tie-dyed1 Regulates carbohydrate accumulation in maize leaves

Acquisition of cell identity requires communication among neighboring cells. To dissect the genetic pathways regulating cell signaling in later leaf development, a screen was performed to identify mutants with chloroplast pigmentation sectors that violate cell lineage boundaries in maize (Zea mays) leaves. We have characterized a recessive mutant, tie-dyed1 (tdy1), which develops stable, nonclonal variegated yellow and green leaf sectors. Sector formation requires high light, occurs during a limited developmental time, and is restricted to leaf blade tissue. Yellow tdy1 sectors accumulate excessive soluble sugars and starch, whereas green sectors appear unaffected. Significantly, starch accumulation precedes chlorosis in cells that will become a yellow sector. Retention of carbohydrates in tdy1 leaves is associated with a delay in reproductive maturity, decreased stature, and reduced yield. To explain the tdy1 sectoring pattern, we propose a threshold model that incorporates the light requirement and the hyperaccumulation of photoassimilates. A possible function consistent with this model is that TDY1 acts as a sugar sensor to regulate an inducible sugar export pathway as leaves develop under high light conditions.     

tie-dyed1 Functions non-cell autonomously to control carbohydrate accumulation in maize leaves

The tie-dyed1 (tdy1) mutant of maize (Zea mays) produces chlorotic, anthocyanin-accumulating regions in leaves due to the hyperaccumulation of carbohydrates. Based on the nonclonal pattern, we propose that the accumulation of sucrose (Suc) or another sugar induces the tdy1 phenotype. The boundaries of regions expressing the tdy1 phenotype frequently occur at lateral veins. This suggests that lateral veins act to limit the expansion of tdy1 phenotypic regions by transporting Suc out of the tissue. Double mutant studies between tdy1 and chloroplast-impaired mutants demonstrate that functional chloroplasts are needed to generate the Suc that induces the tdy1 phenotype. However, we also found that albino cells can express the tdy1 phenotype and overaccumulate Suc imported from neighboring green tissues. To characterize the site and mode of action of Tdy1, we performed a clonal mosaic analysis. In the transverse dimension, we localized the function of Tdy1 to the innermost leaf layer. Additionally, we determined that if this layer lacks Tdy1, Suc can accumulate, move into adjacent genetically wild-type layers, and induce tdy1 phenotypic expression. In the lateral dimension, we observed that a tdy1 phenotypic region did not reach the mosaic sector boundary, suggesting that wild-type Tdy1 acts non-cell autonomously and exerts a short-range compensatory effect on neighboring mutant tissue. A model proposing that Tdy1 functions in the vasculature to sense high concentrations of sugar, up-regulate Suc transport into veins, and promote tissue differentiation and function is discussed.     

<Emphasis Type="Italic">Tie-dyed1</Emphasis> and <Emphasis Type="Italic">Sucrose export defective1</Emphasis> act independently to promote carbohydrate export from maize leaves

tie-dyed1 (tdy1) and sucrose export defective1 (sxd1) are recessive maize (Zea mays) mutants with nonclonal chlorotic leaf sectors that hyperaccumulate starch and soluble sugars. In addition, both mutants display similar growth-related defects such as reduced plant height and inflorescence development due to the retention of carbohydrates in leaves. As tdy1 and sxd1 are the only variegated leaf mutants known to accumulate carbohydrates in any plant, we investigated whether Tdy1 and Sxd1 function in the same pathway. Using aniline blue staining for callose and transmission electron microscopy to inspect plasmodesmatal ultrastructure, we determined that tdy1 does not have any physical blockage or alteration along the symplastic transport pathway as found in sxd1 mutants. To test whether the two genes function in the same genetic pathway, we constructed F₂ families segregating both mutations. Double mutant plants showed an additive interaction for growth related phenotypes and soluble sugar accumulation, and expressed the leaf variegation pattern of both single mutants indicating that Tdy1 and Sxd1 act in separate genetic pathways. Although sxd1 mutants lack tocopherols, we determined that tdy1 mutants have wild-type tocopherol levels, indicating that Tdy1 does not function in the same biochemical pathway as Sxd1. From these and other data we conclude that Tdy1 and Sxd1 function independently to promote carbon export from leaves. Our genetic and cytological studies implicate Tdy1 functioning in veins, and a model discussing possible functions of TDY1 is presented. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tie-dyed1 encodes a novel, phloem-expressed transmembrane protein that functions in carbohydrate partitioning

