Targeted Knockout of CYP79A1 Reduces Cyanogenic Potential in Grain Sorghum.
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Journal Article↗
Biomedical subjects
Peggy G Lemaux
Publications and source records attributed to Peggy G Lemaux.
Multi-season analysis reveals hundreds of drought-responsive genes in sorghum.
Persistent drought affects global crop production and is becoming more severe in many parts of the world in recent decades. Deciphering how plants respond to drought will facilitate the development of flexible mitigation strategies. Sorghum bicolor L. Moench (sorghum), a major cereal crop and an emerging bioenergy crop, exhibits remarkable resilience to drought. To better understand the molecular traits that underlie sorghum's remarkable drought tolerance, we undertook a large-scale sorghum gene expression profiling effort, totaling nearly 1500 transcriptome profiles, across a 3-year field study with replicated plots in California's Central Valley. This study included time-resolved gene expression data from roots and leaves of two sorghum genotypes, BTx642 and RTx430, with different pre-flowering and post-flowering drought-tolerance adaptations under control and drought conditions. Quantification of genotype-specific drought tolerance effects was enabled by de novo sequencing, assembly, and annotation of both BTx642 and RTx430 genomes. These reference-quality genomes were used to construct a pangene set for characterizing conserved and genotype-specific expression. By integrating time-resolved transcriptomic responses to drought in the field across three consecutive years, we identified a set of 726 drought-responsive genes that responded similarly in all 3 years of our field study. Functional enrichment analysis identified abiotic stress, secondary cell wall-related processes and metabolism as particularly affected under both types of drought stress. We also found that some glyoxylate cycle pathway genes, including malate synthase and isocitrate lyase, are differentially regulated particularly during post-flowering drought stress, implicating this pathway as potentially important for drought responsiveness. This expansive dataset represents a unique resource for sorghum and drought research communities and provides a methodological framework for the integration of multi-faceted time-resolved transcriptomic datasets.
Sorghum↗
High-frequency Ds remobilization over multiple generations in barley facilitates gene tagging in large genome cereals.
Transposable elements have certain advantages over other approaches for identifying and determining gene function in large genome cereals. Different strategies have been used to exploit the maize Activator/dissociation (Ac/Ds) transposon system for functional genomics in heterologous species. Either large numbers of independent Ds insertion lines or transposants (TNPs) are generated and screened phenotypically, or smaller numbers of TNPs are produced, Ds locations mapped and remobilized for localized gene targeting. It is imperative to characterize key features of the system in order to utilize the latter strategy, which is more feasible in large genome cereals like barley and wheat. In barley, we generated greater than 100 single-copy Ds TNPs and determined remobilization frequencies of primary, secondary, and tertiary TNPs with intact terminal inverted repeats (TIRs); frequencies ranged from 11.8 to 17.1%. In 16% of TNPs that had damaged TIRs no transposition was detected among progeny of crosses using those TNPs as parental lines. In half of the greater than 100 TNP lines, the nature of flanking sequences and status of the 11 bp TIRs and 8-bp direct repeats were determined. BLAST searches using a gene prediction program revealed that 86% of TNP flanking sequences matched either known or putative genes, indicating preferential Ds insertion into genic regions, critical in large genome species. Observed remobilization frequencies of primary, secondary, tertiary, and quaternary TNPs, coupled with the tendency for localized Ds transposition, validates a saturation mutagenesis approach using Ds to tag and characterize genes linked to Ds in large genome cereals like barley and wheat.
Base Sequence↗
Nature of stress and transgene locus influences transgene expression stability in barley.
