Plant secretomics

Abstract

Plant secretomes are the proteins secreted by the plant cells and are involved in the maintenance of cell wall structure, relationship between host and pathogen, communication between different cells in the plant, etc. Amalgamation of methodologies like bioinformatics, biochemical, and proteomics are used to separate, classify, and outline secretomes by means of harmonizing in planta systems and in vitro suspension cultured cell system (SSCs). We summed up and explained the meaning of secretome, methods used for the identification and isolation of secreted proteins from extracellular space and methods for the assessment of purity of secretome proteins in this review. Two D PAGE method and HPLC based methods for the analysis together with different bioinformatics tools used for the prediction of secretome proteins are also discussed. Biological significance of secretome proteins under different environmental stresses, i.e., salt stress, drought stress, oxidative stress, etc., defense responses and plant interactions with environment are also explained in detail.

Keywords: :

• Proteomics
• secretome
• secretory pathways
• signal peptide

Introduction

Most important biological functions of proteins secreted by plants include maintenance of cell wall structure, relationship between host and pathogen, and communication between different cells in the plant.¹ One of the vital biological mechanisms among them is cell secretion. Cell secretions basically involve release of intracellular products like proteins and metabolites, into the extracellular environment. These intracellular products sense the environmental changes. Extracellular space (ECS) is composed of everything that is outside the plasma membrane while region inside the ECS is generally called apoplastic fluid (APF). This fluid is involved in the interaction and communication of plant cell with its environment. Transportation and communication between the intracellular and extracellular environment is also provided by APF. Several biological and physiological processes are regulated by secretory proteins present in APF, e.g., signaling, cell expansion, response to stress condition and interaction between cells.^2-5 Word secretome was for the first time utilized to explain the signal peptide dependent secreted proteins and secretory proteins in a gram positive bacterium, Bacillus subtilis.⁶ Secretome is referred to as the rich, complex set of molecules secreted from living cells.⁷ There is still a debate in progress that how can we define secretomics in plants. This definition of secretome is constructive but still has inadequacy to give a global vision of secretome.⁸ Proteins involved in the secretory pathway are sometimes immingled/included within secretome⁹ but to be most precise, it involves only secreted proteins from an organism.^10-12

Secretome is now defined as “A group of proteins secreted into the extracellular matrix from a cell, tissue, organelle or organism by some known or unknown mechanism at a particular time.”⁸ Therefore, plant secretomes include all proteins secreted into the extracellular space, i.e., outside the plasma membrane of plant cells and tissues. These secreted proteins are then isolated from the extracellular space and further analyzed by using different proteomic approaches.

Genomes of various plant species have been sequenced completely so far because of the advancement and progress in the sequencing technology. For the functional annotation of genomes it is necessary to predict/identify genes that encode proteins and subcellular locations of those encoded proteins. For the better prediction and analyzation of plant secretome, secreted proteins are manually curated and annotated in uniprot database and the information gathered by using this analysis method assists in accurate prediction of secreted proteins which are then used for the creation of database of plant secretome.¹³

In eukaryotes, classical protein secretion is highly conserved mechanism where proteins with a signal peptide are transported by the help of golgi apparatus. It was predicted that 18 percent of proteins from the genome of Arabidopsis must be secreted.¹⁴ But from the secretome studies, it was seen that about 40 to 70 percent of the identified proteins lack signal peptide and such proteins are included in the class of leaderless secreted proteins (LSPs). It is also possible that proteins from other cell compartments contaminate proteins. Majority of the secretome proteins identified so far are deficient in signal peptides and little is known about the unconventional protein secretion (UPS).¹⁵ APF is composed of 2 different types of secreted proteins depending upon how they are being secreted. One of them is secreted upon environmental signal and the other is being secreted constitutively into the ECF.

In multicellular organisms, endoplasmic reticulum/golgi pathway is very eminent and broadly categorized secretory pathway.¹⁶ This method of conventional protein secretion is achieved by the help of endomembrane system in which N-terminal signal peptide mediates the insertion of secreted proteins into endomembrane system. It follows the ER to the golgi pathway and takes its route to plasma membrane or extracellular apoplastic space.¹⁷ It was proposed that some of them evade golgi apparatus on their journey to vacuole¹⁶ but a recent study carried on GFPChi confirmed that transport of this protein from ER to vacuole is not fully dependent on the golgi apparatus. There is also evidence for the presence of unconventional secretion mechanisms of protein secretion in plants. Because of the presence of increased number of leaderless proteins, identified through proteomics, unconventional method of protein secretion in plants is getting importance. In connection to this, novel approaches like vesicle proteomics and chemical genomics will be able to provide new insights for unconventional protein mechanism.¹⁸ Exocyst positive organelle (EXPO), a plants specific compartment discovered late, is able to mediate unconventional protein secretion (UPS).¹⁹

By using several approaches such as biochemical analysis of subcellular fractions and imaging immunolabelled components an immense contribution has been made from past few decades toward the understanding of fundamental importance of secretory proteins and secretory systems.^20-22

Proteomics covers identification and categorization of proteins expressed in cells, tissues or organelles.²³ In order to quantify great array of proteins, specific techniques have been developed and employed including both gel free and gel based approaches. Gel based approaches includes 1-DE and 2-DE which are also used in combination with mass spectrometry (MS). Gel-free approaches include multi-dimensional protein identification technology or semi continuous multi-dimensional protein identification technology and isobaric tags for relative and absolute quantitation (iTRAQ). These proteomic approaches can be better used for investigation of secretome on large scale because of their compatibility and preference. Minor part of the total secretome is predicted by using particular software packages and isolated by means of techniques like differential extraction. Proteomics is also used for the study of plant pathogen interaction where more than one genome products (i.e., proteins) are present in ECS. Furthermore during plant pathogen interaction if we take example of APF, instead of genes, proteins are secreted by plants and the inhabiting organisms into APF, for that proteomics is used to characterize these proteins.⁸ Since the secretomics has become the center of attention in biological research, and is considered as a complex sub-field of proteomics.⁸ The aim of this review is to: 1) explain in detail about basics of plant secretome, 2) to give details about the methods of identification, isolation, purity assessment and bioinformatic tools used for the prediction of secretome proteins, and 3) to give details about the biological significance of secretome proteins under different conditions.

