Altered expression of SELF-PRUNING disrupts homeostasis and facilitates signal delivery to meristems.

Meristem maintenance, achieved through the highly conserved CLAVATA-WUSCHEL (CLV-WUS) regulatory circuit, is fundamental in balancing stem cell proliferation with cellular differentiation. Disruptions to meristem homeostasis can alter meristem size, leading to enlarged organs. Cotton (Gossypium spp.), the world's most important fiber crop, shows inherent variation in fruit size, presenting opportunities to explore the networks regulating meristem homeostasis and to impact fruit size and crop value. We identified and characterized the cotton orthologs of genes functioning in the CLV-WUS circuit. Using virus-based gene manipulation in cotton, we altered the expression of each gene to perturb meristem regulation and increase fruit size. Targeted alteration of individual components of the CLV-WUS circuit modestly fasciated flowers and fruits. Unexpectedly, controlled expression of meristem regulator SELF-PRUNING (SP) increased the impacts of altered CLV-WUS expression on flower and fruit fasciation. Meristem transcriptomics showed SP and genes of the CLV-WUS circuit are expressed independently from each other, suggesting these gene products are not acting in the same path. Virus-induced silencing of GhSP facilitated the delivery of other signals to the meristem to alter organ specification. SP has a role in cotton meristem homeostasis, and changes in GhSP expression increased access of virus-derived signals to the meristem.

Cotton phloem loads from the apoplast using a single member of its nine-member sucrose transporter gene family.

Phloem loading and transport are fundamental processes for allocating carbon from source organs to sink tissues. Cotton (Gossypium spp.) has a high sink demand for the cellulosic fibers that grow on the seed coat and for the storage reserves in the developing embryo, along with the demands of new growth in the shoots and roots. Addressing how cotton mobilizes resources from source leaves to sink organs provides insight into processes contributing to fiber and seed yield. Plasmodesmata frequencies between companion cells and flanking parenchyma in minor veins are higher than expected for an apoplastic loader, and cotton's close relatedness to Tilia spp. hints at passive loading. Suc was the only canonical transport sugar in leaves and constituted 87% of 14C-labeled photoassimilate being actively transported. [14C]Suc uptake coupled with autoradiography indicated active [14C]Suc accumulation in minor veins, suggesting Suc loading from the apoplast; esculin, a fluorescent Suc analog, did not accumulate in minor veins. Of the nine sucrose transporter (SUT) genes identified per diploid genome, only GhSUT1-L2 showed appreciable expression in mature leaves, and silencing GhSUT1-L2 yielded phenotypes characteristic of blocked phloem transport. Furthermore, only GhSUT1-L2 cDNA stimulated esculin and [14C]Suc uptake into yeast, and only the GhSUT1-L2 promoter caused uidA (ß-glucuronidase) reporter gene expression in minor vein phloem of Arabidopsis thaliana. Collectively, these results argue that apoplastic phloem loading mediated by GhSUT1-L2 is the dominant mode of phloem loading in cotton.

Cotton architecture: examining the roles of SINGLE FLOWER TRUSS and SELF-PRUNING in regulating growth habits of a woody perennial crop.

By specifying patterns of determinate and indeterminate growth, FLOWERING LOCUS T/SINGLE FLOWER TRUSS (SFT) and TERMINAL FLOWER 1/SELF-PRUNING (SP) regulate plant architecture. Though well characterized in Arabidopsis, the impacts of these genes on the architectures of diverse crops cultivated in different environments, and their potential to enhance crop productivity and management, are less well addressed. Cotton (Gossypium spp.) is naturally a short-day photoperiodic perennial that is now grown primarily as a day-neutral, annual row crop. Different environments and cultivation practices favor specific growth habits to optimize yield, and in cotton, especially in regions that rely heavily on mechanized harvest, the trend has been to more determinate varieties. Identifying and functionally characterizing SFT and SP homologs in cotton uncovered new aspects of how ratios of indeterminate and determinate growth are balanced, and unraveling their genetic networks emphasized how broadly these gene products affect cotton growth habits.

SINGLE FLOWER TRUSS and SELF-PRUNING signal developmental and metabolic networks to guide cotton architectures.

