My Alexander von Humboldt postdoctoral project

Role of pectin-related cell wall loosening in the regulation of endosperm weakening and embryo growth during Brassicaceae seed germination - collaboration with PD Dr. Gerhard Leubner (Germany).

 

The embryo of a typical angiosperm seed is surrounded by two covering layers: the endosperm (nutritive tissue, living cells) and the testa (seed coat; maternal tissue, dead cells). The rupture of both structures is two sequential events which occur during germination of Brassicaceae seeds. The control of radicle protrusion of that type of germinating seeds is mediated, at least in part, by endosperm weakening. Lepidium sativum (‘cress’) is a member of Brassicaceae family with big seeds, and is closely related to Arabidopsis thaliana (tiny seeds). Cress seeds provide an excellent model system which can be used in experiments for studying endosperm weakening at the molecular and tissue-specific level. For the endosperm weakening, a process cell separation is required and a pectin hydrolysation has been considered to play an important role in it. The challenge of the present study is to generate transgenic Lepidium seeds altered in candidate weakening gene expression in the endosperm. This reverse genetics approach will enable me to investigate, new molecular mechanisms of endosperm weakening in germinating seeds and reach causal conclusions. The combination of direct biomechanical measurements with this reverse genetics approach, together with the transcriptome and proteome results will provide a novel insight into the role of pectin-related cell wall loosening in the regulation of embryo growth and endosperm weakening during the germination of Brassicaceae seeds.

Myrigalone A Inhibits Lepidium sativum Seed Germination by Interference with Gibberellin Metabolism and Apoplastic Superoxide Production Required for Embryo Extension Growth and Endosperm Rupture - Oracz et al. 2012 PCP 53 (1): 81–95.

Myrica gale L. (sweet gale) fruit leachate contains myrigalone A (MyA), a rare C-methylated dihydrochalcone and putative allelochemical, which is known to be a phytotoxin impeding seedling growth. We found that MyA inhibited Lepidium sativum L. seed germination in a dose-dependent manner. MyA did not affect testa rupture, but inhibited endosperm rupture and the transition to subsequent seedling growth. MyA inhibited micropylar endosperm cap (CAP) weakening and the increase in the growth potential of the radical/hypocotyl region (RAD) of the embryo, both being key processes required for endosperm rupture.  There was compared the contents of abscisic acid (ABA) and gibberellins (GA) in the tissues and found that the major bioactive forms of GA in L. sativum seed tissues were GA4 and GA6, while GA8 and GA13 were abundant inactive metabolites (Figure 1).

 

 

Figure 1. The tissue-specific (RAD, CAP) effect of myrigalone A (MyA) on gibberellin metabolism and gibberellin-related transcript abundance during germination of Lepidium sativum seeds incubated in continuous white light.
(A) The 13-non-hydroxylated (in blue) and 13-hydroxylated (in red) gibberellin biosynthesis and inactivation pathways and important metabolites detected in L. sativum seeds. (B–D) Contents of bioactive gibberellin such as GA1, GA4 and GA6, and inactive forms such as GA24, GA9, GA34, GA8 and GA13 quantified in RADs and CAPs excised from control (CON) and MyA-treated seeds incubated for 15 h. (E) Normalized transcript abundance quantified by qRT–PCR in the RAD and CAP of CON and MyA-treated seeds incubated for 15 h for GA3 oxidase (GA3ox1, GA3ox2, GA3ox3 and GA3ox4) and GA2 oxidase (GA2ox7) genes, whose products catalyze activation and inactivation steps of bioactive gibberellin as indicated in A. (F) Normalized transcript abundance for gibberellin receptors of the GID1ac (LesaGID1a and LesaGID1c) and GID1b (LesaGID1b) groups. Mean values ± SE of four biological replicates.


MyA did not appreciably affect the ABA contents, but severely interfered with GA metabolism and signalling by inhibiting important steps catalysed by GA3 oxidase, as well as by interfering with the GID1-type GA signalling pathway. The hormonally and developmentally regulated formation of apoplastic superoxide radical is important for embryo growth. Specific zones within the RAD were associated with accumulation of apoplastic superoxide radical and endoreduplication indicative of embryo cell extension (Figure 2). MyA negatively affected both of these processes and acted as a scavenger of apoplastic reactive oxygen species. We propose that MyA is an allelochemical with a novel mode of action on seed germination.

 

 

Figure 2. Effect of MyA on the endoreduplication in distinct zones within the RAD during the germination of Lepidium sativum seeds incubated in continuous white light. (A) A uniformly NBT-histostained RAD of control embryo (left) and three distinct visible zones of apoplastic superoxide (O-2) accumulation within the RAD region of a MyA-treated embryo (right) at 22 h. (B) Effect of MyA on the (4C + 8C + 16C)/2C ratios in three distinct zones of the RAD isolated from seeds imbibed for 15 and 22 h.

 

 

Figure 3. The cover of the Special Issue – Seed Biology, 2012 PCP 53 (1)

 

Seeds are efficient plant dispersal units and to this end have evolved a number of unique survival strategies, including the ability to remain dormant without losing viability. The complex mechanisms of dormancy are probably the consequence of natural selections on mutations, or ‘trials and errors’, which seed-bearing plants have undergone during the course of evolution. The mechanisms controlling dormancy and seedling emergence via germination are regulated through changes in hormonal, metabolic, and gene expression patterns. This Special Focus Issue on Seed Biology introduces recent progress in these fields, and provides perspectives about future seed science research including its potential applications. One of the many topics in focus is seeds of parasitic weeds, which have adopted tactics to germinate on the host species when attracted by its exuded substance(s). In contrast, seeds or fruits can produce inhibitors to prevent seed germination in other species. The cover image reveals the effects of an allelochemical and putative natural herbicide, Myrigalone A (MyA), on seed germination. Upper right panel shows suppressed germination of garden cress (Lepidium sativum) seeds (light brown) by MyA leaked from sweet gale (Mirica gale) fruits (dark brown). Middle panel and scheme show nitroblue tetrazolium staining of superoxide radicals in cress embryos and active GA biosynthesis, both of which are affected by MyA (see Oracz et al. 2012 PCP 53 (1): 81–95 for details).