Kenneth L. Poff

Characterization of the Auxin-Inducible SAUR-AC1 Gene for Use as a Molecular Genetic Tool in Arabidopsis

The small auxin up RNA (SAUR) genes were originally characterized in soybean, where they encode a set of unstable transcripts that are rapidly induced by auxin. In this report, the isolation of a SAUR gene, designated SAUR-AC1, from Arabidopsis thaliana (L.) Heynh. ecotype Columbia is described. The promoter of the SAUR-AC1 gene contains putative regulatory motifs conserved among soybean SAUR promoters, as well as sequences implicated in the regulation of other genes in response to auxin. The transcribed region is approximately 500 bp in length and contains no introns. Highly conserved sequences located within the SAUR-AC1 transcript include the central portion of the coding region and a putative mRNA instability sequence (DST) located in the 3[prime] untranslated region. Accumulation of SAUR-AC1 mRNA is readily induced by natural and synthetic auxins and by the translational inhibitor cycloheximide. Moreover, several auxin- and gravity-response mutants of Arabidopsis exhibit decreased accumulation of the SAUR-AC1 mRNA in elongating etiolated seedlings. In particular, in the axr2–1 mutant the SAUR-AC1 transcript accumulates to less than 5% of wild-type levels. These studies indicate that SAUR-AC1 will be a useful probe of auxin-induced gene expression in Arabidopsis and will facilitate the functional analysis of both transcriptional and posttranscriptional regulatory elements.

Mutants of Arabidopsis thaliana with altered phototropism.

Thirty five strains of Arabidopsis thaliana (L.) Heynh. have been identified with altered phototropic responses to 450-nm light. Four of these mutants have been more thoroughly characterized. Strain JK224 shows normal gravitropism and “second positive” phototropism. However, while the amplitude for “first positive” phototropism is the same as that in the wild-type, the threshold and fluence for the maximum response in “first positive” phototropism are shifted to higher fluence by a factor of 20–30. This mutant may represent an alteration in the photoreceptor pigment for phototropism. Strain JK218 exhibits no curvature to light at any fluence from 1 μmol·m-2 to 2700 μmol·m-2, but shows normal gravitropism. Strain JK345 shows no “first positive” phototropism, and reduced gravitropism and “second positive” phototropism. Strain JK229 shows no measurable “first positive” phototropism, but normal gravitropism and “second positive” phototropism. Based on these data, it is suggested that: 1. gravitropism and phototropism contain at least one common element; 2. “first positive” and “second positive” phototropism contain at least one common element; and 3. “first positive” phototropism can be substantially altered without any apparent alteration of “second positive” phototropism.

PHOTOTACTIC ORIENTATION BY THE CILIATE, STENTOR COERULEUS

Stentor coeruleus exhibits negative phototaxis to visible light, in addition to a step‐up photophobic response. The negative phototaxis was established by demonstrating the swimming of Stentor toward a focused beam away from the light source. The action spectrum showed a maximum at 610–620 nm and is essentially identical to that of the step‐up photophobic response. Proton uncouplers such as micromolar concentrations of FCCP and TPMP+ inhibited the negative phototaxis.

Multiple Phytochromes Are Involved in Red-Light-Induced Enhancement of First-Positive Phototropism in Arabidopsis thaliana

The amplitude of phototropic curvature to blue light is enhanced by a prior exposure of seedlings to red light. This enhancement is mediated by phytochrome. Fluence-response relationships have been constructed for red-light-induced enhancement in the phytochrome A (phyA) null mutant, the phytochrome B- (phyB) deficient mutant, and in two transgenic lines of Arabidopsis thaliana that overexpress either phyA or phyB. These fluence-response relationships demonstrate the existence of two responses in enhancement, a response in the very-low-to-low-fluence range, and a response in the high-fluence range. Only the response in the high-fluence range is present in the phyA null mutant. In contrast, the phyB-deficient mutant is indistinguishable from the wild-type parent in red-light responsiveness. These data indicate that phyA is necessary for the very-low-to-low but not the high-fluence response, and that phyB is not necessary for either response range. Based on these results, the high-fluence response, if controlled by a single phytochrome, must be controlled by a phytochrome other than phyA or phyB. Overexpression of phyA has a negative effect and overexpression of phyB has an enhancing effect in the high-fluence range. These results suggest that overexpression of either phytochrome perturbs the function of the endogenous photoreceptor system in an unpredictable fashion.

