JXB Advance Access originally published online on August 28, 2007
Journal of Experimental Botany 2007 58(12):3155-3169; doi:10.1093/jxb/erm153
© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Involvement of the sucrose transporter, OsSUT1, in the long-distance pathway for assimilate transport in rice
1CSIRO Plant Industry, Canberra, ACT 2601, Australia
2Department of Rice Research, National Agricultural Research Center, Joetsu, Niigata, 943-0193 Japan
To whom correspondence should be addressed. E-mail: robert.furbank{at}csiro.au
Received 1 May 2007; Revised 6 June 2007 Accepted 11 June 2007
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Abstract |
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The roles of the rice sucrose transporter, OsSUT1, have previously been examined in filling grain, germination, and early seedling growth. In the current work, the role that OsSUT1 plays in the transport of assimilate along the entire long-distance pathway, from the flag leaf blade to the base of the filling grain, was investigated. OsSUT1 promoter::GUS (β-glucuronidase) reporter gene analysis and immunolocalization revealed that both OsSUT1 promoter::GUS activity and OsSUT protein were present in the mature phloem of all the vegetative tissues involved in the long-distance assimilate transport pathway during grain filling. In addition, expression was observed in the flag leaf blade and sheath prior to heading. The OsSUT1 promoter::GUS activity appeared to be largely confined to the companion cells within the phloem, whereas the protein localized to both the sieve tubes and the companion cells. RT-PCR analysis confirmed that the OsSUT1 transcript is expressed in the uppermost internode of the rice plant (internode-1). These OsSUT localization data were related to measurements of starch and soluble sugar content of these tissues, and localization of the carbohydrate reserves stored in the stem. Results from dye feeding experiments, to examine cellular connections, revealed a symplastic continuity between the phloem and surrounding parenchyma in the flag leaf blade, sheath, and internode-1 tissues. It is proposed that OsSUT1 may primarily play a role in phloem loading of sucrose retrieved from the apoplasm along the transport pathway.
Key words: Long-distance pathway, OsSUT1, rice, starch remobilization, sucrose transport, transport phloem
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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Sucrose is the main form in which assimilate, produced by photosynthetic source tissues such as the flag leaf blade, is transported via the long-distance vascular pathway to sink tissues. In sink tissue, sucrose may be used directly for metabolism or may be temporarily stored prior to remobilization for use at a later stage of the plant's development. The primary sink tissue in cereal species is the filling grain of the panicle, in which the carbohydrate accumulates as starch in the endosperm and embryo. However, other tissues such as the leaf sheath and stem may also function as temporary sink tissues prior to heading. The sucrose is transported to these various sink tissues via a continuous mature transport phloem network (Fig. 1). Phloem from the flag leaf blade is continuous with that present in the flag leaf sheath, which in turn connects to the main stem at a node. At the node, a complex architecture exists (Chonan, 1993, and references therein) that links the sheath phloem elements to those of internode-1. The phloem elements in internode-1 are continuous through both the region that is enclosed by the leaf sheath and the exposed region, and continue into the panicle. The final step in the pathway is via the pedicel, the stalk that supports the spikelet on the panicle rachis branch, which directly enters the rachilla at the base of the filling grain. The main vasculature from the pedicel is continuous with the dorsal vascular bundle of the pericarp (Zee, 1972).
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During vegetative growth, prior to heading and anthesis, rice, like other cereal species, accumulates carbohydrate reserves within the leaf sheath and stem tissue (Perez et al., 1971; Watanabe et al., 1997; Hirose et al., 1999; Yang et al., 2001; Ishimaru et al., 2004; Takahashi et al., 2005). In rice, these temporary reserves are laid down in the form of starch granules that are located within the sheath and stem tissues, which act as temporary sinks. In comparison, temperate cereal species such as wheat and barley store carbohydrate predominantly as water-soluble fructans in their stem tissues (Gebbing, 2003, and references therein). After heading, these reserves can be remobilized, and the starch or fructan is converted back to sucrose and returned to the long-distance pathway for transport to the filling grain on the panicle. In rice, it has been suggested that up to 24–27% of the carbohydrate in the grain originates from stem starch reserves (Cock and Yoshida, 1972), thus making it an important ‘source’ during grain filling. In wheat, it has been suggested that as much as 50% of the photoassimilate deposited in grains is temporarily stored before remobilization to the filling grain (Schnyder, 1993). Under unfavourable conditions for photosynthesis, cereal species have an increased reliance on these temporary reserves to complete grain filling (Yang et al., 2001, 2002, 2003, and references therein). The pathways and mechanisms for transport of assimilate into and out of these temporary storage tissues are not fully understood. In addition, the signalling mechanisms that control the switch between the storage and remobilization phases have yet to be elucidated.
