Mutants of Circadian-Associated PRR Genes Display a Novel and Visible Phenotype as to Light Responses during De-Etiolation of Arabidopsis thaliana Seedlings

In Arabidopsis thaliana, it is currently accepted that certain mutants with lesions in clock-associated genes commonly display hallmarked phenotypes with regard to three characteristic biological events: (i) altered rhythmic expression of circadian-controlled genes, (ii) changes in flowering time, and (iii) altered sensitivity to red light in elongation of hypocotyls. During the course of examination of the clock-associated mutants of PSEUDO-RESPONSE REGULATORS, PRRs, including TOC1 (PRR1), we found that they commonly show another visible phenotype of anomalous greening responses upon the onset to light exposure of etiolated seedlings. These findings are indicative of a novel link between circadian rhythms and chloroplast development.     

Mutants of Arabidopsis thaliana with altered responses to auxins and gravity

Auxin-resistant mutants of Arabidopsis have been induced and isolated by screening for survivors on a medium containing the herbicide 2,4-D. Thirty independently arisen mutants have been isolated in this way and one of them, P 83, has been investigated in detail. When wild type and P 83 are compared in concentration/response curves, where the response is the inhibition of root growth, the ED₅₀ values of the auxins, 2,4-D and IAA, are 14-fold higher for the mutant. The mutant also responds differently to gravity: its roots do not show positive geotropism, but tend to grow with a clockwise curvature on agar surfaces. The seedling roots of the mutant also grow more rapidly than those of the wild type in the absence of 2,4-D, following faster germination. The F₁ between P 83 and wild type is similar to the latter, but has a slightly increased resistance to 2,4-D. Results obtained from the F₂, F₃ and backcross generations suggest monofactorial inheritance. Most of the other 29 mutants have the P 83 phenotype, but at least five are different. Four have lower levels of resistance to 2,4-D and P 83, and their roots appear to respond normally to gravity. One mutant has an abnormal georesponse and a much higher level of resistance to 2,4-D than P 83.     

Characterization of circadian-associated pseudo-response regulators: II. The function of PRR5 and its molecular dissection in Arabidopsis thaliana

Together with PRR1/TOC1, PRR5 belongs to the small family of Pseudo-Response Regulators (PRRs), which function as clock components of Arabidopsis thaliana. We employed a set of transgenic lines, each of which was designed to misexpress a truncated form of the PRR5 molecule, together with the original transgenic line (named PRR5-ox) that misexpresses the entire PRR5 polypeptide. The results of genetic analysis suggested that PRR5-ox seedlings showed a phenotype of hypersensitivity to red light during early photomorphogenesis in a manner dependent on red light photoreceptors (PhyA and PhyB), but independent of PRR1/TOC1. The set of newly constructed transgenic lines (named PRR5-N-ox and PRR5-C-ox) were also characterized in terms of circadian-associated phenotypes. The results suggest that the N-terminal pseudo-receiver domain of the PRR5 molecule seems to be dispensable for the misexpressed PRR5 molecule to bring about the phenotype of red light sensitivity. However, PRR5-N-ox plants, misexpressing only the pseudo-receiver domain, showed a phenotype of long period of free-running circadian rhythms of certain clock-controlled genes. Considering these and other results, we discuss the structure and function of PRR5 in the context of current views of the circadian clock in higher plants.     

Comparative proteomics and physiological characterization of Arabidopsis thaliana seedlings in responses to Ochratoxin A

Ochratoxin A (OTA) is a mycotoxin that is primarily produced by Aspergillus ochraceus and Penicillium verrucosum. This mycotoxin is a contaminant of food and feedstock worldwide and may induce cell death in plants. To investigate the dynamic growth process of Arabidopsis seedlings in response to OTA stress and to obtain a better understanding of the mechanism of OTA toxicity towards Arabidopsis, a comparative proteomics study using 2-DE and MALDI-TOF/TOF MS/MS was performed. Mass spectrometry analysis identified 59 and 51 differentially expressed proteins in seedlings exposed to 25 and 45 μM OTA for 7 days, respectively. OTA treatment decreased root elongation and leaf area, increased anthocyanin accumulation, damaged the photosynthetic apparatus and inhibited photosynthesis. Treatment of the seedlings with 25 μM OTA enhanced energy metabolism, whereas higher concentration of OTA (45 μM) inhibited energy metabolism in the seedlings. OTA treatment caused an increase of ROS, an enhancement of antioxidant enzyme defense responses, disturbance of redox homeostasis and activation of lipid oxidation. Glutamine and S-adenosylmethionine metabolism may also play important roles in the response to OTA. In conclusion, our study provided novel insights regarding the response of Arabidopsis to OTA at the level of the proteome. These results are expected to be highly useful for understanding the physiological responses and dissecting the OTA response pathways in higher plants.     

cDNA-AFLP display for the isolation of Peronospora parasitica genes expressed during infection in Arabidopsis thaliana

To identify genes from the obligatory biotrophic oomycete Peronospora parasitica that are expressed during infection in Arabidopsis thaliana we employed cDNA-amplified fragment length polymorphism (AFLP) display. cDNA-AFLP fragments from infected and non-infected leaves were separated in parallel by gel electrophoresis and displayed by autoradiography. Most differential gene fragments were derived from P. parasitica.     

