Edgar Spalding | University of Wisconsin-Madison (original) (raw)
Papers by Edgar Spalding
TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik, Jan 13, 2017
This is the first quantitative estimation of spontaneous polyploidy in cucumber and we detected 2... more This is the first quantitative estimation of spontaneous polyploidy in cucumber and we detected 2.2% polyploids in a greenhouse study. We provide evidence that polyploidization is consistent with endoreduplication and is an on-going process during plant growth. Cucumber occasionally produces polyploid plants, which are problematic for growers because these plants produce misshaped fruits with non-viable seeds. In this study, we undertook the first quantitative study to estimate the relative frequency of spontaneous polyploids in cucumber. Seeds of recombinant inbred lines were produced in different environments, plants were grown in the field and greenhouse, and flow cytometry was used to establish ploidies. From 1422 greenhouse-grown plants, the overall relative frequency of spontaneous polyploidy was 2.2%. Plants possessed nuclei of different ploidies in the same leaves (mosaic) and on different parts of the same plant (chimeric). Our results provide evidence of endoreduplication ...
The Plant Journal, 2016
Grain yield of the maize plant depends on the sizes, shapes, and numbers of ears and the kernels ... more Grain yield of the maize plant depends on the sizes, shapes, and numbers of ears and the kernels they bear. An automated pipeline that can measure these components of yield from easilyobtained digital images is needed to advance our understanding of this globally important crop. Here we present three custom algorithms designed to compute such yield components automatically from digital images acquired by a low-cost platform. One algorithm determines the average space each kernel occupies along the cob axis using a sliding-window Fourier transform analysis of image intensity features. A second counts individual kernels removed from ears, including those in clusters. A third measures each kernel's major and minor axis after a Bayesian analysis of contour points identifies the kernel tip. Dimensionless ear and kernel shape traits that may interrelate yield components are measured by principal components analysis of contour point sets. Increased objectivity and speed compared to typical manual methods are achieved without loss of accuracy as evidenced by high correlations with ground truth measurements and simulated data. Millimeter-scale differences among ear, cob, and kernel traits that ranged more than 2.5-fold across a diverse group of inbred maize lines were resolved. This system for measuring maize ear, cob, and kernel attributes is being used by multiple research groups as an automated Web service running on community high-throughput computing and distributed data storage infrastructure. Users may create their own workflow using the source code that is staged for download on a public repository.
Plant Systems Biology, 2009
Morphological phenotypes due to mutations frequently provide key information about the biological... more Morphological phenotypes due to mutations frequently provide key information about the biological function of the affected genes. This has long been true of the plant Arabidopsis thaliana, though phenotypes are known for only a minority of this model organism's approximately 25,000 genes. One common explanation for lack of phenotype in a given mutant is that a genetic redundancy masks the effect of the missing gene. Another possibility is that a phenotype escaped detection or manifests itself only in a certain unexamined condition. Addressing this potentially nettlesome alternative requires the development of more sophisticated tools for studying morphological development. Computer vision is a technical field that holds much promise in this regard. This chapter explains in general terms how computer algorithms can extract quantitative information from images of plant structures undergoing development. Automation is a central feature of a successful computer vision application as it enables more conditions and more dependencies to be characterized. This in turn expands the concept of phenotype into a point set in multidimensional condition space. New ways of measuring and thinking about phenotypes, and therefore the functions of genes, are expected to result from expanding the role of computer vision in plant biology.
Plant Journal, 2013
The present study identified a family of six A. thaliana genes that share five limited regions of... more The present study identified a family of six A. thaliana genes that share five limited regions of sequence similarity with LAZY1, a gene in Oryza sativa (rice) shown to participate in the early gravity signaling for shoot gravitropism. A T-DNA insertion into the Arabidopsis gene (At5g14090) most similar to LAZY1 increased the inflorescence branch angle to 81° from the wild type value of 42°. RNA interference lines and molecular rescue experiments confirmed the linkage between the branch-angle phenotype and the gene consequently named AtLAZY1. Time-resolved gravitropism measurements of atlazy1 hypocotyls and primary inflorescence stems showed a significantly reduced bending rate during the first hour of response. The subcellular localization of AtLAZY1 protein was investigated to determine if the nuclear localization predicted from the gene sequence was observable and important to its function in shoot gravity responses. AtLAZY1 fused to green fluorescent protein largely rescued the branch-angle phenotype of atlazy1, and was observed by confocal microscopy at the cell periphery and within the nucleus. Mutation of the nuclear localization signal prevented detectable levels of AtLAZY1 in the nucleus without affecting the ability of the gene to rescue the atlazy1 branch-angle phenotype. These results indicate that AtLAZY1 functions in gravity signaling during shoot gravitropism, being a functional ortholog of rice LAZY1. The nuclear pool of the protein appears to be unnecessary for this function, which instead relies on a pool that appears to reside at the cell periphery.
