Growth and Development of higher plants

Both P1C and P5 have in the past been involved in defining zones of root elongation leaning on either mathematically underpinned methods for whole root studies or focusing on cell elongation of single cells in a file. Combination of methods and new developments (see integrated advanced techniques) will deliver data with an unrivalled level of resolution in cell elongation activity along the root, ready to be fed into the modelling (WP6), and will serve as topological basis for the alignment of characteristics in the following aspects of cell biology. Cell wall composition will be studied making use of confocal microscopy, FESEM (see Integrated Advanced Techniques) and FT-IR (see under WP2.3.). Cytoskeleton and vacuole analysis will de done in transgenic plant lines expressing constructs of GFP-tubulin, -fimbrin (actin binding protein) and –TIP (vacuolar aquaporin), using confocal microscopy. Nuclear ploidy level will be defined by flow cytometry/sorting and in situ cytometry (link with WP1). P1, P1A, P1D and P5 have proven experience in these matters.

H+-ATPases have been proposed to be involved in cell elongation by lowering the external pH and activating proteins that loosen the wall. This is supported by the observation of a lower external pH along the elongation zone as well as the activating effect of auxin and fusicoccin on ATPase activity, external pH and cell elongation. However, these chemicals were added externally and might not reflect a physiological condition. The acid-growth theory has therefore remained very controversial. We will challenge it by internally modifying the H+-ATPase concentration and/or activity. This will be achieved in transgenic plants expressing genes corresponding to either the wild-type H+-ATPase or a constitutively activated H+-ATPase deleted of its auto-inhibitory domain. These genes will be controlled by either an estradiol-activated or cell-type specific promoters. Another question is whether H+-ATPase activation in the elongation zone depends on a change in enzyme amount or activity. We dispose of antibodies that specifically recognize the phosphorylated Thr of the activated H+-ATPase form. Aquaporins might be involved in the control of cell expansion that requires continuous uptake of water to maintain cell turgor pressure. Evidence of aquaporin involvement in this process is indicated by analysis of their expression patterns in plant organs, tissues and cells. We will investigate the role of plasma membrane aquaporin (PIPs) by analyzing cell elongation in plant silenced in PIP genes  (collaboration with P5). In addition, aquaporins activity is regulated by post-translational modifications such as phosphorylation, glycosylation, heteromerization and by cellular factors such as pH and Ca++. There is evidence that some H+-ATPase and aquaporin isoforms show an asymmetrical distribution in root epidermal cells (P4-unpublished data). We want to examine how this asymmetrical localization in the cell plasma membrane might be related to cell polarity, cell elongation and root development. Similar cycling and asymmetrical localization of AtPIN2, an auxin transporter, H+-ATPases and AtPIP2, a plasma membrane aquaporin, have been recently shown in response to auxin or gravitropism. The role of aquaporin and H+-ATPase polarisation in auxin response will be studied. H+-ATPases and aquaporins fused to different GFP variants and specific antibodies are available and could be used for this purpose (collaboration with P1 and P5).

As mentioned in the introduction, in the root postmitotic cells first go through a longer period of slow growth and then through a short period of fast elongation. This biphasic development might well be innate to elongation in Arabidopsis as a similar sequence of slow and fast cell growth is described for hypocotyl elongation in darkness. In the light of the ongoing discussion on the link between cytoskeleton and cell expansion/elongation it is intriguing that fast cell elongation is not sensitive to changes in cell wall architecture nor to the presence of functional microtubules, while the preceding period of slow elongation, on the contrary, is very sensitive to microtubule inhibitors. Root cells treated from the slow elongation stage on indeed develop aberrant cell shapes during the subsequent fast elongation phase and have aberrant patterns of differentiation later on. We hypothesize that it is precisely during this period of “preparative” slow cell growth that specific features of the cell wall, essential for subsequent successful fast elongation, are deposited. We will therefore define the role of the cytoskeleton and the cell wall in the cells prior to fast elongation on the size and shape realized during the final stages of elongation. For this purpose we will use plants expressing GFP-labelled tubulin and -cellulose synthases for in vivo observation, FESEM (Field emission scanning electron microscopy) and polarization confocal microscopy  for analysis of cell wall architecture, and FT-IR (Fourier transform infrared spectroscopy – collaboration with Dr. H. Höfte, INRA Versailles) for analysis of cell wall composition.
P5 and P1D have shown in the past that the control of fast elongation is instrumental to the plant as it allows a rapid and continuous reaction to stresses, reducing or even stopping elongation within minutes, e.g. by ethylene. This fast reduction of cell elongation is caused by the cross-linking of hydroxyproline rich glycoproteins (HRGPs) in the cell wall under the influence of reactive oxygen species (ROS), in an environment with an increased apoplastic pH due to blockage of H+-ATPases in a low activity state (De Cnodder et al., 2005). It is not known if this sequence of activities proceeds via modulation of the activity of existing proteins or if new and specific transcription needs to be activated. From transcriptome analyses (unpublished results from P5 and P1D) we know that the expression of genes coding for proteins that could be involved in cell wall modification (H+-ATPases, peroxidases, HRGPs, expansins, XTHs, PIP2 aquaporins, and cellulose synthases), is altered during the ethylene response. We will elucidate the role of these gene products in the inhibition of cell elongation via the analysis of the phenotype of overproducing and knock-out mutants and the regulation of the expression pattern of promoter::uidA constructs (P5 and P1D).

In the past P1D, P5 and EU1 have published work on hormone signalling and elongation. The greater part of hormone signalling related to root development is covered and summarized in WP3. As gravitropic bending has been included in several approaches to the study of the elongation phenomenon, its link to the more general background of hormone signal interactions is highlighted here. Unequivocal evidence for auxin being a mobile signal transported via the lateral root cap and root epidermis in the control of gravitropic response has recently been established. Ethylene also has been implicated in this process, possible modulating the rate of differential auxin accumulation. A number of lines that aberrantly express F-box proteins implicated in hormonal responses (as those for ethylene, gibberellins, or auxins) in a cell-type specific manner (epidermis, cortex, endodermis) will be created under WP3 (P1D in collaboration with EU1). The gravitropic response of these lines will be studied using time lapse imaging (P1C), and will be compared with the wild type. In addition, mutant target lines will be crossed with reporter lines specific for other hormones. Alterations in expression patterns upon gravistimulation will be investigated (collaboration P1D – EU1).