Mass spectrometric resolution of reversible protein phosphorylation in photosynthetic membranes of Arabidopsis thaliana

The use of mass spectrometry to characterize the phosphorylome, i.e. the constituents of the proteome that become phosphorylated, was demonstrated using the reversible phosphorylation of chloroplast thylakoid proteins as an example. From the analysis of tryptic peptides released from the surface of Arabidopsis thylakoids, the principal phosphoproteins were identified by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. These studies revealed that the D1, D2, and CP43 proteins of the photosystem II core are phosphorylated at their N-terminal threonines (Thr), the peripheral PsbH protein is phosphorylated at Thr-2, and the mature light-harvesting polypeptides LCHII are phosphorylated at Thr-3. In addition, a doubly phosphorylated form of PsbH modified at both Thr-2 and Thr-4 was detected. By comparing the levels of phospho- and nonphosphopeptides, the in vivo phosphorylation states of these proteins were analyzed under different physiological conditions. None of these thylakoid proteins were completely phosphorylated in the steady state conditions of continuous light or completely dephosphorylated after a long dark adaptation. However, rapid reversible hyperphosphorylation of PsbH at Thr-4 in response to growth in light/dark transitions and a pronounced specific dephosphorylation of the D1, D2, and CP43 proteins during heat shock was detected. Collectively, our data indicate that changes in the phosphorylation of photosynthetic proteins are more rapid during heat stress than during normal light/dark transitions. These mass spectrometry methods offer a new approach to assess the stoichiometry of in vivo protein phosphorylation in complex samples.     

ATG7 contributes to plant basal immunity towards fungal infection

Autophagy has an important function in cellular homeostasis. In recent years autophagy has been implicated in plant basal immunity and assigned negative (“anti-death”) and positive (“pro-death”) regulatory functions in controlling cell death programs that establish sufficient immunity to microbial infection. We recently showed that Arabidopsis mutants lacking the autophagy-associated (ATG) genes ATG5, ATG10 and ATG18a are compromised in their resistance towards infection with necrotrophic fungal pathogens but display an enhanced resistance towards biotrophic bacterial invaders. Thus, the function of autophagy as either being pro-death or anti-death depends critically on the lifestyle and infection strategy of invading microbes. Here we show that ATG7 contributes to resistance to fungal pathogens. Genetic inactivation of ATG7 results in elevated susceptibility towards the necrotrophic fungal pathogen, Alternaria brassicicola, with atg7 mutants developing spreading necrosis accompanied by production of reactive oxygen intermediates. Likewise, treatment with the fungal toxin fumonisin B1 causes spreading lesion formation in the atg7 mutant. We conclude that ATG7-dependent autophagy constitutes an “anti-death” (“pro-survival”) plant mechanism to control the containment of cell death and immunity to necrophic fungal infection.Key words: autophagy, ATG7, basal immunity, fungal resistance, arabidopsisPlants have evolved a bipartite plant immune system to cope with microbial infections. The first layer of defense relies on the recognition of pathogen-associated molecular patterns (PAMP) by pattern-recognition receptors (PAMP-triggered immunity, PTI).^1,2 To overcome this defense strategy, successful pathogens deliver so-called effector proteins into plant cells to modify host cellular processes and to suppress immune responses to enhance virulence. The presence or activities of these microbial effectors is sensed by plant resistance proteins and triggers the second layer of defense, the effector-triggered immunity (ETI).^1,2 In contrast to PTI, ETI is most often accompanied by programmed host cell death (PCD) at the site of attempted microbial invasion; however the molecular basis of this apoptosis-like hypersensitive response (HR) is largely unknown.In recent years evidence accumulated that a non-apoptotic form of cell death called autophagy is not only involved in animal PCD and innate immunity³ but is also an important component in the plant basal immune response.⁴ Generally, autophagy (auto, meaning “self” and phagy, “to eat”) is a cytoplasmic bulk degradation process in which cellular components are targeted to lysosomal or vacuolar degradation. This process is ubiquitous in eukaryotic organisms and is considered to aid cellular survival, differentiation, development and homeostasis by nutrient recycling or removal of damaged or toxic materials.^5–7     

Regulator and substrate: Dual roles for the ATG1-ATG13 kinase complex during autophagic recycling in Arabidopsis

Like other organisms, plants rely on autophagy to recycle intracellular components needed for development, new growth and survival during nutrient stress. This 'self eating' is a catabolic process by which unwanted cytoplasmic materials and dysfunctional organelles are sequestered into vesicles and subsequently delivered to the vacuole for breakdown. The process is tightly regulated by the autophagy-related 1(ATG1)-ATG13 kinase complex which is controlled by multiple nutrient-responsive upstream regulators that integrate nutrient demand with availability. To further appreciate how autophagy is controlled in plants, we recently examined the functions of the ATG1-ATG13 complex in Arabidopsis thaliana. Our data revealed a dual role for the ATG1-ATG13 complex, first as a regulator of plant autophagy, and second as a substrate of this recycling process.     

