Ecophysiology of phloem loading in source leaves

The nature of phloem loading of photosynthesis products – either symplastic or apoplastic – has been a matter of debate over the last two decades. This controversy was reconciled by proposing a multiprogrammed loading mechanism. Different modes of phloem loading were distinguished on the basis of the variety of plasmodesmatal connectivity between the minor vein elements. Physiological evidence for at least two phloem loading mechanisms as well as recent support for coincidence between plasmodesmatal connectivity and the loading mechanism is shortly reviewed. The present paper attempts to correlate the plasmodesmatal connectivity between sieve element/companion cell complex and the adjacent cells (the minor vein configuration) – and implicitly the associate phloem loading mechanisms – with different types of climate. The minor vein configuration is a family characteristic. This enables one to relate vein configuration with ecosystem using the family distribution over the globe. The uneven distribution of vein types between terrestrial ecosystems indicates that apoplastic phloem loading predominates in cold and dry climate zones. Projection of the minor vein configuration on the Takhtajan system of flowering plants suggests evolution from apoplastic to symplastic phloem loading. Accordingly, the distribution of minor vein configurations suggests that drought and temperature stress have led to the transformation of the ancient symplastic mode into the more advanced apoplastic mode of loading.     

Saccharose and sorbitol transporters from plasmalemma membrane vesicles of peach tree leaves

The mechanisms of saccharose and sorbitol transport in Prunus persica leaves were investigated in plasma membrane vesicles purified by aqueous 2-phase partitioning and equilibrated in pH 7.5 buffer containing K+. The imposition of an artificial proton motive force energized an active uptake of both saccharose and sorbitol. The maximum uptake rate of saccharose was 2.5 times higher than that of sorbitol. Saccharose and sorbitol uptake exhibited saturation kinetics suggesting they were carrier-mediated. Apparent Km for the saccharose and the sorbitol uptake were 0.36 and 0.67 mM, respectively. Active absorption of saccharose was completely inhibited by a non-permeant thiol reagent, PCMBS, contrary to sorbitol absorption. These results suggested that saccharose and sorbitol were transported at least by two different carriers. This revised version was published online in July 2006 with corrections to the Cover Date.     

Competitive inhibition of phosphoglucose isomerase of apple leaves by sorbitol 6-phosphate

Apple leaf cytosolic phosphoglucose isomerase (PGI, EC 5.3.1.9) was purified to an apparent homogeneity with a specific activity of 2456units/mg protein, and chloroplastic PGI was partially purified to a specific activity of 72units/mg protein to characterize their biochemical properties. These two isoforms showed differential responses to heat treatment; incubation at 50 degrees C for 10min resulted in a complete loss of the chloroplastic PGI activity, whereas the cytosolic PGI only lost 50% of its activity. Apple cytosolic PGI is a dimeric enzyme with a molecular mass of 66kDa for each monomer. The activity of both isoforms was strongly inhibited by erythrose 4-phosphate (E4P) with a K(i) of 1.2 and 3.0muM for the cytosolic PGI and chloroplastic PGI, respectively. Sorbitol 6-phosphate (Sor6P), an intermediate in sorbitol biosynthesis, was found to be a competitive inhibitor for both cytosolic and chloroplastic PGIs with a K(i) of 61 and 40muM, respectively. PGIs from both spinach and tomato leaves were also inhibited by Sor6P in a similar manner. The possible physiological significance of this finding is discussed.     

