The export of amino acid in the phloem is altered in wheat plants lacking the short arm of chromosome 7B

Grain protein content is one of the major determinants of the baking and nutritional quality of wheat. It has previously been reported that the ditelosomic line of wheat (Triticum aestivum L.) CSDT7BL, where the short arm of chromosome 7B is missing, shows a lower grain protein concentration than the normal line, but a similar grain yield. In the present paper the growth and nitrogen (N) metabolism of wheat plants cv. Chinese Spring (CS) and its ditelosomic line CSDT7BL were compared. When plants were grown to maturity in pots with different N supplements, the wild-type line showed a higher grain protein concentration and a lower straw N concentration than the ditelosomic line at every N level analysed, suggesting a deficiency in the N remobilization capacity. When 15-d-old plants were grown in a growth cabinet in pots with sand, and supplied with nutrient solutions of different nitrate concentrations, the ditelosomic line showed no differences in N uptake per unit of root dry weight, nitrate reductase activity, nitrate, total N concentration or free amino acid concentration. However, the ditelosomic line showed a decreased capacity to export amino acids in the phloem under high N, independently of the N source. This deficiency was also observed under dark-induced senescence. The diminished export of amino acids to the phloem was principally caused by a decrease in the export of Glu, Asp, and Gln. It is suggested that the decrease in grain protein concentration in the ditelosomic line is a consequence of defective export in the phloem of these amino acids.     

Amino acid contents and transport in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) under different nitrogen conditions

Oilseed rape (Brassica napus L.) needs very high nitrogen fertilizer inputs. Significant amounts of this nitrogen are lost during early leaf shedding and are a source of environmental and economic concern. The objective of this study was to investigate whether the remobilization of leaf amino acids could be limiting for nitrogen use efficiency. Therefore, amino acid concentrations were analyzed in subcellular compartments of leaf mesophyll cells of plants grown under low (0.5 mM NO₃^–) and high (4 mM NO₃^–) nitrogen supply. With high nitrogen supply, young leaves showed an elevated amino acid content, mainly in vacuoles. In old leaves, however, subcellular concentrations were similar under high and low nitrogen conditions, showing that the excess nitrogen had been exported during leaf development. The phloem sap contained up to 650 mM amino acids, more than four times as much than the cytosol of mesophyll cells, indicating a very efficient phloem-loading process. Three amino acid permeases, BnAAP1, BnAAP2, and BnAAP6, were identified and characterized. BnAAP1 and BnAAP6 mediated uptake of neutral and acidic amino acids into Xenopus laevis oocytes at the actual apoplastic substrate concentrations. All three transporters were expressed in leaves and the expression was still detectable during leaf senescence, with BnAAP1 and BnAAP2 mRNA levels increasing from mature to old leaves. We conclude that phloem loading of amino acids is not limiting for nitrogen remobilization from senescing leaves in oilseed rape.     

Regulation of glutamine synthetase 1 and amino acids transport in the phloem of young wheat plants

The possible regulation of amino acid remobilization via the phloem in wheat (Triticum aestivum L.) by the primary enzyme in nitrogen (N) assimilation and re-assimilation, glutamine synthetase (GS, E.C. 6.3.1.2) was studied using two conditions known to alter N phloem transport, N deficiency and cytokinins. The plants were grown for 15 days in controlled conditions with optimum N supply and then N was depleted from and/or 6-benzylaminopurine was added to the nutrient solution. Both treatments generated an induction of GS1, monitored at the level of gene expression, protein accumulation and enzyme activity, and a decrease in the exudation of amino acids to the phloem, obtained with EDTA technique, which correlated negatively. GS inhibition by metionine sulfoximide (MSX) produced an increase of amino acids exudation and the inhibitor successfully reversed the effect of N deficiency and cytokinin addition over phloem exudation. Our results point to an important physiological role for GS1 in the modulation of amino acids export levels in wheat plants.     