Movement of sucrose into and out of the temporary sink tissues of the stem, and throughout the long-distance transport phloem to the filling grain, may involve apoplastic pathways that require sucrose transporters (SUTs) to facilitate sucrose movement between cells. Evidence to support this arises from previously reported observation of expression of the transcripts for the type II SUT, OsSUT1, and the other members of the rice SUT gene family within tissues of the long-distance transport pathway (Hirose et al., 1997, 1999; Aoki et al., 2003). RT-PCR analysis indicated that all five known members of the rice SUT family are expressed in source leaf blade (flag leaf) and source leaf sheath tissues (second leaf below the flag leaf) (Aoki et al., 2003); however, their expression in internode tissue was not examined. OsSUT1 transcript was also detected in the rachis branch leading to the spikelet (Hirose et al., 1997). The expression of SUTs in various tissues of the long-distance pathway has also been reported in other cereal species including wheat (Aoki et al., 2002, 2004), barley (Weschke et al., 2000), and maize (Aoki et al., 1999). TaSUT1, the wheat orthologue of OsSUT1, was found in flag leaf blade, sheath, and stem tissue (Aoki et al., 2004). The authors concluded that TaSUT1 was involved both in phloem loading of sucrose and in retrieving sucrose leaked to the phloem apoplasm. In sugarcane, a C4 plant species, Casu et al. (2003) and Rae et al. (2005) reported the expression of a SUT in sink stem tissue, and in the latter work provide evidence for the expression of more than one SUT in the stem.
Sucrose uptake by members of the monocot type II group of SUTs has been demonstrated by expression in yeast cells, including OsSUT1 (Hirose et al., 1997; Aoki et al., 2003), HvSUTI (Weschke et al., 2000), and TaSUT1 (Aoki et al., 2006). More recently, through expression in Xenopus oocytes, it has been shown that the monocot type II SUTs HvSUT1 and ShSUT1 have high substrate specificity for sucrose (Sivitz et al., 2005, 2007). In dicots, the type II SUTs, Arabidopsis AtSUT2/SUC3 and tomato LeSUT2, are most closely related to four of the rice SUT genes (Aoki et al., 2003). The AtSUT2 promoter has been reported to be expressed in the transport but not the collection phloem of leaves, and the LeSUT2 protein has been observed in sieve elements of tomato stem (Barker et al., 2000). In addition, Meyer et al. (2004) showed that AtSUC3 (SUT2) is expressed in a number of sink tissues and suggested a role for AtSUC3 (SUT2) in sucrose delivery to these sinks.
In the current work, OsSUT1 promoter activity and OsSUT protein localization have been examined in all of the tissues in the long-distance sugar transport pathway from the flag leaf blade to the base of the filling grain. In addition, transcript expression profiles for all the members of the OsSUT gene family in internode-1 tissue have been determined. Carbohydrate analysis and starch localization were performed in these various tissues, and dye feeding experiments were carried out to examine intercellular connections. These data are discussed in relation to assimilate transport and transport of sucrose remobilized from starch reserves in temporary sink tissues, and the intercellular connections within these tissues.
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Material and methods |
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Plants and growth conditions
Rice plants of Oryza sativa L. ssp. japonica cv. Taipei 309 and O. sativa L. ssp. japonica cv. Nipponbare were used, either untransformed or from T1 seeds harvested from the OsSUT1 promoter::GUS (β-glucuronidase) T0 regenerated lines. Plants were grown to maturity in flooded paddy tanks in a glasshouse, in 75% compost, 25% perlite supplemented with Osmocote slow-release fertilizer. Growth was under natural light with a daytime temperature of 28 °C and a night-time temperature of 25 °C. To provide samples for carbohydrate and RT-PCR analysis, rice plants (O. sativa L. ssp. japonica cv. Nipponbare) were grown under field conditions in 4.0 l plastic pots filled with soil from the paddy field at the NARC, Joetsu, Japan.
Sampling
Samples of tissues involved in the long-distance assimilate transport pathway from the flag leaf blade to the base of the filling grain, including flag leaf blade and sheath, enclosed and exposed internode-1, rachis branch, and pedicel, were examined in this study. The relative positions of these tissues within the plant are illustrated in Fig. 1.
Carbohydrate and chlorophyll determination
Samples of internode-1 and the flag leaf sheaths were harvested on 15 September 2006 corresponding to the twelfth day after heading (dah). In order to obtain profiles for carbohydrate and chlorophyll content in the flag leaf sheath and internode-1, the tissues were divided into segments. The flag leaf sheath was divided into three equal segments corresponding to the bottom, middle, and top of the sheath. The internode-1 samples were initially divided into two parts, the lower part enclosed by the flag leaf sheath and the upper exposed part. The enclosed region was further divided into five equal segments, whilst the exposed region was divided into two equal segments. After sampling, the sheath and internode-1 segments were immediately weighed, frozen in liquid nitrogen, and stored at –80 °C until use. To determine the carbohydrate and chlorophyll content, the flag leaf sheath and internode-1 samples were initially ground to a fine powder using a mixer mill (MM-300, Qiagen, Hilden, Germany) under cryogenic conditions. For carbohydrate determination, the ground samples were extracted twice with 80% ethanol at 80 °C. After centrifugation at 10 000 g for 5 min, the supernatant was dried under vacuum, dissolved in distilled water, and used in assays for soluble sugars using a Dionex DX500 chromatography system equipped with a CarboPac PA-1 column (Sunnyvale, CA, USA). An isocratic elution with 100 mM sodium hydroxide was employed at a flow rate of 1 ml min–1, and integrated pulsed amperometric detection was performed following the manufacturer's recommended programme. For starch determination, the pellet was resuspended in distilled water, boiled for 8 h, digested with amyloglucosidase (Roche Diagnostics, Mannheim, Germany) for 20 min at 55 °C, and the resultant glucose was determined enzymatically with the F-kit for D-glucose (Roche Diagnostics, Manheim, Germany) following the manufacturer's instructions. To determine chlorophyll content, the ground samples were extracted with 99.5% ethanol, and chlorophyll was assayed spectrophotometrically following the method of Wintermans et al. (1965).
