Our contribution to AtGenExpress

Details about the seed and silique samples generated by the Weisshaar group for AtGenExpress

This page has been created in 2004 to describe the biological material that has been used to extract the RNA for the seeds and siliques RNA developmental series. It is addressing only the RNA samples provided by the Weisshaar group to Detlef Weigel's lab. Labelling, hybridrisation and chip reading was done at the MPI in Tübingen.

Added in 2010 Finally, there is a paper which describes the RNA we contributed:

AtGenExpress is a multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana. The general initiative and the German contribution has been coordinated by Thomas Altmann, Lutz Nover and Detlef Weigel.

AtGenExpress links

Process

1) Stage definition

The reference for the stage definitions we used is: Bowman, J. (1994). Arabidopsis: An atlas of morphology and development; Springer; New York.

Stage No. Description
(main stage of embryogenesis)
Hours after flowering Sample harvested AtGE No.
1 up to four cells 12 - 24 - (Weigel)
2 early globular to mid globular 24 - 48 -
3 mid globular to early heart 48 - 66 siliques containing seeds ATGE_76
4 early heart to late heart 66 - 84 siliques containing seeds ATGE_77
5 late heart to mid torpedo 84 - 90 siliques containing seeds ATGE_78
6 mid torpedo to late torpedo 90 - 96 isolated seeds ATGE_79
7 late torpedo to early walking-stick 96 - 108 isolated seeds ATGE_81
8 walking-stick to early curled cotyledons 108 - 120 isolated seeds ATGE_82
9 curled cotyledons to early green cotyledons 120 - 144 isolated seeds ATGE_83
10 green cotyledons 144 - 192 isolated seeds ATGE_84

We did try to rely more on the embryo stage than on timing, but the hours given is what we observed under our greenhouse conditions. Pictures of the stages are included below (scroll down).

2) Growth conditions

Arabidopsis thaliana seeds (accession Columbia-0) were sown on soil (70% ground white peat; 20% "Vermiculite" 3-6 mm; 10% fiercely washed sand; 100 g/m3 iron chelate; 100 g/m3 "Radigen" trace elements). After sowing, seeds were kept in the dark for 3 days at 4°C for stratification. Subsequently, pots were transferred to the greenhouse. Plants were grown under short day conditions (8h light/16h dark cycles) for 3 weeks and then shifted to long day conditions (16h light/8h dark cycles) with additional illumination using SIL-lamps (Na- and Hg-steam) at 180 - 200 µE. (humidity: 50 - 60%; temperature: about 18°C; age of plants at harvest: 8 weeks after germination)

3) Harvest of siliques

Batches of 100 mg of young siliques or seeds isolated from opened siliques were harvested. Plant material was collected on dry ice and subsequently transferred into liquid nitrogen. The developmental stages of seeds were confirmed by light microscopy. Seeds originating from at least 4 different siliques per developmental stage were cleared prior to microscopy using a mixture of chloral hydrate/glycerol/water (8:2:1); incubation was carried out at room temperature for at least 2 h. Microscopy was performed on a Nikon Eclipse E600 microscope system.

4) RNA extraction

Extraction of total RNA was performed essentially according to the protocol of Ruuska and Ohlrogge: "Protocol for small-scale RNA-Isolation and transcriptional profiling of developing Arabidopsis seeds." BioTechniques 31: 752-758 (October 2001) with the following modifications: the extraction buffer contained water instead of tri-isopropylnaphtalene sulphonic acid, and the 2-butoxyethanol/isopropanol precipitation was performed only once. The nucleic acids resulting from this extraction procedure were subsequently purified using Qiagen's "RNeasy Mini spin columns".

Many THANKS to Ute Tartler, Frank Mehrtens and all the people who helped to grow the plants and harvest the material!

Stage
No.
picture of silique stage pictures of respective seed stage
0
1
2
3
4
5
6
7
8
9
10