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Clay J. Carter - One of the best experts on this subject based on the ideXlab platform.
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the role of alanine synthesis and nitrate induced nitric oxide production during hypoxia stress in cucurbita pepo Nectaries
Plant Journal, 2021Co-Authors: Erik M Solhaug, Rahul Roy, Rodney T Venterea, Clay J. CarterAbstract:Floral nectar is a sugary solution produced by Nectaries to attract and reward pollinators. Nectar metabolites, such as sugars, are synthesized within the nectary during secretion from both pre-stored and direct phloem-derived precursors. In addition to sugars, nectars contain nitrogenous compounds such as amino acids; however, little is known about the role(s) of nitrogen (N) compounds in nectary function. In this study, we investigated N metabolism in Cucurbita pepo (squash) floral Nectaries in order to understand how various N-containing compounds are produced and determine the role of N metabolism in nectar secretion. The expression and activity of key enzymes involved in primary N assimilation, including nitrate reductase (NR) and alanine aminotransferase (AlaAT), were induced during secretion in C. pepo Nectaries. Alanine (Ala) accumulated to about 35% of total amino acids in Nectaries and nectar during peak secretion; however, alteration of vascular nitrate supply had no impact on Ala accumulation during secretion, suggesting that nectar(y) amino acids are produced by precursors other than nitrate. In addition, nitric oxide (NO) is produced from nitrate and nitrite, at least partially by NR, in Nectaries and nectar. Hypoxia-related processes are induced in Nectaries during secretion, including lactic acid and ethanolic fermentation. Finally, treatments that alter nitrate supply affect levels of hypoxic metabolites, nectar volume and nectar sugar composition. The induction of N metabolism in C. pepo Nectaries thus plays an important role in the synthesis and secretion of nectar sugar.
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systems analyses of key metabolic modules of floral and extrafloral Nectaries of cotton
bioRxiv, 2019Co-Authors: Elizabeth C Chatt, Clay J. Carter, Marshall Hampton, Peter M Klinkenberg, Sitinabilla Mahalim, Nuraziatull Mohdfadzil, Rahul Roy, Harry T Horner, Basil J. NikolauAbstract:Abstract Nectar is a primary reward mediating plant-animal mutualisms to improve plant fitness and reproductive success. In Gossypium hirsutum (cotton), four distinct trichomatic Nectaries develop, one floral and three extrafloral. The secreted floral and extrafloral nectars serve different purposes, with the floral nectar attracting bees to promote pollination and the extrafloral nectar attracting predatory insects as a means of indirect resistance from herbivores. Cotton therefore provides an ideal system to contrast mechanisms of nectar production and nectar composition between floral and extrafloral Nectaries. Here, we report the transcriptome, ultrastructure, and metabolite spatial distribution using mass spectrometric imaging of the four cotton nectary types throughout development. Additionally, the secreted nectar metabolomes were defined and were jointly composed of 197 analytes, 60 of which were identified. Integration of theses datasets support the coordination of merocrine-based and eccrine-based models of nectar synthesis. The nectary ultrastructure supports the merocrine-based model due to the abundance of rough endoplasmic reticulum positioned parallel to the cell walls and profusion of vesicles fusing to the plasma membranes. The eccrine-based model which consist of a progression from starch synthesis to starch degradation and to sucrose biosynthesis was supported by gene expression data. This demonstrates conservation of the eccrine-based model for the first time in both trichomatic and extrafloral Nectaries. Lastly, nectary gene expression data provided evidence to support de novo synthesis of amino acids detected in the secreted nectars. One sentence summary The eccrine-based model of nectar synthesis and secretion is conserved in both trichomatic and extrafloral Nectaries determined by a system-based comparison of cotton (Gossypium hirsutum) Nectaries.