Interactions of light and ethylene in hypocotyl hook maintenance in Arabidopsis thaliana seedlings

Etiolated seedlings frequently display a hypocotyl or epicotyl hook which opens on exposure to light. Ethylene has been shown to be necessary for maintenance of the hook in a number of plants in darkness. We investigated the interaction of ethylene and light in the regulation of hypocotyl hook opening in Arabidopsis thaliana . We found that hooks of Arabidopsis open in response to continuous red, far-red or blue light in the presence of up to 100 μl l⁻¹ ethylene. Thus a change in sensitivity to ethylene is likely to be responsible for hook opening in Arabidopsis, rather than a decrease in ethylene production in hook tissues. We used photomorphogenic mutants of Arabidopsis to demonstrate the involvement of both blue light and phytochrome photosensory systems in light-induced hook opening in the presence of ethylene. In addition we used ethylene mutants and inhibitors of ethylene action to investigate the role of ethylene in hook maintenance in seedlings grown in light and darkness.     

Acclimation of Arabidopsis thaliana to the light environment: the existence of separate low light and high light responses

The capacity for photosynthetic acclimation in Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta was assessed during growth over a broad range of irradiance. Discontinuities in the response to growth irradiance were revealed for the light- and CO2-saturated rate of photosynthesis (Pmax) and the ratio of chlorophyll a to chlorophyll b (Chl a/b). Three separate phases in the response of Pmax and Chl a/b to growth light were evident, with increases at low and high irradiance ranges and a plateau at intermediate irradiance. By measuring all chlorophyll-containing components of the thylakoid membrane that contribute to Chl a/b we reveal that distinct strategies for growth at low and high irradiance underlie the discontinuous response. These strategies include, in addition to changes in the major light-harvesting complexes of photosystem II (LHCII), large shifts in the amounts of both reaction centres as well as significant changes in the levels of minor LHCII and LHCI components.     

Transgenic AEQUORIN reveals organ-specific cytosolic Ca2+ responses to anoxia and Arabidopsis thaliana seedlings.

Using the transgenic AEQUORIN system, we showed that the cotyledons and leaves of Arabidopsis thaliana seedlings developed a biphasic luminescence response to anoxia, indicating changes in cytosolic Ca2+ levels. A fast and transient luminescence peak occurred within minutes of anoxia, followed by a second, prolonged luminescence response that lasted 1.5 to 4 h. The Ca2+ channel blockers Gd3+, La3+, and ruthenium red (RR) partially inhibited the first response and promoted a larger and earlier second response, suggesting different origins for these responses. Both Gd3+ and RR also partially inhibited anaerobic induction of alcohol dehydrogenase gene expression. However, although anaerobic alcohol dehydrogenase gene induction occurred in seedlings exposed to water-agar medium and in roots, related luminescence responses were absent. Upon return to normoxia, the luminescence of cotyledons, leaves, and roots dropped quickly, before increasing again in a Gd3+, La3+, ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid-, and RR-sensitive fashion.     

Defects in leaf carbohydrate metabolism compromise acclimation to high light and lead to a high chlorophyll fluorescence phenotype in Arabidopsis thaliana

Background

We have studied the impact of carbohydrate-starvation on the acclimation response to high light using Arabidopsis thaliana double mutants strongly impaired in the day- and night path of photoassimilate export from the chloroplast. A complete knock-out mutant of the triose phosphate/phosphate translocator (TPT; tpt-2 mutant) was crossed to mutants defective in (i) starch biosynthesis (adg1-1, pgm1 and pgi1-1; knock-outs of ADP-glucose pyrophosphorylase, plastidial phosphoglucomutase and phosphoglucose isomerase) or (ii) starch mobilization (sex1-3, knock-out of glucan water dikinase) as well as in (iii) maltose export from the chloroplast (mex1-2).

