Additional file 5: of Comparative proteomic analysis reveals a dynamic pollen plasma membrane protein map and the membrane landscape of receptor-like kinases and transporters important for pollen tube growth and interaction with pistils in rice

Plasma membrane-related proteins. (XLSX 240 kb

Differential Analysis of Proteomes and Metabolomes Reveals Additively Balanced Networking for Metabolism in Maize Heterosis

A century ago, dominance and overdominance hypotheses were developed to explain the phenomenon of heterosis, both hypotheses were in a nonadditive pattern. Here, a principal component analysis (PCA) of maize seed proteomes was used for representative inbreds of five heterotic germplasms and three classes of hybrid. Hybrids congregated in the center region of inbreds, forming an additive distribution with hybrids in the middle of their parents. Principal components 1 and 2 indicated biased distributions of proteins with functions of amino acid–protein or carbohydrate–energy metabolisms, respectively, after loading analysis and MS identification of proteins. Then, GC–MS was used to examine free amino acids, carbohydrates, and organic acids. A lower level of these metabolites were found in hybrids than inbreds. Further, we performed similar analyses of germinating seeds of a parent–F1 triad and three F2 segregants and confirmed these results. Therefore, an additive pattern of protein abundances for an unimpeded flow of metabolites was established in heterotic hybrids. That is, an additively balanced networking but not the nonadditive dominance or overdominance regulates heterosis. The less expensive metabolism in hybrids suggested the evolution of sexual reproduction. The Mendelian phenotypic ratio can be better explained based on this additive pattern than dominance

Comparative Proteomic Analysis of Generative and Sperm Cells Reveals Molecular Characteristics Associated with Sperm Development and Function Specialization

In flowering plants, two sperm cells (SCs) are generated from a generative cell (GC) in the developing pollen grain or growing pollen tube and are then delivered to the embryo sac to initiate double fertilization. SC development and function specialization involve the strict control of the protein (gene) expression program and coordination of diverse cellular processes. However, because methods for collecting a large amount of highly purified GCs and SCs for proteomic and transcriptomic studies from a plant are not available, molecular information about the program and the interconnections is lacking. Here, we describe a method for obtaining a large quantity of highly purified GCs and SCs from just-germinated lily pollen grains and growing pollen tubes for proteomic analysis. Our observation showed that SCs had less condensed chromatin and more vacuole-like structures than GCs and that mature SCs were arrested at the G2 phase. Comparison of SC and GC proteomes revealed 101 proteins differentially expressed in the two proteomes. These proteins are involved in diverse cellular and metabolic processes, with preferential involvement in metabolism, the cell cycle, signaling, the ubiquitin/proteasome pathway, and chromatin remodeling. Impressively, almost all proteins in SCF complex-mediated proteolysis and the cell cycle were up-regulated in SCs, whereas those in chromatin remodeling and stress response were down-regulated. Our data also reveal the coordination of SCF complex-mediated proteolysis, cell cycle progression, and DNA repair in SC development and function specialization. This study revealed for the first time a difference in protein profiles between GCs and SCs

Comparative Proteomic Analysis of Generative and Sperm Cells Reveals Molecular Characteristics Associated with Sperm Development and Function Specialization

In flowering plants, two sperm cells (SCs) are generated from a generative cell (GC) in the developing pollen grain or growing pollen tube and are then delivered to the embryo sac to initiate double fertilization. SC development and function specialization involve the strict control of the protein (gene) expression program and coordination of diverse cellular processes. However, because methods for collecting a large amount of highly purified GCs and SCs for proteomic and transcriptomic studies from a plant are not available, molecular information about the program and the interconnections is lacking. Here, we describe a method for obtaining a large quantity of highly purified GCs and SCs from just-germinated lily pollen grains and growing pollen tubes for proteomic analysis. Our observation showed that SCs had less condensed chromatin and more vacuole-like structures than GCs and that mature SCs were arrested at the G2 phase. Comparison of SC and GC proteomes revealed 101 proteins differentially expressed in the two proteomes. These proteins are involved in diverse cellular and metabolic processes, with preferential involvement in metabolism, the cell cycle, signaling, the ubiquitin/proteasome pathway, and chromatin remodeling. Impressively, almost all proteins in SCF complex-mediated proteolysis and the cell cycle were up-regulated in SCs, whereas those in chromatin remodeling and stress response were down-regulated. Our data also reveal the coordination of SCF complex-mediated proteolysis, cell cycle progression, and DNA repair in SC development and function specialization. This study revealed for the first time a difference in protein profiles between GCs and SCs

