Isolation and Characterization of Ice-Binding Proteins from Higher Plants.

The characterization of ice-binding proteins (IBPs) from plants can involve many techniques, a few of which are presented here. Chief among these methods are tests for ice recrystallization inhibition, an activity characteristic of plant IBPs. Two related procedures are described, both of which can be used to demonstrate and quantify ice-binding activity. First, is the traditional "splat" assay, which can easily be set up using common laboratory equipment, and second, is our modification of this method using superhydrophobic coated sapphire for analysis of multiple samples in tandem. Thermal hysteresis is described as another method for quantifying ice-binding activity, during which ice crystal morphology observations can be used to provide clues about ice-plane binding. Once ice-binding activity has been evaluated, it is necessary to verify IBP identity. We detail two methods for enriching IBPs from complex mixtures using ice-affinity purification, the "ice-finger" and "ice-shell" methods, and we highlight their advantages and limitations for the isolation of plant IBPs. Recombinant IBP expression, necessary for detailed ice-binding analysis, can present challenges. Here, a strategy for recovery of soluble, active protein is described. Lastly, verification of function in planta borrows from standard protocols, but with an additional screen applicable to IBPs. Together, these methods, and a few considerations critical to success, can be used to assist researchers wishing to isolate and characterize IBPs from plants.

Ice-binding proteins confer freezing tolerance in transgenic Arabidopsis thaliana.

Lolium perenne is a freeze-tolerant perennial ryegrass capable of withstanding temperatures below -13 °C. Ice-binding proteins (IBPs) presumably help prevent damage associated with freezing by restricting the growth of ice crystals in the apoplast. We have investigated the expression, localization and in planta freezing protection capabilities of two L. perenne IBP isoforms, LpIRI2 and LpIRI3, as well as a processed IBP (LpAFP). One of these isoforms, LpIRI2, lacks a conventional signal peptide and was assumed to be a pseudogene. Nevertheless, both LpIRI2 and LpIRI3 transcripts were up-regulated following cold acclimation. LpIRI2 also demonstrated ice-binding activity when produced recombinantly in Escherichia coli. Both the LpIRI3 and LpIRI2 isoforms appeared to accumulate in the apoplast of transgenic Arabidopsis thaliana plants. In contrast, the fully processed isoform, LpAFP, remained intracellular. Transgenic plants expressing either LpIRI2 or LpIRI3 showed reduced ion leakage (12%-39%) after low-temperature treatments, and significantly improved freezing survival, while transgenic LpAFP-expressing lines did not confer substantial subzero protection. Freeze protection was further enhanced by with the introduction of more than one IBP isoform; ion leakage was reduced 26%-35% and 10% of plants survived temperatures as low as -8 °C. Our results demonstrate that apoplastic expression of multiple L. perenne IBP isoforms shows promise for providing protection to crops susceptible to freeze-induced damage.

Knockdown of Ice-Binding Proteins in Brachypodium distachyon Demonstrates Their Role in Freeze Protection.

Sub-zero temperatures pose a major threat to the survival of cold-climate perennials. Some of these freeze-tolerant plants produce ice-binding proteins (IBPs) that offer frost protection by restricting ice crystal growth and preventing expansion-induced lysis of the plasma membranes. Despite the extensive in vitro characterization of such proteins, the importance of IBPs in the freezing stress response has not been investigated. Using the freeze-tolerant grass and model crop, Brachypodium distachyon, we characterized putative IBPs (BdIRIs) and generated the first 'IBP-knockdowns'. Seven IBP sequences were identified and expressed in Escherichia coli, with all of the recombinant proteins demonstrating moderate to high levels of ice-recrystallization inhibition (IRI) activity, low levels of thermal hysteresis (TH) activity (0.03-0.09°C at 1 mg/mL) and apparent adsorption to ice primary prism planes. Following plant cold acclimation, IBPs purified from wild-type B. distachyon cell lysates similarly showed high levels of IRI activity, hexagonal ice-shaping, and low levels of TH activity (0.15°C at 0.5 mg/mL total protein). The transfer of a microRNA construct to wild-type plants resulted in the attenuation of IBP activity. The resulting knockdown mutant plants had reduced ability to restrict ice-crystal growth and a 63% reduction in TH activity. Additionally, all transgenic lines were significantly more vulnerable to electrolyte leakage after freezing to -10°C, showing a 13-22% increase in released ions compared to wild-type. IBP-knockdown lines also demonstrated a significant decrease in viability following freezing to -8°C, with some lines showing only two-thirds the survival seen in control lines. These results underscore the vital role IBPs play in the development of a freeze-tolerant phenotype and suggests that expression of these proteins in frost-susceptible plants could be valuable for the production of more winter-hardy crops.

