UMAMIT44 is a key player in glutamate export from Arabidopsis chloroplasts.

Selective partitioning of amino acids among organelles, cells, tissues, and organs is essential for cellular metabolism and plant growth. Nitrogen assimilation into glutamine and glutamate and de novo biosynthesis of most protein amino acids occur in chloroplasts; therefore, various transport mechanisms must exist to accommodate their directional efflux from the stroma to the cytosol and feed the amino acids into the extraplastidial metabolic and long-distance transport pathways. Yet, Arabidopsis (Arabidopsis thaliana) transporters functioning in plastidial export of amino acids remained undiscovered. Here, USUALLY MULTIPLE ACIDS MOVE IN AND OUT TRANSPORTER 44 (UMAMIT44) was identified and shown to function in glutamate export from Arabidopsis chloroplasts. UMAMIT44 controls glutamate homeostasis within and outside of chloroplasts and influences nitrogen partitioning from leaves to sinks. Glutamate imbalances in chloroplasts and leaves of umamit44 mutants impact cellular redox state, nitrogen and carbon metabolism, and amino acid (AA) and sucrose supply of growing sinks, leading to negative effects on plant growth. Nonetheless, the mutant lines adjust to some extent by upregulating alternative pathways for glutamate synthesis outside the plastids and by mitigating oxidative stress through the production of other amino acids and antioxidants. Overall, this study establishes that the role of UMAMIT44 in glutamate export from chloroplasts is vital for controlling nitrogen availability within source leaf cells and for sink nutrition, with an impact on growth and seed yield.

Multi-scale analysis provides insights into the roles of ureide permeases in wheat nitrogen use efficiency.

The ureides allantoin and allantoate serve as nitrogen (N) transport compounds in plants, and more recently, allantoin has been shown to play a role in signaling. In planta, tissue ureide levels are controlled by the activity of enzymes of the purine degradation pathway and by ureide transporters called ureide permeases (UPS). Little is known about the physiological function of UPS proteins in crop plants, and especially in monocotyledon species. Here, we identified 13 TaUPS genes in the wheat (Triticum aestivum L.) genome. Phylogenetic and genome location analyses revealed a close relationship of wheat UPSs to orthologues in other grasses and a division into TaUPS1, TaUPS2.1, and TaUPS2.2 groups, each consisting of three homeologs, with a total of four tandem duplications. Expression, localization, and biochemical analyses resolved spatio-temporal expression patterns of TaUPS genes, transporter localization at the plasma membrane, and a role for TaUPS2.1 proteins in cellular import of ureides and phloem and seed loading. In addition, positive correlations between TaUPS1 and TaUPS2.1 transcripts and ureide levels were found. Together the data support that TaUPSs function in regulating ureide pools at source and sink, along with source-to-sink transport. Moreover, comparative studies between wheat cultivars grown at low and high N strengthened a role for TaUPS1 and TaUPS2.1 transporters in efficient N use and in controlling primary metabolism. Co-expression, protein-protein interaction, and haplotype analyses further support TaUPS involvement in N partitioning, N use efficiency, and domestication. Overall, this work provides a new understanding on UPS transporters in grasses as well as insights for breeding resilient wheat varieties with improved N use efficiency.

Altering ureide transport in nodulated soybean results in whole-plant adjustments of metabolism, assimilate partitioning, and sink strength.

Legumes develop a symbiotic relationship with bacteria that are housed in root nodules and fix atmospheric di-nitrogen (N2) to ammonia. In soybean (Glycine max (L.) Merr.) nodules, the final products of nitrogen (N) fixation are amino acids, and the ureides allantoin and allantoic acid that also serve as the major long-distance N transport forms. Recently, we have shown that increased expression of UPS1 (ureide permease 1) in soybean nodules results in enhanced ureide export from nodules with positive effects on N fixation and seed yield. Here, we demonstrate that changes in the ureide transport processes trigger alterations in allantoin and allantoic acid pools and partitioning throughout the transgenic plants. They further result in adjustments in amino acid availability in, and translocation to, root and shoot sinks. In addition, leaf carbon (C) capture, assimilation and allocation to sinks are improved, accommodating the increased nodule function, and root and shoot growth. Overall, we demonstrate that enhanced ureide partitioning in nodulated soybean leads to a complex rebalancing of N and C acquisition, metabolism, and transport processes with positive consequences for above- and below-ground vegetative biomass, and whole-plant N and C gains.

Role of source-to-sink transport of methionine in establishing seed protein quantity and quality in legumes.

