Sugar loading is not required for phloem sap flow in maize plants.

Phloem transport of photoassimilates from leaves to non-photosynthetic organs, such as the root and shoot apices and reproductive organs, is crucial to plant growth and yield. For nearly 90 years, evidence has been generally consistent with the theory of a pressure-flow mechanism of phloem transport. Central to this hypothesis is the loading of osmolytes, principally sugars, into the phloem to generate the osmotic pressure that propels bulk flow. Here we used genetic and light manipulations to test whether sugar import into the phloem is required as the driving force for phloem sap flow. Using carbon-11 radiotracer, we show that a maize sucrose transporter1 (sut1) loss-of-function mutant has severely reduced export of carbon from photosynthetic leaves (only ~4% of the wild type level). Yet, the mutant remarkably maintains phloem pressure at ~100% and sap flow speeds at ~50-75% of those of wild type. Potassium (K+) abundance in the phloem was elevated in sut1 mutant leaves. Fluid dynamic modelling supports the conclusion that increased K+ loading compensated for decreased sucrose loading to maintain phloem pressure, and thereby maintained phloem transport via the pressure-flow mechanism. Furthermore, these results suggest that sap flow and transport of other phloem-mobile nutrients and signalling molecules could be regulated independently of sugar loading into the phloem, potentially influencing carbon-nutrient homoeostasis and the distribution of signalling molecules in plants encountering different environmental conditions.

Physiology and whole-plant carbon partitioning during stem sugar accumulation in sweet dwarf sorghum.

MAIN CONCLUSION: A greater rate of phloem unloading and storage in the stem, not a higher rate of sugar production by photosynthesis or sugar export from leaves, is the main factor that results in sugar accumulation in sweet dwarf sorghum compared to grain sorghum. At maturity, the stem internodes of sweet sorghum varieties accumulate high concentrations of fermentable sugars and represent an efficient feedstock for bioethanol production. Although stem sugar accumulation is a heritable trait, additional factors that drive sugar accumulation in sorghum have not been identified. To identify the constraints on stem sugar accumulation in sweet sorghum, we used a combination of carbon-11 (11C) radiotracer, physiological and biochemical approaches, and compared a grain sorghum and sweet dwarf sorghum line that have similar growth characteristics including height. Photosynthesis did not increase during development or differ between the sorghum lines. During the developmental transition to the reproductive stage, export of 11C from leaves approximately doubled in both sorghum lines, but 11C export in the sweet dwarf line did not exceed that of the grain sorghum. Defoliation to manipulate relative sink demand did not result in increased photosynthetic rates, indicating that the combined accumulation of C by all sink tissues was limited by the maximum photosynthetic capacity of source leaves. Nearly 3/4 of the 11C exported from leaves was transported to the lower stem in sweet sorghum within 2 h, whereas in grain sorghum nearly 3/4 of the 11C was in the panicle. Accordingly, the transcripts of several sucrose transporter (SUT) genes were more abundant in the stem internodes of the sweet dwarf line compared to the grain sorghum. Overall, these results indicate that sugar accumulation in sweet sorghum stems is influenced by the interplay of different sink tissues for the same sugars, but is likely driven by elevated sugar phloem unloading and uptake capacity in mature stem internodes.

Three NPF genes in Arabidopsis are necessary for normal nitrogen cycling under low nitrogen stress.

Internal nitrogen (N) cycling is crucial to N use efficiency. For example, N may be remobilized from older, shaded leaves to young leaves near the apex that receive more direct sunlight, where the N can be used more effectively for photosynthesis. Yet our understanding of the mechanisms and regulation of N transport is limited. To identify relevant transporters in Arabidopsis, fifteen transporter knockout mutants were screened for defects in leaf N export using nitrogen-13 (13N) administered as 13NH3 gas to leaves. We found that three nitrate/peptide transporter family (NPF) genes were necessary for normal leaf N export under low N but not adequate soil N availability, including AtNPF7.1, which has not been previously characterized. High-throughput phenotyping revealed altered leaf area and chlorophyll fluorescence relative to wild-type plants. High AtNPF7.1 expression in flowers and large flower stalks of Atnpf7.1 mutants in low N suggests that AtNPF7.1 influences leaf N export via sink-to-source feedback, perhaps via a role in sensing plant internal N-status. We also identified previously unreported phenotypes for the mutants of the other two NPF transporters that indicate possible roles in N sensing networks.