Quantitative Proteomics and Cytology of Rice Pollen Sterol-Rich Membrane Domains Reveals Pre-established Cell Polarity Cues in Mature Pollen

Cell polarity is essential for generating diverse cell functions. The underlying mechanisms of how a cell establishes, maintains, and changes its polarity are poorly understood. Recently, sterol-rich membrane microdomains are found to be associated with these processes. However, both its exact characteristics and importance are still unclear. Here we show microdomains change dynamically in developing and germinating rice pollen with selective enrichment in the aperture and the tip of newly born pollen tubes by use of the sterol-specific probe filipin. Using the sterol extraction sensitivities of microdomain proteins and quantitative proteomics, we identified 237 microdomain-associated proteins from 934 identified pollen detergent resistant membrane proteins. This proteome includes almost all of the known key regulators comprising the polar growth network, and it shows more similarity to front–back polarized HeLa cells than nonpolarized <i>Arabidopsis</i> suspension cells. We immunolocalize flotilin-like protein, a representative of these sterol-dependent proteins and directly visualize microdomains in pollen. These results indicate the presence of microdomains in pollen and pre-established cell polarity around the aperture during pollen maturation. Our findings reveal an atlas of the microdomain-associated proteome in pollen. This work provides useful resources and knowledge needed to further dissect the mechanisms for the establishment and maintenance of cell polarity

Proteomic Analysis Reveals an Aflatoxin-Triggered Immune Response in Cotyledons of <i>Arachis hypogaea</i> Infected with <i>Aspergillus flavus</i>

An immune response is triggered in host cells when host receptors recognize conserved molecular motifs, pathogen-associated molecular patterns (PAMPs), such as β-glucans, and chitin at the cell surface of a pathogen. Effector-triggered immunity occurs when pathogens deliver effectors into the host cell to suppress the first immune signaling. Using a differential proteomic approach, we identified an array of proteins responding to aflatoxins in cotyledons of peanut (<i>Arachis hypogaea</i>) infected with aflatoxin-producing (toxigenic) but not nonaflatoxin-producing (atoxigenic) strains of <i>Aspergillus flavus</i>. These proteins are involved in immune signaling and PAMP perception, DNA and RNA stabilization, induction of defense, innate immunity, hypersensitive response, biosynthesis of phytoalexins, cell wall responses, peptidoglycan assembly, penetration resistance, condensed tannin synthesis, detoxification, and metabolic regulation. Gene expression analysis confirmed the differential abundance of proteins in peanut cotyledons supplemented with aflatoxins, with or without infection with the atoxigenic strain. Similarly, peanut germination and <i>A. flavus</i> growth were altered in response to aflatoxin B1. These findings show an additional immunity initiated by aflatoxins. With the PAMP- and effector-triggered immune responses, this immunity constitutes the third immune response of the immune system in peanut cotyledon cells. The system is also a three-grade coevolution of plant–pathogen interaction

Quantitative Proteomics and Cytology of Rice Pollen Sterol-Rich Membrane Domains Reveals Pre-established Cell Polarity Cues in Mature Pollen

Cell polarity is essential for generating diverse cell functions. The underlying mechanisms of how a cell establishes, maintains, and changes its polarity are poorly understood. Recently, sterol-rich membrane microdomains are found to be associated with these processes. However, both its exact characteristics and importance are still unclear. Here we show microdomains change dynamically in developing and germinating rice pollen with selective enrichment in the aperture and the tip of newly born pollen tubes by use of the sterol-specific probe filipin. Using the sterol extraction sensitivities of microdomain proteins and quantitative proteomics, we identified 237 microdomain-associated proteins from 934 identified pollen detergent resistant membrane proteins. This proteome includes almost all of the known key regulators comprising the polar growth network, and it shows more similarity to front–back polarized HeLa cells than nonpolarized <i>Arabidopsis</i> suspension cells. We immunolocalize flotilin-like protein, a representative of these sterol-dependent proteins and directly visualize microdomains in pollen. These results indicate the presence of microdomains in pollen and pre-established cell polarity around the aperture during pollen maturation. Our findings reveal an atlas of the microdomain-associated proteome in pollen. This work provides useful resources and knowledge needed to further dissect the mechanisms for the establishment and maintenance of cell polarity

