Sugar beet PMT5a and STP13 carriers suitable for proton-driven plasma membrane sucrose and glucose import in taproots.

Sugar beet (Beta vulgaris) is the major sugar-producing crop in Europe and Northern America, as the taproot stores sucrose at a concentration of around 20%. Genome sequence analysis together with biochemical and electrophysiological approaches led to the identification and characterization of the TST sucrose transporter driving vacuolar sugar accumulation in the taproot. However, the sugar transporters mediating sucrose uptake across the plasma membrane of taproot parenchyma cells remained unknown. As with glucose, sucrose stimulation of taproot parenchyma cells caused inward proton fluxes and plasma membrane depolarization, indicating a sugar/proton symport mechanism. To decipher the nature of the corresponding proton-driven sugar transporters, we performed taproot transcriptomic profiling and identified the cold-induced PMT5a and STP13 transporters. When expressed in Xenopus laevis oocytes, BvPMT5a was characterized as a voltage- and H+-driven low-affinity glucose transporter, which does not transport sucrose. In contrast, BvSTP13 operated as a high-affinity H+/sugar symporter, transporting glucose better than sucrose, and being more cold-tolerant than BvPMT5a. Modeling of the BvSTP13 structure with bound mono- and disaccharides suggests plasticity of the binding cleft to accommodate the different saccharides. The identification of BvPMT5a and BvSTP13 as taproot sugar transporters could improve breeding of sugar beet to provide a sustainable energy crop.

Symplasmic phloem loading and subcellular transport in storage roots are key factors for carbon allocation in cassava.

Cassava (Manihot esculenta) is a deciduous woody perennial shrub that stores large amounts of carbon and water in its storage roots. Previous studies have shown that assimilate unloading into storage roots happens symplasmically once secondary anatomy is established. However, mechanisms controlling phloem loading and overall carbon partitioning to different cassava tissues remain unclear. Here, we used a combination of histological, transcriptional, and biochemical analyses on different cassava tissues and at different timepoints to better understand source-sink carbon allocation. We found that cassava likely utilizes a predominantly passive symplasmic phloem loading strategy, indicated by the lack of expression of genes coding for key players of sucrose transport, the existence of branched plasmodesmata in the companion cell/bundle sheath interface of minor leaf veins, and very high leaf sucrose concentrations. Furthermore, we showed that tissue-specific changes in anatomy and non-structural carbohydrate (NSC) contents are associated with tissue-specific modification in gene expression for sucrose cleavage/synthesis, as well as subcellular compartmentalization of sugars. Overall, our data suggest that carbon allocation during storage root filling is mostly facilitated symplasmically and is likely mostly regulated by local tissue demand and subcellular compartmentalization.

The novel chloroplast glucose transporter pGlcT2 affects adaptation to extended light periods.

Intracellular sugar compartmentation is critical in plant development and acclimation to challenging environmental conditions. Sugar transport proteins are present in plasma membranes and in membranes of organelles such as vacuoles, the Golgi apparatus, and plastids. However, there may exist other transport proteins with uncharacterized roles in sugar compartmentation. Here we report one such novel transporter of the Monosaccharide Transporter Family, the closest phylogenetic homolog of which is the chloroplast-localized glucose transporter pGlcT and that we therefore term plastidic glucose transporter 2 (pGlcT2). We show, using gene-complemented glucose uptake deficiency of an Escherichia coli ptsG/manXYZ mutant strain and biochemical characterization, that this protein specifically facilitates glucose transport, whereas other sugars do not serve as substrates. In addition, we demonstrate pGlcT2-GFP localized to the chloroplast envelope and that pGlcT2 is mainly produced in seedlings and in the rosette center of mature Arabidopsis plants. Therefore, in conjunction with molecular and metabolic data, we propose pGlcT2 acts as a glucose importer that can limit cytosolic glucose availability in developing pGlcT2-overexpressing seedlings. Finally, we show both overexpression and deletion of pGlcT2 resulted in impaired growth efficiency under long day and continuous light conditions, suggesting pGlcT2 contributes to a release of glucose derived from starch mobilization late in the light phase. Together, these data indicate the facilitator pGlcT2 changes the direction in which it transports glucose during plant development and suggest the activity of pGlcT2 must be controlled spatially and temporarily in order to prevent developmental defects during adaptation to periods of extended light.

