The following student awards were presented at the 2018  In Vitro Biology Meeting in Saint Louis, MO. Information on additional awardees at the 2018 Meeting will be presented in the next issue of the In Vitro Report. Information related to the available specific student awards can be found on the SIVB website (www.sivb.org) or by contacting the SIVB Business Office at sivb@sivb.org.

2018 HONOR B. FELL AWARD and 2018 SIVB STUDENT TRAVEL AWARD

The Role of Kar2/Scj1 Complex in the DNA Damage Response

Hsp70 is a well-conserved molecular chaperone involved in the folding, stabilization, and eventual degradation of many “client” proteins. Hsp70 is regulated by a suite of co-chaperone molecules that assist in Hsp70-client interaction and stimulate the intrinsic ATPase of Hsp70. While previous studies have shown the anticancer target ribonucleotide reductase (RNR) is a client of Hsp70 in yeast and mammalian cells, the regulatory co-chaperones involved remain to be determined. To identify co-chaperone(s) involved in RNR activity, 30 yeast Hsp70 co-chaperone knockout mutants were screened for sensitivity to the RNR-perturbing agent Hydroxyurea (HU). Interestingly, cells lacking the ER-resident co-chaperone Scj1 were sensitive to HU. Scj1 is an ER-based co-chaperone that binds the ER Hsp70 isoform Kar2 (homologue of BiP). Strikingly, removal of the C-terminal KDEL ER localization sequence from Scj1 only produced mild sensitivity to HU. To determine the functional role for Scj1 in mediating resistance to HU, we undertook a multicopy suppressor screen for genes that when overexpressed could rescue the HU-sensitive phenotype of of scj1∆ cells. Aside from RNR4 and SCJ1, identified GRX2, a glutaredoxin known to support RNR through hydrogen ion donation. Taken together, these findings suggest a novel indirect role for Scj1/Kar2 in the cellular response to replicative stress.

Laura Knighton, UNC Charlotte, Charlotte, NC. In Vitro Cellular and Developmental Biology, 54:S26, 2018

 


2018 HOPE E. HOPPS AWARD 2018 SIVB STUDENT TRAVEL AWARD

Effect of Oxalic Acid (OA) Phytotoxins of (Sclerotium Cepivorum) Fungi on Onion Plants and Contents of Some Biochemical Compound

In some plant diseases phytotoxins may play a critical role in the development of the disease symptoms. (OA) ranks among the strongest organic acids and has been shown to decrease the pH of woody tissues from 5.5 to 2.8, not only acidifies the plant tissue but also chelates Ca++ from the cell wall, rendering the stressed tissue susceptible to a battery of fungal degradative enzymes. The objective of this study was to identify a suitable protocol for in vitro selection of Allium white rot disease (Sclerotium cepivorum) tolerance in commercial Egyptian onion varieties, namely G20 (Giza 20), G6 (Giza 6) and BR (Beheri Red). Oxalic acid (OA), the phytotoxin produced by Sclerotium cepivorum, was used as the selective agent. Seeds of the three varieties were germinated on four concentrations (0.0, 0.02, 0.2, 2 and 20 mM) of Oxalic acid. Among the tested cultivars, BR had the highest germination frequency (52%) at all concentrations tested. Cotyledon explants from the varieties were cultured on toxic medium, supplemented with 0, 3, 6 and 12 mM OA to use the calli induction time as additional selection cycle. The survival of calli on free toxic medium was 70.7% for all tested cultivars; however, medium, with 3 mM OA reduced the viable calli to 42.1%. The highest OA concentration (12 mM) completely inhibited calli induction from cotyledons explants. In in vitro Selection Protocol a medium supplement with 3 mM OA retarded 80% of calli growth.  Biochemical Pungency and Total phenolic were determined in the donor plants, putative resistant calli and regenerated plants of three genotypes. The BR cultivar exhibited highest pungency contents than other two cultivars (G20 and G6). The data showed that total phenol ranged between 8.8 mg/g dry tissue to 28.2 mg/g dry tissue. The highest mean content (20.85 mg/g dry tissue) of total phenol found in calli which grown in high level (6 mM OA) of toxic. While the low content (11.2 mg/g dry tissue) in calli grown in OA free medium. The content of polyphenol in normal leaves mean 31.98 mg/g dry tissue, highest than in regenerated plants leaf mean 25.4 mg/g dry tissue.

