2University of Missouri, Missouri Research Reactor Center, Department of Chemistry, Columbia, MO, 65211
3Present address: Miltenyi Biotech GmbH, Robert-Koch-Str. 1, 17166, Teterow, Germany
Keywords: Intercellular sugar trafficking; cellulose synthesis; developmental aging; carbon-11; 18FDG;
Growing or expanding cells are typically surrounded by a primary wall comprised largely of polysaccharide macromolecules which contribute to the structural integrity of the cell and to cell adhesion. This structure provides an important conduit for solute transfer including growth resources and key signal molecules that help maintain cellular function [3].
In many cells, this wall can be thickened and further strengthened by the addition of a secondary wall containing lignin, or polyaromatic macromolecules that help maintain cellular water conductance. As plant cells continue to grow, becoming more specialized in function and differentiated by the tissue type, the walls that encase them often can possess a vast array of compositional differences that help serve the diverse cellular functions across the plant.
Plants are sessile organisms, and as such have evolved with the capacity to be highly plastic in their responses to growth conditions imposed by a changing environment. This unique feature empowers them with the ability not only to survive harsh conditions, but also to survive predation by herbivores and attack by pathogens. Their ability to respond in this capacity requires that the cell wall structure, and its composition, be continually modified providing physical barriers as a mechanism to ward off attack by predators. For example, strategically situated parenchyma cells will rapidly develop invaginations or reinforcements of their cell wall as a consequence of plant defense [4]. Jasmonic acid (JA) and it’s methyl ester are key hormones that trigger this response by stimulating re-programming of key biochemical pathways providing essential biosynthetic precursors for lignin as a physical barrier [5,6]. In parallel, JAs also mediate reduced biosynthesis of cellulose, the most abundant polysaccharide macromolecular component of cell walls, and likely the most nutritious to feeding herbivores [7, 8].
The highly dynamic nature of cellulose biosynthesis attests to the complexity of the underpinnings that regulate cell wall construction. To this day, that regulation is still not entirely understood, especially in the context of changing cellular functions that coincide with normal plant growth and development [2,3,9,10 and 11]. To properly generate or modify the cell wall, proteins involved in the process, including cellulose synthesis, glucan synthesis and pectin methyl esterase, are transported intracellular via membrane trafficking to the plasma membrane, or the extracellular space [12,13]. It is presumed that such processes must be tightly regulated [14]. Recent studies even go as far as to suggest that the diversification of membrane trafficking is what contributes to cell wall differentiation across all plant cell types [15]. Even so, there remain many open questions concerning the molecular mechanisms that underlie these transport processes. For example, some proteins at the plasma membrane may become endocytosed into the cytoplasm in response to feedback signals associated with extracellular conditions [16].
Unfortunately, very little attention has been given to understanding the dynamics underpinning the exchange of the simple sugar building blocks that are important to cell wall construction and/or modification. Herein, we report on the use of a short-lived radiocarbon isotope, carbon-11 (t½ 20.4 min), administered to intact leaves in Nicotiana tabacum as 11CO2 to explore the effect of leaf age on the intracellular partitioning of “new carbon” into cell wall cellulose [17]. We also report on the use of the fluorine-18 (t½ 110 min) radioisotope, administered to plants as 2-[18F]fluoro-2-deoxy-D-glucose (18FDG), a radioactive glucose surrogate, to examine the role of intercellular sugar trafficking in cellulose biosynthesis using developmental leaf aging as a model [18]. Over the years, fluorine- 18 has been extremely useful for radio-labeling molecules of interest for use in animal and human research, but in recent years this isotope has found its way into plant science with the radio synthesis of 18F-labeled sucrose analogues to study sugar transport [19, 20]. Additionally, 18FDG has also been shown to actively transport in plant vasculature [21-26] and utilized in secondary metabolism of essential defense compounds [27]. Here, we showed evidence that plants will metabolically assimilate 18FDG into cell wall cellulose and the tracer can be used as a marker for quantifying extracellular glucose flux into cellulose.
Treatments: All chemicals used in these studies were obtained from Sigma Aldrich (St. Louis, MO, USA) and were used without any further purification. Isoxaben (ISX), N-(3’[1-ethyl-lmethylpropyl]- 5-isoxazolyl)-2,6-dimethoxybenzamide is a preemergence, broad leaf herbicide used primarily on small grains, turf grasses and ornamental plants [28].
