Pollination triggers female gametophyte development in immature Nicotiana tabacum flowers

Abstract

In Nicotiana tabacum, female gametophytes are not fully developed at anthesis, but flower buds pollinated 12 h before anthesis produce mature embryo sacs. We investigated several pollination-associated parameters in N. tabacum flower buds to determine the developmental timing of important events in preparation for successful fertilization. First, we performed hand pollinations in flowers from stages 4 to 11 to study at which developmental stage pollination would produce fruits. A Peroxtesmo test was performed to correlate peroxidase activity on the stigma surface, indicative of stigma receptivity, with fruit set. Pollen tube growth and female gametophyte development were microscopically analyzed in pistils of different developmental stages. Fruits were obtained only after pollinations of flower buds at late stage 7 and older; fruit weight and seed germination capacity increased as the developmental stage of the pollinated flower approached anthesis. Despite positive peroxidase activity and pollen tube growth, pistils at stages 5 and 6 were unable to produce fruits. At late stage 7, female gametophytes were undergoing first mitotic division. After 24 h, female gametophytes of unpollinated pistils were still in the end of the first division, whereas those of pollinated pistils showed egg cells. RT-qPCR assay showed that the expression of the NtEC1 gene, a marker of egg cell development, is considerably higher in pollinated late stage 7 ovaries compared with unpollinated ovaries. To test whether ethylene is the signal eliciting female gametophyte maturation, the expression of ACC synthase was examined in unpollinated and pollinated stage 6 and late stage 7 stigmas/styles. Pollination induced NtACS expression in stage 6 pistils, which are unable to produce fruits. Our results show that pollination is a stimulus capable of triggering female gametophyte development in immature tobacco flowers and suggests the existence of a yet undefined signal sensed by the pistil.

Pollination Type Recognition from a Distance by the Ovary Is Revealed Through a Global Transcriptomic Analysis

“…Moreover, transcripts up-regulated in both conspecific pollinations (CCP and CIP) were specifically enriched in several proteins similar to known regulators of ovule specification and development: ARGONAUTE 4 proteins [66], as well as the AGAMOUS-LIKE 11 [67] and AUXIN RESPONSE FACTOR 5 [68] transcription factors (Dataset S4d). This points to a possible role of conspecific pollination as a signal triggering ovule and female gametophyte development, possibly mediated by ethylene and auxin, as demonstrated previously in other species [38,39,40,41].…”

“…More recently, additional roles were discovered for ethylene signaling in the context of ovule and PT function. While pollination-induced ethylene accumulation in immature tobacco flowers was shown to be correlated to female gametophyte maturation [41], ethylene was also demonstrated to control PT elongation in Arabidopsis by affecting the organization of actin microfilaments [72]. Furthermore, the over-accumulation of the ethylene signal-transducing protein ETHYLENE INSENSITIVE 3 (EIN3) in synergid cells was shown to lead to PT attraction defects in Arabidopsis [73].…”

“…Long-distance signaling during plant reproduction was described almost 150 years ago with the discovery of pollination-induced ovule maturation in orchid species [34,35,36], which was later found to be associated with interorgan ethylene signaling [37,38]. Pollination was also shown to be required to complete female gametophyte development in other species such as almond [39], maize [40], and tobacco [41]. Moreover, pollination is known to trigger several other physiological responses in the flower [42], including petal senescence in orchids [43] and Petunia [44], or changes in floral scent, for example in thistles [45].…”

“…Such responses in distal organs require the modulation of genes at a distance following pollination, well before PTs reach the ovules. Indeed, several studies revealed that pollination induces the expression of ethylene biosynthesis genes in the flowers of orchids [38,46], tomato [47], and tobacco [41]. Moreover, Lantin et al [48] showed that the SPP2 gene from the wild potato species Solanum chacoense , which encodes a dioxygenase, is activated at a distance in the ovary by both compatible pollination and stigma wounding.…”

Enhanced Nicotiana benthamiana immune responses caused by heterologous plant genes from Pinellia ternata

“…About 60 h after pollination, corolla fell down together with protective soda drinking straw. After 20–25 days of pollination, seeds were collected and sun-dried [32].…”