A New Method for Direct Introduction of Chemicals into a Single

Plant Cell Physiol. 41(1): 124-128 (2000)JSPP © 2000

A New Method for Direct Introduction of Chemicals into a Single Sieve Tubeof Intact Rice Plants

Shu Fujimaki!> 3, Toru Fujiwaral> 2 and Hiroaki Hayashi*1 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo,

113-8657 Japan2 PRESTO, JST, Japan

We present a novel method for direct introduction ofchemicals into a sieve tube of intact rice plants—namely,the method of micro-introduction using stylet of insect (theMUSI method). Fluorescent dyes were successfully in-troduced into a sieve tube through a severed stylet ofplanthopper and the distribution of the dyes was observed.

Key words: Insect stylectomy — Microinjection — Micro-introduction — Rice {Oryza sativa L.) — Sieve tube.

The sieve tubes of phloem tissue in higher plants,which are responsible for long-distance transport of nutri-ents, have been often compared to the blood vessel systemof animals. Recently, a new hypothesis has been raised thatthe phloem is also "an information superhighway" thatenables the inter-tissue/-organ trafficking of informationmolecules (Lucas and Wolf 1999). Therefore, the studieson the dynamics of various molecules which are loadedinto, functioning in, translocated through and unloadedfrom the sieve tubes are becoming more important. Amethod that allows direct introduction of chemicals into asieve tube should provide us with great possibilities onthese studies as well as an injection into the blood vessel ofanimals and humans. To our knowledge, two microinjec-tion methods feasible for such direct introduction into asingle sieve tube have been published, one with excisedtissues containing phloem (van der Schoot and van Bel1989, Pradel et al. 1996, Botha and Cross 1997), and theother with a leaf attached to an intact plant (Rhodes et al.1996, Knoblauch and van Bel 1998). Both of these methodsrequire the removal of hindering tissues (e.g., epidermis)prior to the microinjection. There have been no reports onthe insertion of a glass capillary to sieve tubes without suchpretreatments. A sieve tube is very narrow (approx. 10 /j.min diameter in rice, for example), and it runs under thelayers of cells with a thick cell wall. These features make itdifficult to identify the sieve tubes microscopically, and tolocate and insert a capillary into the sieve tube without

Abbreviations: FITC, fluorescein isothiocyanate; TMR,tetramethylrhodamine.3 To whom correspondence should be addressed. Fax: + 81-3-5841-8032; E-mail: [email protected].

removal of tissues.Brown planthopper is a phloem sap-feeding insect,

whose feeding process can be considered as a natural"reverse microinjection" (Lucas 1997). It was applied tothe insect laser technique, with which pure phloem sap ofrice is collected through the severed insect stylet that hasbeen inserted into a sieve tube (Kawabe et al. 1980). Ricephloem sap has been collected by this technique andits chemical composition (Fukumorita and Chino 1982,Hayashi and Chino 1985, 1990), proteins (Nakamura et al.1993, Ishiwatari et al. 1995) and mRNAs (Sasaki et al.1998) have been well analyzed.

Here, we present a novel method for direct introduc-tion of chemicals into a sieve tube of intact rice plants. Thismethod, the method of micro-introduction using stylet ofinsects (the MUSI method), is the reverse application of theinsect stylectomy—not to collect phloem sap, but to in-troduce solutions into a sieve tube through the severedstylet. Thus, neither excision nor removal of tissues is re-quired prior to the introduction.

Materials—Rice {Oryza sativa L. cv. Kantou) plantswere grown on hydroponic culture as described earlier(Nakamura et al. 1993). Five weeks after sowing, plantswere transplanted onto the hydroponic culture solution inflasks and used for the experiments. The buffer for themixed dye solution was prepared to have a similar com-position to that of rice phloem sap (Fukumorita and Chino1982). The buffer contained 0.9 M sucrose, 30 mM asparticacid, 50 mM asparagine, 20 mM glutamic acid, and 40 mMglutamine in distilled water. The pH was adjusted to 13with KOH. The buffer was sterilized by nitration. Fluores-cein isothiocyanate (FITC)-dextran (42 kDa) (Sigma, St.Louis, MO, U.S.A.) was nitrated with a centrifugal filterunit with a nominal molecular weight limit of 30,000(UFC3LTK; Millipore, Bedford, MA, U.S.A.) to removelow molecular weight dyes. Similarly, tetramethylrhoda-mine (TMR)-dextran (3 kDa) (Molecular Probes, Eugene,OR, U.S.A.) was gel-filtrated with Sephadex G-25 Fine(Pharmacia Biotech, Uppsala, Sweden). These dyes weredried and added to the buffer to make the mixed dye so-lution containing 3% (w/v) of FITC-dextran and 1.5%(w/v) of TMR-dextran, at the final concentration.

