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 the early stages of embryo emergence from the seed coat
(Kieber and Schaller, 2014, Hedden and Sponsel 2015). The exogenous addition of ethylene with cytokinins or gibberellins resulted in an increase in germination in lettuce seeds (Stewart and Freebair, 1969, Fu and Yang, 1983). Abscisic acid serves as an inhibitor to germination but upon treatment with a combination of the ethylene releasing compound ethephon and abscisic acid, seedling germination was promoted once again (Rao et al.,
1975, Dunlap and Morgan, 1977). Consequently, ethylene is an important factor in seed germination in conjunction with other signaling pathways that effect cell division, cell differentiation, and cell elongation.
Seedling Development
In etiolated seedlings ethylene plays an important role in development, directly affecting cell elongation, cell wall synthesis, and radial expansion (Mondal, 1975, Abeles et al., 1992, Hao et al., 2017). Early work with the effects of ethylene on etiolated sweet pea plants led to the observation of inhibition of cell elongation in the presence of ethylene
(Neljubov, 1901). This was the first characteristic of the ethylene triple response reported
2 and the additional observations from other scientists led to the inclusion of horizontal growth and radial swelling as characteristic of an etiolated plant’s response to ethylene
(Knight et al., 1910, Harvey, 1915, Abeles et al., 1992). The ethylene triple response consists of inhibited hypocotyl and root elongation (inhibition of cell elongation), increased radial swelling, and the formation of an apical hook. The ethylene triple response has been particularly helpful in identifying mutations important for ethylene signaling (Iqbal et al.,
2017). The exposure to ethylene leads to changes in cell wall composition. There is a decrease in insoluble cell wall material while simple soluble materials in the extracellular matrix increases (Harvey, 1915, Mondal, 1975, Abeles et al., 1992). The reorganization of microtubule formation occurs as cellulose microfibrils begin to favor longitudinal growth over transverse growth as the hypocotyl begins to swell radially instead of expand longitudinally (Eisinger and Burg, 1972, Burg, 1973). Ethylene and auxin – another plant hormone – come together to effect radial expansion which requires the reorganization of plant cell walls in a notable way (Liberman, 1979).
Inhibition of cell elongation also occurs in the roots of etiolated seedlings. Auxin and ethylene have shown linked roles in the growth and development of roots, reflecting the complicated signaling pathways involved in the development of plant tissues. Ethylene perception inhibitors – Ag+, cis-2-butene, cyclic olefins, diazocyclopentidine – reversed the inhibition to cell elongation seen in ethylene treatment of etiolated seedlings (Beyer,
1976a, Sisler and Yang, 1984a, Sisler and Blankenship 1993). The effect of ethylene perception while inhibiting elongation promotes adventitious root formation and increased development of root hairs (Mattoo and Suttle, 1991, Abeles et al., 1992). The removal of
3 exogenous ethylene application on etiolated seedlings reversed the inhibition to cell elongation in the hypocotyl and roots (Jackson et al., 1981, Sisler and Yang, 1984b).
The formation of an apical hook in response to ethylene serves to protect the shoot apical meristem and cotyledons as seedlings push through the soil after germination. At the shoot apex the cotyledons curl back protecting the meristematic cells and this apical hook is maintained until light exposure leads to new developmental responses in photomorphogenesis (Raz and Ecker, 1999). Differing rates of cell elongation on the inside and outside of the apical hook region are responsible for how the apical hook is formed as cells elongate faster and curl the cotyledons back on themselves (Silk and Erickson, 1978,
Lehman et al., 1996). Apical hook reversal occurred through slower elongating cells increasing the rate of elongation to straighten the hypocotyl at the shoot apex. The complex regulation of cell elongation was mostly coordinated by ethylene and auxin signaling (Silk and Erickson, 1978, Ecker and Theologis, 1994, Lehman et al., 1996).
Plant maturation and aging
Upon emergence from the soil, etiolated seedlings transition into adult plants where ethylene plays key roles in several processes including leaf development, flower ontogeny, and eventual senescence and deterioration of plant tissues (Hao et al., 2017). Leaf development differentiation is regulated by ethylene, which works to inhibit leaf expansion
(Mattoo and Suttle, 1991, Abeles et al., 1992). Overall rosette size of plants is affected by a variety of inputs but there is a strong correlation with ethylene mutants to suggest that ethylene effects rosette size. The ethylene insensitive Arabidopsis mutants, etr1 and ein2, exhibit larger that wild type leaves while constitutively activated ethylene pathway mutant,
4 ctr1, exhibits a smaller rosette and leaf size (Bleecker et al., 1988, Kieber et al, 1993,
Alonso et al., 1999). In maturing adult plants the abscission zone in Arabidopsis and other plants distinguishes a region around which plant organs – leaves, flowers, sepals, petals, fruits, and seeds – undergo separation from a plant. This can be caused by a variety of situations including biotic and abiotic stressors as well as developmental aging in the plant but is tightly regulated by hormone processes (Gonzalez-Carranza et al., 2012, Woo et al.,
2018). Ethylene plays a critical role in abscission and particularly high concentrations of ethylene influence leaf shedding during senescence (Mattoo and Suttle, 1991). The exogenous application of ethylene can lead to premature senescence and there is accelerated chlorophyll loss for wild type plants in comparison to ethylene insensitive plants in high ethylene environments (Zacarias and Reid, 1990, Bleecker et al., 1988).
Across a variety of plants including pineapples and mangos, ethylene was observed to promote flowering (Rodriguez, 1932, Galang and Agati, 1936, Abeles et al., 1992, Iqbal et al., 2017). In Arabidopsis, where many loss-of-function mutations in ethylene signaling factors have been characterized, the flowering time for ethylene insensitive mutants etr1, ein2, and ein3 is delayed in comparison to wild type (Bleecker et al., 1988 Ogawara et al.,
2003). The floral transition is considered an important step in the progress of the life cycle of plants, and ethylene clearly plays a key role in transitioning to flowering and essentially going from a vegetative phase to a reproductive stage (Simpson and Dean, 2002, Ogawara et al., 2003, Lin et al., 2009). Ethylene affects flower gender determination of cucurbits with exogenous ethylene treatment leading to feminization (Abeles et al., 1992). Flower senescence has been extensively studied in carnation flowers where ethylene biosynthesis
5 is increased until a climax of ethylene production and respiration after which flowers senesce rapidly (Woodson and Lawton, 1988, Jones et al., 1995, Larsen et al., 1995).
Stress Response
As plants acquire nutrients and progress through their life cycle there are a variety of situations that challenge a plant’s capability to reproduce and thrive. These threats can be biotic or abiotic in nature and plants respond to these stressors in a variety of ways.
Abiotic stressors are hot or cold temperatures, challenging chemical/nutrient conditions, drought conditions or flooding, radiation, and mechanical pressures or forces that induce bending. Biotic stressors include herbivory or infections by bacteria, fungi, or viruses
(Abeles et al., 1992, Schaller and Kieber 2002, Woo et al., 2018). Many of these stress conditions have been shown to induce the biosynthesis of ethylene and the expression of ethylene response transcription factors by ozone, cold, drought, salt stress, wounding, and pathogens (Fujimoto et al., 2000, Wang et al., 2002). Extensive work has gone into investigating different pathogens to determine what role ethylene plays in plant defense with results different from pathogen to pathogen. When ethylene responsiveness is changed there are certain pathogens that become less virulent and yet others have much more severe symptoms (Hoffman et al., 1999). Interestingly, in the Arabidopsis ethylene insensitive mutant ein2, enhanced disease tolerance is seen against virulent P. syringae pv tomato or Xanthomonas campestris pv campestris, yet the ein2 mutant also displays enhanced susceptibility to the necrotrophic fungus Botrytis cinereal (Bent et al.,
1992, Thomma et al., 1999, Wang et al., 2002). Plant-pathogen interaction and research into innate plant defense pathways have indicated a few ways plants respond to biotic
6 stressors brought on by pathogens. Systemic acquired resistance is an example of an induced immune mechanism that can lead to local programmed cell death, which is associated with increased salicylic acid (SA) production (Ryals et al., 1994, Saltveit, 1999,
Fu and Dong, 2013). Another implicated mechanism for plant defense responses is through an ethylene and jasmonic acid (JA) mediated response presumably initiated by ethylene and jasmonic acid biosynthesis being upregulated in response to pathogens (Wang et al.,
2002). Other plant-pathogen interactions that do not fit into these categories have suggested alternative mechanisms by which plants respond to biotic factors, one prominent experimental set coming from identification of numerous powdery mildew resistant (PMR) genes (Vogel and Somerville, 2000, Vogel et al., 2002). PMR6 is a putative pectate lyase that is required for PM disease development yet as of now, no mechanism by which powdery mildew resistance is conferred by loss of PMR6 has been demonstrated (Vogel et al., 2002). Another pathway linked to immune and stress response is connected with production of reactive oxygen species (ROS), which often will result in ethylene production thus representing another mechanism by which ethylene levels are modulated and can lead to direct effects of ethylene in plant immune response (Kimura et al., 2017).
ROS production can often be linked to more than just biotic factors with abiotic factors contributing to an increase in ROS production (Wang et al., 2002, Kimura et al., 2017).
Fruit ripening
Ethylene also plays an important role in the ripening of fruits in angiosperms. This has highlighted the role of ethylene as agriculturally important for understanding the tissue development for commercial applications. Within fruit and flower development,
7 climacteric plants are a subset of plants that develop fruiting bodies from flowers for reproduction and seed dispersal (Osorio et al., 2013). Ethylene regulation of climacteric fruit ripening consists of two systems for ethylene production (McMurchie et al. 1972).
System I is responsible for basal levels of ethylene production and functions during normal plant growth and development. Under System I, ethylene production is regulated in an auto-inhibitory manner where treatment with exogenous ethylene does not lead to further synthesis of ethylene (Paul and Pandey, 2011). System II relates to autocatalytic ethylene production, most seen during the ripening of climacteric fruit and floral senescence
(Alexander and Grierson, 2002, Paul and Pandey, 2011). System II is characterized by a sudden onset – auto-catalytic – of ethylene production, especially during fruit ripening of climacteric fruits, like in tomato ripening (Oetiker and Yang, 1995, Lelievre et al., 1997,
Nakatsuka et al., 1998, Yamane et al., 2007). Fruits are categorized as climacteric when ripening is dependent on a sharp increase in ethylene biosynthesis and an increased rate of respiration. Associated with fruit ripening are other events – loss of chlorophyll, formation of pigments, flavor and aroma synthesis, softening of the flesh, and the eventual abscission of the fruit (Goldschmidt et al., 1993, Bleecker and Kende, 2000). Because of the agricultural implications of these biochemical processes, the effect of the degradation of cell walls in fruits resulting in change to the firmness of fruit has been studied for longer storage and retaining flavors of fruits (Paul and Pandey, 2011). Fruit firmness is altered mostly through the activity of cellulase polygalacturonases in the extracellular space of plants that alter cell wall composition (Qian et al., 2011). Polygalacturnonases, are also involved since higher levels of polygalacturonase were found in soft cultivar extractions of
8 tomato in comparison to firm cultivars (Abeles et al., 1992). Numerous mutations have been identified in tomato plants that effect fruit ripening through fruit softening and reduced polygalacturoanse activity (Hobson, 1967, Ng and Tigchelaar, 1977, Pressey and
Avants, 1982, Jarret et al., 1984, Biggs and Handa, 1989). In conjunction with understanding fruit ripening through these mutations, the inhibition of native ethylene production proved to suppress ripening which could be rescued by the addition of exogenous ethylene (Hamilton et al., 1990, Oeller et al., 1991).
9 Chapter 2 – Ethylene Biosynthesis
Ethylene biosynthesis can be observed in plant species and some bacteria and fungi.
The simple alkene is produced in higher plants through the conversion of the amino acid
L-methionine into 1-aminocylopropane-1-carboxylic acid (ACC). The first recognized step is the conversion of L-methionine and adenosine triphosphate (ATP) into S- adenosylmethionine (SAM) by SAM synthetase. SAM is subsequently converted to 5′- methylthioadenosine (MTA) and ACC by ACC synthase (ACS). ACS requires pyridoxal phosphate and MTA is eventually recycled back to L-methionine by the Methionine
Salvage Cycle (Yang Cycle). ACS is also the rate limiting step in ethylene biosynthesis.
ACC is converted into ethylene by ACC oxidase (ACO). Recycling back MTA and maintaining a high rate of ethylene biosynthesis occurs even when free methionine levels are low (Miyazaki and Yang, 1987, Adams and Yang, 1979, Sauter et al., 2013).
The rate limiting step of Ethylene synthesis, ACC synthase (ACS), catalyzes the conversion of SAM into ACC. ACS was first identified for the formation of ACC in 1979 where the production of ACC had been established as a precursor for ethylene production in plants (Boller et al., 1979, Yu et al., 1979). The requirement for SAM as a substrate and use of pyridoxal phosphate as a cofactor led to further studies confirming its mode of action was similar to other pyridoxal phosphate requiring enzymes (Yip et al., 1990). This reaction mechanism explains the affect of aminoethoxyvinylglycine (AVG), a competitive inhibitor for pyridoxal phosphate-linked enzymes, which inhibits ACS activity (Yu et al.,
1979). Through a combination of purification and crystallography studies, functional and active ACS enzyme occurred as a homodimer (Bleecker et al., 1986, Capitani et al., 1999).
10
Figure 1: Ethylene biosynthesis displayed as part of the larger Methionine salvage cycle (Yang Cycle). ACC Synthase and ACC Oxidase convert S-adenosyl-methionine into ACC and subsequently ethylene in a two-step process.
11 Multiple ACS isoforms were identified based on sequenced plant genome studies and eventually Arabidopsis ACS isoforms were demonstrated to form functional heterodimers with one another (Tsuchisaka and Theologis, 2004). Expression of different isoforms presents opportunities for different regulatory mechanisms and the ACS family has been studied across a variety of plant species (Johnson and Ecker, 1998). Arabidopsis specifically has 12 ACS-like genes of which 9 which were organized into three distinct branches (Yamagami et al., 2003, Lin et al., 2009). These three classes of ACS genes have been studied to establish which were functional and for their individual roles in ethylene biosynthesis. In Arabidopsis different isoforms have been differentially expressed in different points of growth, development, hormone signaling, and in response to various stress stimuli (Yamagami et al., 2003, Tsuchisaka and Theologis, 2004, Peng et al., 2005).
Indole acetic acid (IAA), a common plant auxin induces transcription of ACS 2, 4, 5, 6, 8, and 11, wounding in the hypocotyl increases expression of ACS2, 4, 6, 7, and 8 while inhibiting expression of ACS1 and 5 (Lin et al., 2009, Tsuchisaka and Theologis, 2004).
ACS2 is suspected to play a role in negative feedback regulation as prolonged exposure to exogenous ethylene results in decreased expression (Liang et al., 1996, Wang et al., 2002).
In tomato different ACS genes are seen before the onset of ripening and have different expression patterns in fruit tissue (Barry et al., 2000, Alexander and Grierson, 2002). ACC synthase has also been seen to be regulated post-translationally with some ethylene overproducing mutants being linked back to ACS activity. A dominant mutation characterized as ethylene overproduction 2 (eto2) was discovered to be a single base change in ACS5 altering the carboxy-terminus and enhancing activity (Vogel et al., 1998).
12 This constitutive response phenotype could be reversed by treatment with AVG. The mRNA levels for ACS5 in eto2 was equivalent to wild type levels of transcription but seedlings were producing 20-fold more ethylene suggesting enzymatic activity had been altered. ACS expression and activity is a critical component to understanding native ethylene production for higher plants and while a deeper understanding has been developed there is still a lot to be understood by how ethylene production is modified and modulated by other stimuli that the plant is experiencing. It is clear that a major factor in regulation of ethylene production is the presence of biotic and abiotic stressors.
1-aminocylopropane-1-carboxylic acid (ACC) functions as a stable intermediate in native ethylene production in higher plants. In an experiment where fruit was exposed to nitrogen ethylene biosynthesis was halted, and upon air being reintroduced a burst in ethylene production rapidly followed. This was revealed to be due to an accumulation of
ACC as an intermediate and ACC’s dependence on oxygen for conversion to ethylene
(Yang and Hoffman, 1984). Excess ACC if not converted into ethylene is eventually metabolized into N-malonyl-ACC which is considered a biologically inactive end product of ACC metabolism (Tophof et al., 1989, Yang and Hoffman, 1984).
The intermediate molecule ACC is converted into ethylene through oxidation by
ACC oxidase in the final step of ethylene biosynthesis. ACC oxidase has also been referred to as ethylene forming enzyme but as the understanding for how ethylene biosynthesis required oxygen it was changed. The ACO family of enzymes has been studied in a variety of plant species backgrounds with ACO1 through ACO5 being identified in the Arabidopsis background. In tomato there are also five members LeACO1 through LeACO5 and they
13 have been shown to have enhanced activity in the presence of carbon dioxide suggesting an increase in ethylene production follows the spike in respiration of climacteric fruit
(Dong et al., 1992, Smith and John, 1993). Expression of ACO1 are upregulated in response to leaf senescence, wounding, and fruit ripening. The tomato genes see differential expression mostly in the tissue types in which they are expressed (Barry et al., 1996). While
ACS activity is the rate limiting step for ethylene biosynthesis, ACO gene expression and function can be modulated and serve as a regulator of biosynthesis (Liu et al., 1985).
