Technische Universität München
PLANT EMBRYO DEVELOPMENT
Molecular genetic analysis of plant root development.!
WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
The root: structure and RAM.
QC
The root meristem (RAM) organises as a group of initial cells around the quiescent center (QC).
WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
Endodermis
The endodermis is a tissue with a special water repellent suberin layer. The forced passage of water and other solubles through the cell offers a possibility to control this passage.
Starch granules
The position of starch granules (statoliths) in the columella is though to impact the auxin flow in the root tip, which in turn promotes or inhibits the growth of root cells. WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
The root auxin maximum is early elaborated and stronger than the cotyledon maxima.
In contrast to the apical embryo region, in the basal region the auxin flux is organised by several PINs.
PIN1 PIN7 PIN3 suspensor cell
hypophysis, RAM precursor! heart stage
Treml et al., Development, 2005
RAM! torpedo stage
The root auxin maximum (AM) is stronger than the cotyledon AMs.
Weijers et al., Dev Cell 2006! Long & Benfey, Curr Op Cell Biol 2006 !
WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
MONOPTEROS is an auxin responsive transcription factor (ARF). BODENLOS is a so called IAA/AUX protein and works as a repressor of MP in the nucleus. Both organise the correct generation of the root apical meristem (RAM). !
hypophysis derivatives
WT! WT!
mp !
mp !
The disturbed division of the hypophysis is one of the first visible defects of mp and bdl mutants.!
bdl !
2xt-bdl9 ! transgenic!
WT! The phenotype of recessive mp and dominant bdl mutants is similar.!
Berleth and Jürgens, Development, 1994; Mayer et al., 1997; Hardtke und Berleth, EMBO, 1998
MP and BDL both localise to the nucleus.(particle bombardment
with MP, transient expression in onion epidermis)!
WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
BDL mRNA
!
MP mRNA!
BDL and MP expression domains overlap in the embryo (in situ hybridisation analysis).!
The Arabidopsis thaliana genome codes for about two dozen ARF and IAA/AUX proteins. The significance of the majority of possible ARF_IAA/AUX combinations is not known!
Yeast-Two-Hybrid interaction assay.! 1. BDL with C terminus of MP! 2. GNOM with C terminus of MP (control)! 3. BDL with itself (control)! 4. BDL with empty prey vector!
BDL interacts with MP (=ARF5)!
Hamann et al., Genes & Dev, 2002 WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
The (classical) Yeast-Two_Hybrid System finds physical interactions between proteins.
WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
The TIR-subunit of the SCF protein degradation/ubiquitination machinery has been identified as an auxin receptor!! The binding of auxin to TIR activates SCF, which specifically targets IAA/AUX proteins.! ! Dharmasiri et al., Nature 2005! Kepinski et al., Nature 2005!
WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
Model of of ARF_IAA/AUX (inter-)action and ARF liberation; example: MP and BDL.!
BDL!
MP!
AUXIN!
activates
TIR/! -SCF!
ubiquitinates
The F-box protein! TIR is an auxin-receptor!! Dharmasiri et al., Nature 2005! Kepinski et al., Nature 2005!
X! UB Dominant IAA-protein mutantions in IAA12(BDL), IAA3 and IAA7 exhibit a point mutation in the conserved domain II required for protein degradation via the ubiquitinproteasome pathway.!
MP! released!
X!
Trans-! criptional! activation!
targets
and degrades
Proteasome!
=> root mutant!
Hamann et al., Genes & Dev, 2002 WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
Origin of the RAM
WT
TMO7::GFP The RAM consists of ordered cell layers!
mp
TMO7::GFP
Feedback-loop regulation for RAM specification MP & BDL interact in the pro-embryo but not in the hypophysis -> MP positively regulates PIN1 expression in the young embryo -> PIN1 is the major player in accumulating auxin (auxin maximum, AM) in the upper suspensor cell, which becomes the hypophysis cell -> the AM and the mobile TF TARGET OF MONOPTEROS7 (TMO7), which is also controlled by MP, are the signals, which trigger hypophysis specification (indicated by an asymmetric division producing a lens shaped cell). Weijers et al., Dev Cell 2006; Long & Benfey, Curr Op Cell Biol 2006; Schlereth et al., Nature, 2010 ! WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
The non-cell autonomous effect of MP on hypophysis formation results from the activation of TMO7, a mobile bHLH transcription factor.
TPL in situ TPL:GFP
Suppression of shoot fate in the topless mutant. TOPLESS resembles known transcriptional corepressors, which prevents root formation ! in the shoot pole.!
Suppression of root fate by ectopic overexpression of HDZIPIIIs.
WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
The development of the embryonic root involves further factors: a feedback regulation of PIN and PLETHORA genes !
1347 & 2347 = pin1pin3pin4pin7 ! & pin2pin3pin4pin7 !
Blilou et al., Nature 2005 !
In early embryo development ! PIN genes restrict ! PLETHORA (PLT) ! mRNA expression ! and root identity ! to the basal embryo pole. ! ! PLTs are putative ! AP2 transcription factors.!
The comparison of PLT and STM, WUS expression in PIN mutant background shows that only the PLTs are impacted by PINs!! WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
Where do the different root cells and tissues originate from?! Fate mapping of the root apex identify quiescent center (QC) cells and stem cells (initial cells) for the root tissues.! late sector
early sector
Heterologous Ac-! transposon in ! 35S:GUS!