Grain legumes such as pea (Pisum sativum L.) are highly valued as a staple source of protein for human and animal nutrition. However, their seeds often contain limited amounts of high-quality, sulfur (S) rich proteins, caused by a shortage of the S-amino acids cysteine and methionine. It was hypothesized that legume seed quality is directly linked to the amount of organic S transported from leaves to seeds, and imported into the growing embryo. We expressed a high-affinity yeast (Saccharomyces cerevisiae) methionine/cysteine transporter (Methionine UPtake 1) in both the pea leaf phloem and seed cotyledons and found source-to-sink transport of methionine but not cysteine increased. Changes in methionine phloem loading triggered improvements in S uptake and assimilation and long-distance transport of the S compounds, S-methylmethionine and glutathione. In addition, nitrogen and carbon assimilation and source-to-sink allocation were upregulated, together resulting in increased plant biomass and seed yield. Further, methionine and amino acid delivery to individual seeds and uptake by the cotyledons improved, leading to increased accumulation of storage proteins by up to 23%, due to both higher levels of S-poor and, most importantly, S-rich proteins. Sulfate delivery to the embryo and S assimilation in the cotyledons were also upregulated, further contributing to the improved S-rich storage protein pools and seed quality. Overall, this work demonstrates that methionine transporter function in source and sink tissues presents a bottleneck in S allocation to seeds and that its targeted manipulation is essential for overcoming limitations in the accumulation of high-quality seed storage proteins.

Concurrent overexpression of amino acid permease AAP1(3a) and SUT1 sucrose transporter in pea resulted in increased seed number and changed cytokinin and protein levels.

Using pea as our model crop, we sought to understand the regulatory control over the import of sugars and amino acids into the developing seeds and its importance for seed yield and quality. Transgenic peas simultaneously overexpressing a sucrose transporter and an amino acid transporter were developed. Pod walls, seed coats, and cotyledons were analysed separately, as well as leaves subtending developing pods. Sucrose, starch, protein, free amino acids, and endogenous cytokinins were measured during development. Temporal gene expression analyses (RT-qPCR) of amino acid (AAP), sucrose (SUT), and SWEET transporter family members, and those from cell wall invertase, cytokinin biosynthetic (IPT) and degradation (CKX) gene families indicated a strong effect of the transgenes on gene expression. In seed coats of the double transgenics, increased content and prolonged presence of cytokinin was particularly noticeable. The transgenes effectively promoted transition of young sink leaves into source leaves. We suggest the increased flux of sucrose and amino acids from source to sink, along with increased interaction between cytokinin and cell wall invertase in developing seed coats led to enhanced sink activity, resulting in higher cotyledon sucrose at process pea harvest, and increased seed number and protein content at maturity.

Transcriptomic, proteomic, and physiological studies reveal key players in wheat nitrogen use efficiency under both high and low nitrogen supply.

The effective use of available nitrogen (N) to improve crop grain yields provides an important strategy to reduce environmental N pollution and promote sustainable agriculture. However, little is known about the common genetic basis of N use efficiency (NUE) at varying N availability. Two wheat (Triticum aestivum L.) cultivars were grown in the field with high, moderate, and low N supply. Cultivar Zhoumai 27 outperformed Aikang 58 independent of the N supply and showed improved growth, canopy leaf area index, flag leaf surface area, grain number, and yield, and enhanced NUE due to both higher N uptake and utilization efficiency. Further, transcriptome and proteome analyses were performed using flag leaves that provide assimilates for grain growth. The results showed that many genes or proteins that are up- or down-regulated under all N regimes are associated with N and carbon metabolism and transport. This was reinforced by cultivar differences in photosynthesis, assimilate phloem transport, and grain protein/starch yield. Overall, our study establishes that improving NUE at both high and low N supply requires distinct adjustments in leaf metabolism and assimilate partitioning. Identified key genes/proteins may individually or concurrently regulate NUE and are promising targets for maximizing crop NUE irrespective of the N supply.

Oryza sativa Lysine-Histidine-type Transporter 1 functions in root uptake and root-to-shoot allocation of amino acids in rice.