Semi-quantitative RT-PCR analysis
Total RNA was isolated from flag leaf sheath and internode-1 segments using the method of Chang et al. (1993) but with the polyvinylpyrrolidone and spermidine omitted from the extraction buffer. The isolated total RNA (500 ng) was reverse-transcribed (SuperScript II; Invitrogen) using an oligo(dT)13 primer. An aliquot of the first-strand cDNA mixture corresponding to 10 ng of the total RNA was used as a template for semi-quantitative PCR analysis. The PCR (20 µl total volume) was performed using 0.2 U of Taq polymerase (ExTaq; Takara Bio Inc., Shiga, Japan). The gene-specific primers used for semi-quantitative PCR are listed in Table 1. The amount of template cDNA and the number of PCR cycles were determined for each gene to ensure that amplification occurred in the linear range and allowed accurate quantification of the amplified products. The amplified bands were cloned and sequenced to confirm that they were fragments of the intended gene. For each template cDNA, PCR was also carried out using a primer set that covers an intron-containing region in a rice cell wall invertase gene (OsCIN1; Hirose et al., 2002) to verify that there was no genomic DNA contamination.
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Real-time PCR analysis
To determine the transcript levels of OsSUT1 in the internode-1 tissue samples, real-time PCR was performed using a Smart Cycler System (Cepheid Co., Sunnyvale, CA, USA) with the SYBER pre-mix ExTaq (Takara Bio, Inc.) according to the manufacturer's instructions. The cDNA templates used for real-time analysis were the same as those used for the semi-quantitative RT-PCR. Nucleotide sequences for the gene-specific primers are listed in Table 1. The results obtained for the different cDNAs were normalized to the expression level of a rice polyubiquitin gene (RUBQ1; Wang et al., 2000; accession number U37687). Wang et al. (2000) reported highly constitutive expression of this gene in rice tissues. The amplified bands were cloned and sequenced to confirm that they were fragments of the intended gene. A calibration curve for each gene was obtained by performing real-time PCR with several dilutions of the cloned cDNA fragment. The specificity of the individual PCR amplification was confirmed using a heat dissociation protocol from 65 °C to 95 °C following the final cycle of the PCR.
OsSUT1 promoter::GUS activity localization
An OsSUT1 promoter::GUS construct was prepared and rice tissue transformed as described in Scofield et al. (2007). Several independent lines were regenerated, three of which, lines 1, 107, and 120, were grown on for further studies. A population of T2 transgenic plants were grown in the glasshouse, and samples of flag leaf blade, flag leaf sheath, enclosed and exposed internode-1, and the pedicel were harvested at 12 dah. Similar tissue samples were collected from an untransformed Nipponbare plant as the control. The samples were incubated in X-gluc solution at 37 °C in the dark either for 5 h or overnight, following the method described in Scofield et al. (2007). The tissue samples were examined and scored for GUS activity using a Leica MZ7.5 stereomicroscope. Representative samples of each tissue were embedded in LR White resin. Thin sections of between 0.5 µm and 2 µm in thickness were cut using an ultramicrotome and were collected onto glass slides. For visualization of the GUS expression, the sections were examined under Nomarski illumination using a Leica DMR microscope, and representative images were recorded using a Leica DC500 digital camera.
Immunolocalization
Samples of flag leaf blade, flag leaf sheath, enclosed and exposed internode-1, and the pedicel and base of filling grain were harvested from glasshouse-grown Nipponbare plants at 12 dah. In addition, flag leaf blade and flag leaf sheath samples were also collected from a plant estimated to be between 5 d and 8 d pre-heading. The samples were fixed, dehydrated, and embedded in paraffin wax as described in Scofield et al. (2007). Transverse sections were cut, collected, and immunolabelled with an anti-OsSUT1 antibody (see Scofield et al., 2007), used directly without purification, at a dilution of 1 in 200 in blocking solution for 20 h at 4 °C. A pre-immune serum was used, at a similar dilution, for the control.
Starch localization
Starch localization was carried out on similar sections to those used for the immunolabelling experiments. After de-waxing, re-hydrating, and washing in three changes of water, the sections were stained in a solution of 0.5% (w/v) iodine in 5% aqueous potassium iodide for 10 min prior to examination under brightfield illumination with a Leica DMR microscope.
5,6-CFDA feeding experiments
To examine intercellular connections in the various tissues of the long-distance pathway, feeding experiments using 5,6-carboxyfluoescein diacetate (CFDA), a low molecular weight dye, were carried out essentially as described in Aoki et al. (2004). Rice plants grown in glasshouse conditions were used, with feeding experiments taking place in the glasshouse. The dye was fed to rice plants, via the tip of the flag leaf blade, as described in Aoki et al. (2004), for either 2 h or 5 h periods under natural illumination; controls were fed with water alone. Sections of the dye-fed tissues were prepared and examined using a confocal laser scanning microscope as described.
