arabidopsis ath1 affymetrix microarray  (Thermo Fisher)

Journal: Genome Biology

Article Title: Quantitative proteomics of the tobacco pollen tube secretome identifies novel pollen tube guidance proteins important for fertilization

doi: 10.1186/s13059-016-0928-x

Figure Lengend Snippet: Label-free, high-throughput LC-MS/MS quantification of the SIV pollen tube secretome. a Numbers of peptides identified by LC-MS/MS. Box plots summarize peptide number distribution per protein accession within a single sample replicate with the median value designated by the solid red line . Appended pie charts show the distribution of peptide counts in arbitrary bins. b SIV-PS samples showing the reproducibility and dynamics of the secreted protein groups (threefold or more abundant in at least two SIV-PS samples relative to unpollinated controls and identified by at least three or more peptides). c Size distribution of pollen tube-secreted proteins, showing predominant bias towards small secreted proteins. SIV-PS1–2 are used as representatives of all four replicates. d In silico analysis of pollen tube-secreted proteins using SMART and Pfam databases of overrepresented protein families and domains. The v ertical white line indicates significance cutoff ( p < 0.05). e Classified pollen tube-secreted proteins based on the Top3 algorithm showing the dominant presence of unconventionally secreted proteins, although these have comparable protein abundance to conventionally secreted proteins. f Heatmap derived from the Agilent tobacco microarray [ , ] of transcripts encoding identified pollen tube-secreted proteins showing predominantly gametophytic enrichment. g Validated expression profile by semi-quantitative RT-PCR of selected pollen tube-secreted proteins assessed in this study. All samples analyzed were from N. tabacum. NtPsCRP1/2 Nicotiana tabacum cysteine-rich polypeptide protein 1 and 2, EIG-E80 elicitor inducible gene subE80, LLG3 Lorelei-like GPI-anchored protein 3, PT semi in vivo pollen tubes, Ov unfertilized ovules, Inf inflorescence

Article Snippet: Alignment showing the predicted 23 amino acid N-terminal signal peptide ( blue highlighting ), the conserved eight cysteine residues ( boxed ), and N-glycosylation sites ( grey highlighting ). f Expression profiles of pollen receptor kinase 2 (PRK2) and pollen receptor kinase 4 (PRK4) derived from Agilent 44 K Tobacco Genome Array [ ] and Arabidopsis Affymetrix ATH1 microarray [ ] data. g Semi-RT-PCR verification of PRK2 and PRK4 expression in tobacco mature pollen ( MP ), 4 h ( PT4 ) and 24 h ( PT24 ) in vitro pollen tubes, leaves ( LF ), roots ( RT ), SIV pollen tubes, and unfertilized ovules. h Topology of the receptor modules, PRK2 and PRK4, and the ligand module LAT52.

Techniques: High Throughput Screening Assay, Liquid Chromatography with Mass Spectroscopy, In Silico, Derivative Assay, Microarray, Expressing, Quantitative RT-PCR, In Vivo

Journal: Genome Biology

Article Title: Quantitative proteomics of the tobacco pollen tube secretome identifies novel pollen tube guidance proteins important for fertilization

doi: 10.1186/s13059-016-0928-x

Figure Lengend Snippet: Semi-in vivo transcriptome–secretome correlation. Pollen tube-secreted proteins showed moderate positive ( a ) and negative ( b ) correlation, but not linear correlation, with transcripts of the Arabidopsis SIV pollen tube transcriptome . To the right of each heatmap are scatter plots with Pearson correlation coefficient scores and confidence values at p < 0.01. c Comparison of tobacco SIV-PS with downstream targets of MYB97, 101, and 120 identified in [ , ]

Article Snippet: Alignment showing the predicted 23 amino acid N-terminal signal peptide ( blue highlighting ), the conserved eight cysteine residues ( boxed ), and N-glycosylation sites ( grey highlighting ). f Expression profiles of pollen receptor kinase 2 (PRK2) and pollen receptor kinase 4 (PRK4) derived from Agilent 44 K Tobacco Genome Array [ ] and Arabidopsis Affymetrix ATH1 microarray [ ] data. g Semi-RT-PCR verification of PRK2 and PRK4 expression in tobacco mature pollen ( MP ), 4 h ( PT4 ) and 24 h ( PT24 ) in vitro pollen tubes, leaves ( LF ), roots ( RT ), SIV pollen tubes, and unfertilized ovules. h Topology of the receptor modules, PRK2 and PRK4, and the ligand module LAT52.

Techniques: In Vivo

Journal: Genome Biology

Article Title: Quantitative proteomics of the tobacco pollen tube secretome identifies novel pollen tube guidance proteins important for fertilization

