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Kevin M Folta

Publications and source records attributed to Kevin M Folta.

11 recordsLinked to original sources
PubMed2006

Green light adjusts the plastid transcriptome during early photomorphogenic development.

During the transition from darkness to light, a suite of light sensors guides gene expression, biochemistry, and morphology to optimize acclimation to the new environment. Ultraviolet, blue, red, and far-red light all have demonstrated roles in modulating light responses, such as changes in gene expression and suppression of stem growth rate. However, green wavebands induce stem growth elongation, a response not likely mediated by known photosensors. In this study, etiolated Arabidopsis (Arabidopsis thaliana) seedlings were treated with a short, dim, single pulse of green light comparable in fluence and duration to that previously shown to excite robust stem elongation. Genome microarrays were then used to monitor coincident changes in gene expression. As anticipated, phytochrome A-regulated, nuclear-encoded transcripts were induced, confirming proper function of the sensitive phytochrome system. In addition, a suite of plastid-encoded transcripts decreased in abundance, including several typically up-regulated after phytochrome and/or cryptochrome activation. Further analyses using RNA gel-blot experiments demonstrated that the response is specific to green light, fluence dependent, and detectable within 30 min. The response obeys reciprocity and persists in the absence of known photosensors. Plastid transcript down-regulation was also observed in tobacco (Nicotiana tabacum) with similar temporal and fluence-response kinetics. Together, the down-regulation of plastid transcripts and increase in stem growth rate represent a mechanism that tempers progression of early commitment to the light environment, helping tailor seedling development during the critical process of establishment.

Arabidopsis↗
PubMed2006

Rapid and accurate pyrosequencing of angiosperm plastid genomes.

BACKGROUND: Plastid genome sequence information is vital to several disciplines in plant biology, including phylogenetics and molecular biology. The past five years have witnessed a dramatic increase in the number of completely sequenced plastid genomes, fuelled largely by advances in conventional Sanger sequencing technology. Here we report a further significant reduction in time and cost for plastid genome sequencing through the successful use of a newly available pyrosequencing platform, the Genome Sequencer 20 (GS 20) System (454 Life Sciences Corporation), to rapidly and accurately sequence the whole plastid genomes of the basal eudicot angiosperms Nandina domestica (Berberidaceae) and Platanus occidentalis (Platanaceae). RESULTS: More than 99.75% of each plastid genome was simultaneously obtained during two GS 20 sequence runs, to an average depth of coverage of 24.6x in Nandina and 17.3x in Platanus. The Nandina and Platanus plastid genomes shared essentially identical gene complements and possessed the typical angiosperm plastid structure and gene arrangement. To assess the accuracy of the GS 20 sequence, over 45 kilobases of sequence were generated for each genome using conventional sequencing. Overall error rates of 0.043% and 0.031% were observed in GS 20 sequence for Nandina and Platanus, respectively. More than 97% of all observed errors were associated with homopolymer runs, with approximately 60% of all errors associated with homopolymer runs of 5 or more nucleotides and approximately 50% of all errors associated with regions of extensive homopolymer runs. No substitution errors were present in either genome. Error rates were generally higher in the single-copy and noncoding regions of both plastid genomes relative to the inverted repeat and coding regions. CONCLUSION: Highly accurate and essentially complete sequence information was obtained for the Nandina and Platanus plastid genomes using the GS 20 System. More importantly, the high accuracy observed in the GS 20 plastid genome sequence was generated for a significant reduction in time and cost over traditional shotgun-based genome sequencing techniques, although with approximately half the coverage of previously reported GS 20 de novo genome sequence. The GS 20 should be broadly applicable to angiosperm plastid genome sequencing, and therefore promises to expand the scale of plant genetic and phylogenetic research dramatically.

Base Sequence↗
PubMed2006

Characterization of LF9, an octoploid strawberry genotype selected for rapid regeneration and transformation.

Cultivated strawberry (Fragaria xananassa) is a valuable crop, yet the absence of a rapid, high-throughput transgenic system has precluded meaningful application of biotechnology and translation of information from plant models to this crop. A new octoploid strawberry genetic line Laboratory Festival #9 has been identified, selected solely for its rapid regeneration and efficient transformation. Direct organogenesis has been achieved from all tissues tested, with rapidly-growing shoot initials visible in as few as 13 days. The conditions for optimal shoot regeneration, transformant selection, root generation, and plant acclimatization are presented. The progression from explant to plant in soil can be achieved in about 60 days. The development of transformation protocols in this rapid-cycling genotype allows high-throughput studies of gene function in the octoploid strawberry genetic background.

Acclimatization↗
PubMed2005

ASAP: amplification, sequencing & annotation of plastomes.

