Yiming Wang

Depletion of abundant plant RuBisCO protein using the protamine sulfate precipitation method

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant plant leaf protein, hampering deep analysis of the leaf proteome. Here, we describe a novel protamine sulfate precipitation (PSP) method for the depletion of RuBisCO. For this purpose, soybean leaf total proteins were extracted using Tris‐Mg/NP‐40 extraction buffer. Obtained clear supernatant was subjected to the PSP method, followed by 13% SDS‐PAGE analysis of total, PS‐supernatant and ‐precipitation derived protein samples. In a dose‐dependent experiment, 0.1% w/v PS was found to be sufficient for precipitating RuBisCO large and small subunits (LSU and SSU). Western blot analysis confirmed no detection of RuBisCO LSU in the PS‐supernatant proteins. Application of this method to Arabidopsis, rice, and maize leaf proteins revealed results similar to soybean. Furthermore, 2DE analyses of PS‐treated soybean leaf displayed enriched protein profile for the protein sample derived from the PS‐supernatant than total proteins. Some enriched 2D spots were subjected to MALDI‐TOF‐TOF analysis and were successfully assigned for their protein identity. Hence, the PSP method is: (i) simple, fast, economical, and reproducible for RuBisCO precipitation from the plant leaf sample; (ii) applicable to both dicot and monocot plants; and (iii) suitable for downstream proteomics analysis.

Understanding the plant-pathogen interactions in the context of proteomics-generated apoplastic proteins inventory

The extracellular space between cell wall and plasma membrane acts as the first battle field between plants and pathogens. Bacteria, fungi, and oomycetes that colonize the living plant tissues are encased in this narrow region in the initial step of infection. Therefore, the apoplastic region is believed to be an interface which mediates the first crosstalk between host and pathogen. The secreted proteins and other metabolites, derived from both host and pathogen, interact in this apoplastic region and govern the final relationship between them. Hence, investigation of protein secretion and apoplastic interaction could provide a better understanding of plant-microbe interaction. Here, we are briefly discussing the methods available for the isolation and normalization of the apoplastic proteins, as well as the current state of secretome studies focused on the in-planta interaction between the host and the pathogen.

Proteomics of rice and Cochliobolus miyabeanus fungal interaction: Insight into proteins at intracellular and extracellular spaces

Necrotrophic fungal pathogen Cochliobolus miyabeanus causes brown spot disease in rice leaves upon infection, resulting in critical rice yield loss. To better understand the rice–C. miyabeanus interaction, we employed proteomic approaches to establish differential proteomes of total and secreted proteins from the inoculated leaves. The 2DE approach after PEG‐fractionation of total proteins coupled with MS (MALDI‐TOF/TOF and nESI‐LC‐MS/MS) analyses led to identification of 49 unique proteins out of 63 differential spots. SDS‐PAGE in combination with nESI‐LC‐MS/MS shotgun approach was applied to identify secreted proteins in the leaf apoplast upon infection and resulted in cataloging of 501 unique proteins, of which 470 and 31 proteins were secreted from rice and C. miyabeanus, respectively. Proteins mapped onto metabolic pathways implied their reprogramming upon infection. The enzymes involved in Calvin cycle and glycolysis decreased in their protein abundance, whereas enzymes in the TCA cycle, amino acids, and ethylene biosynthesis increased. Differential proteomes also generated distribution of identified proteins in the intracellular and extracellular spaces, providing a better insight into defense responses of proteins in rice against C. miyabeanus. Established proteome of the rice–C. miyabeanus interaction serves not only as a good resource for the scientific community but also highlights its significance from biological aspects.

