Adjustment of photosynthetic activity to drought and fluctuating light in wheat.

Drought is a major cause of losses in crop yield. Under field conditions, plants exposed to drought are usually also experiencing rapid changes in light intensity. Accordingly, plants need to acclimate to both, drought and light stress. Two crucial mechanisms in plant acclimation to changes in light conditions comprise thylakoid protein phosphorylation and dissipation of light energy as heat by non-photochemical quenching (NPQ). Here, we analyzed the acclimation efficacy of two different wheat varieties, by applying fluctuating light for analysis of plants, which had been subjected to a slowly developing drought stress as it usually occurs in the field. This novel approach allowed us to distinguish four drought phases, which are critical for grain yield, and to discover acclimatory responses which are independent of photodamage. In short-term, under fluctuating light, the slowdown of NPQ relaxation adjusts the photosynthetic activity to the reduced metabolic capacity. In long-term, the photosynthetic machinery acquires a drought-specific configuration by changing the PSII-LHCII phosphorylation pattern together with protein stoichiometry. Therefore, the fine-tuning of NPQ relaxation and PSII-LHCII phosphorylation pattern represent promising traits for future crop breeding strategies.

Protein sorting into protein bodies during barley endosperm development is putatively regulated by cytoskeleton members, MVBs and the HvSNF7s.

Cereal endosperm is a short-lived tissue adapted for nutrient storage, containing specialized organelles, such as protein bodies (PBs) and protein storage vacuoles (PSVs), for the accumulation of storage proteins. During development, protein trafficking and storage require an extensive reorganization of the endomembrane system. Consequently, endomembrane-modifying proteins will influence the final grain quality and yield. However, little is known about the molecular mechanism underlying endomembrane system remodeling during barley grain development. By using label-free quantitative proteomics profiling, we quantified 1,822 proteins across developing barley grains. Based on proteome annotation and a homology search, 94 proteins associated with the endomembrane system were identified that exhibited significant changes in abundance during grain development. Clustering analysis allowed characterization of three different development phases; notably, integration of proteomics data with in situ subcellular microscopic analyses showed a high abundance of cytoskeleton proteins associated with acidified PBs at the early development stages. Moreover, endosomal sorting complex required for transport (ESCRT)-related proteins and their transcripts are most abundant at early and mid-development. Specifically, multivesicular bodies (MVBs), and the ESCRT-III HvSNF7 proteins are associated with PBs during barley endosperm development. Together our data identified promising targets to be genetically engineered to modulate seed storage protein accumulation that have a growing role in health and nutritional issues.

The High Light Response in Arabidopsis Requires the Calcium Sensor Protein CAS, a Target of STN7- and STN8-Mediated Phosphorylation.

Reversible phosphorylation of thylakoid proteins contributes to photoacclimation responses in photosynthetic organisms, enabling the fine-tuning of light harvesting under changing light conditions and promoting the onset of photoprotective processes. However, the precise functional role of many of the described phosphorylation events on thylakoid proteins remains elusive. The calcium sensor receptor protein (CAS) has previously been indicated as one of the targets of the state transition kinase 8 (STN8). Here we show that in Arabidopsis thaliana, CAS is also phosphorylated by the state transition kinase 7 (STN7), as well as by another, so-far unknown, Ca2+-dependent kinase. Phosphoproteomics analysis and in vitro phosphorylation assays on CAS variants identified the phylogenetically conserved residues Thr-376, Ser-378, and Thr-380 as the major phosphorylation sites of the STN kinases. Spectroscopic analyses of chlorophyll fluorescence emission at 77K further showed that, while the cas mutant is not affected in state transition, it displays a persistent strong excitation of PSI under high light exposure, similar to the phenotype previously observed in other mutants defective in photoacclimation mechanisms. Together with the observation of a strong concomitant phosphorylation of light harvesting complex II (LHCII) and photosynthetic core proteins under high irradiance in the cas mutant this suggests a role for CAS in the STN7/STN8/TAP38 network of phosphorylation-mediated photoacclimation processes in Arabidopsis.

Quantitative Phosphoproteomic and System-Level Analysis of TOR Inhibition Unravel Distinct Organellar Acclimation in Chlamydomonas reinhardtii.