Regulation of nitrogen partitioning in field-grown almond trees: Effects of fruit load and foliar nitrogen applications

Two treatments were employed to influence the amount of amino nitrogen (N) transport in phloem. In walnut trees (Juglans regia L.), developing fruit significantly reduced the efflux of foliar-applied ¹⁵N-enriched urea from treated spurs over a 33-day period in comparison with similarly-treated defruited spurs. Those data suggest that local aboveground demand for N influences vascular transport of amino N. In another experiment, a 1% urea solution was applied foliarly to 5-year old `Mission' almond trees [Prunus dulcis (Mill.) D. A. Webb] to increase the concentration of amino N in the phloem. The effect of foliar N treatments on a) the transport and distribution of labelled urea N within the trees over the experimental period and b) the uptake of soil-applied labelled N were determined by replicated whole tree excavation, fractionation into various tree components and mass spectrometric analyses of the ¹⁴N/¹⁵N ratios. Concentrations and composition of amino acids in the phloem and xylem saps of control trees and trees receiving foliar-applied urea were also determined. In foliar urea-treated trees, the amino acid concentrations increased significantly in leaf and bark phloem exudate, within 24 and 96 h, respectively. Foliar-applied urea N was translocated to the roots of almond trees over the experimental period and decreased soil N uptake. The results of these experiments are consistent with the hypothesis that aboveground N demand affects the amount of amino N cycling between shoots and roots, and may be involved in the regulation of soil N uptake. This revised version was published online in June 2006 with corrections to the Cover Date.     

Increased phloem transport of S-methylmethionine positively affects sulfur and nitrogen metabolism and seed development in pea plants

Seeds of grain legumes are important energy and food sources for humans and animals. However, the yield and quality of legume seeds are limited by the amount of sulfur (S) partitioned to the seeds. The amino acid S-methylmethionine (SMM), a methionine derivative, has been proposed to be an important long-distance transport form of reduced S, and we analyzed whether SMM phloem loading and source-sink translocation are important for the metabolism and growth of pea (Pisum sativum) plants. Transgenic plants were produced in which the expression of a yeast SMM transporter, S-Methylmethionine Permease1 (MMP1, YLL061W), was targeted to the phloem and seeds. Phloem exudate analysis showed that concentrations of SMM are elevated in MMP1 plants, suggesting increased phloem loading. Furthermore, expression studies of genes involved in S transport and metabolism in source organs, as well as xylem sap analyses, support that S uptake and assimilation are positively affected in MMP1 roots. Concomitantly, nitrogen (N) assimilation in root and leaf and xylem amino acid profiles were changed, resulting in increased phloem loading of amino acids. When investigating the effects of increased S and N phloem transport on seed metabolism, we found that protein levels were improved in MMP1 seeds. In addition, changes in SMM phloem loading affected plant growth and seed number, leading to an overall increase in seed S, N, and protein content in MMP1 plants. Together, these results suggest that phloem loading and source-sink partitioning of SMM are important for plant S and N metabolism and transport as well as seed set.     

Phloem loading and unloading of sugars and amino acids

In terrestrial higher plants, phloem transport delivers most nutrients required for growth and storage processes. Some 90% of plant biomass, transported as sugars and amino nitrogen (N) compounds in a bulk flow of solution, is propelled though the phloem by osmotically generated hydrostatic pressure differences between source (net nutrient export) and sink (net nutrient import) ends of phloem paths. Source loading and sink unloading of sugars, amino N compounds and potassium largely account for phloem sap osmotic concentrations and hence pressure differences. A symplasmic component is characteristic of most loading and unloading pathways which, in some circumstances, may be interrupted by an apoplasmic step. Raffinose series sugars appear to be loaded symplasmically. However, sucrose, and probably certain amino acids, are loaded into minor veins from source leaf apoplasms by proton symporters localized to plasma membranes of their sieve element/companion cell (se/cc) complexes. Sucrose transporters, with complementary kinetic properties, are conceived to function as membrane transporter complexes that respond to alterations in source/sink balance. In contrast, symplasmic unloading is common for many sink types. Intervention of an apoplasmic step, distal from importing phloem, is reserved for special situations. Effluxers that release sucrose and amino acids to the surrounding apoplasm in phloem loading and unloading are yet to be cloned. The physiological behaviour of effluxers is consistent with facilitated membrane transport that can be energy coupled. Roles of sucrose and amino acid transporters in phloem unloading remain to be discovered along with mechanisms regulating symplasmic transport. The latter is hypothesized to exert significant control over phloem unloading and, in some circumstances, phloem loading.     