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Results |
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Flag leaf blade
Transverse sections of flag leaf blade tissue, harvested at 12 dah, from plants expressing the OsSUT1 promoter::GUS construct were examined to localize GUS activity (Fig. 2A–D). GUS activity was found in the phloem of all the vascular bundles across the leaf blade (Fig. 2A, B), in the companion cells, and in cells at the periphery of the phloem that resemble thick-walled sieve elements and/or phloem parenchyma cells. In addition, GUS activity was observed in the minor vascular bundles of the leaf's mid-rib region (Fig. 2C, D), but was absent from the phloem of one of the major vascular bundles in this region (data not shown). Similar GUS activity results were obtained from plants derived from all three independent transformed lines examined, and was absent in sections from untransformed control plants (data not shown). Staining transverse sections of flag leaf blade tissue, from both pre-heading and 12 dah samples, with iodine solution revealed an absence of starch grains (data not shown).
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Labelling of OsSUT epitopes by the anti-OsSUT1 antibody was confined to the phloem of both the major and minor vascular bundles across the entire width of the flag leaf blade (Fig. 2E, G), including all those within the mid-rib region (Fig. 2I, K). Within the phloem, the labelling was present in both the companion cells and the sieve elements. The results were reproducible in serial sections. Labelling was absent from adjacent sections probed with the pre-immune antibody (Fig. 2F, H, J, L). Some occasional background coloration was present in the mesophyll, fibre cells, and aerenchyma borders, on sections probed with the anti-OsSUT1 antibody and the pre-immune antibody, which was diminished with the addition of levamisole to the development solution, thus suggesting that it was due to endogenous alkaline phosphatase activity. Colour development was absent from other tissues in the sections. Transverse sections of flag leaf blade samples harvested from plants prior to heading produced identical results when probed with the anti-OsSUT1 or pre-immune sera (data not shown).
Flag leaf sheath
In transverse sections of flag leaf sheath tissue, from both pre-heading samples and those 12 dah, staining revealed starch grains present exclusively in the parenchyma cells adjacent to both the large and the small vascular bundles and extending down the inter-aerenchyma columns, and absent from all other cell types within the tissue (Fig. 3A–C). In transverse sections of flag leaf sheath tissue, GUS activity was largely confined to the phloem of both the large and the small vascular bundles, where it appeared to be present in companion cells and also in cells around the periphery of the phloem which resemble thick-walled sieve elements and/or phloem parenchyma (Fig. 3D, E). Repeatable results were obtained in sequential sections and in separate samples of flag leaf sheath tissue. Occasionally GUS activity was also present in some of the xylem parenchyma cells surrounding xylem vessels. Similar GUS activity was observed in plants derived from all three independent transformed lines examined, and was absent in sections from untransformed control plants (data not shown).
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Transverse sections probed with the anti-OsSUT1 antibody (Fig. 3F, H) showed labelling confined exclusively to the phloem of both the large and small vascular bundles and absent from all other cell types within this tissue. Within the phloem, labelling was present in the sieve elements, the companion cells, and also in peripheral cells resembling thick-walled sieve elements and/or phloem parenchyma. No labelling was observed in the xylem parenchyma cells. Labelling was absent from adjacent sections that were probed with the pre-immune serum (Fig. 3G, I). Serial transverse sections of flag leaf sheath tissue, harvested from rice plants prior to heading, probed with either the anti-OsSUT1 or pre-immune sera, produced similar results to post-heading samples (data not shown).
To examine the role of the flag leaf sheath in carbohydrate storage, the starch and soluble sugar content of segments of the flag leaf sheath was determined (Fig. 4). The results were subjected to statistical analysis, using Tukey's test to determine the significance of differences. The starch content of the flag leaf sheath (Fig. 4A) was low compared with that of the enclosed internode-1 (Fig. 7A), with no significant difference in content between the top and middle of the sheath, but gradually increased towards the bottom of the sheath, to be approximately three times greater at the bottom compared with the top of the sheath, suggestive of an increased starch storage capacity at the bottom of the sheath. Levels of the hexoses, glucose and fructose, were low and remained relatively unchanged in all three segments of the sheath (Fig. 4B). In comparison, levels of sucrose were considerably higher and decreased towards the bottom of the sheath, but with no significant difference between the middle and bottom. Levels of maltose were extremely low compared with sucrose, and remained unchanged in all three segments.
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Internode-1
Transcript expression profiles were determined for the enclosed and exposed regions of internode-1 samples by semi-quantitative RT-PCR analysis (Fig. 5A). OsSUT1 and OsSUT4 transcripts were present in all the segments of both the enclosed and exposed internode. The OsSUT2 transcript was found to be preferentially expressed in the enclosed region of internode-1 and declining in or absent from the exposed region, whereas OsSUT3 transcript expression exhibited the reverse distribution. OsSUT5 transcript could not be detected in any of the internode-1 segments. To examine expression levels of the OsSUT1 transcript in internode-1 in more detail, real-time PCR analysis was performed (Fig. 5B). In the enclosed region, segments 1–5 inclusive, OsSUT1 transcript was expressed throughout the tissue, with higher levels of expression in the bottom and top segments and lower expression in the middle segments. Expression in the bottom segment of the exposed region of internode-1 was equivalent to the expression in the top segment of the enclosed region; however, the OsSUT1 transcript was expressed most highly in the top segment of the exposed region, with an approximately 2-fold or more expression level compared with any other part of internode-1.