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The pennycress (Thlaspi arvense L.) nectary: structural and transcriptomic characterization
BMC Plant Biology, 2017Co-Authors: Jason B. Thomas, Marshall E. Hampton, M. David Marks, Kevin M. Dorn, Clay J. CarterAbstract:BackgroundPennycress [Thlaspi arvense L (Brassicaceae)] is being domesticated as a renewable biodiesel feedstock that also provides crucial ecosystems services, including as a nutritional resource for pollinators. However, its flowers produce significantly less nectar than other crop relatives in the Brassicaceae. This study was undertaken to understand the basic biology of the pennycress nectary as an initial step toward the possibility of enhancing nectar output from its flowers.ResultsPennycress flowers contain four equivalent Nectaries located extrastaminally at the base of the insertion sites of short and long stamens. Like other Brassicaceae, the Nectaries have open stomates on their surface, which likely serve as the sites of nectar secretion. The Nectaries produce four distinct nectar droplets that accumulate in concave structures at the base of each of the four petals. To understand the molecular biology of the pennycress nectary, RNA was isolated from ‘immature’ (pre-secretory) and ‘mature’ (secretory) Nectaries and subjected to RNA-seq. Approximately 184 M paired-end reads (368 M total reads) were de novo assembled into a total of 16,074 independent contigs, which mapped to 12,335 unique genes in the pennycress genome. Nearly 3700 genes were found to be differentially expressed between immature and mature Nectaries and subjected to gene ontology and metabolic pathway analyses. Lastly, in silico analyses identified 158 pennycress orthologs to Arabidopsis genes with known enriched expression in Nectaries. These nectary-enriched expression patterns were verified for select pennycress loci by semi-quantitative RT-PCR.ConclusionsPennycress Nectaries are unique relative to those of other agriculturally important Brassicaceae, as they contain four equivalent Nectaries that present their nectar in specialized cup-shaped structures at the base of the petals. In spite of these morphological differences, the genes underlying the regulation and production of nectar appear to be largely conserved between pennycress and Arabidopsis thaliana. These results provide a starting point for using forward and reverse genetics approaches to enhance nectar synthesis and secretion in pennycress.
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The pennycress (Thlaspi arvense L.) nectary: structural and transcriptomic characterization.
BMC Plant Biology, 2017Co-Authors: Jason B. Thomas, Kevin M. Dorn, Marshall Hampton, M. David Marks, Clay J. CarterAbstract:Pennycress [Thlaspi arvense L (Brassicaceae)] is being domesticated as a renewable biodiesel feedstock that also provides crucial ecosystems services, including as a nutritional resource for pollinators. However, its flowers produce significantly less nectar than other crop relatives in the Brassicaceae. This study was undertaken to understand the basic biology of the pennycress nectary as an initial step toward the possibility of enhancing nectar output from its flowers. Pennycress flowers contain four equivalent Nectaries located extrastaminally at the base of the insertion sites of short and long stamens. Like other Brassicaceae, the Nectaries have open stomates on their surface, which likely serve as the sites of nectar secretion. The Nectaries produce four distinct nectar droplets that accumulate in concave structures at the base of each of the four petals. To understand the molecular biology of the pennycress nectary, RNA was isolated from ‘immature’ (pre-secretory) and ‘mature’ (secretory) Nectaries and subjected to RNA-seq. Approximately 184 M paired-end reads (368 M total reads) were de novo assembled into a total of 16,074 independent contigs, which mapped to 12,335 unique genes in the pennycress genome. Nearly 3700 genes were found to be differentially expressed between immature and mature Nectaries and subjected to gene ontology and metabolic pathway analyses. Lastly, in silico analyses identified 158 pennycress orthologs to Arabidopsis genes with known enriched expression in Nectaries. These nectary-enriched expression patterns were verified for select pennycress loci by semi-quantitative RT-PCR. Pennycress Nectaries are unique relative to those of other agriculturally important Brassicaceae, as they contain four equivalent Nectaries that present their nectar in specialized cup-shaped structures at the base of the petals. In spite of these morphological differences, the genes underlying the regulation and production of nectar appear to be largely conserved between pennycress and Arabidopsis thaliana. These results provide a starting point for using forward and reverse genetics approaches to enhance nectar synthesis and secretion in pennycress.
