A Plasma Membrane-Anchored Fluorescent Protein Fusion Illuminates Sieve Element Plasma Membranes in Arabidopsis and Tobacco1[W][OA]

By Thompson, Matthew V Wolniak, Stephen M

Rapid acquisition of quantitative anatomical data from the sieve tubes of angiosperm phloem has been confounded by their small size, their distance from organ surfaces, and the time-consuming nature of traditional methods, such as transmission electron microscopy. To improve access to these cells, for which good anatomical data are critical, a monomeric yellow fluorescent protein (mCitrine) was N- terminally fused to a small (approximately 6 kD) membrane protein (AtRCI2A) and stably expressed in Arabidopsis thaliana (Columbia-0 ecotype) and Nicotiana tabacum (‘Samsun’) under the control of a companion cell-specific promoter (AtSUC2p). The construct, called by its abbreviation SUmCR, yielded stable sieve element (SE) plasma membrane fluorescence labeling, even after plastic (methacrylate) embedding. In conjunction with wide-field fluorescence measurements of sieve pore number and position using aniline blue-stained callose, mCitrine-labeled material was used to calculate rough estimates of sieve tube-specific conductivity for both species. The SUmCR construct also revealed a hitherto unknown expression domain of the AtSUC2 Suc-H^sup +^ symporter in the epidermis of the cell division zone of developing root tips. The success of this construct in targeting plasma membrane-anchored fluorescent proteins to SEs could be attributable to the small size of AtRCI2A or to the presence of other signals innate to AtRCI2A that permit the protein to be trafficked to SEs. The construct provides a hitherto unique entree into companion cell-to-SE protein targeting, as well as a new tool for studying whole-plant phloem anatomy and architecture. (ProQuest: … denotes formulae omitted.)

Recent theoretical work has highlighted the lack of good quantitative, geometrical measurements of sieve tubes in the phloem vasculature of plants (Thompson and Holbrook, 2003, 2004; Thompson, 2006). The demand for such measurements is acute because quantitative anatomy determines fluid mechanical conductivity, which, in turn, sets the osmotic context for the molecular control of local solute exchange throughout the phloem. A complete understanding of phloem function is unlikely to emerge without attention to multiscale integration of processes operating at both long and short distances; anatomy is a key element of this integration.

The phloem is seldom accurately visualized from organ surfaces and is not always easily identified or measured with transmission light microscopy (Esau, 1969). Transmission electron microscopy is a potential alternative (Kuhn et al., 1997; Ehlers et al., 2000; Turgeon et al., 2001) but lacks the three-dimensional perspective needed for good quantitative measurements, it requires more time to implement than light microscopy, and it is inappropriate for statistical sampling of large amounts of tissue. Likewise, exogenously applied fluorochromes (Oparka et al., 1995; Knoblauch and van Bel, 1998; Knoblauch et al., 2001; Martens et al., 2006; Bauby et al., 2007) and sieve element (SE) specific antibodies, like RS6 (Khan et al., 2007), have proven their utility, but none of the fluorochromes is specific to the phloem and the use of antibodies is expensive and time consuming, lessening the opportunity for fast and direct observation of phloem cells alone.

Despite the time and cost of the development of stably transgenic plants, the study of phloem anatomy and architecture is likely best and ultimately most easily served by plants that express markers in phloem cells under the control of developmentally or physiologically significant promoters (Imlau et al., 1999; Stadler et al., 2005; Bauby et al., 2007). For present purposes, an ideal marker is one that fluorescently labels the plasma membranes of conductive SEs and sieve pores with an endogenous marker and allows quick measurements of the quantitative geometry of SEs (their radius and length) and sieve plates (the radius and length and number of sieve pores in each sieve plate) down to the resolution limits of light microscopy. Designing SE-specific markers in mature phloem represents a significant challenge. Membrane proteins expressed in immature SEs that are still nucleate, such as the early nodulin (ENOD)-like protein of Arabidopsis (Arabidopsis thaliana; Khan et al., 2007), are likely diluted or degraded as the SEs expand and mature, making them potentially poor targets for fluorescence tagging. Mature SEs lose the capacity for protein expression, delegating it to their sister cells, the companion cells (CCs; van Bel and Knoblauch, 2000). Thus, CC-expressed membrane-anchored proteins remain the strongest candidate for continuous SE membrane labeling in mature phloem.

To date, however, the only fluorescent proteins (FPs), FP fusions, or virus-associated FPs (Casper and Holt, 1996; Cheng et al., 2000) known to traffic from nucleate CCs to enucleate SEs are soluble, whereas membrane-anchored FPs fail to traffic at all (Imlau et al., 1999; Lalonde et al., 2003; Stadler et al., 2005; Martens et al., 2006). Past attempts at SE membrane targeting may have failed for the following reasons, which at this time remain speculative: (1) the FP interfered with signal peptides necessary for CC-to-SE plasmodesma-tal trafficking (Lalonde et al., 2003; Stadler et al., 2005); (2) the Stokes radius of the FP membrane protein fusion was greater than the size exclusion limit of the CC-to-SE plasmodesmata (Stadler et al., 2005); (3) the promoter driving expression was not strong enough to reach a threshold for plasmodesmatal trafficking (Lalonde et al., 2003); (4) cDNA clones were used that lacked the full genomic intronic and 3′-untranslated region (UTR) sequences, some of which might be important for intra- and intercellular targeting (Lalonde et al., 2003); or (5) membrane proteins were used that are not normally targeted to SEs, such as the SUT1/ SUC2 Suc- H^sup +^ symporters (Lalonde et al., 2003; Stadler et al., 2005).

Here, a variety of constructs were developed and tested in Arabidopsis with these potential limitations in mind. The strong CC- specific promoter AtSUC2p (Truernit and Sauer, 1995; Imlau et al., 1999) was used to drive FP fusions (Cutler et al., 2000) of proteins that are known to be targeted to plasma membranes (PIP subfamily of aquaporins; Fraysse et al., 2005) or are potentially small enough to sneak through the plas-modesmata as an FP fusion (AtRCI2A and AtRCI2B; Medina et al., 2001; Nylander et al., 2001). N-terminal FP fusions were employed to avoid interruption of potentially important 3′-signaling sequences in the mature mRNA, and a monomeric, environmentally stable form of yellow fluorescent protein (YFP;mCitrine) was used to avoid FP dimerization (Griesbeck et al., 2001; Zacharias et al., 2002). Where possible, cDNA clones (including 3′-UTR) and genomic clones (introns plus 3′-UTR) of each membrane protein gene were tested. At least one of these constructs was found to unequivocally and vigorously label SE plasma membranes in both Arabidopsis and tobacco (Nicotiana tabacum).