Patterns of indeterminate and determinate growth specify plant architecture and influence crop productivity. In cotton (Gossypium hirsutum), SINGLE FLOWER TRUSS (SFT) stimulates the transition to flowering and determinate growth, while its closely related antagonist SELF-PRUNING (SP) maintains meristems in indeterminate states to favor vegetative growth. Overexpressing GhSFT while simultaneously silencing GhSP produces highly determinate cotton with reduced foliage and synchronous fruiting. These findings suggest that GhSFT, GhSP, and genes in these signaling networks hold promise for enhancing 'annualized' growth patterns and improving cotton productivity and management. To identify the molecular programs underlying cotton growth habits, we used comparative co-expression networks, differential gene expression, and phenotypic analyses in cotton varieties expressing altered levels of GhSFT or GhSP. Using multiple cotton and tomato datasets, we identified diverse genetic modules highly correlated with SFT or SP orthologs which shared related Gene Ontologies in different crop species. Notably, altering GhSFT or GhSP levels in cotton affected the expression of genes regulating meristem fate and metabolic pathways. Further phenotypic analyses of gene products involved in photosynthesis, secondary metabolism, and cell wall biosynthesis showed that early changes in GhSFT and GhSP levels profoundly impacted later development in distal tissues. Identifying the molecular underpinnings of GhSFT and GhSP activities emphasizes their broad actions in regulating cotton architecture.

Improved Yield and Photosynthate Partitioning in AVP1 Expressing Wheat (Triticum aestivum) Plants.

A fundamental factor to improve crop productivity involves the optimization of reduced carbon translocation from source to sink tissues. Here, we present data consistent with the positive effect that the expression of the Arabidopsis thaliana H+-PPase (AVP1) has on reduced carbon partitioning and yield increases in wheat. Immunohistochemical localization of H+-PPases (TaVP) in spring wheat Bobwhite L. revealed the presence of this conserved enzyme in wheat vasculature and sink tissues. Of note, immunogold imaging showed a plasma membrane localization of TaVP in sieve element- companion cell complexes of Bobwhite source leaves. These data together with the distribution patterns of a fluorescent tracer and [U14C]-sucrose are consistent with an apoplasmic phloem-loading model in wheat. Interestingly, 14C-labeling experiments provided evidence for enhanced carbon partitioning between shoots and roots, and between flag leaves and milk stage kernels in AVP1 expressing Bobwhite lines. In keeping, there is a significant yield improvement triggered by the expression of AVP1 in these lines. Green house and field grown transgenic wheat expressing AVP1 also produced higher grain yield and number of seeds per plant, and exhibited an increase in root biomass when compared to null segregants. Another agriculturally desirable phenotype showed by AVP1 Bobwhite plants is a robust establishment of seedlings.

Assessing Long-Distance Carbon Partitioning from Photosynthetic Source Leaves to Heterotrophic Sink Organs with Photoassimilated [14C]CO2.

Phloem loading and long-distance transport of photoassimilate from source leaves to sink organs are essential physiological processes that contribute to plant growth and yield. At a minimum, three steps are involved: phloem loading in source organs, transport along the phloem path, and phloem unloading in sink organs. Each of these can have variable rates contingent on the physiological state of the plant, and thereby influence the overall transport rate. In addition to these phloem transport steps, rates of photosynthesis and photosynthate movement in the pre-phloem path, as well as photosynthate utilization in post phloem tissues of sink organs also contribute to phloem transport. The protocol described here estimates carbon allocation along the entire path from initial carbon fixation to delivery to sink organs after a labeling pulse: [14C]CO2 is photoassimilated in source leaves and loading and transport of the 14C label to heterotrophic sink organs (roots) is quantified by scintillation counting. This method is flexible and can be adapted to quantify long-distance transport in many plant species.

Cotton CENTRORADIALIS/TERMINAL FLOWER 1/SELF-PRUNING genes functionally diverged to differentially impact plant architecture.

