Tuan-Nan Wen

iTRAQ Protein Profile Analysis of Arabidopsis Roots Reveals New Aspects Critical for Iron Homeostasis

Iron (Fe) deficiency is a major constraint for plant growth and affects the quality of edible plant parts. To investigate the mechanisms underlying Fe homeostasis in plants, Fe deficiency-induced changes in the protein profile of Arabidopsis (Arabidopsis thaliana) roots were comprehensively analyzed using iTRAQ (Isobaric Tag for Relative and Absolute Quantification) differential liquid chromatography-tandem mass spectrometry on a LTQ-Orbitrap with high-energy collision dissociation. A total of 4,454 proteins were identified with a false discovery rate of less than 1.1%, and 2,882 were reliably quantified. A subset of 101 proteins was differentially expressed upon Fe deficiency. The changes in protein profiles upon Fe deficiency show low congruency with previously reported alterations in transcript levels, indicating posttranscriptional changes, and provide complementary information on Fe deficiency-induced processes. The abundance of proteins involved in the synthesis/regeneration of S-adenosylmethionine, the phenylpropanoid pathway, the response to oxidative stress, and respiration was highly increased by Fe deficiency. Using Fe-responsive proteins as bait, genome-wide fishing for partners with predictable or confirmed interologs revealed that RNA processing and ribonucleoprotein complex assembly may represent critical processes that contribute to the regulation of root responses to Fe deficiency, possibly by biasing translation efficiency.

Genomics and proteomics of immune modulatory effects of a butanol fraction of echinacea purpurea in human dendritic cells

BackgroundEchinacea spp. extracts and the derived phytocompounds have been shown to induce specific immune cell activities and are popularly used as food supplements or nutraceuticals for immuno-modulatory functions. Dendritic cells (DCs), the most potent antigen presenting cells, play an important role in both innate and adaptive immunities. In this study, we investigated the specific and differential gene expression in human immature DCs (iDCs) in response to treatment with a butanol fraction containing defined bioactive phytocompounds extracted from stems and leaves of Echinacea purpurea, that we denoted [BF/S+L/Ep].ResultsAffymetrix DNA microarray results showed significant up regulation of specific genes for cytokines (IL-8, IL-1β, and IL-18) and chemokines (CXCL 2, CCL 5, and CCL 2) within 4 h after [BF/S+L/Ep] treatment of iDCs. Bioinformatics analysis of genes expressed in [BF/S+L/Ep]-treated DCs revealed a key-signaling network involving a number of immune-modulatory molecules leading to the activation of a downstream molecule, adenylate cyclase 8. Proteomic analysis showed increased expression of antioxidant and cytoskeletal proteins after treatment with [BF/S+L/Ep] and cichoric acid.ConclusionThis study provides information on candidate target molecules and molecular signaling mechanisms for future systematic research into the immune-modulatory activities of an important traditional medicinal herb and its derived phytocompounds.

Quantitative phosphoproteome profiling of iron-deficient Arabidopsis roots

Iron (Fe) is an essential mineral nutrient for plants, but often it is not available in sufficient quantities to sustain optimal growth. To gain insights into adaptive processes to low Fe availability at the posttranslational level, we conducted a quantitative analysis of Fe deficiency-induced changes in the phosphoproteome profile of Arabidopsis (Arabidopsis thaliana) roots. Isobaric tags for relative and absolute quantitation-labeled phosphopeptides were analyzed by liquid chromatography-tandem mass spectrometry on an LTQ-Orbitrap with collision-induced dissociation and high-energy collision dissociation capabilities. Using a combination of titanium dioxide and immobilized metal affinity chromatography to enrich phosphopeptides, we extracted 849 uniquely identified phosphopeptides corresponding to 425 proteins and identified several not previously described phosphorylation motifs. A subset of 45 phosphoproteins was defined as being significantly changed in abundance upon Fe deficiency. Kinase motifs in Fe-responsive proteins matched to protein kinase A/calcium calmodulin-dependent kinase II, casein kinase II, and proline-directed kinase, indicating a possible critical function of these kinase classes in Fe homeostasis. To validate our analysis, we conducted site-directed mutagenesis on IAA-CONJUGATE-RESISTANT4 (IAR4), a protein putatively functioning in auxin homeostasis. iar4 mutants showed compromised root hair formation and developed shorter primary roots. Changing serine-296 in IAR4 to alanine resulted in a phenotype intermediate between mutant and wild type, whereas acidic substitution to aspartate to mimic phosphorylation was either lethal or caused an extreme dwarf phenotype, supporting the critical importance of this residue in Fe homeostasis. Our analyses further disclose substantial changes in the abundance of phosphoproteins involved in primary carbohydrate metabolism upon Fe deficiency, complementing the picture derived from previous proteomic and transcriptomic profiling studies.

