A single positive phototropic response induced with pulsed light in hypocotyls of Arabidopsis thaliana seedlings

The fluence-response curves were measured for phototropic curvature in response to unilateral 450-nm light in hypocotyls of Arabidopsis thaliana (L.) Heynh. These show the classical first positive (peak curvature of 9–10°), indifferent and second positive phototropic response. Reciprocity is valid only for the first positive response; the fluence requirements for its induction are similar to those for induction of the first positive phototropic response of coleoptiles. Large angles of curvature also may be induced by multiple pulses if the individual pulses are separated by an optimum dark period of about 15 min. The curvature induced by a given fluence, whether applied in continuous irradiation or a sequence of pulses, is a linear function of the duration of continuous irradiation or the duration between first and last pulse, respectively. For a given fluence applied in a sequence of pulses, reciprocity remains valid provided the duration between first and last exposure is kept constant. When the duration between first and last pulse is sufficiently long, the fluence required for high phototropic curvature falls in the first positive fluence range. These results are interpreted to indicate the existence of a kinetic limitation in the transduction sequence, and a relatively short lifetime of an initial physiologically active photoproduct. The apparent existence of more than one positive response may have resulted from these characteristics of the transduction sequence.     

Restoration of phototropic responsiveness in decapitated maize coleoptiles

The literature indicates that the tip of maize (Zea mays L.) coleoptiles has the localized functions of producing auxin for growth and perceiving unilateral light stimuli and translocating auxin laterally for phototropism. There is evidence that the auxinproducing function of the tip is restored in decapitated coleoptiles. We examined whether the functions for phototropism are also restored by using blue-light conditions that induced a first pulse-induced positive phototropism (fPIPP) and a time-dependent phototropism (TDP). When the apical 5 mm, in which photosensing predominantly takes place, was removed, no detectable fPIPP occurred even if indole-3-acetic acid (lanolin mixture) was applied to the cut end. However, when the blue-light stimulation was delayed after decapitation, fPIPP became inducible in the coleoptile stumps supplied with indole-3-acetic-acid/lanolin (0.01 mg g-1), indicating that phototropic responsiveness was restored. This restoration progressed 1 to 2 h after decapitation, and the curvature response became comparable to that of intact coleoptiles. The results for TDP were qualitatively similar, but some quantitative differences were observed. It appeared that the overall TDP was based on a major photosensing mechanism specific to the tip and on at least one additional mechanism not specific to the tip, and that the tip-specific TDP was restored in decapitated coleoptiles with kinetics similar to that for fPIPP. It is suggested that the photoreceptor system, which accounts for fPIPP and a substantial part of TDP, is regenerated in decapitated coleoptiles, perhaps together with the mechanism for lateral auxin translocation.     

Phytochrome A regulates red-light induction of phototropic enhancement in Arabidopsis.

Phytochrome A (phyA) and phytochrome B photoreceptors have distinct roles in the regulation of plant growth and development. Studies using specific photomorphogenic mutants and transgenic plants overexpressing phytochrome have supported an evolving picture in which phyA and phytochrome B are responsive to continuous far-red and red light, respectively. Photomorphogenic mutants of Arabidopsis thaliana that had been selected for their inability to respond to continuous irradiance conditions were tested for their ability to carry out red-light-induced enhancement of phototropism, which is an inductive phytochrome response. We conclude that phyA is the primary photoreceptor regulating this response and provide evidence suggesting that a common regulatory domain in the phyA polypeptide functions for both high-irradiance and inductive phytochrome responses.     

RPT2. A signal transducer of the phototropic response in Arabidopsis

The blue light receptor NPH1 (for nonphototropic hypocotyl) has been considered to be the only UV-A/blue light receptor that induces a phototropic response by the hypocotyl and root of Arabidopsis. By analysis of root phototropism (rpt) mutants, we show, however, the involvement of another blue light receptor as well as the existence of two separate signaling pathways working downstream of these receptors in the phototropic response. A newly isolated gene, RPT2, controls one of these pathways. The RPT2 gene is light inducible; encodes a novel protein with putative phosphorylation sites, a nuclear localization signal, a BTB/POZ domain, and a coiled-coil domain; and belongs to a large gene family that includes the recently isolated NPH3 gene. From genetic, physiological, and biochemical evidence, we propose a genetic model of the signaling pathways that induce the phototropic response in Arabidopsis.     

Gene targeting in Arabidopsis thaliana.

Summary Gene targeting of a chromosomally integrated transgene in Arabidopsis thaliana is reported. A chimeric gene consisting of the promoter of the 35S RNA of CaMV, the polyadenylation signal of the octopine synthase gene and the coding region of the bacterial hygromycin phosphotransferase gene (hpt), which was rendered non-functional by deletion of 19 bp, was introduced into the genome of A. thaliana using Agrobacterium-mediated gene transfer. A total of 3.46 x 10⁸ protoplasts isolated from 17 independent transgenic Arabidopsis lines harbouring the defective chimeric hpt gene were transformed via direct gene transfer using various DNA forms containing only the intact coding region of the hpt gene. Out of 150 hygromycin-resistant colonies appearing in the course of these experiments, four were the result of targeted recombination of the incoming DNA with the defective chromosomal locus as revealed by PCR and Southern blot analysis. Comparison with the number of transformants obtained when an hpt gene controlled by a promoter and terminator from the nopaline synthase gene was employed results in a maximal ratio of homologous to non-homologous transformation in A. thaliana of 1 x 10^–4.     