Genes of the CENTRORADIALIS/TERMINAL FLOWER 1/SELF-PRUNING (CETS) family influence meristem identity by controlling the balance between indeterminate and determinate growth, thereby profoundly impacting plant architecture. Artificial selection during cotton (Gossypium hirsutum) domestication converted photoperiodic trees to the day-neutral shrubs widely cultivated today. To understand the regulation of cotton architecture and exploit these principles to enhance crop productivity, we characterized the CETS gene family from tetraploid cotton. We demonstrate that genes of the TERMINAL FLOWER 1 (TFL1)-like clade show different roles in regulating growth patterns. Cotton has five TFL1-like genes: SELF-PRUNING (GhSP) is a single gene whereas there are two TFL1-like and BROTHER OF FT (BFT)-like genes, and these duplications are specific to the cotton lineage. All genes of the cotton TFL1-like clade delay flowering when ectopically expressed in transgenic Arabidopsis, with the strongest phenotypes failing to produce functional flowers. GhSP, GhTFL1-L2, and GhBFT-L2 rescue the early flowering Attfl1-14 mutant phenotype, and the encoded polypeptides interact with a cotton FD protein. Heterologous promoter::GUS fusions illustrate differences in the regulation of these genes, suggesting that genes of the GhTFL1-like clade may not act redundantly. Characterizations of the GhCETS family provide strategies for nuanced control of plant growth.

Elicitors and defense gene induction in plants with altered lignin compositions.

A reduction in the lignin content in transgenic plants induces the ectopic expression of defense genes, but the importance of altered lignin composition in such phenomena remains unclear. Two Arabidopsis lines with similar lignin contents, but strikingly different lignin compositions, exhibited different quantitative and qualitative transcriptional responses. Plants with lignin composed primarily of guaiacyl units overexpressed genes responsive to oomycete and bacterial pathogen attack, whereas plants with lignin composed primarily of syringyl units expressed a far greater number of defense genes, including some associated with cis-jasmone-mediated responses to aphids; these plants exhibited altered responsiveness to bacterial and aphid inoculation. Several of the defense genes were differentially induced by water-soluble extracts from cell walls of plants of the two lines. Glycome profiling, fractionation and enzymatic digestion studies indicated that the different lignin compositions led to differential extractability of a range of heterogeneous oligosaccharide epitopes, with elicitor activity originating from different cell wall polymers. Alteration of lignin composition affects interactions with plant cell wall matrix polysaccharides to alter the sequestration of multiple latent defense signal molecules with an impact on biotic stress responses.

Assessing Rates of Long-distance Carbon Transport in Arabidopsis by Collecting Phloem Exudations into EDTA Solutions after Photosynthetic Labeling with [14C]CO2.

Phloem loading and transport of photoassimilate from photoautotrophic source leaves to heterotrophic sink organs are essential physiological processes that help the disparate organs of a plant function as a single, unified organism. We present three protocols we routinely use in combination with each other to assess (1) the relative rates of sucrose (Suc) loading into the phloem vascular system of mature leaves ( Yadav et al., 2017a ), (2) the relative rates of carbon loading and transport through the phloem (this protocol), and (3) the relative rates of carbon unloading into heterotrophic sink organs, specifically roots, after long-distance transport ( Yadav et al., 2017b ), We propose that conducting all three protocols on experimental and control plants provides a reliable comparison of whole-plant carbon partitioning, and minimizes ambiguities associated with a single protocol conducted in isolation ( Dasgupta et al., 2014 ; Khadilkar et al., 2016 ). In this protocol, [14C]CO2 is photoassimilated in source leaves and phloem loading and transport of photoassimilate is quantified by collecting phloem exudates into an EDTA solution followed by scintillation counting.

Assessing Long-distance Transport from Photosynthetic Source Leaves to Heterotrophic Sink Organs with [14C]CO2.

Phloem loading and transport of photoassimilate from photoautotrophic source leaves to heterotrophic sink organs are essential physiological processes that help the disparate organs of a plant function as a single, unified organism. We present three protocols we routinely use in combination with each other to assess (1) the relative rates of sucrose (Suc) loading into the phloem vascular system of mature leaves ( Yadav et al., 2017a ), (2) the relative rates of carbon loading and transport through the phloem ( Yadav et al., 2017b ), and (3) the relative rates of carbon unloading into heterotrophic sink organs, specifically roots, after long-distance transport (this protocol). We propose that conducting all three protocols on experimental and control plants provides a reliable comparison of whole-plant carbon partitioning, and minimizes ambiguities associated with a single protocol conducted in isolation ( Dasgupta et al., 2014 ; Khadilkar et al., 2016 ). In this protocol, [14C]CO2 is photoassimilated in source leaves and phloem loading and transport of the 14C label to heterotrophic sink organs, particularly roots, is quantified by scintillation counting. Using this protocol, we demonstrated that overexpression of sucrose transporters and a vacuolar proton pumping pyrophosphatase in the companion cells of Arabidopsis enhanced transport of 14C label photoassimilates to sink organs ( Dasgupta et al., 2014 ; Khadilkar et al., 2016 ). This method can be adapted to quantify long-distance transport in other plant species.