The activation of an anion channel in the plasma membrane of Arabidopsis fhaliana hypocotyls by b... more The activation of an anion channel in the plasma membrane of Arabidopsis fhaliana hypocotyls by blue light (BL) is believed to be a signal-transducing event leading to growth inhibition. Here we report that the open probability of this particular anion channel depends on cytoplasmic Ca2+ ([CaZ'],,,) within the concentration range of 1 to 10 p~, raising the possibility that BL activates the anion channel by increasing [Ca2+],,,. Arabidopsis seedlings cytoplasmically expressing aequorin were generated to test this possibility. Aequorin luminescence did not increase during or after BL, providing evidence that Ca2+ does not play a second-messenger role in the activation of anion channels. However, cold shock simultaneously triggered a large increase in [Caz+Icyt and a 110-mV transient depolarization of the plasma membrane. A blocker of the anion channel, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, blocked 61 of the cold-induced depolarization without affecting the increase in [Ca2+Icy,. These data led us to propose that cold shock opens Ca2+ channels at the plasma membrane, allowing an inward, depolarizing CaZ+ current. The resulting large increase in [Ca2+l,,,, activates the anion channel, which further depolarizes the membrane. Although an increase in [Ca2+],,, may activate anion channels in response to cold, it appears that BL does so via a Ca2+-independent pathway.
The Quarterly Review of Biology, 1998
The Plant Journal, 2009
ABCB19 of Arabidopsis thaliana (formerly known as MDR1 and PGP19) belongs to the Multidrug Resist... more ABCB19 of Arabidopsis thaliana (formerly known as MDR1 and PGP19) belongs to the Multidrug Resistance-like (MDR) or B group of the ATP-binding cassette (ABC) transporter superfamily, and mediates polar auxin transport in stems and roots. Here we have investigated the role of ABCB19 and auxin distribution in cotyledon development. During embryogenesis, confocal microscopy showed ABCB19 protein to be present in cotyledons during their main growth phase, but not later. Analysis of ProDR5:GFP expression patterns showed a significantly diminished and restricted auxin distribution pattern in abcb19 cotyledons. Nonetheless, development of abcb19 embryonic cotyledons was very similar to that of wild-type. Post-germination, ABCB19 was present in the plasma membrane of cotyledon epidermal, mesophyll and petiole cells during blade expansion. Post-germination cotyledon blade expansion in abcb19 was 65% slower than in wild-type, although the epidermal cell area was reduced by only 17%. The growth rate reduction quantitatively correlated with reduced auxin levels rather than auxin sensitivity as indicated by quantitative ProDR5:GUS assays and direct auxin measurements, and may be explained by the 50% reduction in the import of auxin through the petioles of abcb19 cotyledons during the period of maximum expansion. Taken together, these data indicate that cotyledon expansion during the establishment of photoautotrophic growth depends on ABCB19-mediated auxin import.
The Plant Journal, 2001
Blue light (BL) rapidly and strongly inhibits hypocotyl elongation during the photomorphogenic re... more Blue light (BL) rapidly and strongly inhibits hypocotyl elongation during the photomorphogenic response known as de-etiolation, the transformation of a dark-grown seedling into a pigmented, photoautotrophic organism. In Arabidopsis thaliana, high-resolution studies of hypocotyl growth accomplished by computer-assisted electronic image capture and analysis revealed that inhibition occurs in two genetically independent phases, the ®rst beginning within 30 sec of illumination. The present work demonstrates that phototropin (nph1), the photoreceptor responsible for phototropism, is largely responsible for the initial, rapid inhibition. Signaling from phototropin during the curvature response is dependent upon interaction with NPH3, but the results presented here demonstrate that NPH3 is not necessary for phototropin-dependent growth inhibition. Activation of anion channels, which transiently depolarizes the plasma membrane within seconds of BL, is an early event in the cryptochrome signaling pathway leading to a phase of growth inhibition that replaces the transient phototropin-dependent phase after approximately 30 min of BL. Surprisingly, cry1 and cry2 were found to contribute equally and non-redundantly to anion-channel activation and to growth inhibition between 30 and 120 min of BL. Inspection of the inhibition kinetics displayed by nph1 and nph1cry1 mutants revealed that the cryptochrome phase of inhibition is delayed in seedlings lacking phototropin. This result indicates that BL-activation of phototropin in¯uences cryptochrome signaling leading to growth inhibition. Mutations in the NPQ1 gene, which inhibit BL-induced stomatal opening, do not affect any aspect of the growth inhibition within the ®rst 120 min examined here, and NPQ1 does not affect the activation of anion channels.
The Plant Journal, 2003
Blue light inhibits elongation of etiolated Arabidopsis thaliana hypocotyls during the ®rst 30 mi... more Blue light inhibits elongation of etiolated Arabidopsis thaliana hypocotyls during the ®rst 30 min of irradiation by a mechanism that depends on the phototropin 1 (phot1) photoreceptor. The cryptochrome 1 (cry1) photoreceptor begins to exert control after 30 min. To identify genes responsible for the cry1 phase of growth inhibition, mRNA expression pro®les of cry1 and wild-type seedlings were compared using DNA microarrays. Of the roughly 420 genes found to be differentially expressed at the point of cry1 response incipience, approximately half were expressed higher and half lower in cry1 relative to the wild type. Many of the cry1-dependent genes encoded kinases, transcription factors, cell cycle regulators, cell wall metabolism enzymes, gibberellic acid (GA) biosynthesis enzymes, and auxin response factors. High-resolution growth studies supported the hypothesis that genes in the last two categories were indeed relevant to cry1-mediated growth control. Inhibiting GA 4 biosynthesis with a 3b-hydroxylase inhibitor (Ca-prohexadione) restored wild-type response kinetics in cry1 and completely suppressed its long-hypocotyl phenotype in blue light. Co-treatment of cry1 seedlings with Ca-prohexadione plus GA 4 completely reversed the effects of the inhibitor, restoring the long-hypocotyl phenotype typical of the mutant. Treatment of wild-type seedlings with GA 4 was not suf®cient to phenocopy cry1 seedlings, but cotreatment with IAA plus GA 4 produced cry1-like growth kinetics for a period of approximately 5 h. The genomic and physiological data together indicate that blue light acting through cry1 quickly affects the expression of many genes, a subset of which suppresses stem growth by repressing GA and auxin levels and/or sensitivity.