RPT2 is a signal transducer involved in phototropic response and stomatal opening by association with phototropin 1 in Arabidopsis thaliana

Phototropin 1 (phot1) and phot2, which are blue light receptor kinases, function in blue light-induced hypocotyl phototropism, chloroplast relocation, and stomatal opening in Arabidopsis (Arabidopsis thaliana). Previous studies have shown that the proteins RPT2 (for ROOT PHOTOTROPISM2) and NPH3 (for NONPHOTOTROPIC HYPOCOTYL3) transduce signals downstream of phototropins to induce the phototropic response. However, the involvement of RPT2 and NPH3 in stomatal opening and in chloroplast relocation mediated by phot1 and phot2 was unknown. Genetic analysis of the rpt2 mutant and of a series of double mutants indicates that RPT2 is involved in the phot1-induced phototropic response and stomatal opening but not in chloroplast relocation or phot2-induced movements. Biochemical analyses indicate that RPT2 is purified in the crude microsomal fraction, as well as phot1 and NPH3, and that RPT2 makes a complex with phot1 in vivo. On the other hand, NPH3 is not necessary for stomatal opening or chloroplast relocation. Thus, these results suggest that phot1 and phot2 choose different signal transducers to induce three responses: phototropic response of hypocotyl, stomatal opening, and chloroplast relocation.     

Photoinhibition of photosystem I at chilling temperature and subsequent recovery in Arabidopsis thaliana

Chilling-induced photoinhibition and subsequent recovery was studied in Arabidopsis thaliana exposed to 4 degrees C and 150 micromol photons m(-2) s(-1). PSII showed progressive damage with a 14% decrease in quantum yield after 8 h exposure. In contrast, the damage to PSI leveled off after 8 h with a decrease in in vitro NADP+ photoreduction activity of around 32%. In vivo P700 measurements demonstrated that antenna efficiency was decreased by the photoinhibitory treatment. Measurements of P700 and immunoblotting demonstrated that the damaged PSI was not degraded during the 8 h light-chilling treatment, but after 12 h recovery at 20 degrees C, no damaged PSI remained in the thylakoids. Thus, degradation of damaged PSI is a step in the recovery and not a direct result of photodamage. Unlike photodamaged PSII, the PSI core complex is not repaired but completely degraded. In contrast, light harvesting complex I proteins have a slow turnover. PSII recovered completely within 8 h after transfer to 20 degrees C whereas PSI activity recovered very slowly, and the amount of PSI on a leaf area basis remained low even after 1 week at 20 degrees C. The results show that damage, protein turnover and recovery are well separated processes in Arabidopsis.     

Metabolic recovery of Arabidopsis thaliana roots following cessation of oxidative stress

To cope with the various environmental stresses resulting in reactive oxygen species (ROS) production plant metabolism is known to be altered specifically under different stresses. After overcoming the stress the metabolism should be reconfigured to recover basal operation however knowledge concerning how this is achieved is cursory. To investigate the metabolic recovery of roots following oxidative stress, changes in metabolite abundance and carbon flow were analysed. Arabidopsis roots were treated by menadione to elicit oxidative stress. Roots were fed with ¹³C labelled glucose and the redistribution of isotope was determined in order to study carbon flow. The label redistribution through many pathways such as glycolysis, the tricarboxylic acid (TCA) cycle and amino acid metabolism were reduced under oxidative stress. After menadione removal many of the stress-related changes reverted back to basal levels. Decreases in amounts of hexose phosphates, malate, 2-oxoglutarate, glutamate and aspartate were fully recovered or even increased to above the control level. However, some metabolites such as pentose phosphates and citrate did not recover but maintained their levels or even increased further. The alteration in label redistribution largely correlated with that in metabolite abundance. Glycolytic carbon flow reverted to the control level only 18 h after menadione removal although the TCA cycle and some amino acids such as aspartate and glutamate took longer to recover. Taken together, plant root metabolism was demonstrated to be able to overcome menadione-induced oxidative stress with the differential time period required by independent pathways suggestive of the involvement of pathway specific regulatory processes.     

Flavonoid-specific staining of Arabidopsis thaliana.

Crop yields may be threatened by increases in UV-B radiation resulting from depletion of the ozone layer. In higher plants, the presence of flavonols provides a protective mechanism, and we report a novel staining procedure for the visualization of such protectants in plant tissue. It is shown that the proposed technique provides sensitive and specific fluorescence of flavonoids in chlorophyll-bleached tissue of Arabidopsis thaliana.     

Copper-sensitive mutant of Arabidopsis thaliana.