Carbon is partitioned between export from the leaf and retention within the leaf, and this process is essential for all aspects of plant growth and development. In most plants, sucrose is loaded into the phloem of carbon-exporting leaves (sources), transported through the veins, and unloaded into carbon-importing tissues (sinks). We have taken a genetic approach to identify genes regulating carbon partitioning in maize (Zea mays). We identified a collection of mutants, called the tie-dyed (tdy) loci, that hyperaccumulate carbohydrates in regions of their leaves. To understand the molecular function of Tdy1, we cloned the gene. Tdy1 encodes a novel transmembrane protein present only in grasses, although two protein domains are conserved across angiosperms. We found that Tdy1 is expressed exclusively in phloem cells of both source and sink tissues, suggesting that Tdy1 may play a role in phloem loading and unloading processes. In addition, Tdy1 RNA accumulates in protophloem cells upon differentiation, suggesting that Tdy1 may function as soon as phloem cells become competent to transport assimilates. Monitoring the movement of a fluorescent, soluble dye showed that tdy1 leaves have retarded phloem loading. However, once the dye entered into the phloem, solute transport appeared equal in wild-type and tdy1 mutant plants, suggesting that tdy1 plants are not defective in phloem unloading. Therefore, even though Tdy1 RNA accumulates in source and sink tissues, we propose that TDY1 functions in carbon partitioning by promoting phloem loading. Possible roles for TDY1 are discussed.     

Reactivation of phloem export in mature maize leaves after a dark period

Mature leaves of corn plants (Zea mays L. cv. Prior) which were darkened for 48 h contain neither bundle-sheath starch nor glucose, and their sucrose content is below 5 M. In such leaves phloem export has ceased. When re-illuminated, photosynthetic sucrose production starts without delay, but the sucrose: glucose ratio is 1.25:1. Obviously, most of the new-formed sugar is utilized locally. Labeling with ¹⁴CO₂ has shown that phloen export starts 30 to 40 min after the onset of photosynthesis, when the sucrose: glucose ratio has increased to 13:1. The first newly formed starch can be detected when phloem export is reactivated. Glucose content remains constantly low af about 2 M for at least 2 h, and it never exceeds 10 M. Radioactivity in the exporting veins is about five times higher after 2 to 7 h of re-illumination than in the 14-h-day plant. Therefore, phloem export is either intensified during the period of reactivation or exported assimilates are partly unloaded along their way. Comparison of photosynthetic activity of equal-sized leaf strips has shown that both accumulation of photosynthates and radioactivity of exporting veins are about three times higher in the detached strip than in the strip which remained attached to the mother plant.     

Trends in carbohydrate depletion, respiratory carbon loss, and assimilate export from soybean leaves at night

To evaluate assimilate export from soybean (Glycine max [L.] Merrill) leaves at night, rates of respiratory CO₂ loss, specific leaf weight loss, starch mobilization, and changes in sucrose concentration were measured during a 10-hour dark period in leaves of pod-bearing `Amsoy 71' and `Wells II' plants in a controlled environment. Lateral leaflets were removed at various times between 2200 hours (beginning dark period) and 0800 hours (ending dark period) for dry weight determination and carbohydrate analyses. Respiratory CO₂ loss was measured throughout the 10-hour dark period. Rate of export was estimated from the rate of loss in specific leaf weight and rate of CO₂ efflux. Rate of assimilate export was not constant. Rate of export was relatively low during the beginning of the dark period, peaked during the middle of the dark period, and then decreased to near zero by the end of darkness. Rate of assimilate export was associated with rate of starch mobilization and amount of starch reserves available for export. Leaves of Amsoy 71 had a higher maximum export rate in conjunction with a greater total change in starch concentration than did leaves of Wells II. Sucrose concentration rapidly declined during the first hour of darkness and then remained constant throughout the rest of the night in leaves of both cultivars. Rate of assimilate export was not associated with leaf sucrose concentration.     