The Plant Journal, 2001
The cryptochrome 1 (cry1) photoreceptor is responsible for the majority of the inhibitory effect ... more The cryptochrome 1 (cry1) photoreceptor is responsible for the majority of the inhibitory effect of blue light on hypocotyl elongation, but phytochrome photoreceptors also contribute to the response through a phenomenon known as coaction. In Arabidopsis thaliana the participation of phytochromes A and B (phyA and phyB) in the early phase of cry1 action was investigated by determining the effects of phyA, phyB and hy1 mutations on a cry1-dependent membrane depolarization, which is caused by the activation of plasma-membrane anion channels within seconds of blue light treatment. High-resolution growth measurements were also performed to determine the timing of the requirement for phytochrome in cry1-mediated growth inhibition, which is causally linked to the preceding anionchannel activation. A null mutation in PHYA impaired the membrane depolarization and prevented the early cry1-dependent phase of growth inhibition as effectively and with the same time course as mutations in CRY1. Thus, phyA is necessary for cry1/cry2 to activate anion channels within the ®rst few seconds of blue light and to suppress hypocotyl elongation for at least 120 min. This ®nding furthers the notion of an intimate mechanistic association between the cry and phy receptors in mediating light responses. The absence of phyB did not affect the depolarization or growth inhibition during this time frame. Instead, double mutant analyses showed that the phyB mutation suppressed the early growth phenotypes of both phyA and cry1 seedlings. This result is consistent with the emerging view that the prevailing growth rate of a stem is a compromise between light-dependent inhibitory and promotive in¯uences. It appears that phyB opposes the cry1/phyA-mediated inhibition by promoting growth during at least the ®rst 120 min of blue light treatment.
The Plant Journal, 2010
During seedling establishment, blue and red light suppress hypocotyl growth through the cryptochr... more During seedling establishment, blue and red light suppress hypocotyl growth through the cryptochrome 1 (cry1) and phytochrome B (phyB) photosensory pathways, respectively. How these photosensory pathways integrate with growth control mechanisms to achieve the appropriate degree of stem elongation was investigated by combining cry1 and phyB photoreceptor mutations with genetic manipulations of a multidrug resistance-like membrane protein known as ABCB19 that influenced auxin distribution within the plant, as evidenced by a combination of reporter gene assays and direct auxin measurements. Auxin signaling and ABCB19 protein levels, hypocotyl growth rates, and apical hook opening were measured in mutant and wildtype seedlings exposed to a range of red and blue light conditions. Ectopic/overexpression of ABCB19 (B19OE) greatly increased auxin in the hypocotyl, which reduced the sensitivity of hypocotyl growth specifically to blue light in long-term assays and red light in high-resolution, short-term assays. Loss of ABCB19 partially suppressed the cry1 hypocotyl growth phenotype in blue light. Hypocotyl growth of B19OE seedlings in red light was very similar to phyB mutants. Altered auxin distribution in B19OE seedlings also affected the opening of the apical hook. The cry1 and phyB photoreceptor mutations both increased ABCB19 protein levels at the plasma membrane, as measured by confocal microscopy. The B19OE plant proved to be a useful tool for determining aspects of the mechanism by which light, acting through cry1 or phyB, influences the auxin transport process to control hypocotyl growth during de-etiolation.
The Plant Journal, 2007
Measuring the effects of mutation, natural variation or treatment on the development of plant for... more Measuring the effects of mutation, natural variation or treatment on the development of plant form is often complicated by the shapes, dynamics or small size of the organismal structures under study. This limits accuracy and throughput of measurement and thereby limits progress toward understanding the underlying gene networks and signaling systems. A computer-vision platform based on electronic image capture and shape-analysis algorithms was developed as an alternative to the mostly manual methods of measuring seedling development currently in use. The spatial and temporal resolution of the method is in the range of microns and minutes, respectively. The algorithm simultaneously quantifies apical hook opening and inhibition of hypocotyl elongation during photomorphogenesis of Arabidopsis thaliana seedlings. It can determine when and where gravitropic curvature develops along the root axis in A. thaliana and Medicago truncatula seedlings. Novel features of gravitropic curvature development were discovered as a result of the high resolution. The computer-vision algorithms developed and demonstrated here could be used to study mutant phenotypes in detail, to form the basis of a high-throughput screening platform, or to quantify natural variation in a population of plants.
