Sonia Hem

Regulation of Arabidopsis Leaf Hydraulics Involves Light-Dependent Phosphorylation of Aquaporins in Veins

The function and regulation of water channel proteins (aquaporins) in plant leaves was dissected by combined genetic, physiological, and proteomic approaches, and their limiting role for water transport in the living cells of veins was established. Phosphorylation of a single aquaporin isoform at two sites was shown to be necessary for enhancement of leaf water permeability in response to darkness. The water status of plant leaves depends on the efficiency of the water supply, from the vasculature to inner tissues. This process is under hormonal and environmental regulation and involves aquaporin water channels. In Arabidopsis thaliana, the rosette hydraulic conductivity (Kros) is higher in darkness than it is during the day. Knockout plants showed that three plasma membrane intrinsic proteins (PIPs) sharing expression in veins (PIP1;2, PIP2;1, and PIP2;6) contribute to rosette water transport, and PIP2;1 can fully account for Kros responsiveness to darkness. Directed expression of PIP2;1 in veins of a pip2;1 mutant was sufficient to restore Kros. In addition, a positive correlation, in both wild-type and PIP2;1-overexpressing plants, was found between Kros and the osmotic water permeability of protoplasts from the veins but not from the mesophyll. Thus, living cells in veins form a major hydraulic resistance in leaves. Quantitative proteomic analyses showed that light-dependent regulation of Kros is linked to diphosphorylation of PIP2;1 at Ser-280 and Ser-283. Expression in pip2;1 of phosphomimetic and phosphorylation-deficient forms of PIP2;1 demonstrated that phosphorylation at these two sites is necessary for Kros enhancement under darkness. These findings establish how regulation of a single aquaporin isoform in leaf veins critically determines leaf hydraulics.

Convergent Evolution of Endosymbiont Differentiation in Dalbergioid and Inverted Repeat-Lacking Clade Legumes Mediated by Nodule-Specific Cysteine-Rich Peptides

Several species from an ancient legume lineage independently evolved a novel class of cysteine-rich peptides to impose a differentiation process on their endosymbionts. Nutritional symbiotic interactions require the housing of large numbers of microbial symbionts, which produce essential compounds for the growth of the host. In the legume-rhizobium nitrogen-fixing symbiosis, thousands of rhizobium microsymbionts, called bacteroids, are confined intracellularly within highly specialized symbiotic host cells. In Inverted Repeat-Lacking Clade (IRLC) legumes such as Medicago spp., the bacteroids are kept under control by an arsenal of nodule-specific cysteine-rich (NCR) peptides, which induce the bacteria in an irreversible, strongly elongated, and polyploid state. Here, we show that in Aeschynomene spp. legumes belonging to the more ancient Dalbergioid lineage, bacteroids are elongated or spherical depending on the Aeschynomene spp. and that these bacteroids are terminally differentiated and polyploid, similar to bacteroids in IRLC legumes. Transcriptome, in situ hybridization, and proteome analyses demonstrated that the symbiotic cells in the Aeschynomene spp. nodules produce a large diversity of NCR-like peptides, which are transported to the bacteroids. Blocking NCR transport by RNA interference-mediated inactivation of the secretory pathway inhibits bacteroid differentiation. Together, our results support the view that bacteroid differentiation in the Dalbergioid clade, which likely evolved independently from the bacteroid differentiation in the IRLC clade, is based on very similar mechanisms used by IRLC legumes.

Towards the profiling of the Arabidopsis thaliana plasma membrane transportome by targeted proteomics

Plant membranes bear a variety of transporters belonging to multigene families that are affected by environmental and nutritional conditions. In addition, they often display high‐sequence identity, making difficult in‐depth investigation by current shot‐gun strategies. In this study, we set up a targeted proteomics approach aimed at identifying and quantifying within single experiments the five major proton pumps of the autoinhibited H+ATPases (AHA) family, the 13 plasma membrane intrinsic proteins (PIP) water channels (PIPs), and ten members of ammonium transporters (AMTs) and nitrate transporter (NRT) families. Proteotypic peptides were selected and isotopically labeled heavy versions were used for technical optimization and for quantification of the corresponding light version in biological samples. This approach allowed to quantify simultaneously nine PIPs in leaf membranes and 13 PIPs together with three autoinhibited H+ATPases, two ammonium transporters, and two NRTs in root membranes. Similarly, it was used to investigate the effect of a salt stress on the expression of these latter 20 transporters in roots. These novel isoform‐specific data were compared with published transcriptome information and revealed a close correlation between PIP isoforms and transcripts levels. The obtained resource is reusable and can be expanded to other transporter families for large‐scale profiling of membrane transporters.

