A comparative structural analysis of direct and indirect shoot ...

60 M. Volgger et al.
other candidates (as mentioned above) will have to be
re-considered.
The Hechtian reticulum may play a role in deplasmolysis
In fact, not only punctuate Hechtian strand adhesion sites
remain attached to the inner surface of the cell wall in
plasmolysed plant cells. A network that reminds of cortical
ER, the Hechtian reticulum, was observed in plasmolysed
onion inner epidermal cells after staining with DiOC 6 (Oparka
et al. 1990; Oparka 1994; Lang-Pauluzzi and Gunning 2000).
It is similar to reticulum that lined the inner walls of the root
hairs after staining for plasma membrane and ER. It was
suggested that the same membrane-spanning linkages which
attach the plasma membrane to the wall on its external face
might also anchor the ER to its internal face (Oparka 1994).
Elements of the actin cytoskeleton could be such candidates;
they are closely associated with the plasma membrane and
the cortical ER (Lichtscheidl and Url 1990; Lichtscheidl and
Hepler 1996; Overall et al. 2001; Baluška et al. 2003),
possibly providing a scaffold for the re-affixture or realignment
of the protoplast during deplasmolysis. In the root hairs
that we investigated here, deplasmolysis usually led to
disruption of the cells. Obviously, the newly synthesised
wall at the tip is too weak and misses a “rescue pattern” to
withstand that pressure.
Roots adapt to hypertonic conditions
Exposure of roots to hypertonic media stopped root hair
growth immediately. However, during prolonged treatment,
roots themselves continued to grow, and they even developed
new root hairs in mannitol solutions as high as 450 mOsm. The
adapted roots were shorter, and also the newly formed root
hairs were shorter, although the velocity of their growth and
their polar organisation were identical to the control (Fig. 6).
Their osmotic value was significantly higher; 250 mOsm
corresponds to the onset of proper plasmolysis and detachment
of the protoplast from the cell wall in T. aestivum.
Accordingly, it needed much higher osmotic values to induce
plasmolysis. Obviously, osmotically active substances were
mobilised that help to adjust to the surrounding medium.
From the curve in Fig. 7a, we conclude that such elevation of
the osmotic value of the wheat root hair is triggered by a
plasmolysing concentration of 300 mOsm and above.
A putative role of aquaporins during plasmolysis
Aquaporins may play a key role during osmotic stress
situations, and both types of aquaporins, plasma membrane
intrinsic proteins and tonoplast intrinsic proteins (TIPs),
have been shown to occur in plants (for review, see
Johansson et al. 2000 and/or Tyerman et al. 2002).
Recently, osmotic water permeability of plasma and
vacuolar membranes was elegantly measured in radish
(Raphanus sativus) protoplasts and high aquaporin activity
observed in both the plasma membrane and the tonoplast
(Murai-Hatano and Kuwagata 2007). At present, all
examined plant aquaporins have enhanced the osmotic
water permeability, although aquaporin function, dependent
on osmotic gradients, is a passive element. Thus, the rate of
aquaporin-mediated water transport also depends on other
active transporters and ion channels. However, vacuolar
membranes containing abundant TIPs are effective to
prevent plasmolysis (Hara-Nishimura and Maeshima 2000).
At the protein level, aquaporins are not the only group of
targeted candidates. Several protein kinases are also known
to be involved in both salt and osmotic stress as well as in
wounding and drought, e.g. SIMK and SAMK (Jonak et al.
1996; Meskiene and Hirt 2000; Šamaj et al. 2002; Ovečka
et al. 2008b). Furthermore, plant responses to abiotic
stresses like drought stress, osmotic stress or wounding
are closely related to signalling roles of plant hormones
ABA and jasmonic acid (Denekamp and Smeekens 2003).
Using mutant analysis, many Arabidopsis genes have been
identified that affect the process of tip growth (Parker et al.
2000). One of them is the Arabidopsis sos4 mutant, salt
overly sensitive 4, that was isolated by screening for NaClhypersensitive
growth. The SOS4 gene encodes a pyridoxal
kinase involved in the production of pyridoxal-5-phosphate.
The general phenotype of sos4 is salt hypersensitivity, but
additionally, sos4 roots failed to form root hairs, and
rhizodermis showed only a few bulges (Shi and Zhu
2002). Further analysis of the salt overly sensitive mutants
showed a dose-dependent reduction of root hair density by
salt treatments (Wang et al. 2008b). The authors found that
Na + ,K + and Li + , but neither the closely related Cs + nor
mannitol stress, caused inhibition of root hair development.
By contrast, osmotic stress caused by 200 mM mannitol
increased root hair density and promoted root hair growth
rate in stressed Arabidopsis roots. This suggests that the
inhibitory effects of salt on root hair development were
caused by ion disequilibrium and not by osmotic effects. In
the present study, we therefore deliberately focussed on
plasmolytic solutions that do not exhibit ion stress on top of
osmotic stress.
Conclusions
Cell wall deposition in tip-growing root hairs is independent
of turgor pressure because it occurs under every
osmotic condition tested. Roots can adapt to osmotic
conditions to a certain extent and can form root hairs also
in hypertonic media by elevation of the osmotic value of
the cells. In future experiments, we hope to gain more