Export of amino acids to the phloem in relation to N supply in wheat

The effect of different N supply on amino acid export to the phloem was studied in young plants of wheat (Triticum aestivum L. cv. Klein Chamaco), using the exudation in EDTA technique. Plants were grown in a growth cabinet in pots with sand, and supplied with nutrient solutions of different NO₃^? concentrations. When plants were grown for 15 days with nutrient solutions containing 1.0, 3.0, 5.0, 10.0, 15.0 or 20.0 mM KNO₃, the exudation rate of sugars from the phloem was unaffected by N supply, but sugars accumulated in the leaf tissue when the N supply was limiting for growth. On the other hand, the rate of exudation of amino acids was proportional to the NO₃^? concentration in the nutrient solution. When the supply of N to plants grown for 15 days with 15.0 mM NO₃^? was interrupted, the exudation of sugars was again unaffected, but there was a fast decrease in the amount of amino acids exudated, and of the concentration of amino acids and nitrogen in the tissues. Also, when 10-day-old plants grown without N were supplied with 15.0 mM NO₃^?, there was a sharp increase in the exudation of amino acids, without changes in the amount of sugar exudated. The rate of exudation of amino acids to the phloem was independent of the concentration of free amino acids in the leaves in all three types of experiment. Asp was the most abundant amino acid in the leaf tissue, while Glu was the one most abundant in the phloem exudate. Asp and Ala were exported to the phloem at a rate lower than expected from their leaf tissue concentrations, indicating some discrimination. On the contrary, Glu showed a preferential export at low N supply. It is concluded that the rate of amino acid export from the leaf to the phloem is dependent on the N available to the plant. This N is used for synthesis of leaf protein when the supply is low, exported to the phloem when supply is adequate, and accumulated in the storage pool when supply is above plant demand.     

Further Studies of the Phloem Loading Process in Leaves of Barley and Spinach. The Comparison of Metabolite Concentrations in the Apoplastic Compartment with those in the Cytosolic Compartment and in the Sieve Tubes1

The concentrations of sucrose, amino acids, nitrate and malate in the apoplastic compartment of illuminated leaves of barley and spinach were determined and compared with the corresponding concentrations in the cytosolic compartment of mesophyll cells and in the phloem sap, as measured previously with plants grown under identical conditions. The concentrations of sucrose and amino acids in the apoplast are found to be much lower than in the cytosol and in the phloem sap, indicating that not only the uptake into the phloem of sucrose, but also of amino acids, requires transport against a concentration gradient. The gradient of sucrose and amino acids between the cytosol and the apoplast was maintained when phloem transport had been blocked by cold girdling. Apparently, the efflux of sucrose and amino acids from the source cells to the apoplast is regulated in such a way that it meets the requirements of phloem transport. The percentages of the single amino acids as part of the total amino acids are quite similar in the cytosol, apoplast and phloem sap. The ratio of sucrose to the total amino acids in the cytosol is similar to that in the apoplast but about five times higher in the phloem sap. It appears from these results that the preferential extraction of sucrose over amino acids from the source cells to the phloem is due to the uptake from the apoplast into the phloem.     

Bread Wheat (Triticum aestivum L.) Grain Protein Concentration Is Related to Early Post-Flowering Nitrate Uptake under Putative Control of Plant Satiety Level

The strong negative correlation between grain protein concentration (GPC) and grain yield (GY) in bread wheat complicates the simultaneous improvement of these traits. However, earlier studies have concluded that the deviation from this relationship (grain protein deviation or GPD) has strong genetic basis. Genotypes with positive GPD have an increased ability to uptake nitrogen (N) during the post-flowering period independently of the amount of N taken up before flowering, suggesting that genetic variability for N satiety could enable the breakage of the negative relationship. This study is based on two genotypes markedly contrasted for GPD grown under semi-hydroponic conditions differentiated for nitrate availability both before and after flowering. This allows exploration of the genetic determinants of post-flowering N uptake (PANU) by combining whole plant sampling and targeted gene expression approaches. The results highlights the correlation (r² = 0.81) with GPC of PANU occurring early during grain development (flowering–flowering + 250 degree-days) independently of GY. Early PANU was in turn correlated (r² = 0.80) to the stem-biomass increment after flowering through its effect on N sink activity. Differences in early PANU between genotypes, despite comparable N statuses at flowering, suggest that genetic differences in N satiety could be involved in the establishment of the GPC. Through its strong negative correlation with genes implied in N assimilation, root nitrate concentration appears to be a good marker for evaluating instantaneous plant N demand, and may provide valuable information on the genotypic N satiety level. This trait may help breeders to identify genotypes having high GPC independently of their GY.     

