www.seedbiology.de The combined use of Arabidopsis thaliana and Lepidium sativum to find conserved mechanisms of seed germination within the Brassicaceae family Linkies, A., Müller, K., Morris, K., Gräber, K., Tintelnot, S., Finch-Savage, W. and Leubner-Metzger, G. Freiburg University, Germany
Lepidium sativum and Arabidopsis thaliana as model plants to investigate seed germination Arabidopsis thaliana Lepidium sativum - Arabidopsis well established model plant - Lepidium sativum: bigger seeds than Arabidopsis, single tissue analysis possible - use of molecular data from Arabidopsis for research with Lepidium - similar seed structure: a thin endosperm that regulates germination timing
2-step germination of L. sativum and A. thaliana: 1. testa rupture 2. endosperm rupture Endosperm rupture is defined as the completion of germination. In between testa rupture and endosperm rupture the radicle elongates and endosperm weakening takes place. Müller et al. (2006) Plant and Cell Phys. 47: 864-877
Endosperm weakening can be measured in L. sativum. Puncture force measurements: Seeds are cut in half, the embryo is carefully removed. A metal probe is slowly lowered into the empty endosperm cap. Puncture force = force it takes to rupture endosperm.
Endosperm weakening is strictly associated with the progression of germination and is regulated by hormones. Abscisic acid (ABA) inhibits seed germination. Gibberellins (GA), cytokinins, ethylene and brassinosteroids promote germination. The ABA-GA antagonism during germination is well described, knowledge about the other hormones is still scarce. How is endosperm weakening in the Brassicaceae regulated? Linkies et al. (2009) Plant Cell 21: 3803-3822
Microarrays as a tool to analyse broad-scale gene expression - RNA extraction - cdna synthesis - labelling with fluorescent dye - hybridisation on gene chips - signal intensity is correlated with gene expression in RNA sample - cross-species microarrays can be carried out with CATMA chips
Cross-species CATMA microarrays using micropylar endosperm and radicle tissue of L. sativum at different times during germination CON 8h= just after testa rupture 18h= just prior to endosperm rupture ABA (10µM ABA) ABA delays endosperm weakening and rupture. 1. earlier physiological times 2. hormone interactions Linkies et al. (2009) Plant Cell 21: 3803-3822
TAGGIT analyses indicate the importance of ethylene for the germination process. TAGGIT workflow: annotation of datasets into functional categories associated with seed maturation, dormancy and germination. (Carrera et al. (2007), Plant Physiol. 143) The abundance of ethylenerelated genes changes during germination in a tissue- and time-related manner. Linkies et al. (2009) Plant Cell 21: 3803-3822
Ethylene promotes germination of A. thaliana and L. sativum by promoting endosperm rupture. ACC oxidases (ACOs) mediate the rate-limiting step of ethylene-biosynthesis in seed germination. 2,5-norbornadiene (NBD) inhibits ethylene action by binding to the ethylene receptors. L. sativum Ethylene partly reverts the inhibitory effects of ABA and NBD. Linkies et al. (2009) Plant Cell 21: 3803-3822
puncture force (mn) ACC promotes endosperm weakening via ethylene signal transduction. 100 75 50 25 0 Control NBD NBD+ACC - NBD (2,5-norbornadiene) is an ethylene action inhibitor - at 18h puncture force measured - NBD delays endosperm weakening - effect of NBD is partially reverted by addition of ACC. Linkies et al. (2009) Plant Cell 21: 3803-3822
ACC oxidase (ACO) activity increases during germination in a tissue- dependent manner. ACC added to individual seed tissues, ethylene evolution measured Radicle (blue): ACO activity increases during germination. Increase in ACO activity is delayed by ABA. Micropylar endosperm (red): ACO activity in endosperm tissue increases only slightly, no ABA effects.
fold increase e.r. after adding ACC ACC oxidase 2 (ACO2) shows germinationrelated expression and is important for reverting ABA-effects. 5 4 3 WT aco2 2 1 LesaACO2 expression increases in radicle and micropylar endosperm during germination. This increase is delayed by ABA in endosperm tissue. 0 5 15 50 % endosperm rupture ABA Linkies et al. (2009) Plant Cell 21: 3803-3822
Interaction between radicle and micropylar endosperm during germination during germination ethylene mainly produced by ACO2. ethylene production mainly in the radicle ethylene biosynthesis inhibited by ABA ethylene perception and signaling determine -at least partlyspeed of endosperm weakening Linkies et al. (2009) Plant Cell 21: 3803-3822
Many thanks to... www.seedbiology.de AG Leubner - Bill Finch-Savage, Karl Morris, James Lynn (Warwick University, UK) - Françoise Corbineau (Université Pierre et Marie Curie, Paris, France)