Rapamycin is an inhibitor of the evolutionary conserved Target of Rapamycin (TOR) kinase which promotes and coordinates translation with cell growth and division. In heterotrophic organisms, TOR regulation is based on intra- and extracellular stimuli such as amino acids level and insulin perception. However, how plant TOR pathways have evolved to integrate plastid endosymbiosis is a remaining question. Despite the close association of the TOR signaling with the coordination between protein turn-over and growth, proteome and phosphoproteome acclimation to a rapamycin treatment have not yet been thoroughly investigated in Chlamydomonas reinhardtii. In this study, we have used in vivo label-free phospho-proteomic analysis to profile both protein and phosphorylation changes at 0, 24, and 48 h in Chlamydomonas cells treated with rapamycin. Using multivariate statistics we highlight the impact of TOR inhibition on both the proteome and the phosphoproteome. Two-way ANOVA distinguished differential levels of proteins and phosphoproteins in response either to culture duration and rapamycin treatment or combined effects. Finally, protein-protein interaction networks and functional enrichment analysis underlined the relation between plastid and mitochondrial metabolism. Prominent changes of proteins involved in sulfur, cysteine, and methionine as well as nucleotide metabolism on the one hand, and changes in the TCA cycle on the other highlight the interplay of chloroplast and mitochondria metabolism. Furthermore, TOR inhibition revealed changes in the endomembrane trafficking system. Phosphoproteomics data, on the other hand, highlighted specific differentially regulated phosphorylation sites for calcium-regulated protein kinases as well as ATG7, S6K, and PP2C. To conclude we provide a first combined Chlamydomonas proteomics and phosphoproteomics dataset in response to TOR inhibition, which will support further investigations.

Microscopic and Proteomic Analysis of Dissected Developing Barley Endosperm Layers Reveals the Starchy Endosperm as Prominent Storage Tissue for ER-Derived Hordeins Alongside the Accumulation of Barley Protein Disulfide Isomerase (HvPDIL1-1).

Barley (Hordeum vulgare) is one of the major food sources for humans and forage sources for animal livestock. The average grain protein content (GPC) of barley ranges between 8 and 12%. Barley hordeins (i.e., prolamins) account for more than 50% of GPC in mature seeds and are important for both grain and flour quality. Barley endosperm is structured into three distinct cell layers: the starchy endosperm, which acts essentially as storage tissue for starch; the subaleurone, which is characterized by a high accumulation of seed storage proteins (SSPs); and the aleurone, which has a prominent role during seed germination. Prolamins accumulate in distinct, ER-derived protein bodies (PBs) and their trafficking route is spatio-temporally regulated. The protein disulfide isomerase (PDI) has been shown to be involved in PB formation. Here, we unravel the spatio-temporal proteome regulation in barley aleurone, subaleurone, and starchy endosperm for the optimization of end-product quality in barley. We used laser microdissection (LMD) for subsequent nanoLC-MS/MS proteomic analyses in two experiments: in Experiment One, we investigated the proteomes of dissected barley endosperm layers at 12 and at ≥20 days after pollination (DAP). We found a set of 10 proteins that were present in all tissues at both time points. Among these proteins, the relative protein abundance of D-hordein, B3-hordein and HvPDIL1-1 significantly increased in starchy endosperm between 12 and ≥20 DAP, identifying the starchy endosperm as putative major storage tissue. In Experiment Two, we specifically compared the starchy endosperm proteome at 6, 12, and ≥20 DAP. Whereas the relative protein abundance of D-hordein and B3-hordein increased between 6 and ≥20 DAP, HvPDIL1-1 increased between 6 and 12 DAP, but remained constant at ≥20 DAP. Microscopic observations showed that these relative protein abundance alterations were accompanied by additional localization of hordeins at the periphery of starch granules and a partial re-localization of HvPDIL1-1 from PBs to the periphery of starch granules. Our data indicate a spatio-temporal regulation of hordeins and HvPDIL1-1. These results are discussed in relation to the putative role of HvPDIL1-1 in end-product quality in barley.

Using RT-qPCR, Proteomics, and Microscopy to Unravel the Spatio-Temporal Expression and Subcellular Localization of Hordoindolines Across Development in Barley Endosperm.

Hordeum vulgare (barley) hordoindolines (HINs), HINa, HINb1, and HINb2, are orthologous proteins of wheat puroindolines (PINs) that are small, basic, cysteine-rich seed-specific proteins and responsible for grain hardness. Grain hardness is, next to its protein content, a major quality trait. In barley, HINb is most highly expressed in the mid-stage developed endosperm and is associated with both major endosperm texture and grain hardness. However, data required to understand the spatio-temporal dynamics of HIN transcripts and HIN protein regulation during grain filling processes are missing. Using reverse transcription quantitative PCR (RT-qPCR) and proteomics, we analyzed HIN transcript and HIN protein abundance from whole seeds (WSs) at four [6 days after pollination (dap), 10, 12, and ≥20 dap] as well as from aleurone, subaleurone, and starchy endosperm at two (12 and ≥20 dap) developmental stages. At the WS level, results from RT-qPCR, proteomics, and western blot showed a continuous increase of HIN transcript and HIN protein abundance across these four developmental stages. Miroscopic studies revealed HIN localization mainly at the vacuolar membrane in the aleurone, at protein bodies (PBs) in subaleurone and at the periphery of starch granules in the starchy endosperm. Laser microdissetion (LMD) proteomic analyses identified HINb2 as the most prominent HIN protein in starchy endosperm at ≥20 dap. Additionally, our quantification data revealed a poor correlation between transcript and protein levels of HINs in subaleurone during development. Here, we correlated data achieved by RT-qPCR, proteomics, and microscopy that reveal different expression and localization pattern of HINs in each layer during barley endosperm development. This indicates a contribution of each tissue to the regulation of HINs during grain filling. The effect of the high protein abundance of HINs in the starchy endosperm and their localization at the periphery of starch granules at late development stages at the cereal-based end-product quality is discussed. Understanding the spatio-temporal regulated HINs is essential to improve barley quality traits for high end-product quality, as hard texture of the barley grain is regulated by the ratio between HINb/HINa.