Stylectomy and dye-application—All procedures ofstylectomy and dye-application were carried out in a roomat 25°C, and 60% relative humidity. Small cages, approx.

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The MUSI method 125

2 cm in height and 1 cm in diameter, made of plastic wrapand kitchen paper were attached to leaf sheaths of each riceplant. Two brown planthoppers (Nilaparvata lugens STAL)were put into each cages, and allowed to feed on thephloem sap for 1 to 2 h. Then the stylets were severed witha YAG laser beam. These procedures of stylectomy weredescribed in detail by Kawabe et al. (1980) and Nakamuraet al. (1993). After droplets were formed on some severedstylets, they were confirmed to be phloem sap by checkingthat their pH was around 8.0 (Fukumorita and Chino 1982)with pH test paper. Roughly the same volume (around 0.5//I) of the mixed dye solution was applied onto the dropletswith a micropipet (Fig. 1). These plants were left for a fewhours or more until the droplets appeared dried out andsolidified.

Microscopy and transverse sectioning—Each plantwas removed from the flask, and its root was covered withplastic wrap to keep wet. The droplet of phloem sap withthe dyes on the surface of the leaf was rinsed well withdistilled water. The precise point of the severed stylet wasidentified under a fluorescence microscope, and imagesfrom the surface were obtained through a video camera(see below). For the observation of transverse sections, apart of the leaf sheath (approx. 2 cm long) including thesevered stylet was excised and mounted in liquid agar (ap-prox. 45°C; 5% (w/v) of agar powder (Nacalai Tesque,Kyoto, Japan) in distilled water) in an aluminum tube, andit was cooled on ice immediately. Then this agar block wassliced with a razor blade manually to make sections ap-prox. 1 mm in thickness, and they were subjected to mi-croscopy.

All the equipment for microscopy was purchased fromOlympus (Tokyo, Japan). A video camera (M-3204C) isattached to the fluorescence microscope system (BX50WI,BX-FLA) to obtain the microscopic images. The light fillercubes used for the fluorescence microscopy shown in thefigures are as follows. Fig. 2a, 3b: U-MWIBA (460-490 nmband path filter for excitation of FITC, 515-550 nm filterfor specific detection of FITC). Fig. 2b, 3c: U-MWIG(520-550 nm band path filter for excitation of TMR, 580-nm filter for specific detection of TMR). Fig. 3a: U-MWU(330-385 nm band path filter for excitation, 420-nm filterfor observation). To make each image of Figure 2, we ob-tained eight images in the same field of view, adjusted theirbrightness and combined them on a computer with Pho-toshop software (Adobe Systems, San Jose, CA, U.S.A.).

Movement of applied dyes—In some of the dye-ap-plied leaf sheaths, fluorescent signals of FITC and TMRwere found extended bidirectionally from the remainingsevered stylet into the vascular bundle. Figure 2 shows anexample with a mature leaf. Both dyes moved into thevascular bundle a few millimeters from the point of thesevered stylet. In general, the length of a sieve element isseveral times of its width (Parthasarathy 1975). Given that

the width of a sieve element in this tissue is 20 jum at most(Fig. 3a), the range of extension of the dyes may include tensieve elements at least (Fig. 2a, b). These images were ob-tained from the direct observation of the intact leaf; thus,the dye-movement did not result from artifacts producedduring the preparation for the microscopy.

To answer the question whether the dyes movedthrough sieve tube(s) or xylem vessel(s) in the vascularbundle, transverse sections of the same sample were made.A transverse section showed that the fluorescence of FITCwas detected predominantly in a single cell in the phloemregion of the large vascular bundle, but no fluorescence ofFITC was detected in the xylem region (Fig. 3b). Thus, weconsider that the cell with the fluorescence was a sieve ele-ment, and FITC-dextran moved through the single sievetube.

In addition, this cell was one of the largest cells in thephloem region and was located in the middle of there(Fig. 3a, b), which may support the identification that thiswas a sieve element. Chonan et al. (1984) observed themiddle of the phloem region of a large vascular bundle ofa rice mature leaf sheath by electron microscopy, andidentified all the large cells as sieve elements and the otherneighboring small cells as companion cells. Ishiwatari et al.(1998) also showed the same region of a transverse sectionwith the same identifications of cells.

From these observations, we conclude that a newmethod for direct introduction of chemicals into a singlesieve tube of an intact rice plant is developed.

Selective distribution of dyes depending on mol wt—Figures 3b and 3 c indicate that the TMR-dextran (3 kDa)passed the cell wall and moved transversely from the sieveelement to neighboring cells, but FITC-dextran (42 kDa)did not. These dyes were introduced into the same sieveelement as a mixture; thus, this differential movement mustbe attributed to the difference in the molecular size of thedyes. Sieve elements in rice leaf sheaths have symplasticconnections by plasmodesmata only with companion cells(Chonan et al. 1984). Therefore, these data suggest that thesize exclusion limit between a sieve element and a com-panion cell is between 3 and 42 kDa in mature rice leafsheaths.