This herbicide is extremely active, with IC50 values in the nanomolar range [29]. Isoxaben treatment has been shown to selectively inhibit 14C-glucose incorporation into the acid insoluble cellulosic cell wall fraction [30,31] and so has become a powerful tool enabling the chemical inhibition of cellulose biosynthesis in vivo.
Isoxaben interferes with rosette formation, and by blocking the demonization of CesA polypeptides 130 it inhibits cellulose biosynthesis [32]. Jasmonic acid (JA), including its methyl ester (MeJA), is ubiquitous in all higher plants filling numerous roles in plant growth and development [33] and especially plant defense hormones eliciting rapid reprogramming of plant metabolism when applied topically to tissues [5,34]. Like ISX, MeJA will also inhibit cellulose biosynthesis in vivo. In all tracer flux measurements involving measuring radio labeled cellulose, we applied either a 5 nm solution of ISX or a 5 μM solution of MeJA as topical treatments to upper intact leaf surfaces. Differences in treatment concentrations were based on perceived substrate biological activities. Treatments were applied to a small single layer section of Kim Wipe™ for 1 hr prior to tracer administration (i.e. 11CO2 to leaves or 18FDG to roots). In special studies designed to assess the effect of treatment dose on 11C-photoassimilate transport, we administered treatments through the cut petiole of an excised leaf. The amount of treatment taken up by the leaf tissue was quantified by measuring the volume of solution assimilated over time. Treatment solutions were significantly reduced for this experimental protocol because a significant volume of solution was always assimilated by the cut leaf over a 1hr time course.
11CO2 Production and Leaf Administration: 11CO2 was produced via the 14N(p, α)11C nuclear transformation from a 20 ml target filled with high-purity nitrogen gas (400 ml @ STP) using 18MeV protons from the TR-19 (Ebco Industries Ltd, Richmond, BC, Canada) cyclotron at BNL, and captured on a molecular sieve (4Å). The 11CO2 that was trapped on the molecular sieve was desorbed and quickly released into an air stream at 200 ml/min as a discrete pulse for labeling a leaf affixed within a 5 x 10 cm lighted (350 μmol m-2 s-1) leaf cell at 21°C to ensure a steady level of fixation [35, 36]. For mature source leaves, only a portion of the leaf was affixed within the cell. The leaf was positioned in the cell so as to capture part of the outside leaf edge. This configuration allowed efficient air/tracer flow around the upper side of the leaf as well as the underside. The load leaf tissue was exposed to 11CO2 tracer for 1 minute as a transient pulse in the air stream, and then was chased with clean air for the duration of the exposure. A PIN diode radiation detector (Carroll Ramsey Associates, Inc, Berkeley, CA, USA) affixed to the bottom of the leaf cell enabled continuous measurement of radioactivity levels within the cell during the pulse.
18FDG Synthesis and Root Administration: Fluorine-18 as fluoride anion (18F-) was produced using an 18 MeV proton beam on the TR-19 cyclotron (Ebco Industries Ltd, Richmond, BC, Canada). This beam was focused onto an oxygen-18 enriched water target causing the 18O (p, n) 18F nuclear reaction to occur. After irradiation, 18F-fluoride was collected on a QMA sep-pak (Waters Accell Plus QMA) located at the target station, then extracted and transferred under pressure to the radiochemistry laboratory using a dilute solution of acetonitrile and potassium bicarbonate (0.1 N). The contents of the transfer were received into a FDG-Plus™ Automated Radiochemistry Module (Bioscan, Inc., Washington, DC, USA) where the 18F-fluoride was dried under vacuum with Kryptofix 2.2.2. 18F-Fluoride was then reacted with mannose trifoliate (1, 3, 4, 6-tetra-O-acetyl-2-O-trifluoromethane sulfonyl-β-D-mannopyrannose). Reaction by nucleo philic displacement with the trifoliate starting material yielded an intermediate radio labeled product in high yield that could be hydrolyzed easily in base and at room temperature to yield 2-deoxy-2- [18F]fluoro-D-glucose (18FDG). The reaction mixture was processed through a C18 Sep-Pak cartridge to remove the phase-transfer catalyst, Kryptofix 2.2.2. The final product was formulated in approximately 1 ml of de-ionized water yielding approximately 5 mCi per dose of purified product at end-ofsynthesis. Doses were measured using a CRC-12 Dose Calibrator (Capintec, Inc., Ramsey, NJ, USA).
