Consistency of polyamine profiles and expression of arginine decarboxylase in mitosis during zygotic embryogenesis of Scots pine

Abstract

In this study, we show that both arginine decarboxylase (ADC) protein and mRNA transcript are present at different phases of mitosis in Scots pine (Pinus sylvestris) zygotic embryogenesis. We also examined the consistency of polyamine (PA) profiles with the effective temperature sum, the latter indicating the developmental stage of the embryos. PA metabolism was analyzed by fitting statistical regression models to the data of free and soluble conjugated PAs, to the enzyme activities of ADC and ornithine decarboxylase (ODC), as well as to the gene expression of ADC. According to the fitted models, PAs typically had the tendency to increase at the early stages but decrease at the late stages of embryogenesis. Only the free putrescine fraction remained stable during embryo development. The PA biosynthesis strongly preferred the ADC pathway. Both ADC gene expression and ADC enzyme activity were substantially higher than putative ODC gene expression or ODC enzyme activity, respectively. ADC gene expression and enzyme activity increased during embryogenesis, which suggests the involvement of transcriptional regulation in the expression of ADC. Both ADC mRNA and ADC protein localized in dividing cells of embryo meristems and more specifically within the mitotic spindle apparatus and close to the chromosomes, respectively. The results suggest the essential role of ADC in the mitosis of plant cells.

Figure 1.

The localization of ADC and ODC mRNAs and ADC enzyme in developing zygotic embryos of Scots pine. ADC gene expression was localized by in situ hybridization with DIG-labeled RNA probes (blue signal) and ADC protein by immunolocalization (brown signal). The embryo in A was from sampling date II and the embryos in E, F, and M were from sampling date III. The embryos in B to D, G to L, N to P, and Q were from sampling date IV. A, ADC gene expression was localized in the cytoplasm of the cells in both dominant and subordinate early embryos. B and C, ADC gene expression localized in the cells of primary shoot and root meristem of late embryo. D, ADC gene expression in the mitotic cells of shoot meristem in the late embryo at the cotyledonary stage. E, No label was detected in the sections hybridized with the sense ADC probe. F, No mRNAs of a putative ODC gene were found in the developing early embryo. G, H, I, J, and K, ADC mRNA transcripts during the mitotic stages in the cells of late embryos. L, No label was detected in the mitotic cells in the sections hybridized with the ADC sense probe. M and N, ADC protein was localized in the nuclei of embryos. O and P, ADC protein was located with the chromosomes in the mitotic cells. Q, No signal was detected in the control sections incubated with preimmune serum.

Figure 2.

The fluctuation of free and soluble conjugated PAs (Put, Spd, and Spm) in developing zygotic embryos of Scots pine. Immature embryos were collected four times in 2001 and 2003 throughout the period of embryo development from two Scots pine clones, K818 and K884. The free and soluble conjugated PA concentrations are presented relative to the effective temperature sum (d.d.) and the year (• = 2003, ○ = 2001). The mean values of the four replicates pertaining to the same clone, year, and sampling date are connected by solid lines for 2003 and dashed lines for 2001.

Figure 3.

The expression of ADC gene in developing zygotic embryos of Scots pine in 2003. The expression was quantified by real-time PCR and normalized by the expression of the ACT gene. Observed relative ADC gene expression is presented relative to the effective temperature sum (d.d.) in clones K884 (•) and K818 (○) with the fitted regression lines (solid for K884, dashed for K818). There are three replicates per clone and per sampling date, except the missing data set of clone K818 on sampling date II.

Figure 4.

ADC and ODC enzyme activities in developing zygotic embryos of Scots pine presented in relation to the effective temperature sum (d.d.) in the four sampling dates in 2003. Enzyme activities were analyzed by measuring the ¹⁴CO₂ evolution of decarboxylated radiolabeled Arg and Orn. The black symbols (• and ▪) represent the activity measurements from clone K884 and the open symbols (○, □, ▵, and ▿) those from clone K818. At each sampling date, the replicate measurements (two or three) done from the same protein extraction are marked with the same symbol. Protein extractions per clone and per sampling date varied from one to four. The locations of the symbols are horizontally jittered about the exact x coordinate to separate overlapping values. The geometric mean values for each clone at different sampling dates are connected with solid line for K884 and dashed line for K818.