doi: 10.1186/s13059-016-0928-x

Figure Lengend Snippet: Simultaneous knockdown of YIP4A/B perturbed NtPsCRP2 secretion in seedlings. a An Arabidopsis seedling showing regions analyzed in this study. b, c NtPsCRP2-GFP localized less frequently in young root hairs but more frequently in mature root hairs, both in the wild type and in the yip4a-1;yip4b double mutant. d NtPsCRP2 secretion in 4-day-old etiolated wild-type seedlings showing localization in epidermal cells of the hypocotyl. The secretion was restricted at the hypocotyl–root junction. Propidium iodide ( red ) marks the beginning of the root. e Close-up of NtPsCRP2-GFP in elongated epidermal cells of the hypocotyl showing accumulation in spherical, aggregated endosomal-like vesicles. f, g Conversely, in yip4a-1;yip4b seedlings, NtPsCRP2 secretion was severely distorted, showing diffuse localization and non-uniform protein aggregates ( arrowheads ). h The spherical marked endosomal aggregates observed in the wild type were substituted with rod-like labeled organelles in yip4a-1;yip4b hypocotyl. i NtPsCRP2 localization at the root tip of wild-type seedlings marking secretory organelles. EPI epidermis, CX cortex. j Magnified root epidermal cells from wild type showing NtPsCRP2-marked organelles constrained to the vicinity of the plasma membrane ( white rectangle ) and also localized in ER-body-like organelles ( inset ). k, l In yip4a-1;yip4b , NtPsCRP2-GFP localization in secretory organelles at the root apical meristem appeared largely disorganized, showing severe organelle aggregations resembling BFA-like compartments ( arrowheads ). The ER-body-like localization ( arrow ) was maintained, suggesting no effect on ER-body biogenesis in yip4a-1;yip4b mutants. m–o Perturbed secretion of NtPsCRP2-GFP was also recapitulated following BFA or wortmannin treatment. Arrowheads show NtPsCRP2-GFP BFA compartments, arrows point to diffusely aggregated NtPsCRP2-GFP aberrant secretion. RC lateral root cap PI propidium iodide stain, WT wild type. Scale bars = 10 μM

Article Snippet: Alignment showing the predicted 23 amino acid N-terminal signal peptide ( blue highlighting ), the conserved eight cysteine residues ( boxed ), and N-glycosylation sites ( grey highlighting ). f Expression profiles of pollen receptor kinase 2 (PRK2) and pollen receptor kinase 4 (PRK4) derived from Agilent 44 K Tobacco Genome Array [ ] and Arabidopsis Affymetrix ATH1 microarray [ ] data. g Semi-RT-PCR verification of PRK2 and PRK4 expression in tobacco mature pollen ( MP ), 4 h ( PT4 ) and 24 h ( PT24 ) in vitro pollen tubes, leaves ( LF ), roots ( RT ), SIV pollen tubes, and unfertilized ovules. h Topology of the receptor modules, PRK2 and PRK4, and the ligand module LAT52.

Techniques: Mutagenesis, Labeling, Staining

Journal: Genome Biology

Article Title: Quantitative proteomics of the tobacco pollen tube secretome identifies novel pollen tube guidance proteins important for fertilization

doi: 10.1186/s13059-016-0928-x

Figure Lengend Snippet: LLG3 secretion is compromised in yip4a-1;yip4b mutant pollen and pollen tubes. a Subcellular localization of LLG3-mRFP under native promoter showing cytosolic foci-like aggregates. vn vegetative cell nucleus, sn sperm cell nuclei. b Top : in the yip4a-1;yip4b mutant, the majority of the pollen grains displayed distinct cytosolic aggregates different from those observed in the wild type. Bottom : z-stack projection of 25 confocal slices showing distinct vegetative cell “bird cage-like” localization that was not observed in wild-type pollen grains. c Localization of LLG3-mRFP in wild-type pollen tubes grown in vitro for 16 h showing localization in likely secretory vesicles of uniform size and partially in the pollen tube ER ( top three panels ). In contrast, yip4a-1;yip4b mutant pollen tubes displayed a significantly higher frequency of LLG3-mRFP-marked endomembrane aggregated vesicles of variable sizes ( bottom three panels ). However, the ER localization was not greatly affected. d LLG3-mRFP also specifically accumulated at the subapical domain ( top panel , arrow ). This accumulation was also not significantly affected in yip4a-1;yip4b mutant pollen tubes ( bottom panel ). e In tobacco pollen tubes, similar localization in secretory vesicles was also observed ( top panel , arrows ) and occasionally LLG3-mRFP as well as LLG3-sGFP showed pollen tube tip-specific localization ( bottom panels , arrowheads ). f Absence of LLG3-mRFP protein in Arabidopsis unfertilized ovules and embryos soon after fertilization (18 h after pollination). Rarely, LLG3-mRFP could be detected at the embryo proper ( EP ) zone in some fertilized ovules. MC micropylar, EP endosperm proper, CE chalaza end endosperm. Scale bars for a and b = 30 μM and for c–e = 10 μM

Article Snippet: Alignment showing the predicted 23 amino acid N-terminal signal peptide ( blue highlighting ), the conserved eight cysteine residues ( boxed ), and N-glycosylation sites ( grey highlighting ). f Expression profiles of pollen receptor kinase 2 (PRK2) and pollen receptor kinase 4 (PRK4) derived from Agilent 44 K Tobacco Genome Array [ ] and Arabidopsis Affymetrix ATH1 microarray [ ] data. g Semi-RT-PCR verification of PRK2 and PRK4 expression in tobacco mature pollen ( MP ), 4 h ( PT4 ) and 24 h ( PT24 ) in vitro pollen tubes, leaves ( LF ), roots ( RT ), SIV pollen tubes, and unfertilized ovules. h Topology of the receptor modules, PRK2 and PRK4, and the ligand module LAT52.

Techniques: Mutagenesis, In Vitro