Impairment of sugar transport in the vascular system acts on nitrogen remobilization and nitrogen use efficiency in Arabidopsis.

Carbon (C) and nitrogen (N) metabolisms have long been known to be coupled, and this is required for adjusting nitrogen use efficiency (NUE). Despite this intricate relationship, it is still unclear how deregulation of sugar transport impacts N allocation. Here, we investigated in Arabidopsis the consequences of the simultaneous downregulation of the genes coding for the sugar transporters SWEET11, SWEET12, SWEET16, and SWEET17 on various anatomical and physiological traits ranging from the stem's vascular system development to plant biomass production, seed yield, and N remobilization and use efficiency. Our results show that intracellular sugar exchanges mediated by SWEET16 and SWEET17 proteins specifically impact vascular development but do not play a significant role in the distribution of N. Most importantly, we showed that the double mutant swt11 swt12, which has an impacted vascular development, displays an improved NUE and nitrogen remobilization to the seeds. In addition, a significant negative correlation between sugar and amino acids contents and the inflorescence stem radial growth exists, highlighting the complex interaction between the maintenance of C/N homeostasis and the inflorescence stem development. Our results thus deepen the link between sugar transport, C/N allocation, and vascular system development.

Natural variation in the long-distance transport of nutrients and photoassimilates in response to N availability.

Phloem and xylem tissues are necessary for the allocation of nutrients and photoassimilates. However, how the long-distance transport of carbon (C) and nitrogen (N) is coordinated with the central metabolism is largely unknown. To better understand how the genetic and environmental factors influence C and N transport, we analysed the metabolite profiles of phloem exudates and xylem saps of five Arabidopsis thaliana accessions grown in low or non-limiting N supply. We observed that xylem saps were composed of 46 or 56% carbohydrates, 27 or 45% amino acids, and 5 or 13% organic acids in low or non-limiting N supply, respectively. In contrast, phloem exudates were composed of 76 or 86% carbohydrates, 7 or 18% amino acids, and 5 or 6% organic acids. Variation in N supply impacted amino acid, organic acid and sugar contents. When comparing low N and non-limiting N, the most striking differences were variations of glutamine, aspartate, and succinate abundance in the xylem saps and citrate and fumarate abundance in phloem exudates. In addition, we observed a substantial variation of metabolite content between genotypes, particularly under high N. The content of several organic acids, such as malate, citrate, fumarate, and succinate was affected by the genotype alone or by the interaction between genotype and N supply. This study confirmed that the response of the transport of nutrients in the phloem and the xylem to N availability is associated with the regulation of the central metabolism and could be an adaptive trait.

Delving deeper into the link between sugar transport, sugar signaling, and vascular system development.

Plant growth and development rely on the transport and use of sugars produced during photosynthesis. Sugars have a dual function as nutrients and signal molecules in the cell. Many factors maintaining sugar homeostasis and signaling are now identified, but our understanding of the mechanisms involved in coordinating intracellular and intercellular sugar translocation is still limited. We also know little about the interplay between sugar transport and signaling and the formation of the vascular system, which controls long-distance sugar translocation. Sugar signaling has been proposed to play a role; however, evidence to support this hypothesis is still limited. Here, we exploited recent transcriptomics datasets produced in aerial organs of Arabidopsis to identify genes coding for sugar transporters or signaling components expressed in the vascular cells. We identified genes belonging to sugar transport and signaling for which no information is available regarding a role in vasculature development. In addition, the transcriptomics datasets obtained from sugar-treated Arabidopsis seedlings were used to assess the sugar-responsiveness of known genes involved in vascular differentiation. Interestingly, several key regulators of vascular development were found to be regulated by either sucrose or glucose. Especially CLE41, which controls the procambial cell fate, was oppositely regulated by sucrose or glucose in these datasets. Even if more experimental data are necessary to confirm these findings, this survey supports a link between sugar transport/signaling and vascular system development.

A vacuolar hexose transport is required for xylem development in the inflorescence stem.