Efficient sugar utilization and transition from oxidative to substrate-level phosphorylation in high starch storage roots of African cassava genotypes.

Cassava's storage roots represent one of the most important sources of nutritional carbohydrates worldwide. Particularly, smallholder farmers in sub-Saharan Africa depend on this crop plant, where resilient and yield-improved varieties are of vital importance to support steadily increasing populations. Aided by a growing understanding of the plant's metabolism and physiology, targeted improvement concepts already led to visible gains in recent years. To expand our knowledge and to contribute to these successes, we investigated storage roots of eight cassava genotypes with differential dry matter content from three successive field trials for their proteomic and metabolic profiles. At large, the metabolic focus in storage roots transitioned from cellular growth processes toward carbohydrate and nitrogen storage with increasing dry matter content. This is reflected in higher abundance of proteins related to nucleotide synthesis, protein turnover, and vacuolar energization in low starch genotypes, while proteins involved in sugar conversion and glycolysis were more prevalent in high dry matter genotypes. This shift in metabolic orientation was underlined by a clear transition from oxidative- to substrate-level phosphorylation in high dry matter genotypes. Our analyses highlight metabolic patterns that are consistently and quantitatively associated with high dry matter accumulation in cassava storage roots, providing fundamental understanding of cassava's metabolism as well as a data resource for targeted genetic improvement.

Vacuolar sugar transporter EARLY RESPONSE TO DEHYDRATION6-LIKE4 affects fructose signaling and plant growth.

Regulation of intracellular sugar homeostasis is maintained by regulation of activities of sugar import and export proteins residing at the tonoplast. We show here that the EARLY RESPONSE TO DEHYDRATION6-LIKE4 (ERDL4) protein, a member of the monosaccharide transporter family, resides in the vacuolar membrane in Arabidopsis (Arabidopsis thaliana). Gene expression and subcellular fractionation studies indicated that ERDL4 participates in fructose allocation across the tonoplast. Overexpression of ERDL4 increased total sugar levels in leaves due to a concomitantly induced stimulation of TONOPLAST SUGAR TRANSPORTER 2 (TST2) expression, coding for the major vacuolar sugar loader. This conclusion is supported by the finding that tst1-2 knockout lines overexpressing ERDL4 lack increased cellular sugar levels. ERDL4 activity contributing to the coordination of cellular sugar homeostasis is also indicated by 2 further observations. First, ERDL4 and TST genes exhibit an opposite regulation during a diurnal rhythm, and second, the ERDL4 gene is markedly expressed during cold acclimation, representing a situation in which TST activity needs to be upregulated. Moreover, ERDL4-overexpressing plants show larger rosettes and roots, a delayed flowering time, and increased total seed yield. Consistently, erdl4 knockout plants show impaired cold acclimation and freezing tolerance along with reduced plant biomass. In summary, we show that modification of cytosolic fructose levels influences plant organ development and stress tolerance.

Genome-wide association study unveils ascorbate regulation by PAS/LOV PROTEIN during high light acclimation.