Studying AMPK in an Evolutionary Context.

The AMPK protein kinase forms the heart of a complex network controlling the metabolic activities in a eukaryotic cell. Unraveling the steps by which this pathway evolved from its primordial roots in the last eukaryotic common ancestor to its present status in contemporary species has the potential to shed light on the evolution of eukaryotes. A homolog search for the proteins interacting in this pathway is considerably straightforward. However, interpreting the results, when reconstructing the evolutionary history of the pathway over larger evolutionary distances, bears a number of pitfalls. With this in mind, we present a protocol to trace a metabolic pathway across contemporary species and backward in evolutionary time. Alongside the individual analysis steps, we provide guidelines for data interpretation generalizing beyond the analysis of AMPK.

Quantitative in vivo phosphoproteomics reveals reversible signaling processes during nitrogen starvation and recovery in the biofuel model organism Chlamydomonas reinhardtii.

BACKGROUND: Nitrogen deprivation and replenishment induces massive changes at the physiological and molecular level in the green alga Chlamydomonas reinhardtii, including reversible starch and lipid accumulation. Stress signal perception and acclimation involves transient protein phosphorylation. This study aims to provide the first experimental phosphoprotein dataset for the adaptation of C. reinhardtii during nitrogen depletion and recovery growth phases and its impact on lipid accumulation. RESULTS: To decipher the signaling pathways involved in this dynamic process, we applied a label-free in vivo shotgun phosphoproteomics analysis on nitrogen-depleted and recovered samples. 1227 phosphopeptides belonging to 732 phosphoproteins were identified and quantified. 470 phosphopeptides showed a significant change across the experimental set-up. Multivariate statistics revealed the reversible phosphorylation process and the time/condition-dependent dynamic rearrangement of the phosphoproteome. Protein-protein interaction analysis of differentially regulated phosphoproteins identified protein kinases and phosphatases, such as DYRKP and an AtGRIK1 orthologue, called CDPKK2, as central players in the coordination of translational, photosynthetic, proteomic and metabolomic activity. Phosphorylation of RPS6, ATG13, and NNK1 proteins points toward a specific regulation of the TOR pathway under nitrogen deprivation. Differential phosphorylation pattern of several eukaryotic initiation factor proteins (EIF) suggests a major control on protein translation and turnover. CONCLUSION: This work provides the first phosphoproteomics dataset obtained for Chlamydomonas responses to nitrogen availability, revealing multifactorial signaling pathways and their regulatory function for biofuel production. The reproducibility of the experimental set-up allows direct comparison with proteomics and metabolomics datasets and refines therefore the current model of Chlamydomonas acclimation to various nitrogen levels. Integration of physiological, proteomics, metabolomics, and phosphoproteomics data reveals three phases of acclimation to N availability: (i) a rapid response triggering starch accumulation as well as energy metabolism while chloroplast structure is conserved followed by (ii) chloroplast degradation combined with cell autophagy and lipid accumulation and finally (iii) chloroplast regeneration and cell growth activation after nitrogen replenishment. Plastid development seems to be further interconnected with primary metabolism and energy stress signaling in order to coordinate cellular mechanism to nitrogen availability stress.

Redox state-dependent modulation of plant SnRK1 kinase activity differs from AMPK regulation in animals.

The evolutionarily highly conserved SNF1-related protein kinase (SnRK1) protein kinase is a metabolic master regulator in plants, balancing the critical energy consumption between growth- and stress response-related metabolic pathways. While the regulation of the mammalian [AMP-activated protein kinase (AMPK)] and yeast (SNF1) orthologues of SnRK1 is well-characterised, the regulation of SnRK1 kinase activity in plants is still an open question. Here we report that the activity and T-loop phosphorylation of AKIN10, the kinase subunit of the SnRK1 complex, is regulated by the redox status. Although this regulation is dependent on a conserved cysteine residue, the underlying mechanism is different to the redox regulation of animal AMPK and has functional implications for the regulation of the kinase complex in plants under stress conditions.

