Hyoung Seok Kim

Calcium/calmodulin inhibition of the Arabidopsis BRASSINOSTEROID-INSENSITIVE 1 receptor kinase provides a possible link between calcium and brassinosteroid signalling

The receptor kinase BRI1 (BRASSINOSTEROID-INSENSITIVE 1) is a key component in BR (brassinosteroid) perception and signal transduction, and has a broad impact on plant growth and development. In the present study, we demonstrate that Arabidopsis CaM (calmodulin) binds to the recombinant cytoplasmic domain of BRI1 in a Ca2+-dependent manner in vitro. In silico analysis predicted binding to Helix E of the BRI1 kinase subdomain VIa and a synthetic peptide based on this sequence interacted with Ca2+/CaM. Co-expression of CaM with the cytoplasmic domain of BRI1 in Escherichia coli strongly reduced autophosphorylation of BRI1, in particular on tyrosine residues, and also reduced the BRI1-mediated transphosphorylation of E. coli proteins on tyrosine, threonine and presumably serine residues. Several isoforms of CaM and CMLs (CaM-like proteins) were more effective (AtCaM6, AtCaM7 and AtCML8, where At is Arabidopsis thaliana) than others (AtCaM2, AtCaM4 and AtCML11) when co-expressed with BRI1 in E. coli. These results establish a novel assay for recombinant BRI1 transphosphorylation activity and collectively uncover a possible new link between Ca2+ and BR signalling.

Chromosome doubling of the bioenergy crop, Miscanthus × giganteus.

The perennial grass, Miscanthus×giganteus is a sterile triploid, which due to its growth rate and biomass accumulation has significant economic potential as a new bioenergy crop. The sterility associated with the triploid genome of this accession requires labor‐intensive vegetative, instead of seed propagation for potential commercial production. Chromosome doubling was used to produce hexaploid plants in an effort to restore fertility to M×giganteus. Tissue culture derived calli from immature inflorescences were treated with the antimitotic agents, colchicine and oryzalin in liquid and solid media. Calli survival rate decreased with increasing concentrations and durations of colchicine or oryzalin treatments and ranged from 0% to 100%. Nuclear DNA content, as determined by flow cytometry, indicated that the frequency of chromosome‐doubled calli varied between compounds and concentrations with the greatest proportion of callus doubling observed using 2‐day treatments of 15 μm oryzalin (78%) or 939 μm colchicine (67%). Liquid media treatments were more effective than solid gels for chromosome doubling. Although oryzalin was effective at chromosome doubling, it inhibited callus growth and plant regeneration frequency. Seven hexaploid plants with doubled DNA content were generated, which displayed increased stomata size (30.0±0.2 μm) compared with regenerated triploid M. ×giganteus plants (24.3±1.0 μm). Following clonal replication these plants will be evaluated for growth rate, biomass accumulation, and pollen viability. Successful chromosome doubling and plant regeneration of M.×giganteus suggests that ploidy manipulation of this plant and its parental species (Miscanthus sinensis and Miscanthus sacchariflorus) could be a means to access genetic variability for the improvement of Miscanthus as a biofuel/bioenergy crop.

Miscanthus×giganteus plant regeneration: effect of callus types, ages and culture methods on regeneration competence

The perennial rhizomatous grass, Miscanthus×giganteus is an ideal biomass crop due to its rapid vegetative growth and high biomass yield potential. As a naturally occurring sterile hybrid, M. ×giganteus must be propagated vegetatively by mechanically divided rhizomes or from micropropagated plantlets. Plant regeneration through somatic embryogenesis is a viable approach to achieve large‐scale production of plantlets in tissue culture. Effect of the callus types, ages and culture methods on the regeneration competence was studied to improve regeneration efficiency and shorten the period of tissue culture in M. ×giganteus. Shoot‐forming calli having a yellow or white compact callus with light‐green shoot‐like structures showed the highest regeneration frequency. Percentage of shoot‐forming callus induction from immature inflorescence explants was 41% on callus induction medium containing 13.6 μM 2,4‐d and 0.44 μM benzyladenine (BA). The use of a regeneration medium containing 1.3 μM NAA and 22 μM BA was effective at shortening the incubation period required for plantlet regeneration, with 69% of total regenerated plantlets obtained within 1 month of incubation on regeneration medium. Embryogenic‐like callus morphotype could maintain regeneration competency for up to 1 year as suspension cultures. Field grown regenerated plants showed normal phenotypic development with DNA content and plant heights comparable to rhizome propagated plants. Winter survival rates of the regenerated plants planted in 2006 and 2007 at the University of Illinois South Farm, Urbana‐Champaign, Illinois, were 78% and 56%, respectively.