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identification of differential gene expression in brassica rapa Nectaries through expressed sequence tag analysis
PLOS ONE, 2010Co-Authors: Marshall Hampton, Brian W Kram, Emily M Chambers, Jerad S Ehrnriter, Jonathan H Gralewski, Teresa Joyal, Clay J. CarterAbstract:Background Nectaries are the floral organs responsible for the synthesis and secretion of nectar. Despite their central roles in pollination biology, very little is understood about the molecular mechanisms underlying nectar production. This project was undertaken to identify genes potentially involved in mediating nectary form and function in Brassica rapa. Methodology and Principal Findings Four cDNA libraries were created using RNA isolated from the median and lateral Nectaries of B. rapa flowers, with one normalized and one non-normalized library being generated from each tissue. Approximately 3,000 clones from each library were randomly sequenced from the 5′ end to generate a total of 11,101 high quality expressed sequence tags (ESTs). Sequence assembly of all ESTs together allowed the identification of 1,453 contigs and 4,403 singleton sequences, with the Basic Localized Alignment Search Tool (BLAST) being used to identify 4,138 presumptive orthologs to Arabidopsis thaliana genes. Several genes differentially expressed between median and lateral Nectaries were initially identified based upon the number of BLAST hits represented by independent ESTs, and later confirmed via reverse transcription polymerase chain reaction (RT PCR). RT PCR was also used to verify the expression patterns of eight putative orthologs to known Arabidopsis nectary-enriched genes. Conclusions/Significance This work provided a snapshot of gene expression in actively secreting B. rapa Nectaries, and also allowed the identification of differential gene expression between median and lateral Nectaries. Moreover, 207 orthologs to known nectary-enriched genes from Arabidopsis were identified through this analysis. The results suggest that genes involved in nectar production are conserved amongst the Brassicaceae, and also supply clones and sequence information that can be used to probe nectary function in B. rapa.
Jason B. Thomas - One of the best experts on this subject based on the ideXlab platform.
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The pennycress (Thlaspi arvense L.) nectary: structural and transcriptomic characterization
BMC Plant Biology, 2017Co-Authors: Jason B. Thomas, Marshall E. Hampton, M. David Marks, Kevin M. Dorn, Clay J. CarterAbstract:BackgroundPennycress [Thlaspi arvense L (Brassicaceae)] is being domesticated as a renewable biodiesel feedstock that also provides crucial ecosystems services, including as a nutritional resource for pollinators. However, its flowers produce significantly less nectar than other crop relatives in the Brassicaceae. This study was undertaken to understand the basic biology of the pennycress nectary as an initial step toward the possibility of enhancing nectar output from its flowers.ResultsPennycress flowers contain four equivalent Nectaries located extrastaminally at the base of the insertion sites of short and long stamens. Like other Brassicaceae, the Nectaries have open stomates on their surface, which likely serve as the sites of nectar secretion. The Nectaries produce four distinct nectar droplets that accumulate in concave structures at the base of each of the four petals. To understand the molecular biology of the pennycress nectary, RNA was isolated from ‘immature’ (pre-secretory) and ‘mature’ (secretory) Nectaries and subjected to RNA-seq. Approximately 184 M paired-end reads (368 M total reads) were de novo assembled into a total of 16,074 independent contigs, which mapped to 12,335 unique genes in the pennycress genome. Nearly 3700 genes were found to be differentially expressed between immature and mature Nectaries and subjected to gene ontology and metabolic pathway analyses. Lastly, in silico analyses identified 158 pennycress orthologs to Arabidopsis genes with known enriched expression in Nectaries. These nectary-enriched expression patterns were verified for select pennycress loci by semi-quantitative RT-PCR.ConclusionsPennycress Nectaries are unique relative to those of other agriculturally important Brassicaceae, as they contain four equivalent Nectaries that present their nectar in specialized cup-shaped structures at the base of the petals. In spite of these morphological differences, the genes underlying the regulation and production of nectar appear to be largely conserved between pennycress and Arabidopsis thaliana. These results provide a starting point for using forward and reverse genetics approaches to enhance nectar synthesis and secretion in pennycress.
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The pennycress (Thlaspi arvense L.) nectary: structural and transcriptomic characterization.
