Randy Allen

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Expression of 11s Seed Storage Protein Genes of Helianthus Annuus L

Typescript (photocopy).Many genes are expressed exclusively during plant embryo development. Amon... more Typescript (photocopy).Many genes are expressed exclusively during plant embryo development. Among these, seed storage protein genes are of primary interest because they are expressed at very high levels only during specific periods of embryo maturation. Helianthinin is the major globulin storage protein of sunflower seeds. The physico-chemical characteristics and subunit composition of helianthinin resembles the 11S protein of pea seeds (legumin). Antiserum prepared against purified helianthinin was used to study storage protein accumulation in developing sunflower embryos. Synthesis of helianthinin begins within 7 days post fertilization (DPF) and it accumulates rapidly until 19 DPF. Storage protein synthesis then slows until seed maturation at 30 DPF. Anti-helianthinin was also used to select specific cDNA recombinants from a bacteriophage λgt11 cDNA expression library prepared from immature sunflower embryo polyadenylated RNA. One of these recombinants, λHa3, contained a 300 base pair (bp) cDNA insert which was used as a hybridization probe to select a recombinant containing a 1200 bp cDNA insert (λHa2). An additional helianthinin specific cDNA recombinant (λHa10) that is not homologous with Ha2 or Ha3 was selected from the library by differential screening. Ha2 and Ha3 cDNAs hybridize with an approximately 1.9 kilobase (kb) sunflower embryo transcript on RNA gel blots. Ha10 reacts with a slightly smaller transcript than Ha2 or Ha3. Helianthinin mRNA homologous to both Ha2/Ha3 and Ha10 is present in embryos 7 DPF and accumulates to maximum prevalence at 12 DPF which corresponds to the period of maximum helianthinin accumulation. Soon after 15 DPF, helianthinin RNA begins to decrease. Full length helianthinin mRNA is not detectable in mature seeds or in germinated seedlings. These results indicate that synthesis of the 11S sunflower seed storage protein occurs during a specific stage of embryogenesis and is regulated by the accumulation of helianthinin mRNA. Genomic DNA blot hybridizations indicate the presence of at least three helianthinin seed protein genes of the Ha2/Ha3 type and two of the Ha10 type in the sunflower genome..

Armstrong et al.: Germin Like Protein and Beet Armyworm Development 693 DIETARY EFFECTS OF COTTON TISSUE EXPRESSING GERMIN LIKE PROTEIN ON BEET ARMYWORM (LEPIDOPTERA: NOCTUIDAE) GROWTH, SURVIVAL AND PUPATION

Transgenic cotton lines that ectopically express a cotton germin-like protein (GLP) were screened... more Transgenic cotton lines that ectopically express a cotton germin-like protein (GLP) were screened for resistance/tolerance factors to the beet armyworm (BAW) Spodoptera exigua (Hübner) via feeding assays. The number of BAW eggs that successfully hatched was not statistically different at 72 h after infestation for wild-type cotton plants (Gossypium hirsutum L. c.v. 'Coker 312') or plants of 4 independent transgenic lines (ABP-A, ABP-B and ABP-C and ABP-D). However, the damage caused by these same larvae at 72 h was higher for 'Coker 312' and line ABP-D when compared to ABP-A, ABP-B and ABP-C transgenic plants. Larval live weights were also significantly higher for Coker 312 and ABP-D at 5, 7, and 14 d when compared to ABP-A, ABP-B and ABP-C. The percentage of larvae that successfully completed pupation was significantly higher for BAW larvae fed 'Coker 312' and ABP-D tissue compared to the other 3 lines. These feeding bioassays show the potential for using co...

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Arabidopsis stress associated protein 9 mediates biotic and abiotic stress responsive ABA signaling via the proteasome pathway

Plant, Cell & Environment, 2017

Arabidopsis thaliana Stress Associated Protein 9 (AtSAP9) is a member of the A20/AN1 zinc finger ... more Arabidopsis thaliana Stress Associated Protein 9 (AtSAP9) is a member of the A20/AN1 zinc finger protein family known to play important roles in plant stress responses and in the mammalian immune response. Although SAPs of several plant species were shown to be involved in abiotic stress responses, the underlying molecular mechanisms are largely unknown, and little is known about the involvement of SAPs in plant disease responses. Expression of SAP9 in Arabidopsis is up‐regulated in response to dehydration, cold, salinity and abscisic acid (ABA), as well as pathogen infection. Constitutive expression of AtSAP9 in Arabidopsis leads to increased sensitivity to ABA and osmotic stress during germination and post‐germinative development. Plants that overexpress AtSAP9 also showed increased susceptibility to infection by non‐host pathogen Pseudomonas syringae pv. phaseolicola, indicating a potential role of AtSAP9 in disease resistance. AtSAP9 was found to interact with RADIATION SENSITIV...