Stress and the nature of the transgene locus can affect transgene expression stability. These effects were studied in two, stably expressing, T6 populations of barley (Hordeum vulgare): bombardment-mediated, multi-copy lines with ubiquitin-driven bar and uidA or single-copy lines from Ds-mediated gene delivery with ubiquitin-driven bar alone. Imposing the environmental stresses, water and nutrient deprivation and heat shock, did not reproducibly affect transgene expression stability; however, high frequencies of heritable transcriptional gene silencing (TGS) occurred following in vitro culture after six generations of stable expression in the multi-copy subline, T3#30, but not in the other lines studied. T3#30 plants with complete TGS had epigenetic modification patterns exactly like those in an identical sibling subline, T3#31, which had significant reduction in transgene expression in the T3 generation and was completely transcriptionally silenced in the absence of imposed stresses in the T6 generation. Complete TGS in T3#30 plants correlated with methylation in the 5'UTR and intron of the ubi1 promoter complex and condensation of chromatin around the transgenes; DNA methylation likely occurred prior to chromatin condensation. Partial TGS in T3#30 also correlated with methylation of the ubi1 promoter complex, as occurred with complete TGS. T3#30 has a complex transgene structure with inverted repeat transgene fragments and a 3'-LTR from a barley retrotransposon, and therefore the transgene locus itself may affect its tendency to silence after in vitro culture and transgene silencing might result from host defense mechanisms activated by changes in plant developmental programming and/or stresses imposed during in vitro growth.
Base Sequence↗
Leafy cotyledon genes are essential for induction of somatic embryogenesis of Arabidopsis.
The capacity for somatic embryogenesis was studied in lec1, lec2 and fus3 mutants of Arabidopsis thaliana (L.) Heynh. It was found that contrary to the response of wild-type cultures, which produced somatic embryos via an efficient, direct process (65-94% of responding explants), lec mutants were strongly impaired in their embryogenic response. Cultures of the mutants formed somatic embryos at a low frequency, ranging from 0.0 to 3.9%. Moreover, somatic embryos were formed from callus tissue through an indirect route in the lec mutants. Total repression of embryogenic potential was observed in double (lec1 lec2, lec1 fus3, lec2 fus3) and triple (fus3 lec1 lec2) mutants. Additionally, mutants were found to exhibit efficient shoot regenerability via organogenesis from root explants. These results provide evidence that, besides their key role in controlling many different aspects of Arabidopsis zygotic embryogenesis, LEC/FUS genes are also essential for in vitro somatic embryogenesis induction. Furthermore, temporal and spatial patterns of auxin distribution during somatic embryogenesis induction were analyzed using transgenic Arabidopsis plants expressing GUS driven by the DR5 promoter. Analysis of data indicated auxin accumulation was rapid in all tissues of the explants of both wild type and the lec2-1 mutant, cultured on somatic embryogenesis induction medium containing 2,4-D. This observation suggests that loss of embryogenic potential in the lec2 mutant in vitro is not related to the distribution of exogenously applied auxin and LEC genes likely function downstream in auxin-induced somatic embryogenesis.
Arabidopsis↗
Evolutionary expansion, gene structure, and expression of the rice wall-associated kinase gene family.
The wall-associated kinase (WAK) gene family, one of the receptor-like kinase (RLK) gene families in plants, plays important roles in cell expansion, pathogen resistance, and heavy-metal stress tolerance in Arabidopsis (Arabidopsis thaliana). Through a reiterative database search and manual reannotation, we identified 125 OsWAK gene family members from rice (Oryza sativa) japonica cv Nipponbare; 37 (approximately 30%) OsWAKs were corrected/reannotated from earlier automated annotations. Of the 125 OsWAKs, 67 are receptor-like kinases, 28 receptor-like cytoplasmic kinases, 13 receptor-like proteins, 12 short genes, and five pseudogenes. The two-intron gene structure of the Arabidopsis WAK/WAK-Likes is generally conserved in OsWAKs; however, extra/missed introns were observed in some OsWAKs either in extracellular regions or in protein kinase domains. In addition to the 38 OsWAKs with full-length cDNA sequences and the 11 with rice expressed sequence tag sequences, gene expression analyses, using tiling-microarray analysis of the 20 OsWAKs on chromosome 10 and reverse transcription-PCR analysis for five OsWAKs, indicate that the majority of identified OsWAKs are likely expressed in rice. Phylogenetic analyses of OsWAKs, Arabidopsis WAK/WAK-Likes, and barley (Hordeum vulgare) HvWAKs show that the OsWAK gene family expanded in the rice genome due to lineage-specific expansion of the family in monocots. Localized gene duplications appear to be the primary genetic event in OsWAK gene family expansion and the 125 OsWAKs, present on all 12 chromosomes, are mostly clustered.