RESULTS

Isolation of Successful Transgenic Lines

Sixteen N-terminal mCitrine fusion constructs, all under the control of the AtSUC2 promoter (Table I; Fig. 1, A and B), were generated and tested in Arabi-dopsis (Columbia-0 [Col-0] ecotype). At least 20 T0 plants were screened for each of the 16 constructs, and representative plants from each were found to express mCitrine to varying degrees in the veins of mature source leaves (data not shown). Proper transformation of each construct was confirmed by reverse transcription (RT)-PCR and sequencing (Fig. 1C). In general, those constructs that contained genomic clones generated a stronger vein-level mCitrine signal, but not consistently. Both mCitrine- SUC2 lines failed to label SE membranes, which is consistent with previous results (Lalonde et al., 2003; Stadler et al., 2005). The only construct that yielded strong and consistent SE labeling in Arabidopsis was AtSUC2p::mCitrine- AtRCI2A-genomic (Figs. 2, B and E, and 3, A and B).

AtRCI2A is a gene with no known phloem function, but which appears to be generally important for drought, cold, and salt tolerance, and for abscisic acid response signaling (Nylander et al., 2001; Kroemer et al., 2004; Medina et al., 2005; Mitsuya et al., 2006; Dai et al., 2007). The gene possesses orthologs in a variety of plant species as well as three Pseudomonas species, Neurospora crassa, and Caenorhabditis elegans (Kroemer et al., 2004). For this study, the gene was selected for the small size of the protein it expresses (Fig. 1B; Table I) and because it is known to target the plasma membrane (Cutler et al., 2000). The mCitrine- RCI2A fusion is predicted to have two transmembrane domains (Nylander et al., 2001; Kroemer et al., 2004) with the relatively large barrel-can mCitrine on the cyto-plasmic face (Fig. 1D). Two strongly expressing homozygous seed lines, 19.07 and 11.07, were isolated at the T2 generation under phosphinothricin selection. Two CC-only control lines, one expressing the construct AtSUC2p::mCitrine-AtPIP1;3-cDNA (line 08) and the other the construct AtSUC2p::mCitrine-AtPIP2;2- genomic (line 33), both of which yielded strong CC expression but no SE expression, were isolated at the T1 generation.

Tobacco (‘Samsun’) was also transformed with the SUmCR construct (an abbreviation of AtSUC2p:: mCitrine-AtRCI2A-genomic). Over 20 independent transgenic lines were screened, and one line (17), which strongly expressed mCitrine in the leaf vasculature and also in the roots (Fig. 2, D and F), was selected. This line is currently in the T1 generation. mCitrine Labeling of SE Membranes

Live-cell imaging of Arabidopsis SUmCR-19.07 and tobacco SUmCR- 17 demonstrated a strong mCitrine signal in SE plasma membranes (Figs. 3, A-D, and 4, A-E and H-O). The same was demonstrated in the SE plasma membranes of thin-sectioned, methacrylate-embedded Arabidopsis inflorescence stem phloem (Fig. 3, E, F, I, and J), as well as in the membranes lining the sieve pores (Fig. 4G). SE labeling was confirmed by aniline blue counterstaining against sieve plate callose (Fig. 3, A-D). In control transgenic lines (AtPIP1;3- cDNA line 08 and AtPIP2;2-genomic line 33), mCitrine label was absent or undetectable in SE plasma membranes, despite the presence of strong CC labeling (e.g. Figs. 3, G and H, and 4F).

A space between the opposing membrane faces of individual sieve plates was regularly discernible (Figs. 3, A, C, D, and J, and 4, A- E, G, and K-N). The scattering inherent to imaging through intact, entire Arabidopsis root tissue made it impossible to resolve double membranes in root phloem sieve plates (Fig. 4, H-J). Unidentified, nonplasmalemma, mCitrine-labeled membrane structures were also labeled in the SEs of both species (Figs. 3C and 4, A, D, E, G, and L-O). The increased brightness often observed in one face of a sieve plate relative to the other could be due to surging (Knoblauch and van Bel, 1998) of these free mCitrine-labeled membrane structures following fixation or hand sectioning (Fig. 4, A, C, andM).

The faces of transversely or obliquely oriented Arabi-dopsis sieve plates were observed in both longitudinal and cross sections in methacrylate-embedded material (Fig. 3, E and I). These sieve plates were often textured in appearance, but there was no clear evidence of unoccluded or open sieve pores, possibly due to the accumulation of free, mCitrine-labeled membrane structures at the face. Thus far, the only unequivocal evidence of sieve pore mCitrine labeling comes from anti-GFP antibody AlexaFluor 555 staining of longitudinal, thin-sectioned, methacrylate-embedded Arabidopsis stem material (Fig. 4G). Such images are rare, however, making precise, quantitative measurements of sieve pore shape and size difficult to replicate in this species using this method.

The Survival of mCitrine Label in Methacrylate

Thin sectioning of methacrylate-embedded SUmCR material was first undertaken to allow immunofluo-rescent labeling of the membrane- bound mCitrine with an AlexaFluor 555-conjugated anti-GFP antibody, under the assumption that the mCitrine signal would be quenched during fixation, dehydration, and embedding. However, in addition to preserving antigenicity (Figs. 3, F and H, and 4G), the methacrylate- embedded material yielded a strong mCitrine fluorescence signal (Fig. 3, E, G, I, and J). The mCitrine identity of this signal is confirmed by the strong overlap between the mCitrine and AlexaFluor 555 signals (Fig. 3, E-H). Specificity of the antibody for mCitrine is demonstrated by the absence of AlexaFluor 555 label in the SEs of the AtSUC2p::mCitrine-PIP1;3-cDNA transgenic line, which yields an mCitrine label only in CCs (Fig. 3, G and H, as compared to Fig. 3, E and F).