Hypotheses for mechanism of dye-entrance—Howcould these dyes move into a sieve tube against the highpressure? We suppose that they moved into a sieve tube bysimple diffusion because we did not give them any drivingforce like pressure. For this to be true, the rate of thediffusion has to exceed that of the exuding flow. Whenphloem sap is collected by the insect stylectomy with a glasscapillary in our laboratory, the exudation often continuesfor a few hours and then finally stops (data not shown).This fact suggests that it often takes a few hours for thesieve pores near the inserted point to be completely closed.In Figures 2a and 2b, the movement of the dyes (especially


126 The MUSI method

YAG laser beam

Stylet

Fluorescent Dye

Planthopper

ice Leafve Tube

Fig. 1 A schematic diagram of the MUSI method.

Fig. 2 The surface view of an intact leaf sheath to which themixed dye solution was applied by the MUSI method. The cubesfor specific detection of FITC (a) and TMR (b) were used for theobservation. The white arrow indicates the remaining severedstylet. Bar = 100//m.

Fig. 3 A transverse section of the leaf sheath shown in Fig. 2. The dye-applied vascular bundle is magnified. These images are in thesame field of view, (a): UV light excitation (autofluorescence of the cell wall), (b): Fluorescence of FITC. (c): Fluorescence of TMR. Thered fluorescence in the phloem region in (a) is due to the large amount of TMR-dextran. SE, sieve element; XV, xylem vessel; MS,mestome sheath cell; BS, bundle sheath cell. Bar


The MUSI method 127

of FITC-dextran with a larger molecular size) seems to bepartially obstructed at some sieve plates. Thus, we supposethat the sieve pores gradually got narrower after theseverance of the stylet on some sieve plates near the in-serted point, which gradually decreased the pressure andthe rate of the exuding flow. When the flow rate becamesufficiently slow and the sieve pores were not completelyclosed yet, the dyes might begin to enter the sieve tube bydiffusion.

Unlike the insect stylectomy mentioned above, thedroplets of phloem sap with dyes often dried out easily onthe MUSI method, probably because nothing like a col-lecting capillary was put onto them. Because of the highconcentration of sucrose in the phloem sap and the dyesolution, the surface of the droplets solidified. There was acase that by rinsing out the solidified droplet, the phloemsap began to exude again even eight hours after the sever-ance of the stylet (data not shown). This suggests that thesolidified surface can stop the exuding flow against thepressure from the sieve tube and it also played a pivotalrole to enable the dyes to diffuse toward the sieve tube.

Merits of the MUSI method—First, it provides directintroduction of chemicals into a sieve tube. There existseveral indirect introducing methods. For instance, foliarspraying is feasible to introduce phloem-mobile chemicals,such as some insecticides, into the phloem. A protein ofinterest can be introduced into the sieve tubes by makingtransgenic plants that express the gene of the protein intheir companion cells (Fukuda et al. 1999). However, thedirect methods (i.e., the MUSI method and the microin-jection methods) have many advantages from the indirectones. For example, (1) even high molecular weight molec-ules can be introduced. (2) More than two chemicals can beintroduced simultaneously (Fig. 2a, b, 3b, c). (3) The ex-periment can be finished in a short term. In the case of theMUSI method, once the plants and the insects are pre-pared, the injection will be finished in one day. (4) Chem-icals are introduced to a very small area. This is essential totrace the fine movement of the chemical of interest in asieve tube.

Secondly, it can be easily done (even though it requireskeeping insects). Unlike the existing microinjection tech-niques, the MUSI method does not require microscopicalidentification of a sieve tube before the application of so-lutions. It is widely accepted that an insect identifies a sievetube and inserts its own microtip through thick-walled ep-idermis all by itself. The ratio between the success andfailure of trials with the MUSI method varies dependingon the conditions of the insects and plants. In our recentfour trials, the ratio of (number of the cases with success-ful dye-introduction)/(number of the planthoppers used)ranged from 1/44 to 4/38. Fisher (unpublished data) hasshown a method to introduce a solution into a single sieveelement via a stylet of aphid by pressure injection. How-

ever, it would be technically more difficult than the MUSImethod because it requires fixing a glass microcapillaryonto the severed stylet with an adhesive agent under a mi-croscope.

Lastly, this method gives little damage to the plant.Excision of tissues is not necessary.

All of the molecules detected in the rice phloem sapcollected by the insect stylectomy can be introduced into asieve tube by the MUSI method. Introduction of xeno-biotics such as dextran dyes would also be a good meansfor the studies on the phloem.