Quantifying the Capacity of Phloem Loading in Leaf Disks with [14C]Sucrose.

Phloem loading and transport of photoassimilate from photoautotrophic source leaves to heterotrophic sink organs are essential physiological processes that help the disparate organs of a plant function as a single, unified organism. We present three protocols we routinely use in combination with each other to assess (1) the relative rates of sucrose (Suc) loading into the phloem vascular system of mature leaves (this protocol), (2) the relative rates of carbon loading and transport through the phloem ( Yadav et al., 2017a ), and (3) the relative rates of carbon unloading into heterotrophic sink organs, specifically roots, after long-distance transport ( Yadav et al., 2017b ). We propose that conducting all three protocols on experimental and control plants provides a reliable comparison of whole-plant carbon partitioning, and minimizes ambiguities associated with a single protocol conducted in isolation ( Dasgupta et al., 2014 ; Khadilkar et al., 2016 ). In this protocol, Arabidopsis leaf disks isolated from mature rosette leaves are infiltrated with a buffered solution containing [14C]Suc. Suc transporters (SUCs or SUTs) load Suc into the phloem and excess, unloaded Suc in the leaf disk is then washed away. Loading of labeled Suc into the veins is visualized by autoradiography of lyophilized leaf disks and quantified by scintillation counting. Results are expressed as disintegration per minute per unit of leaf disk fresh weight or area.

Monopodial and sympodial branching architecture in cotton is differentially regulated by the Gossypium hirsutum SINGLE FLOWER TRUSS and SELF-PRUNING orthologs.

Domestication of upland cotton (Gossypium hirsutum) converted it from a lanky photoperiodic perennial to a day-neutral annual row-crop. Residual perennial traits, however, complicate irrigation and crop management, and more determinate architectures are desired. Cotton simultaneously maintains robust monopodial indeterminate shoots and sympodial determinate shoots. We questioned if and how the FLOWERING LOCUS T/SINGLE FLOWER TRUSS (SFT)-like and TERMINAL FLOWER1/SELF-PRUNING (SP)-like genes control the balance of monopodial and sympodial growth in a woody perennial with complex growth habit. Virus-based manipulation of GhSP and GhSFT expression enabled unprecedented functional analysis of cotton development. GhSP maintains growth in all apices; in its absence, both monopodial and sympodial branch systems terminate precociously. GhSFT encodes a florigenic signal stimulating rapid onset of sympodial branching and flowering in side shoots of wild photoperiodic and modern day-neutral accessions. High florigen concentrations did not alter monopodial apices, implying that once a cotton apex is SP-determined, it cannot be reset by florigen. GhSP is also essential to establish and maintain cambial activity. Dynamic changes in GhSFT and GhSP levels navigate meristems between monopodial and sympodial programs in a single plant. SFT and SP influenced cotton domestication and are ideal targets for further agricultural optimization.

Constitutive and Companion Cell-Specific Overexpression of AVP1, Encoding a Proton-Pumping Pyrophosphatase, Enhances Biomass Accumulation, Phloem Loading, and Long-Distance Transport.

Plant productivity is determined in large part by the partitioning of assimilates between the sites of production and the sites of utilization. Proton-pumping pyrophosphatases (H(+)-PPases) are shown to participate in many energetic plant processes, including general growth and biomass accumulation, CO2 fixation, nutrient acquisition, and stress responses. H(+)-PPases have a well-documented role in hydrolyzing pyrophosphate (PPi) and capturing the released energy to pump H(+) across the tonoplast and endomembranes to create proton motive force (pmf). Recently, an additional role for H(+)-PPases in phloem loading and biomass partitioning was proposed. In companion cells (CCs) of the phloem, H(+)-PPases localize to the plasma membrane rather than endomembranes, and rather than hydrolyzing PPi to create pmf, pmf is utilized to synthesize PPi. Additional PPi in the CCs promotes sucrose oxidation and ATP synthesis, which the plasma membrane P-type ATPase in turn uses to create more pmf for phloem loading of sucrose via sucrose-H(+) symporters. To test this model, transgenic Arabidopsis (Arabidopsis thaliana) plants were generated with constitutive and CC-specific overexpression of AVP1, encoding type 1 ARABIDOPSIS VACUOLAR PYROPHOSPHATASE1. Plants with both constitutive and CC-specific overexpression accumulated more biomass in shoot and root systems. (14)C-labeling experiments showed enhanced photosynthesis, phloem loading, phloem transport, and delivery to sink organs. The results obtained with constitutive and CC-specific promoters were very similar, such that the growth enhancement mediated by AVP1 overexpression can be attributed to its role in phloem CCs. This supports the model for H(+)-PPases functioning as PPi synthases in the phloem by arguing that the increases in biomass observed with AVP1 overexpression stem from improved phloem loading and transport.

