Phloem Loading Of Amino Acids Research Articles

Ethephon-Induced Ethylene Enhances Protein Degradation in Source Leaves, but Its High Endogenous Level Inhibits the Development of Regenerative Organs in Brassica napus.

To investigate the regulatory role of ethylene in the source-sink relationship for nitrogen remobilization, short-term effects of treatment with different concentrations (0, 25, 50, and 75 ppm) of ethephon (2-chloroethylphosphonic acid, an ethylene inducing agent) for 10 days (EXP 1) and long-term effects at 20 days (Day 30) after treatment with 100 ppm for 10 days (EXP 2) on protein degradation and amino acid transport in foliar sprayed mature leaves of Brassica napus (cv. Mosa) were determined. In EXP 1, endogenous ethylene concentration gradually increased in response to the treated ethephon concentration, leading to the upregulation of senescence-associated gene 12 (SAG12) expression and downregulation of chlorophyll a/b-binding protein (CAB) expression. Further, the increase in ethylene concentration caused a reduction in protein, Rubisco, and amino acid contents in the mature leaves. However, the activity of protease and expression of amino acid transporter (AAP6), an amino acid transport gene, were not significantly affected or slightly suppressed between the treatments with 50 and 75 ppm. In EXP 2, the enhanced ethylene level reduced photosynthetic pigments, leading to an inhibition of flower development without any pod development. A significant increase in protease activity, confirmed using in-gel staining of protease, was also observed in the ethephon-treated mature leaves. Ethephon application enhanced the expression of four amino acid transporter genes (AAP1, AAP2, AAP4, and AAP6) and the phloem loading of amino acids. Significant correlations between ethylene level, induced by ethephon application, and the descriptive parameters of protein degradation and amino acid transport were revealed. These results indicated that an increase in ethylene upregulated nitrogen remobilization in the mature leaves (source), which was accompanied by an increase in proteolytic activity and amino acid transport, but had no benefit to pod (sink) development.

Implications of nitrogen phloem loading for carbon metabolism and transport during Arabidopsis development.

Metabolite transport processes and primary metabolism are highly interconnected. This study examined the importance of source-to-sink nitrogen partitioning, and associated nitrogen metabolism for carbon capture, transport and usage. Specifically, Arabidopsis aap8 (AMINO ACID PERMEASE 8) mutant lines were analyzed to resolve the consequences of reduced amino acid phloem loading for source leaf carbon metabolism, sucrose phloem transport and sink development during vegetative and reproductive growth phase. Results showed that decreased amino acid transport had a negative effect on sink development of aap8 lines throughout the life cycle, leading to an overall decrease in plant biomass. During vegetative stage, photosynthesis and carbohydrate levels were decreased in aap8 leaves, while expression of carbon metabolism and transport genes, as well as sucrose phloem transport were not affected despite reduced sink strength. However, when aap8 plants transitioned to reproductive phase, carbon fixation and assimilation as well as sucrose partitioning to siliques were strongly decreased. Overall, this work demonstrates that phloem loading of nitrogen has varying implications for carbon fixation, assimilation and source-to-sink allocation depending on plant growth stage. It further suggests alterations in source-sink relationships, and regulation of carbon metabolism and transport by sink strength in a development-dependent manner.

Connecting Source with Sink: The Role of Arabidopsis AAP8 in Phloem Loading of Amino Acids.

Allocation of large amounts of nitrogen to developing organs occurs in the phloem and is essential for plant growth and seed development. In Arabidopsis (Arabidopsis thaliana) and many other plant species, amino acids represent the dominant nitrogen transport forms in the phloem, and they are mainly synthesized in photosynthetically active source leaves. Following their synthesis, a broad spectrum of the amino nitrogen is actively loaded into the phloem of leaf minor veins and transported within the phloem sap to sinks such as developing leaves, fruits, or seeds. Controlled regulation of the source-to-sink transport of amino acids has long been postulated; however, the molecular mechanism of amino acid phloem loading was still unknown. In this study, Arabidopsis AMINO ACID PERMEASE8 (AAP8) was shown to be expressed in the source leaf phloem and localized to the plasma membrane, suggesting its function in phloem loading. This was further supported by transport studies with aap8 mutants fed with radiolabeled amino acids and by leaf exudate analyses. In addition, biochemical and molecular analyses revealed alterations in leaf nitrogen pools and metabolism dependent on the developmental stage of the mutants. Decreased amino acid phloem loading and partitioning to sinks led to decreased silique and seed numbers, but seed protein levels were unchanged, demonstrating the importance of AAP8 function for sink development rather than seed quality. Overall, these results show that AAP8 plays an important role in source-to-sink partitioning of nitrogen and that its function affects source leaf physiology and seed yield.

