The sugar transporter ZmSUGCAR1 of the nitrate transporter 1/peptide transporter family is critical for maize grain filling.

Maternal-to-filial nutrition transfer is central to grain development and yield. nitrate transporter 1/peptide transporter (NRT1-PTR)-type transporters typically transport nitrate, peptides, and ions. Here, we report the identification of a maize (Zea mays) NRT1-PTR-type transporter that transports sucrose and glucose. The activity of this sugar transporter, named Sucrose and Glucose Carrier 1 (SUGCAR1), was systematically verified by tracer-labeled sugar uptake and serial electrophysiological studies including two-electrode voltage-clamp, non-invasive microelectrode ion flux estimation assays in Xenopus laevis oocytes and patch clamping in HEK293T cells. ZmSUGCAR1 is specifically expressed in the basal endosperm transfer layer and loss-of-function mutation of ZmSUGCAR1 caused significantly decreased sucrose and glucose contents and subsequent shrinkage of maize kernels. Notably, the ZmSUGCAR1 orthologs SbSUGCAR1 (from Sorghum bicolor) and TaSUGCAR1 (from Triticum aestivum) displayed similar sugar transport activities in oocytes, supporting the functional conservation of SUGCAR1 in closely related cereal species. Thus, the discovery of ZmSUGCAR1 uncovers a type of sugar transporter essential for grain development and opens potential avenues for genetic improvement of seed-filling and yield in maize and other grain crops.

Maize Brittle Stalk2-Like3, encoding a COBRA protein, functions in cell wall formation and carbohydrate partitioning.

Carbohydrate partitioning from leaves to sink tissues is essential for plant growth and development. The maize (Zea mays) recessive carbohydrate partitioning defective28 (cpd28) and cpd47 mutants exhibit leaf chlorosis and accumulation of starch and soluble sugars. Transport studies with 14C-sucrose (Suc) found drastically decreased export from mature leaves in cpd28 and cpd47 mutants relative to wild-type siblings. Consistent with decreased Suc export, cpd28 mutants exhibited decreased phloem pressure in mature leaves, and altered phloem cell wall ultrastructure in immature and mature leaves. We identified the causative mutations in the Brittle Stalk2-Like3 (Bk2L3) gene, a member of the COBRA family, which is involved in cell wall development across angiosperms. None of the previously characterized COBRA genes are reported to affect carbohydrate export. Consistent with other characterized COBRA members, the BK2L3 protein localized to the plasma membrane, and the mutants condition a dwarf phenotype in dark-grown shoots and primary roots, as well as the loss of anisotropic cell elongation in the root elongation zone. Likewise, both mutants exhibit a significant cellulose deficiency in mature leaves. Therefore, Bk2L3 functions in tissue growth and cell wall development, and this work elucidates a unique connection between cellulose deposition in the phloem and whole-plant carbohydrate partitioning.

A Functionalizable Analog of the Yariv Reagent for AGP Imaging using Fluorescence Microscopy.

Small molecule fluorescent probes that bind selectively to plant cell wall polysaccharides have been instrumental in elucidating the localization and function of these glycans. Arabinogalactan proteins (AGPs) are cell wall proteoglycans implicated in essential functions such as cell signaling, plant growth, and programmed cell death. There is currently no small molecule probe capable of fluorescently labeling AGPs. The Yariv reagents are the only small molecules that bind AGPs, and have been used to study AGP function and isolate AGPs via precipitation of an AGP-Yariv complex. However, the Yariv reagents are not fluorescent, rendering them ineffective for localization studies using fluorescence microscopy. A fluorescent version of a Yariv reagent that is capable of both binding as well as imaging AGPs would provide a powerful tool for studying AGPs in planta. Herein, we describe the synthesis of an azido analog of the Yariv reagent that can be further functionalized with a fluorophore to provide a glycoconjugate that binds AGPs and is fluorescent. We show that the modified reagent binds gum arabic in in vitro binding assays when used in conjunction with the ßGlcYariv reagent. Fluorescent imaging of AGPs in fixed maize leaf tissue enables localization of AGPs to cell walls in the leaf. Significantly, imaging can also be carried out using fresh tissue. This represents the first small molecule probe that can be used to visualize AGPs using fluorescence microscopy.