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Protein Bodies in Dormant, Imbibed and Germinated Sunflower Cotyledons

Food Structure, 1982

Scanning electron microscopy was used to observe the morphology and structure of protein bodies i... more Scanning electron microscopy was used to observe the morphology and structure of protein bodies in dormant and imbibed sunflower cotyledons and to document the morpho 1 og i ca 1 changes in protein bodies during germination and seedling growth. In order to clearly visualize dormant seed structure, anhydrous fixation techniques were employed. Definite differences in cellular structure are seen in comparisons of dry and imbibed seed tissues. As germination proceeds, protein bodies lose their smooth spherical shape and become indented and pitted. Protein body coalescence and fusion precedes the formation of a central protein vacuole. As protein is hydrolyzed, protein vacuole density decreases, and its surface becomes granular, then fibrous, in appearance. Removal of protein from the protein vacuole appears to proceed more rapidly in cells closest to the embryonic axis. The protein vacuole becomes the main cell vacuole as remaining storage protein is hydrolyzed. The cotyledon ce 11 s undergo a gradua 1 change in function from a quiescent storage stage, through a major exporting phase and to their final function of photosynthesis.

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Increased resistance to oxidative stress in transgenic plants that overexpress chloroplastic Cu/Zn superoxide dismutase

Proceedings of the National Academy of Sciences of the United States of America, Feb 15, 1993

Transgenic tobacco plants that express a chimeric gene that encodes chloroplast-localized Cu/Zn s... more Transgenic tobacco plants that express a chimeric gene that encodes chloroplast-localized Cu/Zn superoxide dismutase (SOD) from pea have been developed. To investigate whether increased expression of chloroplasttargeted SOD could alter the resistance of photosynthesis to environmental stress, these plants were subjected to chilling temperatures and moderate (500 pmmol of quanta per m2 per s) or high (1500 pmol of quanta per m2 per s) light intensity. During exposure to moderate stress, transgenic SOD plants retained rates of photosynthesis-20% higher than untransformed tobacco plants, implicating active oxygen species in the reduction of photosynthesis during chilling. Unlike untransformed plants, transgenic SOD plants were capable of maintaining nearly 90% of their photosynthetic capacity (determined by their photosynthetic rates at 25C) foflowing exposure to chilling at high light intensity for 4 hr. These plants also showed reduced levels of light-mediated cellular damage from the superoxide-generating herbicide methyl viologen. These results demonstrate that SOD is a critical component of the active-oxygen-scavenging system of plant chloroplasts and indicate that modification of SOD expression in transgenic plants can improve plant stress tolerance.

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Regulation of Gene Expression in the Sunflower Embryo

ACS Symposium Series, 1988

Use of Transgenic Plants for Studies of Seed-Specific Gene Expression

Recent Advances in the Development and Germination of Seeds, 1989

In studying the regulated patterns of gene expression in higher plants, molecular biologists empl... more In studying the regulated patterns of gene expression in higher plants, molecular biologists employ a variety ofin vitro tools. The accumulation, distribution and decay of various gene products are monitored through the use of immunological and electrophoretic techniques. The interactions of these gene products can sometimes be observed. Cloned copies of interesting and important genes can be used to follow the accumulation and disappearance of specific messenger RNAs. But in many ways, such studies are merely descriptive. In order to achieve full understanding of the mechanisms which regulate gene expression, researchers need a means to determine cause and effect, a means to introduce a change at the molecular level and to observe its effect(s) on gene expression.

