Metabolic Engineering in Plants to Control Source/sink Relationship and Biomass Distribution

Metabolic Engineering in Plants to Control Source/sink Relationship and Biomass Distribution

Date: August 2013
Creator: Lahiri, Ipsita
Description: Traditional methods like pruning and breeding have historically been used in crop production to divert photoassimilates to harvested organs, but molecular biotechnology is now poised to significantly increase yield by manipulating resource partitioning. It was hypothesized that metabolic engineering in targeted sink tissues can favor resource partitioning to increase harvest. Raffinose Family Oligosaccharides (RFOs) are naturally occurring oligosaccharides that are widespread in plants and are responsible for carbon transport, storage and protection against cold and drought stress. Transgenic plants (GRS47, GRS63) were engineered to generate and transport more RFOs through the phloem than the wild type plants. The transgenic lines produced more RFOs and the RFOs were also detected in their phloem exudates. But the 14CO2 labeling and subsequent thin layer chromatography analysis showed that the RFOs were most likely sequestered in an inactive pool and accumulate over time. Crossing GRS47 and GRS63 lines with MIPS1 plants (that produces more myo-inositol, a substrate in the RFO biosynthetic pathway) did not significantly increase the RFOs in the crossed lines. For future manipulation of RFO degradation in sink organs, the roles of the endogenous α-galactosidases were analyzed. The alkaline α-galactosidases (AtSIP1 and AtSIP2 in Arabidopsis) are most likely responsible for digesting RFOs ...
Contributing Partner: UNT Libraries
Metabolic Engineering of Raffinose-Family Oligosaccharides in the Phloem Reveals Alterations in Patterns of Carbon Partitioning and Enhances Resistance to Green Peach Aphid

Metabolic Engineering of Raffinose-Family Oligosaccharides in the Phloem Reveals Alterations in Patterns of Carbon Partitioning and Enhances Resistance to Green Peach Aphid

Date: August 2010
Creator: Cao, Te
Description: Phloem transport is along hydrostatic pressure gradients generated by differences in solute concentration between source and sink tissues. Numerous species accumulate raffinose-family oligosaccharides (RFOs) in the phloem of mature leaves to accentuate the pressure gradient between source and sinks. In this study, metabolic engineering was used to generate RFOs at the inception of the translocation stream of Arabidopsis thaliana, which transports predominantly sucrose. To do this, three genes, GALACTINOL SYNTHASE, RAFFINOSE SYNTHASE and STACHYOSE SYNTHASE, were expressed from promoters specific to the companion cells of minor veins. Two transgenic lines homozygous for all three genes (GRS63 and GRS47) were selected for further analysis. Sugars were extracted and quantified by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and 21-day old plants of both lines had levels of galactinol, raffinose, and stachyose approaching 50% of total soluble sugar. All three exotic sugars were also identified in phloem exudates from excised leaves of transgenic plants whereas levels were negligible in exudates from wild type leaves. Differences in starch accumulation or degradation between wild type and GRS63 and GRS47 lines were not observed. Similarly, there were no differences in vegetative growth between wild type and engineered plants, but engineered plants flowered ...
Contributing Partner: UNT Libraries
Molecular and Functional Characterization of Medicago Truncatula Npf17 Gene