A series of studies that have ethylene perception and machinery mutations connected with climacteric fruit ripening in tomato suggest ACS and ACO activities are further regulated in developmental transitions of fruit tissues. The mutation of Never ripe
(Nr) results in a lack of ethylene perception while another mutation ripening inhibitor (rin) results in a lack of autocatalytic ethylene production (Lanahan et al., 1994, Herner and
Sink, 1973). Nr and rin mutants were used in a study to link LeACS1 and LeACS6 to system
I ethylene production and a transition to system II ethylene production is dependent on
LeACS2 (Barry et al., 2000).
The transcriptional control of ACS and ACO appear to be the key ways for regulating the biosynthesis of ethylene in higher plants. Beyond the autocatalytic affect of ethylene biosynthesis being promoted by ethylene detection, ACO and ACS activity is promoted by other plant hormones through cross-talk pathways. Oftentimes an increase in
ACO activity precedes an increase in ACS activity (Liu et al., 1985, Manning, 1986). A critical note in studying the cross-talk with plant hormone pathways is distinguishing
14 between the promotion of biosynthesis and factors that interact with members of the ethylene signaling pathway.
Plant hormones play a role in regulating ethylene biosynthesis affect growth and development through gene’s regulated by ethylene signaling. Auxin (IAA) is closely linked with ethylene signaling through promoting the production of ethylene in addition to the auxin signaling cascade. IAA promotes the synthesis of ACC from SAM through affecting
ACS activity (Yu and Yang, 1979). Auxin and a series of synthetic analogs reaching a threshold concentration of 1 uM increases ACS activity, ACS expression, and subsequently ethylene production (Abeles and Rubinstein, 1964, Burg and Burg 1966, Kang and Ray,
1969, Sakai and Imaseki, 1971, Robles et al., 2013). The inhibition of auxin transport, auxin binding, and auxin action results in a reduction in ethylene production linking ethylene biosynthesis with auxin signaling (Tsai and Arteca, 1984, Trebitsh and Riov,
1987). Eight out of the nine ACS genes have a connection with auxin being transcriptionally upregulated by auxin signaling and there is canonical auxin response elements in select ACS promoters (Stepanova et al.,2005, Tsuchisaka and Theologis,
2004). In addition to auxin, the plant hormones cytokinins and brassinosteroids affect the stability of ACS5 and increase the rates of ethylene production (Vogel et al., 1998, Hansen et al., 2009). The stabilization and promotion of ACC synthesis and conversion to ethylene is not limited to only plant hormones promoting its biosynthesis but different biotic, abiotic,
15 and environmental factors influence the rate of ethylene biosynthesis and the aspects of plant growth and development associated with ethylene signaling.
Temperature plays a role in ethylene production – the optimal temperature for ethylene production is 30 oC with higher and lower growth temperatures leading to a decline in the rate of production (Burg and Thimann,1959, Hansen, 1945). Chilling and freezing temperatures however have been shown to increase ethylene biosynthesis in citrus fruit and promoted survival and recovery for injured cells from the damage (Young, 1972).
Environmental stimuli mimicking wind through mechanical stimulation
(thigmomorphogensis) increasing ethylene production after the stimulus is applied returning to normal after 4 hours (Irvine and Osborne, 1973). Gravitropism is linked with the production and detection of ethylene as previously discussed. While not completely regulated by ethylene the detection of even trace levels of ethylene could restore normal vertical growth to diageotropica mutant (dgt) in tomato (Zobel, 1973, Jackson, 1979). The role of ethylene in gravitropism and an explanation as to how low levels of exogenous ethylene restored the normal vertical growth pattern in this mutant get to the heart of how ethylene and other plant hormone pathways – auxin in this case – are engaged in cross-talk to affect plant growth and development (Zobel, 2016). Deficiencies in some inorganic nutrients including calcium, potassium, and magnesium will lead to elevated ethylene production (Barker and Corey, 1988). Salinity stress from plants grown in the presence of sodium leads to increased production of ACC in tomato leaves preceding the onset of oxidative stress and leaf senescence in response to Na+ accumulation (Albacete et al.,
2009).
16
Figure 2: Many biotic and abiotic factors play a role in promoting ethylene production and effecting ethylene response (Yoo et al., 2009).
17 Biotic factors can lead to the promotion of ethylene signaling mostly by promoting ethylene production. One stress response in a localized region is the production of Reactive
Oxygen Species (ROS), free radicals like H2O2 in the extracellular space. While not only limited to the detection of bacterial, fungal, and herbivory stimuli, ROS accumulation and ethylene production are a common response to biotic stressors (Wi et al., 2010, Yang et al.,
2015). While these biotic factors represent complex situations, there are PAMP (pattern- associated molecular pattern) and DAMP (damage-associated molecular patterns) recognized by membrane bound receptors which promote defense responses – usually by directly promoting ethylene biosynthesis in addition to other plant hormones. This is explored in a further section after having introduced ethylene signaling. A telltale connection of ethylene biosynthesis being promoted is the accumulation of ACC and upregulation of ACS expression and activity like the abiotic and environmental factors that promote ethylene production. Tissue damage and wounding either through viral infection or physical damage promote ethylene biosynthesis in a manner proportional to the amount of tissue damaged (Ketring and Melouk, 1982, Hebard and Shain, 1988, Spanu and Boller,
1989). The production of ethylene in wounded and damaged tissue has two affects – the damaged tissue would undergo senescence and neighboring cells and tissues will have prepare plant defense responses linked with ethylene signaling.
The growth and development regulated by ethylene signaling in plants and fruits is critical for multiple developmental points from seed emergence to controlled senescence of tissues. The biosynthesis of this plant hormone is hormone is only a three-step process
18 but the regulation and other factors that contribute to the timing and rate of production indicate that the regulation is quite complex.
19 Chapter 3 – The Ethylene Signaling Pathway
Ethylene functions as a plant hormone and is produced and effects growth and development in a variety of situations. Ethylene affects a variety of developmental programs including germination, senescence, flowering, abscission, and stress responses
(Hao et al, 2017). Ethylene’s mechanism of action has been extensively studied since the screening method for seedlings with altered triple response phenotypes identified an ethylene insensitive mutant etr1 in 1988 (Bleecker et al., 1988). Screening for additional ethylene signaling mutants have pieced together key components in the ethylene signaling network.
Ethylene signaling mutants that have been characterized through ethylene response screens have typically fallen into three main categories phenotypically. The first category involves phenotypes of ethylene insensitivity which includes the ethylene receptor family
(subfamily I members ETR1, ETR2, and EIN4 and subfamily II members ERS1 and ERS2)
(Ju and Chang 2015). Also included are a few other key signaling factors ein2, ein3, ein5, and ein6 (Ju and Chang 2015). This first category is largely insensitive to ethylene at any concentration. The second category of mutants involves constitutively active phenotypes in the absence of ethylene and includes the negative regulator of signaling constitutive triple response ctr1 and the responsive to antagonist ran1. The third category of enhanced ethylene response include mutations that are required for proper ETR1 function reversion- to-ethylene-sensitivity RTE1, mutations resulting in ethylene overproduction eto1, eto2, eto3, and F-box proteins for ethylene factor turnover EIN2 targeting protein ETP1 and
ETP2 and EIN3 binding F-box EBF1 and EBF2 (An et al., 2010). This third category often
20 exhibit normal growth in the absence of ethylene perception but are especially sensitive to or overproduce ethylene giving them an enhanced ethylene response. In addition to these proteins is a separate category of additional enhanced ethylene response eer mutants
(Larsen and Chang, 2001). These mutants exhibit normal growth in the absence of ethylene perception such as in the presence of AgNO3, but have ethylene hypersensitivity and extreme response phenotypes to saturating levels of ethylene.
Ethylene Perception
The first event in the process of ethylene’s signal transduction cascade is binding of ethylene by the ethylene receptor family. This group of five receptors includes
ETHYLENE RECEPTOR 1 (ETR1), ETHYLENE RECEPTOR 2 (ETR2), ETHYLENE
RESPONSE SENSOR 1 (ERS1), ETHYLENE RESPONSE SENSOR 2 (ERS2), and
ETHYLENE INSENSITIVE 4 (EIN4). While all five of these receptors have the ability to bind ethylene (Schaller and Bleecker, 1995, O’Malley et al., 2005), this receptor family is further divided into two subfamilies based on C-terminus structural domains. Ethylene binding takes place at the amino terminus of each protein and is coordinated by a copper cofactor that can interact with ethylene. The etr1-1 mutation C65Y disrupts copper association in ETR1 and highlights that copper is required for normal ethylene perception and response (Schaller and Bleecker, 1995, Rodriguez et al., 1999). Ag+ served to inhibit transduction of the ethylene signaling pathway through occupying the same hydrophobic pocket of the copper ion in the ethylene receptors but is incapable of transducing the signal.
As a result of this it serves as an inhibitor of ethylene action and has been utilized with other ethylene perception inhibitors to block ethylene signaling (Rodriguez et al., 1999).
21
Figure 3: The core of the ethylene signaling pathway. Arrows indicate activation, and T- bars indicate repression of the pathway.
22 Ethylene binding at the N-terminus of the ethylene receptor heterodimer ligand and copper interactions leads to a rearrangement of ligands coordinated with the copper and the conformational changes leading to an ethylene response. The manner in which 1-MCP and other ethylene analogues are expected to bind to the copper suggests that the conformational change dictate if an ethylene signaling response will take place (Sisler and
Serek, 2003).
Ethylene Signaling Cascade
After binding of ethylene by the ethylene receptors, a Raf-like serine/threonine kinase CONSTITUATIVE TRIPLE RESPONSE 1 (CTR1) is shown to interact with the histidine kinase domains of ETR1 and ERS1 (Clark et al., 1998). Loss of function mutants in ctr1 plants result in the ethylene pathway being constitutively activated and a seedling triple response phenotype even in the absence of ethylene. This factor therefore is a negative regulator of ethylene response (Kieber et al., 1993) and the ethylene receptors and
CTR1 form a complex in the absence of ethylene that negatively regulates ethylene responses until ethylene binds to these receptors. CTR1 has long been suspected to be the
MAPKKK in a kinase cascade but downstream kinases are yet to be shown as associating with CTR1 as part of a cascade. CTR1 has been shown to directly phosphorylate the next protein factor of note in the ethylene signaling pathway, ETHYLENE INSENSITIVE 2
(EIN2). EIN2 encodes an ER membrane-localized Nramp homolog that positively regulates ethylene responses. CTR1’s interaction with EIN2 occurs at a large, hydrophilic domain on the C-terminus side of the polypeptide and phosphorylation is at least four different sites across the polypeptide in vivo (Ju et al., 2012). The proteolytic degradation
23 of EIN2 is an important step in determining whether there is ethylene response or not at a given time. The protein levels of EIN2 are tightly regulated by ETP1 and ETP2 – two F- box proteins – that interact with the cytoplasmic domain and target it for degradation most likely through ubiquitination and proteasome mediated degradation (Qiao et al. 2009). The presence of this EIN2 cytoplasmic domain is required for proper ethylene response so affecting the accumulation leads to constitutive activation or insensitivity to ethylene
(Alonso et al., 1999). Overexpression of the C-terminal domain of EIN2 results in a plant with constitutively active ethylene response (Alonso et al., 1999, Ji and Guo et al., 2013).
Conversely, overexpression of ETP1 and ETP2 levels leads to a strong ethylene insensitivity phenotype because EIN2 protein levels are decreased.
In the absence of ethylene, the interaction of CTR1 with the ethylene receptor family keeps EIN2 phosphorylated and the F-box proteins recognize that modify the c- terminus for protease degradation. Disruption of this phosphorylation leads to the accumulation of EIN2 and triggers the downstream signaling events. The phosphorylation sites of two important serine residues are important for the stabilization of EIN2 and the site directed mutagenesis of these serine residues leads to partial constitutive activation individually suggesting the importance of these sites for recognition by ETP1 and ETP2 for turnover. Upon ethylene binding, the CTR1 and EIN2 interaction and consequent phosphorylation are disrupted, thus causing EIN2 accumulation. An as yet identified mechanism or protein factor is responsible for the proteolytic cleavage of the C-terminal cytoplasmic domain of EIN2 from the N-terminal membrane domain and the non-anchored
EIN2 CTERM then relocalizes to the nucleus in a manner dependent on a nuclear localizing
24 signal which is necessary for ethylene response (Qiao et al., 2012, Ju et al., 2012, Wen et al., 2012). The proteolytic cleavage and translocation of the EIN2 into the nucleus promotes the stabilization of EIN3 and EIN3-Like transcription factors (EIL1, EIL2, and
EIL3), which are involved in regulation of expression of ethylene responsive genes (An et al., 2010). These transcription factors are partially redundant and act as positive regulators of ethylene signaling. Null ein3 mutations confer partial ethylene insensitivity and the overexpression of EIN3 or EIL1 in an ein2 mutant display constitutive ethylene response.
EIN2 plays an important role in the stabilization of these transcription factors in vivo but they are themselves sufficient for ethylene-dependent gene expression (An et al., 2010).
EIN3 and the EIN3-Like transcription factors are tightly regulated through the stabilization of the protein in the absence or presence of EIN2 in the nucleus. These transcription factor protein levels are controlled by F-box proteins EBF1 and EBF2 (Guo and Ecker, 2003, Potuschak et al., 2003). In the absence of ethylene these transcription factors are degraded through a ubiquitin-mediated pathway to the 26S proteasome. The presence of ethylene leads to the accumulation of these transcription factors in the nucleus.
These F-box proteins are also tightly regulated; the overexpression of EBF1 results in an ethylene insensitive phenotype while ebf1 and ebf2 mutants inappropriately accumulate
EIN3 and have a constitutive ethylene response. Changes made to the stabilization of EBF1 and EBF2 also result in effects to the ethylene pathway. EIN5 (XRN4), is an exoribonuclease that promotes the turnover of EBF1 and EBF2 mRNA (Olmedo et al.,
2006, Potuschak et al., 2006). The ein5 mutant is ethylene insensitive due to
25 overaccumulation of EBF1 and EBF2 proteins, which suppresses gene expression by promoting the turnover of the EIN3 family of transcription factors.
The stabilization of these transcription factors through other means has been suggested as an alternate approach for initiating ethylene-related gene expression independent of EIN2 stabilization and translocation. A MAP kinase cascade consisting of
MKK7/9 and MAPK3/6 was thought to stabilize EIN3 independent of EIN2 (Yoo et al.,
2008). However, this cascade was reported to promote ethylene production rather than ethylene response in a manner separate from the established ethylene signaling pathway
(Xu et al., 2008). The stabilization of these transcription factors thus far still looks to require EIN2 as a master factor as there are as yet any EIN2 independent signaling cascades to have been identified.
As a result of EIN3 stabilization, ethylene dependent gene expression begins with
EIN3 recognizing a primary ethylene response element (ERE) in promoter sequences. The
ERF1 gene was the first identified transcriptional target with the ERE consisting of a GCC box that is typical of promoters in genes associated with pathogen and disease resistance
(Solano et al., 1998, Ohmetakagi and Shinshi, 1995). EIN3 induces ERF1 expression leading to a subset of ethylene genes being expressed. ERF1 is also a transcription factor which binds to ERE promoters and is part of a ERE binding protein (EREBP) family. The overexpression of ERF1 can promote expression of these genes and it can be seen as an important factor downstream of the main transcription factors in the ethylene signaling cascade (Solano et al., 1998). ERF1 represents an important crosstalk point for the integration of other hormone pathways as its expression is not specific only to ethylene.
26 Jasmonic acid is one plant hormone capable of upregulating ERF1 gene expression and represents one way that ERF1 expression can be controlled in an EIN3-independent manner. ERF1 and other plant defense-related genes can either have synergistic expression or require the presence of both hormones for their expression. Unfortunately, the cross talk of plant hormones makes this dynamic difficult to fully understand (Lorenzo et al., 2003).
While the ethylene signaling cascade has been identified to rely on a fairly linear sequence of factors to result in ethylene dependent gene expression, several different ethylene signaling mutants have been identified suggesting more complexity to how ethylene response occurs. Some of these novel factors have been seen to have an enhanced ethylene response to low concentrations of ethylene or an enhanced ethylene response with exaggerated response at low or saturating amounts of ethylene.
A protein auxiliary factor related to the ethylene signaling pathway was identified using a screen for mutants sensitive to subthreshold levels of ethylene which gave an enhanced ethylene response (Larsen and Chang, 2001). This eer1 mutant led to the loss of a regulatory subunit in PP2A (Larsen and Cancel 2003). This protein phosphatase is suspected to be an important regulator of CTR1 kinase activity as the triple response phenotype to ethylene is exaggerated when combined with ctr1-3. The inability to regulate
CTR1 leading to ethylene sensitivity suggests these other protein factors at least play a role in maintaining ethylene signaling and are potential targets for affecting the ethylene pathway indirectly by other hormone pathways.
