Daniel R. Bush

Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem

A proton–sucrose symporter mediates the key step in carbon export from leaves of most plants. Sucrose transport activity and steady-state mRNA levels of BvSUT1, a sugar beet leaf sucrose symporter, are negatively regulated specifically by sucrose. Results reported here show that BvSUT1 mRNA was localized to companion cells of the leafs vascular system, which supports its role in the systemic distribution of photoassimilate. Immunoblot analysis showed that decreased transport activity was caused by a reduction in the abundance of symporter protein. RNA gel blot analysis of the leaf symporter revealed that message levels also declined, and nuclear run-on experiments demonstrated that this was the result of decreased transcription. Further analysis showed that symporter protein and message are both degraded rapidly. Taken together, these data show that phloem loading is regulated by means of sucrose-mediated changes in transcription of a phloem-specific sucrose symporter gene in a regulatory system that may play a pivotal role in balancing photosynthetic activity with resource utilization.

Carbohydrate Export from the Leaf: A Highly Regulated Process and Target to Enhance Photosynthesis and Productivity

The phloem is a central component of the plant9s complex vascular system that plays a vital role in moving photoassimilates from sites of primary acquisition to the heterotrophic tissues and organs of the plant. Indeed, as much as 50-80% of the CO2 photoassimilated in a mature leaf is transported out of the leaf in the phloem to satisfy the needs of the non-photosynthetic organs of the plant (Kalt-Torres et al., 1987). In recent years, new data has shown that the phloem also plays a key role in moving information molecules that coordinate many facets of plant growth and development (Turgeon and Wolf, 2009). This update will focus on phloem loading9s contribution to assimilate partitioning, and its role in balancing photosynthetic activity with sink utilization of photoassimilates.

Amino acid transporters in plants

Amino acid transporters are essential participants in the resource allocation processes that support plant growth and development. Recent results have identified several new transporters that contribute to a wide array of physiological activities, and detailed molecular analysis has provided fundamental insights into the structure, function and regulation of these integral membrane proteins.

LHT1, A Lysine- and Histidine-Specific Amino Acid Transporter in Arabidopsis

We have identified a new amino acid transporter from the Arabidopsis thaliana expressed sequence tag cDNA collection by functional complementation of a yeast amino acid transport mutant. Transport analysis of the expressed protein in yeast shows that it is a high-affinity transporter for both lysine (Lys) and histidine with Michaelis constant values of 175 and 400 [mu]M, respectively. This transporter (LHT1, lysine histidine transporter) has little affinity for arginine when measured directly in uptake experiments or indirectly with substrate competition. The cDNA is 1.7 kb with an open reading frame that codes for a protein with 446 amino acids and a calculated molecular mass of 50.5 kD. Hydropathy analysis shows that LHT1 is an integral membrane protein with 9 to 10 putative membrane-spanning domains. Southern-blot analysis suggests that LHT1 is a single-copy gene in the Arabidopsis genome. RNA gel-blot analysis shows that this transporter is present in all tissues, with the strongest expression in young leaves, flowers, and siliques. Wholemount, in situ hybridization revealed that expression is further localized on the surface of roots in young seedlings and in pollen. Overall, LHT1 belongs to a new class of amino acid transporter that is specific for Lys and histidine, and, given its substrate specificity, it has significant promise as a tool for improving the Lys content of Lys-deficient grains.

Expression and transcriptional regulation of amino acid transporters in plants.

Summary.Recent studies have shown that there are more than 50 amino acid transporter genes in the Arabidopsis genome. This abundance of amino acid transporters implies that they play a multitude of fundamental roles in plant growth and development. Current research on the expression and regulation (i.e., tissue-specific expression and regulation of expression in response to nutrient and environmental changes) of these genes has provided useful information about the functional significance of plant amino acid transport systems.

Plant feeding site selection on soybean by the facultatively phytophagous predator Orius insidiosus

Experiments were conducted to test whether the facultatively phytophagous predator Orius insidiosus (Say) (Heteroptera: Anthocoridae) ingested phloem, xylem or mesophyll contents from soybean plants (Glycine max L.). Potential uptake of phloem sap was examined by radiolabeling photosynthate with 14CO2 and then measuring the accumulation of radiolabeled metabolites in feeding animals. Most O. insidiosus feeding on radiolabeled plants ingested no or very low levels of label; only 3% ingested small amounts of label, indicating the experimental insects fed very little, if at all, on the phloem. In contrast, well known phloem feeding insects used as positive controls accumulated substantial levels of labeled metabolites after feeding on known host plants. O. insidiosus did feed on xylem contents, as shown by ingestion of safranin‐labeled xylem fluid. A few of the insects showed signs of feeding on the mesophyll, as indicated by the presence of chloroplasts in the gut. However, the small diameter of the food canal may cause limited passage of chloroplasts, which would contribute to an underestimation of the frequency of mesophyll feeding. Some radiolabeled metabolites remain in the mesophyll so those insects that ingested low levels of radiolabel probably ingested label from the mesophyll, which supports the notion that some level of mesophyll feeding occurred. Feeding site determines the nutrients ingested during phytophagy. These insects obtain water from the xylem, and may ingest small amounts of starches, sugars, and amino acids from the mesophyll. The results suggest that facultative phytophagy by this heteropteran predator primarily provides the insect with water, but also may provide some nutrients that supplement a prey diet and help the predator survive periods when prey are scarce.

