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.

Sexual dimorphism in PAR2-dependent regulation of primitive colonic cells.

BACKGROUND: Sexual dimorphism in biological responses is a critical knowledge for therapeutic proposals. However, gender differences in intestinal stem cell physiology have been poorly studied. Given the important role of the protease-activated receptor PAR2 in the control of colon epithelial primitive cells and cell cycle genes, we have performed a sex-based comparison of its expression and of the effects of PAR2 activation or knockout on cell proliferation and survival functions. METHODS: Epithelial primitive cells isolated from colons from male and female mice were cultured as colonoids, and their number and size were measured. PAR2 activation was triggered by the addition of SLIGRL agonist peptide in the culture medium. PAR2-deficient mice were used to study the impact of PAR2 expression on colon epithelial cell culture and gene expression. RESULTS: Colonoids from female mice were more abundant and larger compared to males, and these differences were further increased after PAR2 activation by specific PAR2 agonist peptide. The proliferation of male epithelial cells was lower compared to females but was specifically increased in PAR2 knockout male cells. PAR2 expression was higher in male colon cells compared to females and controlled the gene expression and activation of key negative signals of the primitive cell proliferation. This PAR2-dependent brake on the proliferation of male colon primitive cells was correlated with stress resistance. CONCLUSIONS: Altogether, these data demonstrate that there is a sexual dimorphism in the PAR2-dependent regulation of primitive cells of the colon crypt.

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.

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.