Methacrylate-embedded material was mounted in Vectashield mounting medium (see “Materials and Methods”). With wide-field fluorescence microscopy, the mCitrine signal in methacrylate- embedded material appeared to be at least as photostable as the mCitrine signal in fresh material (tens of minutes). With confocal microscopy, mCitrine was bleached much more quickly in methacrylate (seconds) than in fresh material (minutes).

mCitrine Signal in Root Tips

Stadler et al. (2005) showed that GFP-membrane protein fusions under the control of the AtSUC2 promoter accumulated no further than the metaphloem CCs of developing roots (their constructs: tmGFP2 and tmGFP9). In contrast, the SUmCR Arabidopsis and tobacco plants described here demonstrate significant root tip mCitrine accumulation (Figs. 2 and 5A). Furthermore, accumulation of the mCitrine signal is specific to the epidermis of the cell division zone of the root tip (Fig. 5, B and C). This pattern of expression was observed in all SUmCR primary root tips regardless of the presence or absence of Suc in the growth medium, and was evident in emerging lateral root tips prior to the appearance of a strong lateral root phloem signal.

Analysis of SE Geometry and Estimates of Fluid Mechanical Conductivity

Thompson and Holbrook (2003) derived an equation that yields a rough estimate of the conductivity of sieve tubes, called sieve tube- specific conductivity or k ([mu]m^sup 2^), based on basic fluid mechanical principles for stationary, laminar, low Reynolds number flow:

… (1)

where N^sub p^ is the number of sieve pores per sieve plate, r ([mu]m) is SE radius, l ([mu]m) is SE length, r^sub p^ ([mu]m) is sieve pore radius, and l^sub p^ is sieve plate thickness ([mu]m; Fig. 6C).

One goal of this study was to provide image data that could be used to measure each of the geometric parameters included in Equation 1. SE radius, plate thickness, and SE length are easily measured with confocal and wide-field fluorescence images of Arabi- dopsis and tobacco SEs (Table II), but because sieve pores were either too small or occluded to be measured with an mCitrine signal, it was necessary to measure sieve pore number and radius by alternative means.

Aniline blue-stained callose in sieve plates (Fig. 6A) provided a rough means of estimating both the number and radius of sieve pores. In longitudinal sections, individual callose-filled or callose- coated pores are regularly visible as banding patterns in side views of both Arabidopsis and tobacco sieve plates, although more clearly in those of Arabidopsis. Assuming several things relevant to wide- field fluorescence microscopy- that the visible pores are those that cross the focal plane, that the depth of field is of roughly the same order as pore diameter if not thinner, that all pores are of even size and distribution, that the largest visible pore provides a measure of typical pore radius, and that the pores and plate are circular in circumference-a rough estimate of the number of pores per sieve plate can be derived in terms of parameters easily measured from the UV-excited sieve plate images (Fig. 6B):

… (2)

(See supplemental data for derivation.) This method, applied to the cartoon, 40-pore plate in Figure 6B, yields an estimate of 35 pores. The principal anticipated bias of this method is that more pores are visible than actually cross the focal plane, making Equation 2 an overestimate of N^sub p^. A single sieve pore diameter (D^sub pore^) measurement is taken from each image. Sieve pore radius is similar between the species (Table II), whereas tobacco has twice the number of pores as Arabidopsis (despite having approximately 4 times the sieve plate area).

Even the most limited datasets (Table II) can provide useful estimates of k (Eq. 1). Arabidopsis has a k value on the order of 0.28 [mu]m^sup 2^, whereas tobacco has a k value on the order of 0.45 [mu]m^sup 2^. For both species, these estimates are most sensitive to error in our measurement of sieve pore radius, r^sub p^ (Fig. 6, D and E). In Arabidopsis, k is secondarily most sensitive to errors in our measurement of SE radius, r, whereas in tobacco errors in estimates of r are less important.

DISCUSSION

Mapping Anatomy and Architecture of the Phloem

The goal of this work was to provide a fluorescent, SE plasma membrane marker for rapid studies of sieve tube anatomy and whole- plant architecture. The SUmCR construct meets this goal in both Arabidopsis and tobacco. The label is easily visualized in hand- sectioned material under wide-field fluorescence and confocal microscopy, is nonmobile (i.e. the root tip signal is limited to specific cell layers), and requires no treatment or incubation prior to visualization.

The mCitrine label can also be used in methacrylate-embedded material. It is not yet known how the mCitrine fluorophore survives fixation, dehydration, infiltration, and embedding, although it is possible that the dithiothreitol (DTT) included in each dehydration and infiltration step ameliorates quenching of the already fairly stable mCitrine barrel-can structure. GFP has been shown to survive paraformaldehyde fixation in Xenopus oocytes (Hughes et al., 2001), as well as glycol methacrylate embedding in mice (Ohta et al., 2000) and LR White embedding in zebrafish (Luby-Phelpsa et al., 2003).

The complete overlap of theAlexaFluor 555-conjugated anti-GFP antibody signal confirms the origin of the putative mCitrine signal as a GFP derivative (Fig. 3, E and F), and the absence of the AlexaFluor 555 signal from SEs in a transgenic line that lacks a SE mCitrine signal (Fig. 3, G and H) confirms that the anti-GFP antibody is unbiased toward SEs. The AlexaFluor 555 conjugate further confirms that mCitrine is localized to sieve pore membranes (Fig. 4G).