A Cu-sensitive mutant, cup1-1, of Arabidopsis thaliana has a pattern of heavy-metal sensitivity different from that of the cad1 and cad2 mutants, which are deficient in phytochelatin biosynthesis. The latter are significantly sensitive to Cd and Hg and only slightly sensitive to Cu, whereas the cup1-1 mutant is significantly sensitive to Cu, slightly sensitive to Cd, and not more sensitive to Hg, compared to the wild type. Genetic analysis has shown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus, which has been mapped to chromosome 1. Genetic and biochemical studies demonstrate that the cup1-1 mutant is not affected in phytochelatin biosynthesis or function. The sensitive phenotype of the cup1-1 mutant is associated with, and probably due to, increased accumulation of higher levels of Cd and Cu compared with the wild type. Consistent with this, a Cu-inducible, root-specific metallothionein gene, MT2a, is expressed in cup1-1 roots under conditions in which it is not expressed in the wild type. Undifferentiated cup1-1 callus tissue did not show the Cu-sensitive phenotype, suggesting that the mutant phenotype, in contrast to cad1 and cad2, is not expressed at the cellular level.     

Mutations of Arabidopsis in potential transduction and response components of the phototropic signaling pathway.

Four genetic loci were recently identified by mutations that affect phototropism in Arabidopsis thaliana (L.) Heyhn. seedlings. It was hypothesized that one of these loci, NPH1, encodes the apoprotein for a phototropic photoreceptor. All of the alleles at the other three mutant loci (nph2, nph3, and nph4) contained wild-type levels of the putative NPH1 protein and exhibited normal blue-light-dependent phosphorylation of the NPH1 protein. This indicated that the NPH2, NPH3, and NPH4 proteins likely function downstream of NPH1 photoactivation. We show here that, although the nph2, nph3, and nph4 mutants are all altered with respect to their phototropic responses, only the nph4 mutants are also altered in their gravitropic responsiveness. Thus, NPH2 and NPH3 appear to act as signal carriers in a phototropism-specific pathway, whereas NPH4 is required for both phototropism and gravitropism and thus may function directly in the differential growth response. Despite their altered phototropic responses in blue and green light as etiolated seedlings, the nph2 and nph4 mutants exhibited less dramatic mutant phenotypes as de-etiolated seedlings and when etiolated seedlings were irradiated with unilateral ultraviolet-A (UV-A) light. Examination of the phototropic responses of a mutant deficient in biologically active phytochromes, hy1-100, indicated that phytochrome transformation by UV-A light mediates an increase in phototropic responsiveness, accounting for the greater phototropic curvature of the nph2 and nph4 mutants to UV-A light than to blue light.     

Phosphatidylglycerophosphate synthases from Arabidopsis thaliana.

Two Arabidopsis thaliana genes were shown to encode phosphatidylglycerophosphate synthases (PGPS) of 25.4 and 32.2 kDa, respectively. Apart from their N-terminal regions, the two proteins exhibit high sequence similarity. Functional expression studies in yeast provided evidence that the 25.4 kDa protein is a microsomal PGPS while the 32.2 kDa protein represents a preprotein which can be imported into yeast mitochondria and processed to a mature PGPS. The two isozymes were solubilized and purified as fusion proteins carrying a His tag at their C-terminus. Enzyme assays with both membrane fractions and purified enzyme fractions revealed that the two A. thaliana isozymes have similar properties but differ in their CDP-diacylglycerol species specificity.     

Differential degradation of extraplastidic and plastidic lipids during freezing and post-freezing recovery in Arabidopsis thaliana

Changes in membrane lipid composition play important roles in plant adaptation to and survival after freezing. Plant response to cold and freezing involves three distinct phases: cold acclimation, freezing, and post-freezing recovery. Considerable progress has been made toward understanding lipid changes during cold acclimation and freezing, but little is known about lipid alteration during post-freezing recovery. We previously showed that phospholipase D (PLD) is involved in lipid hydrolysis and Arabidopsis thaliana freezing tolerance. This study was undertaken to determine how lipid species change during post-freezing recovery and to determine the effect of two PLDs, PLDalpha1 and PLDdelta, on lipid changes during post-freezing recovery. During post-freezing recovery, hydrolysis of plastidic lipids, monogalactosyldiacylglycerol and plastidic phosphatidylglycerol, is the most prominent change. In contrast, during freezing, hydrolysis of extraplastidic phospholipids, phosphatidylcholine and phosphatidylethanolamine, occurs. Suppression of PLDalpha1 decreased phospholipid hydrolysis and phosphatidic acid production in both the freezing and post-freezing phases, whereas ablation of PLDdelta increased lipid hydrolysis and phosphatidic acid production during post-freezing recovery. Thus, distinctly different changes in lipid hydrolysis occur in freezing and post-freezing recovery. The presence of PLDalpha1 correlates with phospholipid hydrolysis in both freezing and post-freezing phases, whereas the presence of PLDdelta correlates with reduced lipid hydrolysis during post-freezing recovery. These data suggest a negative role for PLDalpha1 and a positive role for PLDdelta in freezing tolerance.