A group of eer mutants involved with gene expression and transcription factor stability demonstrate that the level of gene expression and gene targets are important for
27 the ethylene signaling sequence and its resetting. Originally characterized as EER3,
AtPHB3 is a prohibitin that is involved in transcriptional regulation (Christians and Larsen,
2007). The eer phenotype from loss of AtPHB3 results in the loss of an unidentified subset of genes involved in regulating ethylene response. Based on this, AtPHB3 is considered a positive regulator of genes required for normal growth. A TFIID-interacting transcription factor, EER4, is shown to regulate the expression of ethylene-dependent genes (Robles et al., 2007). EER4 localizes in the nucleus and interacts directly with EIN3 to express genes including ERF1 and potentially genes involved in dampening or resetting the ethylene response pathway after a signaling cascade occurs. In the nucleus, the EIN2 C-terminus is capable of interacting with EER5, and eer5 mutants exhibit a more severe ethylene response than wild type plants (Christians et al., 2008). EER5 shares homology with components of the COP9 signalosome, suggesting that it participates in protein turnover by the 26S proteasome. Improper turnover of transcription factors and proteins involved in ethylene response by eer5 indicates this factor and the regulation of its activity potentially contribute to regulating when and how quickly ethylene-dependent proteins are degraded, overall regulating the amplitude of response to ethylene.
The continued identification of novel ethylene mutants indicates that the complexity of the ethylene signaling pathway has yet to be fully understood. How hormone pathways can crosstalk with one another and how the ethylene pathway is able to return to a pre-ethylene condition state are important areas to study in order to understand how plants respond to variable conditions in the environment.
28 Chapter 4 - Plant Cell Wall Biosynthesis, Modification, and Signaling
Plant cell walls are complex three-dimensional landscapes that provide structural support along with a barrier for defense, the latter of which requires these to be dynamic and responsive to the local environment of a plant cell. They are mainly composed of carbohydrates and proteins that produce an intricate network of polymers interacting with other carbohydrate chains within the cell and from neighboring cells. Carbohydrate polymers used in cell wall synthesis are primarily comprised of three groups – cellulose, hemicellulose, and pectin – each with varying properties that control how they interact
(Kong et al., 2018). Pectins specifically form ~35% of the dry mass of dicot cell walls and are also quite abundant in gymnosperm cell walls (Micheli, 2001). Pectins are most abundant in the outermost structure of cell walls, which is known as the middle lamella, with these interacting in a complex manner based on covalent modifications, chain length, and the presence of calcium ions (Caffall and Mohnen, 2009).
Cell wall biosynthesis and pectin modification
Pectin is a polymer chain of polygalacturonic acid (PGA), which consists of repeating D-galacturonic acid monosaccharides joined to one another covalently by -(1-
4) glycosidic bonds (McNaught 1997). PGA is further subdivided into four main groups: homogalacturonan (HG), rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and xylogalacturonan (Burton et al., 2010, Harholt et al., 2010). The different groups vary in composition of sugar residues, linkages across their backbones, and substitution/addition of functional groups making the study of how these molecules interact with one another
29
Figure 4: Homogalacturonan pectin structure depicting methyl- (C6) and acetyl- (O2 and O3) modifications to galacturonic acid residues. Addition of Xylose sugar residues would result in Xylogalacturonan. Additional modifications to pectin structure result in complex carbohydrate structures categorized as Rhamnogalacturonan I and Rhamnogalacturonan II (Scheller et al. 2007).
30 still an active area of research of cell wall integrity and synthesis (Burton et al., 2010, Park and Cosgrove, 2015, Sénéchal, 2014).
The overall synthesis of pectins requires activated nucleotide sugars including uracil diphosphate (UDP)-glucose and UDP-galactose (Caffall and Mohnen, 2009).
Extracellular cell wall components undergo assembly and modification mostly in the Golgi apparatus but may initiate in the endoplasmic reticulum (Atmodjo et al., 2013). Ongoing work has looked at how pectin has been assembled in the cis and medial areas of the Golgi with further esterification and branching taking place in the trans Golgi. The diverse and numerous types of pectin found in assembled cell walls represents an area where relatively little research has gone into understanding how and why modifications are made to the cell wall. Some of the modifications take place in Golgi body prior to exporting to the extracellular space while other modifications are made after the assembly of cell walls
(Ridley et al., 2001). The final assembly and delivery of pectins to the extracellular space goes through the trans Golgi where they are packaged into vesicles for export through the plasma membrane (Caffall and Mohnen, 2009).
When assembled in the cell membrane, PGA chains interact with cations through interacting across their own GA backbone or with neighboring chains in order to bridge the gaps between the carbohydrate chains. Ca2+ ions are common counter ions that participate in these interactions formed between the chains (Braccini and Perez, 2001). The establishment of ionic interactions between pectin and these cations changes the solubility of these polymers, resulting in formation of gels or solids (Micheli, 2001). Specifically for unsubstituted GalA chains, these Ca2+ ions interact with the negative charge of uronic acid
31 moieties with the carboxylic acid on C6 and form antiparallel chains of PGA together
(Liners et al., 1992, Scavetta et al., 1999). The effect of substituted groups, methylation, chain length, and branch points is still being studied as to the effect they have on these pectic chains interacting with one another with regards to strength, flexibility, and functionality in the cell wall (Micheli, 2001, Caffall and Mohnen, 2009). While the synthesis of the cell walls in the cell before export plays a role in the characteristics of the pectin, it is also important to note that there are a variety of enzymes that remain to be identified, studied, and characterized regarding how they modify and affect pectin in the extracellular space.
The proteins that are directly involved with the synthesis of pectin in the middle lamella and Golgi have been elusive to identify. There are many genes with sequence homology to pectin synthesizing factors that have not been confirmed to play a role or have had their role defined, possibly due to redundancy (Harholt et al., 2010). One class of pectin biosynthetic enzymes that have been identified are pectin biosynthetic glycosyltransferases found in the Golgi apparatus, with these possibly being key players in conjunction with other proteins to synthesize pectin polymers (Driouich et al., 2012, Atmodjo et al., 2013,
Takenaka et al., 2018). In addition to the synthesis of pectin, a variety of other proteins modify the polymers in several unique ways through glycosyltransferase, methyltransferase, or acetyltransferase activities (Mohenen, 2008). GalA residues are most often modified through methyl-esterification on the C6 carboxyl group or are acetylated at the O2 or O3 hydroxyl groups on the carbohydrate ring (Ridley et al., 2001). Methyl- esterification is a very common modification for pectins with usually 80% of C6 groups
32 methylated. The heavy degree of methylation that occurs in dicots and the variety of different PGA motifs may be one reason for the number of PMEs in the genome (Sénéchal et al., 2014). While methylation of C6 carboxyl groups masks the negative charges on this group and affects the ability for these PGA chains to interact with Ca2+, methylation may also serve other roles including as binding sites for proteins or by affecting interactions of pectin chains without mediation by Ca2+ (Liners et al., 1992, Micheli, 2001). This has led to the expectation that methyl-esterification is a tightly regulated process for growth and development (Willats et al., 2001, Harholt et al., 2010). Pectin acetylation is less common with up to 10% of O2 or O3 groups acetylated (Wolf et al., 2009b, Gou et al., 2012).
Arabidopsis thaliana and other dicot species have a large number of pectin methylesterases
(PME) with 66 putative full-length open reading frames having been annotated (Sénéchal et al., 2014). Comparatively fewer pectin acetyltransferases have been found, consisting of only 12 putative full-length open reading frames (Sénéchal et al., 2014). PMR5 is one pectin acetylating protein that has been studied as loss-of-function mutants were first identified as powdery mildew resistant and exhibited altered cell wall composition (Vogel et al., 2004). PMR5 was shown to be important to the amount of pectin esterification and acetylation in cell walls by comparison to wild type, and purified recombinant protein was able to acetylate galacturonic acid (GalA) oligosaccharides (Vogel et al., 2004, Chiniquy et al., 2019). The effect of changes in cell wall acetylation in pmr5 mutants affected not only morphology and cell wall composition, but also sensitivity to infection from different pathogen types – necrotrophs and biotrophs (Chiniquy et al., 2019). pmr5 demonstrates increased resistance to the biotroph powdery mildew while it is more susceptible to
33 infection from the necrotrophic pathogen Botrytis cinerea (Chiniquy et al., 2019). The
Reduced Wall Acetylation (RWA) family of 4 identified proteins significantly effect the amount of acetylation in Arabidopsis (Manabe et al., 2013). RWA2 is associated with loss- of-function mutants having an increased resistance to B. cinerea in addition to reduced cell wall acetylation (Manabe et al., 2011). The effect of a pmr5;rwa2 double mutant suppresses the powdery mildew resistance phenotype from pmr5 suggesting that not only the amount of acetylation but likely where acetylation occurs on pectin chains and how it affects the accessibility of pectin degrading enzymes from pathogens are both important
(Lionetti et al., 2017, Chiniquy et al., 2019). Changes to the modification of pectin and cell wall integrity can have effects on the resistance of plants to pathogens (Bellincampi et al.,
2014) and specific methylesterases are implicated in susceptibility to particular pathogens
(Raiola et al., 2011, Lionetti et al., 2017). Hydrolyzed partially demethylesterified homogalacturonan has been shown to release short galacturonan chains – oligogalacturonides (OGs) – with signaling functions (Lionetti et al., 2007; Osorio et al.,
2008, Davidsson et al., 2017). The modification of pectin, stability of its chains, and interactions it has with other biomolecules and environments has so far been difficult to understand but seems to be critical to plant development, response to various stimuli including both biotic and abiotic, and homeostasis.
Beyond the types of covalent modifications that can be made enzymatically to pectin, there are also a variety of proteins involved in degradation and assembly of the chains in the dynamic cell wall environment. Cell walls in growing plants require continual remodeling to allow for cell expansion as well as integration of changes in response to
34
Figure 5: Glycoside hydrolase and pectate lyase enzyme reactions for the cleavage of glycosidic bonds of galacturonic acid molecules (S Kongruang, 2003).
35 various stress and growth factors that change cell environments. For pectin there are two different types of enzymes that can be involved in degradation or cleavage of glycosidic bonds – hydrolases and lyases (Kameshwar and Qin, 2018). Glycoside hydrolases use a molecule of water to hydrolyze the glycosidic bond, which leaves a hydroxyl group on both carbohydrate carbons that were originally part of the bond (Davies and Henrissat, 1995, S
Kongruang, 2003). For pectin degradation, polygalacturonase (PG) enzymes carry out the hydrolysis reaction and usually prefer an acidic pH for the enzymatic constraints of their reactions (Tucker and Seymour, 2002, S Kongruang, 2003). There are 68 putative
Arabidopsis thaliana PG enzymes as the different types of PGA residue carbohydrate compositions require PG enzymes for HG, RG-I, RG-II, and xylogalacturonan (Sénéchal et al., 2014). Polysaccharide lyases cleave the glycosidic linkage through a beta- elimination reaction, leaving one of the carbohydrate rings previously involved in the bond with a double bond between C4 and C5 (Herron et al., 2000, S Kongruang, 2003). For pectin degradation pectate lyases usually prefer a basic pH, and there are 26 putative pectate lyases and pectate lyase like proteins in Arabidopsis (Whitaker, 1994, S
Kongruang, 2003, Palusa et al., 2007). This class of pectate lyases includes a number of proteins with undefined roles including several endo-pectate lyases whose catalytic products are separate polysaccharide chains rather than monosaccharides (Herron et al.,
2000, Cao, 2012).
The middle lamella is a complex matrix that makes up the outermost spaces between plant cells. It requires constant intensive maintenance that in large part consists of enzyme action to control assembly, response to environmental changes/hormones, and
36 disassembly (Caffall and Mohnen, 2009, Zhong et al., 2018). While these processes are key to normal plant growth and development, they are still poorly understood. It should be noted that modification and degradation of cell walls also takes place in fleshy fruit ripening (Seymour and Gross, 1996, Smith et al., 1990), which is a horticulturally important process that has received considerable attention. Research into the dependency of tomato fruit ripening on polygalacturonase (PG) activity/expression revealed different stages when PG activity plays a large role in later stages of fruit ripening (Hadfield and
Bennett, 1998). PG expression and activity is upregulated in the pericarp of tomato (Marin-
Rodriguez et al., 2002) and pectate lyase activity was demonstrated from isolates of banana pulp during ripening (Marin-Rodriguez et al., 2003) both of which are consistent with the prominent role of cell wall degradation in fruit ripening. The actual process by which this fruit softening and ripening takes place involves a variety of yet to be fully understood enzymes that are expressed at specific developmental times. The plant-tissue specific gene- expression of 26 putative pectate lyase-like (PLL) genes in Arabidopsis were investigated for how they would affect growth and development (Palusa et al., 2007). These genes displayed some PLLs expressed in different tissues while others were expressed throughout the plant suggesting they play individual roles. Some pectate lyases were inducible by hormones or different types of stress treatment linking this pectate lyase activity to critical steps for the cell wall changing in growth and development (Palusa et al., 2007). Another important cell wall function is to serve as the first line of defense from pathogenic microorganisms and fungi. A subset of these plant pathogens produce cell wall modifying and degrading enzymes in order to establish an infection (Goto and Okabe, 1958, Khare et
37 al., 1994). In order for these pathogens to access the nutrients of the plant, they must degrade the barriers from the plant and evade a defense response from the plant.
Plant cell walls change in response to stimuli such as signals from biotic and abiotic events. Cell wall modification in response to threats to plant survival are detected by plant signaling pathways (Houston et al., 2016). The plant hormones jasmonic acid, ethylene, abscisic acid, and salicylic acid all serve as stress hormones, triggering and modulating defense related genes (Li et al., 2002; Schmelz et al., 2006). Herbivory and recognition of
Pathogen-Associated Molecular Patterns (PAMPs) and Damage-Associated Molecular
Patterns (DAMPs) lead to callose biosynthesis and deposition in leaf epidermal cells
(Ellinger and Voigt, 2014). These PAMPs and DAMPs function through induction of genes connected to innate immune responses in plants (Denoux et al., 2008). These biotic/abiotic stressors and signals affect cell wall size, depth, and carbohydrate/protein consistency; the cell wall is monitored with cell wall fragments serving as signaling molecules that can affect gene expression (Vallarino and Osorio, 2012, Houston et al., 2016).
DAMPs are a category of biological signals representing aberrantly located endogenous molecules that are still being discovered and classified in both animal and plant systems. In plant backgrounds, much less is known about DAMPs compared to the many identified in animals although there are recent connections between Arabidopsis
High Mobility Group Box 3 (AtHMGB3) proteins and similar HMGB proteins that have been identified in animal systems as damage associated elicitors (Choi et al., 2016).
DAMPs recognized by plants include extracellular nucleotides, glutathione, apoplastic peptides and proteins, and a variety of cell wall polysaccharide fragments and associated
38 degraded products including oligogalacturonides (OGs), representing varying chain lengths of galacturonic acid residues with a variety of modifications (Choi and Klessig,
2016, Hou et al., 2019).
The detection of specific molecular signals in plants has been extensively studied with regards to wounding where it has been recorded that short peptide chains are released by neighboring cells at a wound site and activate plant defense genes as seen with systemin
(Ryan 2000). The release of this polypeptide defense signal not only resulted in a localized defense response but also activate plant defense responses in distant tissues over the course of time through migration of the polypeptide (Schaller and Ryan, 1996). Other short polypeptides have been identified in plant species functioning in a similar role as a DAMP or signaling molecule leading to modifications in extracellular conditions, cell differentiation, and growth upon detection in plants (Ryan et al., 2002). The movement of small biomolecules in plants to have plant wide effects from growth and development to defense responses creates a very important precedent for how plants may be responding to wounding or infection to increase survivability.
Innate immunity describes the conserved mechanism for plant defense that occurs through the detection of PAMPs upon an attempt at colonization by microbes (Medzhitov and Janeway, 2002). Within plants, the detection of exogenous and endogenous signals is critical for induction of defense genes to oppose infection. In Arabidopsis cell surface receptors with leucine-rich repeats are commonly found to function in identifying PAMPs and inducing defense related gene expression. FLS2 (Flagellin-Sensitive 2) is a cell wall receptor that detects a portion of flagellin (FLG2) in bacterial flagellum, which initiates a
39 downstream defense response and alters growth of Arabidopsis seedlings (Gomez-Gomez and Boller, 2000). Flagellin perception has become a paradigm for innate immunity in plants and demonstrates how similarly PAMP detection between animals and plants is through the homology of the receptors responsible for detection of the molecular patterns
(Gomez-Gomez and Boller, 2002). With the FLS2 signaling pathway, perception of the
FLG22 flagellin fragment results in an influx of Ca2+ ions into the cell, the production of reactive oxygen species (ROS), and the initiation of MAPK and CDPK kinases which regulate a number of WRKY transcription factors – affecting gene expression (Robatzek and Wirthmueller, 2012, Bakshi and Oelmüller, 2014). The detection of pathogen or molecular patterns is critical for the corresponding response to be initiated in plants and often the pattern recognition receptor functions as a kinase to initiate a MAPK-mediated cascade. (Gomez-Gomez and Boller 2002, Ryan et al., 2007, Newman et al., 2013).
