Phytomicrobiome Interactions and Sustainable Agriculture. Группа авторов

their utility as important agricultural tools, there are inherent disadvantages to many of them and the search for novel, rapid and reliable techniques is an ongoing process within the scientific and agricultural communities.

The preparations needed for proteome analysis requires one to understand as to what stage is the analysis required. Proteome studies necessarily begin with whole protein extract which is subsequently subjected to gel‐based or non‐gel–based separation techniques. Gel‐based approaches use two‐dimensional gel electrophoresis (2DE) as well as differential gel electrophoresis (DIGE). Then there are few non‐gel–based techniques of protein separation such as isobaric technique for relative and absolute quantification (ITRAQ), isotope‐coded affinity tag (ICAT), multidimensional protein identification technique (mud PIT) and stable isotope labeling with the aid of amino acids in cell culture (SILAC). This is succeeded by the method of identification and quantification using tandem mass spectrometry (Quirino et al. 2010). Subsequent to the assimilation of the data on proteins, they are then subjected to a run through the protein databases in order to estimate the proper protein identification which includes the family it belongs to, and the similarity check to get a fair idea about its structural and functional tendencies. The techniques of protein separation can be broadly classified into gel‐based and non‐gel–based techniques which have been elaborated in the following section.

      2.4.1 Gel‐Based Protein Separation Techniques

      The most frequently used technique with gel‐based technology is the 2DE as this method involves utilizing several characteristic features of proteins for separation such as proteins, isoelectric point, molecular mass, as well as solubility (Joshi 2017).

      Here in the foremost step, the proteins are subjected to a pH gradation throughout their gel run. Hence proteins separate and precipitate when their isoelectric constant is achieved. At this point at a particular pH, the overall charge of the protein neutralizes thereby precipitating the proteins. This is also known as isoelectric focusing (IEF). IEF is followed by 2DE separation where the proteins are segregated based upon their molecular weight done by the SDS‐PAGE technique (Gorg et al. 2004). The protein separation profile is then visualized using certain staining methods that stain the proteins against the gel. Then with the comparison with a standard set of proteins, one can verify and characterize the isolated proteins. The major setback of this technology is limited reproducibility and lower sensitivity, hence only a well abundant and most characterized protein separation is recommended (Bunai and Yamane 2005). 2D fluorescence differential gel electrophoresis (2DDIGE) is an alternative method where the separation of proteins is done on the same gel using differential fluorescent dyes and a comparative is set based upon their spot intensity (Dunn 1993).

      2.4.2 Non‐Gel Protein Separation Techniques

      A non‐gel separation technique is usotope coded affinity tag (ICAT) is in principle used to compare the two protein‐separated samples. It consists of a tag having one biotin group containing an isotope‐coded linker, which forms the heavy and light sections of a tag while the other group is a thiol. This technique is conveniently utilized in the integral membrane protein identification (Gygi et al. 1999). Isobaric tagging of relative and absolute quantification of protein (iTRAQ) uses the N terminus of the peptide chain along with the side chain which is tagged by the isobaric‐labeled tags. Within a given sample, this technique permits quantification (relative and absolute) of peptides from varied sources all at once (Ross et al. 2004).

Another non‐gel technique is SILAC (stable isotope labeling by amino acid in cell culture). In this, the cell population consisting of protein of interest is cultivated in either N14‐ or N15‐containing cell medium. Cellular proteins are lysed, fractionated, and separated using 2DE. The role of N15 in this technique causes a shift and creates two peaks for each peptide. The ratio of intensities of peaks is substantially indicative of the amount of expression of proteins (Oda et al. 1999; Ong et al. 2002). SILAC is sensitive to the detection of the smallest of changes that may occur in a protein thus making this technique precise and a reliable method for quantification within MS. There is a relative assessment of the protein with the slightest change (Blagoev et al. 2004). Mud PIT (multidimensional protein identification technique) is another non‐gel–based method which utilizes principles of chromatography to separate the protein are directly associated to tandem mass spectrometry (Washburn et al. 2001; Wolters et al. 2001; McDonald et al. 2002). Quantification of protein is enabled by analysis through mass spectrometry. Upon retrieval of masses of proteins, a comparative of absolute masses is established using bioinformatics tools and computer programs such as OMSSA, MASCOT, and Phenyx (Ross et al. 2004). The path followed using these approaches for the detection of phytomicrobiome relationships is outlined in Figure 2.1.

Figure 2.1 A schematic flowchart of proteomic approaches for phytomicrobiome analysis.

2.5 Analysis of Phytomicrobial Interactions Using Proteomics Approaches

      Thus, microbes can be considered for positive exploitation of the plant growth promotion to enhance the growth of the plant. Microbes also make plants sustainable toward the abiotic as well as biotic stresses looking into progressive climate change. There have been many such types of research, where the positive associations have been well characterized using proteomic analysis and have further been utilized for the enhancement of a natural process.

One such aspect of microbial plant growth promotion is when such an association has been utilized for the purpose of phytoremediation. Phytoremediation is of great help in case of heavy metal remediation. The presence of metal‐solubilizing bacteria enhances the ability of the host plant to tolerate metal‐induced stress. The best case scenario can be observed in the bioethanol producers, such as H. tuberosus, a high biomass yielding crop that acquires enhancement in the accumulation and sustenance of a high concentration of zinc and cadmium. Similarly, the microbial population has been found to be colonizing the internal region of the roots as an endophyte, thereby resulting in increased assimilation of cadmium (Montalbán et al. 2017).

      Enhancement of drought and salt resistance observed for the enhanced salt and drought resistance owing to the effects of plant growth–promoting bacteria as studied in L. perenne, also known as ryegrass. Unfortunately for this perennially relevant grass as turf and forage, it is highly sensitive to drought‐induced stress and high salinity. However, B. amyloliquefaciens, combined with hydrogels to prevent the soil from eroding, can help the grass sustain the drought‐induced stress by the plant (Su et al. 2017).

      In another example, a species of plant growth‐promoting bacteria that have been found to be colonizing the roots, stems, and leaf parts of the sugarcane is the species Gluconacetobacter diazotrophicus. This is one such example where the bacteria serve as a legume endophyte. However, the proteome interaction studies of this bacterium with the sugar cane are still not very deep. A study was performed to investigate the molecular aspects using MS proteomic analysis using N15 metabolic labeling of bacterial, root samples, and co‐cultures. Over 400 proteins were assessed out of which near about 78 combinations of involved proteins appeared relevant among the interaction model. A comparative analysis of the proteomic data thus derived revealed the proteins involved in fundamental roles of protein recognition. Additionally, 30 bacterial active proteins have been identified where 9 were categorically induced by plant signaling pathways. This is the first line of study for the G. diazotrophicus and sugar cane (Schirawski and Perlin 2018).

      The microbial action on plants can work both ways. Certain interactions work for the enhancement of the plant, however, some have a negative impact owing to such associations. Any plant maybe rendered vulnerable to disease owing to an undesirable microbial association or any abiotic stress situation which throws the plants defenses out of order. In the case of the pathogen associate