Combined transcript, proteome, and metabolite analysis of transgenic maize seeds engineered for enhanced carotenoid synthesis reveals pleotropic effects in core metabolism

Highlight Metabolomic, proteomic, and transcriptomic analysis of a maize line genetically engineered for enhanced seed carotenoid biosynthesis revealed how the sugar metabolism adapted to meet the additional precursor supply.

FUBP1: a new protagonist in splicing regulation of the DMD gene.

We investigated the molecular mechanisms for in-frame skipping of DMD exon 39 caused by the nonsense c.5480T>A mutation in a patient with Becker muscular dystrophy. RNase-assisted pull down assay coupled with mass spectrometry revealed that the mutant RNA probe specifically recruits hnRNPA1, hnRNPA2/B1 and DAZAP1. Functional studies in a human myoblast cell line transfected with DMD minigenes confirmed the splicing inhibitory activity of hnRNPA1 and hnRNPA2/B1, and showed that DAZAP1, also known to activate splicing, acts negatively in the context of the mutated exon 39. Furthermore, we uncovered that recognition of endogenous DMD exon 39 in muscle cells is promoted by FUSE binding protein 1 (FUBP1), a multifunctional DNA- and RNA-binding protein whose role in splicing is largely unknown. By serial deletion and mutagenesis studies in minigenes, we delineated a functional intronic splicing enhancer (ISE) in intron 38. FUBP1 recruitment to the RNA sequence containing the ISE was established by RNA pull down and RNA EMSA, and further confirmed by RNA-ChIP on endogenous DMD pre-mRNA. This study provides new insights about the splicing regulation of DMD exon 39, highlighting the emerging role of FUBP1 in splicing and describing the first ISE for constitutive exon inclusion in the mature DMD transcript.

Phosphorylation dynamics of membrane proteins from Arabidopsis roots submitted to salt stress

An excess of NaCl in the soil is detrimental for plant growth. It interferes with mineral nutrition and water uptake and leads to accumulation of toxic ions in the plant. Understanding the response of roots to NaCl stress may facilitate the development of crops with increased tolerance to this and other stresses. Since controls achieved at the posttranslational level are of critical importance for regulating protein function, the present work used a robust label‐free quantitative proteomic methodology to quantify phosphorylation events that affect root membrane proteins in Arabidopsis, in response to short‐term (up to 2 h) NaCl treatments. This work identified 302 proteotypic phosphopeptides including 77 novel phosphorylated sites. NaCl treatment significantly altered the abundance of 74 phosphopeptides, giving novel insights into the regulation of major classes of membrane proteins, including ATPases, sodium transporters, and aquaporins. The data provide a unique access to phosphorylation reprogramming of ionic equilibrium in plant cells under NaCl stress. The use of predictive bioinformatic tools for kinase motifs suggested that root membrane proteins are substrates of cAMP‐dependent protein kinase, cGMP‐dependent protein kinase, and protein kinase C families, also called AGC kinases, arguing for an important role of lipid signaling in abiotic stress responses. It also pointed to cross‐talks between protein kinase families during NaCl stress.

Accumulation of cyclophilin A isoforms in conditioned medium of irradiated breast cancer cells

Secreted proteins play a key role in cell signaling and communication. We recently showed that ionizing radiations induced a delayed cell death of breast cancer cells, mediated by the death receptor pathways through the expression of soluble forms of “death ligands.” Using the same cell model, the objective of our work was the identification of diffusible factors, secreted following cell irradiation, potentially involved in cell death signaling. Differential proteomic analysis of conditioned media using 2DE resulted in detection of numerous spots that were significantly modulated following cell irradiation. The corresponding proteins were identified using MALDI‐TOF MS and LC‐MS/MS approaches. Interestingly, five isoforms of cyclophilin A were observed as increased in conditioned medium of irradiated cells. These isoforms differed in isoelectric points and in accumulation levels. An increase of cyclophilin A secretion was confirmed by Western blotting of conditioned media of irradiated or radiosentive mammary cells. These isoforms displayed an interesting pattern of protein maturation and post‐translational modifications, including an alternating removal of N‐terminal methionine, associated with a combination of acetylations and methylations. The role of the protein is discussed in relation with its potential involvement in the mechanisms of intercells relationships and radiosensitivity.

