Presumable Role of Plasmodesmata in Floral Signal Transduction in Shoot Apical Meristems of Rudbeckia and Perilla Plants

ISS 1021-4437, Russian Journal of Plant Physiology, 2007, ol. 54, o. 4, pp. 498 506. Pleiades Publishing, Ltd., 2007. Original Russian Text E.L. Milyaeva, 2007, published in Fiziologiya Rastenii, 2007, ol. 54, o. 4, pp. 562 571. RESEARCH PAPS Presumable Role of Plasmodesmata in Floral Signal Transduction in Shoot Apical Meristems of Rudbeckia and Perilla Plants E. L. Milyaeva Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia; fax: 7 (495) 977-8018; e-mail: e_milyaeva@mail.ru Received July 24, 2006 Abstract The number of plasmodesmata was calculated per 1 µm of cell wall length in the central and medullar zones of shoot apical meristems (SAM) in the course of floral transition in a long-day (LD) plant Rudbeckia bicolor utt. and a short-day plant Perilla nankinensis Lour. Under the day length unfavorable for flowering (control), the numbers of plasmodesmata differed in the central and medullar zones of SAM, which produce the reproductive organs and stems, respectively. Besides, the numbers of plasmodesmata in the central zone of perilla SAM considerably differed between the anticlinal and periclinal cell walls of the first and second cell layers. Following the photoperiodic induction (PI) with eight LD in rudbeckia and twelve SD in perilla favorable for floral transition, the numbers of plasmodesmata considerably increased in the anticlinal and periclinal cell walls of the first and second cell layers of the central zone; meanwhile in the medullar zone, the numbers of plasmodesmata dropped down following PI. These data show that floral transition presumably involves the activation of cell-to-cell interactions and enhances the signal transduction in SAM. DOI: 10.1134/S1021443707040103 Key words: Rudbeckia bicolor - Perilla nankinensis - shoot apical meristems - plasmodesmata - floral signals ITRODUCTIO Flowering is a major event in the plant life cycle because it provides for plant reproduction in the series of generations. Floral transition is caused by floral gene expression induced by a complex system of interacting external and internal signals. According to the florigen theory developed by M.Kh. Chailakhyan 70 years ago, the external signals, such as photoperiod, vernalization, etc., induce the formation of leaves florigen, a characteristic signal factor transported via the phloem from leaves into shoot apical meristems (SAM); as a result, some changes occur in SAM resulting in the formation of reproductive organs. The outcome of the long-standing studies by Chailakhyan was the concept of florigen as a multicomponent factor of hormonal nature [1]. The major support to the florigen theory came from the classic Chailakhyan s experiments with the leaves from floralinduced photoperiodically-sensitive plants grafted on noninduced plants; as a result, the latter flowered. The fact that the grafts between the plants belonging to different species and genera resulted in rootstock flowering led to the conclusion that floral signals were universal in all plant species [1]. Later, however, many researchers expressed doubt as to the universality of Abbreviations: LD long day; PI photoperiodic induction; SAM shoot apical meristems; SD short day. floral signals and suggested that each plant species employs its specific signals for floral transition [2]. In this context, Chailakhyan s followers, including the author, have systematically focused on the comparative studies of plant species that differed in their photoperiodic responses. In particular, our studies demonstrated that the floral transition in response to the floral signal involved similar changes in the SAM of widely diverse plant species; these data were an additional, although indirect, proof to the concept of the universal floral signal [3]. According to the Chailakhyan s theory, floral signals arise in leaves and are translocated for long distances via the phloem into SAM. Therefore the hormonal system in control of flowering should be defined as a long-distance signal system [4, 5]. Recent progress in molecular biology has provided numerous proofs for the Chailakhyan s theory at new, molecular level [6]. Thus, an extensive and sophisticated network of interacting genes and the products of their expression was found to participate in plant transition from the vegetative to reproductive state [7 9]. In particular, the genes expressed in response to the photoperiodic induction (PI) were found in leaves. The products of such expression were shown to move via the phloem unto SAM where they activated other genes that were immediately involved in the inflorescence and flower morphogenesis and in the formation of particu- 498