Abdelrahem Yousef, Agriculture Research Centre, 9 El-Gamaa St., Giza, Egypt. In Vitro Cellular and Developmental Biology, 54:S52-53, 2018


2018 JOSEPH F. MORGAN AWARD 2018 SIVB STUDENT TRAVEL AWARD

Tryptophan Mediates Morphogenesis in St. John’s Wort (Hypericum perforatum L.) Via Interplay Between Auxin- and Indoleamine-dependent and Independent Mechanisms 

It has been more than 60 years since Skoog and Miller reported the redirection of plant growth to root production by exposure to auxins in culture media but the biochemical mechanisms that control regeneration are still not completely understood. One challenge with understanding regeneration mechanisms is the potential that applied growth regulators may act indirectly or may be metabolized to active growth regulators. For example, it is possible that the effects attributed to auxin may actually be the result of auxin metabolism to tryptophan or melatonin. We hypothesized that tryptophan is metabolized to auxin, melatonin or serotonin inducing de novo organogenesis in St. John’s wort (Hypericum perforatum L.).  Root explants from two germplasm lines of St. John’s wort with altered tryptophan to melatonin metabolism and a wildtype control were incubated with 0 or 10 μM auxin or tryptophan for 0, 24, 48 or 72 h.  Incubation with tryptophan increased de novo shoot organogenesis while incubation with auxin led to de novo root regeneration. Auxin-induced roots were short, pale and had significant root hair development vs longer dark brown roots developed on control or tryptophan medium. Quantification of tryptophan, auxin, serotonin and melatonin did not show a ubiquitous response, but levels varied both by line and duration of exposure. Additionally, while tryptophan showed differential effects across the three lines tested, auxin did not show variable growth between the three lines. These results suggest tryptophan mediates plant growth in a tryptophan-specific manner which cannot be solely explained by increasing precursor supply and flux through established plant growth regulator pathways.

Lauren Alexandra Elizabeth Erland, University of Guelph, Guelph, ON, CANADA. In Vitro Cellular and Developmental Biology, 54:S28-29, 2018


2018 PHILIP R. WHITE AWARD

Engineering Plant Cell Wall with “Designer” Glycopeptides for Improved Biomass Processability

Engineering the plant cell wall with thermostable cell wall depolymerizing (CWD) enzymes represents a promising solution to reduce the overall cost of plant biomass processing for the production of biofuels and/or other biobased products. However, many in planta-expressed CWD enzymes targeted for secretion were mainly retained inside the cytoplasm membrane instead of being secreted into the cell wall matrix. This project aims to leverage an innovative strategy unique to plants − hydroxyproline (Hyp)-O-glycosylation − for de novo design and engineering of novel Hyp-O-glycosylated peptides (HypGPs) that can function as a molecular carrier for the heterologous CWD enzymes expressed in planta to maximize their functions in tailoring cell wall composition and architecture. Different designs of HypGP tags, including an extensin-based (SP4)18 module and some arabinogalactan proteins (AGP)-based (SP)32, (AP)20 and (TP)20 modules, were engineered into tobacco plants as fusion with a reporter protein (GFP) to characterize the Hyp-O-glycosylation and molecular carrier function of the designer HypGP modules, and to assess the phenotypic change of the transgenic plants. While the (SP4)18 module was constitutively Hyp-O-glycosylated but with a rate-limiting glycosylation process, none of the engineered AGP-based modules were Hyp-O-glycosylated. A novel O-glycosylation signal peptide tag facilitating the full glycosylation of the designer modules was finally identified. To establish a proof of concept of the HypGP engineering utilized for cell wall reconstruction, a thermostable CWD enzyme, E1 endoglucanase from Acidothermus cellulolyticus was engineered into tobacco plants with/without a HypGP tag. The engineered HypGP tag improved the enzyme accumulation in planta and increased the biomass saccharification efficiency by 3.2-fold (compared with the wild type plant) without significantly affecting the biomass accumulation yields.

Tristen Dewayne Wright, Arkansas Biosciences Institute, State University, AR. In Vitro Cellular and Developmental Biology, 54:S52, 2018