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.

The Kernel Size-Related Quantitative Trait Locus qKW9 Encodes a Pentatricopeptide Repeat Protein That Aaffects Photosynthesis and Grain Filling.

In maize (Zea mays), kernel weight is an important component of yield that has been selected during domestication. Many genes associated with kernel weight have been identified through mutant analysis. Most are involved in the biogenesis and functional maintenance of organelles or other fundamental cellular activities. However, few quantitative trait loci (QTLs) underlying quantitative variation in kernel weight have been cloned. Here, we characterize a QTL, qKW9, associated with maize kernel weight. This QTL encodes a DYW motif pentatricopeptide repeat protein involved in C-to-U editing of ndhB, a subunit of the chloroplast NADH dehydrogenase-like complex. In a null qkw9 background, C-to-U editing of ndhB was abolished, and photosynthesis was reduced, resulting in less maternal photosynthate available for grain filling. Characterization of qKW9 highlights the importance of optimizing photosynthesis for maize grain yield production.

Maize lateral root developmental plasticity induced by mild water stress. II: Genotype-specific spatio-temporal effects on determinate development.

Maize lateral roots exhibit determinate growth, whereby the meristem is genetically programmed to stop producing new cells. To explore whether lateral root determinacy is modified under water deficits, we studied two maize genotypes (B73 and FR697) with divergent responses of lateral root growth to mild water stress using an experimental system that provided near-stable water potential environments throughout lateral root development. First-order laterals of the primary root system of FR697 exhibited delayed determinacy when grown at a water potential of -0.28 MPa, resulting in longer and wider roots than in well-watered (WW) controls. In B73, in contrast, neither the length nor width of lateral roots was affected by water deficit. In water-stressed FR697, root elongation continued at or above the maximum rate in WW roots for 3 days longer, and was still 45% of maximum when WW roots approached their determinate length. Maintenance of root elongation was associated with sustained rates of cell production. In addition, kinematic analyses showed that reductions in tissue expansion rates with aging were delayed in the longitudinal, radial and tangential planes throughout the root growth zone. Thus, this study reveals large genotypic differences in the interaction of water stress with developmental determinacy of maize lateral roots.

An in Vivo Imaging Assay Detects Spatial Variability in Glucose Release from Plant Roots.

Plants secrete a plethora of metabolites into the rhizosphere that allow them to obtain nutrients necessary for growth and modify microbial communities around the roots. Plants release considerable amounts of photosynthetically fixed carbon into the rhizosphere; hence, it is important to understand how carbon moves from the roots into the rhizosphere. Approaches used previously to address this question involved radioactive tracers, fluorescent probes, and biosensors to study sugar movement in the roots and into the rhizosphere. Although quite effective for studying sugar movement, it has been challenging to obtain data on spatial and temporal variability in sugar exudation using these techniques. In this study, we developed a gel-based enzyme-coupled colorimetric and fluorometric assay to image glucose (Glc) in vivo and used this assay to show that there is spatial variability in Glc release from plant roots. We found that the primary roots of maize (Zea mays) released more Glc from the base of the root than from the root tip and that the Glc release rate is reduced in response to water stress. These findings were confirmed independently by quantifying Glc release in well-watered and water-stressed maize primary roots using high-performance anion-exchange chromatography. Additionally, we demonstrated differential patterns of Glc exudation in different monocot and eudicot plant species. These findings and their implications on root-rhizosphere interactions are discussed.

Maize lateral root developmental plasticity induced by mild water stress. I: Genotypic variation across a high-resolution series of water potentials.