In agricultural soils, amino acids can represent vital nitrogen (N) sources for crop growth and yield. However, the molecular mechanisms underlying amino acid uptake and allocation are poorly understood in crop plants. This study shows that rice (Oryza sativa L.) roots can acquire aspartate at soil concentration, and that japonica subspecies take up this acidic amino acid 1.5-fold more efficiently than indica subspecies. Genetic association analyses with 68 representative japonica or indica germplasms identified rice Lysine-Histidine-type Transporter 1 (OsLHT1) as a candidate gene associated with the aspartate uptake trait. When expressed in yeast, OsLHT1 supported cell growth on a broad spectrum of amino acids, and effectively transported aspartate, asparagine and glutamate. OsLHT1 is localized throughout the rice root, including root hairs, epidermis, cortex and stele, and to the leaf vasculature. Knockout of OsLHT1 in japonica resulted in reduced root uptake of amino acids. Furthermore, in 15 N-amino acid-fed mutants versus wild-type, a higher percentage of 15 N remained in roots instead of being allocated to the shoot. 15 N-ammonium uptake and subsequently the delivery of root-synthesized amino acids to Oslht1 shoots were also significantly decreased, which was accompanied by reduced shoot growth. These results together provide evidence that OsLHT1 functions in both root uptake and root to shoot allocation of a broad spectrum of amino acids in rice.

Role of ureides in source-to-sink transport of photoassimilates in non-fixing soybean.

Nitrogen (N)-fixing soybean plants use the ureides allantoin and allantoic acid as major long-distance transport forms of N, but in non-fixing, non-nodulated plants amino acids mainly serve in source-to-sink N allocation. However, some ureides are still synthesized in roots of non-fixing soybean, and our study addresses the role of ureide transport processes in those plants. In previous work, legume ureide permeases (UPSs) were identified that are involved in cellular import of allantoin and allantoic acid. Here, UPS1 from common bean was expressed in the soybean phloem, which resulted in enhanced source-to-sink transport of ureides in the transgenic plants. This was accompanied by increased ureide synthesis and elevated allantoin and allantoic acid root-to-sink transport. Interestingly, amino acid assimilation, xylem transport, and phloem partitioning to sinks were also strongly up-regulated. In addition, photosynthesis and sucrose phloem transport were improved in the transgenic plants. These combined changes in source physiology and assimilate partitioning resulted in increased vegetative growth and improved seed numbers. Overall, the results support that ureide transport processes in non-fixing plants affect source N and carbon acquisition and assimilation as well as source-to-sink translocation of N and carbon assimilates with consequences for plant growth and seed development.

Synchronization of developmental, molecular and metabolic aspects of source-sink interactions.

Plants have evolved a multitude of strategies to adjust their growth according to external and internal signals. Interconnected metabolic and phytohormonal signalling networks allow adaption to changing environmental and developmental conditions and ensure the survival of species in fluctuating environments. In agricultural ecosystems, many of these adaptive responses are not required or may even limit crop yield, as they prevent plants from realizing their fullest potential. By lifting source and sink activities to their maximum, massive yield increases can be foreseen, potentially closing the future yield gap resulting from an increasing world population and the transition to a carbon-neutral economy. To do so, a better understanding of the interplay between metabolic and developmental processes is required. In the past, these processes have been tackled independently from each other, but coordinated efforts are required to understand the fine mechanics of source-sink relations and thus optimize crop yield. Here, we describe approaches to design high-yielding crop plants utilizing strategies derived from current metabolic concepts and our understanding of the molecular processes determining sink development.

Targeting Nitrogen Metabolism and Transport Processes to Improve Plant Nitrogen Use Efficiency.

In agricultural cropping systems, relatively large amounts of nitrogen (N) are applied for plant growth and development, and to achieve high yields. However, with increasing N application, plant N use efficiency generally decreases, which results in losses of N into the environment and subsequently detrimental consequences for both ecosystems and human health. A strategy for reducing N input and environmental losses while maintaining or increasing plant performance is the development of crops that effectively obtain, distribute, and utilize the available N. Generally, N is acquired from the soil in the inorganic forms of nitrate or ammonium and assimilated in roots or leaves as amino acids. The amino acids may be used within the source organs, but they are also the principal N compounds transported from source to sink in support of metabolism and growth. N uptake, synthesis of amino acids, and their partitioning within sources and toward sinks, as well as N utilization within sinks represent potential bottlenecks in the effective use of N for vegetative and reproductive growth. This review addresses recent discoveries in N metabolism and transport and their relevance for improving N use efficiency under high and low N conditions.

Manipulation of sucrose phloem and embryo loading affects pea leaf metabolism, carbon and nitrogen partitioning to sinks as well as seed storage pools.