BMC Plant Biology, 2017Co-Authors: Jason B. Thomas, Kevin M. Dorn, Marshall Hampton, M. David Marks, Clay J. CarterAbstract:Pennycress [Thlaspi arvense L (Brassicaceae)] is being domesticated as a renewable biodiesel feedstock that also provides crucial ecosystems services, including as a nutritional resource for pollinators. However, its flowers produce significantly less nectar than other crop relatives in the Brassicaceae. This study was undertaken to understand the basic biology of the pennycress nectary as an initial step toward the possibility of enhancing nectar output from its flowers. Pennycress flowers contain four equivalent Nectaries located extrastaminally at the base of the insertion sites of short and long stamens. Like other Brassicaceae, the Nectaries have open stomates on their surface, which likely serve as the sites of nectar secretion. The Nectaries produce four distinct nectar droplets that accumulate in concave structures at the base of each of the four petals. To understand the molecular biology of the pennycress nectary, RNA was isolated from ‘immature’ (pre-secretory) and ‘mature’ (secretory) Nectaries and subjected to RNA-seq. Approximately 184 M paired-end reads (368 M total reads) were de novo assembled into a total of 16,074 independent contigs, which mapped to 12,335 unique genes in the pennycress genome. Nearly 3700 genes were found to be differentially expressed between immature and mature Nectaries and subjected to gene ontology and metabolic pathway analyses. Lastly, in silico analyses identified 158 pennycress orthologs to Arabidopsis genes with known enriched expression in Nectaries. These nectary-enriched expression patterns were verified for select pennycress loci by semi-quantitative RT-PCR. Pennycress Nectaries are unique relative to those of other agriculturally important Brassicaceae, as they contain four equivalent Nectaries that present their nectar in specialized cup-shaped structures at the base of the petals. In spite of these morphological differences, the genes underlying the regulation and production of nectar appear to be largely conserved between pennycress and Arabidopsis thaliana. These results provide a starting point for using forward and reverse genetics approaches to enhance nectar synthesis and secretion in pennycress.
Marshall Hampton - One of the best experts on this subject based on the ideXlab platform.
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systems analyses of key metabolic modules of floral and extrafloral Nectaries of cotton
bioRxiv, 2019Co-Authors: Elizabeth C Chatt, Clay J. Carter, Marshall Hampton, Peter M Klinkenberg, Sitinabilla Mahalim, Nuraziatull Mohdfadzil, Rahul Roy, Harry T Horner, Basil J. NikolauAbstract:Abstract Nectar is a primary reward mediating plant-animal mutualisms to improve plant fitness and reproductive success. In Gossypium hirsutum (cotton), four distinct trichomatic Nectaries develop, one floral and three extrafloral. The secreted floral and extrafloral nectars serve different purposes, with the floral nectar attracting bees to promote pollination and the extrafloral nectar attracting predatory insects as a means of indirect resistance from herbivores. Cotton therefore provides an ideal system to contrast mechanisms of nectar production and nectar composition between floral and extrafloral Nectaries. Here, we report the transcriptome, ultrastructure, and metabolite spatial distribution using mass spectrometric imaging of the four cotton nectary types throughout development. Additionally, the secreted nectar metabolomes were defined and were jointly composed of 197 analytes, 60 of which were identified. Integration of theses datasets support the coordination of merocrine-based and eccrine-based models of nectar synthesis. The nectary ultrastructure supports the merocrine-based model due to the abundance of rough endoplasmic reticulum positioned parallel to the cell walls and profusion of vesicles fusing to the plasma membranes. The eccrine-based model which consist of a progression from starch synthesis to starch degradation and to sucrose biosynthesis was supported by gene expression data. This demonstrates conservation of the eccrine-based model for the first time in both trichomatic and extrafloral Nectaries. Lastly, nectary gene expression data provided evidence to support de novo synthesis of amino acids detected in the secreted nectars. One sentence summary The eccrine-based model of nectar synthesis and secretion is conserved in both trichomatic and extrafloral Nectaries determined by a system-based comparison of cotton (Gossypium hirsutum) Nectaries.
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The pennycress (Thlaspi arvense L.) nectary: structural and transcriptomic characterization.