The respiratory energy requirements involved in nocturnal carbohydrate export from starch-storing mature source leaves and their contribution to leaf dark respiration

The present study explores the potential contribution of theenergy requirements associated with nocturnal carbohydrate exportto (1) the fraction of dark respiration correlating with leafnitrogen concentration and (2) the dark respiration of maturesource leaves. To this end, we determined the nocturnal carbohydrate-exportrates from leaves with an optimal nitrogen supply, and the correlationbetween the nitrogen concentration and the dark respirationof leaves. The specific energy costs of carbohydrate exportfrom starch-storing source leaves were determined both experimentallyand theoretically. The present estimate of the specific energycost involved in carbohydrate export as obtained by linear regression(0.70 mol CO₂ [mol sucrose]^–1), agrees well with bothliterature data obtained by different methods (0.47 to 1.26mol CO₂ [mol sucrose]^–1) and the theoretically calculatedrange for starch-storing species (0.40 to 1.20 mol CO₂ [molsucrose]^–1). The conversion of starch in the chloroplastto sucrose in the cytosol is a major energy-requiring process.Maximally 42 to ‘107’% of the slope of the relationshipbetween respiration rate and organic nitrogen concentrationof primary bean leaves, may be ascribed to the energy costsassociated with nocturnal export of carbohydrates. Total energycosts associated with export were derived from the product ofthe specific costs of carbohydrate export and the export rates,either measured on full-grown (primary) leaves of potato andbean or derived from the literature. These export costs account,on average, for 29% of the dark respiration rate in starch-storingspecies. We conclude that nocturnal carbohydrate export is amajor energy-requiring process in starch-storing species Key words: Carbohydrate export, leaf dark respiration, nitrogen concentration, respiratory costs, specific energy cost     

Polypeptides from maize seedlings with protein kinase functions

Low molecular weight protein kinase from maize seedlings was isolated. The molecular weight of the enzyme was below 10 000. The enzyme preferentially utilized ATP and casein as donor and acceptor of phosphorus, respectively. Casein kinase was a cyclic nucleotide-independent, heparin-sensitive enzyme. The low molecular weight casein kinase was resolved into basic, neutral and slightly acidic peaks of activity by chromatofocussing.     

Carbon partitioning and export from mature cotton leaves

The partitioning of carbon in intact, mature cotton (Gossypium hirsutum L.) leaves was examined by steady-state ¹⁴CO₂ labeling. Plants were exposed to dark periods of varying lengths, followed by similar illuminated labeling periods. These treatments produced leaves with a range of starch and soluble sugar contents, carbon exchange, and carbon export rates. Export during the illuminated periods was neither highly correlated with photosynthesis nor was export during the illuminated periods significantly different among the treatments. In contrast, the rate of subsequent nocturnal carbon export from these leaves varied widely and was found to be highly correlated with leaf starch content at the end of the illumination period (r = 0.934) and with nocturnal leaf respiration (r = 0.954). Leaves which had accumulated the highest levels of starch (about 275 micrograms per square centimeter) by the end of the illumination period exhibited nocturnal export rates very similar to those during the daylight hours. Leaves which accumulated starch to only 50 to 75 micrograms per square centimeter virtually ceased nocturnal carbon export. For leaves with starch accumulations of between 50 and 275 micrograms per square centimeter, nocturnal export was directly proportional to leaf starch at the end of the illumination period. After the nocturnal export rate was established, it continued at a constant rate throughout the night even though leaf starch and sucrose contents declined.     

Effect of water restriction on carbohydrate metabolism and photosynthesis in mature maize leaves

The time course of the modifications induced by a mild water stress has been examined for photosynthesis and several traits of carbohydrate metabolism in adult leaves of two inbred maize lines of North American and European origins, respectively. An early response was a sharp increase of the acid soluble invertase activity in adult leaves, 3–4 d after initiation of water shortage. Accordingly, correlated accumulations of fructose, glucose and to a lesser extent sucrose were observed. In the most responsive genotype, invertase activity finally reached a value > 3 times larger than the control value. By contrast, sucrose phosphate synthase activity, measured either under saturating or limiting substrate conditions, was progressively reduced by 20–40% on the 5th day and by 50–80% on the 7th day, depending on the genotype. Leaf photosynthetic rate was affected at approximately the same time as carbohydrate metabolism and stomatal conductance. Leaf water status, as measured by relative water content, declined afterwards. For all the observed responses, the two genotypes behaved very differently.     