What has become clear through the investigation of plant response to stimuli is the means of detecting and appropriately responding to a foreign or plant derived signal is critical for plant growth and development, defense responses to pathogens, or wounding and damage to plant structure (Boller and Felix, 2009). While many chemically unique structures may initiate a response in plants, the characterized P/DAMP and corresponding
P/DAMP-receptor pairings for plants does not include many receptors for detecting cell wall fragments, even though chemically pure OGAs serve as endogenous elicitors
(D’Ovidio et al., 2004, Boller and Felix, 2009). WALL ASSOCIATED KINASES
(WAKs), which comprise a family of cell wall associated receptors, have demonstrated the ability to bind both polymethyl-esterified pectin and small pectin fragments, preferring
40 chains between 9-16 residues long in a Ca2+ dependent manner (Decreux and Messiaen,
2005, Kohorn 2015). These WAK proteins contain repeating epidermal growth factor
(EGF) repeats in an extracellular domain, traverse the plasma membrane, and contain a mostly conserved kinase domain in the cytosol. While at least WAK2 has been identified as having the ability to bind pectin both in vivo and in vitro (Kohorn et al., 2009), there are several other suspected WAK proteins that contain EGF repeats in an extracellular domain.
In addition to the 5 already identified there are 21 additional putative WAK-like genes in
Arabidopsis yet to be classified but which may bind to other pectin motifs or different OG chain lengths (Verica and He, 2002). These suspected WAK-like (WAKL) genes exhibit tissue specific and developmentally regulated gene expression in Arabidopsis (Verica and
He, 2003). In both rice and Arabidopsis, WAKL genes affect susceptibility to pathogens. In
Arabidopsis dominant mutant alleles resulted in resistance to Fusarium (Diener and
Ausubel, 2005), and in rice there was a decrease in sensitivity to rice blast disease (Li et al., 2009). These WAK proteins and their relationship with cell expansion and pathogen response are an important indicator of an underlying system for detection and response to
OGs (Kohorn and Kohorn, 2012). The chain length and modifications found on OGs is still a place of investigation based on the cell wall’s role in plant defense and signaling. WAK1 and WAK2 bind to de-esterified homogalacturonan (HGA) but not highly esterified HGA,
RG-I, or RG-II (Kohorn et al., 2009). While WAK genes have been implicated in detecting and responding to long OGs, short OGs (3 carbohydrate units) have been shown to induce pathogen resistance-associated gene expression in Arabidopsis (Davidsson et al., 2017). It
41 is very likely that other receptors recognize these shorter OG derived molecular patterns to elicit defense responses and induce hormone response pathways.
42 Chapter 5 – Evidence of a Novel Signaling Event Effecting Ethylene Signaling in pmr6-6
Introduction
The plant hormone ethylene is extensively involved in germination, tissue development and senescence, fruit ripening, and pathogen response (Mattoo and Suttle,
1991, Kazan, 2015, Hao et al., 2017). Its detection by plants initiates a signaling cascade leading to ethylene-responsive gene expression , which has been extensively characterized in Arabidopsis thaliana. The detection of this unsaturated hydrocarbon by a family of five ethylene receptors, including ETHYLENE RECEPTOR 1 (ETR1), in the endoplasmic reticulum leads to the negative regulator and kinase CONSTITUTIVE TRIPLE
RESPONSE 1 (CTR1) being inactivated, no longer phosphorylating ETHYLENE
INSENSITIVE 2 (EIN2) (Mattoo and Suttle, 1991, Hao et al., 2017). Lack of phosphorylation leads to accumulation of EIN2 in the endoplasmic reticulum resulting in proteolytic cleavage and translocation of a cytoplasmic portion of EIN2 to the nucleus, which stabilizes a group of transcription factors (Mattoo and Suttle, 1991, Hao et al., 2017).
The accumulation of ETHYLENE INSENSITIVE 3 (EIN3) and EIN3-like 1 (EIL1) mediate changes in ethylene responsive gene expression including upregulation of additional transcription factors such as ETHYLENE RESPONSE FACTOR 1 (ERF1)
(Mattoo and Suttle, 1991, Hao et al., 2017).
The morphologic changes seen as a result of ethylene signaling include a characteristic triple response phenotype that can be observed in etiolated seedlings following exposure to ethylene. The ethylene triple response in etiolated seedlings includes
(1) shortening and thickening of the hypocotyl, (2) shortening of the roots along with
43 proliferation of root hairs, and (3) exaggerated curvature of the apical hook (Merchante and
Stepanova, 2017). This triple response has been utilized to screen Arabidopsis and other dicot species for mutations that affect ethylene signaling (Merchante and Stepanova, 2017).
Screening mutagenized Arabidopsis seedlings for altered triple response patterns has helped identify four main classes of ethylene mutants: ethylene insensitive (ein), constitutive triple response (ctr), ethylene overproducer (eto), and enhanced ethylene response (eer, ebf, etp, rte). Through a mutagenesis screen for an altered triple response phenotype in Arabidopsis seedlings, a new allele of pmr6 was identified as having an aberrant ethylene response including severe hypocotyl shortening in conjunction with absence of an apical hook. PMR6 encodes a putative pectate lyase first identified as a factor needed for infection by Erysiphe cichoracearum, an Arabidopsis powdery mildew pathogen (Vogel et al., 2002). Preliminary characterization of pmr6-6 suggests that the reduced capability to form an apical hook and increased ethylene sensitivity seen for the mutant may be due to inappropriate buildup of the PMR6 substrate (Alvarez 2014). These results required further investigation to identify the basis of how the PMR6 substrate controls ethylene response.
Results
An important step in the process of characterizing PMR6’s role in growth and development was understanding the nature of the mutation and how it leads to the aberrant triple response phenotype that led to the identification of pmr6-6. In the course of experimental design two ethylene process inhibitors were used - aminoethoxyvinylglycine
(AVG) and AgNO3. AVG limits ethylene production by inhibiting ACC synthase and ACC
44 production, thus lowering the amount of ethylene biosynthesis (Schaller and Binder, 2017).
+2 AgNO3 inhibits ethylene perception by substituting for Cu in the ethylene-binding pocket of ETR1 and other ethylene receptors (Schaller and Binder, 2017). The pmr6-6 mutation demonstrated an aberrant ethylene response that consists of both ethylene hypersensitivity and exaggeration of response to ethylene while at the same time, lack of apical hook formation. It is hypothesized that this results from buildup of a soluble analyte that likely is the substrate of PMR6, which conditions ethylene response.
To determine the severity of the ethylene response phenotype of pmr6-6, Col-0 wt and pmr6-6 seedlings were grown for 4 days in the dark. All growth conditions included
5M AVG in order to block ethylene biosynthesis to allow for examination of response to exogenously added ethylene. Treatments included 2M AgNO3 to block ethylene response and establish the potential for growth in the absence of ethylene perception, air, and increasing concentrations of exogenous ethylene ranging from 0.1 to 100 L/L. The presence of ethylene normally leads to the hypocotyl shortening and thickening in wild type plants along with formation of an apical hook (Merchante and Stepanova, 2017, Hao et al., 2017). For pmr6-6, there is both hypersensitivity and exaggerated response to ethylene specifically for the hypocotyl. In the absence of ethylene preception in the presence of AgNO3, wild type and pmr6-6 etiolated seedlings grow to nearly the same hypocotyl length. In contrast, even low levels of exogenous ethylene lead to severe shortening of the hypocotyl of pmr6-6 seedlings in comparison to wild type. Additionally, pmr6-6 hypocotyls were shorter than wild-type plants even at saturating concentrations of
45
Figure 6: Ethylene responsiveness in Col-0 and pmr6-6 seedlings determined by an ethylene dose-response analysis. 4-day-old etiolated seedlings were grown on PNS media with 5 M AVG. With one condition grown in the presence of 2 M AgNO¬3, blocking ethylene perception, the other conditions had exogenous ethylene environments ranging from 0 (air) to 100 L/L. Hypocotyls were subsequently measured. Mean values ± SD were determined from 30 seedlings.
46
Figure 7: Ethylene responsiveness in Col-0 and pmr6-6 seedlings determined by an ethylene dose-response analysis. 4-day-old etiolated seedlings were grown on PNS media with one condition grown in the presence of 2 M AgNO¬3, blocking ethylene perception, the other conditions had exogenous ethylene environments ranging from 0 (air) to 100 L/L. Roots were subsequently measured. Mean values ± SD values were determined by 30 seedlings.
47
Figure 8: Ethylene responsiveness in Col-0 and pmr6-6 seedlings determined by an ethylene dose-response analysis. 4-day-old etiolated seedlings were grown on PNS media with 5 M AVG. With one condition grown in the presence of 2 M AgNO¬3, blocking ethylene perception, the other conditions had exogenous ethylene environments ranging from 0 (air) to 100 L/L. Apical hook formation was then evaluated.
48
Figure 9: The rate of ethylene production for Col-0 and pmr6-6 4-day-old dark-grown seedlings. Plant media was either PNS or PNS + 5 M AVG Ethylene was collected for 18 hours and determined by gass chromatography. Production rates were calculated based on tissue fresh weight. Mean ± SD values were determined from 10 samples of 100 seedlings each.
49 ethylene, consisting with it also having an exaggerated response. In contrast, compared to wild-type plants ethylene-dependent apical hooks were largely absent for pmr6-6 seedlings treated with saturating concentrations of ethylene. As shown in Figure 8, as the concentration of ethylene increases, the frequency of apical hook formation increases in wild type plants. In contrast, pmr6-6 seedlings have severely compromised apical hook formation with seedlings being almost exclusively unhooked even at saturating concentrations of ethylene.
In order to see whether altered ethylene response is tissue specific, ethylene responsive root growth of the mutant in comparison to wild type was measured. For this analysis, AVG was omitted from the growth media since it has a negative impact on root growth. Seedlings of Col-0 wt and pmr6-6 were grown with 2M AgNO3 to block ethylene perception, air, or a range of increasing concentrations of exogenous ethylene ranging from
0.1 to 100 L/L for 4 days in the dark. As shown in Figures 6 and 7, the aberrant ethylene response seen for pmr6-6 hypocotyls appear to be tissue specific since analysis of the root growth in response to ethylene reveals nearly responses between wild type and pmr6-6 seedlings at all ethylene concentrations tested.
Since it could be argued that differences seen between Col-0 wt and pmr6-6 hypocotyls could be due to altered ethylene biosynthesis, it was important to measure ethylene production by pmr6-6 in comparison to Col-0 wt. For this analysis, Col-0 wt and pmr6-6 seedlings were grown for a total of 96 hours in the dark with ethylene collected for the last 18 hours by capping the collection vials. Following growth, 0.9 mL of headspace from the vials were analyzed through gas chromatography coupled with flame ionization
50 detection (GC-FID). The rate of ethylene production was calculated per gram fresh weight per hour. From this analysis, it was found that similar to other ethylene response mutants pmr6-6 produces more than twice as much ethylene as Col-0 seedlings (Figure 9). Since the dose response analysis was performed in the presence of AVG, it was important to determine whether this had a measurable effect on ethylene biosynthesis rates in order to limit the impact of endogenous ethylene on hypocotyl elongation. A follow up experiment measuring the production of ethylene by GC-FID for pmr6-6 and Col-0 grown as previously described but now in the presence of 5 M AVG resulted in a substantial reduction in the production of ethylene by pmr6-6. Consequently, AVG was deemed to be an important additive for all growth experiments in order to limit the effects of endogenous ethylene in the experimental setup and it was determined that the enhanced ethylene response seen for pmr6-6 at low exogenous ethylene levels likely was not a result of ethylene overproduction.
Additional pmr6 loss-of-function alleles have been characterized and identified previously enabling comparison of the other loss of function mutations with pmr6-6 in order to determine if there is an allelic series with regard to response to ethylene (Vogel et al., 2002). The original study of PMR6’s function in powdery mildew development first identified the mutation in pmr6-1 as being a point mutation that results in substitution of glycine to aspartate at amino acid position 140 (Vogel et al., 2002). pmr6-2 has a mutation at a splice site that disrupts mature mRNA formation (Vogel et al., 2002). Both pmr6-3 and pmr6-4 result from T-DNA insertions in intron sequences that presumably knock down expression of PMR6 (Vogel et al., 2002). pmr6-5 results from substitution of a proline for
51 A:
B: 14
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Hypocotyl Length (mm) Length Hypocotyl 2
0 Col-0 pmr6-1 pmr6-2 pmr6-3 pmr6-5 pmr6-6 Ag 100 ppm Ethylene
Figure 10: (A) adapted from Vogel et al. 2002 with the addition of the pmr6-6 mutation site and effect on the open reading frame of PMR6. Exons are represented by bolded section of line with introns joining them together. (B) Ethylene responsiveness in Col-0, pmr6-1, pmr6-2, pmr6-3, pmr6-5, and pmr6-6 seedlings determined by an ethylene dose-response analysis. 4-day-old etiolated seedlings were grown on PNS media with 5 M AVG. With growth conditions the presence of 2 M AgNO¬3 or 100 L/L ethylene. Apical hook formation was evaluated and reported as a fraction for number of hooked seedlings over total seedlings. Hypocotyl lengths were subsequently measured. Mean values ± SD were determined from 30 seedlings.
52 leucine at amino acid 339 (Vogel et al., 2002). Ethylene responsiveness, including hypocotyl inhibition and apical hook formation, were tested for these mutants in comparison to pmr6-6. For this experiment, Col-0 wt and the pmr6 mutant alleles pmr6-1, pmr6-2, pmr6-3, pmr6-5, and pmr6-6 were grown on 5 M AVG supplemented media either in the absence of ethylene signaling (2M AgNO3) or in the presence of 100 L/L ethylene for 4 days in the dark. Following this the number of apical hooks formed was determined as well as hypocotyl lengths were measured. As shown in Figure 10, in the presence of AgNO3, growth of the various genotypes was effectively indistinguishable from Col-0 wt. The severe hypocotyl shortening in the presence of saturating ethylene that is characteristic of pmr6-6 was not observed for the other alleles, suggesting that – consistent with it being an inappropriate stop codon – pmr6-6 is more severe in nature.
Similar to pmr6-6, apical hook formation was compromised in all genotypes to different degrees likely relative to the strength of the various loss-of-function alleles. This data suggests that hypocotyl shortening, sensitivity to ethylene, and apical hook formation are linked to PMR6 function in Arabidopsis, and that at least in regard to ethylene response pmr6-6 is the most severe mutation in the allelic series. Results with these other alleles support that the compromised formation of apical hooks seen for pmr6-6 results from loss of this protein’s function as the other alleles also have compromised hook formation.
Since the PMR6 substrate may control ethylene responsiveness, it was of interest to test whether overexpression of PMR6 could confer ethylene insensitivity. Using the 35S promoter sequence from Cauliflower Mosaic Virus (CaMV) coupled to the coding sequence of PMR6, transgenic Arabidopsis plants that overexpressed PMR6 were
53
Figure 11: Ethylene responsiveness in seedlings of Col-0, pmr6-6, and three independent lines of PMR6 Overexpressing::pmr6-6 plants determined by an ethylene dose-response analysis. 4-day-old etiolated seedlings were grown on PNS media with 5 M AVG. With one condition grown in the presence of 2 M AgNO¬3, blocking ethylene perception, the other conditions had exogenous ethylene environments ranging of 0.1, 0.2, and 100 L/L. Hypocotyls were subsequently measured. Mean values ± SD were determined from 30 seedlings.
54 generated by transformation of pmr6-6 with Agrobacterium tumefaciens. Three independent insertions were used to investigate how increased PMR6 expression affects ethylene responsiveness. For these PMR6 Overexpressing lines, all three demonstrated increased PMR6 expression based on the radiograph comparison from a northern probe of
RNA samples from Col-0, pmr6-6, and overexpressors 21, 46, and 49. Seedlings of Col-0 wt, pmr6-6, and three independent transgenic lines were grown for 4 days in the dark with
5M AVG either in the presence of AgNO3 or increasing concentrations of ethylene including 100 nL L-1, 200 nL L-1, and 100 L L-1. Following this, hypocotyl lengths were measured and apical hook formation was evaluated. As shown in Figure 11, whereas pmr6-
6 displays ethylene hypersensitivity with no apical hooks, all three PMR6 overexpressing lines showed significant reductions in ethylene response at low concentrations of ethylene in conjunction with full restoration of apical hook formation.
The variety of ethylene mutants available provided an opportunity to investigate how PMR6’s function may be connected to the broader canonical ethylene signaling pathway. The ethylene signaling pathway has well defined components from detection at receptors such as ETR1 through transcription factors including EIN3 and EIL1 that lead to changes in gene expression profiles (An et al., 2010). These mutants have characteristic ethylene triple response phenotypes including ethylene insensitivity for mutants such as etr1-1, ein2-5 and ein3-1;eil1-1, or constitutive triple response in the case of ctr1-3 due to loss of negative regulation of the ethylene signaling pathway (Hao et al., 2017). Double mutants between pmr6-6 and the ethylene signaling mutants etr1-1, ctr1-3, ein2-5, ein3-1, eil1-1 and a triple mutant with ein3-1;eil1-1 were generated to characterize whether the
55
Figure 12: Ethylene response in Col-0, pmr6-6, etr1-1, and etr1-1;pmr6-6 seedlings determined by an ethylene dose-response analysis. 4-day-old etiolated seedlings were grown on PNS media with 5 M AVG. With one condition grown in the presence of 2 M AgNO¬3, blocking ethylene perception, the other condition is an exogenous ethylene environment of 100 L/L. Hypocotyls were subsequently measured. Mean values ± SD were determined from 30 seedlings.
