Ruolo dell'auxina nella risposta delle piante a condizioni spaziali simulate: radiazione neutronica e microgravità
Author(s)
Tassone, Paola
Date Issued
February 18, 2010
Type
Doctoral Thesis
Abstract
Relevant is the interest of the International scientific community to the possibility of germinating and growing plants aboard the Space Stations. Actually, space projects on the biological research are focused on studying the effect of the components which characterize the space environment: microgravity, cosmic radiations, low atmospheric pressure, high CO2 and thermal range. Aim of this work was to answer to some of the not yet clarified questions regarding the plant response to microgravity and neutron radiation. Firstly, we focused our attention on the effects of the ionizing radiations, which are not shielded and are daily absorbed by the astronauts and other organisms on the space shuttles. Even if the scientific relevance of this phenomenon is high, the literature on the space radiations effect on plant is still minimal. One of the limiting factors to do and repeat experiments in this direction is probably the small amount of available space flights. Neutrons are different form the other secondary particles for the absence of the electric charge. This property allows them to easily cross the artificial barriers and shields, causing a of different negative effects. In this work, the attention was focused on the neutron effect on the genes either activated by the phyto-hormone auxin, which regulates the plant morphogenetic processes, or involved in the activation of plant senescence and defense system. The expression profile of three ARF genes (ARF1, ARF2, ARF19), which are known to regulate the main plant morphogenetic and development processes, as well as three AUX/IAA genes (AUX/IAA3, AUX/IAA6, AUX/IAA7), involved in different cell processes, was followed after treating wild-type Arabidopsis plants with 50 mGy of neutrons. The ARF genes were inhibited, and AUX/IAA7 was up-regulated after the treatment. The same expression profile carried out for two mutants defective for the auxin transport (aux1 and eir1), showed different results. In these mutants the ARF genes were up-regulated, while the AUX/IAA7 was either induced in aux1 or inhibited in eir1. It seems that a mutation in the auxin transport may allow a different regulation of the auxin response genes. It was also observed that the senescence related genes SAG12 and SAG13 were up-regulated in both wild-type and eir1 treated plants, but were inhibited in aux1. This result suggests an involvement of auxin transport system in the senescence. Moreover, neutrons induced an increased mRNA expression level of the genes responsible for the catalase biosynthesis (CAT1 and CAT3) and iron superoxide dismutase (FeSOD1), in the wild-type plants. This is consistent with the accelerated senescence and stress
response mechanisms induced by radiation. However, the oxidative stress related genes were inhibited in both aux1 and eir1 mutants. In addition, 20 days after the treatment, both SAG12 and SAG13 genes were still highly overexpressed in the wild-type plants, when compared with controls. However, in the eir1 and arf1-3 mutants, these genes showed lower mRNA transcript level in comparison with the wild-type plants, but still higher than the measured levels in their respective controls. Interestingly, arf2-6 clearly showed a strong down-regulation of the senescence activated genes, also showing a increased juvenile developmental stage, even considering the delayed senescence of this mutant. Data collected 4 weeks after the neutron treatment, suggested an acceleration of the senescence mechanism, in the wild-type as well as in the auxin mutants eir1 and arf1-3. It is known that ARF2 is regulated by auxin and that its mutation allows a delay in the senescence activation. Our results support the hypothesis that ARF2 may help plants to better respond to the characteristic conditions of the space environment (e.g. neutron radiation, microgravity). Further investigation and manipulations on this gene may be useful to generate plants more suitable for the space flights. Another objective of this work was focused on the role of auxin in the plant response to microgravity. Plants orient the root growth direction in response to gravity. This phenomena is known as gravitropic response (or gravitropism). Statoliths, in the columella cells, as well as the endoplasmatic reticulum membranes seem to be involved in the gravitropic stimulus. Recent evidences ascribe to cytoskeleton an important role in plant detection of gravity. It was also shown that knocking down the actinic filaments polymerization allow an altered gravitropic response. Cholodny and Went independently suggested that the phyto-hormone auxin redistribution could be responsible for the differential cell growth, as well as for the phototropic response. Later, experimental evidence demonstrated the central role of auxin in the gravitropic response: a) asymmetrical application of auxin induced a curvature either in stem or in root tissue; b) auxin transport inhibitors inhibited the root apices curvature; c) labeled auxin was accumulated along the gravistimulated organs. Auxin is carried by passive diffusion or via an asymmetric polar active transport. The latter is mediated by two big families of proteins. The auxin efflux carriers are the PIN-FORMED proteins, and the auxin influx carriers are the AUXIN1/LIKE proteins. Moreover, involvement of auxin in the gravitropic response was recently confirmed by different experiments of simulated microgravity (using 3-D clinostats, or random positioning machine, RPM), on both aux1
