Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003
Performing Department
Biochemistry & Molecular Biology
Non Technical Summary
Pollen grains germinate on the stigma, the receptive surface of the female organ pistil. Each pollen grain hydrates and extrudes a pollen tube whose function is to transport two sperm cells carried in its cytoplasm to the female gametophyte inside an ovule, usually located at some distance from the stigma. Recent research in plant reproduction has produced critical insights into how a pollen tube targets the female gametophyte (where the egg apparatus is located) through a female-guided processand how the pollen tube, once inside the female gametophyte, achieves sperm release to enable fertilization. Yet mechanisms that underlie the critical first pollen-pistil interactive steps on the stigma, i.e. adhesion of pollen grains on the stigma, pollen hydration, activation and extrusion of the pollen tube to penetrate the stigmatic tissue, remain unclear. The project utilizes the model plant Arabidopsis to elucidate how these early events are orchestrated. In particular, we examine the contribution by three related receptor kinases (RKs) expressed in the stigma, FERONIA (FER), HERCULES1 (HERK1) and RK7.RKs are transmembrane proteins with an extracellular domain for interactions with other molecules, and a cytoplasmic kinase domain, which modifies molecules to alter their activities, thus mediating signals into cellular actions in response. RKs are important molecules for mediating cell-cell and cell-environment communication, including playing major roles in male-female interactions. FER, HERK1 and RK7 are members of a small RK family (with seventeen members) in Arabidopsis whose importance to plant growth and survival has been established rapidly since reports on its founding members about ten years ago. This is largely due to the potential of these RKs to be functioning at the frontline of interactions with the environment, such as changes in growth conditions or during intrusive growth such as when plants were invaded by pathogens or when pollen tubes penetrate female tissues. Work from our lab and around the world has demonstrated the importance of several members of this group of RKs. In particular FER is crucial throughout the plant's life cycle, including being critically required for female fertility. FER, HERK1 and RK7 are the three most prominently expressed in the stigma among the seventeen membered-protein family. Mutant plants lacking FER produce few seeds, yet the stigmatic function in supporting germination of pollen grains is not impaired at all. On the other hand, loss of HERK1 or RK7 does not induce any notable defects in reproduction. Preliminary results based on higher order mutants, such as loss of FER and RK7 together, indicate that initial pollen grain/tube-stigmatic tissue interaction are compromised. The project will produce information towards understanding how these three related RKs work together to support the initial male-female interactive events of pollination which, only if successful, will lead to fertilization producing grains for food and seeds for future generations.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The project addresses how flowering plants achieve fertilization, which if unsuccessful will result in reproductive failure, devastating agricultural productivity. Pollen grains germinate on the stigma, the receptive surface of the female organ pistil. Each pollen grain hydrates and extrudes a pollen tube whose function is to transport two sperm cells carried in its cytoplasm to the female gametophyte inside an ovule, usually located at some distance from the stigma. Recent research in plant reproduction has produced critical insights into how a pollen tube targets the female gametophyte (where the egg apparatus is located) through a female-guided processand how the pollen tube, once inside the female gametophyte, achieves sperm release to enable fertilization. Yet mechanisms that underlie the critical first pollen-pistil interactive steps on the stigma, i.e. adhesion of pollen grains on the stigma, pollen hydration, activation and extrusion of the pollen tube to penetrate the stigmatic tissue, remain unclear. Thestudy utilizes the model plant Arabidopsis to elucidate how these early events are orchestrated. In particular, we examine the contribution by three related receptor kinases (RKs) expressed in the stigma, FERONIA (FER), HERCULES1 (HERK1) and RK7.RKs are transmembrane proteins with an extracellular domain for interactions with other molecules, and a cytoplasmic kinase domain, which modifies molecules to alter their activities, thus mediating signals into cellular actions in response. RKs are important molecules for mediating cell-cell and cell-environment communication, including playing major roles in male-female interactions. FER, HERK1 and RK7 are members of a small RK family (with seventeen members) in Arabidopsis whose importance to plant growth and survival has been established rapidly since reports on its founding members about ten years ago. This is largely due to the potential of these RKs to be functioning at the frontline of interactions with the environment, such as changes in growth conditions or during intrusive growth such as when plants were invaded by pathogens or when pollen tubes penetrate female tissues. Work from our lab and around the world has demonstrated the importance of several members of this group of RKs. In particular FER is crucial throughout the plant's life cycle, including being critically required for female fertility. FER, HERK1 and RK7 are the three most prominently expressed in the stigma among the seventeen membered-protein family. Mutant plants lacking FER produce few seeds, yet the stigmatic function in supporting germination of pollen grains is not impaired at all. On the other hand, loss of HERK1 or RK7 does not induce any notable defects in reproduction. Preliminary results based on higher order mutants, such as loss of FER and RK7 together, indicate that initial pollen grain/tube-stigmatic tissue interaction are compromised. The project's major goal is to understand how these three related RKs work together to support the initial male-female interactive events of pollination which, only if successful, will lead to fertilization producing grains for food and seeds for future generations. Specifically, we shall (1) establish precisely when the function(s) of FER, HERK1 and RK7 are required during the initial events of pollination;(2) Identify functional partners from both the stigma and pollen that work in concert with FER, HERK1 and RK7 to establish the signal transduction pathway in the stigmatic surface and underlying tissues that orchestrate these early pollination events.