Photosynthate Regulation of the Root System Architecture Mediated by the Heterotrimeric G Protein Complex in Arabidopsis.

Assimilate partitioning to the root system is a desirable developmental trait to control but little is known of the signaling pathway underlying partitioning. A null mutation in the gene encoding the Gß subunit of the heterotrimeric G protein complex, a nexus for a variety of signaling pathways, confers altered sugar partitioning in roots. While fixed carbon rapidly reached the roots of wild type and agb1-2 mutant seedlings, agb1 roots had more of this fixed carbon in the form of glucose, fructose, and sucrose which manifested as a higher lateral root density. Upon glucose treatment, the agb1-2 mutant had abnormal gene expression in the root tip validated by transcriptome analysis. In addition, PIN2 membrane localization was altered in the agb1-2 mutant. The heterotrimeric G protein complex integrates photosynthesis-derived sugar signaling incorporating both membrane-and transcriptional-based mechanisms. The time constants for these signaling mechanisms are in the same range as photosynthate delivery to the root, raising the possibility that root cells are able to use changes in carbon fixation in real time to adjust growth behavior.

Large-Scale Public Transcriptomic Data Mining Reveals a Tight Connection between the Transport of Nitrogen and Other Transport Processes in Arabidopsis.

Movement of nitrogen to the plant tissues where it is needed for growth is an important contribution to nitrogen use efficiency. However, we have very limited knowledge about the mechanisms of nitrogen transport. Loading of nitrogen into the xylem and/or phloem by transporter proteins is likely important, but there are several families of genes that encode transporters of nitrogenous molecules (collectively referred to as N transporters here), each comprised of many gene members. In this study, we leveraged publicly available microarray data of Arabidopsis to investigate the gene networks of N transporters to elucidate their possible biological roles. First, we showed that tissue-specificity of nitrogen (N) transporters was well reflected among the public microarray data. Then, we built coexpression networks of N transporters, which showed relationships between N transporters and particular aspects of plant metabolism, such as phenylpropanoid biosynthesis and carbohydrate metabolism. Furthermore, genes associated with several biological pathways were found to be tightly coexpressed with N transporters in different tissues. Our coexpression networks provide information at the systems-level that will serve as a resource for future investigation of nitrogen transport systems in plants, including candidate gene clusters that may work together in related biological roles.

In vivo quantitative imaging of photoassimilate transport dynamics and allocation in large plants using a commercial positron emission tomography (PET) scanner.

BACKGROUND: Although important aspects of whole-plant carbon allocation in crop plants (e.g., to grain) occur late in development when the plants are large, techniques to study carbon transport and allocation processes have not been adapted for large plants. Positron emission tomography (PET), developed for dynamic imaging in medicine, has been applied in plant studies to measure the transport and allocation patterns of carbohydrates, nutrients, and phytohormones labeled with positron-emitting radioisotopes. However, the cost of PET and its limitation to smaller plants has restricted its use in plant biology. Here we describe the adaptation and optimization of a commercial clinical PET scanner to measure transport dynamics and allocation patterns of (11)C-photoassimilates in large crops. RESULTS: Based on measurements of a phantom, we optimized instrument settings, including use of 3-D mode and attenuation correction to maximize the accuracy of measurements. To demonstrate the utility of PET, we measured (11)C-photoassimilate transport and allocation in Sorghum bicolor, an important staple crop, at vegetative and reproductive stages (40 and 70 days after planting; DAP). The (11)C-photoassimilate transport speed did not change over the two developmental stages. However, within a stem, transport speeds were reduced across nodes, likely due to higher (11)C-photoassimilate unloading in the nodes. Photosynthesis in leaves and the amount of (11)C that was exported to the rest of the plant decreased as plants matured. In young plants, exported (11)C was allocated mostly (88 %) to the roots and stem, but in flowering plants (70 DAP) the majority of the exported (11)C (64 %) was allocated to the apex. CONCLUSIONS: Our results show that commercial PET scanners can be used reliably to measure whole-plant C-allocation in large plants nondestructively including, importantly, allocation to roots in soil. This capability revealed extreme changes in carbon allocation in sorghum plants, as they advanced to maturity. Further, our results suggest that nodes may be important control points for photoassimilate distribution in crops of the family Poaceae. Quantifying real-time carbon allocation and photoassimilate transport dynamics, as demonstrated here, will be important for functional genomic studies to unravel the mechanisms controlling phloem transport in large crop plants, which will provide crucial insights for improving yields.