Varying light conditions elicit metabolic responses as part of acclimation with changes in ascorbate levels being an important component. Here, we adopted a genome-wide association-based approach to characterize the response in ascorbate levels on high light (HL) acclimation in a panel of 315 Arabidopsis (Arabidopsis thaliana) accessions. These studies revealed statistically significant SNPs for total and reduced ascorbate under HL conditions at a locus in chromosome 2. Ascorbate levels under HL and the region upstream and within PAS/LOV PROTEIN (PLP) were strongly associated. Intriguingly, subcellular localization analyses revealed that the PLPA and PLPB splice variants co-localized with VITAMIN C DEFECTIVE2 (VTC2) and VTC5 in both the cytosol and nucleus. Yeast 2-hybrid and bimolecular fluorescence complementation analyses revealed that PLPA and PLPB interact with VTC2 and that blue light diminishes this interaction. Furthermore, PLPB knockout mutants were characterized by 1.5- to 1.7-fold elevations in their ascorbate levels, whereas knockout mutants of the cry2 cryptochromes displayed 1.2- to 1.3-fold elevations compared to WT. Our results collectively indicate that PLP plays a critical role in the elevation of ascorbate levels, which is a signature response of HL acclimation. The results strongly suggest that this is achieved via the release of the inhibitory effect of PLP on VTC2 upon blue light illumination, as the VTC2-PLPB interaction is stronger under darkness. The conditional importance of the cryptochrome receptors under different environmental conditions suggests a complex hierarchy underpinning the environmental control of ascorbate levels. However, the data we present here clearly demonstrate that PLP dominates during HL acclimation.

Carbohydrate distribution via SWEET17 is critical for Arabidopsis inflorescence branching under drought.

Sugars Will Eventually be Exported Transporters (SWEETs) are the most recently discovered family of plant sugar transporters. By acting as uniporters, SWEETs facilitate the diffusion of sugars across cell membranes and play an important role in various physiological processes such as abiotic stress adaptation. AtSWEET17, a vacuolar fructose facilitator, was shown to be involved in the modulation of the root system during drought. In addition, previous studies have shown that overexpression of an apple homolog leads to increased drought tolerance in tomato plants. Therefore, SWEET17 might be a molecular element involved in the plant´s drought response. However, the role and function of SWEET17 in aboveground tissues of Arabidopsis under drought stress remains elusive. By combining gene expression analysis and stem architecture with the sugar profiles of different aboveground tissues, we uncovered a putative role of SWEET17 in carbohydrate supply and thus cauline branch elongation, especially during periods of carbon limitation, as occurs under drought stress. Thus, SWEET17 seems to be involved in maintaining efficient plant reproduction under drought-stress conditions.

Loss of a pyridoxal-phosphate phosphatase rescues Arabidopsis lacking an endoplasmic reticulum ATP carrier.

The endoplasmic reticulum (ER)-located ATP/ADP-antiporter (ER-ANT1) occurs specifically in vascular plants. Structurally different transporters mediate energy provision to the ER, but the cellular function of ER-ANT1 is still unknown. Arabidopsis (Arabidopsis thaliana) mutants lacking ER-ANT1 (er-ant1 plants) exhibit a photorespiratory phenotype accompanied by high glycine levels and stunted growth, pointing to an inhibition of glycine decarboxylase (GDC). To reveal whether it is possible to suppress this marked phenotype, we exploited the power of a forward genetic screen. Absence of a so far uncharacterized member of the HaloAcid Dehalogenase (HAD)-like hydrolase family strongly suppressed the dwarf phenotype of er-ant1 plants. Localization studies suggested that the corresponding protein locates to chloroplasts, and activity assays showed that the enzyme dephosphorylates, with high substrate affinity, the B6 vitamer pyridoxal 5'-phosphate (PLP). Additional physiological experiments identified imbalances in vitamin B6 homeostasis in er-ant1 mutants. Our data suggest that impaired chloroplast metabolism, but not decreased GDC activity, causes the er-ant1 mutant dwarf phenotype. We present a hypothesis, setting transport of PLP by ER-ANT1 and chloroplastic PLP dephosphorylation in the cellular context. With the identification of this HAD-type PLP phosphatase, we also provide insight into B6 vitamer homeostasis.

Hexose translocation mediated by SlSWEET5b is required for pollen maturation in Solanum lycopersicum.