18FDG was administered directly to the hydroponics solution of study plants. In staging these studies, individual plants were removed from their larger hydroponics growth chambers and transferred to individual 50 ml beakers filled with 30 ml de-ionized water. Forced air from an aquarium air pump maintained proper solution aeration and provided good mixing of the tracer. Studies leveraging 18F-fluoride as a water flow tracer were conducted in the same manner as the 18FDG studies. For 18F-fluoride uptake studies, the dried tracer was reconstituted in 1 mL of de-ionized.
Leaf Autoradiography: Counting down from the apex, leaf-2 (a nearly fully expanded source leaf) was subjected to topical treatments while still attached to the plant. MeJA or ISX treatments were applied on half of the leaf’s upper surface, as described above. 2.5 hour following 18F-fluoride or 18FDG tracer administration to the roots, leaf-2 was excised and imaged for quantifying either 18F-fluoride or 18FDG uptake and distribution using autoradiography (Typhoon 7000: GE Healthcare, Piscataway, NJ, USA). Differences in the radioactivity levels between the 190 treated and untreated halves were determined using Image Quant TL 7.0 software (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) where 1 cm diameter circular regions-of interest (ROI) we retraced onto both sides of the leaf’s midrib (placed equidistant from that reference point), and the amount of radioactivity within each ROI was determined from the image intensity. No correction was made for tissue attenuation of radioactivity in the acquired images since it was assumed that each leaf half was identical in nature and thickness to the other.
Autoradiography was also used in 11C tracer studies. In these studies, leaf-2 was detached from the plant to enable treatments to be administered via the cut petiole (see Treatment section for details). Gaseous 11CO2 tracer was administered to the detached leaf in much the same way as in the intact leaf studies and autoradiography was used to quantify 11C-photoassimilate transport(after 11CO2 fixation) as a function of treatment type and treatment dose. Imaging was performed 30 min after 11CO2 fixation to avoid 11C-photoassimilate transport beyond the “fieldof- view” resulting in exudation of tracer out through the petiole cut. Using the same Image Quant software, ROIs were traced around the 11CO2 fixation site, as well as the remaining leaf tissue including petiole outside that site which contained radioactivity. The amount of activity found outside of the fixation site was correlated to transported 11C-photoassimilate.
Magnetic Resonance Imaging: A single intact leaf imaging experiment was conducted using BNL’s 4-Tesla MRI scanner as a way to verify the utility of the 18F-fluoride tracer as a water flow agent in plants. Past studies [37, 38 and 39] have attested to the fact that this tracer can be used as a proxy for tracing the dynamics of water transport in plants. Even so, we designed our own test. In a single experiment we compared leaf autoradiography (Figure. 1) of 18F-fluoride distribution in a ‘masked’ leaf study with that of a proton MRI of the same ‘masked’ leaf. Half of the leaf was masked using an opaque cardboard cover. The other half was illuminated using an LED illuminated fiber optic wand that allowed us to transmit light to the leaf surface while the plant was positioned within the magnet’s bore.
Whole-Plant 18FDG Translocation: The movement of 18FDG from roots up into the shoots and leaves of the plant was monitored using two sodium iodide scintillation detectors (Ortec, Oak Ridge, TN, USA) that were positioned facing perpendicular to the apex area and to leaf-2. Each detector was well-shielded using small tantalum blocks to ensure minimal detector crosstalk.
Detectors were cross-calibrated against a NIST traceable point source of radioactivity prior to each experiment. Each detector was set to report radioactivity levels on 1 min time intervals. The temporal allocation patterns of radiotracer translocation to the apex region and to leaf-2 were based on the amount of tracer assimilated by the plant over the time course of the experiment, and reported as fractional total activities. The amount of tracer assimilated by the plant was determined by measuring the level of radioactivity in the beaker (after the plant was removed at the end of the experiment) and comparing that value to the original dose of administered. All calculations were performed using decay corrected values to account for 18F radioisotope decay over time.
