Discipline: Biological Sciences
Mekiya Fletcher - Virginia State University
Co-Author(s): Irmgard Seidl-Adams and James Tumlinson, Pennsylvania State University, State College, PA Sarah Melissa Witiak, Virginia State University, Petersburg,VA
Green plants produce diverse volatile organic compounds (VOC) in response to herbivory and other stresses. One class of VOC is called green leaf volatiles (GLVs) that facilitate communication within and between plants and other trophic levels. (Holopainen et al. 2012). GLVs consist of six-carbon aldehydes, alcohols and their esters. While we know that plants exposed to Z-3-hexenal (HAL) convert it to Z-3-hexenyl acetate (HAC) and HAL synthesis enzymes are found in the chloroplast envelope, we do not know which cell types sense HAL or HAC in the leaf. Assuming that the conversion and the biosynthesis of GLVs occur in the same location, we hypothesized that GLVs are sensed in mesophyll cells, which have the most direct contact with entering volatile compounds. To test whether and which GLVs are detected by maize mesophyll cells, we developed an assay to isolate mesophyll protoplasts and measure their GLV sensitivity. This information will help us to better understand the signaling and response dynamics of these important compounds. Protoplasts were isolated from 10-day-old corn seedlings (W438). Millimeter thin strips cut from the midsection of four fully expanded leaves were digested by cellulase and maceroenzyme for 3 hours. Intact protoplasts were isolated by differential centrifugation through a sucrose dextran gradient. Protoplasts were concentrated, washed and spread as a thin film on a surface of wash buffer, solidified with agarose in the depression of a depression slide. Protoplasts were then exposed for 20 minutes to 1µg, 5µg or 20µg of Z-3-HAC, all dissolved in 20µL of hexane. Control treatments were no treatment and 20µl hexane alone. Depression slides were placed in glass petri dishes with water on the bottom to create a sealed space. After exposure, total RNA was extracted, DNase treated, and reverse transcribed. To detect plant response to HAC, we measured transcript levels of MYC7, a transcription factor implicated in GLV responses (Engelberth et al 2013). MYC7 transcript levels relative to the expression of two reference genes (APT and CYS) were determined by qRT-PCR. Each treatment was replicated 3 times. Expression levels of MYC7 were compared using a one-way ANOVA. While MYC7 transcript levels were not significantly affected by the hexane treatment or 20µg HAC (f =3.33, p=0.08), 1µg HAC reduced MYC7 transcript levels in two independent experiments by 80%. This variability suggests that the isolation protocol needs to be more precise. For example, corn contains specialized mesophyll cells surrounding the bundle sheath, while other mesophyll cells are found between veins. They can be distinguished according to their sizes. We will improve precision by increasing the sucrose gradient concentration steps to separate these distinct mesophyll types, enabling us to clarify the cellular perception of HAC and other GLVs.
Funder Acknowledgement(s): I want to thank T. Baumgarten, T. McNeilis, SROP and Pennsylvania State University for giving me this opportunity and for their help . I also want to thank A. Ansari for help with funding . I was supported by a grant from AFRI (USDA) subaward to Sarah Melissa Witiak.
Faculty Advisor: Sarah Melissa Witiak, email@example.com
Role: I participated in every aspect of the research. At Penn State, I studied the literature, performed protoplast isolations and assisted with RNA extraction, DNAse treatment and qPCR. I had input on how to change the protocol based on the literature. I also helped with data analysis. I am now performing additional experiments completely on my own at my home university (Virginia State University).