Transgenic approaches to altering carbon and nitrogen partitioning in whole plants: assessing the potential to improve crop yields and nutritional quality.

The principal components of plant productivity and nutritional value, from the standpoint of modern agriculture, are the acquisition and partitioning of organic carbon (C) and nitrogen (N) compounds among the various organs of the plant. The flow of essential organic nutrients among the plant organ systems is mediated by its complex vascular system, and is driven by a series of transport steps including export from sites of primary assimilation, transport into and out of the phloem and xylem, and transport into the various import-dependent organs. Manipulating C and N partitioning to enhance yield of harvested organs is evident in the earliest crop domestication events and continues to be a goal for modern plant biology. Research on the biochemistry, molecular and cellular biology, and physiology of C and N partitioning has now matured to an extent that strategic manipulation of these transport systems through biotechnology are being attempted to improve movement from source to sink tissues in general, but also to target partitioning to specific organs. These nascent efforts are demonstrating the potential of applied biomass targeting but are also identifying interactions between essential nutrients that require further basic research. In this review, we summarize the key transport steps involved in C and N partitioning, and discuss various transgenic approaches for directly manipulating key C and N transporters involved. In addition, we propose several experiments that could enhance biomass accumulation in targeted organs while simultaneously testing current partitioning models.

Arabidopsis type I proton-pumping pyrophosphatase expresses strongly in phloem, where it is required for pyrophosphate metabolism and photosynthate partitioning.

Phloem loading is a critical process in plant physiology. The potential of regulating the translocation of photoassimilates from source to sink tissues represents an opportunity to increase crop yield. Pyrophosphate homeostasis is crucial for normal phloem function in apoplasmic loaders. The involvement of Arabidopsis (Arabidopsis thaliana) type I proton-pumping pyrophosphatase (AVP1) in phloem loading was analyzed at genetic, histochemical, and physiological levels. A transcriptional AVP1 promoter::GUS fusion revealed phloem activity in source leaves. Ubiquitous AVP1 overexpression (35S::AVP1 cassette) enhanced shoot biomass, photoassimilate production and transport, rhizosphere acidification, and expression of sugar-induced root ion transporter genes (POTASSIUM TRANSPORTER2 [KUP2], NITRATE TRANSPORTER2.1 [NRT2.1], NRT2.4, and PHOSPHATE TRANSPORTER1.4 [PHT1.4]). Phloem-specific AVP1 overexpression (Commelina Yellow Mottle Virus promoter [pCOYMV]::AVP1) elicited similar phenotypes. By contrast, phloem-specific AVP1 knockdown (pCoYMV::RNAiAVP1) resulted in stunted seedlings in sucrose-deprived medium. We also present a promoter mutant avp1-2 (SALK046492) with a 70% reduction of expression that did not show severe growth impairment. Interestingly, AVP1 protein in this mutant is prominent in the phloem. Moreover, expression of an Escherichia coli-soluble pyrophosphatase in the phloem (pCoYMV::pyrophosphatase) of avp1-2 plants resulted in severe dwarf phenotype and abnormal leaf morphology. We conclude that the Proton-Pumping Pyrophosphatase AVP1 localized at the plasma membrane of the sieve element-companion cell complexes functions as a synthase, and that this activity is critical for the maintenance of pyrophosphate homeostasis required for phloem function.

Florigen and anti-florigen - a systemic mechanism for coordinating growth and termination in flowering plants.