Mutational analysis of Deinococcus radiodurans bacteriophytochrome reveals key amino acids necessary for the photochromicity and proton exchange cycle of phytochromes

The ability of phytochromes (Phy) to act as photointerconvertible light switches in plants and microorganisms depends on key interactions between the bilin chromophore and the apoprotein that promote bilin attachment and photointerconversion between the spectrally distinct red light-absorbing Pr conformer and far red light-absorbing Pfr conformer. Using structurally guided site-directed mutagenesis combined with several spectroscopic methods, we examined the roles of conserved amino acids within the bilin-binding domain of Deinococcus radiodurans bacteriophytochrome with respect to chromophore ligation and Pr/Pfr photoconversion. Incorporation of biliverdin IXalpha (BV), its structure in the Pr state, and its ability to photoisomerize to the first photocycle intermediate are insensitive to most single mutations, implying that these properties are robust with respect to small structural/electrostatic alterations in the binding pocket. In contrast, photoconversion to Pfr is highly sensitive to the chromophore environment. Many of the variants form spectrally bleached Meta-type intermediates in red light that do not relax to Pfr. Particularly important are Asp-207 and His-260, which are invariant within the Phy superfamily and participate in a unique hydrogen bond matrix involving the A, B, and C pyrrole ring nitrogens of BV and their associated pyrrole water. Resonance Raman spectroscopy demonstrates that substitutions of these residues disrupt the Pr to Pfr protonation cycle of BV with the chromophore locked in a deprotonated Meta-R(c)-like photoconversion intermediate after red light irradiation. Collectively, the data show that a number of contacts contribute to the unique photochromicity of Phy-type photoreceptors. These include residues that fix the bilin in the pocket, coordinate the pyrrole water, and possibly promote the proton exchange cycle during photoconversion.     

Subunit interaction maps for the regulatory particle of the 26S proteasome and the COP9 signalosome.

The 26S proteasome plays a major role in eukaryotic protein breakdown, especially for ubiquitin-tagged proteins. Substrate specificity is conferred by the regulatory particle (RP), which can dissociate into stable lid and base subcomplexes. To help define the molecular organization of the RP, we tested all possible paired interactions among subunits from Saccharomyces cerevisiae by yeast two-hybrid analysis. Within the base, a Rpt4/5/3/6 interaction cluster was evident. Within the lid, a structural cluster formed around Rpn5/11/9/8. Interactions were detected among synonymous subunits (Csn4/5/7/6) from the evolutionarily related COP9 signalosome (CSN) from Arabidopsis, implying a similar quaternary arrangement. No paired interactions were detected between lid, base or core particle subcomplexes, suggesting that stable contacts between them require prior assembly. Mutational analysis defined the ATPase, coiled-coil, PCI and MPN domains as important for RP assembly. A single residue in the vWA domain of Rpn10 is essential for amino acid analog resistance, for degrading a ubiquitin fusion degradation substrate and for stabilizing lid-base association. Comprehensive subunit interaction maps for the 26S proteasome and CSN support the ancestral relationship of these two complexes.     

Homologs of the essential ubiquitin conjugating enzymes UBC1, 4, and 5 in yeast are encoded by a multigene family in Arabidopsis thaliana