In Angiosperms, the development of the vascular system is controlled by a complex network of transcription factors. However, how nutrient availability in the vascular cells affects their development remains to be addressed. At the cellular level, cytosolic sugar availability is regulated mainly by sugar exchanges at the tonoplast through active and/or facilitated transport. In Arabidopsis (Arabidopsis thaliana), among the genes encoding tonoplastic transporters, SUGAR WILL EVENTUALLY BE EXPORTED TRANSPORTER 16 (SWEET16) and SWEET17 expression has been previously detected in the vascular system. Here, using a reverse genetics approach, we propose that sugar exchanges at the tonoplast, regulated by SWEET16, are important for xylem cell division as revealed in particular by the decreased number of xylem cells in the swt16 mutant and the accumulation of SWEET16 at the procambium-xylem boundary. In addition, we demonstrate that transport of hexoses mediated by SWEET16 and/or SWEET17 is required to sustain the formation of the xylem secondary cell wall. This result is in line with a defect in the xylem cell wall composition as measured by Fourier-transformed infrared spectroscopy in the swt16swt17 double mutant and by upregulation of several genes involved in secondary cell wall synthesis. Our work therefore supports a model in which xylem development partially depends on the exchange of hexoses at the tonoplast of xylem-forming cells.

Impacts of environmental conditions, and allelic variation of cytosolic glutamine synthetase on maize hybrid kernel production.

Cytosolic glutamine synthetase (GS1) is the enzyme mainly responsible of ammonium assimilation and reassimilation in maize leaves. The agronomic potential of GS1 in maize kernel production was investigated by examining the impact of an overexpression of the enzyme in the leaf cells. Transgenic hybrids exhibiting a three-fold increase in leaf GS activity were produced and characterized using plants grown in the field. Several independent hybrids overexpressing Gln1-3, a gene encoding cytosolic (GS1), in the leaf and bundle sheath mesophyll cells were grown over five years in different locations. On average, a 3.8% increase in kernel yield was obtained in the transgenic hybrids compared to controls. However, we observed that such an increase was simultaneously dependent upon both the environmental conditions and the transgenic event for a given field trial. Although variable from one environment to another, significant associations were also found between two GS1 genes (Gln1-3 and Gln1-4) polymorphic regions and kernel yield in different locations. We propose that the GS1 enzyme is a potential lead for producing high yielding maize hybrids using either genetic engineering or marker-assisted selection. However, for these hybrids, yield increases will be largely dependent upon the environmental conditions used to grow the plants.

Plant nitrate supply regulates Erwinia amylovora virulence gene expression in Arabidopsis.

We showed previously that nitrogen (N) limitation decreases Arabidopsis resistance to Erwinia amylovora (Ea). We show that decreased resistance to bacteria in low N is correlated with lower apoplastic reactive oxygen species (ROS) accumulation and lower jasmonic acid (JA) pathway expression. Consistently, pretreatment with methyl jasmonate (Me-JA) increased the resistance of plants grown under low N. In parallel, we show that in planta titres of a nonvirulent type III secretion system (T3SS)-deficient Ea mutant were lower than those of wildtype Ea in low N, as expected, but surprisingly not in high N. This lack of difference in high N was consistent with the low expression of the T3SS-encoding hrp virulence genes by wildtype Ea in plants grown in high N compared to plants grown in low N. This suggests that expressing its virulence factors in planta could be a major limiting factor for Ea in the nonhost Arabidopsis. To test this hypothesis, we preincubated Ea in an inducing medium that triggers expression of hrp genes in vitro, prior to inoculation. This preincubation strongly enhanced Ea titres in planta, independently of the plant N status, and was correlated to a significant repression of JA-dependent genes. Finally, we identify two clusters of metabolites associated with resistance or with susceptibility to Ea. Altogether, our data showed that high susceptibility of Arabidopsis to Ea, under low N or following preincubation in hrp-inducing medium, is correlated with high expression of the Ea hrp genes in planta and low expression of the JA signalling pathway, and is correlated with the accumulation of specific metabolites.

Involvement of SUT1 and SUT2 Sugar Transporters in the Impairment of Sugar Transport and Changes in Phloem Exudate Contents in Phytoplasma-Infected Plants.

