UNIVERSITY of CALIFORNIA RIVERSIDE a Cell Wall Fragment Affects Ethylene Response in Arabidopsis a Dissertation Submitted In
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UNIVERSITY OF CALIFORNIA RIVERSIDE A Cell Wall Fragment Affects Ethylene Response in Arabidopsis A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Biochemistry and Molecular Biology by Ryan Caleb Schiefelbein September 2020 Dissertation Committee: Dr. Paul Larsen, Chairperson Dr. Meng Chen Dr. Gregory Alan Barding Copyright by Ryan Caleb Schiefelbein 2020 The Dissertation of Ryan Caleb Schiefelbein is approved: Committee Chairperson University of California, Riverside ABSTRACT OF THE DISSERTATION A Cell Wall Fragment Affects Ethylene Response in Arabidopsis by Ryan Caleb Schiefelbein Doctor of Philosophy, Graduate Program in Biochemistry and Molecular Biology University of California, Riverside, September 2020 Dr. Paul Larsen, Chairperson The plant hormone ethylene is a simple alkene which regulates germination, growth, stress responses, pathogen response, tissue development, fruit ripening, and senescence in plants. In an effort to identify new factors that relate to how plants respond to ethylene, an Arabidopsis thaliana mutagenesis screen revealed a mutation that is hypersensitive to ethylene. The mutation that gave rise to this aberrant ethylene response phenotype was found to caused by a new pmr6 allele. PMR6 (POWDERY MILDEW RESISTANT 6) encodes a putative pectate lyase that was first identified for pmr6 plants being unable to support powdery mildew growth, presumably by effecting the cell wall composition. The identification of PMR6 as a factor involved in ethylene signaling suggested a novel role and the subsequent characterization of this new pmr6-6 allele utilized known ethylene mutants to characterize the aberrant ethylene response. While pmr6-6 plants do produce additional ethylene, ethylene responses are promoted iv independent of ethylene binding to the receptors in pmr6-6 plants. Ethylene response is found to be promoted by soluble metabolites from ethylene treated seedlings present in growth media. The pmr6-6 plant is sensitive to these soluble metabolites and results in shortening of the hypocotyl of etiolated seedlings while wild type plants with functional PMR6 plants do not express an ethylene response at the same concentration. The ethylene response was found to be dependent on EIN2 and the ethylene transcription factors EIN3 and EIL1 to display an ethylene phenotype. These soluble metabolites were also able to promote the stabilization of EIN3:GFP independent of ethylene perception. The PMR6 substrate may play an important role in plant defense response and may be one way that plants detect and respond to changes in the cell wall integrity or to changes in growth that produce cell wall fragments in the extracellular space. v Table of Contents CHAPTER 1 – ETHYLENE AND ITS ROLE IN GROWTH AND DEVELOPMENT ................................ 1 CHAPTER 2 – ETHYLENE BIOSYNTHESIS ............................................................................................ 10 CHAPTER 3 – THE ETHYLENE SIGNALING PATHWAY ..................................................................... 20 CHAPTER 4 – PLANT CELL WALL BIOSYNTHESIS, MODIFICATION AND SIGNALING ............. 29 CHAPTER 5 – EVIDENCE OF A NOVEL SIGNALING EVENT IN PMR6-6 ......................................... 43 CONCLUSION ............................................................................................................................................. 88 MATERIALS AND METHODS .................................................................................................................. 97 LITERATURE CITED................................................................................................................................ 112 vi List of Figures FIGURE 1 – THE METHIONINE SALVAGE PATHWAY WITH ETHYLENE BIOSYNTHESIS ....... 11 FIGURE 2 – BIOTIC AND ABIOTIC FACTORS AFFECT ETHYLENE BIOSYNTHESIS AND ETHYLENE SIGNALING .................................................................................................... 17 FIGURE 3 – THE CORE OF THE ETHYLENE SIGNALING PATHWAY ............................................ 22 FIGURE 4 – HOMOGALACTURONAN PECTIN STRUCTURE ........................................................... 30 FIGURE 5 – GLYCOSIDE HYDROLASE AND PECTATE LYASE ENZYME REACTIONS ............. 35 FIGURE 6 – PMR6-6 ETHYLENE DOSE RESPONSE FOR HYPOCOTYL INHIBITION.................... 46 FIGURE 7 – PMR6-6 ETHYLENE DOSE RESPONSE FOR ROOT INHIBITION ................................. 47 FIGURE 8 – PMR6-6 ETHYLENE DOSE RESPONSE FOR APICAL HOOK FORMATION ............... 48 FIGURE 9 – RATES OF ETHYLENE BIOSYNTHESIS AND NORMALIZATION BY AVG .............. 