Time to dig deep into the plant proteome: a hunt for low-abundance proteins

Two-dimensional gel electrophoresis (2-DGE) has come a long way since its introduction around 40 years by the pioneering work of these three researchers (Klose, 1975; OFarrell, 1975; Scheele, 1975). 2-DGE was one of the major breakthroughs in proteomics, enabling researchers to detect, analyze and identify the whole set of proteins of a cell or tissue, simultaneously. With the advancement in technology, some modifications to this technique like development of immobilized pH gradient (IPG) strips were introduced, which undoubtedly, made this technique more simple, rapid and autonomous (Bjellqvist et al., 1982). After its introduction to the present, 2-DGE has been the method of choice for analyzing the complex proteomes of plants. 2-DGE has been used extensively to investigate the effects of biotic and abiotic stress, role of hormones, and developmental changes of plants, among others (Agrawal and Rakwal, 2008). However, it was slowly realized that identification of the plant proteins led to the repeated detection of high-abundance proteins (HAPs) including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and other housekeeping proteins, which are present at 106-105 order of magnitude (Gygi et al., 2000; Patterson and Aebersold, 2003; Gorg et al., 2004). Signaling and other regulatory proteins are generally present 100 molecules per cells. Subsequently, these proteins are difficult to identify by either gel-based or gel-free proteomic approaches, even with access to the latest mass spectrometers. RuBisCO comprises of a large percentage in the total proteins and thus hinders the absorption of low-abundance proteins (LAPs) on the IPG strips, which subsequently results in the poor detection and identification of LAPs on 2D gels and by mass spectrometry (MS), respectively. Therefore, the time has come for all plant proteomers to realize the need to hunt for the LAPs, moving one step ahead from the present. As RuBisCO is the major HAP in plant leaves, here we recommend the incorporation of a RuBisCO depletion/removal method in every plant protein extraction step to look deeper into the plant proteome. RuBisCO depletion will definitely improve the proteome coverage and will lead to the detection of novel unidentified LAPs.

Transcriptome Analysis of Early Responsive Genes in Rice during Magnaporthe oryzae Infection

Rice blast disease caused by Magnaporthe oryzae is one of the most serious diseases of cultivated rice (Oryza sativa L.) in most rice-growing regions of the world. In order to investigate early response genes in rice, we utilized the transcriptome analysis approach using a 300 K tilling microarray to rice leaves infected with compatible and incompatible M. oryzae strains. Prior to the microarray experiment, total RNA was validated by measuring the differential expression of rice defense-related marker genes (chitinase 2, barwin, PBZ1, and PR-10) by RT-PCR, and phytoalexins (sakuranetin and momilactone A) with HPLC. Microarray analysis revealed that 231 genes were up-regulated (>2 fold change, p < 0.05) in the incompatible interaction compared to the compatible one. Highly expressed genes were functionally characterized into metabolic processes and oxidation-reduction categories. The oxidative stress response was induced in both early and later infection stages. Biotic stress overview from MapMan analysis revealed that the phytohormone ethylene as well as signaling molecules jasmonic acid and salicylic acid is important for defense gene regulation. WRKY and Myb transcription factors were also involved in signal transduction processes. Additionally, receptor-like kinases were more likely associated with the defense response, and their expression patterns were validated by RT-PCR. Our results suggest that candidate genes, including receptor-like protein kinases, may play a key role in disease resistance against M. oryzae attack.

Comparative investigation of seed coats of brown- versus yellow-colored soybean seeds using an integrated proteomics and metabolomics approach

Seed coat color is an important attribute determining consumption of soybean seeds. Soybean cultivar Mallikong (M) has yellow seed coat while its naturally mutated cultivar Mallikong mutant (MM), has brown colored seed coat. We used integrated proteomics and metabolomics approach to investigate the differences between seed coats of M and MM during different stages of seed development (4, 5, and 6 weeks after flowering). 2DE profiling of total seed coat proteins from three stages showed 178 differentially expressed spots between M and MM of which 172 were identified by MALDI‐TOF/TOF. Of these, 62 were upregulated and 105 were downregulated in MM compared with M, while five spots were detected only in MM. Proteins involved in primary metabolism showed downregulation in MM suggesting energy in MM might be utilized for proanthocyanidin biosynthesis via secondary metabolic pathways that leads to the development of brown seed coat color. Besides, downregulation of two isoforms of isoflavone reductase indicated reduced isoflavones in seed coat of MM that was confirmed by quantitative estimation of total and individual isoflavones using HPLC. We propose that low isoflavones level in MM may offer a high substrate for proanthocyanidin production that results in the development of brown seed coat in MM.

Arabidopsis thaliana DM2h (R8) within the Landsberg RPP1-like Resistance Locus Underlies Three Different Cases of EDS1-Conditioned Autoimmunity.

