Cytokinin deficiency causes distinct changes of sink and source parameters in tobacco shoots and roots

Abstract

Cytokinin deficiency causes pleiotropic developmental changes such as reduced shoot and increased root growth. It was investigated whether cytokinin-deficient tobacco plants, which overproduce different cytokinin oxidase/dehydrogenase enzymes, show changes in different sink and source parameters, which could be causally related to the establishment of the cytokinin deficiency syndrome. Ultrastructural analysis revealed distinct changes in differentiating shoot tissues, including an increased vacuolation and an earlier differentiation of plastids, which showed partially disorganized thylakoid structures later in development. A comparison of the ploidy levels revealed an increased population of cells with a 4C DNA content during early stages of leaf development, indicating an inhibited progression from G2 to mitosis. To compare physiological characteristics of sink leaves, source leaves and roots of wild-type and cytokinin-deficient plants, several photosynthetic parameters, content of soluble sugars, starch and adenylates, as well as activities of enzymes of carbon assimilation and dissimilation were determined. Leaves of cytokinin-deficient plants contained less chlorophyll and non-photochemical quenching of young leaves was increased. However, absorption rate, photosynthetic capacity (F(v)/F(m) and J(CO2 max)) and efficiency (Phi CO(2 app)), as well as the content of soluble sugars, were not strongly altered in source leaves, indicating that chlorophyll is not limiting for photoassimilation and suggesting that source strength did not restrict shoot growth. By contrast, shoot sink tissues showed drastically reduced contents of soluble sugars, decreased activities of vacuolar invertases, and a reduced ATP content. These results strongly support a function of cytokinin in regulating shoot sink strength and its reduction may be a cause of the altered shoot phenotype. Roots of cytokinin-deficient plants contained less sugar compared with wild-type. However, this did not negatively affect glycolysis, ATP content, or root development. It is suggested that cytokinin-mediated regulation of the sink strength differs between roots and shoots.

Keywords: Carbohydrates; cell cycle; cytokinin; cytokinin deficiency; differentiation; invertase; meristem; photosynthesis; sink; source.

Fig. 1.

Altered ultrastructure of cells in shoot meristems of cytokinin-deficient tobacco plants. Transmission electron micrographs of cells from the peripheral zone of wild-type (A, B) and 35S:CKX1 transgenic shoot meristems (C–E) are presented at a similar magnification. N, Nucleus; m, mitochondria; p, proplastid; V, vacuole. Arrows in C, D, and E indicate the unusually large tubuli arising from the inner membrane of mitochondria, sectioned transversally (C, D) and longitudinally (E). Scale bars = 2 μm (A, C) and 1 μm (B, D, E).

Fig. 2.

Altered cellular ultrastructure in developing leaves of cytokinin-deficient plants. Transmission electron micrographs of young leaves (leaf 3; see Fig. 3) from wild-type (A–C) and 35S:CKX1-expressing (D–G) tobacco plants. Cross-section of wild-type (A) and 35S:CKX1 (D) leaf. The asterisk indicates a cell in preparation for mitosis with dense cytoplasm and condensed chromatin. (B, E) Comparison of chloroplast structure (arrows indicate grana). (C, F) Comparison of adaxial epidermal cells (arrows point to irregular cell wall thickness). (G) Necrotic cell with pycnotic nuclei and disorganized plastid and membrane. CW, Cell wall; E, epidermis; N, nucleus; PP, palisade parenchyma; SP, spongy parenchyma. Scale bars = 10 μm (A, D), 2 μm (B, E), and 1 μm (C, F, G).

Fig. 3.

Cell cycle parameters are altered in cytokinin-deficient shoot organs. Histograms of nuclear DNA distribution in wild-type and 35S:CKX1-expressing tobacco plants. Pooled apices and pooled leaves of different developmental stages (leaf 1 being the youngest visible leaf) were analysed using juvenile (4-week-old plants with 4–5 leaves) and mature (15-week-old plants with 20–22 leaves) plants. The x- and y-axis show DNA fluorescence (log scale) and the frequency of stained nuclei, respectively. For each sample, 5000–10 000 nuclei were analysed. Samples consisted of pooled tissues from 5 to 10 plants.

Fig. 4.

Maximum quantum yield (F_v/F_m) of PSII in source and sink leaves of wild-type and cytokinin-deficient tobacco plants. Data represent mean values ±SD (n ≥30). Representative results from two independent measurements are shown. Pairwise Student's t-test was used to compare values with the wild type. *, P < 0.05; **, P < 0.01. WT, Wild type.

Fig. 5.

Non-photochemical quenching of chlorophyll fluorescence (qN) in source and sink leaves of wild-type and cytokinin-deficient tobacco plants. Data represent mean values ±SD (n=3–6). Representative results from two independent measurements are shown. Pairwise Student's t-test was used to compare values with the wild type. *, P < 0.05; **, P < 0.01. WT, Wild type.

Fig. 6.

Contents of carbohydrates in sink and source tissues of wild-type and cytokinin-deficient tobacco plants. Data represent mean values ±SD (n=6). Representative results from three independent experiments are shown. Pairwise Student's t-test was used to compare values with the wild type. *, P < 0.05; **, P < 0.01. FW, Fresh weight; WT, wild type.

Fig. 7.

ATP and ADP content in sink and source tissues of wild-type and cytokinin-deficient tobacco plants. Data represent mean values ±SE (n=6–9) of three independent experiments. Pairwise Student's t-test was used to compare values with the wild type. *, P < 0.05; **, P < 0.01. FW, Fresh weight; WT, wild type.

Fig. 8.

Invertase activity in sink and source tissues of wild-type and cytokinin-deficient tobacco plants. Data represent mean values ±SD (n=6). Representative results from three independent experiments are shown. Pairwise Student's t-test was used to compare values with the wild type. *, P < 0.05; **, P < 0.01. C, Cytosolic; CW, cell wall; V, vacuolar; WT, wild type.

Fig. 9.

Activity of PFK, PFP, aldolase, and enolase in sink and source tissues of wild-type and cytokinin-deficient tobacco plants. Data represent mean values ±SD (n=6). Representative results from three independent experiments are shown. Activity of PFP in CKX-expressing source leaves was reduced to detection limit in all replicate measurements. Pairwise Student's t-test was used to compare values with the wild type. *, P < 0.05; **, P < 0.01. WT, Wild type.