Lateral root developmental plasticity induced by mild water stress was examined across a high-resolution series of growth media water potentials (Ψw ) in two genotypes of maize. The suitability of several media for imposing near-stable Ψw treatments on transpiring plants over prolonged growth periods was assessed. Genotypic differences specific to responses of lateral root growth from the primary root system occurred between cultivars FR697 and B73 over a narrow series of water stress treatments ranging in Ψw from -0.25 to -0.40 MPa. In FR697, both the average length and number of first-order lateral roots were substantially enhanced at a Ψw of -0.25 MPa compared with well-watered controls. These effects were separated spatially, occurring primarily in the upper and lower regions of the axial root, respectively. Furthermore, first-order lateral roots progressively increased in diameter with increasing water stress, resulting in a maximum 2.3-fold increase in root volume at a Ψw of -0.40 MPa. In B73, in contrast, the length, diameter, nor number of lateral roots was increased in any of the water stress treatments. The genotype-specific responses observed over this narrow range of Ψw demonstrate the necessity of high-resolution studies at mild stress levels for characterization of lateral root developmental plasticity.

Maize Carbohydrate partitioning defective1 impacts carbohydrate distribution, callose accumulation, and phloem function.

Plants synthesize carbohydrates in photosynthetic tissues, with the majority of plants transporting sucrose to non-photosynthetic tissues to sustain growth and development. While the anatomical, biochemical, and physiological processes regulating sucrose long-distance transport are well characterized, little is known concerning the genes controlling whole-plant carbohydrate partitioning. To identify loci influencing carbon export from leaves, we screened mutagenized maize plants for phenotypes associated with reduced carbohydrate transport, including chlorosis and excessive starch and soluble sugars in leaves. Carbohydrate partitioning defective1 (Cpd1) was identified as a semi-dominant mutant exhibiting these phenotypes. Phloem transport experiments suggested that the hyperaccumulation of starch and soluble sugars in the Cpd1/+ mutant leaves was due to inhibited sucrose export. Interestingly, ectopic callose deposits were observed in the phloem of mutant leaves, and probably underlie the decreased transport. In addition to the carbohydrate hyperaccumulation phenotype, Cpd1/+ mutants overaccumulate benzoxazinoid defense compounds and exhibit increased tolerance when attacked by aphids. However, double mutant studies between Cpd1/+ and benzoxazinoid-less plants indicate that the ectopic callose and carbon hyperaccumulation are independent of benzoxazinoid production. Based on the formation of callose occlusions in the developing phloem, we hypothesize that the cpd1 gene functions early in phloem development, thereby impacting whole-plant carbohydrate partitioning.

Sugar Transporters in Plants: New Insights and Discoveries.

Carbohydrate partitioning is the process of carbon assimilation and distribution from source tissues, such as leaves, to sink tissues, such as stems, roots and seeds. Sucrose, the primary carbohydrate transported long distance in many plant species, is loaded into the phloem and unloaded into distal sink tissues. However, many factors, both genetic and environmental, influence sucrose metabolism and transport. Therefore, understanding the function and regulation of sugar transporters and sucrose metabolic enzymes is key to improving agriculture. In this review, we highlight recent findings that (i) address the path of phloem loading of sucrose in rice and maize leaves; (ii) discuss the phloem unloading pathways in stems and roots and the sugar transporters putatively involved; (iii) describe how heat and drought stress impact carbohydrate partitioning and phloem transport; (iv) shed light on how plant pathogens hijack sugar transporters to obtain carbohydrates for pathogen survival, and how the plant employs sugar transporters to defend against pathogens; and (v) discuss novel roles for sugar transporters in plant biology. These exciting discoveries and insights provide valuable knowledge that will ultimately help mitigate the impending societal challenges due to global climate change and a growing population by improving crop yield and enhancing renewable energy production.

Sucrose Transporter ZmSut1 Expression and Localization Uncover New Insights into Sucrose Phloem Loading.