BACKGROUND: Availability of DNA sequence information is vital for pursuing structural, functional and comparative genomics studies in plastids. Traditionally, the first step in mining the valuable information within a chloroplast genome requires sequencing a chloroplast plasmid library or BAC clones. These activities involve complicated preparatory procedures like chloroplast DNA isolation or identification of the appropriate BAC clones to be sequenced. Rolling circle amplification (RCA) is being used currently to amplify the chloroplast genome from purified chloroplast DNA and the resulting products are sheared and cloned prior to sequencing. Herein we present a universal high-throughput, rapid PCR-based technique to amplify, sequence and assemble plastid genome sequence from diverse species in a short time and at reasonable cost from total plant DNA, using the large inverted repeat region from strawberry and peach as proof of concept. The method exploits the highly conserved coding regions or intergenic regions of plastid genes. Using an informatics approach, chloroplast DNA sequence information from 5 available eudicot plastomes was aligned to identify the most conserved regions. Cognate primer pairs were then designed to generate approximately 1 - 1.2 kb overlapping amplicons from the inverted repeat region in 14 diverse genera. RESULTS: 100% coverage of the inverted repeat region was obtained from Arabidopsis, tobacco, orange, strawberry, peach, lettuce, tomato and Amaranthus. Over 80% coverage was obtained from distant species, including Ginkgo, loblolly pine and Equisetum. Sequence from the inverted repeat region of strawberry and peach plastome was obtained, annotated and analyzed. Additionally, a polymorphic region identified from gel electrophoresis was sequenced from tomato and Amaranthus. Sequence analysis revealed large deletions in these species relative to tobacco plastome thus exhibiting the utility of this method for structural and comparative genomics studies. CONCLUSION: This simple, inexpensive method now allows immediate access to plastid sequence, increasing experimental throughput and serving generally as a universal platform for plastid genome characterization. The method applies well to whole genome studies and speeds assessment of variability across species, making it a useful tool in plastid structural genomics.

Base Sequence↗
PubMed2005

Design and fabrication of adjustable red-green-blue LED light arrays for plant research.

BACKGROUND: Although specific light attributes, such as color and fluence rate, influence plant growth and development, researchers generally cannot control the fine spectral conditions of artificial plant-growth environments. Plant growth chambers are typically outfitted with fluorescent and/or incandescent fixtures that provide a general spectrum that is accommodating to the human eye and not necessarily supportive to plant development. Many studies over the last several decades, primarily in Arabidopsis thaliana, have clearly shown that variation in light quantity, quality and photoperiod can be manipulated to affect growth and control developmental transitions. Light emitting diodes (LEDs) has been used for decades to test plant responses to narrow-bandwidth light. LEDs are particularly well suited for plant growth chambers, as they have an extraordinary life (about 100,000 hours), require little maintenance, and use negligible energy. These factors render LED-based light strategies particularly appropriate for space-biology as well as terrestrial applications. However, there is a need for a versatile and inexpensive LED array platform where individual wavebands can be specifically tuned to produce a series of light combinations consisting of various quantities and qualities of individual wavelengths. Two plans are presented in this report. RESULTS: In this technical report we describe the practical construction of tunable red-green-blue LED arrays to support research in plant growth and development. Two light fixture designs and corresponding circuitry are presented. The first is well suited for a laboratory environment for use in a finite area with small plants, such as Arabidopsis. The second is expandable and appropriate for growth chambers. The application of these arrays to early plant developmental studies has been validated with assays of hypocotyl growth inhibition/promotion and phototropic curvature in Arabidopsis seedlings. CONCLUSION: The presentation of these proven plans for LED array construction allows the teacher, researcher or electronics aficionado a means to inexpensively build efficient, adjustable lighting modules for plant research. These simple and effective designs permit the construction of useful tools by programs short on electronics expertise. These arrays represent a means to modulate precise quality and quantity in experimental settings to test the effect of specific light combinations in regulating plant growth, development and plant-product yield.

Arabidopsis↗
PubMed2005

Expressed sequence tags (ESTs) and simple sequence repeat (SSR) markers from octoploid strawberry (Fragaria x ananassa).

BACKGROUND: Cultivated strawberry (Fragaria x ananassa) represents one of the most valued fruit crops in the United States. Despite its economic importance, the octoploid genome presents a formidable barrier to efficient study of genome structure and molecular mechanisms that underlie agriculturally-relevant traits. Many potentially fruitful research avenues, especially large-scale gene expression surveys and development of molecular genetic markers have been limited by a lack of sequence information in public databases. As a first step to remedy this discrepancy a cDNA library has been developed from salicylate-treated, whole-plant tissues and over 1800 expressed sequence tags (EST's) have been sequenced and analyzed. RESULTS: A putative unigene set of 1304 sequences--133 contigs and 1171 singlets--has been developed, and the transcripts have been functionally annotated. Homology searches indicate that 89.5% of sequences share significant similarity to known/putative proteins or Rosaceae ESTs. The ESTs have been functionally characterized and genes relevant to specific physiological processes of economic importance have been identified. A set of tools useful for SSR development and mapping is presented. CONCLUSION: Sequences derived from this effort may be used to speed gene discovery efforts in Fragaria and the Rosaceae in general and also open avenues of comparative mapping. This report represents a first step in expanding molecular-genetic analyses in strawberry and demonstrates how computational tools can be used to optimally mine a large body of useful information from a relatively small data set.