Pollen fertility is critical for successful fertilization and, accordingly, for crop yield. While sugar unloading affects the growth and development of all types of sink organs, the molecular nature of sugar import to tomato (Solanum lycopersicum) pollen is poorly understood. However, sugar will eventually be exported transporters (SWEETs) have been proposed to be involved in pollen development. Here, reverse transcription-quantitative polymerase chain reaction (PCR) revealed that SlSWEET5b was markedly expressed in flowers when compared to the remaining tomato SlSWEETs, particularly in the stamens of maturing flower buds undergoing mitosis. Distinct accumulation of SlSWEET5b-ß-glucuronidase activities was present in mature flower buds, especially in anther vascular and inner cells, symplasmic isolated microspores (pollen grains), and styles. The demonstration that SlSWEET5b-GFP fusion proteins are located in the plasma membrane supports the idea that the SlSWEET5b carrier functions in apoplasmic sugar translocation during pollen maturation. This is consistent with data from yeast complementation experiments and radiotracer uptake, showing that SlSWEET5b operates as a low-affinity hexose-specific passive facilitator, with a Km of ∼36 mM. Most importantly, RNAi-mediated suppression of SlSWEET5b expression resulted in shrunken nucleus-less pollen cells, impaired germination, and low seed yield. Moreover, stamens from SlSWEET5b-silenced tomato mutants showed significantly lower amounts of sucrose (Suc) and increased invertase activity, indicating reduced carbon supply and perturbed Suc homeostasis in these tissues. Taken together, our findings reveal the essential role of SlSWEET5b in mediating apoplasmic hexose import into phloem unloading cells and into developing pollen cells to support pollen mitosis and maturation in tomato flowers.

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.

The Tapetal Major Facilitator NPF2.8 Is Required for Accumulation of Flavonol Glycosides on the Pollen Surface in Arabidopsis thaliana.

The exine of angiosperm pollen grains is usually covered by a complex mix of metabolites including pollen-specific hydroxycinnamic acid amides (HCAAs) and flavonoid glycosides. Although the biosynthetic pathways resulting in the formation of HCAAs and flavonol glycosides have been characterized, it is unclear how these compounds are transported to the pollen surface. In this report we provide several lines of evidence that a member of the nitrate/peptide transporter family is required for the accumulation and transport of pollen-specific flavonol 3-o-sophorosides, characterized by a glycosidic ß-1,2-linkage, to the pollen surface of Arabidopsis (Arabidopsis thaliana). Ectopic, transient expression in Nicotiana benthamiana epidermal leaf cells demonstrated localization of this flavonol sophoroside transporter (FST1) at the plasmalemma when fused to green fluorescent protein (GFP). We also confirmed the tapetum-specific expression of FST1 by GFP reporter lines driven by the FST1 promoter. In vitro characterization of FST1 activity was achieved by microbial uptake assays based on 14C-labeled flavonol glycosides. Finally, rescue of an fst1 insertion mutant by complementation with an FST1 genomic fragment restored the accumulation of flavonol glycosides in pollen grains to wild-type levels, corroborating the requirement of FST1 for transport of flavonol-3-o-sophorosides from the tapetum to the pollen surface.

Vernalization Alters Sink and Source Identities and Reverses Phloem Translocation from Taproots to Shoots in Sugar Beet.