Planta, 1989
Blue-light (BL)-induced suppression of elongation of etiolated Cucumis sativus L. hypocotyls bega... more Blue-light (BL)-induced suppression of elongation of etiolated Cucumis sativus L. hypocotyls began after a 30-s lag time, which was halved by increasing the fluence rate from 10 to 100 lamol. m-Z-s-1. Prior to the growth suppression, the plasma-membrane of the irradiated cells depolarized by as much as 100 mV, then returned within 2-3 min to near its initial value. The potential difference measured with surface electrodes changed with an identical time course but opposite polarity. The lag time for the change in surface potential showed an inverse dependence on fluence rate, similar to the lag for the growth inhibition. Green light and red light caused neither the electrical response nor the rapid inhibition of growth. The depolarization by BL did not propagate to nonirradiated regions and exhibited a refractory period of about 10 min following a BL pulse. Fluenceresponse relationships for the electrical and growth responses provide correlational evidence that the plasma-membrane depolarization reflects an event in the transduction chain of this light-growth response.
Planta, 1992
A large, transient depolarization of the plasma membrane precedes the rapid blue-light (BL)-induc... more A large, transient depolarization of the plasma membrane precedes the rapid blue-light (BL)-induced growth suppression in etiolated seedlings of Cucumis sativus L. The mechanism of this voltage transient was investigated by applying inhibitors of ion channels and the plasma-membrane H § by manipulating extracellular ion concentrations, and by measuring cell input resistance and ATP levels. The depolarizing phase was not affected by Ca z +-channel blockers (verapamil, La 3+) or by reducing extracellular free Ca/+ by treatment with ethylene glycol-bis(/~-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). However, these treatments did reduce the rate of repolarization, indicating an inward movement of Ca / + is involved. No effects of the K +-channel blocker tetraethylammonium (TEA § were detected. Vanadate and KCN, used to inhibit the H+-ATPase, reduced or completely inhibited the BL-induced depolarization. Levels of ATP increased by 11-26% after 1-2 min of BL. Input resistance of trichome cells, measured with double-barreled microelectrodes, remained constant during the onset of the depolarization but decreased as the membrane voltage became more positive than-90 mV. The results indicate that the depolarization mechanism initially involves inactivation of the H-ATPase with subsequent transient activation of one or more types of ion channels.
Plant, Cell and Environment, 2005
... Both phot1 and phot2 receptors guide the process in a partially redundant manner (Briggs &... more ... Both phot1 and phot2 receptors guide the process in a partially redundant manner (Briggs & Christie 2002). ... 2001; Jarillo et al. 2001b). In higher plants phytochromes serve only in the transition between accumulation and avoidance (DeBlasio et al. 2003). ...
Plant, Cell and Environment, 1993
A variety of electrolytes (10-30 mol m-3) increased the relative growth rate of etiolated cucumbe... more A variety of electrolytes (10-30 mol m-3) increased the relative growth rate of etiolated cucumber (Cucumis sativus L. cv. Burpee's Pickler) hypocotyls by 20-50% relative to water-only controls. The nonelectrolyte mannitol inhibited growth by 10%. All salts tested were effective, regardless of chemical composition or valence. Measurements of cell-sap osmolality ruled out an osmotic mechanism for the growth stimulation by electrolytes. This, and the nonspecificity of the response, indicate that an electrical property of the solutions was responsible for their growth-stimulating activity. Measurements of surface electrical potential supported this reasoning. Treatment with electrolytes also enhanced nutation and altered the pattern of phototropic curvature development. A novel analytical method for quantitating these effects on growth was developed. The evidence indicates that electrolytes influence an electrophysiological parameter that is involved in the control of cell expansion and the coordination of growth underlying tropisms and nutations.
Plant, Cell & Environment, 2010
Plant growth and development is profoundly influenced by environmental conditions that laboratory... more Plant growth and development is profoundly influenced by environmental conditions that laboratory experimentation typically attempts to control. However, growth conditions are not uniform between or even within laboratories and the extent to which these differences influence plant growth and development is unknown. Experiments with wild-type Arabidopsis thaliana were designed to quantify the influences of parental environment and seed size on growth and development in the next generation. A single lot of seed was planted in six environmental chambers and grown to maturity. The seed produced was mechanically sieved into small and large size classes then grown in a common environment and subjected to a set of assays spanning the life cycle. Analysis of variance demonstrated that seed size effects were particularly significant early in development, affecting primary root growth and gravitropism, but also flowering time. Parental environment affected progeny germination time, flowering and weight of seed the progeny produced. In some cases, the parental environment affected the magnitude of (interacted with) the observed seed size effects. These data indicate that life history circumstances of the parental generation can affect growth and development throughout the life cycle of the next generation to an extent that should be considered when performing genetic studies.
Photochemistry and Photobiology, 2008
An apparatus for making high-resolution measurements of electrophysiological changes induced by l... more An apparatus for making high-resolution measurements of electrophysiological changes induced by light in plant cells was constructed. Its main components were a xenon arc lamp, an electronic shutter, a liquid light-guide, a computer equipped with an analog-to-digital converter and a computer program that controlled the shutter and data acquisition. The apparatus was used to examine transient changes in membrane potential (V,) that occur upon illumination in Arabidopsis leaves. Light-on induced a transient hyperpolarization of 4 mV after a lag time of 0.53 s. It was followed by a much larger transient depolarization that peaked 31 s after light-on. The V, returned to near its original value after approximately 3 min. The early changes in V, have been proposed to result from effects of photosynthetically produced ATP on the activities of H+-ATPases and K+ channels at the plasma membrane. The kinetics of the initial hyperpolarization were found to be reasonably consistent with such a mechanism. It is expected that the apparatus described here will be useful in future investigations of this and other electrophysiological responses to light.
TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik, Jan 13, 2017
This is the first quantitative estimation of spontaneous polyploidy in cucumber and we detected 2... more This is the first quantitative estimation of spontaneous polyploidy in cucumber and we detected 2.2% polyploids in a greenhouse study. We provide evidence that polyploidization is consistent with endoreduplication and is an on-going process during plant growth. Cucumber occasionally produces polyploid plants, which are problematic for growers because these plants produce misshaped fruits with non-viable seeds. In this study, we undertook the first quantitative study to estimate the relative frequency of spontaneous polyploids in cucumber. Seeds of recombinant inbred lines were produced in different environments, plants were grown in the field and greenhouse, and flow cytometry was used to establish ploidies. From 1422 greenhouse-grown plants, the overall relative frequency of spontaneous polyploidy was 2.2%. Plants possessed nuclei of different ploidies in the same leaves (mosaic) and on different parts of the same plant (chimeric). Our results provide evidence of endoreduplication ...
The Plant Journal, 2016
Grain yield of the maize plant depends on the sizes, shapes, and numbers of ears and the kernels ... more Grain yield of the maize plant depends on the sizes, shapes, and numbers of ears and the kernels they bear. An automated pipeline that can measure these components of yield from easilyobtained digital images is needed to advance our understanding of this globally important crop. Here we present three custom algorithms designed to compute such yield components automatically from digital images acquired by a low-cost platform. One algorithm determines the average space each kernel occupies along the cob axis using a sliding-window Fourier transform analysis of image intensity features. A second counts individual kernels removed from ears, including those in clusters. A third measures each kernel's major and minor axis after a Bayesian analysis of contour points identifies the kernel tip. Dimensionless ear and kernel shape traits that may interrelate yield components are measured by principal components analysis of contour point sets. Increased objectivity and speed compared to typical manual methods are achieved without loss of accuracy as evidenced by high correlations with ground truth measurements and simulated data. Millimeter-scale differences among ear, cob, and kernel traits that ranged more than 2.5-fold across a diverse group of inbred maize lines were resolved. This system for measuring maize ear, cob, and kernel attributes is being used by multiple research groups as an automated Web service running on community high-throughput computing and distributed data storage infrastructure. Users may create their own workflow using the source code that is staged for download on a public repository.
Plant Systems Biology, 2009
Morphological phenotypes due to mutations frequently provide key information about the biological... more Morphological phenotypes due to mutations frequently provide key information about the biological function of the affected genes. This has long been true of the plant Arabidopsis thaliana, though phenotypes are known for only a minority of this model organism's approximately 25,000 genes. One common explanation for lack of phenotype in a given mutant is that a genetic redundancy masks the effect of the missing gene. Another possibility is that a phenotype escaped detection or manifests itself only in a certain unexamined condition. Addressing this potentially nettlesome alternative requires the development of more sophisticated tools for studying morphological development. Computer vision is a technical field that holds much promise in this regard. This chapter explains in general terms how computer algorithms can extract quantitative information from images of plant structures undergoing development. Automation is a central feature of a successful computer vision application as it enables more conditions and more dependencies to be characterized. This in turn expands the concept of phenotype into a point set in multidimensional condition space. New ways of measuring and thinking about phenotypes, and therefore the functions of genes, are expected to result from expanding the role of computer vision in plant biology.
Plant Journal, 2013
The present study identified a family of six A. thaliana genes that share five limited regions of... more The present study identified a family of six A. thaliana genes that share five limited regions of sequence similarity with LAZY1, a gene in Oryza sativa (rice) shown to participate in the early gravity signaling for shoot gravitropism. A T-DNA insertion into the Arabidopsis gene (At5g14090) most similar to LAZY1 increased the inflorescence branch angle to 81° from the wild type value of 42°. RNA interference lines and molecular rescue experiments confirmed the linkage between the branch-angle phenotype and the gene consequently named AtLAZY1. Time-resolved gravitropism measurements of atlazy1 hypocotyls and primary inflorescence stems showed a significantly reduced bending rate during the first hour of response. The subcellular localization of AtLAZY1 protein was investigated to determine if the nuclear localization predicted from the gene sequence was observable and important to its function in shoot gravity responses. AtLAZY1 fused to green fluorescent protein largely rescued the branch-angle phenotype of atlazy1, and was observed by confocal microscopy at the cell periphery and within the nucleus. Mutation of the nuclear localization signal prevented detectable levels of AtLAZY1 in the nucleus without affecting the ability of the gene to rescue the atlazy1 branch-angle phenotype. These results indicate that AtLAZY1 functions in gravity signaling during shoot gravitropism, being a functional ortholog of rice LAZY1. The nuclear pool of the protein appears to be unnecessary for this function, which instead relies on a pool that appears to reside at the cell periphery.