Nuclear Factors Interact with a Soybean b-Conglycinin Enhancer

The Plant Cell, 1989

Elucidating the unknown transcriptional responses and PHR1 mediated biotic and abiotic stress tolerance during phosphorus-limitation

Phosphorus (P) limitation in the majority of world soils is a major constraint for plant growth a... more Phosphorus (P) limitation in the majority of world soils is a major constraint for plant growth and crop productivity. RNA sequencing was used to discover novel P-responsive gene transcripts (PRGT) in leaves and roots of Arabidopsis. Hisat StringTie and Cufflinks TopHat transcript assembler were used to analyze reads and identify 1,074 PRGTs with a >5-fold altered abundance during P-limitation. Interestingly, 60% of these transcripts were not previously reported. Among the novel PRGT, 106 were from unannotated genes, and some were among the most P-responsive, including At2g36727 which encodes a novel microRNA. Annotated novel PRGTs encode for transcription factors, microRNAs, small signaling peptides, long non-coding RNAs, defense-related proteins, and transporters, along with proteins involved in many biological processes. We identified several genes that undergo alternative splicing during P-limitation, including a novel miR399 resistant splice variant of PHOSPHATE2 (PHO2.2). S...

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WOX9 functions antagonistic to STF and LAM1 to regulate leaf blade expansion inMedicago truncatulaandNicotiana sylvestris

Plant specific WOX family transcription factors are known to regulate embryogenesis, meristem mai... more Plant specific WOX family transcription factors are known to regulate embryogenesis, meristem maintenance and lateral organ development. Modern clade WOX genes function through a transcriptional repression mechanism, and the intermediate clade transcriptional activator WOX9 functions with the repressor WOX genes in embryogenesis and meristems maintenance, but the mechanism of this interaction is unclear.WOX1homologuesSTFandLAM1are required for leaf blade outgrowth inMedicago truncatulaandNicotiana Sylvestris, respectively. Here we show thatWOX9negatively regulates leaf blade outgrowth and functions antagonistically toSTFandLAM1. WhileNsWOX9ectopic expression enhances thelam1mutant phenotype, and antisense expression partially rescues thelam1mutant, both overexpression ofNsWOX9and knockout by CRISPR/Cas9 genome editing inN. sylvestrisresulted in a range of severe leaf blade distortions, indicating that controlled negative regulation by NsWOX9 is required for proper blade development....

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Enhancing Cotton Fiber Elongation and Cellulose Synthesis by Manipulating Fructokinase Activity

a. Objectives (a) Identification and characterization of the cotton fiber FRKs; (b) Generating tr... more a. Objectives (a) Identification and characterization of the cotton fiber FRKs; (b) Generating transgenic cotton plants overproducing either substrate inhibited tomato FRK or tomato FRK without substrate inhibition; (c) Generating transgenic cotton plants with RNAi suppression of fiber expressed FRKs; (d) Generating Arabidopsis plants that over express FRK1, FRK2, or both genes, as additional means to assess the contribution of FRK to cellulose synthesis and biomass production. b. Background to the topic: Cellulose synthesis and fiber elongation are dependent on sugar metabolism. Previous results suggested that FRKs (fructokinase enzymes that specifically phosphorylate fructose) are major players in sugar metabolism and cellulose synthesis. We therefore hypothesized that increasing fructose phosphorylation may enhance fiber elongation and cellulose synthesis in cotton plants. Accordinlgy, the objectives of this research were: c. Major conclusions and achievements: Two cotton FRK...

SPL 7 and SPL 8 represent a novel flowering regulation mechanism in switchgrass

New Phytologist, 2019

The aging pathway in flowering regulation is controlled mainly by microRNA156 (miR156). Studies i... more The aging pathway in flowering regulation is controlled mainly by microRNA156 (miR156). Studies in Arabidopsis thaliana reveal that nine miR156-targeted SQUAMOSA PROMOTER BINDING-LIKE (SPL) genes are involved in the control of flowering. However, the roles of SPLs in flowering remain elusive in grasses. Inflorescence development in switchgrass was characterized using scanning electron microscopy (SEM). Microarray, quantitative reverse transcription polymerase chain reaction (qRT-PCR), chromatin immunoprecipitation (ChIP)-PCR and EMSA were used to identify regulators of phase transition and flowering. Gene function was characterized by downregulation and overexpression of the target genes. Overexpression of SPL7 and SPL8 promotes flowering, whereas downregulation of individual genes moderately delays flowering. Simultaneous downregulation of SPL7/SPL8 results in extremely delayed or nonflowering plants. Furthermore, downregulation of both genes leads to a vegetative-to-reproductive reversion in the inflorescence, a phenomenon that has not been reported in any other grasses. Detailed analyses demonstrate that SPL7 and SPL8 induce phase transition and flowering in grasses by directly upregulating SEPALLATA3 (SEP3) and MADS32. Thus, the SPL7/8 pathway represents a novel regulatory mechanism in grasses that is largely different from that in Arabidopsis. Additionally, genetic modification of SPL7 and SPL8 results in much taller plants with significantly increased biomass yield and sugar release.