Radio-metabolite analysis of carbon-11 biochemical partitioning to non-structural carbohydrates for integrated metabolism and transport studies.

Metabolism and phloem transport of carbohydrates are interactive processes, yet each is often studied in isolation from the other. Carbon-11 ((11)C) has been successfully used to study transport and allocation processes dynamically over time. There is a need for techniques to determine metabolic partitioning of newly fixed carbon that are compatible with existing non-invasive (11)C-based methodologies for the study of phloem transport. In this report, we present methods using (11)C-labeled CO2 to trace carbon partitioning to the major non-structural carbohydrates in leaves-sucrose, glucose, fructose and starch. High-performance thin-layer chromatography (HPTLC) was adapted to provide multisample throughput, raising the possibility of measuring different tissues of the same individual plant, or for screening multiple plants. An additional advantage of HPTLC was that phosphor plate imaging of radioactivity had a much higher sensitivity and broader range of sensitivity than radio-HPLC detection, allowing measurement of (11)C partitioning to starch, which was previously not possible. Because of the high specific activity of (11)C and high sensitivity of detection, our method may have additional applications in the study of rapid metabolic responses to environmental changes that occur on a time scale of minutes. The use of this method in tandem with other (11)C assays for transport dynamics and whole-plant partitioning makes a powerful combination of tools to study carbohydrate metabolism and whole-plant transport as integrated processes.

Initial characterization of shade avoidance response suggests functional diversity between Populus phytochrome B genes.

Shade avoidance signaling involves perception of incident red/far-red (R/FR) light by phytochromes (PHYs) and modulation of downstream transcriptional networks. Although these responses are well studied in Arabidopsis, little is known about the role of PHYs and the transcriptional responses to shade in the woody perennial Populus. Tissue expression and subcellular localization of Populus PHYs was studied by quantitative real-time PCR (qRT-PCR) and protoplast transient assay. Transgenic lines with altered PHYB1 and/or PHYB2 expression were used in phenotypic assays and transcript profiling with qRT-PCR. RNA-Seq was used to identify transcriptional responses to enriched FR light. All three PHYs were differentially expressed among tissue types and PHYBs were targeted to the nucleus under white light. Populus PHYB1 rescued Arabidopsis phyB mutant phenotypes. Phenotypes of Populus transgenic lines and the expression of candidate shade response genes suggested that PHYB1 and PHYB2 have distinct yet overlapping functions. RNA-Seq analysis indicated that genes associated with cell wall modification and brassinosteroid signaling were induced under enriched FR light in Populus. This study is an initial attempt at deciphering the role of Populus PHYs by evaluating transcriptional reprogramming to enriched FR and demonstrates functional diversity and overlap of the Populus PHYB1 and PHYB2 in regulating shade responses.

Pseudomonas fluorescens induces strain-dependent and strain-independent host plant responses in defense networks, primary metabolism, photosynthesis, and fitness.

Colonization of plants by nonpathogenic Pseudomonas fluorescens strains can confer enhanced defense capacity against a broad spectrum of pathogens. Few studies, however, have linked defense pathway regulation to primary metabolism and physiology. In this study, physiological data, metabolites, and transcript profiles are integrated to elucidate how molecular networks initiated at the root-microbe interface influence shoot metabolism and whole-plant performance. Experiments with Arabidopsis thaliana were performed using the newly identified P. fluorescens GM30 or P. fluorescens Pf-5 strains. Co-expression networks indicated that Pf-5 and GM30 induced a subnetwork specific to roots enriched for genes participating in RNA regulation, protein degradation, and hormonal metabolism. In contrast, only GM30 induced a subnetwork enriched for calcium signaling, sugar and nutrient signaling, and auxin metabolism, suggesting strain dependence in network architecture. In addition, one subnetwork present in shoots was enriched for genes in secondary metabolism, photosynthetic light reactions, and hormone metabolism. Metabolite analysis indicated that this network initiated changes in carbohydrate and amino acid metabolism. Consistent with this, we observed strain-specific responses in tryptophan and phenylalanine abundance. Both strains reduced host plant carbon gain and fitness, yet provided a clear fitness benefit when plants were challenged with the pathogen P. syringae DC3000.

Arabidopsis Hexokinase-Like1 and Hexokinase1 form a critical node in mediating plant glucose and ethylene responses.