Developmental control of H+/amino acid permease gene expression during seed development of Arabidopsis

Long distance transport of amino acids is mediated by several families of differentially expressed amino acid transporters. The two genes AAP1 and AAP2 encode broad specificity H⁺-amino acid co-transporters and are expressed to high levels in siliques of Arabidopsis, indicating a potential role in supplying the seeds with organic nitrogen. The expression of both genes is developmentally controlled and is strongly induced in siliques at heart stage of embryogenesis, shortly before induction of storage protein genes. Histochemical analysis of transgenic plants expressing promoter-GUS fusions shows that the genes have non-overlapping expression patterns in siliques. AAP1 is expressed in the endosperm and the cotyledons whereas AAP2 is expressed in the vascular strands of siliques and in funiculi. The endosperm expression of AAP1 during early stages of seed development indicates that the endosperm serves as a transient storage tissue for organic nitrogen. Amino acids are transported in both xylem and phloem but during seed filling are imported only via the phloem. AAP2, which is expressed in the phloem of stems and in the veins supplying seeds, may function in uptake of amino acids assimilated in the green silique tissue, in the retrieval of amino acids leaking passively out of the phloem and in xylem-to-phloem transfer along the path. The promoters provide excellent tools to study developmental, hormonal and metabolic control of nitrogen nutrition during development and may help to manipulate the timing and composition of amino acid import into seeds.     

Grain protein and grain yield of tritordeum in comparison to wheat and triticale

The new species of cereal × Tritordeum Ascherson et Graebner (Hordeum chilense Roem. et Shultz × Triticum ssp.) has a grain protein concentration (GPC) of up to 25%. The relationship between GPC and yield, and the factors responsible for the high GPC of tritordeum were examined and compared in field experiments. Three experimental tritordeum lines, two early and a later released (recombined and secondary tritordeums) were compared to wheat (cv. Cajeme) and triticale cultivars (cv. Trujillo). GPC's were 19%–22% for recombined tritordeums, 16% for the secondary tritordeum, 12–15% for wheats and 11% for triticale. Grain yields of the recombined and secondary tridordeum were 17–33% and 45–57% that of the wheats and triticale, respectively. Reducing grain sink size by spikelet removal resulted in an increased GPC of remaining grains. Considering all species together there were a strong inverse relationship between GPC and grain yield (GY) per main ear (GPC=26–4.76 ln GY; r²=0.82). In another experiment, frost damage to an early sown treatment of wheat reduced sink size. Harvest index (HI) of early sown wheat was reduced from 0.45 to 0.19, values comparable to that of tritordeum. Having similar HI, the GPC of the early sown wheat was the same as an early sown tritordeum (around 18%). Data for total N uptake and the N concentration of plant tissue during the growing season indicated that enhanced N uptake and remobilisation were not responsible for tritordeum's high GPC. These results suggest that the high GPC of the early lines of tritordeum is a consequence of the small grain yield concentrating the grain protein.     

Root phloem-specific expression of the plasma membrane amino acid proton co-transporter AAP3

Amino acids are regarded as the nitrogen 'currency' of plants. Amino acids can be taken up from the soil directly or synthesized from inorganic nitrogen, and then circulated in the plant via phloem and xylem. AtAAP3, a member of the Amino Acid Permease (AAP) family, is mainly expressed in root tissue, suggesting a potential role in the uptake and distribution of amino acids. To determine the spatial expression pattern of AAP3, promoter-reporter gene fusions were introduced into Arabidopsis. Histochemical analysis of AAP3 promoter-GUS expressing plants revealed that AAP3 is preferentially expressed in root phloem. Expression was also detected in stamens, in cotyledons, and in major veins of some mature leaves. GFP-AAP3 fusions and epitope-tagged AAP3 were used to confirm the tissue specificity and to determine the subcellular localization of AtAAP3. When overexpressed in yeast or plant protoplasts, the functional GFP-AAP3 fusion was localized in subcellular organelle-like structures, nuclear membrane, and plasma membrane. Epitope-tagged AAP3 confirmed its localization to the plasma membrane and nuclear membrane of the phloem, consistent with the promoter-GUS study. In addition, epitope-tagged AAP3 protein was localized in endodermal cells in root tips. The intracellular localization suggests trafficking or cycling of the transporter, similar to many metabolite transporters in yeast or mammals, for example, yeast amino acid permease GAP1. Despite the specific expression pattern, knock-out mutants did not show altered phenotypes under various conditions including N-starvation. Microarray analyses revealed that the expression profile of genes involved in amino acid metabolism did not change drastically, indicating potential compensation by other amino acid transporters.     