The covalent attachment of the 76 amino acid protein ubiquitin is an important prerequisite for the degradation of many eukaryotic proteins. The specificity of this ligation is accomplished in part by a family of distinct ubiquitin conjugating enzymes (E2s) working in concert with specific ubiquitin-protein ligases (E3s). Three essential E2s in yeast encoded by ScUBC1, −4 , and − 5 comprise a functionally overlapping E2 subfamily that appears responsible for degrading most abnormal and short-lived proteins. A 15 kDa E2 protein homologous to this family has been identified previously in wheat germ, designated Ta E2_15kDa (Girod and Vierstra (1993) J. Biol. Chem. 268, 955–960). This E2 is responsible for much of the ubiquitin conjugating activity observed in wheat germ extracts and works together with a unique E3 (designated E3γ) for substrate recognition. In this paper, the cloning of five genes encoding E2_15kDa from Arabidiopsis thaliana is described (designated AtUBC8—12 ). They encode 149 amino acid basic proteins 94–98% similar to each other and 88–92% similar to ScUBC4 at the amino acid sequence level. In contrast, AtUBC8—12 are only 55–65% similar to the Arabidopsis E2s encoded by AtUBC1, −4, and − 7 . The At UBC8—12 proteins do not contain N- or C-terminal extensions and have the active site at residue Cys-86, based on their homology with other E2s. Analyses of genomic Southern blots are consistent with the existence of multiple members encoding this E2 subfamily. AtUBC8—12 are transcribed to yield about 800 nucleotide mRNAs that, unlike ScUBC4 and − 5 , are not strongly induced by heat shock. Expression of AtUBC8 in Escherichia coli results in substantial production of functional E2_15kDa that works together with wheat E3γ in conjugating ubiquitin to endogenous or added substrates in vitro .     

Phytochrome A overexpression in transgenic tobacco. Correlation of dwarf phenotype with high concentrations of phytochrome in vascular tissue and attenuated gibberellin levels.

Phytochromes are a family of related chromoproteins that regulate photomorphogenesis in plants. Ectopic overexpression of the phytochrome A in several plant species has pleiotropic effects, including substantial dwarfing, increased pigmentation, and delayed leaf senescence. We show here that the dwarf response is related to a reduction in active gibberellins (GAs) in tobacco (Nicotiana tabacum) overexpressing oat phytochrome A under the control of the cauliflower mosaic virus (CaMV) 35S promoter and can be suppressed by foliar applications of gibberellic acid. In transgenic seedlings, high concentrations of oat phytochrome A were detected in stem and petiole vascular tissue (consistent with the activity of the CaMV 35S promoter), implicating vascular tissue as a potential site of phytochrome A action. To examine the efficacy of this cellular site, oat phytochrome A was also expressed using Arabidopsis chlorophyll a/b-binding protein (CAB) and the Arabidopsis ubiquitin (UBQ1) promoters. Neither promoter was as effective as CaMV 35S in expressing phytochrome in vascular tissue or in inducing the dwarf phenotype. Collectively, these data indicate that the spatial distribution of ectopic phytochrome is important in eliciting the dwarf response and suggest that the phenotype is invoked by elevated levels of the far-red-absorbing form of phytochrome within vascular tissue repressing GA biosynthesis.     

Is the far-red-absorbing form of Avena phytochrome A that is present at the end of the day able to sustain stem-growth inhibition during the night in transgenic tobacco and tomato seedlings?

Avena phytochrome A (phyA) overexpressed in tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon sculentum Mill) was functionally characterised by comparing wild-type (WT) and transgenic seedlings. Different proportions of phytochrome in its far-red-absorbing form (Pfr/P) were provided by end-of-day (EOD) light pulses. Stem-length responses occurred largely in the range of low Pfr/P (3–61%) for WT seedlings and in the range of high Pfr/P (61–87%) for transgenic seedlings. A similar shift was observed when the photoperiod was interrupted by short light pulses providing different Pfr/P ratios and followed by 1 h dark incubation. In other experiments, Avena phyA was allowed to re-accumulate in darkness and subsequently phototransformed to Pfr but no extra inhibition of stem extension growth was observed. In transgenic tomato seedlings the response to EOD far-red light was faster and the response to a far-red light pulse delayed into darkness was larger than in the WT. Avena phyA Pfr remaining at the end of the photoperiod appears intrinsically unable to sustain growth inhibition in subsequent darkness. Avena phyA modifies the sensitivity and the kinetics of EOD responses mediated by native phytochrome.Abbreviations EOD end-of-day - FR far-red light - Pfr/P pro-portion of phytochrome in its FR-absorbing form - phyA phyto-chrome A - phyB phytochrome B - R red light - RFR R to FR ratio - WT wild type We thank Dr Brian Thomas for providing the antibodies used in this work, and Federico Guerendiain for his excellent technical assistance. This work was financially supported by grants UBA AG 040 and Fundacion Antorchas A-12830/1-19 (both to J.J.C.), PID-CONICET (to R.A.S. and J.J.C.), United States Department of Energy DE-FG02-88ER13968 (to R.D.V.).