The activation of an anion channel in the plasma membrane of Arabidopsis fhaliana hypocotyls by b... more The activation of an anion channel in the plasma membrane of Arabidopsis fhaliana hypocotyls by blue light (BL) is believed to be a signal-transducing event leading to growth inhibition. Here we report that the open probability of this particular anion channel depends on cytoplasmic Ca2+ ([CaZ'],,,) within the concentration range of 1 to 10 p~, raising the possibility that BL activates the anion channel by increasing [Ca2+],,,. Arabidopsis seedlings cytoplasmically expressing aequorin were generated to test this possibility. Aequorin luminescence did not increase during or after BL, providing evidence that Ca2+ does not play a second-messenger role in the activation of anion channels. However, cold shock simultaneously triggered a large increase in [Caz+Icyt and a 110-mV transient depolarization of the plasma membrane. A blocker of the anion channel, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, blocked 61 of the cold-induced depolarization without affecting the increase in [Ca2+Icy,. These data led us to propose that cold shock opens Ca2+ channels at the plasma membrane, allowing an inward, depolarizing CaZ+ current. The resulting large increase in [Ca2+l,,,, activates the anion channel, which further depolarizes the membrane. Although an increase in [Ca2+],,, may activate anion channels in response to cold, it appears that BL does so via a Ca2+-independent pathway.
The Quarterly Review of Biology, 1998
The Plant Journal, 2009
ABCB19 of Arabidopsis thaliana (formerly known as MDR1 and PGP19) belongs to the Multidrug Resist... more ABCB19 of Arabidopsis thaliana (formerly known as MDR1 and PGP19) belongs to the Multidrug Resistance-like (MDR) or B group of the ATP-binding cassette (ABC) transporter superfamily, and mediates polar auxin transport in stems and roots. Here we have investigated the role of ABCB19 and auxin distribution in cotyledon development. During embryogenesis, confocal microscopy showed ABCB19 protein to be present in cotyledons during their main growth phase, but not later. Analysis of ProDR5:GFP expression patterns showed a significantly diminished and restricted auxin distribution pattern in abcb19 cotyledons. Nonetheless, development of abcb19 embryonic cotyledons was very similar to that of wild-type. Post-germination, ABCB19 was present in the plasma membrane of cotyledon epidermal, mesophyll and petiole cells during blade expansion. Post-germination cotyledon blade expansion in abcb19 was 65% slower than in wild-type, although the epidermal cell area was reduced by only 17%. The growth rate reduction quantitatively correlated with reduced auxin levels rather than auxin sensitivity as indicated by quantitative ProDR5:GUS assays and direct auxin measurements, and may be explained by the 50% reduction in the import of auxin through the petioles of abcb19 cotyledons during the period of maximum expansion. Taken together, these data indicate that cotyledon expansion during the establishment of photoautotrophic growth depends on ABCB19-mediated auxin import.
The Plant Journal, 2001
Blue light (BL) rapidly and strongly inhibits hypocotyl elongation during the photomorphogenic re... more Blue light (BL) rapidly and strongly inhibits hypocotyl elongation during the photomorphogenic response known as de-etiolation, the transformation of a dark-grown seedling into a pigmented, photoautotrophic organism. In Arabidopsis thaliana, high-resolution studies of hypocotyl growth accomplished by computer-assisted electronic image capture and analysis revealed that inhibition occurs in two genetically independent phases, the ®rst beginning within 30 sec of illumination. The present work demonstrates that phototropin (nph1), the photoreceptor responsible for phototropism, is largely responsible for the initial, rapid inhibition. Signaling from phototropin during the curvature response is dependent upon interaction with NPH3, but the results presented here demonstrate that NPH3 is not necessary for phototropin-dependent growth inhibition. Activation of anion channels, which transiently depolarizes the plasma membrane within seconds of BL, is an early event in the cryptochrome signaling pathway leading to a phase of growth inhibition that replaces the transient phototropin-dependent phase after approximately 30 min of BL. Surprisingly, cry1 and cry2 were found to contribute equally and non-redundantly to anion-channel activation and to growth inhibition between 30 and 120 min of BL. Inspection of the inhibition kinetics displayed by nph1 and nph1cry1 mutants revealed that the cryptochrome phase of inhibition is delayed in seedlings lacking phototropin. This result indicates that BL-activation of phototropin in¯uences cryptochrome signaling leading to growth inhibition. Mutations in the NPQ1 gene, which inhibit BL-induced stomatal opening, do not affect any aspect of the growth inhibition within the ®rst 120 min examined here, and NPQ1 does not affect the activation of anion channels.
The Plant Journal, 2003
Blue light inhibits elongation of etiolated Arabidopsis thaliana hypocotyls during the ®rst 30 mi... more Blue light inhibits elongation of etiolated Arabidopsis thaliana hypocotyls during the ®rst 30 min of irradiation by a mechanism that depends on the phototropin 1 (phot1) photoreceptor. The cryptochrome 1 (cry1) photoreceptor begins to exert control after 30 min. To identify genes responsible for the cry1 phase of growth inhibition, mRNA expression pro®les of cry1 and wild-type seedlings were compared using DNA microarrays. Of the roughly 420 genes found to be differentially expressed at the point of cry1 response incipience, approximately half were expressed higher and half lower in cry1 relative to the wild type. Many of the cry1-dependent genes encoded kinases, transcription factors, cell cycle regulators, cell wall metabolism enzymes, gibberellic acid (GA) biosynthesis enzymes, and auxin response factors. High-resolution growth studies supported the hypothesis that genes in the last two categories were indeed relevant to cry1-mediated growth control. Inhibiting GA 4 biosynthesis with a 3b-hydroxylase inhibitor (Ca-prohexadione) restored wild-type response kinetics in cry1 and completely suppressed its long-hypocotyl phenotype in blue light. Co-treatment of cry1 seedlings with Ca-prohexadione plus GA 4 completely reversed the effects of the inhibitor, restoring the long-hypocotyl phenotype typical of the mutant. Treatment of wild-type seedlings with GA 4 was not suf®cient to phenocopy cry1 seedlings, but cotreatment with IAA plus GA 4 produced cry1-like growth kinetics for a period of approximately 5 h. The genomic and physiological data together indicate that blue light acting through cry1 quickly affects the expression of many genes, a subset of which suppresses stem growth by repressing GA and auxin levels and/or sensitivity.