Hypertonia-linked protein Trak1 functions with mitofusins to promote mitochondrial tethering and fusion

Hypertonia is a neurological dysfunction associated with a number of central nervous system disorders, including cerebral palsy, Parkinson’s disease, dystonia, and epilepsy. Genetic studies have identified a homozygous truncation mutation in Trak1 that causes hypertonia in mice. Moreover, elevated Trak1 protein expression is associated with several types of cancersand variants in Trak1 are linked to childhood absence epilepsy in humans. Despite the importance of Trak1 in health and disease, the mechanisms of Trak1 action remain unclear and the pathogenic effects of Trak1 mutation are unknown. Here we report that Trak1 has a crucial function in regulation of mitochondrial fusion. Depletion of Trak1 inhibits mitochondrial fusion, resulting in mitochondrial fragmentation, whereas overexpression of Trak1 elongates and enlarges mitochondria. Our analyses revealed that Trak1 interacts and colocalizes with mitofusins on the outer mitochondrial membrane and functions with mitofusins to promote mitochondrial tethering and fusion. Furthermore, Trak1 is required for stress-induced mitochondrial hyperfusion and pro-survival response. We found that hypertonia-associated mutation impairs Trak1 mitochondrial localization and its ability to facilitate mitochondrial tethering and fusion. Our findings uncover a novel function of Trak1 as a regulator of mitochondrial fusion and provide evidence linking dysregulated mitochondrial dynamics to hypertonia pathogenesis.     

Putrescine export from leaves in relation to floral transition in Sinapis alba

Sinapis alba L. was induced to flower by either a long day or a displaced short day. Following collection of leaf (phloem) and root (xylem) exudates from induced and non-induced plants, polyamines in the exudates were extracted, separated and analyzed quantitatively. The titers of free and conjugated putrescine the major polyamine fractions in all samples, increased early and markedly in leaf exudates during the floral transition, coinciding closely with movement of the floral stimulus out of the induced leaf. By contrast, putrescine titer in the root exudate did not increase. A spray of difluoromethylornithine (DFMO), an irreversible inhibitor of the putrescine-biosynthetic enzyme ornithine decarboxylase, at hour 8 of the long day considerably reduced the titer of free and conjugated putrescine in leaf exudates, and at the same time, markedly decreased the flowering response of induced plants. This effect of DFMO on flowering was substantially reversed by a simultaneous application of putrescine to the roots. DFMO sprayed on induced plants also suppressed early activation of indices of both mitosis and DNA synthesis in the shoot apical meristem. These results support the view that the extra putrescine synthesized in induced leaves is a necessary component of the floral stimulus in Sinapis.     

The MDM2 RING-finger domain is required to promote p53 nuclear export

MDM2 can bind to p53 and promote its ubiquitination and subsequent degradation by the proteasome. Current models propose that nuclear export of p53 is required for MDM2-mediated degradation, although the function of MDM2 in p53 nuclear export has not been clarified. Here we show that MDM2 can promote the nuclear export of p53 in transiently transfected cells. This activity requires the nuclear-export signal (NES) of p53, but not the NES of MDM2. A mutation within the MDM2 RING-finger domain that inhibits p53 ubiquitination also inhibits the ability of MDM2 to promote p53 nuclear export. Finally, inhibition of nuclear export stabilizes wild-type p53 and leads to accumulation of ubiquitinated p53 in the nucleus. Our results indicate that MDM2-mediated ubiquitination, or other activities associated with the RING-finger domain, can stimulate the export of p53 to the cytoplasm through the activity of the p53 NES.     

Determining the role of Tie-dyed1 in starch metabolism: epistasis analysis with a maize ADP-glucose pyrophosphorylase mutant lacking leaf starch

In regions of their leaves, tdy1-R mutants hyperaccumulate starch. We propose 2 alternative hypotheses to account for the data, that Tdy1 functions in starch catabolism or that Tdy1 promotes sucrose export from leaves. To determine whether Tdy1 might function in starch breakdown, we exposed plants to extended darkness. We found that the tdy1-R mutant leaves retain large amounts of starch on prolonged dark treatment, consistent with a defect in starch catabolism. To further test this hypothesis, we identified a mutant allele of the leaf expressed small subunit of ADP-glucose pyrophosphorylase (agps-m1), an enzyme required for starch synthesis. We determined that the agps-m1 mutant allele is a molecular null and that plants homozygous for the mutation lack transitory leaf starch. Epistasis analysis of tdy1-R; agps-m1 double mutants demonstrates that Tdy1 function is independent of starch metabolism. These data suggest that Tdy1 may function in sucrose export from leaves.     