Molecular Cloning, Immunochemical Localization to the Vacuole, and Expression in Transgenic Yeast and Tobacco of a Putative Sugar Transporter from Sugar Beet

Several plant genes have been cloned that encode members of the sugar transporter subgroup of the major facilitator superfamily of transporters. Here we report the cloning, expression, and membrane localization of one of these porters found in sugar beet (Beta vulgaris L.). This clone, cDNA-1, codes for a protein with 490 amino acids and an estimated molecular mass of 54 kD. The predicted membrane topology and sequence homology suggest that cDNA-1 is a member of the sugar transporter family. RNA gel blot analysis revealed that this putative sugar transporter is expressed in all vegetative tissues and expression increases with development in leaves. DNA gel blot analysis indicated that multiple gene copies exist for this putative sugar transporter in the sugar beet genome. Antibodies directed against small peptides representing the N- and C-terminal domains of the cDNA1 protein identified a 40-kD polypeptide in microsomes isolated from cDNA-1-transformed yeast (Saccharomyces cerevisiae). Moreover, the same protein was identified in sugar beet and transgenic tobacco (Nicotiana tobacum L.) membrane fractions. Detailed analysis of the transporters distribution across linear sucrose gradients and flotation centrifugations showed that it co-migrates with tonoplast membrane markers. We conclude that this carrier is located on the tonoplast membrane and that it may mediate sugar partitioning between the vacuole and cytoplasmic compartments.

Protein phosphorylation plays a key role in sucrose-mediated transcriptional regulation of a phloem-specific proton-sucrose symporter

Assimilate partitioning refers to the systemic distribution of sugars and amino acids from sites of primary assimilation (source tissue) to import-dependent tissues and organs (sinks). One of the defining questions in this area is how plants balance source productivity with sink demand. Recent results from our laboratory showed that sucrose transport activity is directly proportional to the transcription rate of the phloem-specific proton–sucrose symporter BvSUT1 in Beta vulgaris L. Moreover, symporter gene transcription is regulated by sucrose levels in the leaf. Here we show that sucrose-dependent regulation of BvSUT1 transcription is mediated, at least in part, by a protein phosphorylation relay pathway. Protein phosphatase inhibitors decreased sucrose transport activity, symporter protein and mRNA abundance, and the relative transcription rate of the symporter gene. In contrast, protein kinase inhibitors had no effect or increased sucrose transport, protein and mRNA abundance, and transcription. Furthermore, pre-treating leaves with kinase inhibitors before feeding with sucrose blocked the sucrose-dependent decrease in symporter transcription and transport activity. The latter observation provides direct evidence for a protein phosphorylation cascade operating between the sucrose-sensor and the transcriptional regulator that controls BvSUT1 expression and, ultimately, phloem loading.

Chitinase-Like Protein CTL1 Plays a Role in Altering Root System Architecture in Response to Multiple Environmental Conditions

Plant root architecture is highly responsive to changes in nutrient availability. However, the molecular mechanisms governing the adaptability of root systems to changing environmental conditions is poorly understood. A screen for abnormal root architecture responses to high nitrate in the growth medium was carried out for a population of ethyl methanesulfonate-mutagenized Arabidopsis (Arabidopsis thaliana). The growth and root architecture of the arm (for anion altered root morphology) mutant described here was similar to wild-type plants when grown on low to moderate nitrate concentrations, but on high nitrate, arm exhibited reduced primary root elongation, radial swelling, increased numbers of lateral roots, and increased root hair density when compared to the wild-type control. High concentrations of chloride and sucrose induced the same phenotype. In contrast, hypocotyl elongation in the dark was decreased independently of nitrate availability. Positional cloning identified a point mutation in the AtCTL1 gene that encodes a chitinase-related protein, although molecular and biochemical analysis showed that this protein does not possess chitinase enzymatic activity. CTL1 appears to play two roles in plant growth and development based on the constitutive effect of the arm mutation on primary root growth and its conditional impact on root architecture. We hypothesize that CTL1 plays a role in determining cell wall rigidity and that the activity is differentially regulated by pathways that are triggered by environmental conditions. Moreover, we show that mutants of some subunits of the cellulose synthase complex phenocopy the conditional effect on root architecture under nonpermissive conditions, suggesting they are also differentially regulated in response to a changing environment.

TOPOLOGY OF NAT2, A PROTOTYPICAL EXAMPLE OF A NEW FAMILY OF AMINO ACID TRANSPORTERS

Amino acids are the predominant form of nitrogen available to the heterotrophic tissues of plants. These essential organic nutrients are transported across the plasma membrane of plant cells by proton-amino acid symporters. Our lab has cloned an amino acid transporter from Arabidopsis, NAT2/AAP1, that represents the first example of a new class of membrane transporters. We are investigating the structure and function of this porter because it is a member of a large gene family in plants and because its wide expression pattern suggests it plays a central role in resource allocation. In the results reported here, we investigated the topology of NAT2 by engineering a c-myc epitope on either the N or C terminus of the protein. We then used in vitro translation, partial digestion with proteinase K, and immunoprecipitation to identify a group of oriented peptide fragments. We modeled the topology of NAT2 based on the lengths of the peptide fragments that allowed us to estimate the location of protease accessible cleavage sites. We independently identified the location of the N and C termini using immunofluorescence microscopy of NAT2 expressed in COS-1 cells. We also investigated the glycosylation status of several sites of potentialN-linked glycosylation. Based on the combined data, we propose a novel 11 transmembrane domain model with the N terminus in the cytoplasm and C terminus facing outside the cell. This model of protein topology anchors our complementary investigations of porter structure and function using site-directed and random mutagenesis.