Mapping the Functional Roles of Cap Cells in the Response of Arabidopsis Primary Roots to Gravity1

The cap is widely accepted to be the site of gravity sensing in roots because removal of the cap abolishes root curvature. Circumstantial evidence favors the columella cells as the gravisensory cells because amyloplasts (and often other cellular components) are polarized with respect to the gravity vector. However, there has been no functional confirmation of their role. To address this problem, we used laser ablation to remove defined cells in the cap of Arabidopsis primary roots and quantified the response of the roots to gravity using three parameters: time course of curvature, presentation time, and deviation from vertical growth. Ablation of the peripheral cap cells and tip cells did not alter root curvature. Ablation of the innermost columella cells caused the strongest inhibitory effect on root curvature without affecting growth rates. Many of these roots deviated significantly from vertical growth and had a presentation time 6-fold longer than the controls. Among the two inner columella stories, the central cells of story 2 contributed the most to root gravitropism. These cells also exhibited the largest amyloplast sedimentation velocities. Therefore, these results are consistent with the starch-statolith sedimentation hypothesis for gravity sensing.

Two Isoforms of NADPH:Cytochrome P450 Reductase in Arabidopsis thaliana : Gene Structure, Heterologous Expression in Insect Cells, and Differential Regulation

We have investigated two NADPH-cytochrome (Cyt) P450 reductase isoforms encoded by separate genes (AR1 and AR2) in Arabidopsis thaliana. We isolated AR1 and AR2 cDNAs using a mung bean (Phaseolus aureus L.) NADPH-Cyt P450 reductase cDNA as a probe. The recombinant AR1 and AR2 proteins produced using a baculovirus expression system showed similar Km values for Cyt c and NADPH, respectively. In the reconstitution system with a recombinant cinnamate 4-hydroxylase (CYP73A5), the recombinant AR1 and AR2 proteins gave the same level of cinnamate 4-hydroxylase activity (about 70 nmol min−1 nmol−1 P450). The AR2 gene expression was transiently induced by 4- and 3-fold within 1 h of wounding and light treatments, respectively, and the induction time course preceded those of CYP73A5 and a phenylalanine ammonia-lyase (PAL1) gene. On the contrary, the AR1 expression level did not change during the treatments. Analysis of the AR1 and AR2 gene structure revealed that only the AR2 promoter contained three putative sequence motifs (boxes P, A, and L), which are involved in the coordinated expression of CYP73A5 and other phenylpropanoid pathway genes. These results suggest the possibility that AR2 transcription may be functionally linked to the induced levels of phenylpropanoid pathway enzymes.

Aluminum Induces Oxidative Stress Genes in Arabidopsis thaliana1

Changes in gene expression induced by toxic levels of Al were characterized to investigate the nature of Al stress. A cDNA library was constructed from Arabidopsis thaliana seedlings treated with Al for 2 h. We identified five cDNA clones that showed a transient induction of their mRNA levels, four cDNA clones that showed a longer induction period, and two down-regulated genes. Expression of the four long-term-induced genes remained at elevated levels for at least 48 h. The genes encoded peroxidase, glutathione-S-transferase, blue copper-binding protein, and a protein homologous to the reticuline:oxygen oxidoreductase enzyme. Three of these genes are known to be induced by oxidative stresses and the fourth is induced by pathogen treatment. Another oxidative stress gene, superoxide dismutase, and a gene for Bowman-Birk protease inhibitor were also induced by Al in A. thaliana. These results suggested that Al treatment of Arabidopsis induces oxidative stress. In confirmation of this hypothesis, three of four genes induced by Al stress in A. thaliana were also shown to be induced by ozone. Our results demonstrate that oxidative stress is an important component of the plant's reaction to toxic levels of Al.

RIGS (repeat-induced gene silencing) in Arabidopsis is transcriptional and alters chromatin configuration.

We have previously reported repeat-induced gene silencing (RIGS) in Arabidopsis, in which transgene expression may be silenced epigenetically when repeated sequences are present. Among an allelic series of lines comprising a primary transformant and various recombinant progeny carrying different numbers of drug resistance gene copies at the same locus, silencing was found to depend strictly on repeated sequences and to correlate with an absence of steady-state mRNA. We now report characterization, in nuclei isolated from the same transgenic lines, of gene expression by nuclear run-on assay and of chromatin structure by nuclease protection assay. We find that silencing is correlated with absence of run-on transcripts, indicating that expression is silenced at the level of transcription. We find further that silencing is also correlated with increased resistance to both DNase I and micrococcal nuclease, indicating that the silenced state reflects a change in chromatin configuration. We propose that silencing results when a locally paired region of homologous repeated nucleotide sequences is flanked by unpaired heterologous DNA, which leads chromatin to adopt a local configuration that is difficult to transcribe, and possibly akin to heterochromatin.

