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 to the hemiparasitism of these species. The plants represent root hemiparasites which gain mineral nutrients and water from their host species. Recent studies showed that they also can receive up to 50% of their carbon budget from the hosts as xylem-mobile organic compounds (Tešitel et al., 2010). This could explain a need for increased apoplasmic uptake of carbon compounds from the xylem into their minor vein phloem for further transloca­ tion, along with symplasmic loading of mesophyll sugars derived from photosynthesis as has been shown for monocots (Botha et al., 2008). However, the correlation between hemiparasitism and minor vein phloem subtype disappeared when more species were included in the analysis (Table 4). Interestingly, MIC-b displayed not only plasmodesmal fields and cell wall ingrowths at the interface with the bundle sheath, but they also developed cell wall ingrowths at the walls which directly contacted the xylem. The immediate contact of companion cells and even of sieve elements with xylem elements in m inor veins of these species might be a reason for a need to increase the sur­ face of apoplasmic contact between xylem and phloem. It would be interesting to study minor vein structures in these species in course of phloem development and in relation with establishment of hemiparasitism, and to find out which metabolites are present in xylem- and phloem sap, respectively. The important question of the exact mechanisms of symplas­ mic phloem loading can be investigated further using several new models described here, such as CC-b in Apocynaceae, MIC-b in hemiparasitic Orobanchaceae, and ICL which seemingly differ from ICs only by the ability to accumulate starch in leucoplasts. In all these cases, there are asymmetrically branched plasmodes­ mata the exclusion limit of which is probably regulated, analogous to the situation in ICs. The polymer trap model proposing RFO synthesis as a mechanism regulating barrier properties of such plasmodesmata in species with ICs was cast into doubt based on results obtained using the ICs-containing species Alonsoa merid- ionalis (Voitsekhovskaja et al., 2006, 2009); moreover, in leaves of species containing MIC-b and CC-b, no RFOs could be detected by GC-MS which, considering the sensitivity of the method, leads to the conclusion that RFOs cannot be the main transport form in these species, at least not at all developmental stages. In the analysis of the spatial organization of the minor veins and its relation to the types of companion cells and phloem parenchyma within the veins we summarized the complexes of structural features characteristic of types 1 and 2, and introduced Table 4 | Type of minor vein phloem (Supplemental Table 1) and hemiparasitism in Orobanchaceae (Gamalei, 2007). Hemi-parasitic Life form Minor vein genera phloem Bartsia Perennial herb 1-1 Castilleja Annual herb 1-11 Pedicularis Perennial herb 1-11 Euphrasia Annual herb 2-VI Melampyrum Annual herb 2-VI Odontites Annual herb 2-VI Rhinanthus Annual herb 2-VI type 0 for minor veins showing unstable structural characteris­ tics. The spatial organization of m inor vein phloem results from the patterns of the divisions of phloem initials in course of the minor vein development, and thus has an important evolution­ ary meaning; this is supported by the fact that minor veins of types 1 and 2 show strong correlations with the growth forms of trees and herbs, respectively (Gamalei, 1989, 1991). The present study showed that the layout of minor vein phloem in an over­ whelming number of species correlates with the extent of the development of symplasmic connections between bundle sheath cells and phloem companion cells, confirming earlier conclusions (Gamalei, 1989, 1991). However, several exceptions to this rule were found. First, the hemiparasitic Orobanchaceae species are able to form plasmodesmal fields along with cell wall protuber­ ances in their companion cells, and the spatial organization of the m inor veins differed from that in type 1 species. Second, in species of the Campanulaceae and the Convolvulaceae families, the m inor vein layout was similar to that of type 1 veins but the veins contained no ICs or IC-like companion cells, and no plas­ modesmal fields; their companion cells were connected with the bundle sheath by multiple single plasmodesmata. A detailed study of this minor vein type was published by Madore et al. (1986). These examples show that the two features, the anticlinal position of the first division plane of the phloem initial and the ability to develop highly abundant plasmodesmal fields, although coupled in the majority of species studied thus far, can develop indepen­ dently. Interestingly, the position of the Campanulaceae on the phylogenetic tree of Asterales is at the base, where shrubs and small trees occur (Lundberg, 2009), but the Asteraceae family at the top of this tree is represented by herbs with m inor veins of type 2. The classification of type 0 is far from complete, since the subclass Asteridae predominantly comprises species with highly specialized minor veins of types 1 or 2 (Figure 6). Type 0 prob­ ably could be better resolved via a similar study of the subclass Rosidae which is rich in “type 0 species” (Yu. V. Gamalei, M. V. Pakhomova, and D. R. Batashev, unpublished observations). The new nomenclature presented in this study has been devel­ oped for the Asteridae and thus may not be sufficient to cover all dicots; however, it can be easily broadened by adding new subtypes in the course of future studies on the Asteridae or other groups of dicots. Generally, type l-2a in Gamalei’s classi­ fication (Gamalei, 1991) is divided between subtypes 0 and 2-1 in the nomenclature of this study, while Gamalei’s types 1 and 2 keep their positions, but the numbers of subtypes increase (Figure 3). Again, we stress the fact that the 1, l-2a, 2a, 2b nomenclature is still valuable for characterization of the general ability for the sym- or apoplasmic loading mode. Several years ago, a classification was proposed which might be regarded as consensus between functional and structural descriptions. This classification, which was independently proposed by Gamalei (2000) and van Bel and Hafke (2005), is a combination of onto­ genetic and structural views of minor vein phloem, van Bel and Hafke (2005) integrated the functional aspect by including the transport carbohydrates. It includes five subtypes of the minor vein phloem, 0, 1A, IB, 2A, and 2B, where 0, 1, and 2 refer to minor vein spatial organization and the phloem-loading mode, www.frontiersin.org August 2013 | Volume 4 | Article 312 | 11