56
Figure 13: Growth phenotype of Col-0, pmr6-6, ctr1-3, and ctr1-3;pmr6-6 seedlings determined by ambient growth in the dark. 4-day-old etiolated seedlings were grown on PNS media with 5 M AVG in air. Apical hook formation was evaluated and reported as a fraction for number of hooked seedlings over total seedlings. Hypocotyls were subsequently measured. Mean values ± SD were determined from 30 seedlings.
57
Figure 14: Ethylene response in Col-0, pmr6-6, ein2-5, and ein2-5;pmr6-6 seedlings determined by an ethylene dose-response analysis. 4-day-old etiolated seedlings were grown on PNS media with 5 M AVG. With one condition grown in the presence of 2 M AgNO¬3, blocking ethylene perception, the other condition is an exogenous ethylene environment of 100 L/L. Hypocotyls were subsequently measured. Mean values ± SD were determined from 30 seedlings.
58
Figure 15: Ethylene response in Col-0, pmr6-6, ein3-1, eil1-1, ein3-1;eil1-1, ein3-1;pmr6- 6, eil1-1;pmr6-6, and ein3-1;eil1-1;pmr6-6 seedlings determined by an ethylene dose- response analysis. 4-day-old etiolated seedlings were grown on PNS media with 5 M AVG. With one condition grown in the presence of 2 M AgNO¬3, blocking ethylene perception, the other condition is an exogenous ethylene environment of 100 L/L. Hypocotyls were subsequently measured. Mean values ± SD were determined from 30 seedlings.
59 enhanced ethylene response seen for pmr6-6 was indeed dependent on ethylene signaling.
These double and triple mutants were investigated for their responses to ethylene for morphological changes and inhibition of hypocotyl elongation. Experiments were conducted with 5M AVG added to all samples in order to block ethylene biosynthesis.
AgNO3 was included where appropriate to block ethylene perception. For treatments, 100
L/L ethylene was used to give maximal response.
Seedlings of Col-0 wt, etr1-1, pmr6-6 and etr1-1;pmr6-6 were grown for 4 days in the dark in the presence of 5M AVG either with 2M AgNO3 or 100 L/L ethylene, after which hypocotyls were measured. As shown in Figure 12, growth of etr1-1;pmr6-6 hypocotyls in the presence of ethylene was indistinguishable from etr1-1 in comparison to pmr6-6. The pmr6-6 ethylene phenotype is suppressed in the absence of the ethylene perception similar to what is seen when grown on AgNO3. In the presence of saturating ethylene, etr1-1;pmr6-6 hypocotyls remained long similarly to etr1-1.
Since loss of CTR1 leads to activation of ethylene response independent of ethylene, it was of interest to determine whether a ctr1-3;pmr6-6 double mutant would display the same phenotype as ethylene-treated pmr6-6, including exaggerated hypocotyl shortening and loss of an apical hook. For this experiment, Col-0 wt, ctr1-3, pmr6-6 and ctr1-3;pmr6-6 were grown on 5 M AVG supplemented media for 4 days in the dark in air after which hypocotyl lengths were measured and number of apical hooks was determined. ctr1-3 seedlings grown in this way exhibit the ethylene triple response even in the absence of ethylene. Similar to what was seen for ethylene treated pmr6-6, ctr1-3;pmr6-
6 fails to display normal apical hook formation and seedlings were significantly shorter
60 than ctr1-3. Additionally, as for ethylene treated pmr6-6, ctr1-3;pmr6-6 seedlings had no apical hooks.
EIN2 is a transmembrane protein that is critical for proper detection of ethylene as well as other inputs such as jasmonic acid (Alonso et al., 1999). Consequently, it was important to determine whether the increased ethylene response seen for pmr6-6 was dependent on EIN2 function. For this, Col-0 wt, ein2-5, pmr6-6, and ein2-5;pmr6-6 were grown on 5 M AVG supplemented media either in the presence of (2M AgNO3) or 100
L/L ethylene for 4 days in the dark, after which hypocotyls were measured. As shown in
Figure 14, loss of EIN2 function in ein2-5;pmr6-6 resulted in loss of the exaggerated response to ethylene seen for pmr6-6, indicating that the mutant ethylene response phenotype is fully dependent on EIN2.
The ethylene transcription factors EIN3 and EIL1 are required together for ethylene response and when both transcription factors are not functional the plant exhibits strong ethylene insensitivity similar to other ethylene mutants (An et al., 2010). EIN3 is the primary transcription factor for ethylene response and ein3 loss-of-function mutants have a greater degree of ethylene insensitivity than an eil1 null. Double mutants were generated for both ein3-1 and eil1-1 with pmr6-6, along with the triple mutant ein3-1;eil1-1;pmr6-6.
To evaluate ethylene responsiveness Col-0 wt, ein3-1, eil1-1, pmr6-6, ein3-1;pmr6-6, eil1-
1;pmr6-6, and ein3-1;eil1-1;pmr6-6 were grown in medium supplemented with 5 M
AVG either in the presence of 2M AgNO3 or 100 L/L ethylene for 4 days in the dark, after which hypocotyls were measured. As shown in Figure 15, ein3-1;pmr6-6 was more sensitive to ethylene in comparison to ein3-1 alone indicating that loss of PMR6 results in
61 an increased impact of ethylene in this background. A similar effect was seen for eil1-
1;pmr6-6 although the impact of the pmr6-6 mutation on response was significantly less proportionally than what was seen for ein3-1;pmr6-6. Interestingly, ethylene response seen for ein3-1 arguably occurs mostly through EIL1 whereas response in eil1-1 is most likely occurring though EIN3. As will be discussed later, EIN3 has been determined to be the primary response factor for ethylene with only a limited role for EIL1. Based on this and other data, it seems as though EIL1 may be more important for integrating the effects of the pmr6-6’s aberrant ethylene response. Analysis of the growth of ein3-1;eil1-1;pmr6-6 shows almost complete ethylene insensitivity as would be expected for a mutant that was incapable of ethylene response.
Preliminary observations suggested that exaggerated hypocotyl shortening could be promoted for pmr6-6 by feeding back water-soluble cell wall extracts from ethylene treated seedlings. This in contrast to Col-0 wt, which at similar amount of cell wall extracts, showed only minimal hypocotyl inhibition. This suggested that the PMR6 substrate was enriched in the cell wall extracts and that pmr6-6 mutants could not break it down, with it serving as a signaling molecule that controls hypocotyl elongation likely in conjunction with ethylene response. in other words, hyperaccumulation of this molecule, which promotes hypocotyl inhibition, would result in the additive effect that has been observed for ethylene treated pmr6-6 seedlings. Since wild type seedlings possess functional PMR6, they are speculated to be able to metabolize the PMR6 substrate and thus do not experience similar inhibitory effects unless levels of the substrate reaches levels too high to allow for its proper turnover. As shown in Figure 16, this effect is independent of ethylene since it
62
Figure 16: Soluble analyte “SS” responsiveness in Col-0 and pmr6-6 seedlings determined by an ethylene dose-response analysis with resolubilized metabolite extracts from Col-0 ethylene treated seedlings. 4-day-old etiolated seedlings were grown on PNS media with 5 M AVG. With one condition representing a lack of ethylene perception, seedlings were grown in the presence of 2 M AgNO¬3, blocking ethylene perception. The other conditions had an exogenous ethylene environment of 100 L/L. Control represented by 50 L diH2O in 6 mL volume of PNS media while “SS” is representative of 50 L metabolite extracts in 6 mL volume of PNS media. Hypocotyls were subsequently measured. Mean values ± SD were determined from 30 seedlings.
63
Figure 17: Soluble analyte effects on Col-0 and pmr6-6 seedling roots determined by growth on effectively hypocotyl response saturating metabolite growth. 4-day-old etiolated seedlings were grown on PNS media in the presence of 2 M AgNO¬3, blocking ethylene perception. Control condition contains 50 L diH2O in 6 mL volume of PNS media while “SS” is representative of 50 L metabolite extracts in 6 mL volume of PNS media. Roots were subsequently measured. Mean values ± SD values were determined by 30 seedlings.
64 occurs even in the presence of AgNO3 and AVG. In order to verify the preliminary observations and quantify the effects of the presumed PMR6 substrate on growth, soluble material (SE) was extracted from ethylene treated Col-0 wt seedlings. These aqueous extracts were added to the growth media with 5 M AVG and either 2M AgNO3 or 100
L/L ethylene for 4 days in the dark, after which hypocotyls were measured. For all controls without SE, Col-0 wt and pmr6-6 seedlings were grown with an equivalent volume of water added to the growth media. As shown in Figure 16, in media supplemented with
SE had pmr6-6 hypocotyls with nearly a 40% reduction in height while wild type plants remained unaffected. This phenotype was tissue specific since pmr6-6 roots remained unchanged and grew comparably to wild type (Figure 16). Since PMR6 encodes for a putative pectate lyase based on sequence homology (Vogel et al., 2002), it could be argued that ethylene treatment leads to buildup of high levels of PMR6 substrate that the pmr6-6 mutant is unable to breakdown as it lacks functional PMR6. Since this cell wall product had pronounced effects on the hypocotyl, further experiments were conducted to investigate this analyte’s potential role in signal transduction within plants.
The stability of the soluble analyte was an additional point of interest in experimental observation. In some extractions in which the plant tissue for Col-0 plants thawed before lyophilization or during lyophilization, the soluble extracts would lose potency and often be completely ineffective to produce a shortened growth in hypocotyls and as such were kept frozen throughout the process of tissue homogenization and lyophilization. Once tissue had been dried completely the extraction procedure produced robust and repeatable soluble metabolites that were stable when left at room temperature
65 though stored at -20OC as a precaution. In some unreported experiments were significant studies conducted with soluble extract material that had spent significant time at room temperature without loss to potency and it was of interest to investigate if the analyte of interest was heat labile. Soluble extracts were solubilized in sterile H2O and pooled together in a sterile hood. Half of the resuspended soluble extracts were placed in a boiling water bath which maintained a rolling boil for 10 minutes before being permitted to cool again alongside nonboiled soluble extracts for 10 minutes. Col-0 wt and pmr6-6 were grown on media supplemented with 5 M AVG in the presence of 2M AgNO3 with soluble material (SE) either boiled or nonboiled ranging in amounts from 20 L to 100 L, after which hypocotyls were measured. The boiling of these soluble extracts did not lead to a loss of potency for these materials as seen in Figure 18.
The ethylene biochemical precursor, 1-aminocyclopropane-1-carboxylic acid
(ACC), was a potential water-soluble, small molecule that is both naturally produced and based on the measurement of ethylene in pmr6-6 (Figure 9), did appear to have modified production as AVG treatment reduced the amount of ethylene produced. ACC is being investigated for potential signaling mechanisms independent of ethylene (Van de Poel and
Van Der Straeten, 2014) so it was of interest to investigate if ACC were responsible for the hypocotyl shortening in pmr6-6. Col-0 and pmr6-6 seeds were grown in a variety of conditions that soluble extracts had a demonstrated effect for in pmr6-6. In ethylene experiments 100 M ACC is considering saturating to plant responses and as such was chosen as a final concentration in growth medias. As seen in Figure 19 in the presence of saturating ethylene and ethylene inhibition with 2 M AgNO3 coupled with 5 M AVG –
66
Figure 18: Analysis of heat stability of soluble extracts from ethylene treated Col-0 seedlings. Responsiveness in Col-0 and pmr6-6 seedlings determined by growth of plants independent of ethylene perception and with reduced ethylene biosynthesis. Seedlings were grown on PNS media with 2 M AgNO¬3 as an ethylne perception inhibitor and 5 M AVG as an ethylene biosynthesis inhibitor. Soluble extracts were resuspended in sterile H2O and from a single pool of 600 L were split into 300 L aliquots - one of which was placed in a boiling water bath for 10 minutes. Soluble extracts from boiled or nonboiled condition were added to growth media before planting with effective amounts of 20 L, 50 L and 100 L for each condition. After 4 days growth at 20OC hypocotyls were subsequently measured. Mean values ± SD were determined from 30 seedlings.
67
Figure 19: Analysis of ACC (1-aminocyclopropane-1-carboxylic acid) responsiveness in Col-0 and pmr6-6 seedlings determined by an ethylene dose-response analysis and growth of plants independent of ethylene perception and reduced ethylene biosynthesis. 4-day-old etiolated seedlings were grown on PNS media. With one condition representing a lack of ethylene perception, seedlings were grown in the presence of 2 M AgNO¬3 and 5 M AVG as an ethylene biosynthesis inhibitor. The other condition had an exogenous ethylene environment of 100 L/L. For each of these conditions the media either contained a final concentration of 100 M ACC or water was added for the controls for each condition. After 4 days growth at 20OC hypocotyls were subsequently measured. Mean values ± SD were determined from 30 seedlings.
68 treatment with ACC did not display a change in pmr6-6 hypocotyl length as observed in soluble extract treatment. As such ACC alone is not responsible for the shortening of the hypocotyl independent of ethylene signaling.
PMR6 overexpressing lines were subsequently tested for their responsiveness to the cell wall extract, with the expectation being that increased levels of the PMR6 enzyme would boost the capability to degrade the PMR6 substrate and reduce its negative effects on hypocotyl growth. For this analysis, Col-0 wt, pmr6-6, and 3 PMR6 overexpressing lines were grown on media supplemented with 5M AVG in the presence of 2M AgNO3 and soluble material (SE) extracted from ethylene treated Col-0 wt seedlings resuspended in water ranging in amounts from 50 L to 200 L, after which hypocotyls were measured.
As shown in Figure 20, at higher levels of cell wall extract, even Col-0 wt hypocotyls were inhibited suggesting that levels of PMR6 substrate were becoming high enough to overwhelm the enzymatic capacity of PMR6 in the Col-0 wt seedlings. In contrast, the
PMR6 overexpressing lines were significantly less impacted than both pmr6-6 and Col-0 wt, which is consistent with these lines producing more PMR6 enzyme and having increased capacity to degrade its substrate.
It was also of interest to determine whether this hypersensitivity to the cell wall extract could be observed for the previously published pmr6 alleles (Vogel et al., 2002).
For this analysis, Col-0 wt, pmr6-1, pmr6-2, pmr6-3, pmr6-5, and pmr6-6 lines were grown for 4 days in the dark on media supplemented with 5 M AVG in the presence of 2M
AgNO3 with and without 50 L of soluble material extracted from ethylene treated Col-0 wt seedlings, after which hypocotyls were measured. As shown in Figure 21, as with
69
Figure 20: Soluble analyte “SS” responsiveness in Col-0, pmr6-6, and three independent lines of PMR6 Overexpressing::pmr6-6 seedlings determined by increasing amounts of resolubilized metabolite extracts from Col-0 ethylene treated seedlings. 4-day-old etiolated seedlings were grown on PNS media with 5 M AVG and 2 M AgNO¬3. Control represented by 200 L diH2O in 6 mL volume of PNS media while soluble extracts were added to 6 mL volume of PNS media from 50 L to 200 L. Hypocotyls were subsequently measured. Mean values ± SD were determined from 30 seedlings.
70
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Figure 21: Soluble analyte response in Col-0, pmr6-1, pmr6-2, pmr6-3, pmr6-5, and pmr6- 6 seedlings to resolubilized metabolite extracts from Col-0 ethylene treated seedlings. 4- day-old etiolated seedlings were grown on PNS media with 5 M AVG and 2 M AgNO¬3. Control represented by 50 L diH2O in 6 mL volume of PNS media while “SS” is representative of 50 L metabolite extracts in 6 mL volume of PNS media. Hypocotyls were subsequently measured. Mean values ± SD were determined from 30 seedlings.
71 pmr6-6, the other pmr6 loss-of-function alleles were also sensitive to soluble extracts from ethylene treated Col-0 seedlings but similar to what was observed following ethylene treatment (Figure 9) and consistent with the nature of its mutation, pmr6-6 hypocotyls were significantly more sensitive to the SE.
Having established a bioassay for measuring the soluble analyte’s effect on hypocotyl length in the absence of ethylene signaling, it was of interest to determine whether there was any relationship of the inhibitory effects of this molecule to the ethylene signaling pathway. The double and triple mutants for the ethylene signaling factors with pmr6-6 were investigated for their responses to the presumptive PMR6 substrate for morphological changes and inhibition of hypocotyl elongation. For treatments, 50 L of soluble cell wall extracts generated from ethylene treated Col-0 seedlings was used to give maximal response.
Seedlings of Col-0 wt, etr1-1, pmr6-6 and etr1-1;pmr6-6 were grown for 4 days in the dark on media supplemented with 5M AVG in the presence of 2M AgNO3 either in the absence or presence of 50L SE, after which hypocotyls were measured. As shown in
Figure 22, growth of etr1-1;pmr6-6 in the presence of soluble extracts resulted in the same sensitivity to the soluble extract as seen for pmr6-6, indicating that the PMR6 substrate is detected in a manner independent of ETR1.
