Planta (1989)179:506-515 Planta 9 Springer-Verlag 1989 A phloem-specific, lectin-like protein is located in pine sieve-element plastids by immunocytochemistry Alexander Schulz 1, M. Carol Alosi 2 ,, Dinkar D. Sabnis 3, and Roderic B. Park 2 1 Zellenlehre der Universitfit Heidelberg, Im Neuenheimer Feld 230, D-6900 Heidelberg, Federal Republic of Germany 2 Department of Botany, University of California, Berkeley, CA 94720, USA, and 3 Department of Plant Science, University of Aberdeen, Aberdeen AB9 2UD, UK Abstract. Two co-purifying phloem polypeptides of 24 and 25 kilodaltons (kDa) were isolated from ho- mogenates ofPinus sabiniana Dougl. phloem by dif- ferential centrifugation, selective solubilization and electrophoresis, and rabbit antibodies raised against them. The antisera were found to be specific for doublet bands between 23 and 25 kDa in Western blots of whole phloem extracts of Pinus species; no xylem polypeptides were labelled, nor did labelling occur in blots of phloem extracts from other genera in the Pinaceae. Solubilized phloem polypeptides bind strongly to chitin (oligomeric N-acetylglucos- amine) columns and are sensitive to thiol reagents, both characteristics which relate them to phloem- specific lectins isolated from angiosperm species (C. Allen, 1979, Biochem. J. 183, 133-137; A.K. Gietl et al., 1979, Planta 144, 367-371). Fluores- cence microscopy and immuno-gold electron micro- scopic cytochemistry demonstrated antigenic sites specifically associated with protein crystals pecu- liar to the sieve-element plastids of the Pinaceae. Key words: Lectin - Phloem (specific polypeptide) - Pinus - Plastid (in sieve elements) - Sieve-element plastid Introduction In vascular plants, the living cells of the phloem constitute a continuous transport system between * Present address: Genetics Project, U.S.D.A. Forest Service, Pacific Southwest Forest and Range Experiment Station, P.O. Box 254, Berkeley, CA 94701, USA Abbreviations: DAB = diaminobenzidine tetrachloride; FITC = fluorescein isothiocyanate; kDa = kilodalton; PBS = phos- phate-buffered saline; PP = phloem polypeptide(s); SDS- PAGE= sodium dodecyl sulphate-polyacrylamide gel electro- phoresis; Tris = 2-amino-2-(hydroxymethyl)- 1,3-propanediol tissues that export and utilize assimilates. The phloem is protected against injury by possession of a rapid wound response which includes the plug- ging of sieve pores by cytoplasmic material and their further constriction by callose deposition (Evert 1982). The P-protein of angiosperm sieve elements has been frequently located within the sieve pores of severed sieve tubes (Engleman 1965; Anderson and Cronshaw 1969; Schulz 1986) and implicated in the former role. The filamentous, tu- bular and crystalline forms adopted by P-protein, together with biochemical properties that include associated lectin activity (Cronshaw and Sabnis 1989), are consistent with proposals that a major role of this phloem-specific material is to plug in- jured sieve elements and prevent the loss of assimi- lates. Although the phloem of gymnosperms is as vulnerable as that of angiosperms, its sieve cells do not contain P-protein (see Behnke 1983; Evert 1984; Schulz 1989). However, biochemical frac- tionation and analysis of phloem from Pinus sabin- iana Dougl. revealed phloem-specific polypeptides of relative molecular mass (Mr) 24 and 25 kilodal- tons (kDa) (Alosi and Park 1983). They repre- sented a major part of the total polypeptide com- plement of the phloem and were not detected in the xylem. Polyclonal antibodies were raised against the phloem-specific proteins of Pinus sabiniana in order to (i) locate the proteins in situ employing indirect immunofluorescence microscopy and im- munogold electron microscopy, and (ii) to study their distribution in other gymnosperm species us- ing comparative gel electrophoresis and immuno- blotting procedures. Some characteristic biochemi- cal properties of the phloem polypeptides are also reported.