Explanation of secretome

Secretome word is not originated from plant related study. It has been coined while studying peptide signal dependent transport in B. substilis.⁷ According to Hathout¹⁰ in his review “Approaches to the study of the cell secretome” secretome can be defined as “The total learning of proteins that are produced by a cell, tissue or organism at any set point or in particular circumstances.” According to Dr. Irmgard Schwarte-Waldhoff: “Since we have got more information about makeup of sub proteome, it is to be noted that the name secretome does not mean to cover merely the proteins that are produced from the conventional path. So, the secretome in other words involve proteins produced during a variety of processes together with standard secretion, secretion from non-traditional paths and exosomes secretions.”²⁴

A question is being raised, is present definition of secretome enough to cover all of ECS secretory proteins and various organelles that are involved in the specificity and transport of protein. According to these conclusions, we suggest an amended definition of secretome as “The universal set of proteins secreted into extracellular matrix by a cell, tissue, or organ or an individual at a particular instance and circumstances.”

Identification and isolation of secretome proteins

Suspension-cultured cells (SSCs) system

Up to last 3–4 y suspension-cultured cells system (SSCs) was the desired choice for secretome samples and till now most of the studies have been conducted with SSC. First, cell culture filtrate (composed of secreted proteins) of the suspension-cultured cells is obtained and proteins isolated is used for further analysis. Its major advantage over in plantasystem is that cell leakage can easily be estimated, secretome can easily be separated from intact cells by filtration and its centrifugation is easier.²⁵

In planta system

As SSC system does not provide a natural environment for the cells and physiologically similar treatments are facing difficulty for their application, so recent studies prefer in plantasystem. Through this system it is easy to derive organ and developmental specific secretomes by using plant material.²⁵ In planta system secreted proteins are obtained from apoplastic fluid present in extracellular space.

It has been reported by Jung et al.²⁶ that when the rice secretome proteins were used with an overlap of 6 spots out of 222 after resolution by 2D-PAGE showed a prominent difference relying on whether the SSC system is used or in planta is used and there seems a difference of 27% and 76% respectively, in the level of predicted protein. Two secretomes were derived using in planta and SSC system and contamination at low level was observed by means of determining malate dehydrogenase activity. So it is clear that both systems and their mechanism are different thus giving different secretomes. It must be noted that when preparing secretomes from intact plant organs there should be no leakage and breakage of cell. There are 2 methods established for this purpose: Vacuum infiltration centrifugation (VIC) method and gravity extraction method (GEM) method.³

Vacuum infiltration centrifugation (VIC) method

The Vacuum Infiltration Centrifugation method is comprised of 2 stages, i.e., appropriate extraction and speed as well as duration of centrifugation. Modifications in classical VIC method increases the accuracy of secretomic studies which has been in practice from past 50 y.²⁷ However pH of the penetration buffer that disturbs the metabolic composition, osmolarity, and incubation time have minute influence on the elute.²⁸ Because of the difference in the adjustment of sample infiltrability which varies with plant species for example recently a rice method is suggested by Nouchi et al.²⁹ In case of potato leaves, to decrease leaf surface tension first wash the leaves with a buffer and simplify the vacuum infiltration which is reiterated once. In order to avoid the sample from its dilution the leaf surface should quickly be dried. After that transfer the rolled leaves very carefully to 15 ml falcon tube, which contains a washer at the bottom in order to avoid immersion of leaves into the collected APF.³⁰ To avoid cell breakage the centrifugation forces should not exceed 1000 g.³¹

In 1980, the classical VIC method was established which was used to extract APF from epicotyl of pea segments (1–2cm). Expunged sections were crammed upright in plastic syringe (20ml). Crammed tubes were then positioned in refined water that covers the tissue at the extremity of 1–3mm and then washed it with 20mM potassium phosphate buffer in order to eliminate cytoplasmic adulteration from cut sides. Buffer is then removed by washing the sections with ice water and subverted with water, vacuum it for 6 min and 3 min respectively. Suction was supplied to every tube in order to draw the surface water and subsequently placed on top of 12 ml conical centrifuge tubes and these conical tubes with section tubes were positioned in centrifuge tubes of 50 ml followed by centrifugation at 1000 rpm for 15 min to obtain the APF sap.²⁵

The efficiency of VIC method for APF isolation makes scientists to apply this method with certain alterations for secreted proteins. As in planta studies have been beleaguered to create secretomes, it is important to mention that till date no investigation has been carried on the significance/influence of various infiltration buffers on secretome configuration, e.g., if infiltration buffer containing high concentrations of KCl (ionic reagent) is used, it will absorb cell wall proteins which are weakly bounded to cell wall.²⁵

Steps in VIC method were modified in such a way that isolated APF from wheat head infected with Fusarium graminearum are used. Infected wheat crowns were taken, completely inundated in deionized water and after that its vacuum infiltration was done and centrifuged at 1500rpm for about 10 min. At the end, samples obtained were subjected to biochemical and proteomics analyses. In this case one problem emerges that is lack of washing step and usage of somewhat greater gravity force (2000 g) might result in cytoplasmic adulteration to the isolated APF.³²

Gravity extraction method (GEM)

The gravity extraction method (GEM) has not been used/ practiced much so far.³ This method is different from VIC in a way that the vacuum infiltration step is omitted in it and APF is collected directly to reduce cell damage and solubilization of membrane proteins. To remove contamination of interfering compounds like carbohydrates, it is essential to add acetyl trimethyl ammonium bromide (CTAB) for mediating precipitation after sap isolation. Modifications for efficient secretome preparation are essential, e.g., to get apoplast fluid from poplar stem tissue, a water displacement method was developed.³³

The GEM is an innovative and reproducible extraction process for APF and provision of unadulterated secretomes. Its first phase is to get the APF, the second and the last step is to remove interfering compounds especially in case of proteomics analysis. In case of rice plants, the gathered undamaged leaves are placed prudently in a falcon tube of 50 ml followed by centrifugation and APF containing secreted proteins are then collected. At the end the collected APF is purified with CTAB precipitation method in order to remove contamination and interfering compounds that disturb proteomics analysis.⁸