Methyl Jasmonate and 1-Methylcyclopropene Treatment Effects on Quinone Reductase Inducing Activity and Post-Harvest Quality of Broccoli

Effect of pre-harvest methyl jasmonate (MeJA) and post-harvest 1-methylcyclopropene (1-MCP) treatments on broccoli floret glucosinolate (GS) concentrations and quinone reductase (QR, an in vitro anti-cancer biomarker) inducing activity were evaluated two days prior to harvest, at harvest and at 10, 20, and 30 days of post-harvest storage at 4 °C. MeJA treatments four days prior to harvest of broccoli heads was observed to significantly increase floret ethylene biosynthesis resulting in chlorophyll catabolism during post-harvest storage and reduced product quality. Post-harvest treatment with 1-methylcyclopropene (1-MCP), which competitively binds to protein ethylene receptors, maintained post-harvest floret chlorophyll concentrations and product visual quality in both control and MeJA-treated broccoli. Transcript abundance of BoPPH, a gene which is responsible for the synthesis of pheophytinase, the primary enzyme associated with chlorophyll catabolism in broccoli, was reduced by 1-MCP treatment and showed a significant, negative correlation with floret chlorophyll concentrations. The GS, glucobrassicin, neoglucobrassicin, and gluconasturtiin were significantly increased by MeJA treatments. The products of some of the GS from endogenous myrosinase hydrolysis [sulforaphane (SF), neoascorbigen (NeoASG), N-methoxyindole-3-carbinol (NI3C), and phenethyl isothiocyanate (PEITC)] were also quantified and found to be significantly correlated with QR. Sulforaphane, the isothiocyanate hydrolysis product of the GS glucoraphanin, was found to be the most potent QR induction agent. Increased sulforaphane formation from the hydrolysis of glucoraphanin was associated with up-regulated gene expression of myrosinase (BoMyo) and the myrosinase enzyme co-factor gene, epithiospecifier modifier1 (BoESM1). This study demonstrates the combined treatment of MeJA and 1-MCP increased QR activity without post-harvest quality loss.

CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity

GmCDPKβ phosphorylates GmCDPKβ by protein kinase assay (View interaction)

Transphosphorylation of E. coli proteins during production of recombinant protein kinases provides a robust system to characterize kinase specificity

Protein kinase specificity is of fundamental importance to pathway regulation and signal transduction. Here, we report a convenient system to monitor the activity and specificity of recombinant protein kinases expressed in E. coli. We apply this to the study of the cytoplasmic domain of the plant receptor kinase BRASSINOSTEROID-INSENSITIVE 1 (BRI1), which functions in brassinosteroid (BR) signaling. Recombinant BRI1 is catalytically active and both autophosphorylates and transphosphorylates E. coli proteins in situ. Using enrichment approaches followed by LC-MS/MS, phosphosites were identified allowing motifs associated with auto- and transphosphorylation to be characterized. Four lines of evidence suggest that transphosphorylation of E. coli proteins by BRI1 is specific and therefore provides meaningful results: (1) phosphorylation is not correlated with bacterial protein abundance; (2) phosphosite stoichiometry, estimated by spectral counting, is also not related to protein abundance; (3) a transphosphorylation motif emerged with strong preference for basic residues both N- and C-terminal to the phosphosites; and (4) other protein kinases (BAK1, PEPR1, FLS2, and CDPKβ) phosphorylated a distinct set of E. coli proteins and phosphosites. The E. coli transphosphorylation assay can be applied broadly to protein kinases and provides a convenient and powerful system to elucidate kinase specificity.

An automated, high-throughput plant phenotyping system using machine learning-based plant segmentation and image analysis

A high-throughput plant phenotyping system automatically observes and grows many plant samples. Many plant sample images are acquired by the system to determine the characteristics of the plants (populations). Stable image acquisition and processing is very important to accurately determine the characteristics. However, hardware for acquiring plant images rapidly and stably, while minimizing plant stress, is lacking. Moreover, most software cannot adequately handle large-scale plant imaging. To address these problems, we developed a new, automated, high-throughput plant phenotyping system using simple and robust hardware, and an automated plant-imaging-analysis pipeline consisting of machine-learning-based plant segmentation. Our hardware acquires images reliably and quickly and minimizes plant stress. Furthermore, the images are processed automatically. In particular, large-scale plant-image datasets can be segmented precisely using a classifier developed using a superpixel-based machine-learning algorithm (Random Forest), and variations in plant parameters (such as area) over time can be assessed using the segmented images. We performed comparative evaluations to identify an appropriate learning algorithm for our proposed system, and tested three robust learning algorithms. We developed not only an automatic analysis pipeline but also a convenient means of plant-growth analysis that provides a learning data interface and visualization of plant growth trends. Thus, our system allows end-users such as plant biologists to analyze plant growth via large-scale plant image data easily.