We expect this method to be improved and appliedwidely to other plants in which the insect stylectomy (e.g.,aphid technique) is available, and to shed light on unsolvedquestions about the phloem.

This study is supported in part by grants from the Ministry ofEducation, Science, Sports and Culture, Japan (Grant-in-Aid forScientific Research on Priority Areas No. 09274104 and No.09274102) and The Ministry of Agriculture, Forestry and Fisheriesof Japan (Development of novel weed control technology by ap-plying metabolic genes in plant). We also thank Dr. ShigenoriMorita (Univ. of Tokyo) for his helpful advice on the morpho-logical identification of sieve elements.

References

Botha, C.E.J. and Cross, R.H.M. (1997) Plasmodesmatal frequency inrelation to short-distance transport and phloem loading in leaves ofbarley (Hordeum vulgare). Phloem is not loaded directly from thesymplast. Physiol. Plant. 99: 355-362.

Chonan, N., Kawahara, H. and Matsuda, T. (1984) Ultrastructure ofvascular bundles and fundamental parenchyma in relation to movementof photosynthate in leaf sheath of rice. Jpn. J. Crop Sci. 53: 435-444.(In Japanese with English summary and figure legends)

Fukuda, A., Ishiwatari, Y., Abe, K., Chino, M., Fujiwara, T. andHayashi, H. (1999) Control of protein content in the rice phloem sap. InPlant Nutrition—Molecular Biology and Genetics. Edited by Gissel-Nielsen, G. and Jensen, A. pp. 39-45. Kluwer Academic Publishers,Netherlands.

Fukumorita, T. and Chino, M. (1982) Sugar, amino acid and inorganiccontents in rice phloem sap. Plant Cell Physiol. 23: 273-283.

Hayashi, H. and Chino, M. (1985) Nitrate and other anions in the ricephloem sap. Plant Cell Physiol. 26: 1319-1327.

Hayashi, H. and Chino, M. (1990) Chemical composition of phloem sapfrom uppermost internode of the rice plant. Plant Cell Physiol. 31:247-251.

Ishiwatari, Y., Fujiwara, T., McFarland, K.C., Nemoto, K., Hayashi, H.,Chino, M. and Lucas, W.J. (1998) Rice phloem thioredoxin h has thecapacity to mediate its own cell-to-cell transport through plasmodesma-ta. Planta 205: 12-22.

Ishiwatari, Y., Honda, C , Kawashima, I., Nakamura, S., Hirano, H.,Mori, S., Fujiwara, T., Hayashi, H. and Chino, M. (1995) Thioredoxinh is one of the major proteins in rice phloem sap. Planta 195: 456-463.

Kawabe, S., Fukumorita, T. and Chino, M. (1980) Collection of ricephloem sap from stylets of homopterous insects severed by YAG laser.Plant Cell Physiol. 21: 1319-1327.

Knoblauch, M. and van Bel, A. J.E. (1998) Sieve tubes in action. Plant Cell10: 35-50.

Lucas, W.J. (1997) Application of microinjection techniques to plant nu-trition. Plant Soil 196: 175-189.

Lucas, W.J. and Wolf, S. (1999) Connections between virus movement,macromolecular signaling and assimilate allocation. Curr. Opin. Plant


128 The MUSI method

Biol. 2: 192-197.Nakamura, S., Hayashi, H., Mori, S. and Chino, M. (1993) Protein

phosphorylation in the sieve tubes of rice plants. Plant Cell Physiol. 34:927-933.

Parthasarathy, M.V. (1975) Sieve-Element Structure. In Transport inPlants I: Phloem Transport, Encyclopedia of Plant Physiology NewSeries Volume 1. Edited by Zimmermann, M.H. and Milburn, J.A. pp.3-38. Springer-Verlag, Berlin.

Pradel, K.S., Rezmer, C , Krausgrill, S., Rausch, T. and Ullrich, C.I.(1996) Evidence for symplastic phloem unloading with concomitant highactivity of acid cell wall invertase in Agrobacterium tumefaciens-induced

plant tumors. Bot. Ada 109: 397-404.Rhodes, J.D., Thain, J.F. and Wildon, D.C. (1996) The pathway for

systemic electrical signal conduction in the wounded tomato plant.Planta 200: 50-57.

Sasaki, T., Chino, M., Hayashi, H. and Fujiwara, T. (1998) Detection ofseveral mRNA species in rice phloem sap. Plant Cell Physiol. 39: 895-897.

van der Schoot, C. and van Bel, A.J.E. (1989) Glass microelectrodemeasurements of sieve tube membrane potentials in internode discs andpetiole strips of tomato (Solanum lycopersicum L.). Protoplasma 149:144-154

(Received September 22, 1999; Accepted November 4, 1999)


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A New Method for Direct Introduction of Chemicals into a Single