Nathan D. Miller

Metallicities of Old Open Clusters

We present radial velocities and metallicities for a sample of 39 open clusters with ages greater than about 700 million years. For 24 clusters new moderate-resolution spectroscopic data obtained with multiobject spectrographs on the Kitt Peak National Observatory and the Cerro Tololo Inter-American Observatory 4 m telescopes are used to determine radial velocities and mean cluster metallicities. These new results are combined with data published previously by Friel & Janes to provide a sample of 459 giants in 39 old open clusters, which are used to investigate radial abundance gradients in the Galactic disk. Based on an updated abundance calibration of spectroscopic indices measuring Fe and Fe-peak element blends, this larger sample yields an abundance gradient of -0.06 ± 0.01 dex kpc-1 over a range in Galactocentric radius of 7 to 16 kpc. There is a slight suggestion of a steepening of the abundance gradient with increasing cluster age in this sample, but the significance of the result is limited by the restricted distance range for the youngest clusters. The clusters show no correlation of metallicity with age in the solar neighborhood. Consistent with the evidence for a steepening of the gradient with age, the clusters in the outer disk beyond 10 kpc show a suggestion at the 1.5 σ level of a dependence of metallicity on age.

Separating the Roles of Acropetal and Basipetal Auxin Transport on Gravitropism with Mutations in Two Arabidopsis Multidrug Resistance-Like ABC Transporter Genes

Two Arabidopsis thaliana ABC transporter genes linked to auxin transport by various previous results were studied in a reverse-genetic fashion. Mutations in Multidrug Resistance-Like1 (MDR1) reduced acropetal auxin transport in roots by 80% without affecting basipetal transport. Conversely, mutations in MDR4 blocked 50% of basipetal transport without affecting acropetal transport. Developmental and auxin distribution phenotypes associated with these altered auxin flows were studied with a high-resolution morphometric system and confocal microscopy, respectively. Vertically grown mdr1 roots produced positive and negative curvatures threefold greater than the wild type, possibly due to abnormal auxin distribution observed in the elongation zone. However, upon 90° reorientation, mdr1 gravitropism was inseparable from the wild type. Thus, acropetal auxin transport maintains straight growth but contributes surprisingly little to gravitropism. Conversely, vertically maintained mdr4 roots grew as straight as the wild type, but their gravitropism was enhanced. Upon reorientation, curvature in this mutant developed faster, was distributed more basally, and produced a greater total angle than the wild type. An amplified auxin asymmetry may explain the mdr4 hypertropism. Double mutant analysis indicated that the two auxin transport streams are more independent than interdependent. The hypothesis that flavanols regulate MDR-dependent auxin transport was supported by the epistatic relationship of mdr4 to the tt4 phenylpropanoid pathway mutation.

Auxin and ethylene induce flavonol accumulation through distinct transcriptional networks

Auxin and ethylene are key regulators of plant growth and development, and thus the transcriptional networks that mediate responses to these hormones have been the subject of intense research. This study dissected the hormonal cross talk regulating the synthesis of flavonols and examined their impact on root growth and development. We analyzed the effects of auxin and an ethylene precursor on roots of wild-type and hormone-insensitive Arabidopsis (Arabidopsis thaliana) mutants at the transcript, protein, and metabolite levels at high spatial and temporal resolution. Indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) differentially increased flavonol pathway transcripts and flavonol accumulation, altering the relative abundance of quercetin and kaempferol. The IAA, but not ACC, response is lost in the transport inhibitor response1 (tir1) auxin receptor mutant, while ACC responses, but not IAA responses, are lost in ethylene insensitive2 (ein2) and ethylene resistant1 (etr1) ethylene signaling mutants. A kinetic analysis identified increases in transcripts encoding the transcriptional regulators MYB12, Transparent Testa Glabra1, and Production of Anthocyanin Pigment after hormone treatments, which preceded increases in transcripts encoding flavonoid biosynthetic enzymes. In addition, myb12 mutants were insensitive to the effects of auxin and ethylene on flavonol metabolism. The equivalent phenotypes for transparent testa4 (tt4), which makes no flavonols, and tt7, which makes kaempferol but not quercetin, showed that quercetin derivatives are the inhibitors of basipetal root auxin transport, gravitropism, and elongation growth. Collectively, these experiments demonstrate that auxin and ethylene regulate flavonol biosynthesis through distinct signaling networks involving TIR1 and EIN2/ETR1, respectively, both of which converge on MYB12. This study also provides new evidence that quercetin is the flavonol that modulates basipetal auxin transport.

