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This review highlights discoveries made using phage display that impact the use of agricultural products. The contribution phage display made to our fundamental understanding of how various protective molecules serve to safeguard plants and seeds from herbivores and microbes is discussed. The utility of phage display for directed evolution of enzymes with enhanced capacities to degrade the complex polymers of the cell wall into molecules useful for biofuel production is surveyed. Food allergies are often directed against components of seeds; this review emphasizes how phage display has been employed to determine the seed component(s) contributing most to the allergenic reaction and how it has played a central role in novel approaches to mitigate patient response. Finally, an overview of the use of phage display in identifying the mature seed proteome protection and repair mechanisms is provided. The identification of specific classes of proteins preferentially bound by such protection and repair proteins leads to hypotheses concerning the importance of safeguarding the translational apparatus from damage during seed quiescence and environmental perturbations during germination. These examples, it is hoped, will spur the use of phage display in future plant science examining protein-ligand interactions.

In silico analysis of amino acid sequences of some aromatic and aliphatic microbial nitrilases for certain physiochemical properties and specificity to aromatic or aliphatic nitriles has been done. The multiple sequence alignment analysis of amino acid sequences has shown clear differences between aromatic and aliphatic nitrilases in terms of position specific presence of conserved amino acid. Statistical analysis of most of the physiochemical parameters did not show any clear distinction between the two nitrilases. In aromatic nitrilases the conserved amino acid residues besides active site domain triad (Glu, Lys, Cys) were His-129, Asn-168 and Arg-174 and these were replaced by Arg-129, His -168 and Lys-174 in aliphatic nitrilases. The physiochemical properties of these two groups of nitrilases also differed e.g. as compared to aliphatic nitrilases, aromatic nitrilases have lesser number of amino acid residues, lower molecular mass, higher pI values, higher content of Ala and Cys residues.

CELLULOSE SYNTHASE5 (CESA5) synthesizes cellulose necessary for seed mucilage adherence to seed coat epidermal cells of Arabidopsis (Arabidopsis thaliana). The involvement of additional CESA proteins in this process and details concerning the manner in which cellulose is deposited in the mucilage pocket are unknown. Here, we show that both CESA3 and CESA10 are highly expressed in this cell type at the time of mucilage synthesis and localize to the plasma membrane adjacent to the mucilage pocket. The isoxaben resistant1-1 and isoxaben resistant1-2 mutants affecting CESA3 show defects consistent with altered mucilage cellulose biosynthesis. CESA3 can interact with CESA5 in vitro, and green fluorescent protein-tagged CESA5, CESA3, and CESA10 proteins move in a linear, unidirectional fashion around the cytoplasmic column of the cell, parallel with the surface of the seed, in a pattern similar to that of cortical microtubules. Consistent with this movement, cytological evidence suggests that the mucilage is coiled around the columella and unwinds during mucilage extrusion to form a linear ray. Mutations in CESA5 and CESA3 affect the speed of mucilage extrusion and mucilage adherence. These findings imply that cellulose fibrils are synthesized in an ordered helical array around the columella, providing a distinct structure to the mucilage that is important for both mucilage extrusion and adherence.

Relationship of leaf anatomy with photosynthetic acclimation of Valeriana jatamansi was studied under full irradiance [FI, 1 600 μmol(PPFD) m−2 s−1] and net-shade [NS, 650 μmol(PPFD) m−2 s−1]. FI plants had thicker leaves with higher respiration rate (RD), nitrogen content per unit leaf area, chlorophyll a/b ratio, high leaf mass per leaf area unit (LMA), and surface area of mesophyll cell (Smes) and chloroplasts (Sc) facing intercellular space than NS plants. The difference between leaf thickness of FI and NS leaves was about 28 % but difference in photon-saturated rate of photosynthesis per unit leaf area (PNmax) was 50 %. This indicates that PNmax can increase to a larger extent than the leaf thickness with increasing irradiance in V. jatamansi. Anatomical studies showed that the mesophyll cells of FI plants had no open spaces along the mesophyll cell walls (higher Sc), but in NS plants wide open spaces along the mesophyll cell wall (lower Sc) were found. Positive correlation between Sc and PNmax explained the higher PNmax in FI plants. Increase in mesophyll thickness increased the availability of space along the mesophyll cell wall for chloroplasts (increased Sc) and hence PNmax was higher in FI plants. Thus this Himalayan species can acclimate to full sunlight by altering leaf anatomy and therefore may be cultivated in open fields.

