Growth and Development of higher plants

Until recently, hormone action was much viewed as an organ-specific process. Cell type-specific action, elegantly demonstrated for auxins adds a new dimension to the concept of individual and collective hormone action on plant development. The observation that auxins specifically act at the level of the epidermis, and in this way affect root elongation sheds new light on the mechanism of auxin action, particularly on the cell type specificity thereof. Whether this is also true for other hormones is not yet known. Furthermore, 26S proteasome-mediated protein degradation appears to play a central role in different hormone pathways, by perception of the hormone at the F-box component of an E3 ligase, or by destabilization of key factors in the hormone signalling pathway. Hence one can speculate (1) that cell type specificity plays a more general role in hormone action mechanisms, implying that elongation growth requires communication between different cell layers that differentially respond to internal and external signals, and (2) that cell type specificity of F-box factors may play an important role. The specific objectives of WP3 are to unravel cell type-specific effects during elongation growth and to shed light on the signal interactions that drive this process; besides the characterization of a limited number of effector proteins.

As a proof of concept, EU1 and P1C recently demonstrated that auxin regulates differential root growth by inhibiting epidermal cell elongation. This unprecedented level of cellular resolution was achieved by expressing a dominant form of the auxin repressor IAA17 termed axr3-1 in selected root tissues. We will employ the same approach to identify which root tissue(s) perceive other phytohormone signals (s) that control root growth.
Dominant mutant signalling components (gai, etr1-1, abi1-1, a dominant form of BRI1 (EU1) or F-box components, or other regulators of several of these pathways (P1D)) will be expressed in selected root tissues (epidermis/cortex/endodermis/stele) in order to alter their sensitivity towards hormone signals including GA, ethylene, ABA, and brassinosteroids (collaboration P1D and EU1).
In collaboration with P1C, EU1 and P1D will characterize the effect that desensitizing/modulation of sensitivity of selected tissues to each of these hormone signals has on root growth processes inside and outside this targeted cell type/region employing kinematic analysis. The effect on aerial development, shoot growth and flowering will be investigated in collaboration with P2. Together with the analysis of root characteristics, this will allow us to assess possible alterations in root/shoot interactions resulting from cell-type specific alterations in hormone sensitivity. Blocking a root tissue’s responsiveness to one hormone may also disrupt the response to another hormone signal. For example, analysis on lines expressing axr3-1 in elongating root epidermal and cortex cells has observed that roots also became less sensitive to ethylene. Hence, all lines generated during the first phase will be screened for altered sensitivity towards a collection of hormones. EU1 will combine this tissue level information with gene networks developed in house and by other partners to construct an integrated model for hormone regulated root growth in WP6.
To further add on our understanding of hormone interactions, this set of experiments will be complemented by crossing mutant target lines with reporter lines specific for other hormones. All the lines will be analyzed for effects on normal root/shoot development (P1D – EU1) and, whenever relevant, root/shoot relation upon floral induction (P2 in collaboration with P1D – EU1). In addition, a limited set of stress experiments (eg. gravitation/dehydration/light stress) can be conducted, in order to evaluate altered behaviour of the mutant lines as compared to wild type plants. In these experiments, a line with cell type-specific expression of a dominant form of GAI will be included (collaboration P1D – EU1).

Hormonal interactions will also be analyzed at the level of downstream effectors. From recent transcriptome analyses we know that the expression of about 5% of the genes is significantly affected upon response to ethylene. A number of these genes encode proteins potentially involved in cell wall modification. We will focus on well-characterized proteins that play a defined role in the process of cell expansion (H+ ATPases, peroxidases, HRGPs, expansins, aquaporins, XTH’s, glucanases and or cellulose synthases; a selection thereof will be made) (P5, P4 and P1D). The role of specific gene products will first be investigated by analysis of overexpression and/or knock-out lines. For those targets for which the results justify further analysis, misexpression will be done under cell type specific promoters and the behaviour of these lines will be analyzed under normal conditions of growth, as well as under influence of hormone treatments (collaboration P1D - EU1). The latter could be indicative for a yet unidentified point of hormonal interaction. Under WP1, the effects of selected compounds from the chemical library will be investigated on the above-mentioned mutants, and compared to the wild type (P1D in collaboration with P1A).

Within the previous IAP network, P1D was focusing on the ethylene/GA cross-talk. The analysis of double mutants defective in GA and ethylene synthesis or signal transduction, initiated during the previous IAP collaboration, will be finalized. The eto2gai combination will be investigated with specific attention to root development. Differences in root length, number of lateral roots, and root growth rates will be recorded (P1D in collaboration with P1C). The effect of the double deficiency on alterations in the root/shoot ratio will be considered as well.

In summary, a more profound understanding of the interaction of hormone signalling processes both at the level of signalling components and that of effector proteins can be expected. The analysis of cell type specificity of these proteins will add an additional level of insight in the puzzle of signalling cross-talk.