Plants have a large panel of nucleotide-binding/leucine rich repeat (NLR) immune receptors which monitor host interference by diverse pathogen molecules (effectors) and trigger disease resistance pathways. NLR receptor systems are necessarily under tight control to mitigate the trade-off between induced defenses and growth. Hence, mis-regulated NLRs often cause autoimmunity associated with stunting and, in severe cases, necrosis. Nucleocytoplasmic ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) is indispensable for effector-triggered and autoimmune responses governed by a family of Toll-Interleukin1-Receptor-related NLR receptors (TNLs). EDS1 operates coincidently or immediately downstream of TNL activation to transcriptionally reprogram cells for defense. We show here that low levels of nuclear-enforced EDS1 are sufficient for pathogen resistance in Arabidopsis thaliana, without causing negative effects. Plants expressing higher nuclear EDS1 amounts have the genetic, phenotypic and transcriptional hallmarks of TNL autoimmunity. In a screen for genetic suppressors of nuclear EDS1 autoimmunity, we map multiple, independent mutations to one gene, DM2h, lying within the polymorphic DANGEROUS MIX2 cluster of TNL RPP1-like genes from A. thaliana accession Landsberg erecta (Ler). The DM2 locus is a known hotspot for deleterious epistatic interactions leading to immune-related incompatibilities between A. thaliana natural accessions. We find that DM2hLer underlies two further genetic incompatibilities involving the RPP1-likeLer locus and EDS1. We conclude that the DM2hLer TNL protein and nuclear EDS1 cooperate, directly or indirectly, to drive cells into an immune response at the expense of growth. A further conclusion is that regulating the available EDS1 nuclear pool is fundamental for maintaining homeostatic control of TNL immune pathways.

Protamine sulfate precipitation method depletes abundant plant seed-storage proteins: A case study on legume plants

Depletion of abundant proteins is one of the effective ways to improve detection and identification of low‐abundance proteins. Our previous study showed that protamine sulfate precipitation (PSP) method can deplete abundant ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) from leaf proteins and is suitable for their in‐depth proteome investigation. In this study, we provide evidence that the PSP method can also be effectively used for depletion of abundant seed‐storage proteins (SSPs) from the total seed proteins of diverse legume plants including soybean, broad bean, pea, wild soybean, and peanut. The 0.05% protamine sulfate (PS) was sufficient to deplete major SSPs from all legumes tested except for peanut where 0.1% PS was required. SDS‐PAGE, Western blotting and 2DE analyses of PS‐treated soybean and peanut seed proteins showed enriched spots in PS‐supernatant than total proteins. Coefficient of variation percentage (%CV) and principal component analysis of 2DE spots support the reproducibility, suitability, and efficacy of the PSP method for quantitative and comparative seed proteome analysis. MALDI‐TOF‐TOF successfully identified some protein spots from soybean and peanut. Hence, this simple, reproducible, economical PSP method has a broader application in depleting plant abundant proteins including SSPs in addition to RuBisCO, allowing discussion for comprehensive proteome establishment and parallel comparative studies in plants.

Abundant storage protein depletion from tuber proteins using ethanol precipitation method: Suitability to proteomics study

High‐abundance proteins (HAPs) hamper in‐depth proteome study necessitating development of a HAPs depletion method. Here, we report a novel ethanol precipitation method (EPM) for HAPs depletion from total tuber proteins. Ethanol showed a dose‐dependent effect on depletion of sporamin from sweet potato and patatin from potato tubers, respectively. The 50% ethanol was an optimal concentration. 2DE analysis of EPM‐prepared sweet potato proteins also revealed enrichment of storage proteins (SPs) in ethanol supernatant (ES) resulting in detection of new low‐abundance proteins in ethanol pellet (EP), compared to total fraction. The ES fraction showed even higher trypsin inhibitor activity than total proteins, further showing the efficacy of EPM in enrichment of sporamin in ES fraction. Application of this method was demonstrated for comparative proteomics of two sweet potato cultivars (Hwang‐geum and Ho‐bac) and purification of SP (sporamin) in its native form, as examples. Comparative proteomics identified many cultivar specific protein spots and selected spots were confidently assigned for their protein identity using MALDI‐TOF‐TOF analysis. Overall, the EPM is simple, reproducible, and economical for depletion of SPs and is suitable for downstream proteomics study. This study opens a door for its potential application to other tuber crops or fruits rich in carbohydrates.

Expect the Unexpected Enrichment of “Hidden Proteome” of Seeds and Tubers by Depletion of Storage Proteins

Dynamic resolution of seed and tuber protein samples is highly limited due to the presence of high-abundance storage proteins (SPs). These proteins inevitably obscure the low-abundance proteins (LAPs) impeding their identification and characterization. To facilitate the detection of LAPs, several methods have been developed during the past decade, enriching the proteome with extreme proteins. Most of these methods, if not all, are based on the specific removal of SPs which ultimately magnify the proteome coverage. In this mini-review, we summarize the available methods that have been developed over the years for the enrichment of LAPs either from seeds or tubers. Incorporation of these methods during the protein extraction step will be helpful in understanding the seed/tuber biology in greater detail.