18FDG-6-Phosphate Metabolite Analyses: Approximately 100 mg of leaf-2 tissue was extracted 2.5 hr after 18FDG was administered through the roots of the plant. Extraction was carried out in 4x w/v of methanol, briefly vortexes (VWR analog vortex mixer; Sigma-Aldrich Corp. St. Louis, MO, USA) and then sonicated (Branson Bransonic 32; Sigma-Aldrich Corp. St. Louis, MO, USA) in an iced water bath for 10 min. The tubes were centrifuged (Eppendorf Centrifuge 5424) for 2 min at 15,000 rpm and the supernatant was filtered through 0.2 μm Acrodiscs (Gelman Sciences, Ann Arbor, MI, USA). An aliquot of the extract was analyzed by radio-TLC [41] using silica gel– coated plastic sheets (Polygram SIL G/UV254; Macherey-Nagel) and acetonitrile/tetrabutylammonium hydroxide, 9.5 mmol/L, 8:2 (v/v), as eluent. The formation of 18FDG-6-P was confirmed by co-spotting the TLC plate with 245 nonradioactive 19FDG-6-P (Sigma-Aldrich, St. Louis, MO USA).
Doses of pure 18FDG-6-P were prepared using in vitro enzyme chemistry and applied to leaf tissue to test for 18FDG-6-P mobility. To generate these doses, 5 mCi of 18FDG was added to 1 ml NaH2PO4 buffer solution (70 mmol/L) that contained 10 mg (250– 400 units) of hexokinase type IV from bakers’ yeast (EC 2.7.1.1; Sigma-Aldrich, St. Louis, MO USA), 2 mg (3.6 μmol) of adenosine triphosphate (Sigma-Aldrich, St. Louis, MO USA) and 1 mg (5 μmol) MgCl2·6H2O (Sigma-Aldrich, St. Louis, MO USA) and was pH adjusted to 7.4. The solution was stirred at room temperature (30°C) for 60 min and samples taken for radio-TLC analysis of product purity (Figure 2) 18FDG-6-P transport assays were carried out by applying tracer to an abraded leaf surface and measuring the extent of transport over 1 hr using autoradiography.
This same cellulose analysis protocol was used to measure 18F-cellulose derived from 18FDG, and in one study application from 18FDG-6P. Unlike the 11C-tracer studies, leaf tissue samples were harvested for 18F-cellulose analysis 2.5 hr after 18FDG tracer was administered to the roots. We tried to target the same region of leaf tissue and mass as in the 11C-tracer studies. A longer time period was used in the 18F-tracer studies than in the 11C-tracer studies to allow sufficient time for initial uptake and transport of 18F-tracer to the aerial portions of the plant. At the end of the cellulose assay, the acid indigestible portion was dried and further deconstructed using the phenol–sulfuric acid assay [46]. This method involved refluxing the material in a 1:10 solution of phenol (5%) and concentrated sulfuric acid at 100°C until all of the solid material was digested. Aliquots of this extract were neutralized and analyzed by radio HPLC for recovered 18FDG.
Measurement of Leaf Water Potential: In a separate set of measurements, leaf water potentials (Ψ) were determined for a set of different aged leaves by using a Scholander pressure bomb (model 3005, Soil moisture Equipment Corp., Santa Barbara, USA).
Statistical Analysis: Data was subjected to the Student t-test for unpaired samples assuming an unequal variance. Statistical significance levels were assigned to the following rating scale (*, P < . 05; **, P < . 01; ***, P < . 001).
Treatments using ISX and MeJA Impact 18F-Tracer Mobility: Chemical inhibitors for cell-wall cellulose, isoxaben (ISX) and methyl jasmonate (MeJA) were tested for their effects on 18F-tracer mobility derived from 18FDG. Treatments were applied to half of leaf-2, and autoradiography was used to quantify effects of treatment on phloem loading of the 18F-tracer (Figure 4). Data was presented as a ratio of the treated tissue activity divided by the untreated tissue activity (T/U) that was measured across the two halves of same leaves. Control data was derived from application of mock treatments using deionized water. Results from these studies were summarized in (Table 1). This table also contained data on the effect of treatment on leaf water flow where 18F-fluoride was used as a water tracer. We felt that it was important to access treatment effects on water flow as this dynamic had a strong bearing on xylem unloading of 18FDG into leaf-2. Furthermore, Table 1 also contained data on the effect of treatment on 18FDG phosphorylation as this process would impact 18F-tracer mobility in phloem loading, as well as remove 18F-tracer from metabolically active sugar pools used in cellulose
18FDG phosphorylation and it slightly increased 18F-tracer loading into the phloem as reflected by the lower T/U value relative to controls, 0.546 ± 0.373 versus 0.949 ± 0.123, respectively (P=0.0520).
