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

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Abstract

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.

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Fig. 1: 11C export from sut1 maize leaves decreased drastically, but sap flow velocity was only moderately decreased.
Fig. 2: Reduced 11C export is due to sut1 loss-of-function, not an artefact of isotopic dilution.
Fig. 3: WT and sut1 mutant leaves do not differ in leaf vein numbers, lignification patterns, anatomy or transport of CF fluorescent dye through the phloem.
Fig. 4: Imaging of ions in major veins by XRF microscopy shows increased levels of potassium in the sut1 phloem.

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Data availability

All of the data supporting the findings of this study are included within the article and the accompanying supplementary material. Source data are provided with this paper.

Availability of materials

Zea mays sut1 loss-of-function mutant seed may be obtained from the Maize Genetics Stock Center (http://maizecoop.cropsci.uiuc.edu/). Source data are provided with this paper.

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Acknowledgements

This work was supported in part by: the United States Department of Energy, Office of Biological and Environmental Research (contract DE-AC02-98CH10886); a Goldhaber Distinguished Fellowship to B.A.B.; the United States Department of Agriculture, National Institute of Food and Agriculture, McEntire-Stennis project number 1009319 (B.A.B.); a US National Science Foundation Plant Genome Research Program grant (IOS-1025976) to D.M.B.; research grants from Villum Fonden (37475) and the Independent Research Fund Denmark (9064-00069B) to K.H.J.; and a DFG grant to R.H. and S.S. (SCHE 2148/1-1). This research used the X27A Beamline of the National Synchrotron Light Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE- AC02-98CH10886.

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Contributions

B.A.B., A.A.K., D.J.K. and J.R. performed 11C radiotracer experiments. R.F.B. and T.M.T. performed genotyping by PCR, vein counts and carboxyfluorescein experiments. J.K. performed pico-gauge measurements of sieve tube pressures and determination of phloem conductivity. B.A.B. and D.M.B. designed initial 11C experiments. B.A.B., D.M.B., J.K., M.K., K.H.J., R.H. and S.S. designed subsequent experiments. B.A.B. and R.T. performed synchrotron X-ray fluorescence measurements on frozen hydrated tissue and R.T. performed image analysis and quantification. G.L. performed aphid stylectomy and analysis of phloem sap. S.S. and R.H. conducted K+ microelectrode experiments. K.H.J. completed the model of sugar transport, with input from B.A.B., J.K. and M.K. B.A.B. drafted the manuscript with input and editing from all co-authors.

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Correspondence to Benjamin A. Babst or David M. Braun.

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Extended data

Extended Data Fig. 1 Feeding of 11CO2 to a leaf.

Photograph showing Carbon-11 (11C) being administered as 11CO2 to a single leaf of a maize plant, clamped in a leaf cuvette. There was a single radiation detector built into the leaf cuvette, and two other detectors positioned along the leaf, basipetal to the leaf cuvette.

Extended Data Fig. 2 Homozygous sut1 mutant leaves had high carbohydrate concentrations, and low carbohydrate specific activity.

Analysis of leaf extracts for (a) unlabeled (12C) soluble sucrose (Poverall model = 0.0001; Pwt-vs-sut1 = 0.0003; Phet-vs-sut1 = 0.0003; Pwt-vs-het = 1.0), glucose (Poverall model = 0.012; Pwt-vs-sut1 = 0.015; Phet-vs-sut1 = 0.036; Pwt-vs-het = 0.80), fructose (Poverall model < 0.0001; Pwt-vs-sut1 = 0.0002; Phet-vs-sut1 = 0.0002; Pwt-vs-het = 1.0), and starch concentrations (Poverall model = 0.003; Pwt-vs-sut1 = 0.008; Phet-vs-sut1 = 0.005; Pwt-vs-het = 0.92), (b) partitioning of 11C to soluble sucrose (Poverall model = 0.0014; Pwt-vs-sut1 = 0.002; Phet-vs-sut1 = 0.004; Pwt-vs-het = 0.92), glucose (Poverall model = 0.009; Pwt-vs-sut1 = 0.015; Phet-vs-sut1 = 0.02; Pwt-vs-het = 0.97), fructose (Poverall model = 0.054), and starch (Poverall model = 0.16) as a percentage of the total fixed 11C, and (c) specific activity of sucrose (Poverall model = 0.0159; Pwt-vs-sut1 = 0.059; Phet-vs-sut1 = 0.017; Pwt-vs-het = 0.69), glucose (Poverall model = 0.23), fructose (Poverall model = 0.014; Pwt-vs-sut1 = 0.017; Phet-vs-sut1 = 0.043; Pwt-vs-het = 0.79), and starch (Poverall model = 0.022; Pwt-vs-sut1 = 0.66; Phet-vs-sut1 = 0.02; Pwt-vs-het = 0.08) shown as GBq of 11C radioactivity per gram of the sugar or starch, respectively (WT n = 3, het n = 3, sut1 n = 4). Specific activity indicates the proportion of 11C to 12C. Biochemical partitioning of 11C to soluble sugars but not starch was elevated in sut1 mutant leaves. The central line is the median, the box indicates the first and third quartiles, and the whiskers indicate the minimum and maximum values. Statistical significance according to a one-way ANOVA is indicated by *(P<0.05), **(P<0.01), ***(P<0.001), or NS (not significant). Different letters above bars indicate which genotypes were statistically different based on a Tukey’s post hoc test.

Source data

Extended Data Fig. 3 When 11C dilution was mitigated by reducing sugar concentrations in sut1 mutant leaves by dark treatment, 11C export remained extremely low in sut1 mutants, but sap flow velocity remained high by comparison.

