteins.

Carbonylation is an irreversible oxidative modification of proteins induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) or by-products of oxidative stress. Carbonylation leads to the loss of protein function and is used as a marker of oxidative stress. Recent data indicate that carbonylation is not only an unfavorable chance process but may also play a significant role in the control of diverse physiological processes. In plants, carbonylated proteins have been found in all cellular compartments; however, mitochondria, one of the major sources of reactive species, show the highest levels of oxidatively modified proteins under normal or stress conditions. Carbonylated proteins tend to misfold and have to be removed to prevent the formation of harmful insoluble aggregates. Mitochondria have developed several pathways that continuously monitor and remove oxidatively damaged polypeptides, and the mitochondrial protein quality control (mtPQC) system, comprising chaperones and ATP-dependent proteases, is the first line of defense. The Lon protease has been recognized as a key protease involved in the removal of oxidized proteins in yeast and mammalian mitochondria, but not in plants. Recently, it has been reported that the inner-membrane human i-AAA and m-AAA and Arabidopsis i-AAA proteases are crucial components of the defense against accumulation of carbonylated proteins, but the molecular basis of their action is not yet clear. Altogether, the mitochondrial AAA proteases secure the mitochondrial proteome against accumulation of carbonylated proteins.

Proteolytic system of plant mitochondria.

The existence of a proteolytic system which can specifically recognize and cleave proteins in mitochondria is now well established. The components of this system comprise processing peptidases, ATP-dependent peptidases and oligopeptidases. A short overview of experimentally confirmed proteases mainly from Arabidopsis thaliana is provided. The role of the mitochondrial peptidases in plant growth and development is emphasized. We also discuss the possibility of existence of as yet unidentified plant homologs of yeast mitochondrial ATP-independent proteases.

The lack of mitochondrial AtFtsH4 protease alters Arabidopsis leaf morphology at the late stage of rosette development under short-day photoperiod.

AtFtsH4 is one of four inner membrane-bound mitochondrial ATP-dependent metalloproteases in Arabidopsis thaliana, called AAA proteases, whose catalytic site is exposed to the intermembrane space. In the present study, we used a reverse-genetic approach to investigate the physiological role of the AtFtsH4 protease. We found that loss of AtFtsH4 did not significantly affect Arabidopsis growth under optimal conditions (long days); however, severe morphological and developmental abnormalities in late rosette development occurred under short-day conditions. The asymmetric shape and irregular serration of expanding leaf blades were the most striking features of the ftsh4 mutant phenotype. The severe abnormal morphology of the leaf blades was accompanied by ultrastructural changes in mitochondria and chloroplasts. These abnormalities correlated with elevated levels of reactive oxygen species and carbonylated mitochondrial proteins. We found that two classes of molecular chaperones, Hsp70 and prohibitins, were over-expressed in ftsh4 mutants during late vegetative growth under both short- and long-day conditions. Taken together, our data indicate that lack of AtFtsH4 results in impairment of organelle development and Arabidopsis leaf morphology under short-day conditions.