Extraction of Apoplastic Wash Fluids and Leaf Petiole Exudates from Leaves of Arabidopsis thaliana

The long-distance translocation of metabolites and mineral elements is crucial for plant growth and reproduction. In most cases, source-to-sink translocation of metabolites and minerals requires their passage through the apoplast, irrespective whether they are transported via the xylem or the phloem. This apoplast-mediated pathway is of particular importance during plant senescence, when photoassimilates as well as organic, inorganic or chelated forms of nutrients are translocated from leaves to fruits or seeds. Recent genetic and physiological studies revealed the involvement of numerous membrane transporters mediating phloem loading of amino acids, sugars, urea or mineral elements. To evaluate the contribution of individual transporters to xylem unloading or phloem loading, the collection of apoplastic fluids and of phloem sap is essential. Here, we describe a method for the extraction of apoplastic fluids and the collection of leaf petiole exudates from Arabidopsis leaves, the latter representing an approximation to the real composition of the phloem sap.

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids.

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic-active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane-localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element-companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.

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.

Amino acid transporter expression and localisation studies in pea (Pisum sativum).

Expression of the amino acid permeases PsAAP1 and PsAAP2 was analysed in developing pea (Pisum sativum L.) plants. Both transporters were expressed in seed coats and cotyledon epidermal transfer cells and storage parenchyma cells. AAP expression is developmentally regulated and coincides with the onset of storage protein synthesis. Nitrogen was shown to induce AAP expression and AAP transcript levels were upregulated during the photoperiod. Analysis of Arabidopsis thaliana AAP1 promoter activity in pea, using promoter-β-glucuronidase (promotor-GUS) studies, revealed targeting of GUS to seed coats and cotyledon epidermal transfer cells. Expression was found in the nutritious endosperm during the early stages of seed development, whereas GUS staining in embryos was detected from the heart stage onward. In addition, AAP1 expression was observed in the phloem throughout the plant. This finding equally applied to PsAAP1 expression as shown by in situ mRNA hybridisation, which also demonstrated that PsAAP1 expression was localised to companion cells. Overall, PsAAP1 expression patterns and cellular localisation point to a function of the transporter in phloem loading of amino acids for translocation to sinks and in seed loading for development and storage protein accumulation.

Amino acid contents and transport in oilseed rape (Brassica napus 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.

Cycling of nitrogen and potassium between shoot and roots in maize as affected by shoot and root growth

