Razumovskaya A.V. Cytology of the minor-vein phloem in 320 species from the subclass Asteridae suggests a high diversity of phloem-loading modes. Frontiers in Plant Science. 2013, V. 4, Article 312.

Batashev et al Minor vein phloem in Asteridae every species studied) were analyzed. Leaf pieces ( 3 x 4 mm) were infiltrated with cold fixative (3% glutaraldehyde, 3% sucrose in 0.1 M potassium phosphate buffer pH 7.2), incubated in fresh fix­ ative for 6 h and washed in buffer six times for 10 min each. The material was then post-fixed for 16 h in 2 % osmium tetroxide in potassium phosphate buffer at 4°C, dehydrated in 30% and 50% ethanol for 20 min each, contrasted with 1.5% uranyl acetate in 70% ethanol for 2 h, further dehydrated in an ethanol:acetone series and embedded in Epon-Araldite epoxy resin. Ultrathin sections (40-60 nm) were cut with glass knives on an LKB-III microtome (LKB, Stockholm, Sweden), contrasted on grids using 2% lead citrate, and viewed and photographed at 75 kV with a Hitachi H-600 electron microscope (Tokyo, Japan). SUGAR ANALYSIS Leaves of Allamanda cathartica L., Alstonia macrophylla Wall, et G. Don, Phtmelia rubra L. and Thevetia nereifolia Juss. ex Steud. (Apocynaceae), and of Melampyntm sylvaticum L., Euphrasia fennica Kihlm. and Rhinantlms minor L. (Orobanchaceae) were extracted twice with 80% ethanol at 60°C. Seven hundred micro­ liter of each extract (corresponding to approx. 500 mg fresh weight) were vacuum-dried at 40° С in a rotary evaporator (Buechi, Flawil, Switzerland) and subjected to derivatization in a mixture of (N,0-Bis-trimethylsilyl)-trifluoroacetamide:pyridine (1:1, v/v) (Sigma-Aldrich, Deisenhofen, Germany) in a her­ metically closed tube for 15 min at 100°C. A gas chromato­ graph Agilent 6850 (Agilent Technologies, Santa Clara, CA, USA) equipped with a mass selective detector Agilent 5975C was used, supplied with a capillary column HP-5MS (30 m length, 0.25 mm diameter, 0.25 (im film thickness; J&W Scientific, Folsom, CA, USA). Helium was used as a carrier gas at a flow rate of 1.3ml/min. The column was operated at an initial tempera­ ture of 70°C and adjusted to 320°C at 6 °C/min. The temper­ ature of the injector was 330°C by the split flow 50:1. The injected volume was 1 (il. The internal standard used was n- C 23 hydrocarbon (Sigma-Aldrich, Deisenhofen, Germany). The data were collected and processed with the Agilent ChemStation system. Mass spectra were interpreted, and substances identi­ fied, with the AMDIS (Automated Mass-Spectral Deconvolution and Identification System) software using NIST 2008 and Wiley 6 libraries. Quantification of chromatograms was performed using UNICHROM software (New Analytical Systems, Minsk, Belarus). RESULTS CYT0L0GICAL DIVERSITY OF COMPANION CELLS IN ASTERIDAE SPECIES Since companion cells are key players in phloem loading, we first focused on their structural diversity in minor veins of Asteridae. To provide a better overview of observed structures, we analyzed several features which were then used to build a classification. Three independent cytological characteristics of companion cells were selected: ( 1 ) presence vs. absence of plasmodesmal fields connecting companion cells to the bundle sheath; ( 2 ) presence vs. absence of cell wall ingrowths in companion cells; (3) type of plastids present in companion cells (leucoplasts vs. chloro- plasts). Plasmodesmal fields are defined here as aggregations of plasmodesmata, either branched or simple, which can be clearly distinguished from the rest of the cell wall that may contain single plasmodesmata. We decided to use this characteristic as indicative for a considerable potential for symplasmic transport, in contrast to a few single plasmodesmata which are always present in any type of companion cell. Moreover, the distinction (plasmodes­ mal fields vs. single plasmodesmata) is qualitative and does not require elaborative counts of numbers of plasmodesmata per cell surface unit. The development of cell wall ingrowths increases the surface of metabolite exchange via apoplast. Thus, the first two characteristics indicate a high specialization level of compan­ ion cells adapted to symplasmic or apoplasmic phloem loading, respectively. The third feature, the type of plastids in companion cells, is easily distinguishable on micrographs. This feature has proved invaluable for discriminating between companion cells and parenchyma cells in minor veins (Russin and Evert, 1985; Reidel et al., 2009) but so far it has not been considered in relation to the phloem-loading mechanism. However, it should be taken into account that plastid retrograde signaling has been recently shown to be a potent regulator of plasmodesmata development (Burch-Smith et al., 2011). Also, the type of plastids in compan­ ion cells might be related to metabolic specialization of these cells. For instance, the fact that plastids in ICs are always leucoplasts and never chloroplasts might simply reflect the main function of these plastids as myo-inositol depots (Moore et al., 1997; Voitsekhovskaja et al., 2006), as the RFO synthesis by ICs requires high activities of myo-inositol production but not of sucrose production because sucrose is supplied by mesophyll cells. We also included in the analyses two subordinate features, the mode of plasmodesmata branching in plasmodesmal fields and the morphology of cell wall protuberances. Asymmetric branch­ ing of plasmodesmata was shown to be an important diagnostic feature for ICs (Turgeon and Medville, 2004). Two different types of the morphology of cell wall protuberances have been described so far, reticulate and flange morphology (Offler et al., 2003); however, only reticulate protuberances were found in companion cells. Here, we describe as a novelty companion cells with cell wall ingrowths of flange morphology (see below). On the basis of the features listed above, eleven varieties of companion cell were distinguished in Asteridae (Table 1; Figure 1); however, some of them were widespread and oth­ ers rare (family- or species-specific; see below). These structural varieties were ranked according to subtypes and grouped into four major companion cell types: OCs, TCs, ICs and IC-like cells, and CC with plasmodesmal fields/many single plasmodes­ mata including modified intermediary cells (MICs; Turgeon et al., 1993). OCs, sometimes referred to simply as “companion cells,” which are characterized by the absence of both plasmodesmal fields and cell wall ingrowths, contained two subtypes with either leucoplasts (ОС-a; Figure 1A) or chloroplasts (OC-b; Figure IB), respectively (Table 1). TCs were classified on the basis of plas­ tid type and morphology of cell wall ingrowths. The companion cells of TC type described by Pate and Gunning as “A-type trans­ fer cells” contained chloroplasts (Pate and Gunning, 1969); a cell of that type (TC-a in the present classification) is shown in Figure 1C. Companion cells of TC type containing solely leu­ coplasts were observed in phloem endings in two cases: either www.frontiersin.org August 2013 | Volume 4 | Article 312 | 3