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The region of internode-1 enclosed by the sheath contained a high density of starch grains (Fig. 6A). The starch grains were localized exclusively within the storage parenchyma cells, and were absent from both the large and small vascular bundles and the cortical fibre cells. The starch grains were present throughout the parenchyma; however, a high density of grains was present in the parenchyma cells immediately below the cortical fibre cell layer and around the large vascular bundles, with a decreasing gradient away from these regions. In contrast to this, the exposed part of internode-1 contained only a very low density of starch grains (Fig. 6B). Again the starch grains were located exclusively in the parenchyma cells; however, they were present only in the parenchyma cells in close proximity to the large vascular bundles. They were absent from all other cell types including the chlorenchyma tissue that is present in this region of internode-1. Resin sections of internode-1 tissue were examined to localize OsSUT1 promoter::GUS activity at the cellular level (Fig. 6C, D). In both the large and the small vascular bundles, GUS activity was largely confined to the phloem. Within the phloem, GUS activity was present in the companion cells and also in some peripheral cells that are probably thick-walled sieve elements and/or phloem parenchyma cells. Occasionally, GUS activity was also observed in the xylem parenchyma cells. Identical results were observed in the exposed region of internode-1 (data not shown). All three transformed lines gave similar results, and sections from untransformed control plants lacked GUS activity (data not shown).
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OsSUT1 epitopes were labelled exclusively within the phloem in transverse sections from the mid-point of the enclosed internode-1 (Fig. 6E–H), in both the large (Fig. 6E) and the small (Fig. 6G) vascular bundles. The labelling was present in both the companion cells and the sieve elements. Labelling was absent from adjacent sections probed with the pre-immune serum (Fig. 6F, H). Identical results were obtained for immunolabelled sections of the exposed region of internode-1 (data not shown).
Starch, soluble sugar, and chlorophyll contents of the enclosed and exposed internode-1 at 12 dah were determined. Figure 7A shows the starch content in sections of the enclosed (1–5) and exposed (6–7) regions of internode-1 from bottom to top. The enclosed internode-1 had its highest levels of starch through the middle section of the internode, which decreased towards both the base and the top of the enclosed internodal region, but with a greater decline towards the top. In comparison, the exposed internode had an equal starch content in both of the segments into which it was divided, and these levels were between five and seven times lower than the starch levels present in the enclosed internode segments. Soluble sugar contents were measured in the same segments of the enclosed and exposed regions of internode-1 (Fig. 7B). Glucose and fructose levels were low in all the segments and declined from the bottom to the top of the enclosed and exposed regions of internode-1. Sucrose was present in the greatest amounts within the middle segments of the enclosed internode-1, corresponding to the same segments in which starch was present at its highest levels, and declined towards both the bottom and the top of the internode. The lowest level of sucrose was present in the bottom segment of the exposed internode-1 and increased in the top segment. Maltose levels were extremely low in both the enclosed and exposed segments of internode-1. Figure 7C shows the chlorophyll content in segments of the enclosed and exposed internode-1 over a gradient from bottom to top for each tissue. The enclosed internode had low levels of chlorophyll, at 0.1 mg g FW, which remained relatively constant from the bottom to the top of the internode, apart from the final segment at the top which contained approximately twice the amount present in the lower segments. In contrast, the exposed region of internode-1 had a higher chlorophyll content, being six times higher at the bottom and eight times higher at the top compared with all except the top segment of the enclosed region of internode-1.
Pedicel and base of grain
The pedicel is the final part of the long-distance pathway leading to the filling grain that was examined. Each filling grain is subtended on a pedicel which in turn branches off the rachis branch. In the region close to the rachis branch the pedicel had a cylindrical core around which were arranged three distinct lobes. One large vascular bundle ran through the central core for the entire length of the pedicel, and each of the three lobes contained one smaller vascular bundle. Starch grains were absent from this tissue at 12 dah (Fig. 8A). Examination of sections from the OsSUT1 promoter::GUS plant lines revealed that GUS activity was present almost exclusively in the phloem of both the central main large vascular bundle and the three minor small vascular bundles (Fig. 8B, C). Within the phloem, GUS activity was mainly confined to the companion cells; however, occasionally GUS activity was also observed in a few xylem parenchyma cells. Similar results were observed in sections cut further along the pedicel up to the base of the filling grain (data not shown).
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Immunolabelling was carried out on transverse sections along the pedicel from its branch with the rachis up to the base of the filling grain. At the branch end of the pedicel, immunolabelling of OsSUT epitopes was clearly visible within both the large and all three smaller vascular bundles, where it was largely confined to the phloem (Fig. 8D–F). Labelling was absent from the control sections probed with the pre-immune serum (Fig. 8G–I). Where the pedicel neared the base of the filling grain, the three-lobed structure became less pronounced in shape until a more cylindrical outline was present. At the base of the filling grain, additional vascular elements were visible, which connected to the glumes, lemma, palea, lodicule, rachilla, as well as the filling grain. Labelling of OsSUT1 epitopes was visible in some of these additional vascular elements as well as continuing in the main vascular bundles (Fig. 8J–M). Labelling was absent from sections probed with the pre-immune serum (Fig. 8N–Q), though it was noted that two or three specific cells, outside of the vascular bundles, did produce some coloration, presumably due to endogenous alkaline phosphatase activity that was not fully inhibited by addition of levamisole. It was noted that pairs of guard cells present at intervals around the epidermal layer also contained labelling which was absent from the control (Fig. 8R, S). In later sections in the series through the rachilla region at the base of the filling grain, labelling of OsSUT epitopes was also observed in the central vascular bundles of the lemma and palea (data not shown).