Seedlings of Col-0 wt, ein2-5, pmr6-6 and ein2-5;pmr6-6 were grown for 4 days in the dark on media supplemented with 5M AVG in the presence of 2M AgNO3 either with or without 50 L SE, after which hypocotyls were measured. As shown in Figure 23, whereas pmr6-6 hypocotyls showed the expected hypocotyl inhibition in the presence of
72
Figure 22: Soluble analyte response in Col-0, pmr6-6, etr1-1, and etr1-1;pmr6-6 seedlings to resolubilized metabolite extracts from Col-0 ethylene treated seedlings. 4-day-old etiolated seedlings were grown on PNS media with 5 M AVG and 2 M AgNO¬3. Control represented by 50 L diH2O in 6 mL volume of PNS media while “SS” is representative of 50 L metabolite extracts in 6 mL volume of PNS media. Hypocotyls were subsequently measured. Mean values ± SD were determined from 30 seedlings.
73
Figure 23: Soluble analyte response in Col-0, pmr6-6, ein2-5, and ein2-5;pmr6-6 seedlings to resolubilized metabolite extracts from Col-0 ethylene treated seedlings. 4-day-old etiolated seedlings were grown on PNS media with 5 M AVG and 2 M AgNO¬3. Control represented by 50 L diH2O in 6 mL volume of PNS media while “SS” is representative of 20 L or 50 L metabolite extracts in 6 mL volume of PNS media. Hypocotyls were subsequently measured. Mean values ± SD were determined from 30 seedlings.
74
Figure 24: Soluble analyte response in Col-0, pmr6-6, ein3-1, eil1-1, ein3-1;eil1-1, ein3- 1;pmr6-6, eil1-1;pmr6-6, and ein3-1;eil1-1;pmr6-6 seedlings to resolubilized metabolite extracts from Col-0 ethylene treated seedlings. 4-day-old etiolated seedlings were grown on PNS media with 5 M AVG and 2 M AgNO¬3. Control represented by 50 L diH2O in 6 mL volume of PNS media while “SS” is representative of 50 L metabolite extracts in 6 mL volume of PNS media. Hypocotyls were subsequently measured. Mean values ± SD were determined from 30 seedlings.
75 SE, ein2-5;pmr6-6 hypocotyls were found to be completely insensitive to the effects of the
SE indicating that EIN2 is required for hypocotyl inhibition by the presumptive PMR6 substrate.
Seedlings of Col-0 wt, ein3-1, eil1-1, pmr6-6, ein3-1;pmr6-6, eil1-1;pmr6-6, and ein3-1;eil1-1;pmr6-6 were grown for 4 days in the dark on media supplemented with 5 M
AVG in the presence of 2M AgNO3 either with or without 50 L SE in the growth media, after which hypocotyls were measured. As shown in Figure 24, while not as severe as pmr6-6, hypocotyls of ein3-1;pmr6-6 were still inhibited by nearly 30% compared to growth media without soluble analyte. In contrast, eil1-1;pmr6-6 was less sensitive to growth media supplemented with soluble extracts with hypocotyl lengths being reduced by
20% in comparison to media without soluble analyte extracts. As such it seems that EIL1 contributes more to the shortening of the hypocotyl from the soluble analyte perception while ethylene is more dependent on EIN3 for hypocotyl shortening. As seen for ethylene response, ein3-1;eil1-1;pmr6-6 was almost completely insensitive to the SE suggesting that the inhibitory effects of the presumptive PMR6 substrate on hypocotyl elongation is almost completely dependent on these two transcription factors.
The demonstration that these transcription factors are necessary for pmr6-6 dependent hypocotyl shortening based on the growth data of the ethylene and soluble analyte assays indicates that likely the SE stabilizes EIN3 and EIL1, thus causing their accumulation to promote response. Using a transgenic line for GFP (green fluorescent protein) fused to EIN3 (Guo and Ecker et al., 2003), accumulation of the transcription factor can be visualized to determine whether addition of the SE can promote EIN3
76 buildup. In the presence of ethylene, EIN3::GFP is stabilized in the nucleus of plant cells upon exposure to ethylene (Guo and Ecker et al., 2003, An et al., 2010). In order to test whether the SE could also stabilize EIN3, the transgene line was crossed into pmr6-6.
Seedlings of the EIN3::GFP transgenic line and EIN3::GFP in pmr6-6 were grown for 4 days in the dark in the presence of 5M AVG. For control seedlings and those treated with soluble extracts, 2 M AgNO3 was also present in the growth media to inhibit ethylene perception as previously described. Ethylene (100 L/L) or soluble extracts generated from ethylene treated pmr6-6 seedlings were added 3 hours in advance of microscopy as continuous growth in the presence of ethylene or soluble extracts does not lead to EIN3 accumulation. GFP fluorescence was observed on a Keyence BZ-X800E fluorescence microscope with multiple seedlings and multiple excitation times observed for each condition. EIN3::GFP accumulation was observed for both the Col-0 and pmr6-6
EIN3::GFP expressing lines treated with the soluble extract in the absence of ethylene perception or ACC biosynthesis, strongly linking the stabilization of the transcription factor to a process independent of ethylene signaling. The observed accumulation of EIN3 following SE treatment is not as abundant as seen following ethylene exposure. This may in part be due to the analyte being an aqueous molecule that is transported into plant tissues in comparison to gaseous ethylene, which diffuses freely through plant tissues. The stabilization of EIN3 coupled with the growth assays showing dependence upon EIN3 and
EIL1 for SE dependent hypocotyl shortening link the mode of action for the soluble analyte to these transcription factors but in a method independent of ethylene perception. It should be noted that soluble extracts were analyzed for ACC, which could arguably cause a similar
77
Figure 25: Fluorescence microscopy for EIN3::GFP accumulation. Col-0;EIN3::GFP and pmr6-6;EIN3::GFP 4-day-old etiolated seedlings were grown on PNS media with 5 M AVG and with AgNO3 for control condition and soluble analyte condition (blocking ethylene perception). 3 hours prior to fluorescence microscopy jars containing etiolated seedlings were opened in the dark and 2 mL water with 5 M AVG or 2 mL resuspended soluble analyte from ethylene treated pmr6-6 seedlings (300 L soluble extract equivalent) with 5 M AVG was added to media surface where plants were growing. Containers were closed after treatment to retain dark growth conditions. For ethylene treatment ethylene was added for 100 L/L ethylene environment. After 3 hours seedlings were transferred to microscope slides for observation on a Keyence BZ-X800E fluorescence microscope. All samples represent 1 second measurement of light emission at 509 nm. Ag) 2 M AgNO3 no soluble analyte “zero” Col-0;EIN3::GFP hypocotyl showing background fluorescence of hypocotyl cell wall but no distinguishable accumulation of EIN3::GFP. Ethylene) 100 L/L ethylene Col-0;EIN3::GFP hypocotyl showing accumulation of EIN3::GFP in nuclei of plant cells. Soluble extracts) 2 M AgNO3 with soluble analyte treatment of Col- 0;EIN3::GFP hypocotyl showing accumulation of EIN3::GFP in nuclei of plant cells.
78 effect on EIN3 stabilization, but ACC levels were below the level of detection by MS (data not shown).
The implication of transcription factors being critical for at least one mode of action for the PMR6 substrate effects led to investigation of how gene expression changes in response to treatments with the soluble analytes. Plant tissue for analysis of defense gene expression was harvested from 14-day old light grown plants (Col-0 and pmr6-6) that had been treated for 24 hours in different conditions. As a control, plants were exposed to
AgNO3 and AVG to block ethylene response and biosynthesis. Plant defense gene expression as represented by the family of PDF1 (Manners et al., 1998) genes was promoted by treatment of seedlings with 100 M ACC and ± 100 M JA reflecting a saturating ethylene and JA treatment. AVG was present to eliminate endogenous ethylene in the “zero” control. In parallel, Col-0 wt and pmr6-6 seedlings were treated with 100 M
ACC, ± 100 M JA, and 5M AVG along with soluble extracts generated from ethylene- treated pmr6-6 seedlings (50 L equivalents as used in soluble analyte growth analysis).
Following tissue collection, total RNA was extracted from all samples and cDNA was generated for real time PCR (rt-PCR) analysis of genes related to defense response including ERF1, and the PLANT DEFENSIN subset of genes, PDF1.2a, PDF1.2b,
PDF1.2c, and PDF1.3. As shown in Figure 25, PLANT DEFENSIN expression was strongly upregulated in Col-0 wt plants for PDF1.2a, PDF1.2b, PDF1.2c, and PDF1.3 as a result of treatment with JA and ACC (ethylene). For all DEFENSIN genes tested, pmr6-
6 exhibited substantially lower expression of this subset of these plant defense genes in comparison to Col-0 plants. This differential was not seen for ERF1, suggesting that this
79
Figure 26: rt-PCR analysis of PLANT DEFENSIN and ERF1 genes from Col-0 and pmr6- 6 14 day-old light grown seedlings. cDNA was generated from Col-0 and pmr6-6 plants grown hydroponically in PNS for 13 days before being transferred to “control” condition (2 M AgNO3 5 M AVG), “ACC/JA” condition (5 M AVG, 100 M ACC, 100 M JA±), or “JA/ACC/SE” condition (5 M AVG, 100 M ACC, 100 M JA±, 50 L soluble extract equ.). A) rt-PCR data for PDF1.2a B) rt-PCR data for PDF1.2b C) rt-PCR data for PDF1.2c D) rt-PCR data for PDF1.3 E) rt-PCR data for ERF1 rt-PCR was performed on CFX Connect Real-Time PCR Detection System with 3 replicates. Standard deviations and fold change calculated from a double delta Ct analysis of gene expression values.
80 is a specific response. Interestingly treatment of Col-0 and pmr6-6 plants with soluble extracts resulted in Col-0 wt seedlings having a large reduction in expression of the PLANT
DEFENSIN genes with no significant effect on ERF1 expression.
The observation of reduced expression of these DEFENSIN genes in pmr6-6 under the condition of saturating ethylene and JA suggest that there could be a link between the reduced expression of these genes and powdery mildew resistance. The reduction of gene expression in Col-0 plants suggests the affect of this soluble analyte is linked with gene expression in plant defense genes and may be responsible for powdery mildew resistance.
In order to identify additional components of a signaling pathway associated with perception of the soluble analyte, a pmr6-6 suppressor mutagenesis study was undertaken with the goal of identifying non-ethylene pathway components that could suppress the hypersensitivity of pmr6-6 to the presumed PMR6 substrate. This involved mutagenesis of pmr6-6 seeds with ethyl methanesulfonate (EMS) and subsequent screening using the soluble extract supplemented media to identify seedlings with longer hypocotyls than what has been documented for pmr6-6 seedlings. For this, mutagenized pmr6-6 seedlings were grown for 4 days in the dark in the presence of 2 M AgNO3 and 50 L SE generated from ethylene treated Col-0 wt seedlings. Seedlings with longer hypocotyls were identified, ranked, and allowed to self-pollinate to produce progeny for retesting. Further screening and segregation of plants has identified mutants of interest, one of which exhibited a growth phenotype in etiolated seedlings similar to growth characteristics seen for loss-of-function mutants in HOOKLESS 1 (HLS1) (Lehman et al., 1996, Liao et al., 2016). Sequencing of
HLS1 from genomic DNA of the pmr6-6 suppressor mutant 17-6 identified a new loss of
81
Figure 27: EMS mutagenized suppressor of pmr6-6 soluble analyte sensitivity amino acid sequence. Sequencing of HLS1 gene revealed a nonsense mutation at aa 181 as indicated.
82 function allele for HLS1 referred to as hls1-11. The allele arose from a nonsense mutation in HLS1 at aa181 expected to result in loss of HLS1 function. HLS1 is a putative N- acetyltransferase involved in chromatin remodeling that has been implicated in apical hook formation and skotomorphogenesis (Josse and Halliday, 2008, Zhang et al., 2018).
Subsequently, the hls1-11 mutation was backcrossed out of the pmr6-6 background in order to allow for comparisons of hls1-11 to hls1-11;pmr6-6 with regard to response to ethylene and the SE. For the ethylene dose response analysis, seedlings of Col-0 wt, hls1-
11, pmr6-6 and hls1-11;pmr6-6 were grown for 4 days in the dark on 5 M AVG in the presence or absence of 2M AgNO3, air, and increasing concentrations of exogenous ethylene ranging from 0.1 to 100L/L, after which hypocotyls were measured. As shown in Figure 28, it was found that growth of hls1-11 has measurable effects on ethylene dependent growth of pmr6-6. Interestingly, at lower concentrations of ethylene hls1-
11;pmr6-6 seedlings had increased ethylene sensitivity similar to pmr6-6. In contrast, at higher concentrations of ethylene the hls1-11;pmr6-6 hypocotyl length is close to hls1-11 and shows measurable ethylene insensitivity.
For analysis on soluble extract growth, seedlings of Col-0 wt, hls1-11, pmr6-6 and hls1-11;pmr6-6 were also grown for 4 days in the dark on media supplemented with 5 M
AVG supplemented media in the presence of 2M AgNO3 in the absence or presence of
20 L , 50 L , or 100 L of soluble extracts generated from ethylene treated Col-0 wt seedlings, after which hypocotyls were measured. As shown in Figure 29, growth of hls1-
11;pmr6-6 in the presence of soluble extracts was less inhibited compared to pmr6-6 across a range of SE levels. While the hls1-11 only partially suppresses the hypersensitivity to the
83
Figure 28: Ethylene responsiveness in Col-0, hls1-11, pmr6-6, and hls1-11;pmr6-6 seedlings determined by an ethylene dose-response analysis. 4-day-old etiolated seedlings were grown on PNS media with 5 M AVG. With one condition grown in the presence of 2 M AgNO¬3, blocking ethylene perception, the other conditions had exogenous ethylene environments ranging from 0.1 to 100 L/L. Hypocotyls were subsequently measured. Mean values ± SD were determined from 30 seedlings.
84
Figure 29: Soluble extract responsiveness in Col-0, hls1-11, pmr6-6, and hls1-11;pmr6-6 seedlings determined by increasing amounts of resolubilized metabolite extracts from Col- 0 ethylene treated seedlings. 4-day-old etiolated seedlings were grown on PNS media with 5 M AVG and 2 M AgNO¬3. Control represented by diH2O in 6 mL volume of PNS media while soluble extracts were added to 6 mL volume of PNS media from 20 L to 100 L. Hypocotyls were subsequently measured. Mean values ± SD were determined from 30 seedlings.
85
Figure 30: rt-PCR analysis of HOOKLESS1 gene from Col-0, pmr6-6, and PMR6 Overexpressor Line 21 4 day-old dark grown seedlings. cDNA was generated from Col-0 and pmr6-6 plants grown on PNS for 4 days. rt-PCR was performed on CFX Connect Real- Time PCR Detection System with 3 replicates. Standard deviations and fold change calculated from a double delta Ct analysis of gene expression values.
86 PMR6 substrate, it suggests that HLS1-dependent chromatin remodeling may be important for response to the presumptive PMR6 substrate.
HOOKLESS1 expression was investigated as a potential output that could lead to broader downstream changes in gene expression through chromatin remodeling or effect transitions to plant development states, eg. through the initiation of skotomorphogenesis.
HLS1 expression was quantified through RT-PCR from RNA isolated from 4-day old, dark grown Col-0, pmr6-6, and a representative PMR6 Overexpressor line (21) in the absence of ethylene signaling with 2 M AgNO3 and 5 M AVG or with ethylene treatment of 100
L/L with 5 M AVG. As displayed with figure 30, an interesting observation was the lack of HLS1 expression in the PMR6 Overexpressor line. HLS1 gene expression is normally upregulated by ethylene signaling (Lehman et al., 1996) but what was observed in pmr6-6 was the upregulation of HLS1 while hooks did not form morphologically for the plants.
87 Conclusion – PMR6’s substrate serves as an elicitor of plant hormone responses
Our understanding of ethylene response in plants has developed through mutational analysis of genes that are required for a plant to detect and respond to ethylene, yet gaps in our understanding persist. In an effort to identify additional components of ethylene response, EMS-mutagenized Arabidopsis seedlings were screened for those with an aberrant ethylene response. This led to identification of a loss-of-function mutation in the previously characterized PMR6 (Vogel et al., 2002), which encodes a pectate lyase of unknown function that has been linked to normal development of powdery mildew disease.
pmr6-6 displays hypersensitivity to low concentrations of ethylene and showed similar phenotypes in comparison to another class of ethylene mutants - enhanced ethylene response (eer) mutants (Christians and Larsen, 2007, Robles et al., 2007, Christians et al.,
2008, Deslauriers and Larsen, 2010). The mutation responsible for this ethylene hypersensitivity was a single nucelotide change resulting in a premature stop codon in
PMR6. How this mutation leads enhanced ethylene response is not immediately clear, yet the role of PMR6 in ethylene response is supported by similar phenotypes in other loss of function pmr6 alleles. The connection between PMR6 and ethylene response has remained elusive since it was first attributed to ethylene signaling but preliminary results with plant metabolite extracts began to suggest that the PMR6 substrate could be conditioning or interacting with members of the ethylene signaling cascade.