Amino Acid Composition Along the Transport Pathway during Grain Filling in Wheat

The amino acid composition of endosperm cavity sap and of sieve tube saps from the flag leaf, peduncle, rachis, grain pedicel, and grain were determined for wheat plants just past the mid-half of grain filling. On a mole percent basis, glutamine accounted for almost half of the amino acids in sieve tube sap from the peduncle and ear. Other protein amino acids, plug γ-aminobutyrate, were present in varying, but mostly low (a few mole percent) proportions. The amino acid composition of phloem exudate resembled that of the mature wheat grain. The proportions of amino acids in the endosperm cavity were generally similar to those of the sieve tube sap supplying the grain. Cysteine, however, while virtually absent from sieve tube sap, comprised 1 to 2 mole percent of amino acids in the endosperm cavity, suggesting it is transported in a different form. Also, alanine and, to a lesser extent, glutamate were relatively more prominent in endosperm cavity sap than in the sieve tube sap. Thus, while most amino acids were more concentrated in the sieve tube sap than in the endosperm cavity sap, alanine and glutamate appeared to be moving from the sieve tube to the endosperm cavity in the absence of, or perhaps even against, their concentration gradients.     

Modelling postsilking nitrogen fluxes in maize (Zea mays) using 15N-labelling field experiments

In maize (Zea mays), nitrogen (N) remobilization and postflowering N uptake are two processes that provide amino acids for grain protein synthesis. To study the way in which N is allocated to the grain and to the stover, two different 15N-labelling techniques were developed. 15NO(3-) was provided to the soil either at the beginning of stem elongation or after silking. The distribution of 15N in the stover and in the grain was monitored by calculating relative 15N-specific allocation (RSA). A nearly linear relationship between the RSA of the kernels and the RSA of the stover was found as a result of two simultaneous N fluxes: N remobilization from the stover to the grain, and N allocation to the stover and to the grain originating from N uptake. By modelling the 15N fluxes, it was possible to demonstrate that, as a consequence of protein turnover, a large proportion of the amino acids synthesized from the N taken up after silking were integrated into the proteins of the stover, and these proteins were further hydrolysed to provide N to the grain.     

Effects of selected fertilizers on the life history of Bemisia tabaci (Hemiptera: Aleyrodidae) biotype B

We tested the effects among a purportedly sustainable water-soluble fertilizer, a conventional water-soluble fertilizer, an alternation of these, a controlled-release fertilizer, and a clear water control on the life-history traits of sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae; =Bemisia argentifolii Bellows & Perring) biotype B reared on poinsettia (Euphorbia pulcherrima Willdenow ex Klotzch). Free amino acids in petioles were measured to estimate plant nutrient assimilation and phloem nutritional quality for B. tabaci biotype B. The sustainable fertilizer produced plants with the highest concentration of amino acids. In contrast, fecundity of whiteflies was lowest in plants treated with the sustainable fertilizer and the water control. The relationship between total amino acids in phloem and survival was significantly quadratic, with the highest survival at intermediate levels. Fecundity, however, was negatively correlated with total amino acid content of the maternal host plant. Variation in total amino acid concentration in petioles of plants treated within fertilizer treatments makes it difficult to predict whether a particular fertilizer will produce plants with enough amino acids to deleteriously affect both survivorship and fecundity and yet yield a plant of good quality. Despite this limitation, we can conclude that the use of this sustainable fertilizer will not cause increases in whitefly populations relative to plants fertilized with water-soluble and slow-release fertilizers that deliver the same level of nitrogen to the plant.     

Cytokinins in the vascular saps of Ricinus communis

Roots are recognised as the major sites of cytokinin synthesis and shoots receive a continuous supply of cytokinins from the roots. Although reports are available on the xylem mobility of putative free bases and their ribosides, relatively few studies on the phloem mobility of cytokinins have been reported. The origin of phloem-mobile cytokinins is uncertain but there is evidence which implicates a recirculation from the root source. This study is the first report in which zeatin and zeatin riboside from the root pressure exudate and phloem sap of Ricinus have been identified by full-scan GC-MS and quantified by GC-MS selective-ion-monitoring. In this study, the concentration of cytokinins in root pressure exudate was similar, but lower, and in the phloem sap higher than that reported previously. The concentration of cytokinins quantified in the phloem sap confirms their transport in the sieve tubes. The relatively high concentration of zeatin riboside detected in the root pressure exudate and of zeatin detected in the phloem sap indicate a possible vascular recirculation of these hormones.