Metric |
Treatment |
Avg. Ratio (T/U)a |
Sample Size |
Std. Dev. |
Std. Error |
P-Value |
18F-Fluoride Distribution |
Control |
1.036 |
5 |
0.164 |
0.073 |
-- |
ISX |
0.858 |
4 |
0.185 |
0.092 |
0.1685 |
|
MeJA |
0.497 |
4 |
0.219 |
0.109 |
0.0038 |
|
18FDG Distribution |
Control |
0.995 |
3 |
0.096 |
0.055 |
-- |
ISX |
1.993 |
6 |
0.266 |
0.109 |
0.0005 |
|
MeJA |
0.396 |
5 |
0.164 |
0.074 |
0.0013 |
|
18FDG Metabolismb |
Control |
1.013 |
3 |
0.120 |
0.069 |
-- |
ISX |
1.290 |
3 |
0.211 |
0.122 |
0.1195 |
|
MeJA |
1.450 |
3 |
0.257 |
0.148 |
0.0559 |
|
Adjusted |
Control |
0.949 |
-- |
-- |
0.123 |
-- |
ISX |
1.820 |
-- |
-- |
0.116 |
0.0009 |
|
MeJA |
0.546 |
-- |
-- |
0.373 |
0.0520 |
2. 18FDG metabolism was based on the extent of its conversion to the non-transportable 6-phosphorylated sugar.
3. This calculated 18FDG distribution makes adjustments for treatment effects on water input using 18F-fluoride distribution data and adjustments for treatment effects on 18FDG metabolism to its non-transportable form as 6-P-18FDG once in the targeted leaf-2 tissue. Standard propagation of error was applied. Distribution values higher than unity suggest a reduction in phloem reloading.
The distance tracer traveled from the boundary of the 11CO2 leaf cuvette over a fixed period of time was used as the metric of transport. Since treatments were applied via the cut petiole, it was also possible to quantify the dose assimilated by the study leaf during the incubation period and map transport against treatment in a dose dependent manner. Treatments were applied over 107 orders of magnitude ranging from 0.004 pmol • g fresh wt-1 leaf tissue to 50,000 pmol • g fresh wt-1.The results in (Figure 6)were plotted on a logarithmic scale to reflect the exponential
Positive deviation from this ‘zero’ line reflected increased 11C-photoassimilate transport, whereas negative deviation from this line reflected decreased transport. Similar to the 18FDG results, ISX treatment significantly decreased 11C-photoassimilate transport in a dose dependent manner while MeJA significantly increased transport.
This phenomenon would inherently lower the measured values of the cellulose content with increasing leaf age when based on fresh tissue mass, but the magnitude of the observed drop in cellulose content (61%) exceeded the change in water potential (37%), so likely the measured decrease in cellulose with increasing leaf age was real. However, 11C-cellulose content remained unchanged with leaf age while 18F-cellulose decreased significantly from 0.083 ± 0.011 % of tissue radioactivity to 0.010 ± 0.007 % of tissue radioactivity with increasing leaf age.
Leaf type |
11c- Cellulosea |
std.Dev. |
std. |
sample size |
18F |
std. |
std. |
sample size |
cellulose content (%gfw)c |
std. Dev. |
std. |
sample size |
Apex |
0.263 |
0.012 |
0.006 |
4 |
0.083 |
0.033 |
0.011 |
9 |
1.024 |
0.136 |
0.048 |
8 |
Leaf-2 |
0.282 |
0.043 |
0.019 |
5 |
0.042 |
0.010 |
0.003 |
9 |
0.754 |
0.129 |
0.043 |
9 |
Leaf-4 |
0.259 |
0.063 |
0.028 |
5 |
0.029 |
0.018 |
0.008 |
5 |
0.496 |
0.211 |
0.094 |
5 |
Leaf-6 |
0.279 |
0.091 |
0.053 |
3 |
0.01 |
0.013 |
0.007 |
3 |
0.399 |
0.215 |
0.124 |
3 |
b. Calculated by dividing the dried cellulose mass by the grams fresh weight (gfw) of tissue extracted.
c. Presented as the percentage of carbon-11 activity within the extracted tissue.