BMC Plant Biology, 2017Co-Authors: Jason B. Thomas, Kevin M. Dorn, Marshall Hampton, M. David Marks, Clay J. CarterAbstract:Pennycress [Thlaspi arvense L (Brassicaceae)] is being domesticated as a renewable biodiesel feedstock that also provides crucial ecosystems services, including as a nutritional resource for pollinators. However, its flowers produce significantly less nectar than other crop relatives in the Brassicaceae. This study was undertaken to understand the basic biology of the pennycress nectary as an initial step toward the possibility of enhancing nectar output from its flowers. Pennycress flowers contain four equivalent Nectaries located extrastaminally at the base of the insertion sites of short and long stamens. Like other Brassicaceae, the Nectaries have open stomates on their surface, which likely serve as the sites of nectar secretion. The Nectaries produce four distinct nectar droplets that accumulate in concave structures at the base of each of the four petals. To understand the molecular biology of the pennycress nectary, RNA was isolated from ‘immature’ (pre-secretory) and ‘mature’ (secretory) Nectaries and subjected to RNA-seq. Approximately 184 M paired-end reads (368 M total reads) were de novo assembled into a total of 16,074 independent contigs, which mapped to 12,335 unique genes in the pennycress genome. Nearly 3700 genes were found to be differentially expressed between immature and mature Nectaries and subjected to gene ontology and metabolic pathway analyses. Lastly, in silico analyses identified 158 pennycress orthologs to Arabidopsis genes with known enriched expression in Nectaries. These nectary-enriched expression patterns were verified for select pennycress loci by semi-quantitative RT-PCR. Pennycress Nectaries are unique relative to those of other agriculturally important Brassicaceae, as they contain four equivalent Nectaries that present their nectar in specialized cup-shaped structures at the base of the petals. In spite of these morphological differences, the genes underlying the regulation and production of nectar appear to be largely conserved between pennycress and Arabidopsis thaliana. These results provide a starting point for using forward and reverse genetics approaches to enhance nectar synthesis and secretion in pennycress.
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PIN6 is required for nectary auxin response and short stamen development
Plant Journal, 2013Co-Authors: Ricci L. Bender, Marshall Hampton, Peter M Klinkenberg, Megan L. Fekete, Brittany Bauer, Matthew Malecha, Khrystyne Lindgren, Jennifer A. Maki, M. Ann D. N. Perera, Basil J. NikolauAbstract:†‡ SUMMARY The PIN family of proteins is best known for its involvement in polar auxin transport and tropic responses. PIN6 (At1g77110) is one of the remaining PIN family members in Arabidopsis thaliana to which a biological function has not yet been ascribed. Here we report that PIN6 is a nectary-enriched gene whose expression level is positively correlated with total nectar production in Arabidopsis, and whose function is required for the proper development of short stamens. PIN6 accumulates in internal membranes consistent with the ER, and multiple lines of evidence demonstrate that PIN6 is required for auxin-dependent responses in Nectaries. Wild-type plants expressing auxin-responsive DR5:GFP or DR5:GUS reporters displayed intense signal in lateral Nectaries, but pin6 lateral Nectaries showed little or no signal for these reporters. Further, exogenous auxin treatment increased nectar production more than tenfold in wild-type plants, but nectar production was not increased in pin6 mutants when treated with auxin. Conversely, the auxin transport inhibitor N‐1‐naphthylphthalamic acid (NPA) reduced nectar production in wild-type plants by more than twofold, but had no significant effect on pin6 lines. Interestingly, a MYB57 transcription factor mutant, myb57‐2, closely phenocopied the loss-of-function mutant pin6‐2. However, PIN6 expression was not dependent on MYB57, and RNA-seq analyses of pin6‐2 and myb57‐2 mutant Nectaries showed little overlap in terms of differentially expressed genes. Cumulatively, these results demonstrate that PIN6 is required for proper auxin response and nectary function in Arabidopsis. These results also identify auxin as an important factor in the regulation of nectar production, and implicate short stamens in the maturation of lateral Nectaries.