Principle of fate mapping:
Collect sectors (!) (generated by different means),
then consider the approx. stage when the sector was initiated
and
then determine the _position & _number of precursor cells. Scheres et al., Development, 1994 WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
Fate mapping of the root apex (II) !
Scheres et al., Development, 1994 WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
AUXIN is required for the growth of the post-embryonic root.! DR5:GUS construct with synthetic auxin responsive cis-element
(Ulmasov et al., Plant Cell, 1997). PIN1: auxin efflux- , AUX1: auxin influxcarrier
Sabatini et al., Cell, 1999
The PID-molecular switch of PIN1 polarity works also (ectopically) in the adult root. When PID is ectopically overexpressed in the root by the 35S-promoter the root degenerates because the basal PIN1 orientation in the stele is reversed. This reverses the auxin flux, which in turn impedes the formation of the root auxin maximum.!
Benjamins et al., Development, 2001
35S:PID
Degenerated root tip in 35S::PID overexpressor.!
WT WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
Seedling roots incubated for prolonged time with increased concentrations of the auxin transport inhibitor NPA develop “cylindrical roots“ in response to new positional information.!
How the plant interpretes an unusual auxin signal.!
Starch !
Sabatini et al., Cell, 1999
New emerging root hairs orient towards a new and abnormal root tip. NPA induced root mirror-image duplications. WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
The postembryonic root also requires an elaborated set of PIN auxin-transport proteins in order to establish a complex auxin-flux circuit. In an advanced developmental stage, the cell specific expression of PINs is dependent on PLT genes and vice versa.!
Blilou et al., Nature, 2005 !
Model of auxin flux circiut in the root. WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
wt
wt!
!
plt1 plt2!
plt1 plt2!
Later in development, the cell specific expression of PINs is dependent on PLT genes, i. e. PLT1,2 expression! is a prerequisite ! for correct PIN localisation (and ! auxin distribution) in the root. Together PIN and PLT activities establish a feed back loop regulation!!
(Xu et al., Science 2006)!
Blilou et al., Nature 2005 !
PIN4 (and other PINs) are down-regulated in plt-mutants in seedlings and adult plants!! WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
A development & growth model for the Arabidopsis thaliana root.
Factors downstream of PINs, auxin maxima, PLTs and SCR such as WOX5 demonstrate an complex machinery for root development and growth.
SCR
SHR
WOX5
WOX5 is a QC localised transcription factor required for maintenance of root growth. WOX genes are TFs with homology to WUSCHEL. There are WOX genes specific for the apex of the early embryo as well as those specific for the root tip especially WOX5. In WOX5 mutants the root degenerates. Perilli et al., 2012, Current Opinion in Plant biology WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
Root development in maize -! an elaborated system with ! different root types.!
25% of the fine branch roots are shown
primary root (embryonic)! seminal root (embryonic)! lateral root on primary and ! ! seminal roots.! crown roots are adventitious, stem derived roots, ! !up to the 3rd/4th node (called „brace“) !
! WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
rcts: lacks crown roots – ltr1: lacks lateral roots and first (coleoptilar) crown root - the double mutant is additive and lacks both.!
!
rcts
ltr1
!
The elaborated maize root system required the “invention“ of new controlling factors. slr mutants lack lateral roots
drawings by: Miwa Kojima, Schnable lab, ISU
Hochholdinger and Feix, Plant J, 1998 WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
The root tip in maize harbours an auxin maximum.! BrdU control root kept in water!
anti-IAA AB + 2nd AB+AP
Summary (autoradiography 3H Thymidine)!
anti-AAO AB BdrU + 0.1 M ascorbic acid.!
anti-IAA AB + auxin excess!
The primary substrate of
anti- eFα (shows protein synthesis)
ascorbic acid oxidase (AAO) is ascorbic acid (AA). AA is necessary for metabolic pathways, which promote the cell division cycle (CC), in particular G1 -> to S. !
Immunofluorescence shows nuclei of actively dividing cells that had incorporated BrdU into DNA during replication.
Auxin elevates AAO activity. Kerk and Feldman, Development, 1993 WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
Plant cells differentiate with respect to position rather than to cell lineage – insights from laser ablation experiments.!
Seedlings are placed in a chamber made of small coverslips sealed on a larger one, containing propidium iodide. The plants can thus be examined using an inverted microscope and ablated.
van den Berg et al., Nature, 1995, 1997 WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
The Arabidopsis root: laser ablation experiments uncover long range and short range fate determining signals.!
root longitudinal view
Long range signals originating from differentiated cells instruct undifferentiat ed cells to adopt their fate!!
van den Berg et al., Nature, 1995, 1997 WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
Laser ablation of quiescent center- (QC)cells leads to differentiation of neighboured initial cells.!
WT
laser ablati on of cell
Columella initials differentiate upon QC ablation -> differentiated columella cell withstarch granules!
van den Berg et al., Nature, 1995, 1997 WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
Technische Universität München
long range signalling
short range signalling
Cell fate specification is determined by short & long range ! signals originating from differentiated cells.! ! The QC-cells exert an effect promoting undifferentiated ! initial cell and/or inhibiting differentiation.! WZW Lehrstuhl für Genetik Lehrstuhl WZW für Genetik