Carbon partitioning into sorbitol,sucrose, and starch in source and sink apple leaves as affected by elevated CO2

Experiments were conducted in controlled growth chambers to evaluate how increases in CO₂ concentration ([CO₂]) affected carbon metabolism and partitioning into sorbitol, sucrose, and starch in various ages of apple leaves. Apple plants (Malus domestica), 1 year old, were exposed to [CO₂] of 200, 360, 700, 1000, and 1600 μl l⁻¹ up to 8 days. Six groups of leaves (counted from the shoot apex): leaves 1–5 (sink), 6–7 (sink to source transition), 8–9 (sink to source transition), 10–11 (nearly-matured source), 21–22 (mid-age source), and 30–32 (aged source), were sampled at 1, 2, 4, and 8 days after [CO₂] treatments for carbohydrate analysis. Increases in [CO₂] from a sub-ambient (200 μl l⁻¹) to an ambient level (360 μl l⁻¹) significantly increased the concentrations of sorbitol, sucrose, glucose, and fructose tested in all ages of leaves. Continuous increase in [CO₂] from ambient to super-ambient levels up to 1600 μl l⁻¹ also increased sorbitol concentration by ≈50% in source leaves, but not in sink and sink to source transition leaves. Increases in [CO₂] from 360 to 1600 μl l⁻¹, however, had little effect on sucrose content in all ages of leaves. Starch concentrations increased in all ages of leaves as [CO₂] increased. Rapid starch increases (e.g. 5-, 6-, 20-, and 50-fold increases for leaf groups 1–5, 6–7, 10–11, and 21–22, respectively) occurred from 700 to 1600 μl l⁻¹ [CO₂] during which increases in sorbitol concentration either ceased or slowed down. Our results indicate that changes in carbohydrates were much more responsive to CO₂ enrichment in source leaves than in sink and sink to source transition leaves. Carbon partitioning was favored into starch and sorbitol over sucrose in all ages of leaves when [CO₂] was increased from 200 to 700 μl l⁻¹, and was favored into starch over sorbitol from 700 to 1600 μl l⁻¹ [CO₂].     

A sorbitol oxidase that converts sorbitol to glucose in apple leaf

A new type of sorbitol oxidase which converts sorbitol to glucosein the absence of NAD or NADP was found in the leaf of the apple.The partially purified enzyme consumed 1/2 mole of oxygen inconverting one mole of sorbitol to glucose (sorbitol+1/2 O₂ glucose+H₂O). The enzyme had its optimum activity at pH 4.0,and it had a Km value of 100 mM for sorbitol and showed a weakdependency on divalent cations. This sorbitol oxidase seemsto play an important role in the utilization of accumulatedsorbitol. ¹This paper is contribution A-109, Fruit Tree Research Station (Received March 15, 1980; )     

Identification of phloem involved in assimilate loading in leaves by the activity of the galactinol synthase promoter

The definition of "minor" veins in leaves is arbitrary and of uncertain biological significance. Generally, the term refers to the smallest vein classes in the leaf, believed to function in phloem loading. We found that a galactinol synthase promoter, cloned from melon (Cucumis melo), directs expression of the gusA gene to the smallest veins of mature Arabidopsis and cultivated tobacco (Nicotiana tabacum) leaves. This expression pattern is consistent with the role of galactinol synthase in sugar synthesis and phloem loading in cucurbits. The expression pattern in tobacco is especially noteworthy since galactinol is not synthesized in the leaves of this plant. Also, we unexpectedly found that expression in tobacco is limited to two of three companion cells in class-V veins, which are the most extensive in the leaf. Thus, the "minor" vein system is defined and regulated at the genetic level, and there is heterogeneity of response to this system by different companion cells of the same vein.     

Identification of clp genes expressed in senescing Arabidopsis leaves.