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identification of differential gene expression in brassica rapa Nectaries through expressed sequence tag analysis
PLOS ONE, 2010Co-Authors: Marshall Hampton, Brian W Kram, Emily M Chambers, Jerad S Ehrnriter, Jonathan H Gralewski, Teresa Joyal, Clay J. CarterAbstract:Background Nectaries are the floral organs responsible for the synthesis and secretion of nectar. Despite their central roles in pollination biology, very little is understood about the molecular mechanisms underlying nectar production. This project was undertaken to identify genes potentially involved in mediating nectary form and function in Brassica rapa. Methodology and Principal Findings Four cDNA libraries were created using RNA isolated from the median and lateral Nectaries of B. rapa flowers, with one normalized and one non-normalized library being generated from each tissue. Approximately 3,000 clones from each library were randomly sequenced from the 5′ end to generate a total of 11,101 high quality expressed sequence tags (ESTs). Sequence assembly of all ESTs together allowed the identification of 1,453 contigs and 4,403 singleton sequences, with the Basic Localized Alignment Search Tool (BLAST) being used to identify 4,138 presumptive orthologs to Arabidopsis thaliana genes. Several genes differentially expressed between median and lateral Nectaries were initially identified based upon the number of BLAST hits represented by independent ESTs, and later confirmed via reverse transcription polymerase chain reaction (RT PCR). RT PCR was also used to verify the expression patterns of eight putative orthologs to known Arabidopsis nectary-enriched genes. Conclusions/Significance This work provided a snapshot of gene expression in actively secreting B. rapa Nectaries, and also allowed the identification of differential gene expression between median and lateral Nectaries. Moreover, 207 orthologs to known nectary-enriched genes from Arabidopsis were identified through this analysis. The results suggest that genes involved in nectar production are conserved amongst the Brassicaceae, and also supply clones and sequence information that can be used to probe nectary function in B. rapa.
Kevin M. Dorn - One of the best experts on this subject based on the ideXlab platform.
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The pennycress (Thlaspi arvense L.) nectary: structural and transcriptomic characterization
BMC Plant Biology, 2017Co-Authors: Jason B. Thomas, Marshall E. Hampton, M. David Marks, Kevin M. Dorn, Clay J. CarterAbstract:BackgroundPennycress [Thlaspi arvense L (Brassicaceae)] is being domesticated as a renewable biodiesel feedstock that also provides crucial ecosystems services, including as a nutritional resource for pollinators. However, its flowers produce significantly less nectar than other crop relatives in the Brassicaceae. This study was undertaken to understand the basic biology of the pennycress nectary as an initial step toward the possibility of enhancing nectar output from its flowers.ResultsPennycress flowers contain four equivalent Nectaries located extrastaminally at the base of the insertion sites of short and long stamens. Like other Brassicaceae, the Nectaries have open stomates on their surface, which likely serve as the sites of nectar secretion. The Nectaries produce four distinct nectar droplets that accumulate in concave structures at the base of each of the four petals. To understand the molecular biology of the pennycress nectary, RNA was isolated from ‘immature’ (pre-secretory) and ‘mature’ (secretory) Nectaries and subjected to RNA-seq. Approximately 184 M paired-end reads (368 M total reads) were de novo assembled into a total of 16,074 independent contigs, which mapped to 12,335 unique genes in the pennycress genome. Nearly 3700 genes were found to be differentially expressed between immature and mature Nectaries and subjected to gene ontology and metabolic pathway analyses. Lastly, in silico analyses identified 158 pennycress orthologs to Arabidopsis genes with known enriched expression in Nectaries. These nectary-enriched expression patterns were verified for select pennycress loci by semi-quantitative RT-PCR.ConclusionsPennycress Nectaries are unique relative to those of other agriculturally important Brassicaceae, as they contain four equivalent Nectaries that present their nectar in specialized cup-shaped structures at the base of the petals. In spite of these morphological differences, the genes underlying the regulation and production of nectar appear to be largely conserved between pennycress and Arabidopsis thaliana. These results provide a starting point for using forward and reverse genetics approaches to enhance nectar synthesis and secretion in pennycress.
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The pennycress (Thlaspi arvense L.) nectary: structural and transcriptomic characterization.