The ARG1-LIKE2 Gene of Arabidopsis Functions in a Gravity Signal Transduction Pathway That Is Genetically Distinct from the PGM Pathway

The arl2 mutants of Arabidopsis display altered root and hypocotyl gravitropism, whereas their inflorescence stems are fully gravitropic. Interestingly, mutant roots respond like the wild type to phytohormones and an inhibitor of polar auxin transport. Also, their cap columella cells accumulate starch similarly to wild-type cells, and mutant hypocotyls display strong phototropic responses to lateral light stimulation. The ARL2 gene encodes a DnaJ-like protein similar to ARG1, another protein previously implicated in gravity signal transduction in Arabidopsis seedlings. ARL2 is expressed at low levels in all organs of seedlings and plants. arl2-1 arg1-2 double mutant roots display kinetics of gravitropism similar to those of single mutants. However, double mutants carrying both arl2-1 and pgm-1 (a mutation in the starch-biosynthetic gene PHOSPHOGLUCOMUTASE) at the homozygous state display a more pronounced root gravitropic defect than the single mutants. On the other hand, seedlings with a null mutation in ARL1, a paralog of ARG1 and ARL2, behave similarly to the wild type in gravitropism and other related assays. Taken together, the results suggest that ARG1 and ARL2 function in the same gravity signal transduction pathway in the hypocotyl and root of Arabidopsis seedlings, distinct from the pathway involving PGM.

The effects of potassium nitrate and NO-donors on phytochrome A- and phytochrome B-specific induced germination of Arabidopsis thaliana seeds

Nitrogenous compounds, such as potassium nitrate, potentiate germination of different species of light-requiring seeds. Using light-induced Arabidopsis thaliana seed germination as a model system, our data suggested that only phytochrome A (phyA)-specific induced germination was affected after the exogenous application of nitrates, different nitric oxide (NO)-donors (such as organic nitrates) or sodium nitroprusside. The stimulative effect was very pronounced. Treated seed samples reached maximal germination after very short periods of red-light irradiation. To a far lesser extent, these substances affected phytochrome B (phyB)-specific induced germination. In phyB-specific induced germination, potassium nitrate was most effective, but germination percentages never exceeded 50%. The least effective was sodium nitroprusside, which practically did not affect phyB-specific induced germination. These results were confirmed using corresponding phytochrome mutants.

Step-up photophobic response in the ciliate, Stentor coeruleus

The avoidance by Stentor coeruleus of a light trap is caused by a step-up photophobic response. The phobic response invariably consists of a delay of about 200 ms, a stop response, a turn to one side, and resumption of swimming in the new direction. After this the cells enter a refractory period of 1–3 s following a phobic response, during which they will not give a second response. Phobic responses can be elicited by spatial and temporal increases in light intensity. The action spectrum for the step-up photophobic response resembles the absorption spectrum of stentorin, the proposed photoreceptor pigment, and of its chromophore, hypericin.The phobic response is specifically inhibited by the protonophorous uncouplers TPMP+ and FCCP but not by the ionophores gramicidin and A23187. Since the uncouplers block light-induced membrane potential changes at the same concentrations, it has been proposed that the primary photoreception causes a light-induced potential change, which in turn, induces a motor response.

LIGHT-INDUCED ACCUMULATIONS OF DICTYOSTELIUM DISCOIDEUM AMOEBAE

Abstract— Light‐induced accumulations of amoebae of Dictyostelium discoideum in ‘light traps’ have been observed. The greatest accumulations are obtained with cell densities of about 8 × 104 cells/mm2. Accumulations are observed at incident fluence rates over about one decade both for white and for monochromatic light; higher fluence rates cause dispersal from the ‘light trap’. An action spectrum for the photoaccumulation, calculated from fluence‐response curves using the zero thresholds, shows a major peak between 405 and 410 nm and extends through most of the visible spectrum. This action spectrum does not coincide with any of the pigments known to be present in D. discoideum. The cellular basis for the photoaccumulation has been studied. No light effects on cell divisions or cell aggregation are observed during the 2 h duration of an accumulation experiment. Microvideographic analysis of single amoebae is consistent with the hypothesis that the amoebae are positively phototactic and move toward the light scattered from cells in the ‘light trap’, thus accumulating in the trap.

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.

Thermal adaptation of thermosensing and negative thermotaxis in Dictyostelium

Abstract The degree of orientation of the pseudoplasmodia of Dictyostelium discoideum on different temperature gradients has been measured as a function of the temperature of growth and development. Pseudoplasmodia migrate toward the cooler side of a temperature gradient at mid-gradient temperatures >2 °C below the growth/development temperature, and toward the warmer side of the gradient at higher mid-gradient temperatures. The zero-transition between positive and negative thermotaxis occurs at 20.5 °C for pseudoplasmodia on 0.22 °C/cm gradients and at 20 °C on 0.11 °C/cm gradients when the amoebae are grown and pseudoplasmodia permitted to develop at 23.5 °C. The zero-transition is dependent on the temperature of growth and development, being 14.5, 17, 20.5, and 23.5 °C for growth/development temperatures of 16, 18, 23.5, and 27.5 °C respectively. The period of greatest thermal adaptation occurs during the last 5 h of pseudoplasmodium development (when pseudoplasmodia are formed from tight aggregates). As a resuit of the discovery of negative thermotaxis, three thermosensors are postulated for Dictyostelium —one sensor regulating thermal adaptation, and a minimum of two sensors to explain both positive and negative thermotaxis.