Seed development largely depends on the long-distance transport of sucrose from photosynthetically active source leaves to seed sinks. This source-to-sink carbon allocation occurs in the phloem and requires the loading of sucrose into the leaf phloem and, at the sink end, its import into the growing embryo. Both tasks are achieved through the function of SUT sucrose transporters. In this study, we used vegetable peas (Pisum sativum L.), harvested for human consumption as immature seeds, as our model crop and simultaneously overexpressed the endogenous SUT1 transporter in the leaf phloem and in cotyledon epidermal cells where import into the embryo occurs. Using this 'Push-and-Pull' approach, the transgenic SUT1 plants displayed increased sucrose phloem loading and carbon movement from source to sink causing higher sucrose levels in developing pea seeds. The enhanced sucrose partitioning further led to improved photosynthesis rates, increased leaf nitrogen assimilation, and enhanced source-to-sink transport of amino acids. Embryo loading with amino acids was also increased in SUT1-overexpressors resulting in higher protein levels in immature seeds. Further, transgenic plants grown until desiccation produced more seed protein and starch, as well as higher seed yields than the wild-type plants. Together, the results demonstrate that the SUT1-overexpressing plants with enhanced sucrose allocation to sinks adjust leaf carbon and nitrogen metabolism, and amino acid partitioning in order to accommodate the increased assimilate demand of growing seeds. We further provide evidence that the combined Push-and-Pull approach for enhancing carbon transport is a successful strategy for improving seed yields and nutritional quality in legumes.

Function of pea amino acid permease AAP6 in nodule nitrogen metabolism and export, and plant nutrition.

Legumes fix atmospheric nitrogen through a symbiotic relationship with bacteroids in root nodules. Following fixation in pea (Pisum sativum L.) nodules, nitrogen is reduced to amino acids that are exported via the nodule xylem to the shoot, and in the phloem to roots in support of growth. However, the mechanisms involved in amino acid movement towards the nodule vasculature, and their importance for nodule function and plant nutrition, were unknown. We found that in pea nodules the apoplasmic pathway is an essential route for amino acid partitioning from infected cells to the vascular bundles, and that amino acid permease PsAAP6 is a key player in nitrogen retrieval from the apoplasm into inner cortex cells for nodule export. Using an miRNA interference (miR) approach, it was demonstrated that PsAAP6 function in nodules, and probably in roots, and affects both shoot and root nitrogen supply, which were strongly decreased in PsAAP6-miR plants. Further, reduced transporter function resulted in increased nodule levels of ammonium, asparagine, and other amino acids. Surprisingly, nitrogen fixation and nodule metabolism were up-regulated in PsAAP6-miR plants, indicating that under shoot nitrogen deficiency, or when plant nitrogen demand is high, systemic signaling leads to an increase in nodule activity, independent of the nodule nitrogen status.

Leaf Amino Acid Supply Affects Photosynthetic and Plant Nitrogen Use Efficiency under Nitrogen Stress.

The coordinated distribution of nitrogen to source leaves and sinks is essential for supporting leaf metabolism while also supplying sufficient nitrogen to seeds for development. This study aimed to understand how regulated amino acid allocation to leaves affects photosynthesis and overall plant nitrogen use efficiency in Arabidopsis (Arabidopsis thaliana) and how soil nitrogen availability influences these processes. Arabidopsis plants with a knockout of AAP2, encoding an amino acid permease involved in xylem-to-phloem transfer of root-derived amino acids, were grown in low-, moderate-, and high-nitrogen environments. We analyzed nitrogen allocation to shoot tissues, photosynthesis, and photosynthetic and plant nitrogen use efficiency in these knockout plants. Our results demonstrate that, independent of nitrogen conditions, aap2 plants allocate more nitrogen to leaves than wild-type plants. Increased leaf nitrogen supply positively affected chlorophyll and Rubisco levels, photosynthetic nitrogen use efficiency, and carbon assimilation and transport to sinks. The aap2 plants outperformed wild-type plants with respect to growth, seed yield and carbon storage pools, and nitrogen use efficiency in both high and deficient nitrogen environments. Overall, this study demonstrates that increasing nitrogen allocation to leaves represents an effective strategy for improving carbon fixation and photosynthetic nitrogen use efficiency. The results indicate that an optimized coordination of nitrogen and carbon partitioning processes is critical for high oilseed production in Arabidopsis, including in plants exposed to limiting nitrogen conditions.

Source and sink mechanisms of nitrogen transport and use.