Isolation and characterization of ice-binding proteins from higher plants.

The characterization of ice-binding proteins from plants can involve many techniques, only a few of which are presented here. Chief among these methods are tests for ice recrystallization inhibition activity. Two distinct procedures are described; neither is normally used for precise quantitative assays. Thermal hysteresis assays are used for quantitative studies but are also useful for ice crystal morphologies, which are important for the understanding of ice-plane binding. Once the sequence of interest is cloned, recombinant expression, necessary to verify ice-binding protein identity can present challenges, and a strategy for recovery of soluble, active protein is described. Lastly, verification of function in planta borrows from standard protocols, but with an additional screen applicable to ice-binding proteins. Here we have attempted to assist researchers wishing to isolate and characterize ice-binding proteins from plants with a few methods critical to success.

The Arabidopsis calmodulin-like protein, CML39, functions during early seedling establishment.

During Ca(2+) signal transduction, Ca(2+)-binding proteins known as Ca(2+) sensors function to decode stimulus-specific Ca(2+) signals into downstream responses. Plants possess extended families of unique Ca(2+) sensors termed calmodulin-like proteins (CMLs) whose cellular roles are not well understood. CML39 encodes a predicted Ca(2+) sensor whose expression is strongly increased in response to diverse external stimuli. In the present study, we explored the biochemical properties of recombinant CML39, and used a reverse genetics approach to investigate its physiological role. Our data indicate that Ca(2+) binding by CML39 induces a conformational change in the protein that results in an increase in exposed-surface hydrophobicity, a property that is consistent with its predicted function as a Ca(2+) sensor. Loss-of-function cml39 mutants resemble wild-type plants under normal growth conditions but exhibit persistent arrest at the seedling stage if grown in the absence of sucrose or other metabolizable carbon sources. Under short-day conditions, cml39 mutants display increased sucrose-induced hypocotyl elongation. When grown in the dark, cml39 mutants show impaired hypocotyl elongation in the absence of sucrose. Promoter-reporter data indicate that CML39 expression is prominent in the apical hook in dark-grown seedlings. Collectively, our data suggest that CML39 functions in Arabidopsis as a Ca(2+) sensor that plays an important role in the transduction of light signals that promote seedling establishment.

Narrowing down the targets: towards successful genetic engineering of drought-tolerant crops.

Drought is the most important environmental stress affecting agriculture worldwide. Exploiting yield potential and maintaining yield stability of crops in water-limited environments are urgent tasks that must be undertaken in order to guarantee food supply for the increasing world population. Tremendous efforts have been devoted to identifying key regulators in plant drought response through genetic, molecular, and biochemical studies using, in most cases, the model species Arabidopsis thaliana. However, only a small portion of these regulators have been explored as potential candidate genes for their application in the improvement of drought tolerance in crops. Based on biological functions, these genes can be classified into the following three categories: (1) stress-responsive transcriptional regulation (e.g. DREB1, AREB, NF-YB); (2) post-transcriptional RNA or protein modifications such as phosphorylation/dephosphorylation (e.g. SnRK2, ABI1) and farnesylation (e.g. ERA1); and (3) osomoprotectant metabolism or molecular chaperones (e.g. CspB). While continuing down the path to discovery of new target genes, serious efforts are also focused on fine-tuning the expression of the known candidate genes for stress tolerance in specific temporal and spatial patterns to avoid negative effects in plant growth and development. These efforts are starting to bear fruit by showing yield improvements in several crops under a variety of water-deprivation conditions. As most such evaluations have been performed under controlled growth environments, a gap still remains between early success in the laboratory and the application of these techniques to the elite cultivars of staple crops in the field. Nevertheless, significant progress has been made in the identification of signaling pathways and master regulators for drought tolerance. The knowledge acquired will facilitate the genetic engineering of single or multiple targets and quantitative trait loci in key crops to create commercial-grade cultivars with high-yielding potential under both optimal and suboptimal conditions.