Sucrose transporters (SUTs) translocate sucrose (Suc) across cellular membranes, and in eudicots, multiple SUTs are known to function in Suc phloem loading in leaves. In maize (Zea mays), the Sucrose Transporter1 (ZmSut1) gene has been implicated in Suc phloem loading based upon RNA expression in leaves, electrophysiological experiments, and phenotypic analysis of zmsut1 mutant plants. However, no previous studies have examined the cellular expression of ZmSut1 RNA or the subcellular localization of the ZmSUT1 protein to assess the gene's hypothesized function in Suc phloem loading or to evaluate its potential roles, such as phloem unloading, in nonphotosynthetic tissues. To this end, we performed RNA in situ hybridization experiments, promoter-reporter gene analyses, and ZmSUT1 localization studies to elucidate the cellular expression pattern of the ZmSut1 transcript and protein. These data showed that ZmSut1 was expressed in multiple cell types throughout the plant and indicated that it functions in phloem companion cells to load Suc and also in other cell types to retrieve Suc from the apoplasm to prevent its accumulation and loss to the transpiration stream. Additionally, by comparing a phloem-mobile tracer with ZmSut1 expression, we determined that developing maize leaves dynamically switch from symplasmic to apoplasmic phloem unloading, reconciling previously conflicting reports, and suggest that ZmSut1 does not have an apparent function in either unloading process. A model for the dual roles for ZmSut1 function (phloem loading and apoplasmic recycling), Sut1 evolution, and its possible use to enhance Suc export from leaves in engineering C3 grasses for C4 photosynthesis is discussed.

Sucrose transporter2 contributes to maize growth, development, and crop yield.

During daylight, plants produce excess photosynthates, including sucrose, which is temporarily stored in the vacuole. At night, plants remobilize sucrose to sustain metabolism and growth. Based on homology to other sucrose transporter (SUT) proteins, we hypothesized the maize (Zea mays) SUCROSE TRANSPORTER2 (ZmSUT2) protein functions as a sucrose/H+ symporter on the vacuolar membrane to export transiently stored sucrose. To understand the biological role of ZmSut2, we examined its spatial and temporal gene expression, determined the protein subcellular localization, and characterized loss-of-function mutations. ZmSut2 mRNA was ubiquitously expressed and exhibited diurnal cycling in transcript abundance. Expressing a translational fusion of ZmSUT2 fused to a red fluorescent protein in maize mesophyll cell protoplasts revealed that the protein localized to the tonoplast. Under field conditions, zmsut2 mutant plants grew slower, possessed smaller tassels and ears, and produced fewer kernels when compared to wild-type siblings. zmsut2 mutants also accumulated two-fold more sucrose, glucose, and fructose as well as starch in source leaves compared to wild type. These findings suggest (i) ZmSUT2 functions to remobilize sucrose out of the vacuole for subsequent use in growing tissues; and (ii) its function provides an important contribution to maize development and agronomic yield.

Sucrose accumulation in sweet sorghum stems occurs by apoplasmic phloem unloading and does not involve differential Sucrose transporter expression.

