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 vacuoles strongly depend on the physiological condition of the plant (Gamalei et al., 2000). In the present study, we classified the companion cells only on the basis of their stable structural fea­ tures. An example of a companion cell, the structure of which is similar to that of ICs but for which no analyses of transport sug­ ars have been performed is shown in Figure 1Ffor Hamelia patens (Rubiaceae). This cell has asymmetrical branching ofplasmodes­ mata in the fields facing the bundle sheath (Figure IF, detail 1), and possesses leucoplasts. In many species, companion cells with abundant plasmod­ esmal fields in the cell walls facing the bundle sheath could be found which resembled ICs in some respects but differed in others. We refer to these cells here as ICL (intermediary-cells- like). These cells contained multiple plasmodesmal fields with asymmetrical branching of plasmodesmata and leucoplasts but their leucoplasts contained starch while leucoplasts in ICs had never been shown to accumulate starch. To our knowledge, this cell type has not been described before. One example of IC-like cells with starch-containing leucoplasts is shown in Figure 1G for Catesbaea spinosa (Rubiaceae); for this species, no phloem sap analysis has been performed. Interestingly, small vacuoles were present in most cases in such cells. Such companion cells with starch-containing leucoplasts are obviously different from ICs and referred to as ICL in Table 1. Companion cells with multiple plasmodesmal fields, or sometimes many single plasmodesmata (see below) were also found in Asteridae. In representatives of Cornaceae and Hydrangeaceae (Cornales), Griseliniaceae (Apiales, former Cornales) and Eucommiaceae (Garryales), companion cells pos­ sessed leucoplasts without starch and in most cases small vac­ uoles; however, multiple plasmodesmal fields contained only symmetrically branched plasmodesmata (i.e., with similar num­ ber of branches on both sides). Companion cells of closely similar structure have been described, e.g., for poplar (Russin and Evert, 1985) or for Liquidambar styraciflua by Turgeon and Medville (2004); these authors found that they were functionally quite different from ICs in that they were not involved in loading RFOs in the phloem. These cells are referred to as CC-a in Table 1. Furthermore, companion cells with abundant plasmod­ esmal fields which could not be classified as ICs were found in several Apocynaceae species. These companion cells contained chloroplasts; the plasmodesmal fields were found in local thick­ enings of the cell walls facing the bundle sheath which contained both branched (with more branches at the CC side) and sim­ ple plasmodesmata, in contrast to the situation in ICs where only highly branched plasmodesmata with more branches at the IC side were observed in plasmodesmal fields (Figure 1H). In four of these species, Allamanda cathartica , Alstonia macrophylla, Plumeria rubra , and Thevetia nereifolia , leaf sugars were analyzed by GC-MS but no members of the RFO family were detected (data not shown) which, taking into account the high sensitiv­ ity of the method, means that these cells do not synthesize RFO. Such companion cells, to our knowledge, have not been described previously. We refer to these cells as CC-b (Table 1). Quite peculiar companion cells which, to our knowledge, have not been described before were found in minor vein phloem of several hemiparasitic Orobanchaceae species of the genera Euphrasia , Melampyrum , Odontites , and Rhinanthus. These cells contained plasmodesmal fields with numerous asymmetrically branched plasmodesmata, resembling those in ICs, along with highly developed cell wall ingrowths (Figure 1J). The plastids found in such cells were leucoplasts which in some cases con­ tained one or two single thylakoids; the presence of chloroplasts could not be confirmed. Combinations of plasmodesmal fields and cell wall protuberances were previously reported for MICs described for Asarina scandens (Turgeon et al., 1993) and A. bar- claiana (Voitsekhovskaja et al., 2006; Figure II). However, con­ trary to the companion cells ofthe hemiparasitic Orobanchaceae, MIC in Asarina species contained only small plasmodesmal fields and only few cell wall ingrowths. Sugar analyses in leaves of Euphrasia , Melampyrum , and Rhinanthus species revealed high amounts of sucrose and a sugar alcohol galactitol (dulcitol) but no raffinose or stachyose (data not shown). We classified these companion cells as MIC-a for Asarina species, and MIC-b for hemiparasitic Orobanchaceae, respectively (Table 1). SPATIAL ORGANIZATION OF MINOR VEINS IN ASTERIDAE AND CLASSIFICATION OF MINOR VEIN PHLOEM INTO SUBTYPES The analysis of companion cell types in Asteridae raised the questions as to how various subtypes of companion cells can be combined in a phloem ending, and how the newly described structures are related to the spatial organization of minor veins. In type 1 (“open” type) minor veins as described by Gamalei (1989) for Syringa vulgaris , the first divisions ofthe phloem initial are anticlinal, sometimes occurring as an anticlinal bifurcation. Therefore, one initial produces two to three cells at once. Each of them subsequently undergoes one unequal division. The smaller cell differentiates in a sieve element, and the larger cell in a com­ panion cell. As a result, two or three SE-CCCs form an arc around the abaxial pole of the radial xylem ray in a mature minor vein of type 1 (Figure 2A). This sequence of cell divisions resembles the radial divisions characteristic for the cambium of trees. The xylem and phloem parts of the minor vein in type 1 are usually separated by a row of parenchyma cells designated either as vas­ cular parenchyma, or as phloem parenchyma, which is not quite correct because these cells do not originate from the phloem ini­ tial. Here these cells are referred to as vascular parenchyma. In type 2 (“closed” type) minor veins, the first divisions ofxylem and A 8 В В FIGURE 2 I Schemes showing cell division planes in course of minor vein development [redrawn from Gamalei (1990)]. (A) type 1; (B) type 2. Phloem initial cell and mother cell of the phloem complex are shown in pink, SE-CCCs are shown in red, and phloem parenchyma cells are shown in purple. The vascular parenchyma is shown in green. Frontiers in Plant Science | Plant Physiology August 2013 | Volume 4 | Article 312 | 6