Biology Reference
In-Depth Information
Further analysis will shed light on how DUO1 activates its targets, and how
DUO1 itself is activated specifically in the male germline. The identification of
the role of DUO1 in germ cell specification also provides an exciting platform to
develop a detailed regulatory network for male gametogenesis and for compara-
tive studies of the control of sperm cell production. DUO1 homologs are found
throughout the land plants from the non-flowering plants Selaginella moellen-
dorffii and Physcomitrella patens (moss) through to the monocots and dicots.
Exploring the functional conservation of DUO1 in different species will reveal
if DUO1 has a conserved role in male gamete production, in terms of both of
germline mitosis and specification, where DUO1 may regulate the expression of a
similar suite of genes such as the conserved GCS1 protein. Such studies may shed
light on the evolution of regulatory mechanisms in plant germline development
and their significance in double fertilization in flowering plants.
Materials and Methods
Plant Material and Transformation
Arabidopsis plants were grown at 21°C with a 16 h-light and 8 h-dark cycle or with
24 h light, with variable humidity. Experiments were conducted in the duo1-1
(in No-0) or the No-0 backgrounds, except for those involving the inducible ec-
topic expression of mDUO1 and analysis of the DUO1 promoter that were con-
ducted in Col-0. The AtGCS1-AtGCS1::GFP, AtGEX2-GFP and CDG marker
lines are also in Col-0. Plants were transformed with Agrobacterium tumefaciens
(GV3101) using a standard floral dipping method. Transformants were selected
either on Murashige and Skoog (MS) agar containing 50 µg/ml kanamycin or
20 µg/ml hygromycin or on soil with 30 µg/ml BASTA (glufosinate ammonium,
DHAI PROCIDA) fed by sub-irrigation.
Vector Construction
Gateway single and multi-site construction (Invitrogen) was used to generate
most vectors. DNA was amplified from genomic DNA, cDNA or plasmid DNA
by PCR with high fidelity Phusion DNA polymerase (Finnzymes) and primers
with suitable attachment site (attB) adapters. Full-length attB sites were added
to each fragment in a second high fidelity PCR. For site-directed mutagenesis of
the putative GRSF binding site in the DUO1 promoter a two-step recombinant
PCR approach was taken. Two overlapping PCR fragments were generated con-
taining the mutated sequence and the two fragments joined in a stitching PCR.
PCR fragments were cloned into pDONR vectors (Invitrogen; pDONR207 for