Effect of absence of developing grain on carbohydrate content and senescence of maize leaves

In maize (Zea mays L.) grown under normal conditions in Rhodesia, prevention of pollination or removal of the ears after flowering caused premature senescence of the leaves above the ear, preceded by the appearance of a purplish red color. In plants from which the ears had been removed the concentration of sugars and starch increased markedly in both upper and lower leaves, the increase being greater in the upper leaves.     

Sucrose efflux and export from the maize scutellum*

Abstract During incubation of maize scutellum slices in fructose, there was an efflux of sucrose. Efflux was constant for at least 4 h at fructose concentrations of 70 or 100 mol m^?3. Efflux was increased by EDTA, and decreased by Ca²⁺. Efflux was independent of pH after EDTA treatment, but increased from untreated slices when the pH was lowered from 7 to 4. Uranyl ion and PCMBS (p-chloro-mercuribenzenesulfonic acid) abolished sucrose uptake, but were only weak inhibitors of sucrose efflux. These results are consistent with efflux occurring by simple diffusion through aqueous pores, but they do not rule out facilitated diffusion. Rates of sucrose export from the scutellum to the root shoot axis were estimated from measurements of axis respiration and dry weight gain. Sucrose efflux from scutellum slices was only 14-22% of the export rate. Sucrose efflux from the whole scutellum was only 3-4% of the export rate. It is concluded that the observed efflux is from leaky cells and does not represent sucrose on the way to the phloem along a path that includes the apoplast. These results support the idea that the path for sucrose from parenchyma cell to sieve tube in the maize scutellum is entirely symplastic.     

Photosynthetic acclimation in the context of structural constraints to carbon export from leaves

The potential role of foliar carbon export features in the acclimation of photosynthetic capacity to differences and changes in light environment was evaluated. These features included apoplastic vs. symplastic phloem loading, density of loading veins, plasmodesmatal frequency in intermediary cells, and the ratio of loading cells to sieve elements. In initial studies, three apoplastic loaders (spinach, pea, Arabidopsis thaliana) exhibited a completely flexible photosynthetic response to changing light conditions, while two symplastic loaders (pumpkin, Verbascum phoeniceum), although able to adjust to different long-term growth conditions, were more limited in their response when transferred from low (LL) to high (HL) light. This suggested that constraints imposed by the completely physical pathway of sugar export might act as a bottleneck in the export of carbon from LL-acclimated leaves of symplastic loaders. While both symplastic loaders exhibited variable loading vein densities (low in LL and high in HL), none of the three apoplastic loaders initially characterized exhibited such differences. However, an additional apoplastic species (tomato) exhibited similar differences in vein density during continuous growth in different light environments. Furthermore, in contrast to the other apoplastic loaders, photosynthetic acclimation in tomato was not complete following a transfer from LL to HL. This suggests that loading vein density and loading cells per sieve element, and thus apparent loading surface capacity, play a major role in the potential for photosynthetic acclimation to changes in light environment. Photosynthetic acclimation and vein density acclimation were also characterized in the slow-growing, sclerophytic evergreen Monstera deliciosa. This evergreen possessed a lower vein density during growth in LL compared to HL and exhibited a more severely limited potential for photosynthetic acclimation to increases in light environment than the rapidly-growing, mesophytic annuals.     

Interaction of analogues of substrate with NADP-malic enzyme from maize leaves

The effect of structural analogues of l-malate was studied on NADP-malic enzyme purified from Zea mays L. leaves. Among the compounds tested, the organic acids behaved as more potent inhibitors at pH 7.0 than at pH 8.0, suggesting that the dimeric form was more susceptible to the inhibition than the tetrameric form of the enzyme.Oxalate, ketomalonate, hydroxymalonate, malonate, oxaloacetate, tartrate, -hydroxybutyrate, -ketobutyrate, -ketoglutarate and -hydroxyglutarate exhibited linear competitive inhibition with respect to the substrate l-malate at pH 8.0. On the other hand, glyoxylate and glycolate turned out to be non-competitive inhibitors, while glycolaldehyde, succinate, fumarate, maleate and - and -hydroxybutyrate had no effect on the enzyme activity, at the concentrations assayed. These results suggest that the extent of inhibition was dependent on the size of the analogues and that the presence of an 1-carboxyl group along with a 2-hydroxyl or 2-keto group was important for binding of the substrate analogue to the enzyme.