Molecular and Functional Characterization of Medicago Truncatula Npf17 Gene

Access: Use of this item is restricted to the UNT Community.
Date: December 2013
Creator: Salehin, Mohammad
Description: Legumes are unique among plants for their ability to fix atmospheric nitrogen with the help of soil bacteria rhizobia. Medicago truncatula is used as a model legume to study different aspects of symbiotic nitrogen fixation. M. truncatula, in association with its symbiotic partner Sinorhizobium meliloti, fix atmospheric nitrogen into ammonia, which the plant uses for amino acid biosynthesis and the bacteria get reduced photosynthate in return. M. truncatula NPF1.7 previously called MtNIP/LATD is required for symbiotic nitrogen fixing root nodule development and for normal root architecture. Mutations in MtNPF1.7 have defects in these processes. MtNPF1.7 encodes a member of the NPF family of transporters. Experimental results showing that MtNPF1.7 functioning as a high-affinity nitrate transporter are its expression restoring chlorate susceptibility to the Arabidopsis chl1-5 mutant and high nitrate transport in Xenopus laevis oocyte system. However, the weakest Mtnip-3 mutant allele also displays high-affinity nitrate transport in X. laevis oocytes and chlorate susceptibility to the Atchl1-5 mutant, suggesting that MtNPF1.7 might have another biochemical function. Experimental evidence shows that MtNPF1.7 also functions in hormone signaling. Constitutive expression of MtNPF1.7 in several species including M. truncatula results in plants with a robust growth phenotype. Using a synthetic auxin reporter, the presence ...
Contributing Partner: UNT Libraries
Molecular Basis of Plant Defense Against Aphids: Role of the Arabidopsis Thaliana PAD4 and MPL1 Genes

Molecular Basis of Plant Defense Against Aphids: Role of the Arabidopsis Thaliana PAD4 and MPL1 Genes

Date: August 2011
Creator: Louis, Joe
Description: Myzus persicae (Sülzer), commonly known as green peach aphid (GPA), utilizes its slender stylet to penetrate the plant tissues intercellularly and consume copious amounts of photoassimilates present in the phloem sap causing extensive damage to host plants. The compatible interaction between GPA and Arabidopsis thaliana enabled us to characterize plant response to aphid infestation. Upon GPA infestation, Arabidopsis PAD4 (PHYTOALEXIN DEFICIENT4) gene modulates premature leaf senescence, which is involved in the programmed degradation of cellular components and the export of nutrients out of the senescing leaf. Senescence mechanism is utilized by plants to limit aphid growth. In addition, PAD4 provides antixenosis (deters insect settling and feeding) and antibiosis (impair aphid fecundity) against GPA and adversely impact sieve element availability to GPA. Basal expression of PAD4 contributes to antibiosis, and the GPA-induced expression of PAD4 contributes to antixenosis. Mutation in the Arabidopsis stearoyl-ACP desaturase encoding SSI2 (suppressor of SALICYLIC ACID [SA] insensitivity2) gene that results in an accelerated cell death phenotype and dwarfing, also conferred heightened antibiosis to GPA. Results of this study indicate that PAD4 is required for the ssi2-mediated enhanced antibiosis to GPA. The PAD4 protein contains conserved Ser, Asp and His residues that form the catalytic triad of ...
Contributing Partner: UNT Libraries
Manipulating Sucrose Proton Symporters to Understand Phloem Loading

Manipulating Sucrose Proton Symporters to Understand Phloem Loading

Date: August 2013
Creator: Dasgupta, Kasturi
Description: Phloem vascular tissues transport sugars synthesized by photosynthesis in mature leaves by a process called phloem loading in source tissues and unloading in sink tissues. Phloem loading in source leaves is catalyzed by Suc/H+ symporters (SUTs) which are energized by proton motive force. In Arabidopsis the principal and perhaps exclusive SUT catalyzing phloem loading is AtSUC2. In mutant plants harboring a T-DNA insertion in each of the functional SUT-family members, only Atsuc2 mutants demonstrate overtly debilitated phloem transport. Analysis of a mutant allele (Atsuc2-4) of AtSUC2 with a T-DNA insertion in the second intron showed severely stunted phenotype similar to previously analyzed Atsuc2 null alleles. However unlike previous alleles Atsuc2-4 produced viable seeds. Analysis of phloem specific promoters showed that promoter expression was regulated by Suc concentration. Unlike AtSUC2p, heterologous promoter CoYMVp was not repressed under high Suc conc. Further analysis was conducted using CoYMVp to test the capacity of diverse clades in SUT-gene family for transferring Suc in planta in Atsuc2 - / - mutant background. AtSUC1 and ZmSUT1 from maize complemented Atsuc2 mutant plants to the highest level compared to all other transporters. Over-expression of the above SUTs in phloem showed enhanced Suc loading and transport, but against ...
Contributing Partner: UNT Libraries
9-Lipoxygenase Oxylipin Pathway in Plant Response to Biotic Stress