Arabidopsis (Arabidopsis thaliana) Hexokinase-Like1 (HKL1) lacks glucose (Glc) phosphorylation activity and has been shown to act as a negative regulator of plant growth. Interestingly, the protein has a largely conserved Glc-binding domain, and protein overexpression was shown previously to promote seedling tolerance to exogenous 6% (w/v) Glc. Since these phenotypes occur independently of cellular Glc signaling activities, we have tested whether HKL1 might promote cross talk between the normal antagonists Glc and ethylene. We show that repression by 1-aminocyclopropane-1-carboxylic acid (ACC) of the Glc-dependent developmental arrest of wild-type Arabidopsis seedlings requires the HKL1 protein. We also describe an unusual root hair phenotype associated with growth on high Glc medium that occurs prominently in HKL1 overexpression lines and in glucose insensitive 2-1 (gin2-1), a null mutant of Hexokinase1 (HXK1). Seedlings of these lines produce bulbous root hairs with an enlarged base after transfer from agar plates with normal medium to plates with 6% Glc. Seedling transfer to plates with 2% Glc plus ACC mimics the high-Glc effect in the HKL1 overexpression line but not in gin2-1. A similar ACC-stimulated, bulbous root hair phenotype also was observed in wild-type seedlings transferred to plates with 9% Glc. From transcript expression analyses, we found that HKL1 and HXK1 have differential roles in Glc-dependent repression of some ethylene biosynthesis genes. Since we show by coimmunoprecipitation assays that HKL1 and HXK1 can interact, these two proteins likely form a critical node in Glc signaling that mediates overlapping, but also distinct, cellular responses to Glc and ethylene treatments.

The M. truncatula SUNN gene is expressed in vascular tissue, similarly to RDN1, consistent with the role of these nodulation regulation genes in long distance signaling.

Encoding a conserved protein of unknown function, the Medicago truncatula RDN1 gene is involved in autoregulation of nodulation through signaling in the root.  In contrast, the SUNN kinase in M. truncatula has been shown by grafting of mutant scions to control nodule number in the root by communication of a signal from the shoot to the root.  GUS staining patterns resulting from expression of the SUNN promoter fused to uidA showed expression of SUNN in most parts of plant including the root, but confined to the vascular tissue, a pattern that overlaps with that published for RDN1.  Real Time qRT-PCR analysis showed levels of both SUNN RNA and RDN1 RNA did not change significantly during early nodulation signaling (0-72 hours after inoculation).  The similarity in expression in cell types strongly suggests vascular signaling for nodule number regulation, while the lack of changes over early nodule development suggest post transcriptional mechanisms such as protein association or phosphorylation transmit the signal.  

Comparative physiology and transcriptional networks underlying the heat shock response in Populus trichocarpa, Arabidopsis thaliana and Glycine max.

The heat shock response continues to be layered with additional complexity as interactions and crosstalk among heat shock proteins (HSPs), the reactive oxygen network and hormonal signalling are discovered. However, comparative analyses exploring variation in each of these processes among species remain relatively unexplored. In controlled environment experiments, photosynthetic response curves were conducted from 22 to 42 °C and indicated that temperature optimum of light-saturated photosynthesis was greater for Glycine max relative to Arabidopsis thaliana or Populus trichocarpa. Transcript profiles were taken at defined states along the temperature response curves, and inferred pathway analysis revealed species-specific variation in the abiotic stress and the minor carbohydrate raffinose/galactinol pathways. A weighted gene co-expression network approach was used to group individual genes into network modules linking biochemical measures of the antioxidant system to leaf-level photosynthesis among P. trichocarpa, G. max and A. thaliana. Network-enabled results revealed an expansion in the G. max HSP17 protein family and divergence in the regulation of the antioxidant and heat shock modules relative to P. trichocarpa and A. thaliana. These results indicate that although the heat shock response is highly conserved, there is considerable species-specific variation in its regulation.

Function of Arabidopsis hexokinase-like1 as a negative regulator of plant growth.