Treatment |
11C |
Std. Dev. |
Std.Error |
Sample size |
P-Value |
18F-Celluloseb |
Std. Dev. |
Std.Error |
Sample size |
P-Value |
Control |
0.282 |
0.43 |
0.019 |
5 |
-- |
0.042 |
0.010 |
0.003 |
9 |
-- |
ISX |
0.406 |
0.115 |
0.051 |
5 |
0.0527 |
0.021 |
0.006 |
0.001 |
5 |
0.0010 |
MeJA |
0.09 |
0.058 |
0.026 |
6 |
0.0003 |
0.055 |
0.012 |
0.005 |
6 |
0.0496 |
b. Presented as the percentage of fluorine-18 activity within the extracted tissue.
Since the identification of the first eukaryotic monosaccharide transporter in Chlorellakessleri [53], monosaccharide transporters have also been identified in a variety of higher plants [54-57]. For example, in Arabidopsis thaliana, the AtSTP1 and AtSTP3 monosaccharide transporters display both high and low affinity for D-glucose transport, respectively, suggesting functional differences [58, 59]. Marked differences in the pattern of expression have also been noted across tissues, organs and even cell types. For example, AtSTP1 transcripts are present in leaves, stems, flowers, and roots, while AtSTP2 is expressed only in developing pollen [60]. Analysis of expression patterns also suggests that these transporters are highly regulated and responsive to environmental cues, such as in response to pathogen infection or after wounding.
Additionally, from past studies, the monosaccharide transporter, MST1, was isolated from Nicotiana tabaccum and found to be most strongly expressed in sink tissues including roots, flowers and young leaves [61] where it appears to play a role in unloading of sugars. The protein is homologous to those hexose transport proteins found in Arabidopsis thaliana and Chlorella kessleri and possesses 12 putative membrane-spanning domains which can function as a H+/monosaccharide symporter just like SUT1, catalyzing the cellular uptake of hexoses (e.g. D-glucose and D-galactose) or pentoses (e.g. D-xylose). Even so, overwhelming evidence from 14C-tracer studies and detailed phloem sap analyses has led to the general consensus in the phloem transport field that sucrose is the predominant sugar substrate that is carried in the sieve tubes of higher plants and transported over long distances to distal tissues [62]. More specifically, hexose sugars do not transport over such long distances.
Furthermore, to the best of our knowledge, no one has seen evidence for the presence of such monosaccharide transporters in the companion cells or sieve tube elements that would facilitate such long-distance transport of hexose sugars. Of course, we cannot deny the fact that our tracer data clearly showed evidence of phloem loading and long-distance transport of 18F-tracer that was derived from 18FDG after its uptake into source leaf-2. This observation is consistent with past observations indicating phloem loading and long distance transport of 18F-tracer after application of 18FDG to sorghum leaf tips [22]. One possible explanation of these observations is that 18FDG is converted to a 18F-sucrose analog that can be actively transported [19]. Alternatively, conversion of 18FDG to its respective sugar alcohol form might provide a mechanism for moving the 18F-tracer. Polyols are reduced forms of aldoses and ketoses, and can be found in all living forms [63]. In some higher plants these sugar alcohols are, together with sucrose, direct products of photosynthesis and serve similar functions as sucrose such as translocation of carbon skeletons and energy resources between sources and sink organs [64, 65].