Clp protease is a highly selective protease in E. coli, which consists of two types of subunits, the regulatory subunit with ATPase activity, ClpA, and the catalytic subunit, ClpP. In order to examine the possible association of plant Clp protease with the degradation of protein in senescing chloroplasts, we isolated a cDNA clone for ClpC which is a plant homologue of ClpA from Arabidopsis thaliana in addition to ERD1 which we had isolated earlier [Kiyosue et al. (1993) Biochem. Biophys. Res. Commun. 196: 1214]. We also isolated a clone for the plastidic gene, clpP (pclpP) and cDNA clones for putative nuclear clpP genes (nclpP1-6). We analyzed the expression of these clp genes in Arabidopsis leaves after various dark periods and during natural senescence. The expression of erd1 was increased by dark-induced and by natural senescence, as reported earlier [Nakashima et al. (1997) Plant J. 12: 851], while that of AtclpC was decreased. Two catalytic subunits nclpPs (nclpP3 and nclpP5) showed high expression in naturally senescing leaves, but the expression of pclpP and the other nclpPs was not changed. Immunoblot analysis of chloroplast protein and in vitro import analysis demonstrated that both nucleus-encoded regulatory subunits as well as nClpP5 were localized in the chloroplast stroma. These observations suggest that chloroplast Clp protease is composed of very complicated combinations of subunits, and that ERD1, nClpP5 and pClpP have a role in the concerted degradation of protein in senescing chloroplasts.     

Transport of cytokinins mediated by purine transporters of the PUP family expressed in phloem,hydathodes, and pollen of Arabidopsis

Nucleobases and derivatives like cytokinins and caffeine are translocated in the plant vascular system. Transport studies in cultured Arabidopsis cells indicate that adenine and cytokinin are transported by a common H+-coupled high-affinity purine transport system. Transport properties are similar to that of Arabidopsis purine transporters AtPUP1 and 2. When expressed in yeast, AtPUP1 and 2 mediate energy-dependent high-affinity adenine uptake, whereas AtPUP3 activity was not detectable. Similar to the results from cell cultures, purine permeases (PUP) mediated uptake of adenine can be inhibited by cytokinins, indicating that cytokinins are transport substrates. Direct measurements demonstrate that AtPUP1 is capable of mediating uptake of radiolabeled trans-zeatin. Cytokinin uptake is strongly inhibited by adenine and isopentenyladenine but is poorly inhibited by 6-chloropurine. A number of physiological cytokinins including trans- and cis-zeatin are also efficient competitors for AtPUP2-mediated adenine uptake, suggesting that AtPUP2 is also able to mediate cytokinin transport. Furthermore, AtPUP1 mediates transport of caffeine and ribosylated purine derivatives in yeast. Promoter-reporter gene studies point towards AtPUP1 expression in the epithem of hydathodes and the stigma surface of siliques, suggesting a role in retrieval of cytokinins from xylem sap to prevent loss during guttation. The AtPUP2 promoter drives GUS reporter gene activity in the phloem of Arabidopsis leaves, indicating a role in long-distance transport of adenine and cytokinins. Promoter activity of AtPUP3 was only found in pollen. In summary, three closely related PUPs are differentially expressed in Arabidopsis and at least two PUPs have properties similar to the adenine and cytokinin transport system identified in Arabidopsis cell cultures.     

Sucrose transporters and plasmodesmal regulation in passive phloem loading

An essential step for the distribution of carbon throughout the whole plant is the loading of sugars into the phloem in source organs. In many plants,accumulation of sugars in the sieve element-companion cell(SE-CC)complex is mediated and regulated by active processes.However,for poplar and many other tree species,a passive symplasmic mechanism of phloem loading has been proposed,characterized by symplasmic continuity along the pre-phloem pathway and the absence of active sugar accumulation in the SE-CC complex. A high overall leaf sugar concentration is thought to enable diffusion of sucrose into the phloem. In this review,we critically evaluate current evidence regarding the mechanism of passive symplasmic phloem loading,with a focus on the potential influence of active sugar transport and plasmodesmal regulation. The limited experimental data,combined with theoretical considerations,suggest that a concomitant operation of passive symplasmic and active phloem loading in the same minor vein is unlikely.However,active sugar transport could well play an important role in how passively loading plants might modulate the rate of sugar export from leaves. Insights into the operation of this mechanism has direct implications for our understanding of how these plants utilize assimilated carbon.     

