Final Report Summary - STEADYSTATE (Regulators of gene networks in Arabidopsis thaliana that confer signalling steady states of high stress adaptability)
Introduction
In this project we aimed at characterising developmentally conditioned stress tolerance and identifying genetic regulators for this process in Arabidopsis. Developmentally conditioned stress tolerance can be observed in plants exposed to biotic or abiotic stress that induce cell death responses. When exposed to these stresses, older leaves of the plants die, while younger leaves survive. This polarisation between young and old leaf stress tolerance, shows that plants have an inherent mechanism for an increased margin of stress tolerance that could be exploited for agricultural purposes. We reasoned that if the increased stress tolerance mechanisms of the younger leaves could be induced also in older leaves, this would potentially result in an increased total stress tolerance of the plant. To our aid we used advanced and novel in house developed bioinformatic algorithms that identify genetic networks of distinct physiological processes and suggest regulators for those networks.
Using bioinformatic algorithms to identify regulators for complex traits
We compared the output of two of these algorithms, ENIGMA and LeMoNe to a manual selection, looking at the regulator's expression levels. We selected genes involved in regulatory processes in Arabidopsis that are regulated by stress, differentially regulated in young and old leaves, and by plant hormones. In total we selected 58 potential regulators, 21 regulators predicted by ENIGMA, 15 regulators predicted by LeMoNe, and 22 regulators selected manually.
Screening regulators
We devised an in vitro screening assay that induces photorespiratory hydrogen peroxide that leads to cell death in old but not young leaves. We used this assay to test 14 of the ENIGMA genes, 11 LeMoNe regulators, and 12 of the manually selected regulators in the form of transgenic overexpression or knock out lines.
This selection resulted in the verification of a total of 13 stress regulators that had a differential affect on old leaf stress tolerance. We also concluded that ENIGMA was superior at predicting regulators with a function for this complex trait. Interestingly, one of the regulators, for purpose of this summary named XYX1, had a tightly regulated stress induced death of leaf one and two (the two first true leaves in Arabidopsis).
Characterisation of developmentally conditioned stress tolerance
We characterised the developmentally conditioned stress tolerance, using known hormone mutants and molecular expression markers for plants hormones and different stress responses. From this analysis we concluded that the major hormones involved in this process are salicylic acid, ethylene and jasmonic acid. We also found a significant induction of general oxidative stress markers, and surprisingly also markers of heat shock and DNA damage.
We also exposed the confirmed regulators to different stress conditions such as, cold, high light, drought, UV and freezing stress. From these studies we concluded that two of the regulators were involved in the protection to high light stress and that drought stress completely mimicked the phenotype observed in XYX1 overexpression plants.
XYX1
Because of the remarkable phenotype of XYX1 overexpression (XYX1OE) plants, we focused our efforts on a detailed characterisation of the function of this gene.
We found that the specific early induction of senescence in leaf one and two was followed by an increased survival to drought in the XYX1OE. We then proceeded with experimenting on the removal of leaf one and two and found that doing so reduced senescence in younger leaves, reduced variability in plant size induced by variable water availability, and more strikingly, completely abolished the increased survival in XYX1OE plants.
From these data we conclude that we have identified a novel mechanism for the induction of drought stress tolerance and developmentally conditioned stress tolerance. This mechanism consists of the novel function of leaf one and two to monitor water levels and induce senescence that contributes to increased drought survival. A major regulator for this mechanism is XYX1.
We then analyzed genes that were induced by XYX1 overexpression. This analysis resulted in the identification of three regulators of special interest, as well as confirming the indications that salicylic acid may be an important player in the stress induced senescence
In this project we aimed at characterising developmentally conditioned stress tolerance and identifying genetic regulators for this process in Arabidopsis. Developmentally conditioned stress tolerance can be observed in plants exposed to biotic or abiotic stress that induce cell death responses. When exposed to these stresses, older leaves of the plants die, while younger leaves survive. This polarisation between young and old leaf stress tolerance, shows that plants have an inherent mechanism for an increased margin of stress tolerance that could be exploited for agricultural purposes. We reasoned that if the increased stress tolerance mechanisms of the younger leaves could be induced also in older leaves, this would potentially result in an increased total stress tolerance of the plant. To our aid we used advanced and novel in house developed bioinformatic algorithms that identify genetic networks of distinct physiological processes and suggest regulators for those networks.
Using bioinformatic algorithms to identify regulators for complex traits
We compared the output of two of these algorithms, ENIGMA and LeMoNe to a manual selection, looking at the regulator's expression levels. We selected genes involved in regulatory processes in Arabidopsis that are regulated by stress, differentially regulated in young and old leaves, and by plant hormones. In total we selected 58 potential regulators, 21 regulators predicted by ENIGMA, 15 regulators predicted by LeMoNe, and 22 regulators selected manually.
Screening regulators
We devised an in vitro screening assay that induces photorespiratory hydrogen peroxide that leads to cell death in old but not young leaves. We used this assay to test 14 of the ENIGMA genes, 11 LeMoNe regulators, and 12 of the manually selected regulators in the form of transgenic overexpression or knock out lines.
This selection resulted in the verification of a total of 13 stress regulators that had a differential affect on old leaf stress tolerance. We also concluded that ENIGMA was superior at predicting regulators with a function for this complex trait. Interestingly, one of the regulators, for purpose of this summary named XYX1, had a tightly regulated stress induced death of leaf one and two (the two first true leaves in Arabidopsis).
Characterisation of developmentally conditioned stress tolerance
We characterised the developmentally conditioned stress tolerance, using known hormone mutants and molecular expression markers for plants hormones and different stress responses. From this analysis we concluded that the major hormones involved in this process are salicylic acid, ethylene and jasmonic acid. We also found a significant induction of general oxidative stress markers, and surprisingly also markers of heat shock and DNA damage.
We also exposed the confirmed regulators to different stress conditions such as, cold, high light, drought, UV and freezing stress. From these studies we concluded that two of the regulators were involved in the protection to high light stress and that drought stress completely mimicked the phenotype observed in XYX1 overexpression plants.
XYX1
Because of the remarkable phenotype of XYX1 overexpression (XYX1OE) plants, we focused our efforts on a detailed characterisation of the function of this gene.
We found that the specific early induction of senescence in leaf one and two was followed by an increased survival to drought in the XYX1OE. We then proceeded with experimenting on the removal of leaf one and two and found that doing so reduced senescence in younger leaves, reduced variability in plant size induced by variable water availability, and more strikingly, completely abolished the increased survival in XYX1OE plants.
From these data we conclude that we have identified a novel mechanism for the induction of drought stress tolerance and developmentally conditioned stress tolerance. This mechanism consists of the novel function of leaf one and two to monitor water levels and induce senescence that contributes to increased drought survival. A major regulator for this mechanism is XYX1.
We then analyzed genes that were induced by XYX1 overexpression. This analysis resulted in the identification of three regulators of special interest, as well as confirming the indications that salicylic acid may be an important player in the stress induced senescence