It is critical to prepare secreted protein sample from a highly reproducible and simple method. So, attempts should be made to improve this method to get secretomes from different plant organs, tissues, and species. For the scientific community to perform comparative proteomics and share their data with great confidence, it is necessary to have a standard method in all secretome laboratories.⁸

Purity evaluation

To evaluate the purity of secretome fractions, immune blotting and microscopy have been used. Especially in case of in planta studies which involve more rigorous evaluation of purity to guarantee secretome fractions with little intracellular adulteration. To evaluate cytosolic adulteration, enzyme activities of glucose-6-phosphate dehydrogenase, catalase, and malate dehydrogenase are normally measured. On the basis of malate dehydrogenase activity 1–3% of adulteration is usually seen, but up to 10% has been reported till now.³⁴ In destructive methods, in which the fraction of cell wall is isolated, determining the reputed adulteration by plasma membrane is also essential, e.g., by measuring H-ATPase activity.^35,36 Contamination level can also be determined by frequent use of antibodies against malate dehydrogenase and RuBisCo.^33,37 To estimate levels of membrane mutilation, electrolyte leakage and concentration of malondialdehyde have to be measured.³⁸ In case of plant–pathogen relation studies, it should be kept in mind that cell seepage can be caused by direct damage of hyphae or cell wall deliquescence by the biological system itself rather than the isolation procedure. Apart from this plant developmental processes and apoptosis during xylem formation can discharge non-secretary cytosolic proteins into the apoplast.²⁵

Two categories of biochemical investigations are usually conducted to evaluate, whether the equipped portion of secretomes are unrestricted from adulteration of fathomable proteins of cytoplasm or not. They include enzymatic technique and western blotting.⁸

Enzymatic assay

APF adulteration with cytoplasmic proteins obtained in case of the SCCs method is due to apoptosis of the cell. Same is the case with VIC method, in which cell wall and the cell membrane are damaged. Based on the assumption that marker enzymes do not occur normally in AFP, the marker enzyme is selected. If there is a condition of breakage and damage of cell wall or plasma lemma, then marker enzyme are recovered in AFP accordingly. The activity of MDH (cytosolic Malate dehydrogenase) has been used for evaluation of pureness.^28,31 However, the presence of soluble MDH activity in the apoplast of some species of plants is supported by some evidences^39,40 but it does not prevent the use of this activity for the determination of adulteration of cells in the APF.⁴¹ Further enzyme markers of cytoplasm used for pureness evaluation are aldolase and glucose 6-phosphate dehydrogenase. Big magnitude of enzyme markers like HPI (hexose phosphate isomerise) and MDH (70 kDa of MDH and 120 kDa of HPI) is a drawback of this technique.⁸

Western blot analysis

Another method implied to evaluate the wholesomeness of secretomes is western blot analysis. Rubisco, actin, HSP 70, and α-tubulin have been used as intracellular marker proteins for plant secretomes. Usage of both the 2 biochemical procedures aids in better evaluation of APF adulteration with intracellular proteins. Accruing evidence on the composition of plant secretome will make available signs on improved variety of intracellular markers of proteins that are not existent in APF and also in case of the cell wall at enduring stages.⁸

Proteomic analysis

2D-PAGE method

Secretome analysis in plants is done by methods like 2D-PAGE (PAGE), and 1D sodium dodecyl sulfate (SDS)- PAGE.^30,37 2D-PAGE is a well-established and relatively inexpensive method. Some proteins being very large, highly hydrophobic, and transmembrane proteins are very much difficult to analyze by 2D-PAGE method. Thus, apoplast proteins do not belong to this category. The properties present in intact proteins as well as post-translational modifications and splicing will affect migration in 2D-PAGE separation method. However, the process of migration is not required always. From 2D-gel spot, multiple proteins are identified and make unambiguous identification difficult.²⁵

HPLC-based methods

High-performance liquid chromatography (HPLC)-based methods, can identify a large number of proteins. The proteins can be pre-fractionated or directly digested. The proteins that cannot be retained in solution in 2D-PAGE analysis and the small proteins like proteolysis fragments that are commonly found in protease rich apoplast are analyzed by HPLC-based methods. However, more complicated sample preparation and data processing are used in some cases and analysis for individual peptides splice variants and post- translational modifications (PTMs) may still remain unrecognized. In the HPLC-based methods, peptides can be quantified either by isotopic labeling or by label-free methods.^42-44

Peptides are quantified by either label-free methods or by isotopic labeling in HPLC based methods.^42-44 Kaffarnik et al.⁴⁵ used absolute and relative quantitative (iTRAQ) method to analyze secretome of Arabidopsis SSC challenged with Pseudomonas. Peptide mass spectrometrical signal and the number of times it is identified, can be used for obtaining quantitative data in label-free methods. In case of sample preparation and the number of comparisons that can be made are not limited in label-free methods, this can lead to more challenging computation. For detection of selected peptides, monitoring of HPLC column eluate is done for high dynamic range using SRM-MS (Selected reaction monitoring specific mass spectrometry).^46,47 In order to get sensitive absolute quantification, it is combined with isotopically labeled standard peptides. But only pre-selected set of peptides can be measured in this way. More than one organism may be responsible for secreted proteins identified in plant pathogen interactions. Even then, very few proteins of pathogens are identified in interaction studies. While matching the peptides from pathogens, false positive hits should be avoided by taking precaution because most of the times interacting organisms are expected to be very few in number. If plant pathogen protein database is added with a random sequence database, false discovery rate determination can be done.²⁵ A powerful oxidative burst in the secretome occurs due to pathogens and other sources of stress. Enzymatic activity and stability is influenced by the oxidation of protein and progressive development is being made in field of oxidation proteomics.⁴⁸