Biodiesel and poly-unsaturated fatty acids production from algae and crop plants - a rapid and comprehensive workflow for lipid analysis.

Fatty acid methyl ester analysis (FAME) by gas chromatography coupled to mass spectrometry (GC-MS) is a widely used technique in biodiesel/bioproduct (e.g. poly-unsaturated fatty acids, PUFA) research but typically does not allow distinguishing between bound and free fatty acids. To understand and optimize biosynthetic pathways, however, the origin of the fatty acid is an important information. Furthermore the annotation of PUFAs is compromised in classical GC-EI-MS because the precursor molecular ion is missing. In the present protocol an alkaline methyl esterification step with TMS derivatization enabling the simultaneous analysis of bound and free fatty acids but also further lipids such as sterols in one GC-MS chromatogram is combined. This protocol is applied to different lipid extracts from single cell algae to higher plants: Chlorella vulgaris, Chlamydomonas reinhardtii, Coffea arabica, Pisum sativum and Cuscuta japonica. Further, field ionization (GC-FI-MS) is introduced for a better annotation of fatty acids and exact determination of the number of double bonds in PUFAs. The proposed workflow provides a convenient strategy to analyze algae and other plant crop systems with respect to their capacity for third generation biodiesel and high-quality bioproducts for nutrition such as PUFAs.

An evolutionary perspective of AMPK-TOR signaling in the three domains of life.

AMPK and TOR protein kinases are the major control points of energy signaling in eukaryotic cells and organisms. They form the core of a complex regulatory network to co-ordinate metabolic activities in the cytosol with those in the mitochondria and plastids. Despite its relevance, it is still unclear when and how this regulatory pathway was formed during evolution, and to what extent its representations in the major eukaryotic lineages resemble each other. Here we have traced 153 essential proteins forming the human AMPK-TOR pathways across 412 species representing all three domains of life-prokaryotes (bacteria, archaea) and eukaryotes-and reconstructed their evolutionary history. The resulting phylogenetic profiles indicate the presence of primordial core pathways including seven proto-kinases in the last eukaryotic common ancestor. The evolutionary origins of the oldest components of the AMPK pathway, however, extend into the pre-eukaryotic era, and descendants of these ancient proteins can still be found in contemporary prokaryotes. The TOR complex in turn appears as a eukaryotic invention, possibly to aid in retrograde signaling between the mitochondria and the remainder of the cell. Within the eukaryotes, AMPK/TOR showed both a highly conserved core structure and a considerable plasticity. Most notably, KING1, a protein originally assigned as the γ subunit of AMPK in plants, is more closely related to the yeast SDS23 gene family than to the γ subunits in animals or fungi. This suggests its functional difference from a canonical AMPK γ subunit.

Comprehensive tissue-specific proteome analysis of drought stress responses in Pennisetum glaucum (L.) R. Br. (Pearl millet).

UNLABELLED: Pearl millet is the fifth most important cereal crop worldwide and cultivated especially by small holder farmers in arid and semi-arid regions because of its drought and salt tolerance. The molecular mechanisms of drought stress tolerance in Pennisetum remain elusive. We have used a shotgun proteomics approach to investigate protein signatures from different tissues under drought and control conditions. Drought stressed plants showed significant changes in stomatal conductance and increased root growth compared to the control plants. Root, leaf and seed tissues were harvested and 2281 proteins were identified and quantified in total. Leaf tissue showed the largest number of significant changes (120), followed by roots (25) and seeds (10). Increased levels of root proteins involved in cell wall-, lipid-, secondary- and signaling metabolism and the concomitantly observed increased root length point to an impaired shoot-root communication under drought stress. The harvest index (HI) showed a significant reduction under drought stress. Proteins with a high correlation to the HI were identified using sparse partial least square (sPLS) analysis. Considering the importance of Pearl millet as a stress tolerant food crop, this study provides a first reference data set for future investigations of the underlying molecular mechanisms. BIOLOGICAL SIGNIFICANCE: Drought stress is the most limiting factor for plant growth and crop production worldwide. At the same time drought susceptible cereal crops are among the largest producers worldwide. In contrast, Pearl millet is a drought and salt tolerant cereal crop especially used in arid and semi-arid regions by small farmers. The multifactorial molecular mechanisms of this unique drought tolerance are not known. Here, we employ shotgun proteomics for a first characterization of the Pearl millet drought stress proteome. The experimental setup and the data set generated from this study reveal comprehensive physiological and proteomic responses of the drought stressed Pearl millet plants. Our study reveals statistically significant tissue-specific protein signatures during the adaptation to drought conditions. Thus, the work provides a first reference study of the drought stress proteome and related drought responsive proteins (DRP's) in Pearl millet.