Directed Mutagenesis of Specific Active Site Residues onFibrobacter succinogenes1,3–1,4-β-d-Glucanase Significantly Affects Catalysis and Enzyme Structural Stability

The functional and structural significance of amino acid residues Met39, Glu56, Asp58, Glu60, and Gly63 ofFibrobacter succinogenes1,3–1,4-β-d-glucanase was explored by the approach of site-directed mutagenesis, initial rate kinetics, fluorescence spectroscopy, and CD spectrometry. Glu56, Asp58, Glu60, and Gly63 residues are conserved among known primary sequences of the bacterial and fungal enzymes. Kinetic analyses revealed that 240-, 540-, 570-, and 880-fold decreases in k cat were observed for the E56D, E60D, D58N, and D58E mutant enzymes, respectively, with a similar substrate affinity relative to the wild type enzyme. In contrast, no detectable enzymatic activity was observed for the E56A, E56Q, D58A, E60A, and E60Q mutants. These results indicated that the carboxyl side chain at positions 56 and 60 is mandatory for enzyme catalysis. M39F, unlike the other mutants, exhibited a 5-fold increase inK m value. Lower thermostability was found with the G63A mutant when compared with wild type or other mutant forms ofF. succinogenes 1,3–1,4-β-d-glucanase. Denatured wild type and mutant enzymes were, however, recoverable as active enzymes when 8 m urea was employed as the denaturant. Structural modeling and kinetic studies suggest that Glu56, Asp58, and Glu60 residues apparently play important role(s) in the catalysis of F. succinogenes 1,3–1,4-β-d-glucanase.

cDNA cloning and gene expression of the major prolamins of rice.

A full-length cDNA (pS 18) encoding the 16 kDa rice prolamin composed of 158 amino acids was sequenced. Analysis of N-terminal amino acid sequence of a major rice prolamin indicated that an 18 amino acid signal peptide was removed from 16 kDa precursor prolamin to form the 14 kDa prolamin during seed development. Synthesis of the 16 kDa precursor prolamin began around 8 days after flowering (DAF), increased remarkably at 8–11 DAF and gradually reached maximum levels with the maturation of rice seeds.

Post-Transcriptional Coordination of the Arabidopsis Iron Deficiency Response is Partially Dependent on the E3 Ligases RING DOMAIN LIGASE1 (RGLG1) and RING DOMAIN LIGASE2 (RGLG2)

Acclimation to changing environmental conditions is mediated by proteins, the abundance of which is carefully tuned by an elaborate interplay of DNA-templated and post-transcriptional processes. To dissect the mechanisms that control and mediate cellular iron homeostasis, we conducted quantitative high-resolution iTRAQ proteomics and microarray-based transcriptomic profiling of iron-deficient Arabidopsis thaliana plants. A total of 13,706 and 12,124 proteins was identified with a quadrupole-Orbitrap hybrid mass spectrometer in roots and leaves, respectively. This deep proteomic coverage allowed accurate estimates of post-transcriptional regulation in response to iron deficiency. Similarly regulated transcripts were detected in only 13% (roots) and 11% (leaves) of the 886 proteins that differentially accumulated between iron-sufficient and iron-deficient plants, indicating that the majority of the iron-responsive proteins was post-transcriptionally regulated. Mutants harboring defects in the RING DOMAIN LIGASE1 (RGLG1)1 and RING DOMAIN LIGASE2 (RGLG2) showed a pleiotropic phenotype that resembled iron-deficient plants with reduced trichome density and the formation of branched root hairs. Proteomic and transcriptomic profiling of rglg1 rglg2 double mutants revealed that the functional RGLG protein is required for the regulation of a large set of iron-responsive proteins including the coordinated expression of ribosomal proteins. This integrative analysis provides a detailed catalog of post-transcriptionally regulated proteins and allows the concept of a chiefly transcriptionally regulated iron deficiency response to be revisited. Protein data are available via ProteomeXchange with identifier PXD002126.

Structural and Functional Roles of Glycosylation in Fungal Laccase from Lentinus sp.

Laccases are multi-copper oxidases that catalyze the oxidation of various organic and inorganic compounds by reducing O2 to water. Here we report the crystal structure at 1.8 Å resolution of a native laccase (designated nLcc4) isolated from a white-rot fungus Lentinus sp. nLcc4 is composed of three cupredoxin-like domains D1-D3 each folded into a Greek key β-barrel topology. T1 and T2/T3 copper binding sites and three N-glycosylated sites at Asn75, Asn238, and Asn458 were elucidated. Initial rate kinetic analysis revealed that the k cat, K m, and k cat/K m of nLcc4 with substrate ABTS were 3,382 s -1, 65.0 ± 6.5 μM, and 52 s -1μM-1, respectively; and the values with lignosulfonic acid determined using isothermal titration calorimetry were 0.234 s -1, 56.7 ± 3.2 μM, and 0.004 s -1μM-1, respectively. Endo H-deglycosylated nLcc4 (dLcc4), with only one GlcNAc residue remaining at each of the three N-glycosylation sites in the enzyme, exhibited similar kinetic efficiency and thermal stability to that of nLcc4. The isolated Lcc4 gene contains an open reading frame of 1563 bp with a deduced polypeptide of 521 amino acid residues including a predicted signaling peptide of 21 residues at the N-terminus. Recombinant wild-type Lcc4 and mutant enzymes N75D, N238D and N458D were expressed in Pichia pastoris cells to evaluate the effect on enzyme activity by single glycosylation site deficiency. The mutant enzymes secreted in the cultural media of P. pastoris cells were observed to maintain only 4-50% of the activity of the wild-type laccase. Molecular dynamics simulations analyses of various states of (de-)glycosylation in nLcc support the kinetic results and suggest that the local H-bond networks between the domain connecting loop D2-D3 and the glycan moieties play a crucial role in the laccase activity. This study provides new insights into the role of glycosylation in the structure and function of a Basidiomycete fungal laccase.