9-Lipoxygenase Oxylipin Pathway in Plant Response to Biotic Stress

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Date: May 2012
Creator: Nalam, Vamsi J.
Description: The activity of plant 9-lipoxygenases (LOXs) influences the outcome of Arabidopsis thaliana interaction with pathogen and insects. Evidence provided here indicates that in Arabidopsis, 9-LOXs facilitate infestation by Myzus persicae, commonly known as the green peach aphid (GPA), a sap-sucking insect, and infection by the fungal pathogen Fusarium graminearum. in comparison to the wild-type plant, lox5 mutants, which are deficient in a 9-lipoxygenase, GPA population was smaller and the insect spent less time feeding from sieve elements and xylem, thus resulting in reduced water content and fecundity of GPA. LOX5 expression is induced rapidly in roots of GPA-infested plants. This increase in LOX5 expression is paralleled by an increase in LOX5-synthesized oxylipins in the root and petiole exudates of GPA-infested plants. Micrografting experiments demonstrated that GPA population size was smaller on plants in which the roots were of the lox5 mutant genotype. Exogenous treatment of lox5 mutant roots with 9-hydroxyoctadecanoic acid restored water content and population size of GPA on lox5 mutants. Together, these results suggest that LOX5 genotype in roots is critical for facilitating insect infestation of Arabidopsis. in Arabidopsis, 9-LOX function is also required for facilitating infection by F. graminearum, which is a leading cause of Fusarium head ...
Contributing Partner: UNT Libraries
Identification of Three Symbiosome Targeting Domains in the MtENOD8 Protein and Cell-to-cell MtENOD8 mRNA Movement in Nodules

Identification of Three Symbiosome Targeting Domains in the MtENOD8 Protein and Cell-to-cell MtENOD8 mRNA Movement in Nodules

Access: Use of this item is restricted to the UNT Community.
Date: May 2012
Creator: Meckfessel, Matthew Harold
Description: The model legume, Medicago truncatula, is able to enter into a symbiotic relationship with soil bacteria, known as rhizobia. This relationship involves a carbon for nitrogen exchange in which the plant provides reduced carbon from photosynthesis in exchange for reduced, or “fixed” atmospheric nitrogen, which allows the plant to thrive in nitrogen depleted soils. Rhizobia infect and enter plant root organs, known as nodules, where they reside inside the plant cell in a novel organelle, known as the symbiosome where nitrogen fixation occurs. the symbiosome is enriched in plant proteins, however, little is known about the mechanisms that direct plant proteins to the symbiosome. Using the M. truncatula ENOD8 (MtENOD8) protein as a model to explore symbiosome protein targeting, 3-cis domains were identified within MtENOD8 capable of directing green fluorescent protein (GFP) to the symbiosome, including its N-terminal signal peptide (SP). the SP delivered GFP to the vacuole in the absence of nodules suggesting that symbiosome proteins share a common targeting pathway with vacuolar proteins. a time course analysis during nodulation indicated that there is a nodule specific redirection of MtENOD8-SP from the vacuole to the symbiosome in a MtNIP/LATD dependent manner. GFP expression by the MtENOD8 promoter revealed spatial ...
Contributing Partner: UNT Libraries
Studies on Plant-aphid Interactions: a Novel Role for Trehalose Metabolism in Arabidopsis Defense Against Green Peach Aphid

Studies on Plant-aphid Interactions: a Novel Role for Trehalose Metabolism in Arabidopsis Defense Against Green Peach Aphid