Evidence for apoplasmic phloem unloading in developing apple fruit

The phloem unloading pathway remains unclear in fleshy fruits accumulating a high level of soluble sugars. A structural investigation in apple fruit (Malus domestica Borkh. cv Golden Delicious) showed that the sieve element-companion cell complex of the sepal bundles feeding the fruit flesh is symplasmically isolated over fruit development. 14C-autoradiography indicated that the phloem of the sepal bundles was functional for unloading. Confocal laser scanning microscopy imaging of carboxyfluorescein unloading showed that the dye remained confined to the phloem strands of the sepal bundles from the basal to the apical region of the fruit. A 52-kD putative monosaccharide transporter was immunolocalized predominantly in the plasma membrane of both the sieve elements and parenchyma cells and its amount increased during fruit development. A 90-kD plasma membrane H(+)-ATPase was also localized in the plasma membrane of the sieve element-companion cell complex. Studies of [14C]sorbitol unloading suggested that an energy-driven monosaccharide transporter may be functional in phloem unloading. These data provide clear evidence for an apoplasmic phloem unloading pathway in apple fruit and give information on the structural and molecular features involved in this process.     

Symplasmic transport and phloem loading in gymnosperm leaves

Despite more than 130 years of research, phloem loading is far from being understood in gymnosperms. In part this is due to the special architecture of their leaves. They differ from angiosperm leaves among others by having a transfusion tissue between bundle sheath and the axial vascular elements. This article reviews the somewhat inaccessible and/or neglected literature and identifies the key points for pre-phloem transport and loading of photoassimilates. The pre-phloem pathway of assimilates is structurally characterized by a high number of plasmodesmata between all cell types starting in the mesophyll and continuing via bundle sheath, transfusion parenchyma, Strasburger cells up to the sieve elements. Occurrence of median cavities and branching indicates that primary plasmodesmata get secondarily modified and multiplied during expansion growth. Only functional tests can elucidate whether this symplasmic pathway is indeed continuous for assimilates, and if phloem loading in gymnosperms is comparable with the symplasmic loading mode in many angiosperm trees. In contrast to angiosperms, the bundle sheath has properties of an endodermis and is equipped with Casparian strips or other wall modifications that form a domain border for any apoplasmic transport. It constitutes a key point of control for nutrient transport, where the opposing flow of mineral nutrients and photoassimilates has to be accommodated in each single cell, bringing to mind the principle of a revolving door. The review lists a number of experiments needed to elucidate the mode of phloem loading in gymnosperms.     

Mineral partitioning in the phloem during autumn senescence of beech leaves

Summary During the period of leaf senescence in fall, the minerals Mg, Ca, K, P, Cl, S, and Si were compared for occurrence and density in tissue compartments of leaf blade, petiole, and subtending stem of beech (Fagus sylvatica L.). Measurements were made by energy-dispersive X-ray microanalysis. The plant material was collected on 2,9, 16 and 23 October, and showed green, greenyellow, yellow, and red-brown autumn leaf coloration. Mg, K, and P were retrieved from the leaf blade prior to shedding, and deposited mainly in cortex and pith tissues of the stem. S and Ca remained in the leaf, and Si and Cl appeared to accumulate in the leaf prior to shedding. During the four stages of leaf senescence, the phloem compartments of the petiole showed considerable changes in mineral content. In addition, leaf senescence in several cases was accompanied by ion shifting from symplastic to apoplastic compartments and vice versa.     