Phytoplasmas inhabit phloem sieve elements and cause abnormal growth and altered sugar partitioning. However, how they interact with phloem functions is not clearly known. The phloem responses were investigated in tomatoes infected by "Candidatus Phytoplasma solani" at the beginning of the symptomatic stage, the first symptoms appearing in the newly emerged leaf at the stem apex. Antisense lines impaired in the phloem sucrose transporters SUT1 and SUT2 were included. In symptomatic sink leaves, leaf curling was associated with higher starch accumulation and the expression of defense genes. The analysis of leaf midribs of symptomatic leaves indicated that transcript levels for genes acting in the glycolysis and peroxisome metabolism differed from these in noninfected plants. The phytoplasma also multiplied in the three lower source leaves, even if it was not associated with the symptoms. In these leaves, the rate of phloem sucrose exudation was lower for infected plants. Metabolite profiling of phloem sap-enriched exudates revealed that glycolate and aspartate levels were affected by the infection. Their levels were also affected in the noninfected SUT1- and SUT2-antisense lines. The findings suggest the role of sugar transporters in the responses to infection and describe the consequences of impaired sugar transport on the primary metabolism.

Salinity Effects on Sugar Homeostasis and Vascular Anatomy in the Stem of the Arabidopsis Thaliana Inflorescence.

The regulation of sugar metabolism and partitioning plays an essential role for a plant's acclimation to its environment, with specific responses in autotrophic and heterotrophic organs. In this work, we analyzed the effects of high salinity on sugar partitioning and vascular anatomy within the floral stem. Stem sucrose and fructose content increased, while starch reduced, in contrast to the response observed in rosette leaves of the same plants. In the stem, the effects were associated with changes in the expression of SWEET and TMT2 genes encoding sugar transporters, SUSY1 encoding a sucrose synthase and several FRK encoding fructokinases. By contrast, the expression of SUC2, SWEET11 and SWEET12, encoding sugar transporters for phloem loading, remained unchanged in the stem. Both the anatomy of vascular tissues and the composition of xylem secondary cell walls were altered, suggesting that high salinity triggered major readjustments of sugar partitioning in this heterotrophic organ. There were changes in the composition of xylem cell walls, associated with the collapse and deformation of xylem vessels. The data are discussed regarding sugar partitioning and homeostasis of sugars in the vascular tissues of the stem.

Live-Cell Imaging of Fluorescently Tagged Phloem Proteins with Confocal Microscopy.

Confocal laser scanning microscopy can enable observation of phloem cells in living tissues. Here we describe live imaging of phloem cells in the leaves and roots of Arabidopsis thaliana using fluorescently tagged proteins, either expressed in the vasculature using phloem specific promoters or constitutively expressed reference marker lines. Now, the majority of phloem cell types can be identified, allowing a precise cellular and subcellular localization of phloem proteins.

Arabidopsis Natural Accessions Display Adaptations in Inflorescence Growth and Vascular Anatomy to Withstand High Salinity during Reproductive Growth.

Plant responses to abiotic stresses entail adaptive processes that integrate both physiological and developmental cues. However, the adaptive traits that are involved in the responses to a high soil salinity during reproductive growth are still poorly studied. To identify new clues, we studied the halophyte, Thellungiella salsuginea, and three Arabidopsis accessions, known as tolerant or salt-sensitive. We focused on the quantitative traits associated with the stem growth, sugar content, and anatomy of the plants subjected to the salt treatment, with and without a three-day acclimation, applied during the reproductive stage. The stem growth of Thellungiella salsuginea was not affected by the salt stress. By contrast, salt affected all of the Arabidopsis accessions, with a natural variation in the effect of the salt on growth, sugar content, and stem anatomy. In response to the high salinity, irregular xylem vessels were observed, independently of the accession's tolerance to salt treatment, while the diameter of the largest xylem vessels was reduced in the tolerant accessions. The stem height, growth rate, hexoses-to-sucrose ratio, and phloem-to-xylem ratio also varied, in association with both the genotype and its tolerance to salt stress. Our findings indicate that several quantitative traits for salt tolerance are associated with the control of inflorescence growth and the adjustment of the phloem-to-xylem ratio.

Lateral Transport of Organic and Inorganic Solutes.

