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Proteomic analyses of apoplastic proteins from germinating Arabidopsis thaliana pollen.

ABSTRACT: Pollen grains play important roles in the reproductive processes of flowering plants. The roles of apoplastic proteins in pollen germination and in pollen tube growth are comparatively less well understood. To investigate the functions of apoplastic proteins in pollen germination, the global apoplastic proteins of mature and germinated Arabidopsis thaliana pollen grains were prepared for differential analyses by using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE) saturation labeling techniques. One hundred and three proteins differentially expressed (p value?0.01) in pollen germinated for 6h compared with un-germination mature pollen, and 98 spots, which represented 71 proteins, were identified by LC-MS/MS. By bioinformatics analysis, 50 proteins were identified as secreted proteins. These proteins were mainly involved in cell wall modification and remodeling, protein metabolism and signal transduction. Three of the differentially expressed proteins were randomly selected to determine their subcellular localizations by transiently expressing YFP fusion proteins. The results of subcellular localization were identical with the bioinformatics prediction. Based on these data, we proposed a model for apoplastic proteins functioning in pollen germination and pollen tube growth. These results will lead to a better understanding of the mechanisms of pollen germination and pollen tube growth.

SUBMITTER: Ge W

PROVIDER: S-EPMC3214693 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Proteomic analyses of apoplastic proteins from germinating Arabidopsis thaliana pollen.

Ge Weina W Song Yun Y Zhang Cuijun C Zhang Yafang Y Burlingame Alma L AL Guo Yi Y

Biochimica et biophysica acta 20110723 12

Pollen grains play important roles in the reproductive processes of flowering plants. The roles of apoplastic proteins in pollen germination and in pollen tube growth are comparatively less well understood. To investigate the functions of apoplastic proteins in pollen germination, the global apoplastic proteins of mature and germinated Arabidopsis thaliana pollen grains were prepared for differential analyses by using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE) saturatio ...[more]

PMID: 21798377

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Project description:BackgroundMYC2, a basic helix-loop-helix (bHLH) domain-containing transcription factor, participates in the jasmonate (JA) signaling pathway and is involved in the modulation of diverse JA functions. However, a comprehensive list of MYC2-dependent JA-responsive proteins has yet to be defined.ResultsIn this paper, we report the comparative proteomics of wild-type (WT) plants and jin1-9, a MYC2 mutant plant, in response to methyl jasmonate (MeJA) treatment. Proteins from mock/MeJA-treated jin1-9 and WT samples were extracted and separated by two-dimensional gel electrophoresis. Twenty-seven JA-mediated proteins demonstrated differential expression modulated by MYC2. We observed that MYC2 negatively regulates the accumulation of JA-dependent indolic glucosinolate-related proteins and exhibits opposite effects on the biosynthetic enzymes involved aliphatic glucosinolate pathways. In addition, proteins involved in the tricarboxylic acid cycle and a majority of the MeJA-inducible proteins that are involved in multiple protective systems against oxidative stress were reduced in jin1-9/myc2 sample compared to the WT sample. These results support a positive role for MYC2 in regulating JA-mediated carbohydrate metabolism and oxidative stress tolerance.ConclusionsWe have identified MYC2-dependent jasmonate-regulated proteins in Arabidopsis thaliana by performing two-dimensional gel electrophoresis and MALDI-TOF/TOF MS analysis. The observed pattern of protein expression suggests that MYC2 has opposite effects on the biosynthetic enzymes of indolic and aliphatic glucosinolate pathways and positively regulates JA-mediated carbohydrate metabolism and oxidative stress tolerance-related proteins. Furthermore, it is very interesting to note that MYC2 plays opposite roles in the modulation of a subset of JA-regulated photosynthetic proteins during short-term and long-term JA signaling. This study will enhance our understanding of the function of MYC2 in JA signaling in Arabidopsis thaliana.

Project description:In sexual reproduction, a proper communication and cooperation between male and female organs and tissue are essential for male and female gametes to unite. In flowering plants, female sporophytic tissues and gametophyte direct a male pollen tube towards an egg apparatus, which consists of an egg cell and two synergid cells. The cell-cell communication between the pollen tube and the egg apparatus, such as the reception of a signal from the egg apparatus at the pollen tube, makes the tip of pollen tube rapture to release the sperm cell. To isolate male factors involved in the interaction between a pollen tube and an egg apparatus, we focused on receptor-like kinases (RLKs), which are extensively diversified in the flowering plant lineage to comprise a large monophyletic gene family. Approximately 620 members were found in the Arabidopsis thaliana genome. Expression patterns of 558 RLKs were analyzed using an Affymetrix ATH1 microarray of A. thaliana. We focused on two RLKs, ANXUR1 (ANX1) and ANXUR2 (ANX2), and characterized their function. Here we report that pollen tubes of anx1/anx2 ruptured before arriving at the egg apparatus, suggesting that ANX1 and ANX2 are male factors controlling pollen tube behavior with directing rupture at proper timing. Furthermore, ANX1 and ANX2 were the most closely related paralogs to a female factor FERONIA/SIRENE controlling pollen tube behavior expressed in synergid cells. Our finding shows that the coordinated behaviors of female and male reproductive apparatuses are regulated by the sister genes, whose duplication might play a role in the evolution of fertilization system in flowering plants. Experiment Overall Design: Arabidopsis thaliana Col-0 was grown at 18Â°C for 8 hr in the dark and at 22Â°C for 16 hr in the light. To collect germinated pollen with pollen tubes, opened flowers were touched to cellophane layered on the pollen germination medium containing 13.5% sucrose, 10 mM CaCl2, 0.4 mM boric acid, 1 mM KCl, 0.001% myo-inositol, and 0.6% agarose. The released pollen was incubated at 25Â°C for 6 hr in the dark. For collection of seedlings, seeds were sown on Murashige-Skoog medium containing 1x Murashige and Skoog Plant Salt Mixture (Wako), 1x Gamborgâs vitamin solution (Wako), 1% sucrose, and 0.8% agar. Seedlings including roots with one or two rosette leaves were collected for RNA extraction. RNA was extracted with Isogene (Nippon Gene), precipitated with LiCl, and further purified with RNeasy. The ATH1 genechip (Affymetrix) was used for expression analysis using 10 Âµg each of total RNA following the manufacturer's instructions. Array data were processed and analyzed with the GCOS1.2 program (Affymetrix) and Excel 2004 (Microsoft). Hybridization with germinating pollen RNA was duplicated and the results were compared to that with seedling RNA.

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Proteomic analyses of apoplastic proteins from germinating Arabidopsis thaliana pollen.

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Proteomic analyses of apoplastic proteins from germinating Arabidopsis thaliana pollen.

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