5,6-CFDA feeding
The source leaves of rice plants were fed with the low molecular weight dye 5,6-CFDA, in order to examine symplastic continuity between cells in the pathway of phloem loading/unloading in various tissues of the vascular system. In sections of flag leaf blade tissue, from a plant at 4–5 d pre-heading (Fig. 9A–C), 5,6-CF fluorescence was observed to have moved out from the sieve element–companion cell complex, both to other phloem cells and to the surrounding tissue including the mesophyll. Similar results were obtained in flag leaf blade tissue sampled from plants 8 dah (Fig. 9D). In the flag leaf sheath surrounding the mid-region of the enclosed internode-1, the dye had moved out of the phloem of both the large and the small vascular bundles and was present in the parenchyma cells but was absent from the chlorenchyma (Fig. 9E). In the enclosed region of internode-1, the dye had extensively moved out of the phloem into the surrounding parenchyma cells (Fig. 9F). Similarly, in the exposed region of internode-1 (Fig. 9G), the dye had again moved out of the phloem of the inner vascular bundles; however, dye movement appeared to be less extensive than in the enclosed region of internode-1. No fluorescence was observed within the chlorenchyma layer of the exposed internode-1. Controls fed with water alone exhibited no fluorescence (data not shown).
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Discussion |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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The current work has investigated the expression of OsSUT1 in the vegetative tissues that comprise the entire long-distance assimilate pathway, from the flag leaf blade to the base of the filling grain. From a combination of expression analysis, starch and soluble sugar determinations, and analysis of cellular connectivity within these tissues, roles for OsSUT1 in the retrieval of leaked sucrose to the mature transport phloem, phloem loading of assimilate, and delivery to and reloading of carbohydrate from temporary storage tissues and may be inferred.
In the long-distance sugar transport pathway tissues examined in the current study there was close correlation between OsSUT1 promoter::GUS activity and OsSUT protein localization. However, in some cases, GUS activity was also noted in some of the xylem parenchyma cells surrounding the xylem vessels, whereas OsSUT protein localized exclusively to the phloem. This may be the result of movement of either the GUS protein or the GUS reaction product prior to crystallization, or it may indicate the occurrence of transcript that is not translated into OsSUT protein. Fukuda et al. (2005) reported similar GUS staining in xylem parenchyma cells as well as sieve elements, despite using a companion cell-specific promoter, and suggested that the GUS protein might move though plasmodesmatal connections facilitated by protein unfolding. In the present study, there was, however, no evidence of movement of the GUS protein or product into surrounding tissues, as was reported for the coleoptile and primary leaf tissues of young seedlings (Scofield et al., 2007).
The antibody used in the current work was raised against three peptide regions from OsSUT1 (Furbank et al., 2001; Scofield et al., 2007). The specificity of this antibody was discussed previously (see Scofield et al., 2007). In assessing the immunolabelling results presented in this work, it should be taken into consideration that OsSUTs 3, 4, and 5 may have sufficient homology (73, 68, and 78% identity, respectively), across only one of the three peptides to which the OsSUT1 antibody was raised, for co-labelling to occur. The availability and abundance of this epitope region for labelling, in preference to the other two epitopes, in a given section should be considered; co-labelling is likely to be low but the possibility of its occurrence cannot be excluded. The transcripts for OsSUTs 3, 4, and 5 are expressed in the flag leaf blade and flag leaf sheath post-heading, though the OsSUT4 transcript level is reduced compared with its level when the flag leaf blade is still a sink (Aoki et al., 2003). In the current work, it has been shown that the transcript for OsSUT1 along with those for OsSUTs 2, 3, and 4 are all expressed in internode-1 tissue. Transcript expression of the five members of the OsSUT family has not been examined in pedicel tissue. The phloem-specific labelling pattern, with protein detected in both companion cells and sieve elements, observed in the rice vegetative tissues in this work is consistent with results obtained previously using this antibody in germinating rice seedlings (Scofield et al., 2007) and also in wheat (Aoki et al., 2004, 2006) and sugar cane (Rae et al., 2005).
The OsSUT1 promoter::GUS activity and OsSUT protein were present in the flag leaf blade tissue, both before and after heading, consistent with previous OsSUT1 transcript analysis (Hirose et al., 1997; Aoki et al., 2003). In contrast to the dicot type II AtSUT2 promoter, which is expressed only in transport phloem in source tissues and not in minor veins (Baker et al., 2000), the OsSUT1 transcript and OsSUT protein were observed in major and minor veins. These data would suggest that OsSUT1 may play a role in phloem loading of assimilate within the flag leaf blade. However, Ishimaru et al. (2001) and Scofield et al. (2002) independently reported that transgenic rice with antisense suppression of OsSUT1, under the control of different promoters, did not exhibit an accumulation of sucrose or an effect on the photosynthetic rate in source leaves. These data suggest that OsSUT1 may not play a major role in phloem loading of sucrose in this tissue, or that this function is redundant due to the action of other members of the gene family such as OsSUT3 and OsSUT5, the transcripts for which are both expressed in source flag leaf blade tissue (Aoki et al., 2003). Alternatively, phloem loading in rice flag leaf tissue may follow a symplastic pathway. Evidence in support of this latter view was reported by Kaneko et al. (1980) and Chonan et al. (1981). From ultrastructural studies of small and large vascular bundles in rice leaf blades, respectively, these authors reported numerous plasmodesmatal connections between cells in the assimilate loading pathway, and concluded that a potential symplastic phloem loading pathway exists.