When oxidized amino acid peptides are searched by global analyses, or by enrichment based methods, we can identify the oxidation products. Examples of such methods are enrichment of carbonylated⁴⁹ or nitrosylated peptides.⁵⁰ Sample preparation methods are made to reduce oxidation state as oxidative modifications are quite labile.⁵¹ As buffer composition depends on possible enrichment strategies, it should be considered during experimental design, e.g., isolation of modified cysteine residues.⁵⁰ In the secretome, proteomic knowledge of oxidation is lacking but it can still reveal interesting information about targets and extent of oxidation burst.²⁵

Bioinformatical databases and tools for prediction of secretome proteins

In order to predict the composition of plant secretome, various prediction tools are freely available. By the application of different prediction tools like TMHMM, SignalP, and TargetP on the data sets generated by proteomics it was reported that a considerable amount of secreted proteins from plants have no signal peptides.^3,52

SignalP has been extensively used for in vitro prediction of signal peptides.⁵³ This prediction tool is used to indicate the occurrence and location of cleavage site in a signal peptide. It consists of 2 different signal programs, Signal-NN and SignalP-HMM.^54,55 SignalP software is used to find out signal sequences and cleavage site on these sequences. In order to identify that a secreted protein has a signal sequence or not, this software is used. For the identification of LSPs from prokaryotic and eukaryotic organisms, SecretomeP software was developed.^56,57 For the identification of LSPs, in SecretomeP a neural network is generated. This neural network uses sequence derived feature for identification, e.g., existence of predicted pro peptides, predicted secondary structure, regions of low complexity, etc. This network is used for the identification of many LSPs, e.g., thioredoxin, interleukin-1, etc.^56,57 SecretomeP is used in the identification of LSPs in plants, e.g., 37 LSPs were identified from secretome of Arabidopsis.⁵⁸

Sequences which are present outside the signal peptides of secreted proteins are also predicted by SecretomeP.⁵⁵ This software is basically used for proteins from bacteria and mammals. But there is an interesting fact that about 60 percent of the identified LSPs in Arabidopsis are reported by SecretomeP.⁵⁸ LocTree2 is another promising tools to predict secretome proteins and has high prediction success.⁵⁹

On the basis of MS/MS identification, 471 proteins from Arabidopsis are considered to be extracellular in SUBA3 database.⁶⁰ Among these 471 proteins, less than half are considered to be present in extracellular compartment and less than 10% which are not predicted by TargetP are considered as extracellular proteins. There were 1704 proteins from plants which were identified in UniProt database. By making use of 3 different prediction tools, 97.5% of these proteins were identified to have a signaling peptide.⁶¹ Plant Sec KB is a database specifically for secreted proteins of plants which has been recently introduced in the field of Secretomics.⁶¹ Softwares and tools for the prediction of different proteins are given below.

Prediction of transmembrane domains

TMHMM server v.2.0 predicts helices and transmembrane domains in identified proteins.⁶² There are several prediction programs used to predict transmembrane domain. One of them is HMMTOP, available at http://www.enzim.hu/hmmtop. THUMBPUP is another example available at http://theory.med.buffalo.edu/SoftwaresServicesfiles/thumbup.htm. For the prediction of transmembrane protein from vegetative vacuole proteome of Arabidopsis, these prediction programs are used.⁶³

Prediction of mitochondrial localization

For the prediction of mitochondrial localization, TargetP is used.^56,57 For the purpose of general localization prediction, WoLF PSORT is applied.⁵⁵

Prediction of gpi anchored proteins

There are 2 programs used for the prediction of glycosylphosphatidyl inositol anchored proteins. One of them is PredGPI prediction server available at http://gpcr2.biocomp.unibo.it/gpipe/index.htm and the other is BIG-PI Predictor available at http://mendel.imp.ac.at/gpi/gpi_server.html. In PredGPI, specificity must be greater than 99% and in BIG-PI P or S must be the quality of site, while predicting glycosylphosphatidyl inositol anchored proteins.

Prediction of conserved domains

Pfam program available at http://pfam.sanger.ac.uk/ and InterproScan available at http://www.ebi.ac.uk/Tools/Inter-ProScan/ are the programs used to identify conserved domains in identified proteins.

Endoplasmic reticulum (ER) proteins

Presence of HDEL and KDEL sequence in the C-terminus of a protein is responsible for the identification of ER proteins. On the basis of the secretory organelles helping in the transportation of proteins into the ECS prediction programs are selected. Hence if we consider structural characteristics of the secretory organelles and prediction tools, we can define APF’s predicted secretome as proteins that neither contains transmembrane protein nor glycosylphosphatidyl inositol anchored proteins or any other localization signal, but contains signal peptide at N-terminal.⁸

The major challenges of data analysis are chiefly correlated with the amalgamation of biological information from diverse sources. Improvements in databases and developments in software will significantly add to the convenience and reliability of secretome studies.⁶⁴

Biological significance of plant secretome

Plant secretome studies has been done in different conditions for example in normal and stressful conditions. It is used in different cultivars, under nutritional deficiency, hormone treatment, changing temperature, under salt stress and pathogen attack, etc.⁸

Oxidative stress

Hydrogen peroxide (H₂O₂) plays a pivotal role in determining the responsiveness of cells to stress. How the apoplast proteome changes under oxidative condition is largely unknown. In a study, comparative proteomic analysis was initiated to explore H₂O₂ responsive proteins in the apoplast of rice seedling roots.³⁸ Studies were done on the secretome of rice seedlings treated with 0.3 and 0.6 mM of hydrogen peroxide (in order to create oxidative stress) and under such condition 54 proteins were identified from the secretome. The proteomic analysis of these proteins showed that these proteins carry out several functions including signal transduction, cell wall modification, cell defense, redox homeostasis and carbohydrate metabolism. We can say that in the apoplast of seedling roots under H₂O₂ stress, there is a complex regulatory network. Half of these proteins were concerned with carbohydrate metabolism. This study was the first apoplast proteome investigation of plant seedlings in response to H₂O₂ and may be of paramount importance for understanding the plant regulatory network under environmental stresses. Based on the abundant changes in these proteins, together with their putative functions, it was proposed as a possible protein network that provides new insights into oxidative stress response in the rice root apoplast and clues for the further functional research of target proteins associated with H₂O₂ response.³⁸