PRESUMABLE ROLE OF PLASMODESMATA I FLORAL SIGAL TRASDUCTIO 499 lar flower organs, such as corolla, anthers, pistils, etc. [10, 11]. It is noteworthy that the cells of particular SAM zones differ in their cytological and functional characteristics. These zones were previously shown by the author to give rise to definite plant organs: thus, the reproductive organs develop from central zone cells, and stems, from the medullar zone cells [3, 6, 12]. With vascular bundles absent from SAM apical regions, plasmodesmata, the highly specialized intercellular channels characteristic only of multicellular organisms, become the major pathways of the symplastic transport of signal and plastic substances. Plasmodesmata were shown to represent quasi-organelles changing in response to the physiological status of plant tissues and organs and usually sensitive to the signals that control plant growth and development [13, 14]. Within the latter decade, free transport via plasmodesmata, in addition to low-molecular substances, was shown to include protein and ribonucleoprotein macromolecules, various forms of RA, and the transcription factors, which determined cell destinies in developing plant organs [15 17].The long-distance signals that are formed in leaves in response to PI are further transported, in their initial form or following manifold transformations, to SAM wherein they invoke the expression of the complex system of floral genes, and the arising new products (signal factors) are redistributed between the particular zones and cells within SAM. This redistribution represents the system of short-distance signals active within SAM. The transition from the vegetative SAM morphogenesis to the reproductive one brings about the development of flower initials instead of shoot and leaf initials; at the cell level, such transition is seen as the changes in the plane of cell divisions, the shortening the phases of the cell cycle in SAM cells, etc. [2, 18]. The changes in the number of plasmodesmata provide indirect evidence that the rates of interactions between SAM cells are crucial for signal transduction from cell to cell within the shoot apex. Our present goal was to assess the number of plasmodesmata per unit of cell wall length and compare this index in various SAM cell layers and zones in plant species that are opposite in their photoperiodic response: long-day (LD) rudbeckia and short-day (SD) perilla in the course of their floral transition induced by photoperiod. MATIALS AD METHODS LD plants of Rudbeckia bicolor utt. and SD plants of Perilla nankinensis Lour. were grown at day lengths unfavorable for floral transition: rudbeckia, at SD (8/16 h day/night) and perilla, at LD (16/8 h day/night) for three months, to the stage when these plants, as shown by our previous experiments, were most sensitive to PI [19]. ext, the plants were exposed to PI treatments favorable for flowering: eight LD (16/8 h day/night) for rudbeckia and 12 SD (8/16 h day/night) for perilla. Plants kept through the whole experiment under the day length unfavorable for floral transition served as the control group. At the end of the experiments, shoot buds were cut off, and SAM apical parts 2 5 mm 3 in size were fixed in the Carnoy fluid [20], transferred through a series of ethanol solutions, and embedded in paraffin. The longitudinal sections made with a Reichert rotation microtome (Austria) were stained with hematoxylin and the Schiff reagent after Feulgen [20]. The sections were examined with an Amplival light microscope equipped with an mf-matic device (Carl Zeiss, Germany). For electron microscopy, SAM apical parts 1.0 1.5 mm 3 in size were fixed in 3% glutaraldehyde in phosphate buffer containing 25 mg/ml sucrose for 18 h at 4 ë, washed five times in cold buffer, and postfixed in the 1% éso 4 solution for 3 h. Following dehydration in a series of increasing ethanol concentrations, tissue samples were embedded in Epon by the standard protocol. Ultrathin sections cut with an LKB-4800 ultramicrotome (LKB, Sweden) were contrasted with the 1% uranyl acetate solution in 70% ethanol and then with lead citrate after Reynolds. To calculate the number of plasmodesmata, the sections were examined with JEM-100B and Jeol-100 microscopes (Japan). A hundred of nonserial sections (25 sections for each of four combinations listed above) were examined in each treatment. The Image Tool software was used for computer calculation of the number of plasmodesmata in anticlinal and periclinal cell walls in successive layers of the central and medullar zones of SAM using 5 10 ultrathin SAM sections obtained in the experiments performed within a several-year period, and the frequencies of plasmodesmata were defined as their number at the border between two cells per 1 µm of the border as seen on the section. The data were compared and processed statistically in first three cell layers in the central zone of SAM and first two cell layers in the medullar zone in the control and PI plants of rudbeckia and perilla and also in the cell walls that differed in their orientation: periclinal, that is in parallel to the SAM surface, and anticlinal, that is perpendicular to the SAM surface. To compare the frequencies of plasmodesmata in various cell walls, we built plasmodesmograms following the method of an Bell et al. [21] as shown in Fig. 5. In these plasmodesmograms, the frequencies of plasmodesmata correspond to the numbers of lines between two cell images. One line corresponds to the frequencies comprising 11 to 25% of the maximum, two lines, 26 to 35%, three lines, 36 to 45%, four lines, 46 to 70%, and finally six lines, 86 to 100%. RESULTS SAM zoning and its functional meaning have been debated in the literature for several decades, and this discussion was already reviewed in detail by the author [22].