BMC Plant Biology, 2017Co-Authors: Jason B. Thomas, Kevin M. Dorn, Marshall Hampton, M. David Marks, Clay J. CarterAbstract:Pennycress [Thlaspi arvense L (Brassicaceae)] is being domesticated as a renewable biodiesel feedstock that also provides crucial ecosystems services, including as a nutritional resource for pollinators. However, its flowers produce significantly less nectar than other crop relatives in the Brassicaceae. This study was undertaken to understand the basic biology of the pennycress nectary as an initial step toward the possibility of enhancing nectar output from its flowers. Pennycress flowers contain four equivalent Nectaries located extrastaminally at the base of the insertion sites of short and long stamens. Like other Brassicaceae, the Nectaries have open stomates on their surface, which likely serve as the sites of nectar secretion. The Nectaries produce four distinct nectar droplets that accumulate in concave structures at the base of each of the four petals. To understand the molecular biology of the pennycress nectary, RNA was isolated from ‘immature’ (pre-secretory) and ‘mature’ (secretory) Nectaries and subjected to RNA-seq. Approximately 184 M paired-end reads (368 M total reads) were de novo assembled into a total of 16,074 independent contigs, which mapped to 12,335 unique genes in the pennycress genome. Nearly 3700 genes were found to be differentially expressed between immature and mature Nectaries and subjected to gene ontology and metabolic pathway analyses. Lastly, in silico analyses identified 158 pennycress orthologs to Arabidopsis genes with known enriched expression in Nectaries. These nectary-enriched expression patterns were verified for select pennycress loci by semi-quantitative RT-PCR. Pennycress Nectaries are unique relative to those of other agriculturally important Brassicaceae, as they contain four equivalent Nectaries that present their nectar in specialized cup-shaped structures at the base of the petals. In spite of these morphological differences, the genes underlying the regulation and production of nectar appear to be largely conserved between pennycress and Arabidopsis thaliana. These results provide a starting point for using forward and reverse genetics approaches to enhance nectar synthesis and secretion in pennycress.
Brian W Kram - One of the best experts on this subject based on the ideXlab platform.
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identification of differential gene expression in brassica rapa Nectaries through expressed sequence tag analysis
PLOS ONE, 2010Co-Authors: Marshall Hampton, Brian W Kram, Emily M Chambers, Jerad S Ehrnriter, Jonathan H Gralewski, Teresa Joyal, Clay J. CarterAbstract:Background Nectaries are the floral organs responsible for the synthesis and secretion of nectar. Despite their central roles in pollination biology, very little is understood about the molecular mechanisms underlying nectar production. This project was undertaken to identify genes potentially involved in mediating nectary form and function in Brassica rapa. Methodology and Principal Findings Four cDNA libraries were created using RNA isolated from the median and lateral Nectaries of B. rapa flowers, with one normalized and one non-normalized library being generated from each tissue. Approximately 3,000 clones from each library were randomly sequenced from the 5′ end to generate a total of 11,101 high quality expressed sequence tags (ESTs). Sequence assembly of all ESTs together allowed the identification of 1,453 contigs and 4,403 singleton sequences, with the Basic Localized Alignment Search Tool (BLAST) being used to identify 4,138 presumptive orthologs to Arabidopsis thaliana genes. Several genes differentially expressed between median and lateral Nectaries were initially identified based upon the number of BLAST hits represented by independent ESTs, and later confirmed via reverse transcription polymerase chain reaction (RT PCR). RT PCR was also used to verify the expression patterns of eight putative orthologs to known Arabidopsis nectary-enriched genes. Conclusions/Significance This work provided a snapshot of gene expression in actively secreting B. rapa Nectaries, and also allowed the identification of differential gene expression between median and lateral Nectaries. Moreover, 207 orthologs to known nectary-enriched genes from Arabidopsis were identified through this analysis. The results suggest that genes involved in nectar production are conserved amongst the Brassicaceae, and also supply clones and sequence information that can be used to probe nectary function in B. rapa.