A recent analysis of the hexokinase (HXK) gene family from Arabidopsis revealed that three hexokinase-like (HKL) proteins lack catalytic activity, but share about 50% identity with the primary glucose (glc) sensor/transducer protein AtHXK1. Since the AtHKL1 protein is predicted to bind glc, although with a relatively decreased affinity, a reverse genetics approach was used to test whether HKL1 might have a related regulatory function in plant growth. By comparing phenotypes of an HKL1 mutant (hkl1-1), an HXK1 mutant (gin2-1), and transgenic lines that overexpress HKL1 in either wild-type or gin2-1 genetic backgrounds, it is shown that HKL1 is a negative effector of plant growth. Interestingly, phenotypes of HKL1 overexpression lines are generally very similar to those of gin2-1. These are quantified, in part, as reduced seedling sensitivity to high glc concentrations and reduced seedling sensitivity to auxin-induced lateral root formation. However, commonly recognized targets of glc signalling are not apparently altered in any of the HKL1 mutant or transgenic lines. In fact, most, but not all, of the observed phenotypes associated with HKL1 overexpression occur independently of the presence of HXK1 protein. The data indicate that HKL1 mediates cross-talk between glc and other plant hormone response pathways. It is also considered Whether a possibly decreased glc binding affinity of HKL1 could possibly be a feedback mechanism to limit plant growth in the presence of excessive carbohydrate availability is further considered.

Expression and evolutionary features of the hexokinase gene family in Arabidopsis.

Arabidopsis hexokinase1 (HXK1) is a moonlighting protein that has separable functions in glucose signaling and in glucose metabolism. In this study, we have characterized expression features and glucose phosphorylation activities of the six HXK gene family members in Arabidopsis thaliana. Three of the genes encode catalytically active proteins, including a stromal-localized HXK3 protein that is expressed mostly in sink organs. We also show that three of the genes encode hexokinase-like (HKL) proteins, which are about 50% identical to AtHXK1, but do not phosphorylate glucose or fructose. Expression studies indicate that both HKL1 and HKL2 transcripts occur in most, if not all, plant tissues and that both proteins are targeted within cells to mitochondria. The HKL1 and HKL2 proteins have 6-10 amino acid insertions/deletions (indels) at the adenosine binding domain. In contrast, HKL3 transcript was detected only in flowers, the protein lacks the noted indels, and the protein has many other amino acid changes that might compromise its ability even to bind glucose or ATP. Activity measurements of HXKs modified by site-directed mutagenesis suggest that the lack of catalytic activities in the HKL proteins might be attributed to any of numerous existing changes. Sliding windows analyses of coding sequences in A. thaliana and A. lyrata ssp. lyrata revealed a differential accumulation of nonsynonymous changes within exon 8 of both HKL1 and HXK3 orthologs. We further discuss the possibility that the non-catalytic HKL proteins have regulatory functions instead of catalytic functions.

Actin-based cellular framework for glucose signaling by Arabidopsis hexokinase1.

Glucose functions in plants both as a metabolic resource as well as a hormone that regulates expression of many genes. Arabidopsis hexokinase1 (HXK1) is the best understood plant glucose sensor/transducer, yet we are only now appreciating the cellular complexity of its signaling functions. We have recently shown that one of the earliest detectable responses to plant glucose treatments are extensive alterations of cellular F-actin. Interestingly, AtHXK1 is predominantly located on mitochondria, yet also can interact with actin. A normal functioning actin cytoskeleton is required for HXK1 to act as an effector in glucose signaling assays. We have suggested that HXK1 might alter F-actin dynamics and thereby influence the formation and/or stabilization of cytoskeleton-bound polysomes. In this Addendum, we have extended our initial observations on the subcellular targeting of HXK1 and its interaction with F-actin. We then further consider the cellular context in which HXK1 might regulate gene expression.

A role for F-actin in hexokinase-mediated glucose signaling.

HEXOKINASE1 (HXK1) from Arabidopsis (Arabidopsis thaliana) has dual roles in glucose (Glc) signaling and in Glc phosphorylation. The cellular context, though, for HXK1 function in either process is not well understood. Here we have shown that within normal experimental detection limits, AtHXK1 is localized continuously to mitochondria. Two mitochondrial porin proteins were identified as capable of binding to overexpressed HXK1 protein, both in vivo and in vitro. We also found that AtHXK1 can be associated with its structural homolog, F-actin, based on their coimmunoprecipitation from transgenic plants that overexpress HXK1-FLAG or from transient expression assays, and based on their localization in leaf cells after cryofixation. This association might be functionally important because Glc signaling in protoplast transient expression assays is compromised by disruption of F-actin. We also demonstrate that Glc treatment of Arabidopsis seedlings rapidly and reversibly disrupts fine mesh actin filaments. The possible roles of actin in HXK-dependent Glc signaling are discussed.