Some of the conclusions were reached when looking at the cell wall composition in pmr6-1 and pmr6-2, where there was an enrichment of cell galacturonic acid and consequently thicker cell walls. This was originally cited as a reason for pmr6’s ability to
88 prevent powdery mildew disease from developing on leaves of the loss-of-function mutants
(Vogel et al., 2002, Raab et al., 2003). Powdery mildew sensitivity was restored by pmr6 double mutants with rwa2 and tbr1, which are involved in the acetylation of pectin
(Manabe et al., 2011, Chiniquy et al., 2019). The complexity of the extracellular space of plants and how it contributes to plant-pathogen interactions ia difficult to fully characterize, but there is a change in cell wall composition by FTIR analysis of the extracellular space as a result of pmr6 mutation (Vogel et al., 2002). With the change in the cell wall landscape being altered by the pmr6 mutation it was of interest to see if the ethylene hypersensitivity seen for pmr6-6 was linked with the changes to the cell wall landscape. One potential role
PMR6 may play in the extracellular space is the processing of pectin – by reducing the chain length of a pectin molecule through cleavage of a covalent linkage in a longer pectin molecule. It was under this hypothesis that two consequences may occur if PMR6 functioned to break down a pectin chain into smaller fragments – either the substrate of
PMR6 or the product(s) of PMR6 could serve some additional role in the plant such as functioning as a signaling molecule. Loss of PMR6 would result in a compromised capability to process and eliminate a pectin chain in the extracellular space, thus causing inappropriate buildup of this likely ephemeral molecule. If the substrate or product played a role in signaling mechanisms or could simply interfere in normal growth of the plant, this could be what was responsible for the aberrant ethylene response that was observed in pmr6-6.
Pectin as a complex carbohydrate can range from a simple linear chain to one that is highly complex, with a variety of modifications such as methylation and acetylation
89 made by the plant affecting how the carbohydrate chains interact with their environment
(Caffall and Mohnen, 2009). These modifications play an important role in the susceptibility of plants to biotrophic and necrotrophic pathogens (Manabe et al., 2011,
Raiola et al., 2011, Bellincampi et al., 2014, Lionetti et al., 2017, Chiniquy et al., 2019).
Because of these variables, the molecule(s) that may serve as PMR6’s substrate could be simple or complex making the method of identifying it difficult. Research into oligogalcturonic acid signaling mechanisms has often been dependent on galacturonic acid fragments from pectin chemically degraded or enzymatically digested by macerating enzymes and analyzing the effects of smaller pectin fragments mostly based on the chain lengths and not specific functional groups (Davis et al., 1986, Davis and Hahlbrock, 1987
Broekaert and Pneumas, 1988, Ferrari 2013). Since upon treatment with ethylene, pmr6-6 seedlings exhibit clear phenotypical differences from Col-0 wt, it was hypothesized that ethylene treatment may cause release of and accumulation of the PMR6 substrate in planta.
Upon testing, it was observed that pmr6-6 etiolated seedlings demonstrated a severe reduction in hypocotyl length in comparison to Col-0 wt when grown in the presence of soluble extracts in the absence of ethylene perception and biosynthesis (Figure 16). While chemical analysis of these soluble extracts has proved to be quite challenging, these early physiological studies argue that the PMR6 substrate may function to control hypocotyl elongation in a manner that is synergistic yet independent of ethylene perception.
Factors that are known to be components of the ethylene signaling pathway offered opportunities to investigate how hypocotyl shortening is elicited by the PMR6 substrate independent of ethylene perception. For example, the etr1-1;pmr6-6 double mutant
90 demonstrated a response to the soluble extract containing the predicted PMR6 substrate even though this double mutant was ethylene insensitive (Figure 22). In contrast, the inhibitory effects of the predicted PMR6 substrate were largely dependent on both EIN2 and members of the ethylene dependent transcriptional response, EIN3 and EIL1 (Figures
23 & 24). Since EIN2 functions as a point where cross talk occurs between other signaling pathways and the ethylene pathway (Alonso et al., 1999), there may be a similar connection between perception of the PMR6 substrate and the ethylene hypersensitivity observed in pmr6-6 since ethylene responses are promoted through EIN2 and subsequently dependent on canonical ethylene gene expression.
The effect of the soluble extracts and ethylene on the transcription factors associated with ethylene signaling – EIN3 and EIL1 – led to different hypocotyl phenotypes depending on the molecular input. The loss of both transcription factors effectively eliminates ethylene and soluble analyte response in the triple mutant plants (Figure 15 &
24). Some interesting differences arose when looking at the loss of individual transcription factors in pmr6-6 plants that may indicate diverging roles for the transcription factors. EIN3 is understood to represent the dominant transcription factor for ethylene signaling, while
EIL1 was identified in a screen for weakly insensitive ethylene mutants (Solano et al., 1998,
Alonso et al., 2003). Similarly, as reported the double mutants reflected a growth pattern where EIN3 function seemed to play a more significant role in response to ethylene (Figure
15). An interesting picture emerges from the analysis of the transcription factors grown in the presence of soluble extracts, in which EIL1 plays a mores significant role in hypocotyl shortening in response to the soluble analyte (Figure 24). With functional EIL1 present in
91 the ein3-1;pmr6-6 double mutant, there is a more significant effect on hypocotyl length in comparison with functional EIN3 in eil1-1;pmr6-6. Based on this differential response between the two transcription factors, there may be an alternate method for promoting and regulating ethylene response genes by these transcription factors. EIN3 may play the dominant role in ethylene response while EIL1 may play the dominant role in response to this alternative signaling pathway elicited by the accumulation of PMR6’s substrate signaling upstream of EIN2.
The demonstration that the hypocotyl shortening phenotype is dependent on several key members of the ethylene pathway provides some alternative means of investigating how the components of the ethylene pathway are conditioned by the soluble analyte. An
EIN3::GFP fusion protein expressed in Arabidopsis has been used as a reporter for ethylene signaling as the presence of ethylene leads to its stabilization in the nuclei of plant cells
(Guo et al., 2003). Consistent with the role of EIN3 in response to the PMR6 substrate, this transcription factor is stabilized in the nucleus of plant cells. Because of this the promotion of hypocotyl shortening by soluble extracts can be connected to the canonical ethylene signaling pathway independent of ethylene perception and suggests that the PMR6 substrate may be an alternate input into the ethylene response pathway at a junction with
EIN2 through stabilization of the EIN3 and EIL1 transcription factors. This is shown in
Figure 25, which argues that the mechanism/factor responsible for the perception of the
PMR6 substrate plays a critical role in classifying how the ethylene pathway and potentially a defense pathway are regulated by sensing of the cell wall integrity or changes in the extracellular space.
92
Figure 31: A proposed model for how the substrate of PMR6 – a suspected galacturonic acid – interacts with the ethylene signaling cascade. Since EIN2 function is required for the ethylene independent responses from a PMR6 substrate, it is likely that EIN2 accumulation is promoted, which then causes EIN3/EIL1 transcription factors to accumulate in the nucleus and alter gene expression leading to ethylene responses. EIN2 and ETR1 function are not required for powdery mildew resistance in pmr6 plants (reference) so if the resistance is conditioned by PMR6 substrate detection, this would occur independent of EIN2 function.
93 There is additional precedence that the ethylene pathway is not the only pathway affected by pmr6. In ein2 plants with a pmr6-6 background, the inoculation of plants with powdery mildew does not support powdery mildew growth (results not shown). In the original work exploring PMR6 the Sommerville lab observed that loss of ethylene perception through etr1-1 also did not rescue powdery mildew sensitivity in pmr6-1 (Vogel et al., 2002). Even though the loss of EIN2 function results in suppression of the ethylene hypersensitivity of the hypocotyl, the effects on plant-pathogen interactions are unaffected by interruptions to ethylene response and pmr6 plants remain powdery mildew resistant.
While the experiments conducted looking into the explanation of how pmr6-6 was originally identified as an enhanced ethylene response mutation seem to be closely tied with the ethylene pathway there were additional observations made for this mutant. A proposed signaling effect for a PMR6 substrate that elicited a response in the presence of ethylene suggests that there should be a means of recognition and response in plants. The original explanation of pmr6’s powdery mildew resistance was a thicker cell wall that made penetration by the invading pathogen more difficult (Vogel et al., 2002). This paper also indicated a potential immune response pathway could be elicited causing hypersensitivity and inhibition to the growth and acquiring of resources by a powdery mildew infection, but it could not be related to other known plant immune pathways like systemic acquired resistance. pmr6;tbr1 and pmr6;rwa2 plants were susceptible to powdery mildew infection suggesting that affecting pectin modification can restore susceptibility to pmr6 (Chiniquy et al., 2019). How this directly connects with pmr6 resistance to powdery mildew remains a question but OGA treatment of plants before inoculation for infection with powdery
94 mildew effects the susceptibility of wild-type plants, suggesting a role in detection and signaling of OGAs is linked with susceptibility to powdery mildew (Chiniquy et al., 2019).
The investigation into gene expression with the ethylene and jasmonic acid pathway
(Figure 26) was a strong indicator that genes related to plant defense response were modified by treatment with soluble extracts. There was a dramatic change to the expression of all the PLANT DEFENSIN genes in both Col-0 wild type and pmr6-6 backgrounds suggesting a need for further research into how this PMR6 substrate may affect plant immune response in a way that promotes powdery mildew resistance at higher concentrations. Successful powdery mildew infection may require subverting this immune or stress response that could occur from an increase in pectin chains that cells could perceive as damage or indicators of infection. pmr6 mutations may lead to an increase in sensitivity to powdery mildew’s affect on cell wall integrity and a more sensitive immune response could lead to the specific resistance to powdery mildew that is observed. This would seem all the more reasonable since pmr6 demonstrates sensitivity to other pathogens
(Vogel et al., 2002, Chiniquy et al., 2019) which may infect and acquire nutrients in an alternative method that does not elicit or respond to the same PMR6 substrate mediated response.
The mutation in pmr6-6 provides further opportunities to answer questions about how the cell wall plays a role in plant cell signaling and plant defense. The identification of PMR6’s substrate may be one of the first categorized examples of an oligogalacturonide or a recognizable motif that is responded to by plants. This substrate may involve a novel signaling mechanism or novel receptor for higher plants. Pending further study the PMR6
95 substrate may also affect the susceptibility of plants to the powdery mildew pathogen. If confirmed to affect infectivity in Col-0 plants by exogenous application by lowering rates of infection, there may be a strong link for the signaling effects of the molecule to condition powdery mildew resistance. The effect of this substrate and additional oligogalacturonides in ripening fruit may also promote ethylene production and fruit ripening in climacteric fruits. PMR6 may prove to be a useful protein to understand how changes in the dynamics of the plant extracellular space affect growth, development and senescence in plant tissues, and to identify complex carbohydrates that can elicit a response in plants.
96 Materials and Methods
Seed Sterilization
Seeds were surface sterilized in 70% ethanol followed by 4 washes of diH2O. Seeds were then incubated in bleach for 5 minutes with an additional 4 washes with diH2O.
Centrifugation steps at 2000 rpm for 5 seconds occurred between all steps in process. Seeds were then wrapped in foil preventing light exposure and incubated at 4oC for 2-7 days to synchronize germination.
Growth Conditions
Sterilized seeds were planted on Plant Nutrient media and Sucrose (PNS) unless otherwise specified. PNS media had final concentrations of 5 mM KNO3, 2 mM MgSO4,
2 mM Ca(NO3)22H2O, 50 M FeEDTA, 1 M MnSO4, 5 M H3BO3, 50 nM CuSO4, 20 nM NaMoO4, 100 nM CoSO4, 2.5 mM KH2PO4, 0.5% w/v Sucrose, and 0.8 % w/v molecular genetic agar.
For antibiotic resistance analysis Murashige and Skoog medium (MS) media was used with the following final concentration: 4.3 g/L Murashige and Skoog salts, 1X
Gamborg’s Vitamin Solution, 87.6 M Sucrose, pH 5.7, 0.8% agar. For antibiotics the concentrations used for screening for growth of seedlings with Agrobacterium transfections were 30 µg/mL Kanamycin while 250 µg/mL Vancomycin was used for preventing Agrobacterium growth.
All dark grown experiments were planted sterilely and transferred to airtight jars where ethylene or soluble extract could be added to the growth environment as needed.
Etiolated seedlings were grown for 4 days at 20OC in an I-36LLVL Percival Scientific
97 Incubator. For ethylene experiments, control plates for no ethylene response had sterile filtered AgNO3 added to growth media before setting to a final concentration of 2 M in the growth media as needed. Sterile filtered aminoethoxyvinylglycine (AVG) was added as needed to a final concentration of 5 M AVG to prevent ethylene overproduction for growth experiments. These conditions, including AgNO3 and AVG, were also used for soluble analyte assays for analysis of a soluble analyte’s effect on hypocotyl length independent of ethylene.
Soluble Sugar Extraction and Growth conditions
Etiolated seedlings for soluble sugar extractions were generated by planting sterilized seeds on PNS media with the following modifications: sucrose was omitted, and plant agar increased to 1.6% w/v. Seeds were grown on the media surface in a single layer of seeds avoiding walls of 60mm X 15 mm plates (Fisher Scientific, XXX). Plates were grown 2 plates per jar in airtight jars with 100 L/L ethylene for 4 at 20oC. Whole seedlings were collected off plates immediately after removal from light and ethylene growth and
O flash frozen in liquid N2 (-196 C). Average fresh weight for each tube collected was ~600-
700 mg. Frozen tubes could be stored at -80OC or homogenized immediately with no noticeable effect on extract quality.
Frozen seedlings were homogenized with a prechilled mortar and pestle maintaining frozen tissue throughout the entirety of homogenization. Frozen tissue was lyophilized in vacuo (you need manufacturer) until dry in 2 mL tubes, with tissue in tubes having an average dry weight of 30-40 mg after lyophilization. Following this, 1.6 mL methanol:diH2O (1:1) was added to dried plant material and incubated for 10 min at room
98 temperature with intermittent mixing by vortexing. Resuspended plant tissue was centrifuged at 13000 RPM for 10 min to pellet cell debris. The supernatant was then transferred to 50 mL falcon tube and combined with all other extraction supernatants to limit effects of weight variability from collected material. The cell debris pellet was discarded. Supernatant volumes evenly distributed (approx. 1.5 mL) in 2 mL tubes equal to number of homogenized tubes used. Extracted supernatant was dried in vacuo using a
Sorvall Speed-Vac system.
Soluble extracts were reconstituted in 200 L sterile diH2O. Soluble extracts resuspended in H2O may be sterile filtered to remove any other particulates and cell debris from extraction with no loss in potency. 50 L of resuspended soluble extracts are added to 6 mL PNS (0.8% w/v agar) with 2 M AgNO3 and 5 M AVG added to provide a growth media that blocks ethylene perception and biosynthesis. As a control, 50 L sterile diH2O was added to 6 mL of media for plates without soluble extracts. 30 seeds of each line of interest were planted and grown for 4 days at 20OC in the dark in an identical manner to ethylene assays without ethylene present.
Measurement of Ethylene Production
For ethylene production assays, exactly 100 seeds were counted out and placed in
1.5 mL Eppendorf tubes for both Col-0 and pmr6-6. For growth prior to ethylene sampling,
0.5 mL of PNS or 0.5 mL PNS with 5 M AVG were added to 5 mL glass scintillation vials and 100 seeds were planted in each vial. Vials were covered with parafilm and placed in sterile beakers to grow at 20OC in the dark. After 72h, parafilm was removed from vial openings and rubber septum caps were added to seal the vial in the dark. Plants were moved
99 back to the incubator and grown in the dark for an additional 18h. Subsequently 0.9 mL headspace was sampled using a 1 mL syringe and injected into an HP 6850 series flame ionization gas chromatography system (Agilent). Total fresh weight for each sample was measured by the total seedling mass and used to calculate total ethylene production by nL of ethylene per gram fresh weight per hour. Mean and standard deviation was calculated from 10 replicates per condition.
Growth Conditions – ACC and jasmonic acid (JA)
Col-0 and pmr6-6 seedlings were grown under sterile conditions in a hydroponic system at 20oC under continuous light for 14d total post-planting. Sterilized seeds were planted on sterile mesh suspended on the surface of PNS liquid media in compartmentalized petri dishes (Fisher Scientific FB087582). After 13d, fresh PNS liquid media including various conditions being tested was made and added to new compartmentalized petri dishes and the mesh with plants was transferred for 24-hour treatment under each respective condition. Conditions are referenced as “Control” with 2
M AgNO3 and 5 M AVG, “ACC/JA” with 5 M AVG, 100 M ACC, 100 M JA±, or
“JA/ACC/SE” condition with 5 M AVG, 100 M ACC, 100 M JA±, 50 L soluble extract. After 24 hours plants were collected and flash frozen in liquid N2 and stored at -
80OC for RNA isolation from plant material.