Drought stress

Water-deficit or dehydration impairs almost all physiological processes⁶⁵ and greatly influences the geographical distribution of many crop species.⁶⁵ It has been postulated that higher plants rely mostly on induction mechanisms to maintain cellular integrity during stress conditions. Plant cell wall or extracellular matrix (ECM) forms an important conduit for signal transduction between the apoplast and symplast and acts as front-line defense, thereby playing a key role in cell fate decision under various stress conditions. To better understand the molecular mechanism of dehydration response in plants, 4-week-old rice seedlings were subjected to progressive dehydration by withdrawing water and the changes in the ECM proteome were examined using 2-dimensional gel electrophoresis.³⁵ Dehydration-responsive temporal changes revealed 192 proteins that change their intensities by more than 2.5-fold, at one or more time points during dehydration. It was also observed that there was an upregulation in the PR1, a stress response marker. Studies were done on rice leaves in which drought stress was imposed for 8 d. More than 100 proteins were identified which were affected by drought stress. The proteomic analysis of these proteins showed that these proteins are concerned with several functions, including cell defense, carbohydrate metabolism, cell signaling, cell wall modification, etc.³⁵

Salt stress

Plants have evolved sophisticated systems to cope with adverse environmental conditions such as cold, drought, and salinity.^66-74 The function of plant apoplast in plant stress response has been ignored. Studies were done on rice under salt stress for 12 h. 122 spots were identified by LC-MS/MS, and 117 spots representing 69 proteins have also been identified.³⁴ Of these proteins, 37 are apoplastic proteins according to the bioinformatic analysis. The 64 proteins identified are concerned with the processes of oxido-reduction, carbohydrate metabolism, and protein degradation and processing.³⁴ The expression of these proteins showed change in their abundance and the proteins involved in carbohydrate metabolism were the largest fraction of proteins showing abundance. Results of this study showed that apoplast is significant in plant stress signal reception and response.³⁴

In another study, to investigate the role of apoplastic proteins in the salt stress response, 10 d old rice (Oryza sativa) plants were treated with 200 mM NaCl for 1, 3, or 6 h, and the soluble apoplast proteins were extracted for differential analysis using 2-dimensional electrophoresis.⁷⁵ In salt treated seedlings ten protein spots that increased or decreased significantly in abundance were identified by mass spectrometry. These proteins included some well-known biotic and abiotic stress-related proteins. Among them, an apoplastic protein, with extracellular domain-like cysteine-rich motifs (DUF26), O. sativa root meander curling (OsRMC), has shown drastic increase in response to salt stress during the initial phase. OsRMC RNA interference transgenic rice has been generated to assess the function of OsRMC in salt stress response. The results showed that knocking down the expression level of OsRMC in transgenic rice led to insensitive seed germination, enhanced growth inhibition, and improved salt stress tolerance to NaCl than in non transgenic plants.⁷⁵

Low temperature treatment

Studies were done on the secretome of sea buckthorn exposed to low temperature treatment. Putative antifreeze proteins like chitinase and thaumatin like protein were identified from this study. It was reported by Gupta et al.⁷⁶ that at low temperature stress, the yield and circulation of plants are affected negatively. A Himalayan wonder shrub, Hippophae ramnoides stem secretome were investigated by 2-DE-nano-Liquid chromatography–mass spectrometry/mass spectrometry. Glacial stress of −5 °C, frosty acclimatization and ice-cold acclimatization were done on plantlets and vacuum penetration process was used to isolate extracellular proteins. Approximately 245 spots were reproducibly detected in 2-DE gels of LT treated secretome, out of which 61 were LT responsive. Functional categorization of 34 upregulated proteins showed 47% signaling, redox regulated, and defense associated proteins. Protein that was like thaumatin and antifreeze proteins like chitinase were found to be there in secretome where low temperature stress was applied. Six edged ice-crystals were shown by phase microscopy coupled with nanoliter osmometer and inhibition of recrystallization was shown by a splat assay. It confirmed the presence of antifreeze activity in secretome under low temperature stress. Six edged ice-crystal formation and recrystallization inhibition was observed in a poly-galacturonase inhibitor protein (PGIP), when it was filtrated by adsorption chromatography. It was observed that for antifreeze property, the glycosylation process was not compulsory as galacturonase inhibitor protein preserved its antifreeze property even when it was not glycosylated. Secretome adapted with low temperature refinement and examination of antifreeze activity was first time reported from sea-buckthorn. Secretome adapted with low temperature induced signaling were approached from above mentioned conclusions.⁷⁶

Studies carried on poplar (Populus spp. Growing in riverine ecosystem) exposed to multiple stresses revealed that after collecting samples of secretome by using microarray, gene expression was measured and it was noticed that composition of secretome varied depending upon the type of stress and tissue response. Multi stress response in the apoplast contains a vital adaptive feature for poplar to reside in dynamic environments and is also a possible mechanism in many other riverine plant species.³³

Nitrogen depreviation

A blast disease or a blight of rice seedling is caused by this fungal pathogen named Magnaporthe oryzae. Proteins secreted by fungus Magnaporthe oryzae are involved in pathogenicity of fungus and stress response as well, and these proteins are secreted when the early infection gets started.⁷⁷ During this process there prevails starvation of some nutrients like nitrogen, and this takes place before the successful infection gets established. By using 2-dimensional gel electrophoresis along with the ionization technique MALDI-TOF mass spectrometry and as well as micro-LC–ESI–MS/MS, the secreted proteins from Magnaporthe oryzae were investigated in a nitrogen deficient medium. The 2-dimensional gel electrophoresis detected 89 secreted protein which were expressed differentially as protein spots. Out of these 89 protein spots, 14 protein spots were downregulated and 75 protein spot were upregulated, and these spots were responsive to nitrogen starvation. By the result of analyses of mass spectrometry 85 protein spots were identified. Among these identified proteins 22.4% are hydrolase enzymes of cell wall, 24.7% arehydrolases of lipid and protein, 22.4% are detoxifying proteins of ROS (reactive oxygen species), and 14.1% are proteins with unknown functions.⁷⁷

Another analysis suggests the presence of signal peptides that are present in 67% of the proteins identified. This analysis suggested that M. oryzae along with endoplasmic reticulum and golgi apparatus secretory pathway also have other types of secretory pathways which are not identified yet. By the prediction of TatP and SecretomeP these leaderless and non-classical secretory proteins are of 25.9% among the total proteins identified. A good relationship between RNA and protein levels of 7 secreted proteins were responsive toward nitrogen was dected by a technique named as semi quantitative reverse transcriptase PCR.