Annexin-like protein from Arabidopsis thaliana rescues delta oxyR mutant of Escherichia coli from H2O2 stress.

Reactive oxygen species are common causes of cellular damages in all aerobic organisms. In Escherichia coli, the oxyR gene product is a positive regulator of the oxyR regulon that is induced in response to H2O2 stress. To identify genes involved in counteracting oxidative stress in plants, we transformed a delta oxyR mutant of E. coli with an Arabidopsis thaliana cDNA library and selected for clones that restored the ability of the delta oxyR mutant to grow in the presence of H2O2. Using this approach, we isolated a cDNA that has strong homology with the annexin super-gene family. The complemented mutant showed higher catalase activity. mRNA expression of the annexin gene in A. thaliana was higher in roots as compared with other organs and was also increased when the plants were exposed to H2O2 stress or salicylic acid. Based on the results presented in this study, we propose a novel physiological role for annexin in counteracting H2O2 stress.

Phosphate transporters from the higher plant Arabidopsis thaliana.

Two cDNAs (AtPT1 and AtPT2) encoding plant phosphate transporters have been isolated from a library prepared with mRNA extracted from phosphate-starved Arabidopsis thaliana roots, The encoded polypeptides are 78% identical to each other and show high degree of amino acid sequence similarity with high-affinity phosphate transporters of Saccharomyces cerevisiae, Neurospora crassa, and the mycorrhizal fungus Glomus versiforme. The AtPT1 and AtPT2 polypeptides are integral membrane proteins predicted to contain 12 membrane-spanning domains separated into two groups of six by a large charged hydrophilic region. Upon expression, both AtPT1 and AtPT2 were able to complement the pho84 mutant phenotype of yeast strain NS219 lacking the high-affinity phosphate transport activity. AtPT1 and AtPT2 are representatives of two distinct, small gene families in A. thaliana. The transcripts of both genes are expressed in roots and are not detectable in leaves. The steady-state level of their mRNAs increases in response to phosphate starvation.

Environmental stress sensitivity of an ascorbic acid-deficient Arabidopsis mutant.

L-ascorbic acid (vitamin C) is a powerful reducing agent found in millimolar concentrations in plants, and is proposed to play an important role in scavenging free radicals in plants and animals. However, surprisingly little is known about the role of this antioxidant in plant environmental stress adaptation or ascorbate biosynthesis. We report the isolation of soz1, a semi-dominant ozone-sensitive mutant that accumulates only 30% of the normal ascorbate concentration. The results of genetic approaches and feeding studies show that the ascorbate concentration affects foliar resistance to the oxidizing gas ozone. Consistent with the proposed role for ascorbate in reactive oxygen species detoxification, lipid peroxides are elevated in soz1, but not in wild type following ozone fumigation. We show that the soz1 mutant is hypersensitive to both sulfur dioxide and ultraviolet B irradiation, thus implicating ascorbate in defense against varied environmental stresses. In addition to defining the first ascorbate deficient mutant in plants, these results indicate that screening for ozone-sensitive mutants is a powerful method for identifying physiologically important antioxidant mechanisms and signal transduction pathways. Analysis of soz1 should lead to more information about the physiological roles and metabolism of ascorbate.

SPINDLY, a tetratricopeptide repeat protein involved in gibberellin signal transduction in Arabidopsis.

Gibberellins (GAs) are a major class of plant hormones that control many developmental processes, including seed development and germination, flower and fruit development, and flowering time. Genetic studies with Arabidopsis thaliana have identified two genes involved in GA perception or signal transduction. A semidominant mutation at the GIBBERELLIN INSENSITIVE (GAI) locus results in plants resembling GA-deficient mutants but exhibiting reduced sensitivity to GA. Recessive mutations at the SPINDLY (SPY) locus cause a phenotype that is consistent with constitutive activation of GA signal transduction. Here we show that a strong allele of spy is completely epistatic to gai, indicating that SPY acts downstream of GAI. We have cloned the SPY gene and shown that it encodes a new type of signal transduction protein, which contains a tetratricopeptide repeat region, likely serving as a protein interaction domain, and a novel C-terminal region. Mutations in both domains increase GA signal transduction. The presence of a similar gene in Caenorhabditis elegans suggests that SPY represents a class of signal transduction proteins that is present throughout the eukaryotes.

Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development.