The Plant Journal, 2001
The cryptochrome 1 (cry1) photoreceptor is responsible for the majority of the inhibitory effect ... more The cryptochrome 1 (cry1) photoreceptor is responsible for the majority of the inhibitory effect of blue light on hypocotyl elongation, but phytochrome photoreceptors also contribute to the response through a phenomenon known as coaction. In Arabidopsis thaliana the participation of phytochromes A and B (phyA and phyB) in the early phase of cry1 action was investigated by determining the effects of phyA, phyB and hy1 mutations on a cry1-dependent membrane depolarization, which is caused by the activation of plasma-membrane anion channels within seconds of blue light treatment. High-resolution growth measurements were also performed to determine the timing of the requirement for phytochrome in cry1-mediated growth inhibition, which is causally linked to the preceding anionchannel activation. A null mutation in PHYA impaired the membrane depolarization and prevented the early cry1-dependent phase of growth inhibition as effectively and with the same time course as mutations in CRY1. Thus, phyA is necessary for cry1/cry2 to activate anion channels within the ®rst few seconds of blue light and to suppress hypocotyl elongation for at least 120 min. This ®nding furthers the notion of an intimate mechanistic association between the cry and phy receptors in mediating light responses. The absence of phyB did not affect the depolarization or growth inhibition during this time frame. Instead, double mutant analyses showed that the phyB mutation suppressed the early growth phenotypes of both phyA and cry1 seedlings. This result is consistent with the emerging view that the prevailing growth rate of a stem is a compromise between light-dependent inhibitory and promotive in¯uences. It appears that phyB opposes the cry1/phyA-mediated inhibition by promoting growth during at least the ®rst 120 min of blue light treatment.
The Plant Journal, 2010
During seedling establishment, blue and red light suppress hypocotyl growth through the cryptochr... more During seedling establishment, blue and red light suppress hypocotyl growth through the cryptochrome 1 (cry1) and phytochrome B (phyB) photosensory pathways, respectively. How these photosensory pathways integrate with growth control mechanisms to achieve the appropriate degree of stem elongation was investigated by combining cry1 and phyB photoreceptor mutations with genetic manipulations of a multidrug resistance-like membrane protein known as ABCB19 that influenced auxin distribution within the plant, as evidenced by a combination of reporter gene assays and direct auxin measurements. Auxin signaling and ABCB19 protein levels, hypocotyl growth rates, and apical hook opening were measured in mutant and wildtype seedlings exposed to a range of red and blue light conditions. Ectopic/overexpression of ABCB19 (B19OE) greatly increased auxin in the hypocotyl, which reduced the sensitivity of hypocotyl growth specifically to blue light in long-term assays and red light in high-resolution, short-term assays. Loss of ABCB19 partially suppressed the cry1 hypocotyl growth phenotype in blue light. Hypocotyl growth of B19OE seedlings in red light was very similar to phyB mutants. Altered auxin distribution in B19OE seedlings also affected the opening of the apical hook. The cry1 and phyB photoreceptor mutations both increased ABCB19 protein levels at the plasma membrane, as measured by confocal microscopy. The B19OE plant proved to be a useful tool for determining aspects of the mechanism by which light, acting through cry1 or phyB, influences the auxin transport process to control hypocotyl growth during de-etiolation.
The Plant Journal, 2007
Measuring the effects of mutation, natural variation or treatment on the development of plant for... more Measuring the effects of mutation, natural variation or treatment on the development of plant form is often complicated by the shapes, dynamics or small size of the organismal structures under study. This limits accuracy and throughput of measurement and thereby limits progress toward understanding the underlying gene networks and signaling systems. A computer-vision platform based on electronic image capture and shape-analysis algorithms was developed as an alternative to the mostly manual methods of measuring seedling development currently in use. The spatial and temporal resolution of the method is in the range of microns and minutes, respectively. The algorithm simultaneously quantifies apical hook opening and inhibition of hypocotyl elongation during photomorphogenesis of Arabidopsis thaliana seedlings. It can determine when and where gravitropic curvature develops along the root axis in A. thaliana and Medicago truncatula seedlings. Novel features of gravitropic curvature development were discovered as a result of the high resolution. The computer-vision algorithms developed and demonstrated here could be used to study mutant phenotypes in detail, to form the basis of a high-throughput screening platform, or to quantify natural variation in a population of plants.