Organic (e.g., sugars and amino acids) and inorganic (e.g., K⁺, Na⁺, PO42-, and SO42-) solutes are transported long-distance throughout plants. Lateral movement of these compounds between the xylem and the phloem, and vice versa, has also been reported in several plant species since the 1930s, and is believed to be important in the overall resource allocation. Studies of Arabidopsis thaliana have provided us with a better knowledge of the anatomical framework in which the lateral transport takes place, and have highlighted the role of specialized vascular and perivascular cells as an interface for solute exchanges. Important breakthroughs have also been made, mainly in Arabidopsis, in identifying some of the proteins involved in the cell-to-cell translocation of solutes, most notably a range of plasma membrane transporters that act in different cell types. Finally, in the future, state-of-art imaging techniques should help to better characterize the lateral transport of these compounds on a cellular level. This review brings the lateral transport of sugars and inorganic solutes back into focus and highlights its importance in terms of our overall understanding of plant resource allocation.

Three cytosolic glutamine synthetase isoforms localized in different-order veins act together for N remobilization and seed filling in Arabidopsis.

Glutamine synthetase (GS) is central for ammonium assimilation and consists of cytosolic (GS1) and chloroplastic (GS2) isoenzymes. During plant ageing, GS2 protein decreases due to chloroplast degradation, and GS1 activity increases to support glutamine biosynthesis and N remobilization from senescing leaves. The role of the different Arabidopsis GS1 isoforms in nitrogen remobilization was examined using 15N tracing experiments. Only the gln1;1-gln1;2-gln1;3 triple-mutation affecting the three GLN1;1, GLN1;2, and GLN1;3 genes significantly reduced N remobilization, total seed yield, individual seed weight, harvest index, and vegetative biomass. The triple-mutant accumulated a large amount of ammonium that could not be assimilated by GS1. Alternative ammonium assimilation through asparagine biosynthesis was increased and was related to higher ASN2 asparagine synthetase transcript levels. The GS2 transcript, protein, and activity levels were also increased to compensate for the lack of GS1-related glutamine biosynthesis. Localization of the different GLN1 genes showed that they were all expressed in the phloem companion cells but in veins of different order. Our results demonstrate that glutamine biosynthesis for N-remobilization occurs in veins of all orders (major and minor) in leaves, it is mainly catalysed by the three major GS1 isoforms (GLN1;1, GLN1;2, and GLN1;3), and it is alternatively supported by AS2 in the veins and GS2 in the mesophyll cells.

The rendez-vous of mobile sieve-element and abundant companion-cell proteins.

Thousands of sieve tube exudate proteins (STEP) have now been identified and predicted to fulfill a diversity of functions. However, most STEPs should be considered putative, since methods to collect sieve tube exudates have many technical drawbacks, and advanced functional characterization will be required to distinguish contaminant from bonafide proteins, and determine the latter's location and activity in sieve elements (SE). One major challenge is to develop new approaches to elucidate the function of these SE proteins, which in turn, is expected to shed light on intriguing aspects of SE cell biology.

AtbHLH68 transcription factor contributes to the regulation of ABA homeostasis and drought stress tolerance in Arabidopsis thaliana.

Basic helix-loop-helix (bHLH) transcription factors are involved in a wide range of developmental processes and in response to biotic and abiotic stresses. They represent one of the biggest families of transcription factors but only few of them have been functionally characterized. Here we report the characterization of AtbHLH68 and show that, although the knock out mutant did not have an obvious development phenotype, it was slightly more sensitive to drought stress than the Col-0, and AtbHLH68 overexpressing lines displayed defects in lateral root (LR) formation and a significant increased tolerance to drought stress, likely related to an enhanced sensitivity to abscisic acid (ABA) and/or increased ABA content. AtbHLH68 was expressed in the vascular system of Arabidopsis and its expression was modulated by exogenously applied ABA in an organ-specific manner. We showed that the expression of genes involved in ABA metabolism [AtAAO3 (AtALDEHYDE OXIDASE 3) and AtCYP707A3 (AtABSCISIC ACID 8'HYDROXYLASE 3)], in ABA-related response to drought-stress (AtMYC2, AtbHLH122 and AtRD29A) or during LRs development (AtMYC2 and AtABI3) was de-regulated in the overexpressing lines. We propose that AtbHLH68 has a function in the regulation of LR elongation, and in the response to drought stress, likely through an ABA-dependent pathway by regulating directly or indirectly components of ABA signaling and/or metabolism.