The results from the 5,6-CFDA dye feeding experiments in the current work, where the dye was observed to move freely out of the phloem into the surrounding tissues including the mesophyll in the flag leaf blade both pre- and post-heading, further support the possibility of a symplastic pathway in source leaves. This represents a major difference from the pathway in wheat flag leaf blades, in which there is a lack of symplastic continuity between cells in the phloem loading pathway, suggesting that TaSUT1 may be involved in phloem loading (Aoki et al., 2004). Lack of symplastic continuity in the phloem loading pathway was also reported in barley (Botha and Cross, 1997). It should be noted, however, that the mere occurrence of symplastic connectivity between the phloem and surrounding cells does not allow inferences to be drawn on the rates of flux of sucrose via this pathway. Thus, the possibility of concurrent symplastic and apoplastic loading mechanisms cannot be ruled out.
Previously the OsSUT1 transcript was shown to be present in flag leaf sheath tissue prior to heading, with an increase in transcript level post-heading (Hirose et al., 1999). In the current work, although quantitative conclusions cannot be drawn, OsSUT1 promoter::GUS activity and OsSUT protein were both localized in this tissue prior to and post-heading, which is consistent with the previous transcript analysis (Hirose et al., 1999). The starch and soluble sugar measurements from the flag leaf sheath in the current work were also consistent with previously published analysis in this tissue (Hirose et al., 1999; Ishimaru et al., 2004). Since the level of starch in the flag leaf sheath was observed to be low in comparison with that in the sheaths of lower leaves of the rice plant, during both pre-heading and the heading period of development (Hirose et al., 1999; Ishimaru et al., 2004), it seems unlikely that it functions as a major temporary sink tissue. The flag leaf sheath's primary role is more likely to be both as a photosynthetic source tissue and as the transport link between the flag leaf blade and internode-1. In light of the low starch levels in the sheath, the increase in OsSUT1 transcript post-heading reported by Hirose et al. (1999) may be a direct response to an increased flow of sucrose from the sheath itself, as a source tissue, and an elevated requirement for retrieval of leaked sucrose from the apoplasm rather than a requirement for OsSUT1 to load starch-derived sucrose into the phloem. Results from the 5,6-CFDA dye feeding experiments indicated that, as observed in the flag leaf blade, there is a symplastic pathway between the phloem and surrounding parenchyma cells; however, since sucrose would have to be loaded against a concentration gradient, active transport may also be required.
OsSUT1 promoter::GUS activity and OsSUT protein were detected in the phloem of both the enclosed and exposed internode-1. The phloem in this tissue is primarily involved in transporting assimilate to the filling grain in the panicle. However, it also provides a route for transport of assimilate into and out of the internode's parenchyma storage cells. High levels of starch were measured in the enclosed internode-1, and starch grains were present in the storage parenchyma cells in both pre-heading and post-heading samples. From approximately 10 dah, stem starch reserves are remobilized (Yang et al., 2001) and, after conversion back to sucrose, are presumably loaded into the phloem for transport to the filling grain. OsSUT1 may perhaps have a role in phloem loading of the sucrose remobilized from those reserves. However, real-time PCR revealed that the highest levels of OsSUT1 transcript expression in the segments of the enclosed internode-1 did not correspond to those containing the highest levels of sucrose and starch. Furthermore, results from the 5,6-CFDA feeding experiments suggested a symplastic pathway between the storage parenchyma and phloem at the developmental stage examined, implying no strict requirement for SUT activity in the transport of remobilized sucrose. It is difficult, however, to envisage how sucrose could be loaded symplastically into the phloem considering the high sugar content of the sieve elements.
Over the long distances involved in this phloem network it is likely that sucrose is lost to the apoplasm. OsSUT1 may, as has been proposed for the sheath tissue, function in a sucrose retrieval role, recovering and loading leaked sucrose from the apoplasm back into the phloem. In comparison with the enclosed region of internode-1, the starch grain content was much lower in the exposed region of internode-1 and, together with the presence of a band of chlorenchyma tissue with significantly higher chlorophyll content, suggests that this region of internode-1 functions primarily as a source tissue rather than a temporary sink storage tissue. The difference in starch storage observed between the enclosed and exposed regions of rice internode-1 in this work is consistent with the analogous differences in fructan storage in the enclosed versus exposed peduncle of wheat (Gebbing, 2003). The higher level of OsSUT1 transcript expression observed in this region of internode-1 suggests a possible role in direct phloem loading of assimilate. OsSUT1 may also be required to transport sucrose into the panicle to maintain the supply to the filling grain. Results from the semi-quantitative RT-PCR analysis revealed that transcripts for OsSUTs 2, 3, and 4 were also expressed in internode-1 tissue. It is interesting that OsSUT2 is preferentially expressed in the enclosed region of internode-1, a storage tissue, whereas OsSUT3 is preferentially expressed in the exposed region of internode-1, a source tissue, suggesting that they have different roles within internode-1. The OsSUT4 transcript, like that for OsSUT1, is expressed throughout internode-1. OsSUT5 transcript was not detectable in internode-1, indicating that it may have no function in this tissue at the developmental stage examined. Further examination of the role of these SUTs will require specific antibodies and promoter::GUS fusions or in situ hybridization.
