Regulation of developmental pathways in cultured microspores of tobacco and snapdragon by medium pH

Abstract

The regulation of developmental pathways in cultured microspores of tobacco ( Nicotiana tabacum L) and snapdragon ( Antirrhinum majus L) by medium pH is described for the first time. Unicellular tobacco and snapdragon microspores developed into normal, fertile pollen when cultured in media T1 and AT3 at pH 7.0 and 25 degrees C for 6 and 8 days, respectively. First, pollen mitosis was asymmetric and mature pollen grains were filled with starch granules and germinated upon transfer to a germination medium. However, when tobacco and snapdragon microspores were cultured in media T1 and AT3, respectively, at pH 8.0-8.5 for 4-6 days at 25 degrees C, the frequency of symmetric division increased significantly with the formation two nuclei of equal size, and the gametophytic pathway was blocked, as seen by the lack of starch accumulation and the inhibition of pollen germination. The transfer of these microspores to embryogenesis medium AT3 at pH 6.5 resulted in the formation of multicellular structures in both species and, in tobacco, in the formation of embryos and plants. In order to understand the possible mechanisms of the action of high pH, sucrose metabolism was analysed in isolated microspores of tobacco cultured at various pH values. Invertase (EC 3.2.1.26) activity in microspores was maximal at pH 5.0 and strongly decreased at higher pH, leading to a slow-down of sucrose cleavage. At the same time the incorporation of (14)C-labelled sucrose from the medium into microspores was drastically reduced at high pH. These data suggest that isolated microspores are not able to metabolise carbohydrates at high pH and thus undergo starvation stress, which was shown earlier to block the gametophytic pathway and trigger sporophytic development.

Sucrose and starch catabolism in the anther of Lilium during its development: a comparative study among the anther wall, locular fluid and microspore/pollen fractions

“…Singh and Knox (1984) provided the Wrst direct evidence of the concurrence of a wall-bound extracellular invertase in the pollen of lily, recently conWrmed in cultured microspores of tobacco (Barinova et al 2004). In the present work, we have observed that the wall bound-invertase activity signiWcantly increased coincidentally with the microspore/ pollen wall development, suggesting that sucrose cleavage by this enzyme could be a prerequisite before uptake.…”

Transcriptional and metabolic profiles of stress-induced, embryogenic tobacco microspores

“…Polyamines like spermidine are synthesized by amino acid decarboxylation reactions, which consume H + , and it has been suggested that polyamine accumulation may function as part of a homeostatic mechanism to keep intracellular pH at a constant value (Flores et al 1985). Maintaining pH-homeostasis is an important feature of microspore embryogenesis, as the starvation medium has a comparatively high pH (of 7), and high pH alone is able to reprogram microspores (Barinova et al 2004). Furthermore, polyamines play a role in DNA synthesis (Chattopadhyay et al 2002) and in cell proliferation.…”

Brassica napus Rop GTPases and their expression in microspore cultures

“…Microspore embryogenesis occurs in a wide variety of species (Reynolds 1997) and is extensively utilized by commercial Brassica breeding programs to produce homozygous lines by chromosome doubling treatments of the haploids (Powell 1990;Pauls 1996). The developmental process can be induced by stresses applied to cultured microspores, such as elevated temperatures (Keller and Armstrong 1979;Custers et al 1994), ethanol (Pechan and Keller 1989), gamma irradiation (Pechan and Keller 1989), colchicine (Zhao et al 1996), sucrose starvation (Touraev et al 1996), low temperatures (Sunderland and Dunwell 1974;Kasha et al 1995) and media pH (Barinova et al 2004).…”