Chemical treatments using ISX and MeJA were found to impose interesting systematic differences on the way they affected longdistance transport of 18F-tracer and 11C-photoassimilate that suggests they may act universally on H+-symport mechanisms. Specifically, ISX was seen to inhibit both phloems reloading of 18F-tracer, as well as long distance transport of 11C-photoassimilate in a dose dependent manner. Classical sugar transport inhibitors like carbonyl cyanide 3-chlorophenylhydrazone (CCCP) behave as protonophores that can shuttle protons across the lipid bilayer of the cell’s plasma membrane, and in doing so, uncouple the membrane proton gradient from sugar transport thus shutting down active transport [66]. However, protonophoric activity is not restricted to weakly acidic compounds like CCCP. There are claims that basic compounds like (Z)-5-Methyl-2-[2-(1-naphthyl) ethenyl]-4-piperidinopyridine exhibit protonophoric activity likely due to the amine group [67]. As a weakly basic benzamide, ISX may behave in a similar capacity resulting in its ability to universally shut down sugar transport. Furthermore, we note that past studies have shown that MeJA can re-instate sugar transport after treatment with a classical sugar transport inhibitor like CCCP presumably by re-coupling proton symport. Our observation that MeJA increased long-distance 11C-photoassimilate transport, as well as increased phloem loading and long distance transport of 18F-tracer suggests that its ability to act on the membrane proton gradient serves to increase transport activity of sugars in general.
Our present work also showed that intercellular sugar trafficking was an integral part of the regulatory mechanism for cellulose biosynthesis. Here we used developmental leaf aging as a model for exploring changes in cellulose biosynthesis and for measuring changes in the flux of intra and intercellular sugar resources in the process. Our data clearly demonstrated that 18FDG can be taken up by roots and delivered to all leaf types via xylem transport, and unloaded at the parenchyma symplastxylem interface where the sugar could migrate from cell-tocell through the plasmodesmatal cytosolic sleeve in between the plasma membrane and the endoplasmic reticulum. As cells expand and differentiate, their fate determines the extent to which their cytoplasmic connectivity to other cells is maintained [68]. Some cell types, such as those in the leaf mesophyll remain closely connected with their neighboring cells and this connectivity could extend to the parenchyma symplast-xylem interface. However, cytoplasmic continuity is not a constant and will decline with tissue development as simple plasmodesmata (PD) are lost during leaf sink-source transition [69], and the frequency of even the more complex branched PDs decline with maturation [70]. Taken together, we would expect 18FDG’s ability to move cell-to-cell via the symplast to systematically decline with aging, consistent with our finding that 18F-tracer flux into 18F-cellulose decreased with increasing leaf age. Furthermore, we were surprised to find that there was no change in the flux of 11C into 11C-cellulose from intracellular sugar sources. What this implies is the cellulose synthetic machinery remained active regardless of leaf age, and that the diminished supply of intercellular sugar was what controlled the process. We tested this hypothesis using ISX and MeJA treatments where from our earlier discussion it was concluded that ISX inhibited sugar transport (both in long-distance movement through the phloem, and in cell-to-cell movement within same tissues) while MeJA enhanced it. Consistent with this model, the inhibition of sugar transport by ISX increased the flux of 11C-tracer into 11C-cellulose from intracellular sugar while it decreased the flux of 18F-tracer into 18F-cellulose from intercellular sugar. Also consistent with this model, the increase in sugar transport by MeJA decreased the flux of 11C-tracer into 11C-cellulose from intracellular sugar while it increased the flux of 18F-tracer into 18F-cellulose from intercellular sugar.
To date, there exists an extensive body of information providing insight into the structural architecture of plant cell walls which are comprised of cellulosic micro fibrils embedded in a matrix of no cellulosic polysaccharides that include heteroxylans and 1,3; 1,4)-β-glucans [71]. Even so, progress in understanding the mechanisms for regulating the synthesis of these macromolecular structures has been relatively slow. For example, past work has shown that β-glucan synthesis, responsible for 1, 3-β-glucan (callose) synthesis in cell wall construction, decreases in activity with plant tissue age [72]. In contrast to this, our work suggests that cellulose synthesis remains uniformly active; at least in developing leaf tissues, and most importantly that intercellular sugar trafficking is a critical component to regulating cell-wall cellulose biosynthesis. We expect that the use of sugar transport mutants could help shed additional light on this highly regulated process and this will certainly be the focus of future studies.
Finally, an area of keen interest is to understand the responses of the plant cell wall to a biotic stresses including drought, flooding, and temperature, or to biotic stresses including attack by pathogens and feeding insects. The effects of these stresses on cell wall metabolism are complex, but important to unravel if we are to build adaptation and/or resistance traits in future cropping systems.
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