ORIGINAL ARTICLE Distribution of calcium in the stigma and style of tobacco during pollen germination and tube elongation Li Li Ge Æ Chao Tian Xie Æ Hui Qiao Tian Æ Scott D. Russell Received: 23 August 2008 / Accepted: 19 January 2009 / Published online: 14 February 2009 Ó Springer-Verlag 2009 Abstract Potassium antimonate was used to locate loosely bound calcium in the stigma and style of tobacco. The tobacco stigma is wet and covered by a thick layer of glycoprotein exudate at anthesis. The exudate contains abundant vesicles, which are densely labeled with calcium precipitates. When pollen grains arrive at the stigma, become hydrated, and as the pollen swells, Ca 2? precipi- tates accumulate at the aperture. Calcium precipitates that accumulate in pollen cytoplasm are initially concentrated within small vacuoles, but as germination proceeds these appear to fuse, forming prominent, densely labeled vesicles that preferentially accumulate near the proximal region of the growing tube. Although the stigma has abundant particles, few calcium precipitates are observed in the transmitting tissue from anthesis to 11 h after pollination. However, at 22 h after pollination, accumulation of cal- cium increases distally from the stigmatic interface with the transmitting tissue through the length of the style to the ovary. An examination of flowering plants with differing floral biology will be needed to understand the role of loosely bound calcium accumulation and its relationship to tissue-level changes in calcium uptake, maintenance of other calcium pools, including [Ca 2? ] cyt , and in pollen and style maturation during the progamic phase. Keywords Calcium Á Nicotiana tabacum Á Pollen tube Á Stigma Á Style Introduction Brewbaker and Kwack (1963) first described the require- ment of calcium for in vitro pollen germination, which they found could be met by culturing pollen en masse, resulting in a ‘‘population effect,’’ or by adding calcium to the medium. In either case, calcium proved to have an oblig- atory role in all pollen tube elongation and the importance of calcium in reproductive physiology has been the subject of many studies (Ge et al. 2007). In addition to myriad other metabolic roles of calcium, as a coenzyme, secondary messenger and wall-stabilizing cation, pollen tubes cul- tured in vitro could be attracted by the presence of a calcium gradient (Mascarenhas and Machlis 1962). These workers also found that the Ca 2? increased gradually from the stigma to the ovary in Antirrhinum majus, and this correlated well with the attraction of pollen tubes in vitro. Although promising, the role of a calcium gradient in directing pollen tubes has not proved to be universal in other plants (Glenk et al. 1971; Mascarenhas 1975). Afterwards, few reports appeared in the literature and more emphasis focused on the function of Ca 2? during pollen tube elongation in vitro, in a fundamentally different role, as a largely intracellular signal. Pollen tubes cultured in vitro clearly require the absorp- tion of external calcium and a continuous source is necessary to maintain an intracellular Ca 2? gradient at the tip of the pollen tube for their polar elongation (Holdaway-Clarke Communicated by M. Cresti. L. L. Ge Á H. Q. Tian (&) School of Life Science, Xiamen University, 361005 Xiamen, China e-mail: hqtian@xmu.edu.cn C. T. Xie College of Fisheries, Jimei University, 361021 Xiamen, China S. D. Russell Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019, USA 123 Sex Plant Reprod (2009) 22:87–96 DOI 10.1007/s00497-009-0094-3