Carbohydrate availability modifies sorbitol dehydrogenase activity of apple fruit

Complete defoliation of the stem or spur and girdling the phloem (D/G) subtending a single apple ( Malus domestica Borkh.) fruit at 70 or more days after bloom resulted in reduction of fruit growth. Extractable sorbitol dehydrogenase (SDH) (enzyme code, 1.1.1.14) activity and sorbitol and starch content of the cortex tissue of fruit receiving D/G treatment subsequently declined. Co-extraction of control and D/G cortex tissues yielded expected levels of SDH activity, indicating that the loss of extractable SDH activity in D/G fruit was not due to presence of an inhibitor or proteolytic activity. Incubating cortex sections from D/G fruit in a buffered 200 m M sorbitol or glucose solution increased extractable SDH activity, and incubating cortex sections from control fruit in the sorbitol solution maintained the activity. However, neither 200 m M fructose or 27 m M PEG, the latter with the same osmotic potential as the sorbitol solution, affected extractable SDH activity of D/G fruit. The results indicate that carbohydrate availability may affect extractable SDH activity of apple fruit, and that specific carbohydrates such as sorbitol and glucose may be signals for modulating this activity.     

Sugar synthesis and phloem loading in Coleus blumei leaves

Sugar-synthesis and -transport patterns were analyzed in Coleus blumei Benth. leaves to determine where galactinol, raffinose, and stachyose are made and whether phloem loading includes an apoplastic (extracellular) step or occurs entirely within the symplast (plasmodesmata-connected cytoplasm). To clarify the sequence of steps leading to stachyose synthesis, a pulse (15 s) of ¹⁴CO₂ was given to attached leaves followed by a 5-s to 20-min chase: sucrose was rapidly labeled while galactinol, raffinose and stachyose were labeled more slowly and, within the first few minutes, to approximately the same degree. Leaf tissue was exposed to either ¹⁴CO₂ or [¹⁴C]glucose to identify the sites of synthesis of the different sugars. A 2-min exposure of peeled leaf tissue to [¹⁴C]glucose resulted in preferential labeling of the minor veins, as opposed to the mesophyll; galactinol, raffinose and stachyose were more heavily labeled than sucrose in these preparations. In contrast, when leaf tissue was exposed to ¹⁴CO₂ for 2 min for preferential labeling of the mesophyll, sucrose was more heavily labeled than galactinol, raffinose or stachyose. We conclude that sucrose is synthesized in mesophyll cells while galactinol, raffinose and stachyose are made in the minorvein phloem. Competition experiments were performed to test the possibility that phloem loading involves monosaccharide uptake from the apoplast. Two saturable monosaccharide carriers were identified, one for glucose, galactose and 3-O-methyl glucose, and the other for fructose. Washing the apoplast of peeled leaf pieces with buffer or saturating levels of 3-O-methyl glucose, after providing a pulse of ¹⁴CO₂, did not inhibit vein loading or change the composition of labeled sugars, and less than 0.5% of the assimilated label was recovered in the incubation medium. These and previous results (Turgeon and Gowan, 1991, Plant Physiol. 94, 1244–1249) indicate that the phloem loading pathway in Coleus is probably symplastic.Abbreviations 3-OMG 3-O-methyl glucose - PCMBS p-chloromercuribenzenesulfonic acid - SE-CCC sieve-element-companion-cell complex This research was supported by National Science Foundation Grant DCB-9104159, U.S. Department of Agriculture Competetive Grant 90000854, and Hatch funds.     

Evidence for symplastic phloem unloading in sink leaves of barley

The pathway of phloem unloading in sink barley (Hordeum vulgare) leaves was studied using a combination of electron microscopy, carboxyfluorescein transport, and systemic movement of barley stripe mosaic virus expressing the green fluorescent protein. Studies of plasmodesmatal frequencies between the phloem and mesophyll indicated a symplastic sieve element- (SE) unloading pathway involving thick-walled and thin-walled SEs. Phloem-translocated carboxyfluorescein was unloaded rapidly from major longitudinal veins and entered the mesophyll cells of sink leaves. Unloading was "patchy" along the length of a vein, indicating that sieve element unloading may be discontinuous along a single vascular bundle. This pattern was mirrored precisely by the unloading of barley stripe mosaic virus expressing the green fluorescent protein. Transverse veins were not utilized in the unloading process. The data collectively indicate a symplastic mechanism of SE unloading in the sink barley leaf.