In the flag leaf blade, flag leaf sheath, and enclosed and exposed internode-1, it was observed that both OsSUT1 promoter::GUS activity and OsSUT protein were present in phloem cells located towards the xylem. Although these cells could not be categorically identified, it is likely that they are thick-walled sieve elements and/or phloem parenchyma cells. Botha (2005) suggested that vascular parenchyma cells may be involved in solute recovery from the xylem in monocotyledons. Thick-walled sieve tubes are spatially located closely with the xylem, and it has been speculated that they may have a role in a retrieval pathway. Further experiments involving immunolabelling at the electron microscope level, using the anti-OsSUT1 antibody, would be necessary to determine whether SUT epitopes are actually present in the membranes of these cell types and, therefore, whether they may have a role in this putative assimilate retrieval pathway.
The pedicel represents the final step in the long-distance pathway to the filling grain. It is here that the sucrose directly enters the dorsal vascular bundle of the pericarp which was shown to be continuous with that of the pedicel via the rachilla (Zee, 1972). Rapid transport of sucrose out of the pedicel and into the filling grain is desirable to maintain a gradient and, therefore, a flow of sucrose preventing any negative regulatory feedback. High levels of OsSUT expression were observed in the pedicel, localized to the phloem of the main and secondary vascular bundles within this tissue. OsSUT1 may therefore play an important role in transporting sucrose through this tissue to the filling grain, retrieving sucrose from the apoplasm, and maintaining a gradient to ensure a continuous supply of sucrose to the filling grain. Starch grains were not detectable in this tissue, negating the requirement for OsSUT1 involvement in phloem loading of remobilized sucrose. As with the rachis branch, the pedicel is potentially a photosynthetic source tissue, so a role for OsSUT1 in phloem loading in these tissues cannot be ruled out.
Some epitope labelling was observed in guard cells of the pedicel close to the rachilla of the filling grain. However, there was a lack of corresponding OsSUT1 promoter::GUS expression within these cells. This suggests that one of the other members of the OsSUT family, to which the antibody may cross-hybridize, may be expressed within these cells. The involvement of sucrose, and therefore a possible role for SUTs, in guard cell osmotic regulation has been examined in Commelina (Reddy and Das, 1986), Vicia (Talbott and Zeiger, 1996, and references therein), and Pisum (Ritte et al., 1999). Using inhibitors, Reddy and Das (1986) demonstrated that the sucrose uptake by guard cells is energy dependent, and that sucrose uptake is via proton gradient-driven transport across the plasmalemma, providing strong evidence for involvement of a SUT. More recently, in Arabidopsis the localization of a SUT in guard cells was reported by Meyer et al. (2004). Using green fluorescent protein and GUS reporter constructs, the authors demonstrated expression of the type II SUT AtSUC3 (AtSUT2) promoter in the guard cells of very young but not old rosette leaves. AtSUC3 lies within the same cluster as OsSUTs 1, 3, 4, and 5 (all type II SUTs) on an unrooted dendrogram (Aoki et al., 2003), with OsSUT4 being the closest member of the family. Surprisingly, expression of OsSUT protein in guard cells appeared to be confined to those of the pedicel, as expression was not observed in guard cells of either the flag leaf blade, sheath, or exposed internode-1 at the developmental stages examined in the current work. Young rice leaves were not examined in the current work. The lack of SUT expression in flag leaf blade guard cells may be a consequence of the sampling time; presumably there is a turnover of SUT protein dependent on whether the stomata are opening or closing.
In summary, expression of the OsSUT1 promoter and transcript, and of the OsSUT protein was observed in all tissues involved in the long-distance transport pathway from the flag leaf to filling grain. It is proposed that the primary role of OsSUT1 within these tissues is in the retrieval of sucrose from the apoplasm to the phloem, to maintain the supply of assimilate to the filling grain on the panicle. OsSUT1 may also be involved in directly loading assimilate into the phloem in the exposed region of internode-1, which functions as a source tissue rather than a temporary sink tissue. Evidence from this work suggests that OsSUT1 is less likely to have a role in loading of assimilate remobilized from temporary storage reserves within these tissues during grain filling. Furthermore, data were presented to indicate that a member of the rice SUT family is expressed in and may have a function in guard cells of some tissues.
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Acknowledgements |
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We thank Celia Miller for resin embedding and sectioning of GUS-stained rice tissues, Kiiko Takatsuto for assistance with the semi-quantitative and RT-PCR, Carl Davies and Soussanith Nokham for assistance with figure preparation, and Dr Colin Jenkins for very helpful advice with the carbohydrate measurements and analysis, and for critically reading the manuscript.
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Footnotes |
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* Present address: Laboratory of Crop Science Graduate School of Agriculture and Life Science, The University of Tokyo, 1-1-1 Yayoi, Bunko, Tokyo 113-8657, Japan.
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Abbreviations |
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5,6-CFDA, 5,6-carboxyfluorescein diacetate; dah, days after heading; GUS, β-glucuronidase; SUT, sucrose transporter.
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References |
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