Developmental and stimulus-induced expression patterns of Arabidopsis calmodulin-like genes CML37, CML38 and CML39.

Various aspects of plant development and stress physiology are mediated by Ca(2+) signaling. Ca(2+) sensors, such as calmodulin, detect these signals and direct downstream signaling pathways by binding and activating diverse targets. Plants possess many unique, putative Ca(2+) sensors, including a large family (50 in Arabidopsis) of calmodulin-like proteins termed CMLs. Some of these CMLs have been implicated in Ca(2+)-based stress response but most remain unstudied. We generated transgenic plants expressing CML::GUS reporter genes for members of a subfamily of CMLs (CML37, CML38 and CML39) which allowed us to investigate their expression patterns in detail. We found that CML::GUS genes displayed unique tissue, cell-type, and temporal patterns of expression throughout normal development, particularly in the flower, and in response to a variety of stimuli, including biotic and abiotic stress, hormone and chemical treatments. Our findings are supported by semiquantitative reverse-transcription PCR as well as analyses of microarray databases. Analysis of purified, recombinant CMLs demonstrated their ability to bind Ca(2+) in vitro. Collectively, our data suggest that these CMLs likely play important roles as sensors in Ca(2+)-mediated developmental and stress response pathways and provide a framework of spatial and temporal expression to direct future studies aimed at elucidating their physiological roles.

Biochemical and molecular characterization of AtPAP26, a vacuolar purple acid phosphatase up-regulated in phosphate-deprived Arabidopsis suspension cells and seedlings.

A vacuolar acid phosphatase (APase) that accumulates during phosphate (Pi) starvation of Arabidopsis (Arabidopsis thaliana) suspension cells was purified to homogeneity. The final preparation is a purple APase (PAP), as it exhibited a pink color in solution (A(max) = 520 nm). It exists as a 100-kD homodimer composed of 55-kD glycosylated subunits that cross-reacted with an anti-(tomato intracellular PAP)-IgG. BLAST analysis of its 23-amino acid N-terminal sequence revealed that this PAP is encoded by At5g34850 (AtPAP26; one of 29 PAP genes in Arabidopsis) and that a 30-amino acid signal peptide is cleaved from the AtPAP26 preprotein during its translocation into the vacuole. AtPAP26 displays much stronger sequence similarity to orthologs from other plants than to other Arabidopsis PAPs. AtPAP26 exhibited optimal activity at pH 5.6 and broad substrate selectivity. The 5-fold increase in APase activity that occurred in Pi-deprived cells was paralleled by a similar increase in the amount of a 55-kD anti-(tomato PAP or AtPAP26)-IgG immunoreactive polypeptide and a >30-fold reduction in intracellular free Pi concentration. Semiquantitative reverse transcription-PCR indicated that Pi-sufficient, Pi-starved, and Pi-resupplied cells contain similar amounts of AtPAP26 transcripts. Thus, transcriptional controls appear to exert little influence on AtPAP26 levels, relative to translational and/or proteolytic controls. APase activity and AtPAP26 protein levels were also up-regulated in shoots and roots of Pi-deprived Arabidopsis seedlings. We hypothesize that AtPAP26 recycles Pi from intracellular P metabolites in Pi-starved Arabidopsis. As AtPAP26 also exhibited alkaline peroxidase activity, a potential additional role in the metabolism of reactive oxygen species is discussed.