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 and A or В refer to less or more advanced structural specialization, respectively. The data also make clear that in many species with “open” minor vein cytology, i.e., putative symplasmic phloem load­ ers with multiple plasmodesmata at the bundle sheath/phloem boundary, minor veins often contain a SE-CCC specialized for apoplasmic loading. A well-known example is the minor vein phloem in Coleus Ыитеі (Fisher, 1986). The present study pro­ vides a quantitative estimate of the occurrence of species of the Asteridae with more than one type of SE-CCC in their minor veins (Figure 6). About half of the “putative symplasmic phloem loaders” in the Asteridae (subtypes 1-II and 1-III) seem to rely on apoplasmic phloem loading in addition to the symplasmic path­ way. This proportion might be even higher regarding the fact that also “pure symplasmic loaders” of subtype 1-1 can additionally contain minor veins of the 1-II type in their leaves (e.g., Paederia scandens or Scrophularia americana; Supplemental Table 1). The idea that always both symplasmic and apoplasmic mechanisms contribute to phloem loading, one usually being the dominant loading mode, is not new (see e.g., Kursanov, 1984). However, functional evidence for this idea has been provided only recently using a stachyose-translocating species, Alonsoa meridionalis. In minor veins of A. meridionalis which belong to the subtype 1- II according to the classification of this study, expression of the stachyose synthase gene AmSTSl was found only in the two adaxial SE-CCCs containing ICs while the sucrose transporter AfflSUTl was localized at the plasma membrane of the ОС in the abaxial SE-CCC (Voitsekhovskaja et al., 2009). These data indicate that apoplasmic loading operates even in species with “open” minor vein anatomy, at least in a large part of them. Yet ca. 30% of the Asteridae examined in this study (97 out of 315 species belonging to subtypes 1-1 and 1-IV) seem to use no additional structures specialized for the apoplasmic phloem loading. Interestingly, in the IC-containing species Cucumis melo, a switch from symplasmic to apoplasmic loading has been demonstrated upon Cucumber Mosaic Virus infection; more­ over, sucrose uptake activity from the apoplast into m inor veins was detected in healthy C. melo plants (Gil et al., 2011). Further studies are required to understand how symplasmic phloem load­ ing works in plants belonging to subtypes 1-1 and 1-IV, whether apoplasmic loading takes place, and which mechanisms are used in companion cells of type 1-IV to prevent sucrose leakage into the mesophyll via plasmodesmata in the absence of RFO synthesis. In conclusion, the detailed analysis of the minor vein phloem in the large group of species from the subclass Asteridae presented in this study shows that there is more variation in minor vein organization than previously known. This opens up the field to further developmental, phylogenetic, and functional analyses of phloem loading. Several points which deserve further investiga­ tion are: what are transport sugars and loading mechanisms in subtypes 1-1 and 1-IV species (is apoplastic loading involved or not)? What are the mechanisms underlying the strong correla­ tion of type 1/2 minor vein symmetry and the presence/absence of plasmodesmal fields in companion cells? What are phylo­ genetic relationships between different m inor vein types and subtypes? What are the functions of plasmodesmal fields and cell wall ingrowths, respectively, in hemiparasitic Orobanchaceae with MIC-b cells? ACKNOWLEDGMENTS We are indebted to Alexey L. Shavarda and Alexandra N. Ivanova (Komarov Botanical Institute RAS, St. Petersburg, Russia) for the GC-MS analyses of leaf extracts and expert ТЕМ assis­ tance. We wish to thank Bob Turgeon (Cornell University, Ithaca, NY, USA) for stimulating discussions, John Patrick and Tina Offler (University of Newcastle, Newcastle, Australia) for expert comments, Katharina Pawlowski (Stockholm University, Stockholm, Sweden) for critical reading, and Aart Van Bel (University of Giessen, Germany) for invaluable suggestions to improve the manuscript. This study greatly benefited from the discussions and scientific cooperation with Aart Van Bel and the late Hubert Ziegler (Technical University of Munich, Germany). The financial support of the Russian Foundation for Basic Research (1995-2012), of the Ministry of Education and Science of the Russian Federation (grants # P289, #8133) and of the Russian Academy of Sciences Presidium Programme “Biodiversity” to Yuri V. Gamalei and Olga V. Voitsekhovskaja is gratefully acknowledged. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: http://www.frontiersin.org/Plant_Physiology/ 10.3389/fpls.2013.00312/abstract REFERENCES Angiosperm Phylogeny Group. (2009). An update of the Angiosperm Phylogeny Group classifica­ tion for the orders and families of flowering plants: APG III. Bot. J. Linn. Soc. 161, 105-121. doi: 10.1111/j. 1095-8339.2009. 00996.x Batashev, D. R., and Gamalei, Yu. V. (2000). Organization of the m inor vein phloem in representatives of the Gentianaceae. Bot. Zh. 85, 1 - 8 . Batashev, D. R., and Gamalei, Yu. V. (2005). Organization of the m inor vein phloem in representatives of the Apocynaceae. Bot. Zh. 90, 1368-1379. Botha, С. E. J., Aoki, N., Scofield, G. N., Liu, L., Furbank, R. Т., and White, R. G. (2008). A xylem sap retrieval pathway in rice leaf blades: evidence of a role for endocytosis. /. Exp. Bot. 59, 2 9 45-2954. doi: 10.1093/jxb/ ern l50 Burch-Sm ith, Т. 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