Because the concentration of [12C]-sucrose was extremely high in sut1 leaves, and the specific activity was low (Fig. 1F and H, respectively; Extended Data Fig. 2), one could speculate that the reduced 11C export in sut1 might have appeared more drastic than overall 12C export due to isotopic dilution. However, it is possible that much of the sucrose was unavailable for phloem loading in sut1 mutants, for example if much of it is stored in vacuoles or exuded from hydathodes44,74,75. To measure export under more uniform cellular sugar status, we moved sut1 mutant plants into constant darkness for 42 hrs immediately prior to 11C assays, reducing the unlabeled (12C) leaf sucrose, glucose, fructose and starch concentrations (a) to near WT levels. Note the 30 fold difference in Y-axis scaling for sugar concentrations compared to Extended Data Fig. 2a (6 fold for starch). (b) 11C-labeled sucrose (Poverall model <0.0001; Pwt-vs-sut1 <0.0001; Phet-vs-sut1 = 0.0002; Pwt-vs-het = 0.89), glucose (Poverall model = 0.0051; Pwt-vs-sut1 = 0.0056; Phet-vs-sut1 = 0.042; Pwt-vs-het = 0.61), fructose (Poverall model = 0.017; Pwt-vs-sut1 = 0.018; Phet-vs-sut1 = 0.091; Pwt-vs-het = 0.71), and starch. (c) Specific activities of sucrose, glucose, fructose, and starch were similar in WT and sut1 mutant plants after dark treatment. For all panels, the central line is the median, the box indicates the first and third quartiles, and the whiskers indicate the minimum and maximum values (WT n = 5, het n = 4, and sut1 n = 7). Statistical significance according to a one-way ANOVA is indicated by *(P<0.05), **(P<0.01), ***(P<0.001). Different letters above bars in indicate which genotypes were statistically different based on a Tukey’s post hoc test. In these conditions, we confirmed that the greatly reduced 11C-export by sut1 mutant leaves (Figs. 1c and 2b) was not an artifact of isotopic dilution by high [12C]-sucrose concentrations. The reduction in phloem sap flow velocities in the low-carbohydrate, dark-treated sut1 mutant leaves relative to WT remained modest in this experiment (Fig. 2c), compared to the large reduction of 11C-photoassimilate export (Fig. 2b), similar to the experiment with no dark treatment (Fig. 1c-d).

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Extended Data Fig. 4 Maize phloem sap contents did not significantly correlate with phloem sucrose content.

Phloem sap was collected from wild type (blue diamonds) and sut1 mutant (yellow circles) leaves by aphid stylectomy. We tested for a negative correlation between sucrose and (a) potassium, (b) chloride, (c) sodium, (d) total amino acids, (e) magnesium, (f) calcium, (g) total cations, and (h) potassium, sodium, and chloride combined (See Table 1 for P values).

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Extended Data Fig. 5 Phloem sap potassium (K+) concentrations were higher in sut1 mutant leaves than in wild type leaves.

(a) Comparison of wild type (WT) and mutant (sut1) leaf phloem. The central line is the median, and the box and whiskers indicate the first and third quartiles, and the the minimum and maximum values, respectively (n = 13, *** indicates P = 0.0003 for a two-sided Student’s t-test). (b) K+-selective microprobes were calibrated using K+ standard solutions of known concentration. The reference electrode (blue) was hardly affected by changing external K+ concentrations (concentration shown below the graph ranging from 1M to 1 mM), while the K+- selective electrode (red) shows a near Nernstian behavior. Thus, the reference subtracted black line shifts with the given values of K+ indicating a fast and functional K+-selective setup (values used for calibration are given below the black line). (c) Microscopic image of K+-selective electrode (upper) and reference electrode (lower) impaled into a phloem cell. Both electrodes were controlled by micromanipulators under a microscope for impalement, and both electrodes were calibrated before and after impalement. (d) Raw trace of an example measurement before and after impalement (blue arrow indicates time of reference electrode impalement; red arrow indicates timing of K+-selective electrode impalement). Black trace shows the membrane potential (blue trace) subtracted from the K+-selective potential (red trace). We measured the stable selective potential (grey area) for all plants before removing the selective electrode at the end of the measurement.

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Extended Data Fig. 6 No effect of potassium supplementation on 11C-photoassimilate transport dynamics.

Potassium supplementation of the soil medium did not affect 11C export or transport dynamics in either wild type (WT) or sut1 mutant leaves. (a) Severely reduced 11C export rates from sut1 homozygous mutant leaves. (b) Marginally reduced sap flow velocity of 11C-photoassimilates in sut1 mutant leaves was not alleviated by adding 200 mL of 20 mM KCl to each pot once per week, starting 2 weeks after sowing. HK, high potassium; LK, low potassium. For both panels, center line is median, boxes indicate first and third quartiles, and whiskers are minimum and maximum (n = 4 for all bars, except for sap flow velocity for sut1 low K+, which was n = 3). For export (a), the overall model was significant for a two-way ANOVA (P <0.0001), and effects of genotype were significant (P <0.0001), but there were no statistically significant effects of K+ treatment (P = 0.40) or interactions between genotype and potassium treatment (P = 0.53). For sap flow velocity (b), the overall model was not significant for a two-way ANOVA (P = 0.16), the effects of genotype were significant (P = 0.034), but there were no statistically significant effects of K+ treatment (P = 0.84) or interactions between genotype and potassium treatment (P = 0.55). Statistical significance of genotype effects are indicated by *(P<0.05), **(P<0.01), ***(P<0.001).

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Supplementary Tables 1 and 2,. and text.

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Babst, B.A., Braun, D.M., Karve, A.A. et al. Sugar loading is not required for phloem sap flow in maize plants. Nat. Plants 8, 171–180 (2022). https://doi.org/10.1038/s41477-022-01098-x

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