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Ectopic expression of twoAREB/ABForthologs increases drought tolerance in cotton (Gossypium hirsutum)

Plant, Cell & Environment, 2017

Plants have evolved complex molecular, cellular, and physiological mechanisms to respond to envir... more Plants have evolved complex molecular, cellular, and physiological mechanisms to respond to environmental stressors. Due to the inherent complexity of this response, genetic manipulation to substantially improve water deficit tolerance, particularly in agricultural crops, has been largely unsuccessful, as the improvements are frequently accompanied by slower growth and delayed reproduction. Here, we ectopically express two abiotic stress responsive bZIP AREB/ABF transcription factor orthologs, Arabidopsis ABF3 and a Gossypium hirsutum ABF2D, in G. hirsutum, to compare the effects of exogenous and endogenous AREB/ABF transgene over-expression on dehydration resilience. Our results show ectopic expression of each of these orthologs increases dehydration resilience, though these increases are accompanied by slower growth. These phenotypic effects are proportional to the ectopic expression level in the GhABF2D transgenic plants, while the phenotypes of all of the AtABF3 transgenic plants are similar, largely independent of ectopic expression level, possibly indicating differential posttranscriptional regulation of these transgenes. Our results indicate over-expression of exogenous and endogenous ABF homologs in G. hirsutum substantially increase drought resilience, primarily through stomatal regulation, negatively impacting transpiration and photosynthetic productivity.

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The Role of Cellulase in Hyalocyst Pore Formation in the Moss Syrrhopodon texanus

Annals of Botany, 1984

Overexpression of Chloroplastic Cu/Zn Superoxide Dismutase in Plants

Agrobacterium Protocols

Page 1. CHAPTER 27 Overexpression of Chloroplastic Cu/Zn Superoxide Dismutase in Plants Randy D. ... more

담배 Glutathione S-transferase (GST) 유전자의 토마토로의 도입과 과발현

High-Resolution Hydrodynamic Chromatographic Separation of Large DNA Using Narrow, Bare Open Capillaries: A Rapid and Economical Alternative Technology to Pulsed-Field Gel Electrophoresis?

Analytical Chemistry, 2014

A high-resolution, rapid, and economical hydrodynamic chromatographic (HDC) method for large DNA ... more A high-resolution, rapid, and economical hydrodynamic chromatographic (HDC) method for large DNA separations in free solution was developed using narrow (5 μm diameter), bare open capillaries. Size-based separation was achieved in a chromatographic format with larger DNA molecules being eluting faster than smaller ones. Lambda DNA Mono Cut Mix was baseline-separated with the percentage resolutions generally less than 9.0% for all DNA fragments (1.5 to 48.5 kbp) tested in this work. High efficiencies were achieved for large DNA from this chromatographic technique, and the number of theoretical plates reached 3.6 × 10 5 plates for the longest (48.5 kbp) and 3.7 × 10 5 plates for the shortest (1.5 kbp) fragments. HDC parameters and performances were also discussed. The method was further applied for fractionating large DNA fragments from real-world samples (SacII digested Arabidopsis plant bacterial artificial chromosome (BAC) DNA and PmeI digested Rice BAC DNA) to demonstrate its feasibility for BAC DNA finger printing. Rapid separation of PmeI digested Rice BAC DNA covering from 0.44 to 119.041 kbp was achieved in less than 26 min. All DNA fragments of these samples were baseline separated in narrow bare open capillaries, while the smallest fragment (0.44 kbp) was missing in pulsedfield gel electrophoresis (PFGE) separation mode. It is demonstrated that narrow bare open capillary chromatography can realize a rapid separation for a wide size range of DNA mixtures that contain both small and large DNA fragments in a single run.