The very first evidence of unreported 60 proteins and 25 already known proteins was provided by taking together the secretome of M. oryzae that was responsive toward nitrogen starvation. Due to this study of protein inventory, the understanding of the mechanisms of M. oryzae and the process of its invasive growth is quite easy now.⁷⁷

Plant interaction studies

Nicotiana benthamiana leaf was infected by Agrobacterium tumefaciens containing bacterial gene.⁷⁸ A total of 90 proteins were identified in apoplast, among them only 2 were bacterial in nature. It was observed that most abundant proteins were found to be PR proteins and after infection, level of many proteins were increased.⁷⁸Verticillium longisporum (VL) causes one of the most devastating disease in oil crops of family brassicaceae. The fungus lives for most of the time in the extracellular fluid of the vascular system. Therefore it can’t be controlled by using fungicides. In order to get knowledge about the biology of VL-plant interaction, Arabidopsis thaliana’s secretome that consists of the extracellular proteome, metabolome and cell wall characteristics were studied. VL infection resulted in improved production of cell wall material with a changed composition of carbohydrate polymers and amplified lignification.⁷⁹ Seven proteins were identified, showing changed abundance and most of them belong to peroxidases. Results may suggest that VL enhances its virulence by rapid downregulation as well as delay in induction of plant defense genes.⁷⁹ During early and late infections in rice, virulent and avirulent strains of Magnaporthe oryzae were used to compare compatible and incompatible interactions. At 12th hour, it was observed that there was increase in the number of DUF26 domain-containing proteins during compatible interactions. PR proteins were accumulated in incompatible interactions.⁸⁰ In compatible interaction, only one protein from M. Oryzae named acyclophilin was identified.

Effect of phosphate on the secretome of Arabidopsis SCCs

One of the fundamental macronutrient required for the growth and metabolism of plant is phosphate.⁸¹ In order to engineer Pi-efficient crops, work on the development of different molecular tools and strategies is being performed all over the globe. Secretomes from SCCs of Pi sufficient and Pi-deficient Arabidopsis were compared in order to identify Pi-specific protein markers through 2-D gel electrophoresis.⁸²

Wang et al.⁷⁷ also compared the secretomes of culture filtrate proteome of Arabidopsis thaliana having sufficient phosphate (+Pi) and deficient phosphate (-Pi). The secretomes from the +Pi (phosphate sufficient) and –Pi (phosphate deficient) Arabidopsis cells produced different 2-DE maps. When subjected to peptide mass fingerprinting (PMF) and ionization done through MALDI-TOF mass spectrometry, 50 protein spots were identified and out of these 37 discrete proteins showed unique gene identities. Total 24 proteins were Pi-starvation responsive and among these 24 proteins 18 proteins were upregulated and 6 were downregulated.⁷⁷ The proteins which were upregulated by the phosphate deficiency (-Pi) includes ribonuclease which are involved in finding phosphate from extracellular nucleic acid.

Apart from above secreted proteins some isoforms of purple acid phosphatase was also secreted by the phosphate deficient (-Pi) cells and these isoforms have the subunits of 65 and 55 kDa. This phenomenon of isoform subunit secretion was demonstrated by immunoblotting technique and enzyme activity assays. In this way, a relationship is established between relative quantity of selected secretome proteins and mRNA levels through the semi quantitative RT-PCR technique (a technique used to amplify as well as quantify the RNA fragment).

Wang et al.⁷⁷ also reported that there are many factors contributing to the phosphate starvation response of Arabidopsis and among them is the transcriptional control. The result also showed the importance and value of the proteomic and biochemical studies of phosphate deficient Arabidopsis thaliana plants.⁷⁷

Defense responses

Lucia et al.⁸³ observed that several molecular events operating in plant apoplast are involved in several defense responses. In their study on proteins of Medicago, they used suspension culture for isolation and identification of secreted proteins. Different bioinformatics tools, SDS-PAGE, and tandem mass spectrometry are also used to investigate proteins from the suspension culture.

Lucia et al.⁸³ studied the cultures of M. truncatula 2HA, M. Sativa, and M. truncatula sickle, and 39 proteins were identified. Out of these 39 proteins, 34 proteins contain N-terminal signals for secretion and 5 proteins were secreted through non classical route. The proteins isolated from these samples possessed defensive function, e.g., pathogenesis. SIEP1L is a glyprotein which was only present in M. sativa. From M. truncatula, 3 secreted proteins were identified. Serine carboxypeptidase is detected only in 2HA. There were some proteins which were distinctive to a specific cell culture line. In order to determine the expression of mRNA in selected genes which were present parallel to the proteins present only either in sickle or 2HA or both. A RT-PCR analysis was worked out and results from this study showed a relationship with the proteomic data.⁸³ GDSL-lipase gene was present only in 2HA and was regulated by ethylene. This gene was not present in ethylene sensitive mutants. Likewise, PR1a protein was expressed from an ethylene regulated gene. This protein was present in 2HA but absent in sickle. These experiments signifies that the suspension culture systems are constructive to keep away contamination from cytoplasmic proteins, to spot secreted proteins in Medicago, and should have use in other plant systems also.⁸³

