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 abundance of symplasmic connections between companion cells and bundle sheath (Gamalei, 1989, 1991). The first division of the phloem initial was anticlinal in type 1 species but periclinal in type 2 species which resulted in different spatial organiza­ tion of mature minor veins of type 1 and 2 species, respectively. The position of the first division plane showed a striking correla­ tion with the abundance of plasmodesmata at the companion cell side facing the bundle sheath. This allowed designating types 1 and 2 minor veins as “open” and “closed,” respectively (Gamalei, 1989). Minor veins of type 2 could be divided into 2a, 2b, and 2 c; 2 a species contained companion cells without cell wall ingrowths, 2b species contained TCs with cell wall ingrowths, and the 2c group comprised species with Kranz anatomy. It was noticed that type 1 species are often represented by trees or woody shrubs while type 2 species are mostly herbs (Gamalei, 1989). A large group of species designated l-2a showed numbers of plasmodesmata at the companion cell/bundle sheath boundary intermediate between 1 and 2 a, and minor veins without well- defined spatial organization. For this group, growth form and phloem evolution were not clear (Gamalei, 1989). This classification was modified with the focus on the num ­ ber of symplasmic connections at the mesophyll/phloem interface (Gamalei, 1991). Four types were distinguished, two “open” ones (1 and l - 2 a) and two “closed” ones ( 2 a and 2 b); it should be kept in mind that also in this classification, the first numeral 1 or 2 car­ ries information on the degree of symplasmic continuity between mesophyll and phloem as well as on the ontogenesis of the minor vein phloem. Type 1 corresponded to species with num ­ bers of plasmodesmata per square (im cell surface between 100 and 10 , type l - 2 a included species with these numbers between 10 and 1 , types 2 a and 2 b designated plants with plasmodes­ mata numbers per square (im cell surface below 1 , whereas type 2b specified species with TCs where cell wall ingrowths increase apoplasmic transport (Gamalei, 1991). The introduction of these four types has been very helpful for elucidating the evolution of phloem loading mechanisms (Turgeon et al., 2001; Rennie and Turgeon, 2009) as well as for functional studies (e.g., Turgeon et al., 1993; Hoffmann-Thoma et al., 2001; Turgeon and Medville, 2004). Indeed, functional tests on a range of species in the stud­ ies of van Bel et al. using the classification of Gamalei (van Bel et al., 1992) provided compelling evidence for symplasmic phloem loading which was strongly disputed before. At the same time, many plants contain companion cells of more than one structural type in their minor veins, a fact which renders them beyond the scope of a division into “open” and “closed” types and becomes important when analyzing phloem loading mech­ anisms and their relative contribution to phloem transport in a given species. An example is Alonsoa meridionalis, a type 1 species, which contains ICs in its minor veins and was originally classi­ fied as a putative symplasmic loader but was shown to perform also apoplasmic sucrose loading in a second type of companion cells present in its minor veins, the ОС (Voitsekhovskaja et al., 2009). Thus, a comprehensive view of a phloem ending as a func­ tional unit can rely only on the cytology of all cells involved in assimilate loading. However, the possibility of different structural types of sieve element— companion cell complexes (SE-CCCs) to occur in minor veins of one and the same species is neglected in the four-types-scheme which limits its usefulness and may even render it misleading from the functional point of view in some cases. In the present study, the cytology of minor veins was examined in a number of species from the subclass Asteridae (Moore et al., 2010 ), one of the major clades of the core eudicots (for the list of species see Supplemental Table 1). For the abovementioned rea­ sons, we paid attention to the ultrastructure of all cells in minor vein phloem of the species investigated, as well as to the spatial organization of the minor vein. This study, together with data from published sources (Pate and Gunning, 1969; Peterson and Yeung, 1975; Evert, 1980; Madore and Grodzinski, 1984; Madore et al., 1986; Ding et al., 1988; McCauley and Evert, 1989; Fisher, 1991; van Bel et al., 1992; Turgeon et al., 1993; Roberts et al., 1997; Batashev and Gamalei, 2000, 2005; Voitsekhovskaja et al., 2006; Gamalei et al., 2008; Reidel et al., 2009), resulted in the analysis of 320 species belonging to 200 genera. This makes the Asteridae the best investigated subclass of dicots to date in terms of organi­ zation of phloem endings and structure of companion cells. Here, we give an overview of companion cell types in Asteridae and describe some structures not reported previously. We describe the ways how these cells can be combined in a phloem ending. We show that in the overwhelming number of species, spatial organization of m inor veins and symplastic continuity between companion cells and bundle sheath are strictly correlated, con­ firming the conclusions made by Gamalei (1989). However, we also present a few striking exceptions to this rule indicating the need for further categories of minor veins. We also provide a formal classification of minor veins in Asteridae on the basis of their companion cell type(s), spatial organization and a few additional features which maybe potentially important to under­ stand the assimilate pathways during phloem loading. These data lay a foundation for phylogenetic and functional analyses of the phloem in Asteridae, leading to a deeper understanding of the role of plasmodesmata in phloem loading. MATERIALSAND METHODS PLANT MATERIAL Plants were collected (1) from their native habitats during expedi­ tions in Altai, Cola peninsula, Leningrad region, Magadan region; (2) from botanical gardens of the Komarov Botanical Institute RAS (St.-Petersburg, Russia), from the Nikitskiy botanical gar­ den (Crimea, Ukraine) and from the botanical garden at Altai State University (Barnaul, Russia). (3) Tropical species were col­ lected from greenhouses of the botanical garden of the Komarov Botanical Institute RAS, and several Apocynaceae species were collected in parks of Bangkok (Thailand). The list of species stud­ ied is shown in Supplemental Table 1. Altogether, 320 species from 24 families in sensit APGIII, 2009 (295 species originally studied in the laboratory of Yu. V. Gamalei plus 25 species from published studies from other laboratories) were included in this analysis. TRANSMISSION ELECTRON MICROSCOPY (ТЕМ) STUDIES The ultrastructure of the m inor vein phloem (6-7 transverse sec­ tions of veins of the highest orders) was studied by means of ТЕМ. Mature fully expanded leaves (3-4 leaves from different plants for Frontiers in Plant Science | Plant Physiology August 2013 | Volume 4 | Article 312 | 2