The Receptor-like Kinase FERONIA Is Required for Mechanical Signal Transduction in Arabidopsis Seedlings

Among the myriad cues that constantly inform plant growth and development, mechanical forces are unique in that they are an intrinsic result of cellular turgor pressure and also imposed by the environment. Although the key role of mechanical forces in shaping plant architecture from the cellular level to the level of organ formation is well established, the components of the early mechanical signal transduction machinery remain to be defined at the molecular level. Here, we show that an Arabidopsis mutant lacking the receptor-like kinase FERONIA (FER) shows severely altered Ca(2+) signaling and growth responses to different forms of mechanical perturbation. Ca(2+) signals are either abolished or exhibit qualitatively different signatures in feronia (fer) mutants exposed to local touch or bending stimulation. Furthermore, mechanically induced upregulation of known touch-responsive genes is significantly decreased in fer mutants. In addition to these defects in mechanical signaling, fer mutants also exhibit growth phenotypes consistent with impaired mechanical development, including biased root skewing, an inability to penetrate hard agar layers, and abnormal growth responses to impenetrable obstacles. Finally, high-resolution kinematic analysis of root growth revealed that fer mutants show pronounced spatiotemporal fluctuations in root cell expansion profiles with a timescale of minutes. Based on these results, we propose that FER is a key regulator of mechanical Ca(2+) signaling and that FER-dependent mechanical signaling functions to regulate growth in response to external or intrinsic mechanical forces.

Image analysis is driving a renaissance in growth measurement.

The domain of machine vision, in which digital images are acquired automatically in a highly structured environment for the purpose of computationally measuring features in the scene, is applicable to the measurement of plant growth. This article reviews the quickly growing collection of reports in which digital image-processing has been used to measure plant growth, with emphasis on the methodology and adaptations required for high-throughput studies of populations.

Plasticity of Arabidopsis root gravitropism throughout a multidimensional condition space quantified by automated image analysis.

Plant development is genetically determined but it is also plastic, a fundamental duality that can be investigated provided large number of measurements can be made in various conditions. Plasticity of gravitropism in wild-type Arabidopsis (Arabidopsis thaliana) seedling roots was investigated using automated image acquisition and analysis. A bank of computer-controlled charge-coupled device cameras acquired images with high spatiotemporal resolution. Custom image analysis algorithms extracted time course measurements of tip angle and growth rate. Twenty-two discrete conditions defined by seedling age (2, 3, or 4 d), seed size (extra small, small, medium, or large), and growth medium composition (simple or rich) formed the condition space sampled with 1,216 trials. Computational analyses including dimension reduction by principal components analysis, classification by k-means clustering, and differentiation by wavelet convolution showed distinct response patterns within the condition space, i.e. response plasticity. For example, 2-d-old roots (regardless of seed size) displayed a response time course similar to those of roots from large seeds (regardless of age). Enriching the growth medium with nutrients suppressed response plasticity along the seed size and age axes, possibly by ameliorating a mineral deficiency, although analysis of seeds did not identify any elements with low levels on a per weight basis. Characterizing relationships between growth rate and tip swing rate as a function of condition cast gravitropism in a multidimensional response space that provides new mechanistic insights as well as conceptually setting the stage for mutational analysis of plasticity in general and root gravitropism in particular.

Separating parental environment from seed size effects on next generation growth and development in Arabidopsis

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.

Detection of a gravitropism phenotype in glutamate receptor-like 3.3 mutants of Arabidopsis thaliana using machine vision and computation.