Project Methods
Molecular genetics and biochemical approaches will be used to address the research questions.Aim 1: Establish precisely when the function(s) of FER, HERK1 and RK7 are required during the initial events of pollination.(section 1,2)Aim 2.Identify interacting partners from both the stigma and pollen that potentially work in concert with FER, HERK1 and RK7.(section 3,4)For aim 1, we will usepromoter GUS analysis to determine the temporal and spatial expression pattern from FER, HERK1 and RK7 and their responses to pollination. We already have FER promoter:GUS and RK7 promoter:GUS line and their expression patterns are partially characterized. HERK1 promoter GUS line and its seedling expression has been described (20). We will obtain this line, or construct our own if necessary. These will provide very convenient materials to assess the overall developmental regulation of these genes in flowers.Arabidopsis floral developmental stages are well defined. Our focus will be on pistils from stage 13 flowers (mature but not yet pollinated) and initially self-pollinated stage 15 flowers (mature and pollinated) because of the relative ease to obtain experimental materials. Then we would compare developmentally comparable stage 15 pistils, either emasculated and left unpollinated, or emasculated and pollinated to demonstrate if developmental regulation is in place for these RKs. To assess if pollination regulates the expression of these genes, we will sample pistils at close intervals upon germination, e.g. 5, 15, 30 minutes, periods that we believe to be the most critical for this study as events are getting started on the stigma and pollen tubes are just penetrating the stigmatic tissues. Then progressively sampling will be at longer intervals in the later stages since most pollen tubes will have grown past the style/ovary junction by 2-3 hours after pollination.These studies will be accompanied by direct measurement of transcript levels by RT-PCR.Analysis of pollination and seed yield phenotypes in higher order mutants in FER, HERK1 and RK7. Since loss-of-function T-DNA induced mutants of these genes do not display pollination defects on the stigma, higher order mutants of double or triple knockouts are proposed here. Currently we have a double fer-4 rk7 mutant that show loss in seed yields even more severely than in fer-4 alone. This is probably due to few pollen tubes (pollination using wild type pollen grains) grow into the ovarian chamber, and those arriving at the female gametophyte are aborted in their ability to fertilize. We have ascertained that complementation by RK7-GFP restored fer-4 level of female defects. We will generate also fer-4 herk1 double, herk1 rk7 double, and also a triple mutant using the weaker fer-5 allele (which produces a truncated transcript), and mutant plants are generally normal developmentally except for defective root hairs.We will subject these mutants to pollination analysis by observing the behavior of wild type pollen grains on their stigmas. Natural pollination and seed production will provide some baseline suggestions, and reciprocal pollinations using these mutants and female and male parents will establish male- or female- deficiency. But hand-pollination with countable numbers of pollen grains from otherwise normal quartet (qrt) plants (26) will provide quantitative analysis of the efficiency of the stigmas from these mutants to support various stages of the early pollination process, from adhesion, to pollen tube extrusion, penetration of the stigma and growth into the stylar tissue. Results will inform when the most prominent impact occur during the germination and early tube growth processes. Seed yield analysis after corresponding pollination assays will inform whether events later in the pollen tube growth and fertilization process have compounded the severity of the phenotype. We anticipate that in the fer-4 background, we will see a more severe seed yield phenotype relative to defects in the stigmatic/stylar region. We recently showed that autophagy-mediated cytoplasmic deletion is essential for tobacco pollen germination and tube extrusion in vitro. As a first analysis to explore the in vivo process on the stigma, we will pollinate wild type and mutant pistils with Arabidopsis pollen grains expression an autophagy reporter (ATG8-GFP) to determine if the RKs studied here participate in this critical first step.For Aim 2: We will examine interactions between these stigma RKs and the following molecules. We note that the purposes here are not just to establish similar interactive relationships as those already known for the best characterizedFER, but each interaction could inform specific aspects of the pollination process. The first question is whetherthe ECDs of FER, HERK1 and RK7 bind pectic fragments generated by pollen tube penetration of pistil? We have already established that the ECDs of FER, ANXURs and BUPs bind PGA; we will determine if HERK1 and RK7 ECD have similar properties. In our studies with FER, we observed that pollinated pistils produce a pectin-rich exudate, presumably as a result of hydrolases secreted by pollen tubes during the growth process (unpublished). Such exudates will be collected and used in binding assays as carried out inwith FER, HERK1 and RK7. The second question is whetherFER, HERK1 and RK7 targets of pollen-produced RALFs? From our studies of pollen FER-related RKs, we already have several pollen-expressed RALFs, 4 and 19 in particular, in our hands, and procedures are established for their production. We will subject these RALFs and the RKs to protein-protein interactions assays already established in our laboratory.