Contents Summary 35 I. Introduction 35 II. Nitrogen acquisition and assimilation 36 III. Root-to-shoot transport of nitrogen 38 IV. Nitrogen storage pools in vegetative tissues 39 V. Nitrogen transport from source leaf to sink 40 VI. Nitrogen import into sinks 42 VII. Relationship between source and sink nitrogen transport processes and metabolism 43 VIII. Regulation of nitrogen transport 43 IX. Strategies for crop improvement 44 X. Conclusions 46 Acknowledgements 47 References 47 SUMMARY: Nitrogen is an essential nutrient for plant growth. World-wide, large quantities of nitrogenous fertilizer are applied to ensure maximum crop productivity. However, nitrogen fertilizer application is expensive and negatively affects the environment, and subsequently human health. A strategy to address this problem is the development of crops that are efficient in acquiring and using nitrogen and that can achieve high seed yields with reduced nitrogen input. This review integrates the current knowledge regarding inorganic and organic nitrogen management at the whole-plant level, spanning from nitrogen uptake to remobilization and utilization in source and sink organs. Plant partitioning and transient storage of inorganic and organic nitrogen forms are evaluated, as is how they affect nitrogen availability, metabolism and mobilization. Essential functions of nitrogen transporters in source and sink organs and their importance in regulating nitrogen movement in support of metabolism, and vegetative and reproductive growth are assessed. Finally, we discuss recent advances in plant engineering, demonstrating that nitrogen transporters are effective targets to improve crop productivity and nitrogen use efficiency. While inorganic and organic nitrogen transporters were examined separately in these studies, they provide valuable clues about how to successfully combine approaches for future crop engineering.

The way out and in: phloem loading and unloading of amino acids.

Amino acids represent the major transport form of reduced nitrogen in plants. Long-distance transport of amino acids occurs in the xylem and the phloem. However, the phloem is the main transport route for bulk flow of the organic nitrogen from source leaves to sink tissues. Phloem loading in leaves of most annual plant species follows an apoplasmic transport path and requires the coordinated activity of transport protein mediating cellular export or import of amino acids. Phloem unloading of amino acids is generally a symplasmic process but apoplasmic transport is additionally required for efficient post-phloem nitrogen transport. In this review we summarize the current data on the physiology of amino acid phloem loading and unloading, and the molecular players involved. We discuss the implications of amino acid transporters in nitrogen signaling and highlight the necessity to investigate the coordination of symplasmic and apoplasmic transport processes.

Improving Plant Nitrogen Use Efficiency through Alteration of Amino Acid Transport Processes.

Improving the efficiency of nitrogen (N) uptake and utilization in plants could potentially increase crop yields while reducing N fertilization and, subsequently, environmental pollution. Within most plants, N is transported primarily as amino acids. In this study, pea (Pisum sativum) plants overexpressing AMINO ACID PERMEASE1 (AAP1) were used to determine if and how genetic manipulation of amino acid transport from source to sink affects plant N use efficiency. The modified plants were grown under low, moderate, or high N fertilization regimes. The results showed that, independent of the N nutrition, the engineered plants allocate more N via the vasculature to the shoot and seeds and produce more biomass and higher seed yields than wild-type plants. Dependent on the amount of N supplied, the AAP1-overexpressing plants displayed improved N uptake or utilization efficiency, or a combination of the two. They also showed significantly increased N use efficiency in N-deficient as well as in N-rich soils and, impressively, required half the amount of N to produce as many fruits and seeds as control plants. Together, these data support that engineering N allocation from source to sink presents an effective strategy to produce crop plants with improved productivity as well as N use efficiency in a range of N environments.

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.

Increasing Nitrogen Fixation and Seed Development in Soybean Requires Complex Adjustments of Nodule Nitrogen Metabolism and Partitioning Processes.

Legumes are able to access atmospheric di-nitrogen (N2) through a symbiotic relationship with rhizobia that reside within root nodules. In soybean, following N2 fixation by the bacteroids, ammonia is finally reduced in uninfected cells to allantoin and allantoic acid [1]. These ureides present the primary long-distance transport forms of nitrogen (N), and are exported from nodules via the xylem for shoot N supply. Transport of allantoin and allantoic acid out of nodules requires the function of ureide permeases (UPS1) located in cells adjacent to the vasculature [2, 3]. We expressed a common bean UPS1 transporter in cortex and endodermis cells of soybean nodules and found that delivery of N from nodules to shoot, as well as seed set, was significantly increased. In addition, the number of transgenic nodules was increased and symbiotic N2 fixation per nodule was elevated, indicating that transporter function in nodule N export is a limiting step in bacterial N acquisition. Further, the transgenic nodules showed considerable increases in nodule N assimilation, ureide synthesis, and metabolite levels. This suggests complex adjustments of nodule N metabolism and partitioning processes in support of symbiotic N2 fixation. We propose that the transgenic UPS1 plants display metabolic and allocation plasticity to overcome N2 fixation and seed yield limitations. Overall, it is demonstrated that transporter function in N export from nodules is a key step for enhancing atmospheric N2 fixation and nodule function and for improving shoot N nutrition and seed development in legumes.

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.