Regulation of cytokinin oxidase (CKX) activity in relation to enzyme glycosylation and secretion was studied in wild‐type (WT) and transgenic conditionally isopentenyltransferase gene (ipt)‐expressing (IPT) tobacco (Nicotiana tabacum L. cv. Wisconsin 38) cell suspensions, calli and leaves. An increase in endogenous cytokinin content due to the tetracycline (Tc)‐induced derepression of the ipt gene transcription or surface application of N6‐benzylaminopurine (BA) resulted in significant enhancement of CKX activity in all these plant materials. As revealed by Concanavalin A‐Sepharose 4B chromatography the cytokinin‐induced enhancement of CKX activity was associated predominantly with the N‐glycoform of the enzyme (10‐ to 15‐fold increase) in calli and leaves. Application of BA to the culture media of WT and IPT cell suspensions and the derepression of the ipt by Tc substantially enhanced endogenous levels of isoprenoid cytokinins and CKX activity in both cells and the culture medium. Most CKX activity in control, BA‐ and Tc‐treated cells was associated with the non‐glycosylated form of the enzyme, whereas the majority of CKX activity in the culture media was due to the glycosylated form. The pH optimum of CKX in cells (pH 8.5) differed considerably from that in the culture medium (pH 6.0). No significant differences were found in apparent Km(iP) values of CKX between control, BA‐ and Tc‐treated IPT cells and media or between purified glycosylated and non‐glycosylated CKX. These results suggest that cytokinins induce changes in the proportions of glyco‐ and non‐glycoforms of the enzyme in multicellular calli and leaves, and influence its secretion to the cell exterior.

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BackgroundMany flowering plants produce bicellular pollen. The two cells of the pollen grain are destined for separate fates in the male gametophyte, which provides a unique opportunity to study genetic interactions that govern guided single-cell polar expansion of the growing pollen tube and the coordinated control of germ cell division and sperm cell fate specification. We applied the Agilent 44 K tobacco gene chip to conduct the first transcriptomic analysis of the tobacco male gametophyte. In addition, we performed a comparative study of the Arabidopsis root-hair trichoblast transcriptome to evaluate genetic factors and common pathways involved in polarized cell-tip expansion.ResultsProgression of pollen grains from freshly dehisced anthers to pollen tubes 4 h after germination is accompanied with > 5,161 (14.9%) gametophyte-specific expressed probes active in at least one of the developmental stages. In contrast, > 18,821 (54.4%) probes were preferentially expressed in the sporophyte. Our comparative approach identified a subset of 104 pollen tube-expressed genes that overlap with root-hair trichoblasts. Reverse genetic analysis of selected candidates demonstrated that Cu/Zn superoxide dismutase 1 (CSD1), a WD-40 containing protein (BP130384), and Replication factor C1 (NtRFC1) are among the central regulators of pollen-tube tip growth. Extension of our analysis beyond the second haploid mitosis enabled identification of an opposing-dynamic accumulation of core regulators of cell proliferation and cell fate determinants in accordance with the progression of the germ cell cycle.ConclusionsThe current study provides a foundation to isolate conserved regulators of cell tip expansion and those that are unique for pollen tube growth to the female gametophyte. A transcriptomic data set is presented as a benchmark for future functional studies using developing pollen as a model. Our results demonstrated previously unknown functions of certain genes in pollen-tube tip growth. In addition, we highlighted the molecular dynamics of core cell-cycle regulators in the male gametophyte and postulated the first genetic model to account for the differential timing of spermatogenesis among angiosperms and its coordination with female gametogenesis.

A tobacco pollen tube glycoprotein, p69 is encoded by the pollen-specific gene ntp303 that is transcribed during pollen development and pollen tube growth, but it is abundantly translated only after pollen germination. To investigate the translational repression of ntp303 mRNA during pollen development the compartmentation of ntp303 mRNA was examined and compared against another transcript (ntp52), which is efficiently translated during pollen maturation. Three subcellular fractions were isolated: a post-polysomal fraction enriched with messenger ribonucleoprotein particles, a polysomal fraction and a novel fraction of EDTA/ puromycin-resistant particles co-sedimentating with polysomes (EPP). At all developmental stages studied, ntp303 mRNA was found to be present in all fractions. Surprisingly, most of the translationally inactive ntp303 mRNA was localised in the polysomal fraction and EPPs, whereas ntp52 mRNA was distributed between the post-polysomal fraction and polysomes but was virtually undetectable in EPPs. This differential mRNA distribution pattern may help to explain the developmentally regulated translational repression of the ntp303 gene during pollen maturation, highlighting a potential role of EPPs. A model of how this differential mRNA compartmentation pattern regulates ntp303 mRNA translation is proposed.

Tobacco (Nicotiana tabacum L.) microspores at the time of mitosis are characterized by the abundant occurrence of 92- and 98-kDa glycoproteins (GP92 and GP98). GP92 is a soluble protein while GP98 is bound to the insoluble microspore fraction. Both glycoproteins were isolated by affinity chromatography and SDS-PAGE and analysed by MS. Peptide sequences were determined by mu-HPLC/nano-ESI-MS/MS (electrospray ionization tandem MS). GP92 displayed homology to beta-galactosidase (EC 3.2.1.23) and GP98 to beta-xylosidase (EC 3.2.1.37) from Arabidopsis thaliana (L.) Heynh. The activities of the two enzymes in microspore and pollen extracts of tobacco exhibited similar developmental changes to the occurrence of GP92 and GP98, with a maximum around microspore mitosis. These two glycoproteins are the first identified enzymes characteristic of mitotic microspores. Arabidopsis transcriptomic data for five beta-galactosidase and three beta-xylosidase genes abundantly expressed in pollen were verified by reverse transcription-PCR of RNA from different stages of Arabidopsis pollen development and from various parts of the sporophyte. The results showed abundant expression of two genes (At5g20710, At1g31740) homologous to tobacco GP92 in microspores and early pollen, and of three genes (At5g56870, At2g16730 and At4g35010) in maturing pollen. Analysis of beta-xylosidases showed abundant expression of a late pollen-specific gene At3g62710 and low expression of an early gene At5g10560. It is suggested that the early beta-galactosidase and beta-xylosidase genes may participate in cell wall loosening associated with pollen expansion after microspore mitosis and that the products of the late genes may play a role in cell expansion during pollen germination.