Dataset Information

Development of the cell wall in alfalfa

ABSTRACT: In this study, the cell wall proteomes of 3 different regions of alfalfa stems were compared. These three regions correspond to three phases in the stem development. The sequential extraction of cell wall proteins with CaCl2, EGTA and LiCl-complemented buffers was combined with a gel-based proteome approach and multivariate analysis. Although the highest similarities were observed between the apical and intermediate stem regions, the three proteome patterns are characteristic for each region. In the basal stem region, proteins that bind carbohydrates and have proteolytic activity, as well as enzymes involved in glycan remobilization, accumulate. Beta-amylase and ferritin likewise accumulate more in the basal stem segment. Therefore remobilization of nutrients appears to be an important process in the oldest stem segment. The intermediate and the apical region are sites of cell wall polymer remodelling, as suggested by the high abundance of proteins involved in the remodelling of the cell wall such as xyloglucan endoglucosylase, polygalacturonase non-catalytic subunit or beta-galactosidase. The most striking change between the different stem parts is however the strong accumulation of a DUF642-conserved domain containing protein in the apical region of the stem which suggests a particular role of this protein during the early development of stem tissues.

INSTRUMENT(S): AB SCIEX TOF/TOF Series Explorer Software

ORGANISM(S): Medicago Sativa (alfalfa)

TISSUE(S): Plant Cell, Plant Stem Cell, Stem Cell, Shoot

SUBMITTER: kjell sergeant

LAB HEAD: Kjell Sergeant

PROVIDER: PXD010666 | Pride | 2019-11-12

REPOSITORIES: Pride

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Publications

The Dynamics of the Cell Wall Proteome of Developing Alfalfa Stems.

Sergeant Kjell K Printz Bruno B Guerriero Gea G Renaut Jenny J Lutts Stanley S Hausman Jean-Francois JF

Biology 20190819 3

In this study, the cell-wall-enriched subproteomes at three different heights of alfalfa stems were compared. Since these three heights correspond to different states in stem development, a view on the dynamics of the cell wall proteome during cell maturation is obtained. This study of cell wall protein-enriched fractions forms the basis for a description of the development process of the cell wall and the linking cell wall localized proteins with the evolution of cell wall composition and struc ...[more]

PMID: 31430995

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Project description:Data analysis. Spot detection and matching were performed with a comparative cross analysis of all the gels using DeCyder software v.6.5 (GE Healthcare). 178 spots were selected based on 1.15-fold for protein ratio cut-off, allowing for the appearance of the spots in 23 out of 28 gels (69 out of 84 total images). Data from 95 spots were submitted. 93 spots were identified with high confidence. Spot picking and Trypsin digestion. The spots of interest were picked up by Ettan Spot Picker (GE Healthcare) based on the in-gel analysis and spot picking design by DeCyder software. The gel spots were washed a few times then digested in-gel with modified porcine trypsin protease (Promega, Fitchburg, WI). The digested tryptic peptides were desalted using a Zip-tip C18 (Millipore, Billerica, MA). Peptides were eluted from the Zip-tip with 0.5 uL of matrix solution (alpha-cyano-4-hydroxycinnamic acid, 5 mg/mL in 50% acetonitrile, 0.1% trifluoroacetic acid, 25mM ammonium bicarbonate) and spotted on a MALDI plate. Mass Spectrometry. MALDI-TOF MS and TOF/TOF tandem MS/MS were performed on AB SCIEX TOF/TOF 5800 System (AB SCIEX). MALDI-TOF mass spectra were acquired in reflectron positive ion mode, averaging 4000 laser shots per spectrum. TOF/TOF tandem MS fragmentation spectra were acquired for each sample, averaging 4000 laser shots per fragmentation spectrum on each of the 7-10 most abundant ions present in each sample (excluding trypsin autolytic peptides and other known background ions). Database search. Both the resulting peptide mass and the associated fragmentation spectra were submitted to GPS Explorer workstation equipped with MASCOT search engine (Matrix Science, Boston, MA) to search the Swiss-Prot database. Searches were performed without constraining protein molecular weight or isoelectric point, with variable carbamidomethylation of cysteine and oxidation of methionine residues, and with one missed cleavage also allowed in the search parameters. Candidates with either protein score C.I.% or Ion C.I.% greater than 95 were considered significant. When multiple IDs were significant for a given spot, the selection was made by evaluating apparent molecular weight, isoelectric point, the location of the spot in the gel, and the presence of strips of multiple protein isoforms in the adjacent spots.

Project description:The experiment was carried out during two vegetation seasons from 25th of April to 10th of September. Two Polish potato cultivars, the drought-tolerant Gwiazda and drought-sensitive Oberon were used. Selected tubers with transverse diameters of 3 - 4 cm were pre-sprouted for 2 weeks before planting. Plants were grown in a vegetation hall in pots filled with a thin layer of gravel on the bottom and the universal vegetable soil substrate ‘Hollas’ (Agaris Polska Ltd., Poland) produced from peat with the addition of chalk at a pH range of 5.5-6.5. Additionally, in phase 20 of the BBCH-scale of plant development, MIS-3 (Intermag) fertilizer was applied. Water content (WC) in volumetric basis in soil pots was measured according Black (1965). Weather conditions during the years of study were monitored using a Weather Campbell Station (Campbell Scientific Inc.) located in close proximity, and a thermohygrograph placed between pots. Meteorological data of air temperature, the photosynthetically active radiation, and humidity were comparable in the years of study and favorable for potato development. Three weeks after the initiation of the tuberisation phase (56 DAP), plants were divided into 3 groups, each consisting of 6 plants. The first group of plants was subjected to soil drought (remained without irrigation, day/night temperature 22°C/18°C), the second one to high temperature (day/night temperature 38°C/25°C) and the third one was watered according to needs and at optimal temperature (control plants, day/night temperature 22°C/18°C). Stress application lasted 14 days and finished at 70 DAP. During this period, plants were placed in phytotron equipped with six Hortilux Schreder Lamps with Philips light bulbs of 1600 W each. Air humidity was in the range 65-70%. WC under 14 days of soil drought reached 30% (v/v), and remained 80% (v/v) in control and high temperature conditions. During the recovery period, after 14 days of stress treatment, WC reached control levels. Plant root material for proteomic research was collected on the 14th day of stress treatment (70 DAP).

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Dataset Information

Development of the cell wall in alfalfa

Publications

The Dynamics of the Cell Wall Proteome of Developing Alfalfa Stems.

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