Actin in mung bean mitochondria and implications for its function.

This work demonstrates the existence of actin within plant mitochondria, and the import of actin into mitochondria is shown in fluorescent-actin transgenic plants and in wild-type cotyledon mitochondria during germination. The findings suggest that the dynamics of mitochondrial actin can regulate cytochrome C release and, consequently, may initiate programmed cell death of the mung bean cotyledons. Here, a large fraction of plant mitochondrial actin was found to be resistant to protease and high-salt treatments, suggesting it was protected by mitochondrial membranes. A portion of this actin became sensitive to protease or high-salt treatment after removal of the mitochondrial outer membrane, indicating that some actin is located inside the mitochondrial outer membrane. The import of an actin–green fluorescent protein (GFP) fusion protein into the mitochondria in a transgenic plant, actin:GFP, was visualized in living cells and demonstrated by flow cytometry and immunoblot analyses. Polymerized actin was found in mitochondria of actin:GFP plants and in mung bean (Vigna radiata). Notably, actin associated with mitochondria purified from early-developing cotyledons during seed germination was sensitive to high-salt and protease treatments. With cotyledon ageing, mitochondrial actin became more resistant to both treatments. The progressive import of actin into cotyledon mitochondria appeared to occur in concert with the conversion of quiescent mitochondria into active forms during seed germination. The binding of actin to mitochondrial DNA (mtDNA) was demonstrated by liquid chromatography–tandem mass spectrometry analysis. Porin and ADP/ATP carrier proteins were also found in mtDNA-protein complexes. Treatment with an actin depolymerization reagent reduced the mitochondrial membrane potential and triggered the release of cytochrome C. The potential function of mitochondrial actin and a possible actin import pathway are discussed.

Protein Phosphatase 2Cs and Microtubule-Associated Stress Protein 1 Control Microtubule Stability, Plant Growth, and Drought Response

Clade E Growth-Regulating (EGR) phosphatases and Microtubule-Associated Stress Protein 1 (MASP1) modulate cell expansion and drought response via control of microtubule stability and organization. Plant growth is coordinated with environmental factors, including water availability during times of drought. Microtubules influence cell expansion; however, the mechanisms by which environmental signals impinge upon microtubule organization and whether microtubule-related factors limit growth during drought remains unclear. We found that three Clade E Growth-Regulating (EGR) Type 2C protein phosphatases act as negative growth regulators to restrain growth during drought. Quantitative phosphoproteomics indicated that EGRs target cytoskeleton and plasma membrane-associated proteins. Of these, Microtubule-Associated Stress Protein 1 (MASP1), an uncharacterized protein, increased in abundance during stress treatment and could bind, bundle, and stabilize microtubules in vitro. MASP1 overexpression enhanced growth, in vivo microtubule stability, and recovery of microtubule organization during drought acclimation. These MASP1 functions in vivo were dependent on phosphorylation of a single serine. For all EGR and MASP1 mutants and transgenic lines examined, enhanced microtubule recovery and stability were associated with increased growth during drought stress. The EGR-MASP1 system selectively regulates microtubule recovery and stability to adjust plant growth and cell expansion in response to changing environmental conditions. Modification of EGR-MASP1 signaling may be useful to circumvent negative growth regulation limiting plant productivity. EGRs are likely to regulate additional proteins involved in microtubule stability and stress signaling.

Systems-wide analysis of manganese deficiency-induced changes in gene activity of Arabidopsis roots

Manganese (Mn) is pivotal for plant growth and development, but little information is available regarding the strategies that evolved to improve Mn acquisition and cellular homeostasis of Mn. Using an integrated RNA-based transcriptomic and high-throughput shotgun proteomics approach, we generated a comprehensive inventory of transcripts and proteins that showed altered abundance in response to Mn deficiency in roots of the model plant Arabidopsis. A suite of 22,385 transcripts was consistently detected in three RNA-seq runs; LC-MS/MS-based iTRAQ proteomics allowed the unambiguous determination of 11,606 proteins. While high concordance between mRNA and protein expression (R = 0.87) was observed for transcript/protein pairs in which both gene products accumulated differentially upon Mn deficiency, only approximately 10% of the total alterations in the abundance of proteins could be attributed to transcription, indicating a large impact of protein-level regulation. Differentially expressed genes spanned a wide range of biological functions, including the maturation, translation, and transport of mRNAs, as well as primary and secondary metabolic processes. Metabolic analysis by UPLC-qTOF-MS revealed that the steady-state levels of several major glucosinolates were significantly altered upon Mn deficiency in both roots and leaves, possibly as a compensation for increased pathogen susceptibility under conditions of Mn deficiency.