Isolation of Total RNA
Tissue collected from either experiments with 4-day-old dark grown seedlings or
14-day-old plants were flash frozen upon collection in liquid nitrogen and stored at -80oC.
Isolation of total RNA was carried out using an RNeasy Mini Kit (Qiagen) according to
100 manufacturers instructions. Extracted RNA was stored at -80oC and quantifications were carried out using a NanoDrop 2000 UV/Vis spectrophotometer (ThermoFisher). cDNA Synthesis and Quantitative RT-PCR
For cDNA synthesis, 6 µg of total RNA was treated with RQ1 RNase-free DNase
(Promega) for 30 minutes at 37oC followed by a reaction stop step for 5 min at 85oC.
Samples were then quantified again and 1 µg of DNase treated RNA was used for cDNA
Synthesis by reverse transcriptase through SuperScriptIII First-Strand Synthesis System
(Invitrogen) with oligo-dT provided primers.
Synthesized cDNA libraries were diluted 1:20 and quantified to provide a 50 ng/L
DNA concentration by NanoDrop. and 4 L of cDNA libraries were used for reaction volumes of 24 L in each RT-PCR reaction. 2X SYBR Green Master Mix (Bio-Rad) and the following primers were used for amplification of the following gene targets. RT-PCR and detection was conducted on CFX Connect Real-Time PCR Detection System (Bio-
Rad).
ERF1
Forward: 5’-TCGGCGATTCTCAATTTTTC-3'
Reverse: 5’-ACAACCGGAGAACAACCATC-3'
PCR product from cDNA produces a product size of 89 bp.
PDF1.2
Forward: 5’-TTTGCTGCTTTCGACGCAC-3'
Reverse: 5’-CGCAAACCCCTGACCATG-3'
PCR product from cDNA produces a product size of 82 bp.
101 PDF1.2
Forward: 5’-GTTCTCTTTGCAGCTTTTG-3'
Reverse: 5’-GCATGCCTTAAGTCACTCATAG-3'
PCR product from cDNA produces a product size of 286 bp.
PDF1.2c
Forward: 5’-CATCACCTTCCTTTTCGCTGC-3'
Reverse: 5’-TTCCACCATTGTTGGTGC-3'
PCR product from cDNA produces a product size of 64 bp.
PDF1.3
Forward: 5’-AAGCACCGATAATGGTGGAAGCAC-3'
Reverse: 5’-GTATAATTGGTAGTCATTGGTAGC-3'
PCR product from cDNA produces a product size of 203 bp.
HLS1
Forward: 5’-GTGATGGTGTTGTTGGTGATTG-3'
Reverse: 5’-TAGGAGATATTACCTCTTAC-3'
PCR product from cDNA produces a product size of 268 bp.
UBQ5 (as a control)
Forward: 5’-GTTAAGCTCGCTGTTCTTCAGT-3'
Reverse: 5’-TCAAGCTTCAACTCCTTCTTTC-3'
PCR product from cDNA produces a product size of 183 bp.
Fold changes were calculated based on analysis of gene expression after having crossed the cycle threshold (Ct) as determined by the CFX Connect software analysis of
102 RT-PCR results (Livak and Schmittgen, 2001). Expression fold changes (2^-ΔΔCt) were determined by double delta Ct analysis comparing the delta Ct control (UBQ5) to the delta
Ct experimental for a final delta delta Ct (ΔΔCt) value.
DNA Extractions
DNA was extracted from plant tissue through homogenization in lysis buffer containing 0.83M NaCl, 0.15M Sorbitol, 0.125M Tris, 23mM EDTA, 23mM CTAB
(hexadecyltrimethlammonium bromide), 1% Sarkosyl, and 20mM Sodium metabisulfite.
Tubes were incubated at 65oC for 30 min. Equal volume 24:1 chloroform/isoamyl alcohol was added to lysates, vortexed, and centrifuged at 13000 RPM for 5 minutes. The aqueous layer was transferred to a new tube and mixed with an equal volume of ice cold 100% isopropanol. Centrifugation at 13000 RPM for 10 minutes pelleted precipitated genomic
DNA. The pellet was washed with 70% ethanol and thoroughly dried. The DNA pellet was
O resuspended in TE buffer or ddH2O and stored at -20 C.
SSLP and CAPS marker Primers pmr6-5
Forward: 5’-CACGACGAGGTTATGTTGTTG-3’
Reverse: 5’-CTGGATTCATAGAAGATAGGAC-3’
PCR product of 523 bp was sent for Sanger sequencing off site with primer:
5’-GTTCAGAGGATGCCTAGGTG-3’
103 pmr6-6
Forward: 5’-TTCAATCGGCTTCGGACACG-3’
Reverse: 5’-GAGGTTGTGGATGATGATGTGTTG-3’
The resulting PCR products are digested with restriction enzyme TfiI. Col-0 yields a 292 bp product while pmr6-6 yields 205 bp and 87 bp products. etr1-1:
Sequences were amplified with the following primers
Forward: 5’-TGATCTGTCTACGCTACGTTC-3’
Reverse: 5’-ATGAGTCAACATTCTCACATG-3’
PCR product of 563 bp was sent for Sanger sequencing off site with primer:
5’-GTGAGAGAGGAACTATAGTG-3’ ctr1-3:
Sequences were analyzed with the following primers and sent for Sanger sequencing off site
Forward: 5’-CTTGATAGGTATGATAACAAG-3’
Reverse: 5’-GATACTGAGACATGATCAAG-3’ ein2-5:
Forward: 5’-GATCACAGGTATTTATGAGTC-3’
Reverse: 5’-AGCATCATTGCCACCAAGACC-3’
Resulting PCR product of 265 bp digested by NlaIII restriction endonuclease. Col-0 results in 114 bp fragment and 151 bp fragment, ein2-5 results in 265 bp fragment.
104 ein3-1:
Forward: 5’-GGTTTGATCGTAATGGTCCTG-3’
Reverse: 5’-CCTTCTCGAAACCTTCAACATC-3’
After digestion by HaeIII restriction endonuclease, Col-0 results in 334 bp, 218 bp, and 24 bp fragments while ein3-1 results in 334 bp and 242 bp fragments. eil1-1:
Forward: 5’-CTTGATTGTGGTGGAAGAGAC-3’
Reverse: 5’-ACTGATACTTAGCAATAGCAG-3’
WT reaction produces a PCR product of 493 bp. PCR product from transposon containing plant produces ~750 bp product observable in homozygous and heterozygous plants. eil1-1 mutation was selected as positive for 750 bp PCR product without wild type
PCR product. hls1-11:
Forward: 5’-CTGTAACAGGTGGCTGAGATG-3’
Reverse: 5’-GACGAAAGTCCCAAGCGAGAG-3’
Resulting PCR product of 599 bp digested by Fnu4HI restriction endonuclease. Col-0 results in 465 bp fragment and 134 bp fragment, hls1-11 results in 599 bp fragment.
EMS mutagenesis and screening for pmr6-6 suppressors
For EMS mutagenesis, 1 gram of pmr6-6 seeds was soaked in sterile H2O for 10 minutes with rocking. After imbibition, seeds were allowed to settle and excess water was pipetted off. Additional water was added to the falcon tubes to account for 40 mL H2O total water volume and seeds were mixed. After seeds had settled on benchtop, excess water
105 was pipetted off seed surface. 100 mL of 0.25% EMS in sterile H2O solution was made up in fume hood. Seeds were transferred to a plastic container with EMS solution with gentile stirring by stir bar and remained in EMS solution overnight with gentile agitation. After this, the EMS solution with seeds was transferred to a fresh 50 mL falcon tube and seeds were spun down at 1000 RPM for 30 seconds. EMS solution was removed and seeds were washed with 40 mL water volumes 4 times sequentially. Mutagenized seeds were resuspended in 0.1% agarose solution and pipetted evenly across soil surface to allow for growth. Collected seeds from mutagenized plants were classified by pot pools for screening for suppressors of pmr6-6 phenotypes. Mutagenized seeds were surface sterilized and planted on PNS growth media with 2 M AgNO3 to inhibit ethylene perception and 50 L soluble extracts from ethylene treated Col-0 seedlings per 6 mL growth media and grown in the dark for 4 days at 20oC. Col-0 and pmr6-6 seed lines were planted on identical growth media to establish controls to compare for response to soluble extracts. Mutagenized seedlings that demonstrated long hypocotyl growth in the presence of soluble extracts were transferred sterilely from growth media to PNS after 4 days growth before being transferred to pots and allowed to self pollinate, following which seeds were collected. For lines of interest, 30 progeny seeds were planted on PNS growth media with 2 M AgNO3 and 5
M AVG or PNS growth media with 2 M AgNO3, 5 M AVG, and 50 L soluble extracts from ethylene treated Col-0 seedlings for confirmation of pmr6-6 phenotype suppression.
106 Subcloning and Sequencing
For generation of a PMR6 overexpressing line: cDNA synthesized from previous
RNA extractions was used as a template for a PCR reaction with following primers for the
CDS of PMR6, which generated a PCR product of ~1500 bp.
Forward: 5’-AGGATCCATGCTTCTTCAAAACTTCTCC-3’
Reverse: 5’-AGGATCCTCACAATAATAGAGTTGATAAC-3’
The resulting PCR product was sequenced while in pGEMT-Easy vector plasmid. pGEMT-
EZ (Promega) containing PMR6 was digested with BamH1 and PMR6 was isolated by gel electrophoresis with the resulting product being ligated into the pCGN18 vector digested with BamHI. The ligation was transformed into DH5 and the orientation of PMR6 in pCGN18 vector was identified by digestion with HindIII producing a digestion product of
2.7 kbp when in proper orientation and a digestion product of 2 kbp when oriented backwards. The transgene construct was then transformed into Agrobacterium tumefaciens strain AgLo by electroporation using a MicroPulser (Bio-Rad) and the transformants were plated and incubated overnight at 30OC on LB+Gentamycin (50 µg/mL) plates.
DNA Extraction of antibiotic resistant Agrobacterium to confirm to contain the transgene PMR6 construct by growning in LB+Gentamycin (50 µg/mL) overnight at 30C.
Cells were pelleted at 5000 RPM for 5 min. Media was removed and cell pellet was resuspended in 1 mL of resuspension buffer (50 mM glucose, 25 mM Tris-HCl pH 8.0, 10 mM EDTA, and 4 mg/mL lysozyme). Cells were incubated at room temperature for 10 minutes before addition of 0.2 mL of lysis buffer (1% SDS, 0.2 N NaOH) and gentle mixing. Cells were incubated at room temperature for 10 minutes. 0.03 mL of 2:1
107 (phenol:lysis buffer) mixture was added to the suspension and mixed by inversion. 0.15 mL of 3 M sodium acetate (ph 4.8) was added to the suspension and mixed by inversion.
Tubes were incubated at -20OC for 15 minutes and centrifuged at 13000 RPM for 3 minutes. Supernatant is discarded and 0.5 mL of 0.3M acetate at pH 7.0 was added to pellet.
The tube was filled with ice-cold 95% EtOH (-20OC) and incubated at -80oC for 15 minutes. Tubes were centrifuged at 13000 RPM for 3 minutes and the supernatant was completely removed from the pellet. 70% EtOH was added to the pellet and tubes were centrifuged at 13000 RPM for 3 minutes. The supernatant was removed and pellets allowed to dry on ice until the EtOH had evaporated completely. Plasmid DNA was resuspended in sterile H2O and digested with HindIII to confirm plasmid insert and proper orientation in pCGN18 in the Agrobacterium colony.
Agrobacterium-mediated transformation
Confirmed colonies containing PMR6 in pCGN18 were grown overnight in 500 mL of LB+Gentamycin (50 µg/mL) media at 30OC. Agrobacterium was pelleted by centrifugation at 4000 RPM for 15 minutes. Supernatant was removed and cells were resuspended gently and completely in 200 mL of infiltration media (ph 5.7 with NaOH -
50 g/L Sucrose, 2.2 g/L MS salts, 1X Gamborg’s vitamin solution, 44 nM benzylaminopurine, and 200 L/L Silwet L-77). Additional infiltration media was added to a Pyrex square glass dish and Agrobacterium resuspension was added to the dish. pmr6-
6 potted plants having been planted 4 weeks previously were at the early stages of flowering and were dipped into the glass dish with infiltration media twice for 30 seconds each time. Potted plants were wrapped in saran wrap and placed back in growth room at
108 22OC with constant light. Saran wrap was removed after 3 days and seeds were collected after normal plant maturation.
PMR6 Overexpressor screening
Seeds collected from Agrobacterium transformed plants were surface sterilized and cold treated for 2d. Seeds were then planted on MS + Kanamycin (30 µg/mL) and
Vancomycin (250 µg/mL) plates and incubated under constant light at 20oC. Plates were monitored over the course of 14 days as seeds that did not have agrobacterium transformation would not develop true leaves and would have inhibited root growth. As seedlings were identified with kanamycin resistance by development of true leaves and longer roots on the selection they were sterilely transferred to new PNS media plates without antibiotics before being transferred to soil. Isolates displaying kanamycin resistance were allowed to self-pollinate and progeny were screened for kanamycin resistance. All progeny from self-pollinated transformants that grew on MS+Kanamycin
(30 µg/mL) media plates were heterozygous for the insertion so 30 seeds from F2 generation were screened on MS+Kanamycin (30 µg/mL) media plates to identify lines that were segregating for Kanamycin resistance. These heterozygous plants were identified so homozygous insertion lines could be generated and tested for Soluble Extract sensitivity.
Homozygous PMR6 OEX plants were grown with Col-0 and pmr6-6 to compare for decreased observable response to soluble extracts from ethylene treated etiolated Col-
0 seedlings on PNS media with 2 M AgNO3 to inhibit ethylene perception. Transgenic lines were scored based on how effectively suppressed the pmr6-6 phenotype was upon growth on soluble extracts in comparison to the pmr6-6 seedlings. For this analysis, 3
109 independently isolated insertion lines were characterized as inhibiting the pmr6-6 phenotype through this process characterized as PMR6 OEX lines 21, 46, and 49. As further evidence of the expression level for PMR6 being altered by overexpression these lines were investigated by Northern Blot for PMR6 and 16S rRNA.
Northern Analysis
Homozygous kanamycin resistant seed pools from independently isolated insertion lines 21, 46, and 49 were grown (aprox. 100 seeds each) on PNS media for 7 days.
Seedlings were flash frozen and RNA isolated using the Qiagen RNeasy RNA extraction kit for Northern analysis experiments. 10 µg of RNA for each treatment used for total RNA sample. RNA was prepared in Loading buffer – 1X MOPS, 64% Formamide, 23%
Formaldehyde, 10% Glycerol, and 20 L/mL Ethidium Bromide. RNA samples were separated by electrophoresis on a 1% Agarose gel with 1X MOPS and 18% Formaldehyde with running buffer for electrophoresis being 1X MOPS. RNA was transferred from the agarose gel onto Zeta Probe GT Membrane (Bio-Rad) with 20X SSC buffer serving for
RNA transfer to membrane. RNA was crosslinked to the membrane by treatment with 1200 uJ/cm2 UV light using a Spectrolinker XL1000 (Spectronics Corporation).
Stripping of the membrane for before and after probe binding involved a 10-minute boiling of the membrane in 0.1X SSC and 0.5% SDS. Prehybridization of the membrane before binding of probe was performed by incubation at 65oC in 6X SSC, 2X Denhardt’s reagent, 0.1% SDS, and 100 µg/mL herring sperm DNA. Radiolabeled probes were synthesized using RadPrime DNA labeling system (Invitrogen) in accordance with the manufacturer’s instructions with [-32P]dCTP as the integrated labeled nucleotide.
110 Sephadex G-50 (GE Healthcare) was used to separate probes from free nucleotides after labeling. Probe was added to membrane in prehybridization solution and incubated at 65oC overnight. After incubation, the membrane was `washed with increasing stringency of washes for 20 minutes. Wash 1 was 2X SSC and 0.1% SDS, wash 2 was 0.5X SSC and
0.1% SDS, while wash 3 was 0.1X SSC and 0.1% SDS. Blots were then exposed to Kodak
BioMax Light Film.
GFP (green fluorescent protein) Visualization Conditions
For EIN3::GFP analyses, plants were grown on PNS supplemented with 5 M
AVG that were placed in airtight jars and grown in the dark for 4dat 20oC. Ethylene was added to jars 3 hours before GFP visualization through a rubber septum. Ethylene perception was blocked by addition of 2 M AgNO3 and was confirmed to block the accumulation of EIN3::GFP in the presence of 100 L/L Ethylene. Treatment of plants with soluble extracts involved the equivalent of 600 L soluble extracts reconstituted in 2 mL diH2O and added to plates in the dark 3 hours before GFP visualization. Treatment conditions had a final concentration of 2 M AgNO3 and 5 M AVG in the 2 mL Soluble
Extract resuspension. As a control, 2 mL of 2 M AgNO3 and 5 M AVG in diH2O was added to non-ethylene treated plates while 2 mL of 5 M AVG is added before ethylene for exogenous ethylene environments serving as a positive control for EIN3::GFP accumulation. GFP visualization occurred on a Keyence BZ-X800E fluorescence microscope. Excitation of GFP was done with 395 nm while GFP emission was measured at 509 nm.
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