and eir1/pin2/agr1 mutants. The mechanism by which auxin acts is regulated by transcriptional activators and repressors. The ARF proteins activate the expression of auxin response genes; and the AUX/IAA factors inhibit the transcription of auxin related genes. In plants, physiological processes are regulated by endogenous and environmental factors, and auxin seems to be the key regulator, like germination and development, flowering and senescence. Moreover, transcriptional activity of different senescence related genes is repressed by auxin. In the last years, many experiments were done to study the effect of space environment on plant development. Exposure of plants to reduced gravity conditions (microgravity), triggers physiological, cellular and biochemical alterations, with different patterns and in different organs. However, also the primary root movements seems to be altered by microgravity. One of the main aims of this work was to investigate the effect of simulated microgravity on two different mutants in auxin transport, and investigate the maintenance of balance between polymerization/de-polymerization of cellular microtubules (MT) and microfilaments. Microtubules play an central role into the cell, helping the root growth. Quantitative analysis of the mRNA transcript level of the genes responsible for the synthesis of the microtubule sub-unities, showed an up-regulation of both α- and β-tubulin (TUA4, TUA6, TUB2), and actin 8 (ACT8) 3 ours after the RPM treatment in the wild-type plants; while this transcriptional induction was delayed (6 h after the treatment) in the eir1 and aux1 mutants. This result suggested a slower response to microgravity of plants with a defective auxin transport, at least in the regulation between polymerization/de-polymerization of MT. Moreover, microgravity induced, in the wild-type plants, an increase of the β-tubulin protein level 6 h after the RPM treatment, while in the aux1 and eir1 mutants β-tubulin protein level was not significantly changed, if compared to those measured in the control plants. Our results reveals a higher state of alteration in the cytoskeleton of the aux1 and eir1 cells, supporting the hypothesis that a defective auxin transport could cause stress conditions, at cellular level, together with a delayed response to microgravity. Transcriptional analysis of the genes involved in the plant response to oxidative stress is in accordance with this idea. Microgravity induced the expression of both CAT3 and FeSOD1 genes just 3 h after the RPM treatment in the WT plants, while no differences in the transcript levels of these genes was observed in aux1 and eir1 mutants. This result is also consistent with the slower response to microgravity of the auxinic mutants, with respect to the WT plants. In addition, measurements of the
apoptotic apical root cells were performed using a fluorescence microscope before and after the RPM treatment. Treated WT plants showed a higher number of apoptotic cells, if compared to the control, but this microgravity-induced stress condition was much more evident in the aux1 and eir1 apical root cells. Our results show that the most sensitive zone to the effects of microgravity seems to be the columella and the root cap (quiescent cells zone), which is known to be highly sensitive to gravi-stimulation. Moreover, the quiescent cells zone is known to have a high auxin content, and these cells are hypothesized to be the auxin transport initial cells. The putative overlapping between the localization of gravi-perception zone and the initial site of auxin transport is consistent with the results we obtained: 1) aux1 and eir1 (auxin transport mutants) showed a delayed response to microgravity; 2) primary root cells (especially the columella cells) of both aux1 and eir1 plants showed higher microgravity-induced oxidative stress damage, if compared to the treated WT plants. In conclusion, auxin could be involved in the response to the oxidative stress condition, caused by variation of the gravity vector. An altered polar auxin transport is able to inhibit the activation of the plant defense system in response to microgravity. In this work it was shown that a damaged auxin transport is sufficient to alter reorganization of the cytoskeleton microtubules in the cells grown under microgravity. This idea also suggests a larger role of auxin in the regulation of the gravity response system. In addition, our results support the hypothesis that the columella cells are the putative site of both gravi-perception and auxin transport. This hypothesis is also supported by the isolation and characterization of a new partially agravitropic mutant of Arabidopsis, rha1, as described in the last part of this work. RHA1, the gene responsible for the mutation, seems to be involved in some process connected to the auxin transport and action and encodes an heat-shock factor (HSF). It was observed that rha1 shows an altered gravitropic response, as well as a reduced stem and primary root length, a reduced amount of inflorescences and secondary roots. All these characteristics are similar to those observed in other auxinic mutants and, in particular, in aux1 and axr4. Rha1 does not seem to be disturbed in its circumnutation, but in the slanting and gravitropism of the primary root growth pattern.
Additional information
Dottorato di ricerca in Evoluzione biologica e biochimica
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