Proteomic Analysis Reveals an Aflatoxin-Triggered Immune Response in Cotyledons of <i>Arachis hypogaea</i> Infected with <i>Aspergillus flavus</i>

An immune response is triggered in host cells when host receptors recognize conserved molecular motifs, pathogen-associated molecular patterns (PAMPs), such as β-glucans, and chitin at the cell surface of a pathogen. Effector-triggered immunity occurs when pathogens deliver effectors into the host cell to suppress the first immune signaling. Using a differential proteomic approach, we identified an array of proteins responding to aflatoxins in cotyledons of peanut (<i>Arachis hypogaea</i>) infected with aflatoxin-producing (toxigenic) but not nonaflatoxin-producing (atoxigenic) strains of <i>Aspergillus flavus</i>. These proteins are involved in immune signaling and PAMP perception, DNA and RNA stabilization, induction of defense, innate immunity, hypersensitive response, biosynthesis of phytoalexins, cell wall responses, peptidoglycan assembly, penetration resistance, condensed tannin synthesis, detoxification, and metabolic regulation. Gene expression analysis confirmed the differential abundance of proteins in peanut cotyledons supplemented with aflatoxins, with or without infection with the atoxigenic strain. Similarly, peanut germination and <i>A. flavus</i> growth were altered in response to aflatoxin B1. These findings show an additional immunity initiated by aflatoxins. With the PAMP- and effector-triggered immune responses, this immunity constitutes the third immune response of the immune system in peanut cotyledon cells. The system is also a three-grade coevolution of plant–pathogen interaction

Quantitative Proteomics and Cytology of Rice Pollen Sterol-Rich Membrane Domains Reveals Pre-established Cell Polarity Cues in Mature Pollen

Cell polarity is essential for generating diverse cell functions. The underlying mechanisms of how a cell establishes, maintains, and changes its polarity are poorly understood. Recently, sterol-rich membrane microdomains are found to be associated with these processes. However, both its exact characteristics and importance are still unclear. Here we show microdomains change dynamically in developing and germinating rice pollen with selective enrichment in the aperture and the tip of newly born pollen tubes by use of the sterol-specific probe filipin. Using the sterol extraction sensitivities of microdomain proteins and quantitative proteomics, we identified 237 microdomain-associated proteins from 934 identified pollen detergent resistant membrane proteins. This proteome includes almost all of the known key regulators comprising the polar growth network, and it shows more similarity to front–back polarized HeLa cells than nonpolarized <i>Arabidopsis</i> suspension cells. We immunolocalize flotilin-like protein, a representative of these sterol-dependent proteins and directly visualize microdomains in pollen. These results indicate the presence of microdomains in pollen and pre-established cell polarity around the aperture during pollen maturation. Our findings reveal an atlas of the microdomain-associated proteome in pollen. This work provides useful resources and knowledge needed to further dissect the mechanisms for the establishment and maintenance of cell polarity

Proteomic Analysis Reveals an Aflatoxin-Triggered Immune Response in Cotyledons of <i>Arachis hypogaea</i> Infected with <i>Aspergillus flavus</i>

An immune response is triggered in host cells when host receptors recognize conserved molecular motifs, pathogen-associated molecular patterns (PAMPs), such as β-glucans, and chitin at the cell surface of a pathogen. Effector-triggered immunity occurs when pathogens deliver effectors into the host cell to suppress the first immune signaling. Using a differential proteomic approach, we identified an array of proteins responding to aflatoxins in cotyledons of peanut (<i>Arachis hypogaea</i>) infected with aflatoxin-producing (toxigenic) but not nonaflatoxin-producing (atoxigenic) strains of <i>Aspergillus flavus</i>. These proteins are involved in immune signaling and PAMP perception, DNA and RNA stabilization, induction of defense, innate immunity, hypersensitive response, biosynthesis of phytoalexins, cell wall responses, peptidoglycan assembly, penetration resistance, condensed tannin synthesis, detoxification, and metabolic regulation. Gene expression analysis confirmed the differential abundance of proteins in peanut cotyledons supplemented with aflatoxins, with or without infection with the atoxigenic strain. Similarly, peanut germination and <i>A. flavus</i> growth were altered in response to aflatoxin B1. These findings show an additional immunity initiated by aflatoxins. With the PAMP- and effector-triggered immune responses, this immunity constitutes the third immune response of the immune system in peanut cotyledon cells. The system is also a three-grade coevolution of plant–pathogen interaction