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

A group of intrinsically disordered, hydrophilic proteins—Late Embryogenesis Abundant (LEA) proteins—has been linked to survival in plants and animals in periods of stress, putatively through safeguarding enzymatic function and prevention of aggregation in times of dehydration/heat. Yet despite decades of effort, the molecular-level mechanisms defining this protective function remain unknown. A recent effort to understand LEA functionality began with the unique application of phage display, wherein phage display and biopanning over recombinant Seed Maturation Protein homologs fromArabidopsis thalianaandGlycine maxwere used to retrieve client proteins at two different temperatures, with one intended to represent heat stress. From this previous study, we identified 21 client proteins for which clones were recovered, sometimes repeatedly. Here, we use sequence analysis and homology modeling of the client proteins to ascertain common sequence and structural properties that may contribute to binding affinity with the protective LEA protein. Our methods uncover what appears to be a predilection for protein-nucleic acid interactions among LEA client proteins, which is suggestive of subcellular residence. The results from this initial computational study will guide future efforts to uncover the protein protective mechanisms during heat stress, potentially leading to phage-display-directed evolution of synthetic LEA molecules.

Morpho-anatomical leaf traits and photosynthetic activity of two alpine herbs, Podophyllum hexandrum (shade-tolerant) and Rheum emodi (light-requiring), were studied under field (PAR>2 000 µmol m−2 s−1) and greenhouse (PAR 500 µmol m−2 s−1) conditions. Mesophyll thickness, surface area of mesophyll cells facing intercellular spaces (Smes), surface area of chloroplasts facing intercellular spaces (Sc), intercellular spaces of mesophyll cells (porosity), photon-saturated rate of photosynthesis per unit leaf area (P Nmax), and ribulose-1,5-bisphosphate carboxylase/oxygenase activity decreased in the greenhouse with respect to the field and the decreases were significantly higher in R. emodi than in P. hexandrum. P. hexandrum had lower intercellular CO2 concentration than R. emodi under both irradiances. The differences in acclimation of the two alpine herbs to low irradiance were due to their highly unlikely changes in leaf morphology, anatomy, and P Nmax which indicated that the difference in radiant energy requirement related to leaf acclimation had greater impact under low than high irradiance.

Background Improving saccharification efficiency in bioenergy crop species remains an important challenge. Here, we report the characterization of a Sorghum (Sorghum bicolor L.) mutant, named REDforGREEN (RG), as a bioenergy feedstock. Results It was found that RG displayed increased accumulation of lignin in leaves and depletion in the stems, antithetic to the trend observed in wild type. Consistent with these measurements, the RG leaf tissue displayed reduced saccharification efficiency whereas the stem saccharification efficiency increased relative to wild type. Reduced lignin was linked to improved saccharification in RG stems, but a chemical shift to greater S:G ratios in RG stem lignin was also observed. Similarities in cellulose content and structure by XRD-analysis support the correlation between increased saccharification properties and reduced lignin instead of changes in the cellulose composition and/or structure. Conclusion Antithetic lignin accumulation was observed in the RG mutant leaf-and stem-tissue, which resulted in greater saccharification efficiency in the RG stem and differential thermochemical product yield in high lignin leaves. Thus, the red leaf coloration of the RG mutant represents a potential marker for improved conversion of stem cellulose to fermentable sugars in the C4 grass Sorghum.