Browsing by Subject "Nectar"

Now showing 1 - 4 of 4
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    Metabolic and Biochemical Analysis of Cucurbita Pepo Nectaries: Insights Into How Carbon and Nitrogen Metabolites Are Synthesized, Partitioned and Secreted
    (2019-06) Solhaug, Erik
    Floral nectar is a sugary solution produced by plants to entice pollinator visitation. Nectar contains a complex mixture of sugars and other compounds, such as amino acids. Although the mechanism of nectar production is somewhat well known from genetics studies in Arabidopsis, very little is known about how nectar metabolites are produced from a metabolic and biochemical perspective. We used Cucurbita pepo (squash) to examine how carbon and nitrogen metabolites are synthesized, partitioned, and secreted into nectar. Toward this end, we analyzed patterns of global transcript levels, followed by a more in-depth examination of enzymes and metabolites in nectar and nectaries. Here we show that C. pepo nectar is most likely secreted by an eccrine mechanism (facilitated diffusion of sucrose) similar to Arabidopsis. Next, we show evidence that sugar transported directly from the phloem, without prior storage as starch, is important for C. pepo nectar secretion. Similarly, we demonstrate a role for trehalose-based sugar signaling in regulating nectar production. Finally, we have found that nectaries undergo extreme anoxic conditions during nectar secretion and that nitrate reduction to nitric oxide and alanine metabolism are essential for regenerating NAD+ to maintain ATP synthesis for the energetically demanding process of nectar synthesis. These data improve our understanding of how nectar components are produced in an agronomically-relevant species with the potential for use as a model to help us gain insight into the biochemistry and metabolism of nectar secretion in flowering plants.
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    MNC1 Negatively Regulates Nectar Production through Auxin and Jasmonic Acid Response Pathways in Arabidopsis thaliana
    (2014-11) Jia, Mengyuan
    Many flowering plants offer a reward for pollinators in the form of nectar. Despite the central role of pollination in reproduction of plants and the considerable amount of energy a plant devotes to produce nectar, little is known of the molecular mechanism of nectar production and its regulation. Previous reports have suggested a significant role for the plant hormone auxin in regulating nectar production. Recent transcriptome studies have made it possible to focus research on several nectary-specific candidate genes with putative roles in the auxin response. In Arabidopsis thaliana this includes a gene termed MEDIAN NECTARY CUPIN 1 (MNC1; At1g74820), which is highly expressed in median nectaries. MNC1 silenced mutants (mnc1) showed more nectar production and increased auxin response activity while MNC1 overexpresser mutant (MNC1 T6) showed significantly less nectar production and less auxin response activity in nectaries. A comparative sequence analysis of proteins with known function shows that MNC1 is a germin-like protein belonging to the RmlC-like cupins superfamily. MNC1 also has a conserved zinc binding domain with known Auxin Binding Protein1. Thus, we hypothesize that MNC1 negatively regulates nectar production, likely through an auxin dependent pathway. PIN6 (At1g77110), an auxin transporter family protein, has been reported to be an auxin transporter localized to the ER modulating cytoplasmic free auxin concentration in nectaries. Arabidopsis thaliana mutant lines with different combinations of crossed target genes were used to understand the feedback mechanism of auxin, nectar production, PIN6, and MNC1 protein behind nectar regulation and production. Transformed Escherichia coli and Pichia pastoris, a methylotrophic yeast species, expressing MNC1 protein were employed for studying its biochemical nature, including auxin binding activity. Jasmonic acid (JA) was also suggested to be required for nectar production, with possible crosstalk to auxin in regulation of nectar production. COI1-independent JA response pathway was found to regulate nectar production by altering the expression of nectary-specific genes such as SWEET9, a sucrose transporter required for nectar production, CWINV4, a cell wall invertase required for nectar production, and MNC1. A potential auxin-JA crosstalk mechanism was constructed based on results in this study and previous studies.