Gene disruption frequently produces no phenotype in the model plant Arabidopsis thaliana, complicating studies of gene function. Functional redundancy between gene family members is one common explanation but inadequate detection methods could also be responsible. Here, newly developed methods for automated capture and processing of time series of images, followed by computational analysis employing modified linear discriminant analysis (LDA) and wavelet-based differentiation, were employed in a study of mutants lacking the Glutamate Receptor-Like 3.3 gene. Root gravitropism was selected as the process to study with high spatiotemporal resolution because the ligand-gated Ca2+-permeable channel encoded by GLR3.3 may contribute to the ion fluxes associated with gravity signal transduction in roots. Time series of root tip angles were collected from wild type and two different glr3.3 mutants across a grid of seed-size and seedling-age conditions previously found to be important to gravitropism. Statistical tests of average responses detected no significant difference between populations, but LDA separated both mutant alleles from the wild type. After projecting the data onto LDA solution vectors, glr3.3 mutants displayed greater population variance than the wild type in all four conditions. In three conditions the projection means also differed significantly between mutant and wild type. Wavelet analysis of the raw response curves showed that the LDA-detected phenotypes related to an early deceleration and subsequent slower-bending phase in glr3.3 mutants. These statistically significant, heritable, computation-based phenotypes generated insight into functions of GLR3.3 in gravitropism. The methods could be generally applicable to the study of phenotypes and therefore gene function.

The Cyclic Nucleotide-Gated Channel CNGC14 Regulates Root Gravitropism in Arabidopsis thaliana

In plant roots, auxin inhibits cell expansion, and an increase in cellular auxin levels on the lower flanks of gravistimulated roots suppresses growth and thereby causes downward bending. These fundamental features of root growth responses to auxin were first described over 80 years ago, but our understanding of the underlying molecular mechanisms has remained scant. Here, we report that CYCLIC NUCLEOTIDE-GATED CHANNEL 14 (CNGC14) is essential for the earliest phase of auxin-induced ion signaling and growth inhibition in Arabidopsis roots. Using a fluorescence-imaging-based genetic screen, we found that cngc14 mutants exhibit a complete loss of rapid Ca(2+) and pH signaling in response to auxin treatment. Similarly impaired ion signaling was observed upon gravistimulation. We further developed a kinematic analysis approach to study dynamic root growth responses to auxin at high spatiotemporal resolution. These analyses revealed that auxin-induced growth inhibition and gravitropic bending are significantly delayed in cngc14 compared to wild-type roots, where auxin suppresses cell expansion within 1 min of treatment. Finally, we demonstrate that auxin-induced cytosolic Ca(2+) changes are required for rapid growth inhibition. Our results support a direct role for CNGC14-dependent Ca(2+) signaling in regulating the early posttranscriptional phase of auxin growth responses in Arabidopsis roots.

AUXIN UP-REGULATED F-BOX PROTEIN1 regulates the cross talk between auxin transport and cytokinin signaling during plant root growth.

Plant root development is mediated by the concerted action of the auxin and cytokinin phytohormones, with cytokinin serving as an antagonist of auxin transport. Here, we identify the AUXIN UP-REGULATED F-BOX PROTEIN1 (AUF1) and its potential paralog AUF2 as important positive modifiers of root elongation that tether auxin movements to cytokinin signaling in Arabidopsis (Arabidopsis thaliana). The AUF1 mRNA level in roots is strongly up-regulated by auxin but not by other phytohormones. Whereas the auf1 single and auf1 auf2 double mutant roots grow normally without exogenous auxin and respond similarly to the wild type upon auxin application, their growth is hypersensitive to auxin transport inhibitors, with the mutant roots also having reduced basipetal and acropetal auxin transport. The effects of auf1 on auxin movements may be mediated in part by the misexpression of several PIN-FORMED (PIN) auxin efflux proteins, which for PIN2 reduces its abundance on the plasma membrane of root cells. auf1 roots are also hypersensitive to cytokinin and have increased expression of several components of cytokinin signaling. Kinematic analyses of root growth and localization of the cyclin B mitotic marker showed that AUF1 does not affect root cell division but promotes cytokinin-mediated cell expansion in the elongation/differentiation zone. Epistasis analyses implicate the cytokinin regulator ARR1 or its effector(s) as the target of the SKP1-Cullin1-F Box (SCF) ubiquitin ligases assembled with AUF1/2. Given the wide distribution of AUF1/2-type proteins among land plants, we propose that SCFAUF1/2 provides additional cross talk between auxin and cytokinin, which modifies auxin distribution and ultimately root elongation.