The image data presented here provide a useful platform for analyzing SE quantitative anatomy and for providing the datasets necessary to generate reasonable first-order estimates of sieve tube- specific conductivity (k). However, good data for pore radius and pore number continue to depend, at the moment, on the use of aniline blue as a pore-staining proxy (Fig. 6, A and B) because sieve pores are only occasionally discernible using the mCitrine and AlexaFluor 555 signals. This is an optical limitation made worse by the possibility that pores are occluded by free mCitrine-labeled membrane structures (e.g. Figs. 3, E and I, and 4, A and C). Available data for sieve pore radius (Esau, 1969; Thompson and Holbrook, 2003) has long indicated that the pores of most plants are quite small (<0.5 [mu]m). Notable exceptions include the sieve pores of the cucurbits and some legumes. For most species, then, pore number and radius data must be supplemented by secondary methods, such as electron microscopy or aniline blue staining. Higher resolution imaging of aniline blue-stained plates using a UV laser on a single-photon confocal microscope leads to rapid aniline blue bleaching, but imaging could be possible with lower energy multiphoton microscopy (A. Schulz, personal communication), deconvolution methods, and the pore number calculation introduced in Figure 6. The importance of improving estimates of pore radius is highlighted by the fact that it is this variable to which our calculationof conductivity ismost sensitive (Fig. 6,D and E). An additional source of error is the loss of turgor and subsequent reduction in sieve tube radius that inevitably follows fixation or hand sectioning. Sensitivity analysis of our conductivity estimate (Fig. 6, D and E) suggests that radial contraction of the sieve tube will not be as strong a source of error in tobacco as it is in Arabidopsis, but the importance of this error in either species will depend ultimately on the degree of expected radial contraction. Thompson (2005) showed that, given the high values of the drained pore modulus (or elasticity) of phloem tissue and the fact that SEs are bounded by far thicker walls than are cells in the surrounding tissue (Esau and Cheadle, 1958; Esau, 1969), the geometric expansion of sieve tubes following a change in turgor is expected to be relatively small. However, the unnatural conditions imposed by fixation and sectioning impose a far larger drop in turgor than would be expected under the physiological range of changes explored by Thompson (2005). The drained pore modulus of the phloem is known to be high relative to other tissues (in part due to the small size of most phloem cells), but values as low as 2 MPa have been reported for bulk phloem tissue (Wright and Fisher, 1983; Kallarackal and Milburn, 1985). A sieve tube with this low a modulus operating at a turgor of 1 MPa that experienced a sudden drop of turgor to 0 MPa would undergo a radial contraction of up to 40% (assuming linear elastic behavior). It is unlikely, however, given the greater thickness of SE walls, that a modulus of 2 MPa is representative of the SEs themselves. If the SE operated with a modulus of 18 MPa (which is at the high end of measured values for bulk phloem tissue), a sudden 1 MPa drop in turgor would result only in a 5% drop in radius. This implies that a 40% reduction in SE radius in Arabidopsis would mean a 50% drop in k, whereas a 5% reduction would have a negligible effect. In tobacco, where k is less sensitive to changes in r, a 40% reduction in r would only result in a 10% reduction in k. The effect on k of a 5% drop in r would also be negligible. As such, the effect of turgor reduction during tissue preparation appears to be important, but it is one-tail. Thus, any error in our measurement of r due to turgor loss during tissue preparation is expected to yield an underestimate of k.

Regardless of these difficulties, it is clear that tobacco petiole phloem possesses a slightly higher value of k than the inflorescence stem phloem of Arabidop-sis. This has important implications for the distance over which the phloem can maintain certain fluid mechanical properties, such as a small source-sink turgor difference, and the capacity to rapidly transmit changes in local turgor pressure or concentration throughout the rest of the phloem (Thompson, 2006). Plants with higher values of k can translocate sap over longer distances while still maintaining these important control properties; a plant with too low a value of k for a given translocation distance would be required to scale its local molecular control of sap solute exchange in a position-dependent manner (Thompson and Holbrook, 2004; Thompson, 2006).

mCitrine Expressed by the SUmCR Construct Accumulates in the Epidermis of Developing Root Tips

Soluble GFP expressed under the control of the AtSUC2 promoter is known to accumulate in developing root tips (Imlau et al., 1999; Stadler et al., 2005). In this study, a similar, but less intense, mCitrine signal was observed in all primary roots of SUmCR Arabi- dopsis and tobacco plants, as well as in the tips of lateral roots soon after emergence (Figs. 2 and 5). Root tip accumulation of mCitrine could be explained by posttranslational cleavage and subsequent mass transport to root tips, except that the signal is specific to the epidermis of the cell division zone basal to the root cap and is found in no other root cell layers (Fig. 5, B and C).

This pattern is strongly suggestive of an AtSUC2 promoter expression domain. Whereas no such domain has been reported previously in the literature, the promoter of a related Suc-H^sup +^ symporter, AtSUC3p, driving the expression of a membrane protein- GFP fusion, was shown to express strongly in the root tip epidermis (Meyer et al., 2004). The failure to observe a root tip epidermal expression domain in earlier work with AtSUC2p could be due the fact that a shorter, 900-bp fragment of the promoter was employed (a 900- bp fragment from plasmid pEPS1; Stadler et al., 2005), in contrast to the 2,141-bp fragment used here.

The high metabolic demands of these cells-cells that are many cell lengths away from developing protophloem-could partially explain why Suc transporters are needed near the root tip. Enhanced Suc loading in root tip epidermal cells could enhance Suc uptake in those regions that need it most, such as the first 200 to 300 mm of the Arabidopsis root tip where cell division rates are highest (Beemster and Baskin, 1998). The expression pattern was observed regardless of whether the growth medium contained Suc, suggesting that the promoter’s expression pattern is independent of rhizosphere Suc concentration.

Expression of the SUmCR Construct Yields an mCitrine Signal in SE Membranes of Both Arabidopsis and Tobacco

Past attempts to modify plants transgenically to express FP membrane protein fusions in SEs (Lalonde et al., 2003; Yoshimoto et al., 2003; Stadler et al., 2005) have resulted in retention of the fluorescence signal in CCs. At the same time, of course, a wide variety of integral membrane proteins are known and expected to be found in SEs. The target of the SE-specific antibody RS6, raised against the isolated SEs of Strep-tanthus tortuosus (Brassicaceae), was recently found in Arabidopsis to be an ENOD-like integral protein (Khan et al., 2007). Other proteins have been immu- nolocalized to SEs, such as the Suc-H^sup +^ symporter SUT1 in potato (Solanum tuberosum; Kuhn et al., 1997), a putative monosaccharide transporter and a proton ATPase in developing apple (Malus domestica) fruit (Zhang et al., 2004), a proton-amino acid transporter in Arabidopsis roots (Okumoto et al., 2004), a calcium channel-like protein in tobacco and Pistia stratiotes leaves (Volk and Franceschi, 2000), and a plasma membrane aquaporin in spinach (Spinacia oleracea; Fraysse et al., 2005).