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    A Sucrose Transporter and Proper Hormone Response are Essential for Nectary Function in the Brassicaceae
    (2013-12) Klinkenberg, Peter
    Nectar is a reward presented by flowers to attract pollinators to facilitate fertilization. While much is known about the chemical make-up of nectar, little is known about the mechanisms of production and secretion of this pollinator attractant. SWEET9, a nectary enriched gene, was demonstrated to be vital for nectar production in two Brassicaceae species, Arabidopsis thaliana and Brassica rapa as determined by SWEET9pro::GUS histochemical staining and RT-PCR,. The Arabidopsis mutant atsweet9-3, produced no nectar and three independent mutants in B. rapa, (brsweet9-1, -2, and -3) similarly produced no nectar. All four mutants had normal nectary morphology. Transporter assays of SWEET9 expressed in Xenopus oocytes displayed sucrose uniport activity, suggesting a direct role in sugar export. To determine a potential mechanism for the regulation of SWEET9 expression, the plant hormone jasmonic acid (JA) was investigated because it was previously implicated in nectary function. Indeed, JA synthesis (aos-2 and dad1) and response (myb21-4) mutants displayed an absence of floral nectar, in addition to male-sterility. When treated with exogenous MeJA, aos-2 and dad1 mutants regained their nectar production and fertility, while the myb21-4 transcription factor mutant was insensitive to treatment. Significantly, SWEET9 expression was strongly decreased in the JA response mutant myb21-4, in addition to several other genes known to be important in nectary function. For example, all three JA mutants studied displayed decreased expression of PIN6, a nectary enriched gene required for proper auxin homeostasis in the nectaries of Arabidopsis. Additionally auxin response was lost in the JA synthesis mutant aos-2, suggesting an important hormonal crosstalk between JA and auxin. To further investigate the link between JA and the auxin response in nectaries, mutants with altered endogenous auxin levels were created. Mutants with decreased nectary auxin produced 50% less nectar than wild-type plants and had reduced auxin response. Cumulatively, these results identify SWEET9 as a sucrose transporter required for nectar production and that JA plays a major role in the regulation of nectary-specific genes and other hormonal pathways important for nectar production.
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    Understanding and engineering the molecular regulation of nectar production in field pennycress (Thlaspi arvense).
    (2020-09) Thomas, Jason
    Anthropogenic climate change and the growing world population are putting pressure on our agroecosystems. Sustainable farming efforts are needed when intensive agriculture systems are fallow and prone to erosion and nutrient leaching. We can mitigate these issues while increasing farmer income by planting field pennycress (Thlaspi arvense). Pennycress forms penny shaped pods containing oil-rich seeds with diverse uses from jet fuel to cooking oil. As an overwintering cover crop, pennycress grows from fall to late spring, avoiding land-use competition with summer annual cash crops. Pennycress provides an ecosystem service to pollinator populations, which are needed for fruit and vegetable production. Pollinators are suffering due partially to losses of habitat and floral resources. Fortunately, pennycress flowers provide nectar as a floral resource when most agricultural landscapes are barren. Additionally, pollinator visitation increases pennycress seed yield. Therefore, the purpose of the study was to understand the genetics behind nectar production and develop pennycress plants with altered floral traits. Microscopy was performed on pennycress flowers to characterize the structure of the nectar-producing glands called nectaries. Additionally, pennycress nectary transcriptomes were determined using transcriptomic sequencing which led to the identification of genes and metabolic pathways. In both cases, the strong similarity in nectar production was confirmed between pennycress and Arabidopsis thaliana, a model plant and close pennycress relative. Because of the close relationship, it was possible to characterize Arabidopsis genes that can later be used to find orthologs in pennycress. The Arabidopsis gene At5g60760 annotated as ‘a P-LOOP containing nucleoside triphosphate hydrolases superfamily protein’ (hereafter AT5G60760) is a putative inositol kinase highly expressed in nectaries. Through assaying mutant AT5G60760 gene expression and nectar production, it was determined that AT5G60760 negatively regulates nectar production. By using findings from nectary genetics, such as the function of AT5G60760 and the pennycress nectary transcriptome, 13 genes were mutated using CRISPR/Cas9 to alter traits relating to pollinator attraction in field pennycress. We have identified two homozygous mutant lines and conducted phenotyping. The auxin response factor 8 (arf8) mutant flowers and petals are larger and produce more nectar than wild type. The cell wall invertase 4 (cwinv4) mutants do not produce nectar and have greatly reduced invertase activity in nectaries. In the future, these plants can be grown in field settings to test pollinator attraction, assay pollinator health, and measure pennycress seed yield.