During their first year of growth, overwintering biennial plants transport Suc through the phloem from photosynthetic source tissues to storage tissues. In their second year, they mobilize carbon from these storage tissues to fuel new growth and reproduction. However, both the mechanisms driving this shift and the link to reproductive growth remain unclear. During vegetative growth, biennial sugar beet (Beta vulgaris) maintains a steep Suc concentration gradient between the shoot (source) and the taproot (sink). To shift from vegetative to generative growth, they require a chilling phase known as vernalization. We studied sugar beet sink-source dynamics upon vernalization and showed that before flowering, the taproot underwent a reversal from a sink to a source of carbohydrates. This transition was induced by transcriptomic and functional reprogramming of sugar beet tissue, resulting in a reversal of flux direction in the phloem. In this transition, the vacuolar Suc importers and exporters TONOPLAST SUGAR TRANSPORTER2;1 and SUCROSE TRANSPORTER4 were oppositely regulated, leading to the mobilization of sugars from taproot storage vacuoles. Concomitant changes in the expression of floral regulator genes suggest that these processes are a prerequisite for bolting. Our data will help both to dissect the metabolic and developmental triggers for bolting and to identify potential targets for genome editing and breeding.

Acclimation in plants - the Green Hub consortium.

Acclimation is the capacity to adapt to environmental changes within the lifetime of an individual. This ability allows plants to cope with the continuous variation in ambient conditions to which they are exposed as sessile organisms. Because environmental changes and extremes are becoming even more pronounced due to the current period of climate change, enhancing the efficacy of plant acclimation is a promising strategy for mitigating the consequences of global warming on crop yields. At the cellular level, the chloroplast plays a central role in many acclimation responses, acting both as a sensor of environmental change and as a target of cellular acclimation responses. In this Perspective article, we outline the activities of the Green Hub consortium funded by the German Science Foundation. The main aim of this research collaboration is to understand and strategically modify the cellular networks that mediate plant acclimation to adverse environments, employing Arabidopsis, tobacco (Nicotiana tabacum) and Chlamydomonas as model organisms. These efforts will contribute to 'smart breeding' methods designed to create crop plants with improved acclimation properties. To this end, the model oilseed crop Camelina sativa is being used to test modulators of acclimation for their potential to enhance crop yield under adverse environmental conditions. Here we highlight the current state of research on the role of gene expression, metabolism and signalling in acclimation, with a focus on chloroplast-related processes. In addition, further approaches to uncovering acclimation mechanisms derived from systems and computational biology, as well as adaptive laboratory evolution with photosynthetic microbes, are highlighted.

Multi-omics data integration reveals link between epigenetic modifications and gene expression in sugar beet (Beta vulgaris subsp. vulgaris) in response to cold.

BACKGROUND: DNA methylation is thought to influence the expression of genes, especially in response to changing environmental conditions and developmental changes. Sugar beet (Beta vulgaris ssp. vulgaris), and other biennial or perennial plants are inevitably exposed to fluctuating temperatures throughout their lifecycle and might even require such stimulus to acquire floral competence. Therefore, plants such as beets, need to fine-tune their epigenetic makeup to ensure phenotypic plasticity towards changing environmental conditions while at the same time steering essential developmental processes. Different crop species may show opposing reactions towards the same abiotic stress, or, vice versa, identical species may respond differently depending on the specific kind of stress. RESULTS: In this study, we investigated common effects of cold treatment on genome-wide DNA methylation and gene expression of two Beta vulgaris accessions via multi-omics data analysis. Cold exposure resulted in a pronounced reduction of DNA methylation levels, which particularly affected methylation in CHH context (and to a lesser extent CHG) and was accompanied by transcriptional downregulation of the chromomethyltransferase CMT2 and strong upregulation of several genes mediating active DNA demethylation. CONCLUSION: Integration of methylomic and transcriptomic data revealed that, rather than methylation having directly influenced expression, epigenetic modifications correlated with changes in expression of known players involved in DNA (de)methylation. In particular, cold triggered upregulation of genes putatively contributing to DNA demethylation via the ROS1 pathway. Our observations suggest that these transcriptional responses precede the cold-induced global DNA-hypomethylation in non-CpG, preparing beets for additional transcriptional alterations necessary for adapting to upcoming environmental changes.

Transporter SlSWEET15 unloads sucrose from phloem and seed coat for fruit and seed development in tomato.