Arabidopsis plants transformed with an antisense construct of an Arabidopsis methyltransferase cDNA (METI) have reduced cytosine methylation in CG dinucleotides. Methylation levels in progeny of five independent transformants ranged from 10% to 100% of the wild type. Removal of the antisense construct by segregation in sexual crosses did not fully restore methylation patterns in the progeny, indicating that methylation patterns are subject to meiotic inheritance in Arabidopsis. Plants with decreased methylation displayed a number of phenotypic and developmental abnormalities, including reduced apical dominance, smaller plant size, altered leaf size and shape, decreased fertility, and altered flowering time. Floral organs showed homeotic transformations that were associated with ectopic expression of the floral homeotic genes AGAMOUS and APETALA3 in leaf tissue. These observations suggest that DNA methylation plays an important role in regulating many developmental pathways in plants and that the developmental abnormalities seen in the methyltransferase antisense plants may be due to dysregulation of gene expression.

CAX1, an H+/Ca2+ antiporter from Arabidopsis.

Reestablishment of the resting state after stimulus-coupled elevations of cytosolic-free Ca2+ requires the rapid removal of Ca2+ from the cytosol of plant cells. Here we describe the isolation of two genes, CAX1 and CAX2, from Arabidopsis thaliana that suppress a mutant of Saccharomyces cerevisiae that has a defect in vacuolar Ca2+ accumulation. Both genes encode polypeptides showing sequence similarities to microbial H+/Ca2+ antiporters. Experiments on vacuolar membrane-enriched vesicles isolated from yeast expressing CAX1 or CAX2 demonstrate that these genes encode high efficiency and low efficiency H+/Ca2+ exchangers, respectively. The properties of the CAX1 gene product indicate that it is the high capacity transporter responsible for maintaining low cytosolic-free Ca2+ concentrations in plant cells by catalyzing pH gradient-energized vacuolar Ca2+ accumulation.

Action spectra for phytochrome A- and B-specific photoinduction of seed germination in Arabidopsis thaliana.

We have examined the seed germination in Arabidopsis thaliana of wild type (wt), and phytochrome A (PhyA)- and B (PhyB)-mutants in terms of incubation time and environmental light effects. Seed germination of the wt and PhyA-null mutant (phyA) was photoreversibly regulated by red and far-red lights of 10-1,000 micromol m-2 when incubated in darkness for 1-14 hr, but no germination occurred in PhyB-null mutant (phyB). When wt seeds and the phyB mutant seeds were incubated in darkness for 48 hr, they synthesized PhyA during dark incubation and germinated upon exposure to red light of 1-100 nmol m-2 and far-red light of 0.5-10 micromol m-2, whereas the phyA mutant showed no such response. The results indicate that the seed germination is regulated by PhyA and PhyB but not by other phytochromes, and the effects of PhyA and PhyB are separable in this assay. We determined action spectra separately for PhyA- and PhyB-specific induction of seed germination at Okazaki large spectrograph. Action spectra for the PhyA response show that monochromatic 300-780 nm lights of very low fluence induced the germination, and this induction was not photoreversible in the range examined. Action spectra for the PhyB response show that germination was photoreversibly regulated by alternate irradiations with light of 0.01-1 mmol m-2 at wavelengths of 540-690 nm and 695-780 nm. The present work clearly demonstrated that PhyA photoirreversibly triggers the germination upon irradiations with ultraviolet, visible and far-red light of very low fluence, while PhyB controls the photoreversible effects of low fluence.

An anion channel in Arabidopsis hypocotyls activated by blue light.

A rapid, transient depolarization of the plasma membrane in seedling stems is one of the earliest effects of blue light detected in plants. It appears to play a role in transducing blue light into inhibition of hypocotyl (stem) elongation, and perhaps other responses. The possibility that activation of a Cl- conductance is part of the depolarization mechanism was raised previously and addressed here. By patch clamping hypocotyl cells isolated from dark-grown (etiolated) Arabidopsis seedlings, blue light was found to activate an anion channel residing at the plasma membrane. An anion-channel blocker commonly known as NPPB 15-nitro-2-(3-phenylpropylamino)-benzoic acid] potently and reversibly blocked this anion channel. NPPB also blocked the blue-light-induced depolarization in vivo and decreased the inhibitory effect of blue light on hypocotyl elongation. These results indicate that activation of this anion channel plays a role in transducing blue light into growth inhibition.

Identification of transferred DNA insertions within Arabidopsis genes involved in signal transduction and ion transport.

The transferred DNA (T-DNA) of Agrobacterium tumefaciens serves as an insertional mutagen once integrated into a host plant's genome. As a means of facilitating reverse genetic analysis in Arabidopsis thaliana, we have developed a method that allows one to search for plants carrying F-DNA insertions within any sequenced Arabidopsis gene. Using PCR, we screened a collection of 9100 independent T-DNA-transformed Arabidopsis lines and found 17 T-DNA insertions within the 63 genes analyzed. The genes surveyed include members of various gene families involved in signal transduction and ion transport. As an example, data are shown for a T-DNA insertion that was found within CPK-9, a member of the gene family encoding calmodulin-domain protein kinases.