Chromosome Mapping↗
PubMed2004

Green light stimulates early stem elongation, antagonizing light-mediated growth inhibition.

During the transition from darkness to light, the rate of hypocotyl elongation is determined from the integration of light signals sensed through the phototropin, cryptochrome, and phytochrome signaling pathways. In all light conditions studied, from UV to far-red, early hypocotyl growth is rapidly and robustly suppressed within minutes of illumination in a manner dependent upon light quality and quantity. In this study, it is shown that green light (GL) irradiation leads to a rapid increase in the growth rate of etiolated Arabidopsis seedlings. GL-mediated growth promotion was detected in response to constant irradiation or a short, single pulse of light with a similar time course. The response has a threshold between 10(-1) and 10(0) micromol m(-2), is saturated before 10(2) micromol m(-2) and obeys reciprocity. Genetic analyses indicate that the cryptochrome or phototropin photoreceptors do not participate in the response. The major phytochrome receptors influence the normal amplitude and timing of the GL response, yet the GL response is normal in seedlings grown for hours under constant dim-red light. Therefore, phytochrome activation enhances, but is not required for, the GL response. Seedlings grown under green, red, and blue light together are longer than those grown under red and blue alone. These data indicate that a novel GL-activated light sensor promotes early stem elongation that antagonizes growth inhibition.

Arabidopsis↗
PubMed2003

Primary inhibition of hypocotyl growth and phototropism depend differently on phototropin-mediated increases in cytoplasmic calcium induced by blue light.

The phototropin photoreceptors transduce blue-light signals into several physiological and developmental responses in plants. A transient rise in cytoplasmic calcium (Ca2+) that begins within seconds of phototropin 1 (phot1) excitation is believed to be an important element in the transduction pathways leading to one or more of the phot1-dependent responses. The goal of the present work was to determine whether the Ca2+ response was necessary for (a). the inhibition of hypocotyl elongation that develops within minutes of the irradiation, and (b). hypocotyl phototropism (curved growth of the stem in response to asymmetric illumination). After determining that pulses of light delivering photon fluences of between 1 and 1000 micromol m-2 induced growth inhibition mediated by phot1 without significant interference from other photosensory pathways, the effect of blocking the Ca2+ rise was assessed. Treatment of seedlings with a Ca2+ chelator prevented the rise in cytoplasmic Ca2+ and prevented phot1-mediated growth inhibition. However, the same chelator treatment did not impair phot1-mediated phototropism. Thus, it appears that the early, transient rise in cytoplasmic Ca2+ is an important intermediary process in at least one but not all phot1-signaling pathways.

Aequorin↗
PubMed2003

Phototropin 1 is required for high-fluence blue-light-mediated mRNA destabilization.

Irradiation of etiolated wild-type Arabidopsis thaliana seedlings with a single pulse of blue (B) light with a total fluence equal to that of 1 min of sunlight causes the destabilization of nuclear-encoded Lhcb transcripts. Transcript destabilization is not observed in phototropin1 (phot1, formerly nph1) mutant seedlings, indicating that phot1 is likely the photoreceptor mediating this response. Destabilization is also absent in nph3 mutants, but occurs normally in nph4 mutants. The rates of Lhcb transcription and B low-fluence-induced Lhcb transcript accumulation are normal in phot1 seedlings, confirming that photl regulates destabilization, not a change in transcription. A similar pattern of regulation is observed for the chloroplast-encoded rbcL transcript. The lack of destabilization of a second chloroplast encoded transcript, psbD, indicates that the phot1/B-high-fluence system does not result in a general destabilization of all chloroplast transcripts. Localized sequence similarity between the Lhcb 5'-UTR and the rbcL 3'-UTR suggests a similar mechanism of destabilization even though the two transcripts are located in different sub-cellular compartments. The high-fluence threshold of phot1-mediated RNA destabilization contrasts with the low-fluence threshold required for phot1-directed first-positive phototropic curvature. This study indicates that phot1, like phytochrome, can discriminate between several fluence ranges and direct responses in specific tissues or different sub-cellular compartments.

Arabidopsis↗
PubMed2003

Genomic and physiological studies of early cryptochrome 1 action demonstrate roles for auxin and gibberellin in the control of hypocotyl growth by blue light.

Blue light inhibits elongation of etiolated Arabidopsis thaliana hypocotyls during the first 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 profiles 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 GA4 biosynthesis with a 3beta-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 GA4 completely reversed the effects of the inhibitor, restoring the long-hypocotyl phenotype typical of the mutant. Treatment of wild-type seedlings with GA4 was not sufficient to phenocopy cry1 seedlings, but co-treatment with IAA plus GA4 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.

Arabidopsis↗