Arabidopsis↗
Strategies for avoiding reinventing the precollege education and outreach wheel.
The National Science Foundation's recent mandate that all Principal Investigators address the broader impacts of their research has prompted an unprecedented number of scientists to seek opportunities to participate in precollege education and outreach. To help interested geneticists avoid duplicating efforts and make use of existing resources, we examined several precollege genetics, genomics, and biotechnology education efforts and noted the elements that contributed to their success, indicated by program expansion, participant satisfaction, or participant learning. Identifying a specific audience and their needs and resources, involving K-12 teachers in program development, and evaluating program efforts are integral to program success. We highlighted a few innovative programs to illustrate these findings. Challenges that may compromise further development and dissemination of these programs include absence of reward systems for participation in outreach as well as lack of training for scientists doing outreach. Several programs and institutions are tackling these issues in ways that will help sustain outreach efforts while allowing them to be modified to meet the changing needs of their participants, including scientists, teachers, and students. Most importantly, resources and personnel are available to facilitate greater and deeper involvement of scientists in precollege and public education.
Biotechnology↗
Methylation of the exon/intron region in the Ubi1 promoter complex correlates with transgene silencing in barley.
Methylation of plant promoters is often associated with transcriptional gene silencing, while methylation of the transcribed region is associated with post-transcriptional gene silencing. Promoter complexes that include the first untranslated exon and intron, such as maize ubiquitin1 and rice actin1, have been widely used in monocot transformation because they support higher levels of transient transgene expression than the core promoter does. However, persistent problems with transgene silencing driven by these promoter complexes brought up a troubling question: were higher initial levels of transgene expression at the expense of long-term expression stability? Here we report that methylation of an untranslated exon and intron in the promoter complex is correlated with transcriptional transgene silencing in barley. Two sibling sublines, designated T(3)30 and T(3)31, were identified in a homozygous T3 population from a single transgenic parental line. In the T6 generation, all progeny of one subline, T(3)30, expressed ubiquitin-driven bar and uidA, but both transgenes were transcriptionally silenced in the other subline, T(3)31. Although the structure of the transgene locus is complex, no differences in the physical structure or location of the locus were detected between the two sublines. Transcriptional transgene silencing in T(3)31 correlated with two molecular events: methylation of the first untranslated exon and 5' end of the intron of the promoter complex, and condensation of the chromatin in regions containing transgenes. Passage of the non-silenced subline through in vitro culture recreated the silenced phenotype of T(3)31 and the molecular events leading to its silencing.
5' Flanking Region↗
Transgenic barley grain overexpressing thioredoxin shows evidence that the starchy endosperm communicates with the embryo and the aleurone.
Homozygous lines of barley overexpressing a wheat thioredoxin h transgene (up to 30-fold) were generated earlier by using a B(1)-hordein promoter with a signal peptide sequence for targeting to the protein body and found to be enriched in starch debranching enzyme (pullulanase). Here, we describe the effect of biochemically active, overexpressed thioredoxin h on germination and the onset of alpha-amylase activity. Relative to null segregant controls lacking the transgene, homozygotes overexpressing thioredoxin h effected (i) an acceleration in the rate of germination and appearance of alpha-amylase activity with a 1.6- to 2.8-fold increase in gibberellin A(1) (GA(1)) content; (ii) a similar acceleration in the appearance of the alpha-amylase activity in deembryonated transgenic grain incubated with gibberellic acid; (iii) a 35% increase in the ratio of relative reduction (abundance of SH) of the propanol soluble proteins (hordein I fraction); and (iv) an increase in extractable and soluble protein of 5-12% and 11-35%, respectively. Thioredoxin h, which was highly reduced in the dry grain, was degraded in both the null segregant and homozygote after imbibition. The increase in alpha-amylase activity and protein reduction status was accompanied by a shift in the distribution of protein from the insoluble to the soluble fraction. The results provide evidence that thioredoxin h of the starchy endosperm communicates with adjoining tissues, thereby regulating their activities, notably by accelerating germination of the embryo and the appearance of alpha-amylase released by the aleurone.