Oh et al.⁸⁴ reported various proteins in Arabidopsis secretome against Alternaria brassicicola. GLIP1 has its function in disease resistance. Plants with glp1 are more vulnerable to infection by Alternaria brassicicola as compared with other plants without glp1. The recombinant GLIP1 protein contained lipase and antimicrobial actions to facilitate direct disruption of fungal spore viability. Moreover, GLIP1 is also responsible for the activation of systemic resistance signaling in plants when challenged with A. brassicicola. Pre-treatment of the glip1 mutant with recombinant GLIP1 protein repressed A. brassicicola–induced cell death in both peripheral and distal leaves.⁸⁴ Furthermore, glip1 is also responsible for change in expression of ethylene-related defense genes. Ethphon is an ethylene releaser responsible for the increased GLIP1 transcription. Increase in GLIP1 transcription is not observed in case of jasmonic acid and salicylic acid. This study showed that GLIP1 if associated with ethylene signaling might have a significant role in defense against A. brassicicola.⁸⁴

Two DE-based proteomic approaches were used for the first systemic study of differential expression of secretome in SCCs of Arabidopsis to discover SA-responsive secreted proteins.⁸⁴ Thirteen SA responsive proteins were identified comparative to control. Examples of LSP’s identified are jacalin-related and ubiquitin-like proteins. Apart from this, in control sample 91 secreted proteins were identified. It was predicted by signal P analysis that there were signal peptides only in 49 proteins. By the functional analysis of GLIP1 which is one of the SA responsive proteins, it was observed that this protein is involved in defense and resistance against A. brassicicola. It was reported recently that GLIP2, which is another protein belonging to GDSL lipase family also played an important role in defense and resistance against Erwinia carotovora, through negative regulation of auxin signaling.⁸⁵ These studies give us better understanding of targeted function analysis of secretome proteins in Arabdopsis. By using lable free features of LC/MSE instrument, a study on Arabidopsis SCCs are done in which complementary gel-free proteomic approach was applied in order to quantitatively profile the secretion dynamics of SA-responsive proteins.⁸² Results of LC/MSE showed 74 secreted proteins out of which 63 proteins showed 2 times increase in their secretion subsequent to SA treatment. The secretion of proteins attaining an upper limit at 1 h with 26 proteins, 2 h with 14 proteins, 6 h with 14 proteins, and 18 h time-period including 9 proteins. As most secretion occurred in first 2 h of SA treatment, it can be recommended that these proteins are concerned with early stages of plant defense. A number of these richly secreted proteins within 1 h are proteins with signal peptides. Many early secreted proteins were LSPs based on both SignalP and SecretomeP analysis and contributed for more than 55% among the entire identified secreted proteins.⁸² This study showed evaluation of time and dose-effects of SA on protein secretion into the APF.⁸⁴

In grape secretome, expression levels of different proteins like SGNH plant lipase-like, subtilisin-like protease, pathogenesis-related proteins, xyloglucan endo-transglycosylase and peroxidases was affected in response to elicitors methyl jasmonate and methylated cyclodextrins.⁸⁶ These elicitors are responsible for the induction of defense responses that bear a resemblance to those produced by pathogen attack. It was observed by 2D gel analysis that 29 out of 35 spots were encoded by 10 different genes encoding different proteins. Most of these encoded proteins belong to pathogenesis-related type proteins.⁸⁶

In tomato SSC, and many other PR proteins were identified by the application of methyl jasmonate and cyclodextrins.⁸⁷ Study of the extracellular proteome depicted the existence of amino acid sequences homologous to pathogenesis-related 1 and 5 proteins, a biotic cell death-associated protein, and a cationic peroxidase.⁸⁷ It suggested that methyl jasmonate and cyclodextrins could play a part in controlling defense-related gene expression in S. lycopersicum.⁸⁷

Interaction of Magnaporthe grisea and its elicitor with rice SCCs induces predominantly defense-related protein secretion and 21 differentially expressed secreted proteins were identified.⁸⁸ Mainly assigned proteins were concerned with defense, such as chitinases, germin A/oxalate oxidases, domain unknown function 26 secretory proteins and b-expansin. One of the chitin-binding chitinase proteins has strong enzymatic activity in an in-gel assay. Interestingly, their protein abundance correlated well at transcript levels in elicitor-treated cultures as judged by semi-quantitative RT-PCR. Consequently, it is promising to reproduce the in vitro SCC results into the in planta system provided that a cautious experimental design and approach is used.⁸⁸

2-D fluorescence difference gel electrophoresis (2-D DIGE) revealed regulation of apoplastic protein secretion by oligogalacturonides in Arabidopsis.⁸⁹ Oligogalacturonides are elicitors of plant defense responses released after a pathogen attack. APF of Arabidopsis seedlings showed 62 proteins. Out of these 62 proteins, 52 spots were identified, and most of these spots contain cell wall and secretory pathway proteins. Results showed the occurrence of low abundant proteins, novel in their characteristics giving evidence of the convenience of DIGE in expanding the secretome.⁸⁹

ICF of tobacco leaf is rich in peptidases

One of the most important components responsible for defense response against stress in plants is extracellular peptidases^90,91 For the isolation of ICF of N. tabacum, modern VIC method is used.⁹² By this method, 17 peptides were identified and they showed similarity with peptidases from other plants. Some of the examples of these plant peptidases are aspartic peptidases, papain cysteine peptidases, serine carboxypeptidases, cathepsin B-like cysteine peptidases, and subtilisin-like serine peptidases. These plant peptides at some point are specifically involved in different stress and defense responses^90,91

By means of transient overexpression of different SNAREs in tobacco protoplasts, effects on exocytosis and protein transportation were observed in a well designed experiment, even if there are only few proteins identified.⁹³

Secreted proteins from root cap

The primary site or the place where organism like fungi, bacteria, and nematodes initiate their infection is the site of elongation, which is just behind root tip and is the site where new tissues are formed.⁹⁴ There is very little knowledge about the mechanism that protects the root tip from the infection of pathogen. In this regard secretome of pea root cap was established to investigate the protection mechanism.⁵²