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cell wall invertase 4 is required for nectar production in arabidopsis
Journal of Experimental Botany, 2010Co-Authors: Jeffrey M Ruhlmann, Brian W Kram, Clay J. CarterAbstract:To date, no genes have been reported to directly affect the de novo production of floral nectar. In an effort to identify genes involved in nectar production, the Affymetrix® ATH1 GeneChip was previously used to examine global gene expression profiles in Arabidopsis thaliana Nectaries. One of the genes displaying highly enriched expression in Nectaries was CELL WALL INVERTASE 4 (AtCWINV4, At2g36190), which encodes an enzyme that putatively catalyses the hydrolysis of sucrose into glucose and fructose. RT-PCR was used to confirm the nectary-enriched expression of AtCWINV4, as well as an orthologue from Brassica rapa. To probe biological function, two independent Arabidopsis cwinv4 T-DNA mutants were isolated. Unlike wild-type plants, cwinv4 lines did not produce nectar. While overall nectary morphology appeared to be normal, cwinv4 flowers accumulated higher than normal levels of starch in the receptacle, but not within the Nectaries themselves. Conversely, wild-type, but not cwinv4, nectarial stomata stained intensely for starch. Cell wall extracts prepared from mutant flowers displayed greatly reduced invertase activity when compared with wild-type plants, and cwinv4 flowers also accumulated significantly lower levels of total soluble sugar. Cumulatively, these results implicate CWINV4 as an absolutely required factor for nectar production in the Brassicaceae, specifically by maintaining constant sink status within Nectaries, thus allowing them to accumulate the sugars necessary for nectar production. In addition, CWINV4 is probably responsible for the hexose-rich composition observed for many Brassicaceae nectars.
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Arabidopsis thaliana as a model for functional nectary analysis.
Sexual Plant Reproduction, 2009Co-Authors: Brian W Kram, Clay J. CarterAbstract:Nectaries and nectar have received much research attention for well over 200 years due to their central roles in plant-pollinator interactions. Despite this, only a few genes have demonstrated impacts on nectary development, and none have been reported to mediate de novo nectar production. This scarcity of information is largely due to the lack of a model that combines sizeable Nectaries, and high levels of nectar production, along with suitable genomics resources. For example, even though Arabidopsis thaliana has been useful for developmental studies, it has been largely overlooked as a model for studying nectary function due to the small size of its flowers. However, Arabidopsis Nectaries, along with those of related species, are quite operational and can be used to discern molecular mechanisms of nectary form and function. A current understanding of the machinery underlying nectary function in plants is briefly presented, with emphasis placed on the prospects of using Arabidopsis as a model for studying these processes.
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uncovering the arabidopsis thaliana nectary transcriptome investigation of differential gene expression in floral nectariferous tissues
BMC Plant Biology, 2009Co-Authors: Brian W Kram, Clay J. CarterAbstract:Many flowering plants attract pollinators by offering a reward of floral nectar. Remarkably, the molecular events involved in the development of Nectaries, the organs that produce nectar, as well as the synthesis and secretion of nectar itself, are poorly understood. Indeed, to date, no genes have been shown to directly affect the de novo production or quality of floral nectar. To address this gap in knowledge, the ATH1 Affymetrix® GeneChip array was used to systematically investigate the Arabidopsis nectary transcriptome to identify genes and pathways potentially involved in nectar production. In this study, we identified a large number of genes differentially expressed between secretory lateral Nectaries and non-secretory median nectary tissues, as well as between mature lateral Nectaries (post-anthesis) and immature lateral Nectaries (pre-anthesis). Expression within Nectaries was also compared to thirteen non-nectary reference tissues, from which 270 genes were identified as being significantly upregulated in Nectaries. The expression patterns of 14 nectary-enriched genes were also confirmed via RT PCR. Upon looking into functional groups of upregulated genes, pathways involved in gene regulation, carbohydrate metabolism, and lipid metabolism were particularly enriched in Nectaries versus reference tissues. A large number of genes preferentially expressed in Nectaries, as well as between nectary types and developmental stages, were identified. Several hypotheses relating to mechanisms of nectar production and regulation thereof are proposed, and provide a starting point for reverse genetics approaches to determine molecular mechanisms underlying nectar synthesis and secretion.