It is unknown why the SUmCR construct labels SE membranes intensely and exclusively while others do not. At first blush, the strength of the mCitrine signal generated by the SUmCR construct relative to the larger PIP1, PIP2, and SUC2 fusions (Fig. 1; Table I), which demonstrated no SE labeling or labeling that was perhaps too faint to detect (e.g. Figs. 3, G and H, and 4F), would suggest that the principal factor is the size of the protein. Indeed, SUmCR is a smaller fusion than has been developed previously for this purpose; however, while size may be an important factor for the trafficking of soluble proteins, it is unclear how the same argument could be made for membrane-anchored protein fusions. Stadler et al. (2005) found that, whereas relatively large soluble GFP-protein fusions were capable of trafficking between CCs and SEs in Arabidopsis, GFP-integral protein fusions of any size-and even one with a smaller mass (tmGFP9) than one of the trafficked soluble protein fusions (GFP-patatin)- could not. For membrane proteins, size could be largely irrelevant up to a certain limit (Lough and Lucas, 2006). Another possibility is that the SUmCR construct simply expresses protein at a much higher level than do the other constructs; however, the others, which yield no expression in SEs, regularly exhibit the same degree of expression in CCs (e.g. Fig. 4F).

The lack of ribosomes in SEs (although there is ER) implicates two pathways for the trafficking of the mCitrine-AtRCI2A fusion between CCs and SEs. Both necessarily require the expression of the mature, membrane-anchored fusion protein in the CC followed by trafficking to the SE (Schulz, 1999). In the first case, the fusion protein diffuses from the CC plasma membrane through the membrane sleeve of the plasmo-desmatal pore unit (or PPU) directly to the SE plasma membrane. In the second case, the mature protein moves to the PPU via the CC endoplasmic reticulum (ER) and diffuses in the desmotubule membrane to the SE ER, followed by additional vesicular transport to the SE plasma membrane. The ER route seems more likely given the observed mCitrine labeling of nonplasma-lemma membrane structures, but such labeling cannot explicitly rule out the plasma membrane sleeve as an important route for CC-to-SE mCitrine-RCI2A trafficking.

In either case, it is conceivable that gating of some sort is involved (even though lipids freely move between CC and SE via PPUs; Martens et al., 2006). Additional layers of selectivity could exist in the CC ER prior to PPU trafficking. For instance, ER membrane proteins diffuse faster in the membrane than can be explained by passive diffusion alone (Runions et al., 2005), suggesting that the CC ER could be capable of actively shepherding certain membrane proteins to specific ER domains near PPUs. A determination of which suite of trafficking mechanisms predominates is beyond the scope of this study, but experimental modifications to the SUmCR construct could provide a useful entree for learning more. MATERIALS AND METHODS

Additional details of all of the methods presented below can be found in Supplemental Documentation S1.

Plant Strains and Growth Conditions

Arabidopsis (Arabidopsis thaliana) ecotype Col-0, unless otherwise noted, was cultivated in growth chambers under a 14-h day, 200 [mu]mol m^sup -2^ s^sup -1^ illumination with cool-white fluorescent bulbs, 22[degrees]C, 50% relative humidity, and in individual cells on Scott’s LC1 Sunshine soil mix. Plants were watered as needed, and when seedlings had at least six true leaves, they were fertilized by subinfiltration every 2 weeks with one-half- strength solution of 5 mM KNO^sub 3^, 2.5 mM KH^sub 2^PO^sub 4^ (adjusted to pH 5.5), 2 mM MgSO^sub 4^, 2 mM Ca(NO^sub 3^)^sub 2^, 50 [mu]M Fe-EDTA, 70 [mu]M H^sub 3^BO^sub 3^, 14 [mu]M MnCl^sub 2^, 10 [mu]M NaCl, 5 [mu]M CuSO^sub 4^, 1 [mu]M ZnSO^sub 4^, 0.2 [mu]M Na^sub 2^MoO^sub 4^, and 0.01 [mu]M CoCl^sub 2^ (following the laboratory of Dr. Pamela Green, University of Delaware). For root images, seeds were surface sterilized and germinated on Suc-less germination medium (GM/Suc-: Murashige and Skoog basal salts, 4.33 g/ L, M524; Phytotechnology Labora-tories; 1 x Gamborg vitamins, G249; Phytotechnology Laboratories; 0.5 g/L MES; pH 5.7; 0.7% technical agar). Plates were sealed with Micropore surgical tape (3M Healthcare), stratified for 4 d at 4[degrees]C, and then placed in a 14-h day, 22[degrees]C chamber at 50 [mu]Em^sup -2^ s-^sup 1^ until roots of germinated seeds had penetrated the gel surface to the bottom of the dish. Three to seven plants were then transferred to GM/Suc- plates with 1.5% technical agar. Roots were then grown along the gel surface with the plates held at a 90[degrees] angle relative to the horizontal in the same chamber. Tobacco (Nicotiana tabacum) ‘Samsun’ trans-formants were similarly treated, but with 400 [mu]mol m^sup -2^ s^sup -1^ illumination for soil-grown plants. Arabidopsis and tobacco plants were prepared for transformation as described in Supplemental Documentation S1.