49 FIGURE 10 – ETHYLENE TREATMENT OF PMR6 ALLELES .............................................................. 52 FIGURE 11 – ETHYLENE TREATMENT OF 3 PMR6 OVEREXPRESSOR LINES ............................... 54 FIGURE 12 – ETHYLENE TREATMENT OF ETR1-1;PMR6-6................................................................ 56 FIGURE 13 – GROWTH OF CTR1-3;PMR6-6 ............................................................................................ 57 FIGURE 14 – ETHYLENE TREATMENT OF EIN2-5;PMR6-6 ................................................................ 58 FIGURE 15 – ETHYLENE TREATMENT OF EIN3-1;EIL1-1;PMR6-6 .................................................... 59 FIGURE 16 – SOLUBLE ANALYTE HYPOCOTYL GROWTH ANALYSIS OF PMR6-6 ..................... 63 FIGURE 17 – SOLUBLE ANALYTE ROOT GROWTH ANALYSIS OF PMR6-6................................... 64 FIGURE 18 – HEAT STABILITY OF SOLUBLE ANALYTE BY HYPOCOTYL GROWTH ANALYSIS OF PMR6-6 ............................................................................................................................ 67 vii FIGURE 19 – HYPOCOTYL GROWTH UNDER SATURATING ACC CONCENTRATIONS .............. 68 FIGURE 20 – SOLUBLE ANALYTE HYPOCOTYL ANALYSIS OF PMR6 OVEREXPRESSOR ......... 70 FIGURE 21 – SOLUBLE ANALYTE HYPOCOTYL ANALYSIS OF PMR6 ALLELES ......................... 71 FIGURE 22 – SOLUBLE ANALYTE HYPOCOTYL ANALYSIS OF ETR1-1;PMR6-6 .......................... 73 FIGURE 23 – SOLUBLE ANALYTE HYPOCOTYL ANALYSIS OF EIN2-5;PMR6-6 ........................... 74 FIGURE 24 – SOLUBLE ANALYTE HYPOCOTYL ANALYSIS OF EIN3-1;EIL1-1;PMR6-6 .............. 75 FIGURE 25 – EIN3::GFP ACCUMULATION BY ETHYLENE AND SOLUBLE ANALYTE TREATMENT ....................................................................................................................... 78 FIGURE 26 – RT-PCR ANALYSIS OF ETHYLENE AND JASMONIC ACID INDUCIBLE GENES .... 80 FIGURE 27 – HLS1-11 MUTATION BY EMS MUTAGENESIS .............................................................. 82 FIGURE 28 – HLS1-11;PMR6-6 ETHYLENE DOSE RESPONSE FOR HYPOCOTYL INHIBITION .... 84 FIGURE 29 – SOLUBLE ANALYTE HYPOCOTYL ANALYSIS OF HLS1-11;PMR6-6 ........................ 85 FIGURE 30 – RT-PCR ANALYSIS OF HLS1 EXPRESSION IN DARK GROWN SEEDLINGS ............ 86 FIGURE 31 – PROPOSED MODEL FOR PMR6’S SUBSTRATE AFFECTS ON ETHYLENE SIGNALING .......................................................................................................................... 93 viii Chapter 1 – Ethylene and its Role in Growth and Development Ethylene (C2H4) is a simple alkene that plays a role in a variety of processes in the growth and development of plants. The study of how ethylene regulates plant developmental signals began as it was first identified as a plant hormone in 1901 (Neljubow, 1901). It became the first identified plant hormone known to regulate several processes as further study linked ethylene to numerous processes in plant growth and development as well as response to abiotic and biotic stresses (Hao et al, 2017). Over time numerous plant hormones have been identified and the overlapping roles they play has illuminated a complex network by which plants regulate various stages of plant growth and respond to their environment as it changes in a variety of ways (Kazan 2015). Seed Germination Ethylene’s role in plant development begins at the very beginning of a developing plant’s life with seed germination. For land plants, many seeds germinate in a dark environment under the soil with a variety of external conditions beyond the seed coat. The germination process depends on a variety of these conditions: temperature, water, nitrates, and plant hormones like ethylene (Mattoo and Suttle, 1991). Exogenous application of ethylene was linked with increased rates of germination in some plant species (Matilla, 2000, Gianinetti et al. 2007). Ethylene was implicated in the promotion of seed germination of potato tubers whereby ethylene accumulation in seedlings as the seed coat splits connects ethylene with seed emergence from the plant embryo (Vacha and Harvey, 1927, Kucera et al., 2005). When endogenously produced ethylene is removed from the environment of a germinating seed by an ethylene absorbent, germination became delayed 1 indicating a dependence on ethylene accumulation for embryo emergence and cell division and elongation (Mattoo and Suttle, 1991). For the ethylene insensitive Arabidopsis mutant etr, which is a defect in an ethylene receptor that prevents ethylene binding, germination was not induced by treatment with ethylene (Bleecker et al., 1988). Ethylene has been shown to function in conjunction with other plant hormones – namely cytokinins and gibberellins – for effecting seed dormancy in different plant species. Cytokinins and gibberellins are plant hormones that promote seedling germination, cell division, and cell elongation in