Date: May 2012
Creator: Singh, Vijay
Description: Myzus persicae (Sülzer), commonly known as the green peach aphid (GPA), is a polyphagous insect that can infest over 100 families of economically important plants and is major pest for vegetable crops. This study utilizes the Arabidopsis-GPA model system with the aim to elucidate the role of the plant disaccharide trehalose in providing defense against GPA. This study demonstrates a novel role for TPS11 in providing defense against GPA. TPS11 expression was found to be transiently induced in Arabidopsis plants in response to GPA infestation and the TPS11 gene was required for curtailing GPA infestation. TPS11, which encodes for trehalose phosphate synthase and phosphatase activities, contributes to the transient increase in trehalose in the GPA infested tissues. This work suggests that TPS11-dependent trehalose has a signaling function in plant defense against GPA. in addition, trehalose also has a more direct role in curtailing GPA infestation on Arabidopsis. This work also shows that TPS11 is able to modulate both carbohydrate metabolism and plant defenses in response to GPA infestation. the expression of PAD4, an Arabidopsis gene required for phloem-based defenses against GPA, was found to be delayed in GPA infested tps11 mutant plants along with increased sucrose levels and lower starch ...
Contributing Partner: UNT Libraries
Role of N-Acylethanolamines in Plant Defense Responses: Modulation by Pathogens and Commercial Antimicrobial Stressors

Role of N-Acylethanolamines in Plant Defense Responses: Modulation by Pathogens and Commercial Antimicrobial Stressors

Date: August 2010
Creator: Vadapalli, Vatsala
Description: N-acyl ethanolamines (NAEs) are a class of lipids recently recognized as signaling molecules which are controlled, in part, by their degradation by fatty acid amide hydrolase (FAAH). On the basis of previous studies indicating increased NAE levels in a tobacco cell suspension-xylanase elicitor exposure system and the availability of FAAH mutants, overexpressor and knockout (OE and KO) genotypes in Arabidopsis thaliana, further roles of NAEs in A. thaliana plant defense was investigated. The commonly occurring urban antimicrobial contaminant triclosan (TCS) has been shown to suppress lipid signaling associated with plant defense responses. Thus, a second objective of this study was to determine if TCS exposure specifically interferes with NAE levels. No changes in steady state NAE profiles in A. thaliana-Pseudomonas syringae pv. syringae and A. thaliana-flagellin (bacterial peptide, flg22) challenge systems were seen despite evidence that defense responses were activated in these systems. There was a significant drop in enoyl-ACP reductase (ENR) enzyme activity, which catalyzes the last step in the fatty acid biosynthesis pathway in plants, on exposure of the seedlings to TCS at 10 ppm for 24 h and decreased reactive oxygen species (ROS) production due to flg22 in long term exposure of 0.1 ppm and short term ...
Contributing Partner: UNT Libraries
Functional Characterization of Mtnip/latd’s Biochemical and Biological Function

Functional Characterization of Mtnip/latd’s Biochemical and Biological Function

Access: Use of this item is restricted to the UNT Community.
Date: December 2013
Creator: Bagchi, Rammyani
Description: Symbiotic nitrogen fixation occurs in plants harboring nitrogen-fixing bacteria within the plant tissue. The most widely studied association is between the legumes and rhizobia. In this relationship the plant (legumes) provides the bacteria (rhizobia) with reduced carbon derived from photosynthesis in exchange for reduced atmospheric nitrogen. This allows the plant to survive in soil, which is low in available of nitrogen. Rhizobia infect and enter plant root and reside in organs known as nodules. In the nodules the bacteria fix atmospheric nitrogen. The association between the legume, Medicago truncatula and the bacteria Sinorhizobium meliloti, has been studied in detail. Medicago mutants that have defects in nodulation help us understand the process of nitrogen fixation better. One such mutant is the Mtnip-1. Mtnip-1 plants respond to S. meliloti by producing abnormal nodules in which numerous aberrant infection threads are produced, with very rare rhizobial release into host plant cells. The mutant plant Mtnip-1 has an abnormal defense-like response in root nodules as well as defects in lateral root development. Three alleles of the Mtnip/latd mutants, Mtnip-1, Mtlatd and Mtnip-3 show different degrees of severity in their phenotype. Phylogenetic analysis showed that MtNIP/LATD encodes a protein belonging to the NRT1(PTR) family of ...
Contributing Partner: UNT Libraries
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