From the extracellular fluid, more than 100 extracellular proteins were identified. It is not astonishing that a large number of proteins could be assigned the duty of defense or stress. Among the total identified secreted proteins there were 80% of LSPs in correspondence with defense or stress related proteins.⁸ Examples of LSPs include 5 unique proteins, they are 14–3-3, actin, calmodulin, ribosomal proteins (40S, 60S, L2, L3, L10A, RL5, S6, S7, S13, S14, S15A, and S18A), calreticulin, fructose-1,6-bisphosphate aldolase, enolase, adenosylhomocysteinase, glutathione reductase, GST, glyceraldehyde-3-phosphate dehydrogenase, HSP90, methionine adenosyltransferase, ATP citrate lyase, monodehydroascorbate reductase I, S-adenosylmethionine synthetase, b-amylase, nucleoside diphosphate kinase, HSP70, polyubiquitin, porin, SOD, lipoxygenase, glycosylated protein and histone H4.⁸ On the basis of localization the well-known and multifunctional protein in the cell is cytoplasmic 14–3-3 protein so its identification in the extracellular fluid is of great interest. It is confirmed by immunolocalization analysis that 14–3-3 is located in the extracellular fluid. So it was reported by Agrawal et al.⁸ that the secretion of 14–3-3 protein in the rhizosphere environment gives another benefit to the functional role of the protein 14–3-3 in plant cell. It was known that the root tip secretes high molecular weight polysaccharide mucilage and protect the root tip from the infection of pathogens.⁵²

Root-microbe communications

The techniques of gene expression analysis and tiny molecule swap among organisms are used to deliberate the communications among plants and microbes. For the most part, these studies have focused on aboveground interactions and fewer studies have examined these types of processes belowground.⁹⁵ Close interactions can be set up by roots with various microbes in rhizosphere, with both the harmful as well as useful bacteria. It has been discovered that in these interactions the protein swap over is an important task. Such exchanges contain the secretion of resistance proteins from roots, those proteins that can cause movement of bacteria toward or away from chemicals, the proteins that are present in the cells of root margins and those proteins that can trigger native defense mechanism in plants. Recent information about proteomics related to communication between roots and microorganisms can be used to recognize possible areas of improvement and advancement in agriculture.⁹⁵

In rhizosphere, biotic interactions are of great biological significance. But there is very little information about the role of secreted proteins in microbes and root interaction. Studies on Arabidopsis and Medicago sativa interacting with Pseudomonas syringae and Sinorhizobium meliloti respectively were done in order to get knowledge about protein secretion at some point in the communication between plant roots and interacting bacteria.⁹⁶

Rice roots grown in an aseptic hydroculture have about 100 secreted proteins identified so far.⁹⁷ About 55 percent of these proteins consist of predicted signal peptides and they are assumed to perform a very significant role in rhizosphere. Primary maize roots have mucilage from which secreted proteins were collected.⁹⁸

Legume chickpea’s secretome was characterized and 700 proteins has been identified by 1D SDS-PAGE and HPLC-MS/MS. On the basis of sequence homology, secretome is compared with the secretome of Oryza, Medicago, Arabidopsis. In secreted proteins high degree of species specificity was observed with little differences in the apoplast composition between monocots and dicots and different species as well.⁷⁶ Proteins isolated from fruit pericarp of 3 different cultivars of tomoto consist of 50 to 70 percent of signal peptides which makes clear that there is also possibility of cultivar specific secretome composition.⁹⁹

189 proteins were recognized by LC-MS/MS by means of brassica EST along with cDNA sequences. A predicted signal peptide was present in 164 proteins signifying that most proteins of the xylem sap are secreted. Proteases and oxido-reductases were more abundant in the xylem sap proteome, while enzyme inhibitors were uncommon.¹⁰⁰

Proteomic examination of the stigmatic exudates of Olea europaea and Lilium longiflorum led to the recognition of 51 and 57 proteins, correspondingly, most of them are described for the first time in this secreted fluid. These results specify that the stigmatic exudate is a metabolically active extracellular environment, participating in at least 80 different biological processes and 97 molecular functions.¹⁰¹

Conclusion and Future Prospects

Catalogue of recognized secretomes by experiments is enhancing and predicted to propagate at an unparalleled degree in coming future due to the recent heady procedures like GEM. In order to experimentally drench the secretome of plants and to completely apprehend its purpose it may take many years. That’s why, in order to foretell secretome proteins of plants, it would be astute to first use previously obtained genome wide extrapolation approaches. Such extrapolations will aid not only to evaluate the extent of inundation attained experimentally on secretomes, but joint with methodologies determined experimentally and statistics will permit producing theory and emerging finding based plans on the way to drenching and functionally interpreting the secretomes. Recent technical advances such as improved databases aid in the identification of secretome proteins. Proteomics in field-grown material exposed to complex, natural environments, and influenced by multiple organisms. Overall, we are closer than ever to global analyses of plant secretomes similar to what we have seen for some prokaryotes. It is the start of a long voyage to mark each member of the plant secretome family and to be familiar with their interactions before we start to know functions of the plant secretome. This will give us an idea about to gain better understanding on how cells connect the rigorous action of secreted protein network to their interior and exterior environments. We do know that this voyage is difficult and at times frustrating due to the variety and complexity of multicellular processes. But still it is one of the most fascinating and interesting studies in the scientific world.

Abbreviations:
Extracellular space	=	ECS
Apoplastic fluid	=	APF
Leaderless secreted proteins	=	LSPs
Unconventional protein secretion	=	UPS
Endoplasmic reticulum	=	ER
Suspension-cultured cells	=	SSCs
Vacuum infiltration centrifugation	=	VIC
Gravity extraction method	=	GEM
Acetyl trimethyl ammonium bromide	=	CTAB
Malate dehydrogenase	=	MDH
hexose phosphate isomerise	=	HPI
Post- translational modifications	=	PTMs
Selected reaction monitoring specific mass spectrometry	=	SRM-MS
extracellular matrix	=	ECM
Liquid chromatography–mass spectrometry/mass spectrometry	=	LC-MS/MS
O. sativa root meander curling	=	OsRMC
poly galacturonase inhibitor protein	=	PGIP
Matrix Assisted Laser Desorption/Ionization-time of flight/mass spectrometry	=	MALDI-TOF mass spectrometry
Liquid chromatography-Electrospray ionization/ mass spectrometry	=	-LC–ESI–MS/MS
Verticillium longisporum	=	VL
2-D Fluorescence Difference Gel Electrophoresis	=	2-D DIGE

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.