Vector Construction and Plant Transformation

Vectors were prepared following standard protocols (Sambrook et al., 2001) using Escherichia coli strains DH5alpha (Hanahan, 1983) and DB3.1 (Invi-trogen), depending on the absence or presence of a GATEWAY (Invitrogen) cassette, respectively. Agrobacterium tumefaciens strain GV3101 (Holsters et al., 1980) was used for transformation of both plant species.

pGREENII-0229, a plant expression vector carrying the nos-bar (phosphi-nothricin resistance) selection marker (Hellens et al., 2000a, 2000b), was modified to contain a 2,141-bp fragment of the AtSUC2 promoter region upstream of a GATEWAY (Invitrogen) destination cassette and a t35S terminator (Fig. 1A). A GATEWAY entry vector containing a modified YFP reading frame (called mCitrine; Griesbeck et al., 2001; Zacharias et al., 2002; Shaner et al., 2005) was developed from a YFP clone provided courtesy of David Ehrhardt (Carnegie Institution of Washington) upstream of a 10-mer Ala linker followed by a small multiple cloning site (Supplemental Fig. S1). Genomic and cDNA clones of each of the test genes were amplified from genomic DNA and first-strand cDNA, respectively, and subcloned into the multiple cloning site of the entry vector (Fig. 1B). All clones contained the 3′-UTR of each gene and the genomic clones also contained the full set of introns. Entry vectors were then LR recombined using GATEWAY LR clonase II (Invitrogen) with the destination vector to make 16 mCitrine fusion constructs under the control of the AtSUC2 promoter, which were subsequently introduced into GV3101 cells for plant transformation. Detailed cloning steps are provided in Supplemental Documentation S1.

Plants were transformed following standard methods (Horsch et al., 1985; Bent et al., 1994), with modifications detailed in Supplemental Documentation S1. At least 20 Arabidopsis plants were selected per transformation, spraying with 5 mg/L phosphinothricin (i.e. Basta; Crescent Chemical Company; molecular weight 195 g/mol) and Silwet L-77, 0.02% (Lehle Seeds) four times at 4-d intervals following stratification, germination, and the emergence of the first true leaves. Selected plants were then screened for leaf minor vein-specific mCitrine expression under low magnification with a Zeiss Axioskop using a YFP-specific filter set (41028; Chroma Technology). Brightly labeled known homozygote T1 seedlings were selected and bulked into the T2 generation using the same phosphinothricin/Silwet solution.

Total RNA was isolated from each of the expressing Arabidopsis lines. First-strand cDNA was generated from each using a poly-T primer and SuperScript III (Invitrogen) reverse transcriptase. PCR primers were designed to amplify fragments between 200 and 250 bp spanning the Ala linker of each line using a gene-specific reverse primer against a mCitrine-based forward primer, mC-FW(rtpcr). Fragments were TA-cloned and sequenced to confirm construct identity.

Transformed tobacco plants were selected on 8 mg/L phosphinothricin in medium (as described in Supplemental Documentation S1), cultivated as above, and screened with wide- field fluorescent microscopy using mature source leaves as with Arabidopsis.

Imaging

Arabidopsis stem inflorescence stems were glued to a glass microscope slidewith amedical adhesive (Hollister) and then finely hand sectionedwith a sharp blade. Stem strips were stained with 0.01% aqueous aniline blue for 10 min in the dark, and then rinsed, mounted in 10 mM KCl, 10 mM CaCl2 , and 5 mM NaCl, and imaged. The petioles and midribs of mature tobacco source leaves were treated as with Arabidopsis stems, but care was taken to keep track of external versus internal petiole phloem. Undisturbed Arabidopsis root phloem could be imaged through intact tissue with inevitable scattering.

Fresh and sectioned material was imaged using wide-field fluorescence with a Zeiss Axioskop using a YFP-specific filter set (as above) for mCitrine labeling, a narrow-band DAPI/Hoechst/AMCA filter set (31013v2; Chroma) for aniline blue, or a TRITC (Rhodamine)/DiI/Cy3 filter set (31002; Chroma) for the AlexaFluor 555 conjugate dye.

Fresh and sectioned material was imaged with confocal laser- scanning microscopy using a Zeiss LSM 510 confocal microscope with a 488-nm argon excitation band with a 505- to 550-nm emission band- pass filter for mCitrine imaging, and a 543 He-Ne excitation band with a 560- to 615-nm emission band-pass filter for AlexaFluor 555 imaging.No UV laser was available on this microscope for aniline blue confocal imaging.

Material for methacrylate embedding (Baskin et al., 1992, 1996; Baskin and Wilson, 1997) was fixed in a pH 7.0, 4% paraformaldehyde buffer, dehydrated in ethanol with 1mM DTT, and infiltrated in amethacrylate:ethanol series. The methacrylate solution contained 80% n-butyl methacrylate, 20% methyl methacrylate, 5 g/L benzoin ethyl ether catalyst (12,100, 18,800, and 11,280, respectively; Electron Microscopy Sciences), and 10 mM DTT. The series was performed in flat-bottomed embedding capsules (70021; Electron Microscopy Sciences). The methacrylate resin was cured with UV light at 4[degrees]C for 5 h. Sections from embedded material were chloroform and acetone etched (Baskin et al., 1992; Klink and Wolniak, 2001) and mounted in Vectashield (Vector Labs) under cover glasses sealed with nail polish, or etched and stained with a 1:200 dilution of anti-GFP antibody AlexaFluor 555 conjugate (A-31851; Molecular Probes) in phosphate-buffered saline for 2 h at 37[degrees]C in the dark, and then mounted.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Diagram of Ala-10 linker region used in each expression vector.

Supplemental Table S1. List of oligonucleotides used in this study.

Supplemental Documentation S1. Detailed description of vector construction and plant transformation, and a derivation of the sieve pore number equation (Eq. 2).

ACKNOWLEDGMENTS

At the University of Maryland, we thank Amy Beaven of the Visual Imaging Center in the Department of Cell Biology and Molecular Genetics for support with the Zeiss LSM 510 confocal microscope and DNA sequencers, Dr. Gary Coleman and Kuang-Yu Chen for laboratory space and assistance near the growth chambers in the Department of Plant Science and Landscape Architecture, Dr. James Culver of the Center for Biosystems Research for additional tissue culture growth chamber space, Shaun Faulkner for growth chamber management, Elizabeth Halliday and Charles Allen for Arabidopsis and tobacco culture and growth maintenance, Michele Cox for tobacco screening, and the laboratories of Dr. June Kwak, Dr. Heven Sze, Dr. Caren Chang, Dr. Stephen Mount, and Dr. Zhongchi Liu of the Department of Cell Biology and Molecular Genetics for other advice and assistance. The pGREENII backbone was used with the permission of the John Innes Centre, Norwich, UK, and with the kind assistance of Dr. Roger P. Hellens, HortResearch, New Zealand. Our template YFP clone was provided by Dr. David Ehrhardt, Carnegie Institute of Washington, Department of Plant Biology. Special thanks for the patience and advice of Dr. Joe Chappell, Dr. Shunji Takahashi, and Dr. Shuiqin Wu of the Plant and Soil Sciences Department, University of Kentucky, without which none of this would be possible. We further thank Robert Turgeon, Helle Martens, Alexander Schultz, and two anonymous reviewers for helpful comments on the manuscript.