BACKGROUND: Sorghum (Sorghum bicolor L. Moench) cultivars store non-structural carbohydrates predominantly as either starch in seeds (grain sorghums) or sugars in stems (sweet sorghums). Previous research determined that sucrose accumulation in sweet sorghum stems was not correlated with the activities of enzymes functioning in sucrose metabolism, and that an apoplasmic transport step may be involved in stem sucrose accumulation. However, the sucrose unloading pathway from stem phloem to storage parenchyma cells remains unelucidated. Sucrose transporters (SUTs) transport sucrose across membranes, and have been proposed to function in sucrose partitioning differences between sweet and grain sorghums. The purpose of this study was to characterize the key differences in carbohydrate accumulation between a sweet and a grain sorghum, to define the path sucrose may follow for accumulation in sorghum stems, and to determine the roles played by sorghum SUTs in stem sucrose accumulation. RESULTS: Dye tracer studies to determine the sucrose transport route revealed that, for both the sweet sorghum cultivar Wray and grain sorghum cultivar Macia, the phloem in the stem veins was symplasmically isolated from surrounding cells, suggesting sucrose was apoplasmically unloaded. Once in the phloem apoplasm, a soluble tracer diffused from the vein to stem parenchyma cell walls, indicating the lignified mestome sheath encompassing the vein did not prevent apoplasmic flux outside of the vein. To characterize carbohydrate partitioning differences between Wray and Macia, we compared the growth, stem juice volume, solute contents, SbSUTs gene expression, and additional traits. Contrary to previous findings, we detected no significant differences in SbSUTs gene expression within stem tissues. CONCLUSIONS: Phloem sieve tubes within sweet and grain sorghum stems are symplasmically isolated from surrounding cells; hence, unloading from the phloem likely occurs apoplasmically, thereby defining the location of the previously postulated step for sucrose transport. Additionally, no changes in SbSUTs gene expression were detected in sweet vs. grain sorghum stems, suggesting alterations in SbSUT transcript levels do not account for the carbohydrate partitioning differences between cultivars. A model illustrating sucrose phloem unloading and movement to stem storage parenchyma, and highlighting roles for sucrose transport proteins in sorghum stems is discussed.

Understanding and manipulating sucrose phloem loading, unloading, metabolism, and signalling to enhance crop yield and food security.

Sucrose is produced in, and translocated from, photosynthetically active leaves (sources) to support non-photosynthetic tissues (sinks), such as developing seeds, fruits, and tubers. Different plants can utilize distinct mechanisms to transport sucrose into the phloem sieve tubes in source leaves. While phloem loading mechanisms have been extensively studied in dicot plants, there is less information about phloem loading in monocots. Maize and rice are major dietary staples, which have previously been proposed to use different cellular routes to transport sucrose from photosynthetic cells into the translocation stream. The anatomical, physiological, and genetic evidence supporting these conflicting hypotheses is examined. Upon entering sink cells, sucrose often is degraded into hexoses for a wide range of metabolic and storage processes, including biosynthesis of starch, protein, and cellulose, which are all major constituents for food, fibre, and fuel. Sucrose, glucose, fructose, and their derivate, trehalose-6-phosphate, also serve as signalling molecules to regulate gene expression either directly or through cross-talk with other signalling pathways. As such, sugar transport and metabolism play pivotal roles in plant development and realization of crop yield that needs to be increased substantially to meet the projected population demand in the foreseeable future. This review will discuss the current understanding of the control of carbon partitioning from the cellular to whole-plant levels, focusing on (i) the pathways employed for phloem loading in source leaves, particularly in grasses, and the routes used in sink organs for phloem unloading; (ii) the transporter proteins responsible for sugar efflux and influx across plasma membranes; and (iii) the key enzymes regulating sucrose metabolism, signalling, and utilization. Examples of how sugar transport and metabolism can be manipulated to improve crop productivity and stress tolerance are discussed.

Tie-dyed2 encodes a callose synthase that functions in vein development and affects symplastic trafficking within the phloem of maize leaves.

The tie-dyed2 (tdy2) mutant of maize (Zea mays) displays variegated green and yellow leaves. Intriguingly, the yellow leaf tissues hyperaccumulate starch and sucrose, the soluble sugar transported long distance through the phloem of veins. To determine the molecular basis for Tdy2 function, we cloned the gene and found that Tdy2 encodes a callose synthase. RNA in situ hybridizations revealed that in developing leaves, Tdy2 was most highly expressed in the vascular tissue. Comparative expression analysis with the vascular marker maize PINFORMED1a-yellow fluorescent protein confirmed that Tdy2 was expressed in developing vein tissues. To ascertain whether the defect in tdy2 leaves affected the movement of sucrose into the phloem or its long-distance transport, we performed radiolabeled and fluorescent dye tracer assays. The results showed that tdy2 yellow leaf regions were defective in phloem export but competent in long-distance transport. Furthermore, transmission electron microscopy of tdy2 yellow leaf regions showed incomplete vascular differentiation and implicated a defect in cell-to-cell solute movement between phloem companion cells and sieve elements. The disruption of sucrose movement in the phloem in tdy2 mutants provides evidence that the Tdy2 callose synthase functions in vascular maturation and that the vascular defects result in impaired symplastic trafficking into the phloem translocation stream.