Tomato (Solanum lycopersium), an important fruit crop worldwide, requires efficient sugar allocation for fruit development. However, molecular mechanisms for sugar import to fruits remain poorly understood. Expression of sugars will eventually be exported transporters (SWEETs) proteins is closely linked to high fructose/glucose ratios in tomato fruits and may be involved in sugar allocation. Here, we discovered that SlSWEET15 is highly expressed in developing fruits compared to vegetative organs. In situ hybridization and ß-glucuronidase fusion analyses revealed SlSWEET15 proteins accumulate in vascular tissues and seed coats, major sites of sucrose unloading in fruits. Localizing SlSWEET15-green fluorescent protein to the plasma membrane supported its putative role in apoplasmic sucrose unloading. The sucrose transport activity of SlSWEET15 was confirmed by complementary growth assays in a yeast (Saccharomyces cerevisiae) mutant. Elimination of SlSWEET15 function by clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPR-associated protein gene editing significantly decreased average sizes and weights of fruits, with severe defects in seed filling and embryo development. Altogether, our studies suggest a role of SlSWEET15 in mediating sucrose efflux from the releasing phloem cells to the fruit apoplasm and subsequent import into storage parenchyma cells during fruit development. Furthermore, SlSWEET15-mediated sucrose efflux is likely required for sucrose unloading from the seed coat to the developing embryo.

Ectopic maltase alleviates dwarf phenotype and improves plant frost tolerance of maltose transporter mutants.

Maltose, the major product of starch breakdown in Arabidopsis (Arabidopsis thaliana) leaves, exits the chloroplast via the maltose exporter1 MEX1. Consequently, mex1 loss-of-function plants exhibit substantial maltose accumulation, a starch-excess phenotype and a specific chlorotic phenotype during leaf development. Here, we investigated whether the introduction of an alternative metabolic route could suppress the marked developmental defects typical for mex1 loss-of-function mutants. To this end, we ectopically expressed in mex1  chloroplasts a functional maltase (MAL) from baker's yeast (Saccharomyces cerevisiae, chloroplastidial MAL [cpMAL] mutants). Remarkably, the stromal MAL activity substantially alleviates most phenotypic peculiarities typical for mex1 plants. However, the cpMAL lines contained only slightly less maltose than parental mex1 plants and their starch levels were, surprisingly, even higher. These findings point to a threshold level of maltose responsible for the marked developmental defects in mex1. While growth and flowering time were only slightly retarded, cpMAL lines exhibited a substantially improved frost tolerance, when compared to wild-types. In summary, these results demonstrate the possibility to bypass the MEX1 transporter, allow us to differentiate between possible starch-excess and maltose-excess responses, and demonstrate that stromal maltose accumulation prevents frost defects. The latter insight may be instrumental for the development of crop plants with improved frost tolerance.

Contributions of sugar transporters to crop yield and fruit quality.

The flux, distribution, and storage of soluble sugars regulate crop yield in terms of starch, oil, protein, and total carbohydrates, and affect the quality of many horticultural products. Sugar transporters contribute to phloem loading and unloading. The mechanisms of phloem loading have been studied in detail, but the complex and diverse mechanisms of phloem unloading and sugar storage in sink organs are less explored. Unloading and subsequent transport mechanisms for carbohydrates vary in different sink organs. Analyzing the transport and storage mechanisms of carbohydrates in important storage organs, such as cereal seeds, fruits, or stems of sugarcane, will provide information for genetic improvements to increase crop yield and fruit quality. This review discusses current research progress on sugar transporters involved in carbohydrate unloading and storage in sink organs. The roles of sugar transporters in crop yield and the accumulation of sugars are also discussed to highlight their contribution to efficient breeding.

Metabolic profiles of six African cultivars of cassava (Manihot esculenta Crantz) highlight bottlenecks of root yield.