Results

All double mutants were viable and indistinguishable from the wild type when grown under low light conditions, but - except for sex1-3/tpt-2 - developed a high chlorophyll fluorescence (HCF) phenotype and growth retardation when grown in high light. Immunoblots of thylakoid proteins, Blue-Native gel electrophoresis and chlorophyll fluorescence emission analyses at 77 Kelvin with the adg1-1/tpt-2 double mutant revealed that HCF was linked to a specific decrease in plastome-encoded core proteins of both photosystems (with the exception of the PSII component cytochrome b₅₅₉), whereas nuclear-encoded antennae (LHCs) accumulated normally, but were predominantly not attached to their photosystems. Uncoupled antennae are the major cause for HCF of dark-adapted plants. Feeding of sucrose or glucose to high light-grown adg1-1/tpt-2 plants rescued the HCF- and growth phenotypes. Elevated sugar levels induce the expression of the glucose-6-phosphate/phosphate translocator2 (GPT2), which in principle could compensate for the deficiency in the TPT. A triple mutant with an additional defect in GPT2 (adg1-1/tpt-2/gpt2-1) exhibited an identical rescue of the HCF- and growth phenotype in response to sugar feeding as the adg1-1/tpt-2 double mutant, indicating that this rescue is independent from the sugar-triggered induction of GPT2.

Conclusions

We propose that cytosolic carbohydrate availability modulates acclimation to high light in A. thaliana. It is conceivable that the strong relationship between the chloroplast and nucleus with respect to a co-ordinated expression of photosynthesis genes is modified in carbohydrate-starved plants. Hence carbohydrates may be considered as a novel component involved in chloroplast-to-nucleus retrograde signaling, an aspect that will be addressed in future studies.     

Proteomic pattern-based analyses of light responses in Arabidopsis thaliana wild-type and photoreceptor mutants

Light critically affects the physiology of plants. Using two-dimensional gel electrophoresis, we used a proteomics approach to analyze the responses of Arabidopsis thaliana to red (660 nm), far-red (730 nm) and blue (450 nm) light, which are utilized by type II and type I phytochromes, and blue light receptors, respectively. Under specific light treatments, the proteomic profiles of 49 protein spots exhibited over 1.8-fold difference in protein abundance, significant at p <0.05. Most of these proteins were metabolic enzymes, indicating metabolic changes induced by light of specific wavelengths. The differentially-expressed proteins formed seven clusters, reflecting co-regulation. We used the 49 differentially-regulated proteins as molecular markers for plant responses to light, and by developing a procedure that calculates the Pearson correlation distance of cluster-to-cluster similarity in expression changes, we assessed the proteome-based relatedness of light responses for wild-type and phytochrome mutant plants. Overall, this assessment was consistent with the known physiological responses of plants to light. However, we also observed a number of novel responses at the proteomic level, which were not predicted from known physiological changes.     

Phenotypic variability of Arabidopsis thaliana seedlings as a result of inhibition of Hsp90 chaperones

The influence of geldanamycin (GDA)—an inhibitor of Hsp90 chaperones—on the growth and morphogenesis of Arabidopsis thaliana seedlings was studied. It was shown that the treatment of seeds Col with the inhibitor resulted in a dose-dependent increase in the variability of seedling growth rates and phenotypes. GDA treatment of genetic polymorphic seeds of natural A. thaliana populations and UV-B irradiated seeds Col resulted in a significant rise in the amount of nongerminated seeds. The obtained data testify that Hsp90 may restrict stochastic processes, thereby participating in plant development canalization, conceal genetic variations, and maintain cell viability.     

Quantifying latitudinal clines to light responses in natural populations of Arabidopsis thaliana (Brassicaceae)

Evidence of adaptation in Arabidopsis thaliana (Brassicaceae) phenotypic traits has rarely been shown. We demonstrate latitudinal clines in two A. thaliana traits: hypocotyl responses to red and far-red light. Natural populations of A. thaliana were sampled along a latitudinal gradient from southern to northern Norway. Seeds from maternal families within each population were subjected to 1 wk of constant red, far-red, blue, white, and dark treatments. Hypocotyl lengths were measured for each maternal family within each population. Significant variability within and among populations in hypocotyl responses for the various treatments was found. There was a significant latitudinal cline in hypocotyl responses for red and far-red treatments, with northern populations being more de-etiolated than southern populations. These results suggest that northern populations are more responsive to red and far-red light than southern populations. Thus, differentiation of seedling traits in natural populations of A. thaliana seems in part to be mediated by the phytochrome pathway. There was no correlation between hypocotyl responses and flowering time for any treatment. This suggests that flowering time variability and variability in hypocotyl responses may not be governed by genes shared between the pathways, such as those involved in photoreception or the circadian clock.