Desiccation↗
Similarity of expression patterns of knotted1 and ZmLEC1 during somatic and zygotic embryogenesis in maize ( Zea mays L.).
Expression of knotted1 ( kn1) and ZmLEC1, a maize homologue of the Arabidopsis LEAFY COTYLEDON1 ( LEC1) was studied using in situ hybridization during in vitro somatic embryogenesis of maize ( Zea mays L.) genotype Hi-II. Expression of kn1 was initially detected in a small group of cells (5-10) in the somatic embryo proper at the globular stage, in a specific region where the shoot meristem is initiating at the scutellar stage, and specifically in the shoot meristem at the coleoptilar stage. Expression of ZmLEC1 was strongly detected in the entire somatic embryo proper at the globular stage, gradually less in the differentiating scutellum at the scutellar and coleoptilar stages. The results of analyses show that the expression pattern of kn1 during in vitro somatic embryogenesis of maize is similar to that of kn1 observed during zygotic embryo development in maize. The expression pattern of ZmLEC1 in maize during in vitro development is similar to that of LEC1 in Arabidopsis during zygotic embryo development. These observations indicate that in vitro somatic embryogenesis likely proceeds through similar developmental pathways as zygotic embryo development, after somatic cells acquire competence to form embryos. In addition, based on the ZmLEC1 expression pattern, we suggest that expression of ZmLEC1 can be used as a reliable molecular marker for detecting early-stage in vitro somatic embryogenesis in maize.
Arabidopsis Proteins↗
Endosperm-specific expression of green fluorescent protein driven by the hordein promoter is stably inherited in transgenic barley (Hordeum vulgare) plants.
The expression of green fluorescent protein (GFP) and its inheritance were studied in transgenic barley (Hordeum vulgare L.) plants transformed with a synthetic green fluorescent protein gene [sgfp(S65T)] driven by either a rice actin promoter or a barley endosperm-specific d-hordein promoter. The gene encoding phosphinothricin acetyltransferase (bar), driven by the maize ubiquitin promoter and intron, was used as a selectable marker to identify transgenic tissues. Strong GFP expression driven by the rice actin promoter was observed in callus cells and in a variety of tissues of T0 plants transformed with the sgfp(S65T)-containing construct. GFP expression, driven by the rice actin promoter, was observed in 14 out of 17 independent regenerable transgenic callus lines; however, expression was gradually lost in T0 and later generation progeny of diploid lines. Stable GFP expression was observed in T2 progeny from only 6 out of the 14 (43%) independent GFP-expressing callus lines. Four of the 8 lines not expressing GFP in T2 progeny, lost GFP expression during T0 plant regeneration from calli; one lost GFP expression in the transition from the T0 to T1 generations and three lines were sterile. Similarly, expression of bar driven by the maize ubiquitin promoter was lost in T1 progeny; only 21 out of 26 (81%) independent lines were Basta-resistant. In contrast to actin-driven expression, GFP expression driven by the d-hordein promoter exhibited endosperm-specificity. All seven lines transformed with d-hordein-driven GFP (100%) expressed GFP in the T1 and T2 generations, regardless of ploidy levels, and expression segregated in a Mendelian fashion. We conclude that the sgfp(S65T) gene was successfully transformed into barley and that GFP expression driven by the d-hordein promoter was more stable in its inheritance pattern in T1 and T2 progeny than that driven by the rice actin promoter or the bar gene driven by the maize ubiquitin promoter.
Journal Article↗