Genetic studies in Arabidopsis established FLOWERING LOCUS T (FT) as a key flower-promoting gene in photoperiodic systems. Grafting experiments established unequivocal one-to-one relations between SINGLE FLOWER TRUSS (SFT), a tomato homolog of FT, and the hypothetical florigen, in all flowering plants. Additional studies of SFT and SELF PRUNING (SP, homolog of TFL1), two antagonistic genes regulating the architecture of the sympodial shoot system, have suggested that transition to flowering in the day-neutral and perennial tomato is synonymous with "termination." Dosage manipulation of its endogenous and mobile, graft-transmissible levels demonstrated that florigen regulates termination and transition to flowering in an SP-dependent manner and, by the same token, that high florigen levels induce growth arrest and termination in meristems across the tomato shoot system. It was thus proposed that growth balances, and consequently the patterning of the shoot systems in all plants, are mediated by endogenous, meristem-specific dynamic SFT/SP ratios and that shifts to termination by changing SFT/SP ratios are triggered by the imported florigen, the mobile form of SFT. Florigen is a universal plant growth hormone inherently checked by a complementary antagonistic systemic system. Thus, an examination of the endogenous functions of FT-like genes, or of the systemic roles of the mobile florigen in any plant species, that fails to pay careful attention to the balancing antagonistic systems, or to consider its functions in day-neutral or perennial plants, would be incomplete.

Expression of Sucrose Transporter cDNAs Specifically in Companion Cells Enhances Phloem Loading and Long-Distance Transport of Sucrose but Leads to an Inhibition of Growth and the Perception of a Phosphate Limitation.

Sucrose (Suc) is the predominant form of carbon transported through the phloem from source to sink organs and is also a prominent sugar for short-distance transport. In all streptophytes analyzed, Suc transporter genes (SUTs or SUCs) form small families, with different subgroups evolving distinct functions. To gain insight into their capacity for moving Suc in planta, representative members of each clade were first expressed specifically in companion cells of Arabidopsis (Arabidopsis thaliana) and tested for their ability to rescue the phloem-loading defect caused by the Suc transporter mutation, Atsuc2-4. Sequence similarity was a poor indicator of ability: Several genes with high homology to AtSUC2, some of which have phloem-loading functions in other eudicot species, did not rescue the Atsuc2-4 mutation, whereas a more distantly related gene, ZmSUT1 from the monocot Zea mays, did restore phloem loading. Transporter complementary DNAs were also expressed in the companion cells of wild-type Arabidopsis, with the aim of increasing productivity by enhancing Suc transport to growing sink organs and reducing Suc-mediated feedback inhibition on photosynthesis. Although enhanced Suc loading and long-distance transport was achieved, growth was diminished. This growth inhibition was accompanied by increased expression of phosphate (P) starvation-induced genes and was reversed by providing a higher supply of external P. These experiments suggest that efforts to increase productivity by enhancing sugar transport may disrupt the carbon-to-P homeostasis. A model for how the plant perceives and responds to changes in the carbon-to-P balance is presented.

Metabolic engineering of raffinose-family oligosaccharides in the phloem reveals alterations in carbon partitioning and enhances resistance to green peach aphid.

Many plants employ energized loading strategies to accumulate osmotically-active solutes into the phloem of source organs to accentuate the hydrostatic pressure gradients that drive the flow of water, nutrients and signals from source to sinks. Proton-coupled symport of sugars from the apoplasm into the phloem symplasm is the best studied phloem-loading mechanism. As an alternative, numerous species use a polymer trapping mechanism to load through symplasm: sucrose enters the phloem through specialized plasmodesmata and is converted to raffinose-family oligosaccharides (RFOs) which accumulate because of their larger size. In this study, metabolic engineering was used to generate RFOs at the inception of the translocation stream of Arabidopsis thaliana, which loads from the apoplasm and transports predominantly sucrose, and the fate of the sugars throughout the plant determined. 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. Three-week-old plants of both lines had RFO levels approaching 50% of total soluble sugar. RFOs were also identified in exudates from excised leaves of transgenic plants whereas levels were negligible in exudates from wild type (WT) leaves. Differences in starch accumulation between WT and GRS63 and GRS47 lines were not observed. Similarly, there were no differences in vegetative growth between WT and engineered plants, but the latter flowered slightly earlier. Finally, since the sugar composition of the translocation stream appeared altered, we tested for an impact on green peach aphid (Myzus persicae Sulzer) feeding. When given a choice between WT and transgenic plants, green peach aphids preferred settling on the WT plants. Furthermore, green peach aphid fecundity was lower on the transgenic plants compared to the WT plants. When added to an artificial diet, RFOs did not have a negative effect on aphid fecundity, suggesting that although aphid resistance in the transgenic plants is enhanced, it is not due to direct toxicity of RFO toward the insect.