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HSI2/VAL1 Silences AGL15 to Regulate the Developmental Transition from Seed Maturation to Vegetative Growth in Arabidopsis

The Plant Cell, 2018

Gene expression during seed development in Arabidopsis thaliana is controlled by transcription fa... more Gene expression during seed development in Arabidopsis thaliana is controlled by transcription factors including LEAFY COTYLEDON1 (LEC1) and LEC2, ABA INSENSITIVE3 (ABI3), FUSCA3 (FUS3), known as LAFL proteins, and AGAMOUS-LIKE15 (AGL15). The transition from seed maturation to germination and seedling growth requires the transcriptional silencing of these seed maturation-specific factors leading to downregulation of structural genes including those that encode seed storage proteins, oleosins, and dehydrins. During seed germination and vegetative growth, B3-domain protein HSI2/VAL1 is required for the transcriptional silencing of LAFL genes. Here, we report chromatin immunoprecipitation analysis indicating that HSI2/VAL1 binds to the upstream sequences of the AGL15 gene but not at LEC1, ABI3, FUS3, or LEC2 loci. Functional analysis indicates that the HSI2/VAL1 B3 domain interacts with two RY elements upstream of the AGL15 coding region and at least one of them is required for HSI2/VAL1-dependent AGL15 repression. Expression analysis of the major seed maturation regulatory genes LEC1, ABI3, FUS3, and LEC2 in different genetic backgrounds demonstrates that HSI2/VAL1 is epistatic to AGL15 and represses the seed maturation regulatory program through downregulation of AGL15 by deposition of H3K27me3 at this locus. This hypothesis is further supported by results that show that HSI2/VAL1 physically interacts with the Polycomb Repressive Complex 2 component protein MSI1, which is also enriched at the AGL15 locus.

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Evaluation of drought-tolerance strategies in cotton

Research made possible through the use of the wealth of genomic information and genetic resources... more Research made possible through the use of the wealth of genomic information and genetic resources available for model plant species such as Arabidopsis thaliana has led to a substantial increase in our knowledge about how plants respond to stressful environmental conditions. This leap in understanding provides the basis for many new strategies for optimization of stress tolerance in crop plants through both traditional and transgenic approaches. Although, to date, most of these strategies have been tested only in model plants, evaluation of some of them in important crop species, including cotton, is now underway. Application of novel stress-tolerance technologies will require extensive physiological and agronomic evaluation of plants that express these genes to determine their ultimate applicability to crop systems. A broad-based evaluation strategy for candidate genes will be necessary to test their value under a wide variety of field conditions. In cotton, this will include testi...

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HSI2/VAL1 and HSL1/VAL2 function redundantly to regulate seed dormancy by controllingDOG1expression in Arabidopsis

ABSTRACTDELAY OF GERMINATION1(DOG1) represents a major quantitative locus for the genetic regulat... more ABSTRACTDELAY OF GERMINATION1(DOG1) represents a major quantitative locus for the genetic regulation of seed dormancy in Arabidopsis. Accumulation of DOG1 in seeds leads to deep dormancy and delayed germination. Here, we report that the conserved B3 DNA binding domains of the transcriptional repressors HIGH-LEVEL EXPRESSION OF SUGAR INDICIBLE GENE2/ VIVIPAROUS-1/ABSCISIC ACID INSENSITIVE 3-LIKE1 (HSI2/VAL1) and HSI2-LIKE1/ VIVIPAROUS-1/ABSCISIC ACID INSENSITIVE 3-LIKE2 (HSL1/VAL2), which play critical roles in the developmental transition from seed maturation to seedling growth, interact with RY elements in theDOG1proximal promoter leading to repression ofDOG1transcription during germination and seedling establishment.DOG1expression is partially de-repressed inhsi2/val1(hsi2-2) but not inhsl1/val2(hsl1-1) knockout mutants and is strongly upregulated in ahsi2/val1 hsl1/val2double mutant, indicating that HSI2/VAL1 and HSL1/VAL2 act redundantly to repressDOG1expression. HSI2/VAL1 and H...

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