Metabolic engineering of astaxanthin biosynthesis in maize endosperm and characterization of a prototype high oil hybrid

Maize was genetically engineered for the biosynthesis of the high value carotenoid astaxanthin in the kernel endosperm. Introduction of a β-carotene hydroxylase and a β-carotene ketolase into a white maize genetic background extended the carotenoid pathway to astaxanthin. Simultaneously, phytoene synthase, the controlling enzyme of carotenogenesis, was over-expressed for enhanced carotenoid production and lycopene ε-cyclase was knocked-down to direct more precursors into the β-branch of the extended ketocarotenoid pathway which ends with astaxanthin. This astaxanthin-accumulating transgenic line was crossed into a high oil- maize genotype in order to increase the storage capacity for lipophilic astaxanthin. The high oil astaxanthin hybrid was compared to its astaxanthin producing parent. We report an in depth metabolomic and proteomic analysis which revealed major up- or down- regulation of genes involved in primary metabolism. Specifically, amino acid biosynthesis and the citric acid cycle which compete with the synthesis or utilization of pyruvate and glyceraldehyde 3-phosphate, the precursors for carotenogenesis, were down-regulated. Nevertheless, principal component analysis demonstrated that this compositional change is within the range of the two wild type parents used to generate the high oil producing astaxanthin hybrid.

Proteomic demonstration of the recurrent presence of inter-alpha-inhibitor H4 heavy-chain during aspergillosis induced in an animal model

Invasive pulmonary aspergillosis remains a matter of great concern in oncology/haematology, intensive care units and organ transplantation departments. Despite the availability of various diagnostic tools with attractive features, new markers of infection are required for better medical care. We therefore looked for potential pulmonary biomarkers of aspergillosis, by carrying out two-dimensional (2D) gel electrophoresis comparing the proteomes of bronchial-alveolar lavage fluids (BALF) from infected rats and from control rats presenting non-specific inflammation, both immunocompromised. A bioinformatic analysis of the 2D-maps revealed significant differences in the abundance of 20 protein spots (ANOVA P-value<0.01; q-value<0.03; power>0.8). One of these proteins, identified by mass spectrometry, was considered of potential interest: inter-alpha-inhibitor H4 heavy-chain (ITIH4), characterised for the first time in this infectious context. Western blotting confirmed its overabundance in all infected BALF, particularly at early stages of murine aspergillosis. Further investigations were carried on rat serum, and confirmed that ITIH4 levels increased during experimental aspergillosis. Preliminary results in human samples strengthened this trend. To our knowledge, this is the first description of the involvement of ITIH4 in aspergillosis.

Phosphoproteomic analysis reveals major default phosphorylation sites outside long intrinsically disordered regions of Arabidopsis plasma membrane proteins

BackgroundGenome-wide statistics established that long intrinsically disordered regions (over 30 residues) are predicted in a large part of proteins in all eukaryotes, with a higher ratio in trans-membrane proteins. At functional level, such unstructured and flexible regions were suggested for years to favour phosphorylation events. In plants, despite increasing evidence of the regulation of transport and signalling processes by phosphorylation events, only few data are available without specific information regarding plasma membrane proteins, especially at proteome scale.ResultsUsing a dedicated phosphoproteomic workflow, 75 novel and unambiguous phosphorylation sites were identified in Arabidopsis plasma membrane. Bioinformatics analysis showed that this new dataset concerned mostly integral proteins involved in key functions of the plasma membrane (such as transport and signal transduction, including protein phosphorylation). It thus expanded by 15% the directory of phosphosites previously characterized in signalling and transport proteins. Unexpectedly, 66% of phosphorylation sites were predicted to be located outside long intrinsically disordered regions. This result was further corroborated by analysis of publicly available data for the plasma membrane.ConclusionsThe new phosphoproteomics data presented here, with published datasets and functional annotation, suggest a previously unexpected topology of phosphorylation in the plant plasma membrane proteins. The significance of these new insights into the so far overlooked properties of the plant plasma membrane phosphoproteome and the long disordered regions is discussed.