Received November 15, 2007; accepted January 20, 2008; published January 25, 2008.

1 This work was supported by the National Science Foundation (grant no. IOS-0641111 to M.V.T. and grant nos. MCB-0234423 and MCB- 0720486 to S.M.W.). [W] The online version of this article contains Web-only data.

[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.113274

LITERATURE CITED

Baskin TI, Busby CH, Fowke LC, Sammut M, Gubler F (1992) Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa. Planta 187: 405-413

Baskin TI, Miller DD, Vos JW, Wilson JE, Hepler PK (1996) Cryofixing single cells and multicellular specimens enhances structure and immu-nohistochemistry for light microscopy. J Microsc 182: 149-161

Baskin TI,Wilson JE (1997) Inhibitors of protein kinases and phosphatases alter root morphology and disorganize cortical microtubules. Plant Physiol 113: 493-502

Bauby H, Divol F, Truernit E, Grandjean O, Palauqui JC (2007) Proto-phloem differentiation in early Arabidopsis thaliana development. Plant Cell Physiol 48: 97-109

Beemster GTS, Baskin TI (1998) Analysis of cell division and elongation underlying developmental acceleration of root growth in Arabidopsis thaliana. Plant Physiol 116: 1515-1526

Bent AF, Kunkel BN, Dahlbeck D, Schmidt R, Giraudet J, Leung J, Staskawicz BJ (1994) RPS2 of Arabidopsis thaliana: a leucine-rich repeat class of plant disease resistance genes. Science 265: 1856- 1860

Casper SJ, Holt CA (1996) Expression of the green fluorescent protein-encoding gene from a tobacco mosaic virus-based vector. Gene 173: 69-73

Cheng NH, Su CL, Carter SA, Nelson RS (2000) Vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in Nicotiana benthamiana. Plant J 23: 349-362

Cutler SR, Ehrhardt DW, Griffitts JS, Somerville CR (2000) Random GFP:cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proc Natl Acad Sci USA 97: 3718-3723

Dai XY, Xu YY, Ma QB, Xu WY, Wang T, Xue YB, Chong K (2007) Overexpression of an R1R2R3 MYB gene, OsMYB3R-2, increases tolerance to freezing, drought, and salt stress in transgenic Arabidopsis. Plant Physiol 143: 1739-1751

Ehlers K, Knoblauch M, van Bel AJE (2000) Ultrastructural features of well-preserved and injured sieve elements: minute clamps keep the phloem transport conduits free for mass flow. Protoplasma 214: 80-92

Esau K (1969) The Phloem. Handbuch der Pflanzenanatomie Histologie, Band V, Teil 2. Gebruder Borntraeger, Berlin-Stuttgart, Germany

Esau K, Cheadle VI (1958) Wall thickening in sieve elements. Proc Natl Acad Sci USA 44: 546-553

Fraysse LC, Wells B, McCann MC, Kjellbom P (2005) Specific plasma membrane aquaporins of the PIP1 subfamily are expressed in sieve elements and guard cells. Biol Cell 97: 519-534

Griesbeck O, Baird GS, Campbell RE, Zacharias DA, Tsien RY (2001) Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J Biol Chem 276: 29188-29194

Hanahan D (1983) Studies on transformation of E. coli with plasmids. J Mol Biol 166: 557-580

Hellens R, Edwards EA, Leyland NR, Bean S, Mullineaux P (2000a) pGreen: a versatile and flexible binary Ti vector for Agrobacterium- mediated plant transformation. Plant Mol Biol 42: 819-832

Hellens R, Mullineaux P, Klee H (2000b) A guide to Agrobacterium binary Ti vectors. Trends Plant Sci 5: 446-451

Holsters M, Silva B, Van Vliet F, Genetello C, Deblock M, Dhaese P, Depicker A, Inze D, Engler G, Villarroel R, et al (1980) The functional organization of the nopaline Agrobacterium tumefaciens plasmid pTiC58. Plasmid 3: 212-230

Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227: 1229-1231

Hughes TE, Zhang H, Logothetis DE, Berlot CH (2001) Visualization of a functional Gaq-green fluorescent protein fusion in living cells. J Biol Chem 276: 4227-4235

Imlau A, Truernit E, Sauer N (1999) Cell-to-cell and long- distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein in sink tissues. Plant Cell 11: 309-322

Kallarackal J, Milburn JA (1985) Phloem sap exudation in Ricinus communis: elastic responses and anatomical implications. Plant Cell Environ 8: 239-245

Khan JA, Wang Q, Sjolund RD, Schulz A, Thompson GA (2007) An early nodulin-like protein accumulates in the sieve element plasma membrane of Arabidopsis. Plant Physiol 143: 1576-1589

Klink VP, Wolniak SM (2001) Centrin is necessary for the formation of the motile apparatus in spermatids of Marsilea. Mol Biol Cell 12: 761-776

Knoblauch M, Peters WS, Ehlers K, van Bel AJE (2001) Reversible calcium-regulated stopcocks in legume sieve tubes. Plant Cell 13: 1221-1230

Knoblauch M, van Bel AJE (1998) Sieve tubes in action. Plant Cell 10: 35-50

Kroemer K, Reski R, Frank W (2004) Abiotic stress response in the moss Physcomitrella patens: evidence for an evolutionary alteration in signaling pathways in land plants. Plant Cell Rep 22: 864-870

Kuhn C, Franceschi VR, Schulz A, Lemoine R, Frommer WB (1997) Macromolecular trafficking indicated by localization and turnover of sucrose transporters in enucleated sieve elements. Science 275: 1298- 1300

Lalonde S, Weise A, Walsh RP, Ward JM, Frommer WB (2003) Fusion to GFP blocks intercellular trafficking of the sucrose transporter SUT1 leading to accumulation in companion cells. BMC Plant Biol 3: 8