Genetic variability of the phloem sap metabolite content of maize (Zea mays L.) during the kernel-filling period.

Using a metabolomic approach, we have quantified the metabolite composition of the phloem sap exudate of seventeen European and American lines of maize that had been previously classified into five main groups on the basis of molecular marker polymorphisms. In addition to sucrose, glutamate and aspartate, which are abundant in the phloem sap of many plant species, large quantities of aconitate and alanine were also found in the phloem sap exudates of maize. Genetic variability of the phloem sap composition was observed in the different maize lines, although there was no obvious relationship between the phloem sap composition and the five previously classified groups. However, following hierarchical clustering analysis there was a clear relationship between two of the subclusters of lines defined on the basis of the composition of the phloem sap exudate and the earliness of silking date. A comparison between the metabolite contents of the ear leaves and the phloem sap exudates of each genotype, revealed that the relative content of most of the carbon- and nitrogen-containing metabolites was similar. Correlation studies performed between the metabolite content of the phloem sap exudates and yield-related traits also revealed that for some carbohydrates such as arabitol and sucrose there was a negative or positive correlation with kernel yield and kernel weight respectively. A posititive correlation was also found between kernel number and soluble histidine.

Live imaging of companion cells and sieve elements in Arabidopsis leaves.

The phloem is a complex tissue composed of highly specialized cells with unique subcellular structures and a compact organization that is challenging to study in vivo at cellular resolution. We used confocal scanning laser microscopy and subcellular fluorescent markers in companion cells and sieve elements, for live imaging of the phloem in Arabidopsis leaves. This approach provided a simple framework for identifying phloem cell types unambiguously. It highlighted the compactness of the meshed network of organelles within companion cells. By contrast, within the sieve elements, unknown bodies were observed in association with the PP2-A1:GFP, GFP:RTM1 and RTM2:GFP markers at the cell periphery. The phloem lectin PP2-A1:GFP marker was found in the parietal ground matrix. Its location differed from that of the P-protein filaments, which were visualized with SEOR1:GFP and SEOR2:GFP. PP2-A1:GFP surrounded two types of bodies, one of which was identified as mitochondria. This location suggested that it was embedded within the sieve element clamps, specific structures that may fix the organelles to each another or to the plasma membrane in the sieve tubes. GFP:RTM1 was associated with a class of larger bodies, potentially corresponding to plastids. PP2-A1:GFP was soluble in the cytosol of immature sieve elements. The changes in its subcellular localization during differentiation provide an in vivo blueprint for monitoring this process. The subcellular features obtained with these companion cell and sieve element markers can be used as landmarks for exploring the organization and dynamics of phloem cells in vivo.

Increased expression of a phloem membrane protein encoded by NHL26 alters phloem export and sugar partitioning in Arabidopsis.

The complex process of phloem sugar transport involves symplasmic and apoplasmic events. We characterized Arabidopsis thaliana lines ectopically expressing a phloem-specific gene encoding NDR1/HIN1-like26 (NHL26), a putative membrane protein. NHL26 overexpressor plants grew more slowly than wild-type plants, accumulated high levels of carbohydrates in mature leaves, and had a higher shoot biomass, contrasting with slower root growth and a lower seed yield. Similar effects were observed when NHL26 was overexpressed in companion cells, under the control of a companion cell-specific promoter. The soluble sugar content of the phloem sap and sink organs was lower than that in the wild type, providing evidence of a sugar export defect. This was confirmed in a phloem-export assay with the symplastic tracer carboxyfluorescein diacetate. Leaf sugar accumulation was accompanied by higher organic acid, amino acid, and protein contents, whereas analysis of the metabolite profile of phloem sap exudate revealed no change in amino acid or organic acid content, indicating a specific effect on sugar export. NHL26 was found to be located in the phloem plasmodesmata and the endoplasmic reticulum. These findings reveal that NHL26 accumulation affects either the permeability of plasmodesmata or sugar signaling in companion cells, with a specific effect on sugar export.