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 Table 3 I Distribution of minor vein phloem types and subtypes among the families of Asteridae. Family Subtype of the minor vein phloem 1-1 1-11 1-III 1-IV 0 2-I 2-II 2-III 2-IV 2-V 2-VI Acanthaceae + + + Apocynaceae + + + + Asteraceae + + Bignoniaceae + + Boraginaceae + + Campanulaceae + Convolvulaceae + Cornaceae + Eucommiaceae + Gentianaceae + + + Gesneriaceae + Griseliniaceae + Hydrangeaceae + Lamiaceae + + + Oleaceae + Orobanchaceae + + + Pawlowniaceae + Phrymaceae + Plantaginaceae + + + + + + + + (Veronicaceae) Polemoniaceae + + Rubiaceae + + + + Scrophulariaceae + + + Solanaceae + + + Verbenaceae + + DISCUSSION In this study, we analyzed the minor vein anatomy of 320 species from the subclass Asteridae. First, we performed a cytologi- cal analysis of companion cells, and described altogether eleven differentiation forms some of which had not been reported pre­ viously. These structures were classified as subtypes within four major types of companion cells; this classification can be extended by adding newly found structural types of companion cells as the cytological studies of companion cells in angiosperms pro­ ceed. Furthermore, we investigated the anatomical features of minor vein phloem and developed a classification of minor vein structure in the Asteridae. These two classifications can be used independently, as each has its own range of applications. We performed separate classifications for companion cell cytology and for minor vein anatomy, respectively, for several rea­ sons. Obviously, it is the cytology of companion cells that defines the potential for symplasmic or apoplasmic loading and thus is relevant for the functional classification of plants with regard to their phloem loading mode. At the same time, many species have more than one type of companion cells in m inor veins, and more than one mode of phloem loading, but nevertheless are still referred to as “apoplasmic loaders” and “symplasmic loaders,” fol­ lowing the typology of Gamalei (1991) where the possibility for different types of companion cells to be combined in one phloem ending was not taken into account. To solve this problem, a more detailed classification is to be introduced where attention is paid to all types of companion cells present in one phloem ending. However, it would be wrong to reduce the classification of minor vein phloem just to a description of the entire assembly of companion cells as this would neglect the evolutionary aspects of minor vein phloem. As shown in this study, plasmodesmal connections or cell wall ingrowths, respectively, can develop in companion cells of minor veins with different ontogenesis. This was not obvious in early studies because the minor vein types 1 and 2 were originally discovered in species among which anatom­ ical, developmental and cytological features were correlated, a fact later referred to as “symplasmic/apoplasmic syndrome” (Gamalei, 2007). The main features, the patterns of cells divisions that give rise to the minor veins, and the numbers of plasmodesmata con­ necting companion cells and bundle sheath cells showed a very good correlation in that species with ICs (“typical symplasmic loaders”) were always characterized by first anticlinal divisions of the phloem initial and those with TCs (“typical apoplasmic loaders”) by periclinal divisions (Gamalei, 1989). The reasons why in an overwhelming number of species two major types of vein spatial organization correlate with symplasmic continuity remain unclear; however, as shown in this study, there are a few exceptions to this correlation. Thus, the two classifications carry important information, the classification of companion cell ultra­ structure for functional studies, and the classification of minor vein structure for developmental and evolutionary studies. In the first part of our study, we used several structural fea­ tures to describe the observed diversity of companion cells. The development of cell wall ingrowths, the presence of plasmodesmal fields and the type of plastids in companion cells were indepen­ dent features, while the form of plasmodemata branching and the morphology of cell wall ingrowths were subordinate features. These traits were correlated in some cases, but not in others. For instance, leucoplasts were always correlated with the presence of asymmetrically branched plasmodesmal fields (and with the syn­ thesis of RFOs) in ICs, and chloroplasts were always correlated with the development of cell wall ingrowths of reticulate mor­ phology in TCs in type 2 minor veins. Generally, chloroplasts occurred in those companion cells types where no plasmodesmal fields developed; the only remarkable exception were ICL-c cells in Apocynaceae species, where chloroplasts were present together with asymmetrically branched plasmodesmal fields. Altogether these cellular structures represented diagnostic features which helped to distinguish between various companion cell types, while their functional significance requires further investigation. We report here a few novel structures of companion cells. In some representatives of Gentianaceae, TCs showed flange mor­ phology of their cell wall ingrowths, which had been never observed to date in TCs of the phloem origin. Also, new struc­ tural types of companion cells were found containing highly developed plasmodesmal fields. Among them, the most inter­ esting type seems to be MIC-b found in some hemiparasitic Orobanchaceae species. Representatives of genera like Rhinantlms and Melampyrum (subtype 2-VI) provide an interesting model for functional studies because their companion cells possess both well-developed cell wall ingrowths and plasmodesmal fields with no features of plasmodesmal occlusions (Figure 1J, detail 1). The very unusual structure of the companion cells might be related Frontiers in Plant Science | Plant Physiology August 2013 | Volume 4 | Article 312 | 10