Cassava is an important staple crop in sub-Saharan Africa, due to its high productivity even on nutrient poor soils. The metabolic characteristics underlying this high productivity are poorly understood including the mode of photosynthesis, reasons for the high rate of photosynthesis, the extent of source/sink limitation, the impact of environment, and the extent of variation between cultivars. Six commercial African cassava cultivars were grown in a greenhouse in Erlangen, Germany, and in the field in Ibadan, Nigeria. Source leaves, sink leaves, stems and storage roots were harvested during storage root bulking and analyzed for sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, protein, activities of enzymes in central metabolism and yield traits. High ratios of RuBisCO:phosphoenolpyruvate carboxylase activity support a C3 mode of photosynthesis. The high rate of photosynthesis is likely to be attributed to high activities of enzymes in the Calvin-Benson cycle and pathways for sucrose and starch synthesis. Nevertheless, source limitation is indicated because root yield traits correlated with metabolic traits in leaves rather than in the stem or storage roots. This situation was especially so in greenhouse-grown plants, where irradiance will have been low. In the field, plants produced more storage roots. This was associated with higher AGPase activity and lower sucrose in the roots, indicating that feedforward loops enhanced sink capacity in the high light and low nitrogen environment in the field. Overall, these results indicated that carbon assimilation rate, the K battery, root starch synthesis, trehalose, and chlorogenic acid accumulation are potential target traits for genetic improvement.

GeSUT4 mediates sucrose import at the symbiotic interface for carbon allocation of heterotrophic Gastrodia elata (Orchidaceae).

Gastrodia elata, a fully mycoheterotrophic orchid without photosynthetic ability, only grows symbiotically with the fungus Armillaria. The mechanism of carbon distribution in this mycoheterotrophy is unknown. We detected high sucrose concentrations in all stages of Gastrodia tubers, suggesting sucrose may be the major sugar transported between fungus and orchid. Thick symplasm-isolated wall interfaces in colonized and adjacent large cells implied involvement of sucrose importers. Two sucrose transporter (SUT)-like genes, GeSUT4 and GeSUT3, were identified that were highly expressed in young Armillaria-colonized tubers. Yeast complementation and isotope tracer experiments confirmed that GeSUT4 functioned as a high-affinity sucrose-specific proton-dependent importer. Plasma-membrane/tonoplast localization of GeSUT4-GFP fusions and high RNA expression of GeSUT4 in symbiotic and large cells indicated that GeSUT4 likely functions in active sucrose transport for intercellular allocation and intracellular homeostasis. Transgenic Arabidopsis overexpressing GeSUT4 had larger leaves but were sensitive to excess sucrose and roots were colonized with fewer mutualistic Bacillus, supporting the role of GeSUT4 in regulating sugar allocation. This is not only the first documented carbon import system in a mycoheterotrophic interaction but also highlights the evolutionary importance of sucrose transporters for regulation of carbon flow in all types of plant-microbe interactions.

Auxin signaling and vascular cambium formation enable storage metabolism in cassava tuberous roots.

Cassava storage roots are among the most important root crops worldwide, and represent one of the most consumed staple foods in sub-Saharan Africa. The vegetatively propagated tropical shrub can form many starchy tuberous roots from its stem. These storage roots are formed through the activation of secondary root growth processes. However, the underlying genetic regulation of storage root development is largely unknown. Here we report distinct structural and transcriptional changes occurring during the early phases of storage root development. A pronounced increase in auxin-related transcripts and the transcriptional activation of secondary growth factors, as well as a decrease in gibberellin-related transcripts were observed during the early stages of secondary root growth. This was accompanied by increased cell wall biosynthesis, most notably increased during the initial xylem expansion within the root vasculature. Starch storage metabolism was activated only after the formation of the vascular cambium. The formation of non-lignified xylem parenchyma cells and the activation of starch storage metabolism coincided with increased expression of the KNOX/BEL genes KNAT1, PENNYWISE, and POUND-FOOLISH, indicating their importance for proper xylem parenchyma function.