500 MILYAEA Anticlinal cell wall Periclinal cell wall Layer I Layer II Anticlinal cell wall Central zone Medullar zone Lateral zone Periclinal cell wall Layer I Layer II Fig. 1. A scheme representing SAM structure (longitudinal section). Arrows indicate cell layers and cell walls used to calculate the number of plasmodesmata. ( ) (b) (c) (d) Fig. 2. Microphotographs of the longitudinal SAM sections. (a) Rudbeckia plants grown under SD; (b) rudbeckia plant following 8-LD induction; (c) perilla plants grown under LD; (d) perilla plant following 12-SD induction. o morphological changes are found following such induction periods. Magnification 7 10.

PRESUMABLE ROLE OF PLASMODESMATA I FLORAL SIGAL TRASDUCTIO 501 P ( ) 1 µm P PP (b) 1 µm Fig. 3. Ultrastructure of the areas in (a) the central and (b) the medullar SAM zones in the control rudbeckia plants. Plasmodesmata are seen in the periclinal and anticlinal cell walls as dark strands perpendicular to cell walls. cell wall; endoplasmic reticulum; M mitochondrion; nucleus; P plastid; PP proplastid; vacuole. Plasmodesmata are shown with arrows. The experimental data concerning this issue allowed us to conclude that in the course of floral transition, the central zone of SAM gave rise to the reproductive organs, whereas the medullar zone produced stem tissues. This conclusion was used as a basis in our calculations of the number of plasmodesmata in particular cell layers of the central and medullar zones and in the walls of the cells that differed in their orientation towards the apex rather than in SAM as a whole. The position of cell walls used to assess the frequencies of

502 MILYAEA PP ( ) PP 0.5 µm (b) 0.5 µm P (c) 0.5 µm (d) 0.5 µm (e) 1 µm (f) 0.1 µm (g) 0.1 µm Fig. 4. Ultrastructure of the cell wall areas in the second cell layer of (a) the central and (b) the medullar SAM zones in the SDrudbeckia plant; (c) the central and (d) the medullar SAM zones in the rudbeckia plant following 8-LD induction; (e) the central and (f) the medullar SAM zones in the LD-perilla plant; (g) the central zone in the perilla plant following 12-SD induction. (a, c, f, g) Single plasmodesmata; (b, d, e) groups of plasmodesmata; (b, d, e, g) the membranes of endoplasmic reticulum are seen near plasmodesmata. Other designations as in Fig. 3. plasmodesmata is shown schematically with arrows on the SAM longitudinal section (Fig. 1). Figure 2 presents the microphotographs of the SAM longitudinal sections in the control (Figs. 2a, 2c) and photoperiod-induced (Figs. 2b, 2d) plants of rudbeckia and perilla. Following the induction with 8 LD for rudbeckia (Fig. 2b) and 12 SD for perilla (Fig. 2d), no notable morphological changes were recognized in SAM, except some expansion in rudbeckia. Figure 3 presents the microphotographs of the cell areas from the central (Fig. 3a) and medullar (Fig. 3b) zones of SAM in rudbeckia. The plasmodesmata seen as dark threads perpendicular to the cell wall (shown by arrows) are found both in the periclinal and anticlinal cell walls. At higher magnifications (Fig. 4), one can see that plasmodesmata are usually located as single strands (Figs. 4a, 4c, 4f, 4g) or in groups of two three strands (Figs. 4b, 4d, 4e); they connect cytoplasm of adjacent cells involving such cytoskeletal elements as

PRESUMABLE ROLE OF PLASMODESMATA I FLORAL SIGAL TRASDUCTIO 503 microtubules and microfilaments (Figs. 4b, 4d, 4e, 4g) into the intercellular transport. The assessment of the number of plasmodesmata in SAM cells of noninduced (control) cells of rudbeckia showed that this index diverged in the central and medullar zones and differed by several times when the anticlinal and periclinal walls were compared in the first and second cell layers of SAM (Table 1). The highest frequency of plasmodesmata was observed between the cells belonging to the first and also the first and second layers in the SAM medullar zone of the control rudbeckia plants (Table 1). Following PI favorable for floral transition, the number of plasmodesmata per 1 µm of cell wall length considerably increased in both SAM zones and in all cell layers (Table 1). Following floral transition evoked by photoperiod in rudbeckia plants, the number of plasmodesmata notably increased in the cells of the first, second, and third layers of the central zone, whereas the opposite pattern was observed in the medullar zone: following PI, the frequency of plasmodesmata became significantly lower than in the control SAM (Table 1; Fig. 5). In another species such as SD plant perilla, the evocation of flowering increased the number of plasmodesmata per 1 µm of cell wall both in periclinal and anticlinal cell walls of the first and second cell layers (Table 2). Following PI, the number of plasmodesmata increased in the periclinal cell walls of the first and second cell layers of the central zone, whereas in the corresponding cells of the medullar zone similar to the pattern observed in the rudbeckia SAM, the number of plasmodesmata declined (Table 2; Fig. 5). LD 3 layer 3 layer LD 3 layer 3 layer ( ) Central zone Medullar zone (b) Central zone Medullar zone 8 SD 3 layer 3 layer 12 SD 3 layer 3 layer Fig. 5. Plasmodesmatograms illustrating the frequencies of plasmodesmata in cell layers of the central and medullar SAM zones in the control and PI plants. (a) Rudbeckia; (b) perilla. Line numbers between various cell walls and cell layers correspond to the numbers of plasmodesmata (see Materials and Methods). PI increased the numbers of plasmodesmata in the first and second cell layers of the central zone in rudbeckia and perilla and diminished these numbers in the corresponding cell layers in the medullar zone. DISCUSSIO Previously we demonstrated that the exposure to favorable PI used in this study corresponded to the completion of floral evocation in these plant species. Within this period, floral genes were expressed, and some of SAM cytological indices underwent considerable changes, such as tenfold increase in the mitotic activity, shortening cell cycle, the increased number of mitochondria per cell, the enhanced sugar and starch synthesis, and the changes in the volume and structure of nucleoli and the ratio between euchromatin and heterochromatin in nuclei; however, no morphological changes were observed within this period [2, 18, 23, 24]. In the present study, we describe notable changes in the number and distribution of plasmodesmata in cell walls of different orientation in various SAM zones and cell layers in two groups of photoperiodically sensitive plants after the induction of flowering and in the control. The similar and different characteristics in the plasmodesm patterns are best recognized in the plasmodesmograms shown in Fig. 5. Following the floral transition in rudbeckia evoked by eight LD, the number of plasmodesmata increased in the periclinal and anticlinal cell walls between the first, second, and third cell layers in the central SAM zone. At the same time, this index was diminished in the first and second cell layers of the medullar zone. Similar evidence was obtained with perilla plants: following PI, the number of plasmodesmata increased in the central zone and decreased in the medullar zone (Table 2; Fig. 5). These data presume that SAM comprises some symplastic compartments, or domains defined on the basis of the frequency of plasmodesmata. oteworthy is the

504 MILYAEA Table 1. Plasmodesmata distribution in the cell walls of central and medullar zones of rudbeckia SAM Treatment Position of cell walls, position of plasmodesmata Zones central medullar no. plasmodesmata/µm SD periclinal, in layer 1 0.73 ± 0.02 0.91 ± 0.01 anticlinal, between layers 1 and 2 1.36 ± 0.02 0.87 ± 0.06 periclinal, in layer 2 0.33 ± 0.01 0.71 ± 0.03 anticlinal, between layers 2 and 3 0.38 ± 0.01 periclinal, in layer 3 0.41 ± 0.03 8 LD periclinal, in layer 1 0.98 ± 0.04 0.48 ± 0.04 anticlinal, between layers 1 and 2 1.36 ± 0.02 0.51 ± 0.06 periclinal, in layer 2 0.77 ± 0.02 0.61 ± 0.02 anticlinal, between layers 2 and 3 0.84 ± 0.04 periclinal, in layer 3 0.70 ± 0.07 ote: The means of 100 regions of the cell walls examined and their standard errors are presented. Table 2. Plasmodesmata distribution in the cell walls of central and medullar zones of perilla SAM Treatment Position of cell walls, position of plasmodesmata Zones central medullar no. plasmodesmata/µm LD periclinal, in layer 1 0.51 ± 0.03 0.81 ± 0.04 anticlinal, between layers 1 and 2 1.22 ± 0.02 0.73 ± 0.04 periclinal, in layer 2 0.30 ± 0.01 0.76 ± 0.02 anticlinal, between layers 2 and 3 0.25 ± 0.01 periclinal, in layer 3 0.33 ± 0.01 12 SD periclinal, in layer 1 0.91 ± 0.04 0.38 ± 0.01 anticlinal, between layers 1 and 2 0.87 ± 0.03 0.46 ± 0.02 periclinal, in layer 2 0.80 ± 0.01 0.41 ± 0.02 anticlinal, between layers 2 and 3 0.73 ± 0.01 periclinal, in layer 3 0.83 ± 0.01 ote: The means of 100 regions of the cell walls examined and their standard errors are presented. fact that the limits of these domains coincide with the location of SAM zones; this coincidence would provide for the functional activity of particular SAM zones in the course of SAM transition from the vegetative to reproductive state. These data also support indirectly the diverse morphological roles of particular SAM zones in such transition. The increase in the number of plasmodesmata following LD induction was also reported by Ormenese et al. [25] in all cells of the central and lateral SAM zones of an LD plant Sinapis alba. It is noteworthy that floral transition in this plant species occurs following one LD induction, and the changes in SAM proceed very rapidly, within several hours. As a result, the assessments of the changes in the number of plasmodesmata in S. alba are more difficult and less precise than in perilla and rudbeckia; besides, Ormenese et al. did not calculate the number of plasmodesmata in the medullar SAM zone. Gisel et al. [26] used a confocal microscope to follow the transfer of a luminescent dye between SAM cells in another LD plant species, Arabidopsis thaliana, and found an enhanced fluorescence during the floral transition; these authors also reported the complicated pattern of such enhancement in various SAM sites. In spite of the fact that arabidopsis is the major model plant in the studies of floral genetics, this species is not very feasible for the studies of photoperiodic responses: in arabidopsis, the photoperiodic reaction is quantitative, whereas our model plants, rudbeckia and perilla, are qualitatively sensitive to PI. The

PRESUMABLE ROLE OF PLASMODESMATA I FLORAL SIGAL TRASDUCTIO 505 papers cited above concern only two LD species, white mustard and arabidopsis, whereas our study compared for the first time the changes in the numbers of plasmodesmata in two plant species contrasting in their photoperiodic responses: qualitatively LD and qualitatively SD. In both cases, PI produced a similar trend of events: the number of plasmodesmata increased in the central zone and decreased in the medullar zone. We therefore conclude that independent of day length inducing the floral transition, this process is accompanied by the increase of the number of plasmodesmata in the central zone and decrease, in the medullar zone. It follows that, whatever plant species and their photoperiodic responses, the floral transition involves the enhanced interactions between cells of the central SAM zone, which provide for improved intercellular transport and translocation of the floral signal, apparently universal in diverse plant species. Upon the evidence that floral transition involves the diverse changes in the frequencies of plasmodesmata in SAM central and medullar zones, we conclude that the floral transition includes the development of symplastic domains coinciding with these two zones. Within the symplastic domain of the central zone, the number of plasmodesmata increased, whereas in another symplastic domain corresponding to the medullar zone, floral transition led to the diminishing frequency of plasmodesmata, apparently representing the decline in the rate of symplastic transport. It follows that during the floral transition, the symplastic contacts between the central and medullar zones become restricted, and these data are the basis for drawing the boundaries of symplastic domains corresponding to the central and medullar zones of SAM. The different numbers of plasmodesmata in the anticlinal and periclinal cell walls of SAM in the induced rudbeckia and perilla plants seem to imply the different order of development of flower initials in the inflorescences of diverse types, that is, the terminal initials in rudbeckia inflorescences and the lateral initials, in perilla. To conclude, this study demonstrated considerable increase in the number of plasmodesmata in the central zone of SAM of various plant genotypes following the induction of floral transition. This phenomenon is interpreted as enhancement of intercellular exchange with long-distance and short-distance signals critical for plant transition from the vegetative to the reproductive development. 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