A Plate Growth Assay to Quantify Embryonic Root Development of Zea mays.

Murashige-Skoog medium solutions have been used in a variety of plant plate growth assays, yet most research uses Arabidopsis thaliana as the study organism. For larger seeds such as maize (Zea mays), most protocols employ a paper towel roll method for experiments, which often involves wrapping maize seedlings in wet, sterile germination paper. What the paper towel roll method lacks, however, is the ability to image the roots over time without risk of contamination. Here, we describe a sterile plate growth assay that contains Murashige-Skoog medium to grow seedlings starting two days after germination. This protocol uses a section of a paper towel roll method to achieve uniform germination of maize seedlings, which are sterilely transferred onto large acrylic plates for the duration of the experiment. The media can undergo modification to include an assortment of plant hormones, exogenous sugars, and other chemicals. The acrylic plates allow researchers to freely image the plate without disturbing the seedlings and control the environment in which the seedlings are grown, such as modifications in temperature and light. Additionally, the protocol is widely adaptable for use with other cereal crops. Key features • Builds upon plate growth methods routinely used for Arabidopsis seedlings but that are inadequate for maize. • Real-time photographic analysis of seedlings up to two weeks following germination. • Allows for testing of various growth conditions involving an assortment of additives and/or modification of environmental conditions. • Samples are able to be collected for genotype screening.

De novo transcriptome assembly from the nodal root growth zone of hydrated and water-deficit stressed maize inbred line FR697.

Certain cultivars of maize show increased tolerance to water deficit conditions by maintenance of root growth. To better understand the molecular mechanisms related to this adaptation, nodal root growth zone samples were collected from the reference inbred line B73 and inbred line FR697, which exhibits a relatively greater ability to maintain root elongation under water deficits. Plants were grown under various water stress levels in both field and controlled environment settings. FR697-specific RNA-Seq datasets were generated and used for a de novo transcriptome assembly to characterize any genotype-specific genetic features. The assembly was aided by an Iso-Seq library of transcripts generated from various FR697 plant tissue samples. The Necklace pipeline was used to combine a Trinity de novo assembly along with a reference guided assembly and the Viridiplantae proteome to generate an annotated consensus "SuperTranscriptome" assembly of 47,915 transcripts with a N50 of 3152 bp in length. The results were compared by Blastn to maize reference genes, a Benchmarking Universal Single-Copy Orthologs (BUSCO) genome completeness report and compared with three maize reference genomes. The resultant 'SuperTranscriptome' was demonstrated to be of high-quality and will serve as an important reference for analysis of the maize nodal root transcriptomic response to environmental perturbations.

Phloem Loading and Unloading of Sucrose: What a Long, Strange Trip from Source to Sink.

Sucrose is transported from sources (mature leaves) to sinks (importing tissues such as roots, stems, fruits, and seeds) through the phloem tissues in veins. In many herbaceous crop species, sucrose must first be effluxed to the cell wall by a sugar transporter of the SWEET family prior to being taken up into phloem companion cells or sieve elements by a different sugar transporter, called SUT or SUC. The import of sucrose into these cells is termed apoplasmic phloem loading. In sinks, sucrose can similarly exit the phloem apoplasmically or, alternatively, symplasmically through plasmodesmata into connecting parenchyma storage cells. Recent advances describing the regulation and manipulation of sugar transporter expression and activities provide stimulating new insights into sucrose phloem loading in sources and unloading processes in sink tissues. Additionally, new breakthroughs have revealed distinct subpopulations of cells in leaves with different functions pertaining to phloem loading. These and other discoveries in sucrose transport are discussed.