In nitrate-fed plants cycling of nitrogen (N) and potassium (K) may serve several functions including supply of the roots with nutrients needed for growth, signalling of the growth-related shoot demand for nutrients to the roots, and removal of excess K from the shoot. In the present study, cycling and recycling of N and K were estimated in plants showing different rates of shoot and root growth. To induce these variations in growth, the plants were cultured with the same optimal nutrient supply but with the root zone temperature (RZT) at 12°C or 24°C. Additionally at both RZT, the plants were grown with their shoot base including apical shoot meristem at high or low temperature (SBT), Decreasing the RZT to 12°C drastically diminished root growth and accumulation of N and K in the roots. Cycling of N and K were less reduced by low RZT. At both RZT, N and K cycling were markedly reduced at low in comparison to high SBT although root growth was not affected by the SBT. Obviously, N and K cycling from shoot to roots were more affected by shoot growth than by the growth related demand of the roots for nutrients. At both RZT, N and K cycling exceeded accumulation in the roots. It was estimated that at least 20-33% of the N, and 24-51% of the K translocated from the roots to the shoot in the xylem is not directly derived from root uptake but from cycling. Plant culture at low shoot base temperature (SBT) drastically diminished shoot growth, and the accumulation of N and K in the shoot to less than 50% of the values measured in plants grown at high SBT. The low SBT-induced decrease of N accumulation in the shoot, at both RZT was associated with a reduction of K circulation and recirculation rates to less than 50% of those found in plants grown at high SBT. These findings are in accordance with the suggested role of K + for charge balance facilitating the transport of NO 3 - in the xylem and disposal of the negatively charged products of NO 3 - assimilation from shoot to roots in the phloem. In plants cultured at low SBT, net uptake and translocation rates of N and K were diminished to less than 50% of those measured in plants grown at high SBT. This repression was associated with reduced rates of N and K cycling from the shoot to the roots. Obviously, low rates of N and K cycling from the shoot to the roots are not necessarily signals to increase uptake in the roots. It is suggested that for plants adequately supplied with N, high rates of N cycling and recycling might be the consequence of an apparent lack in control of phloem loading of amino acids in the leaves.

Use of Phloem Exudate Technique in the Study of Amino Acid Transport in Pea Plants

The phloem exudation technique using ethylenediaminetetraacetic acid (EDTA) was evaluated in studies of amino acid translocation in Pisum sativum L. seedlings. Exudation of phloem sap from cut petioles of fully expanded leaves was enhanced by EDTA (20 millimolar disodium salt [pH 7.0]). Amino acids (mainly asparagine, homoserine, glutamate, and also aspartate and serine) were present in petiole exudates from EDTA-treated leaves at levels which were commonly 5- to 10-fold (or more) higher compared with water-treated controls. Exudation was greater from darkened leaves, and the pattern of amino acids was markedly different from the more uniform mixture leaking from water-treated controls.After feeding (14)C-labeled amino acids to the leaf blade, distribution of radioactivity in components of the exudate differed from that of the leaf tissue, suggesting selectivity of amino acid loading. [(14)C]Asparagine was converted to 2-hydroxysuccinamic acid and to other amino acids by the leaf, but was recovered in exudate mainly as asparagine (60%) and aspartate (30%). Similarly, in the exudate, 65 to 70% of the label from [(14)C]-aspartate was in amino acids, although in the leaf tissue 50% was in the organic acid fraction and only 11% remained as aspartate. Metabolism of asparagine and aspartate was essentially the same in intact leaf blades as in EDTA-treated leaves. Despite the possibility of EDTA damage in the petiole, phloem loading of amino acids appeared to be relatively unimpaired. Although the amount of labeled material appearing in the exudate is less than the amount translocated in the intact plant, the technique is useful in the study of amino acid transport.

Phloem Loading of Amino Acids from the Petioles of Ricinus Leaves

Phloem Loading of Amino Acids from the Petioles of <i>Ricinus</i> Leaves

Energy-dependent Loading of Amino Acids and Sucrose into the Phloem of Soybean

Radioactive sucrose, l-leucine, l-glutamate, and gamma-aminobutyrate were applied exogenously to abraded areas of soybean leaves. The three amino acids were translocated with similar velocities and mass transfer rates on a molar basis, although they were metabolized differently in the sink tissue. The concentration dependence of leucine translocation showed a triphasic saturation response, while sucrose translocation showed a biphasic saturation response to increasing concentration. Apparent K(m) and V(max) for leucine and sucrose loading in the phloem differed. Both leucine and sucrose translocation were inhibited by uncouplers, high K(+), and p-chloromercuribenzenesulfonic acid. Treatment with 0.8 m sorbitol had little effect on sucrose translocation but stimulated leucine translocation, indicating an apoplastic route of loading for leucine. No effect on mass transfer rates was observed when sucrose and amino acids were applied exogenously together. These data provide evidence that phloem loading of amino acids and sucrose is mediated by different and separate carriers, both being dependent on an energy-requiring mechanism.