Lough TJ, Lucas WJ (2006) Integrative plant biology: role of phloem long-distance macromolecular trafficking. Annu Rev Plant Biol 57: 203-232

Luby-Phelpsa K, Ninga G, Fogertya J, Besharsea JC (2003) Visualization of identified GFP-expressing cells by light and electron microscopy. J Histochem Cytochem 51: 271-274

Martens HJ, Roberts AG, Oparka KJ, Schulz A (2006) Quantification of plasmodesmatal endoplasmic reticulum coupling between sieve elements and companion cells using fluorescence redistribution after photobleaching. Plant Physiol 142: 471-480

Medina J, Catala R, Salinas J (2001) Developmental and stress regulation of RCI2A and RCI2B, two cold-inducible genes of arabidopsis encoding highly conserved hydrophobic proteins. Plant Physiol 125: 1655-1666

Medina J, Rodriguez-Franco M, Penalosa A, Carrascosa MJ, Neuhaus G, Salinas J (2005) Arabidopsis mutants deregulated in RCI2A expression reveal new signaling pathways in abiotic stress responses. Plant J 42: 586-597

Meyer S, Lauterbach C, Niedermeier M, Barth I, Sjolund RD, Sauer N (2004) Wounding enhances expression of AtSUC3, a sucrose transporter from Arabidopsis sieve elements and sink tissues. Plant Physiol 134: 684-693

Mitsuya S, Taniguchi M, Miyake H, Takabe T (2006) Overexpression of RCI2A decreases Na1 uptake and mitigates salinity-induced damages in Arabidopsis thaliana plants. Physiol Plant 128: 95-102

Nylander M, Heino P, Helenius E, Palva ET, Ronne H, Welin BV (2001) The low-temperature and salt-induced RCI2A gene of Arabidopsis complements the sodium sensitivity caused by a deletion of the homologous yeast gene SNA1. Plant Mol Biol 45: 341-352

Ohta H, Yomogida K, Dohmae K, Nishimune Y (2000) Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF. Development 127: 2125-2131

Okumoto S,KochW, Tegeder M, FischerWN, Biehl A, Leister D, Stierhof YD, Frommer WB (2004) Root phloem-specific expression of the plasma membrane amino acid proton co-transporter AAP3. J Exp Bot 55: 2155-2168

Oparka KJ, Prior DAM, Wright KM (1995) Symplastic communication between primary and developing lateral roots of Arabidopsis thaliana. J Exp Bot 46: 187-197

Rhee SY, Beavis W, Berardini TZ, Chen G, Dixon D, Doyle A, Garcia Hernandez M, Huala E, Lander G, Montoya M, et al (2003) The Arabidopsis Information Resource (TAIR): a model organism database providing a centralized, curated gateway to Arabidopsis biology, research materials and community. Nucleic Acids Res 31: 224-228

Runions J, Brach T, Kuhner S, Hawes C (2005) Photoactivation of GFP reveals protein dynamics within the endoplasmic reticulum membrane. J Exp Bot 57: 43-50

Sambrook J, MacCallum P, Russell D (2001) Molecular Cloning: A Laboratory Manual, Ed 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

Schulz A (1999) Physiological control of plasmodesmal gating. In AJE van Bel, WJP van Kesteren, eds, Plasmodesmata: Structure, Function, Role in Cell Communication. Springer, New York, pp 173- 204

Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2: 905-909

Stadler R, Wright KM, Lauterbach C, Amon G, Gahrtz M, Feuerstein A, Oparka KJ, Sauer N (2005) Expression of GFP-fusions in Arabidopsis companion cells reveals non-specific protein trafficking into sieve elements and identifies a novel post-phloem domain in roots. Plant J 41: 319-331

Thompson MV (2005) Scaling phloem transport: elasticity and pressure-concentration waves. J Theor Biol 236: 229-241

Thompson MV (2006) Phloem: the long and the short of it. Trends Plant Sci 11: 26-32

Thompson MV, Holbrook NM (2003) Scaling phloem transport: water potential equilibrium and osmoregulatory flow. Plant Cell Environ 26: 1561-1577

Thompson MV, Holbrook NM (2004) Scaling phloem transport: information transmission. Plant Cell Environ 27: 509-519

Truernit E, Sauer N (1995) The promoter of the Arabidopsis thaliana SUC2 sucrose-H1 symporter gene directs expression of b- glucuronidase to the phloem: Evidence for phloem loading and unloading by SUC2. Planta 196: 564-570

Turgeon R, Medville R, Nixon KC (2001) The evolution of minor vein phloem and phloem loading. Am J Bot 88: 1331-1339

van Bel AJE, Knoblauch M (2000) Sieve element and companion cell: the story of the comatose patient and the hyperactive nurse. Aust J Plant Physiol 27: 477-487

Volk GM, Franceschi VR (2000) Localization of a calcium channel- like protein in the sieve element plasma membrane. Aust J Plant Physiol 27: 779-786

Wright JP, Fisher DB (1983) Estimation of the volumetric elastic modulus and membrane hydraulic conductivity of willow sieve tubes. Plant Physiol 73: 1042-1047 Yoshimoto N, Inoue E, Saito K, Yamaya T, Takahashi H (2003) Phloem-localizing sulfate transporter, Sultr1;3, mediates re-distribution of sulfur from source to sink organs in Arabidopsis. Plant Physiol 131: 1511-1517

Zacharias DA, Violin JD, Newton AC, Tsien RY (2002) Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296: 913-916

Zhang LY, Peng YB, Pelleschi-Travier S, Fan Y, Lu YF, Lu YM, Gao XP, Shen YY, Delrot S, Zhang DP (2004) Evidence for apoplasmic phloem unloading in developing apple fruit. Plant Physiol 135: 574- 586

Matthew V. Thompson* and Stephen M. Wolniak

Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742

* Corresponding author; e-mail mvthom@umd.edu.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Matthew V. Thompson (mvthom@umd.edu).

Copyright American Society of Plant Biologists Apr 2008

(c) 2008 Plant Physiology. Provided by ProQuest Information and Learning. All rights Reserved.