Planta, 1989
Blue-light (BL)-induced suppression of elongation of etiolated Cucumis sativus L. hypocotyls bega... more Blue-light (BL)-induced suppression of elongation of etiolated Cucumis sativus L. hypocotyls began after a 30-s lag time, which was halved by increasing the fluence rate from 10 to 100 lamol. m-Z-s-1. Prior to the growth suppression, the plasma-membrane of the irradiated cells depolarized by as much as 100 mV, then returned within 2-3 min to near its initial value. The potential difference measured with surface electrodes changed with an identical time course but opposite polarity. The lag time for the change in surface potential showed an inverse dependence on fluence rate, similar to the lag for the growth inhibition. Green light and red light caused neither the electrical response nor the rapid inhibition of growth. The depolarization by BL did not propagate to nonirradiated regions and exhibited a refractory period of about 10 min following a BL pulse. Fluenceresponse relationships for the electrical and growth responses provide correlational evidence that the plasma-membrane depolarization reflects an event in the transduction chain of this light-growth response.
Planta, 1992
A large, transient depolarization of the plasma membrane precedes the rapid blue-light (BL)-induc... more A large, transient depolarization of the plasma membrane precedes the rapid blue-light (BL)-induced growth suppression in etiolated seedlings of Cucumis sativus L. The mechanism of this voltage transient was investigated by applying inhibitors of ion channels and the plasma-membrane H § by manipulating extracellular ion concentrations, and by measuring cell input resistance and ATP levels. The depolarizing phase was not affected by Ca z +-channel blockers (verapamil, La 3+) or by reducing extracellular free Ca/+ by treatment with ethylene glycol-bis(/~-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). However, these treatments did reduce the rate of repolarization, indicating an inward movement of Ca / + is involved. No effects of the K +-channel blocker tetraethylammonium (TEA § were detected. Vanadate and KCN, used to inhibit the H+-ATPase, reduced or completely inhibited the BL-induced depolarization. Levels of ATP increased by 11-26% after 1-2 min of BL. Input resistance of trichome cells, measured with double-barreled microelectrodes, remained constant during the onset of the depolarization but decreased as the membrane voltage became more positive than-90 mV. The results indicate that the depolarization mechanism initially involves inactivation of the H-ATPase with subsequent transient activation of one or more types of ion channels.
Plant, Cell and Environment, 2005
... Both phot1 and phot2 receptors guide the process in a partially redundant manner (Briggs &... more ... Both phot1 and phot2 receptors guide the process in a partially redundant manner (Briggs & Christie 2002). ... 2001; Jarillo et al. 2001b). In higher plants phytochromes serve only in the transition between accumulation and avoidance (DeBlasio et al. 2003). ...
Plant, Cell and Environment, 1993
A variety of electrolytes (10-30 mol m-3) increased the relative growth rate of etiolated cucumbe... more A variety of electrolytes (10-30 mol m-3) increased the relative growth rate of etiolated cucumber (Cucumis sativus L. cv. Burpee's Pickler) hypocotyls by 20-50% relative to water-only controls. The nonelectrolyte mannitol inhibited growth by 10%. All salts tested were effective, regardless of chemical composition or valence. Measurements of cell-sap osmolality ruled out an osmotic mechanism for the growth stimulation by electrolytes. This, and the nonspecificity of the response, indicate that an electrical property of the solutions was responsible for their growth-stimulating activity. Measurements of surface electrical potential supported this reasoning. Treatment with electrolytes also enhanced nutation and altered the pattern of phototropic curvature development. A novel analytical method for quantitating these effects on growth was developed. The evidence indicates that electrolytes influence an electrophysiological parameter that is involved in the control of cell expansion and the coordination of growth underlying tropisms and nutations.
Plant, Cell & Environment, 2010
Plant growth and development is profoundly influenced by environmental conditions that laboratory... more Plant growth and development is profoundly influenced by environmental conditions that laboratory experimentation typically attempts to control. However, growth conditions are not uniform between or even within laboratories and the extent to which these differences influence plant growth and development is unknown. Experiments with wild-type Arabidopsis thaliana were designed to quantify the influences of parental environment and seed size on growth and development in the next generation. A single lot of seed was planted in six environmental chambers and grown to maturity. The seed produced was mechanically sieved into small and large size classes then grown in a common environment and subjected to a set of assays spanning the life cycle. Analysis of variance demonstrated that seed size effects were particularly significant early in development, affecting primary root growth and gravitropism, but also flowering time. Parental environment affected progeny germination time, flowering and weight of seed the progeny produced. In some cases, the parental environment affected the magnitude of (interacted with) the observed seed size effects. These data indicate that life history circumstances of the parental generation can affect growth and development throughout the life cycle of the next generation to an extent that should be considered when performing genetic studies.
Photochemistry and Photobiology, 2008
An apparatus for making high-resolution measurements of electrophysiological changes induced by l... more An apparatus for making high-resolution measurements of electrophysiological changes induced by light in plant cells was constructed. Its main components were a xenon arc lamp, an electronic shutter, a liquid light-guide, a computer equipped with an analog-to-digital converter and a computer program that controlled the shutter and data acquisition. The apparatus was used to examine transient changes in membrane potential (V,) that occur upon illumination in Arabidopsis leaves. Light-on induced a transient hyperpolarization of 4 mV after a lag time of 0.53 s. It was followed by a much larger transient depolarization that peaked 31 s after light-on. The V, returned to near its original value after approximately 3 min. The early changes in V, have been proposed to result from effects of photosynthetically produced ATP on the activities of H+-ATPases and K+ channels at the plasma membrane. The kinetics of the initial hyperpolarization were found to be reasonably consistent with such a mechanism. It is expected that the apparatus described here will be useful in future investigations of this and other electrophysiological responses to light.