Regulation of plant growth by cytokinin

Abstract

Cytokinins are a class of plant-specific hormones that play a central role during the cell cycle and influence numerous developmental programs. Because of the lack of biosynthetic and signaling mutants, the regulatory roles of cytokinins are not well understood. We genetically engineered cytokinin oxidase expression in transgenic tobacco plants to reduce their endogenous cytokinin content. Cytokinin-deficient plants developed stunted shoots with smaller apical meristems. The plastochrone was prolonged, and leaf cell production was only 3-4% that of wild type, indicating an absolute requirement of cytokinins for leaf growth. In contrast, root meristems of transgenic plants were enlarged and gave rise to faster growing and more branched roots. These results suggest that cytokinins are an important regulatory factor of plant meristem activity and morphogenesis, with opposing roles in shoots and roots.

Figure 1

AtCKX gene expression and enzyme activity in transgenic tobacco plants and yeast. (A) Northern blots (50 μg total RNA) of individual transformants were probed with gene-specific probes that covered the whole genomic sequences. Only clones with detectable AtCKX transcripts showed a phenotype, and no cross-hybridization with untransformed tobacco (WT) was detected. 25S, hybridization with 25S rRNA as control for loading. (B) Cytokinin oxidase activity in leaves of tobacco plants. Specific activity in extracts of wild type (100%) was 8 ± 0.9 pmol adenine × mg protein⁻¹ × h⁻¹. Bars show SD; n = 3. (C) Cytokinin oxidase activity in yeast cells and medium. Specific activity of the control strain (100%) was 1.16 nmol adenine × mg protein⁻¹ × h⁻¹.

Figure 2

Shoot phenotype of AtCKX1-expressing tobacco plants. (A) Top view of 6-week-old plants. (B) Tobacco plants at the flowering stage. (C) Kinetics of stem elongation. Arrows mark the onset of flowering. Age of plants (days after germination) and leaf number at that stage are indicated above the arrows. Bars indicate SD; n = 12. (D) Number of leaves (n = 12) formed between day 68 and day 100 after germination and final surface area of these leaves (100% of wild type is 3646 ± 144 cm²; n = 3). (E) Comparison of leaf size and senescence. Leaves were from nodes number 4, 9, 12, 16, and 20 from the top (from left to right).

Figure 3

Root phenotype of AtCKX-expressing transgenic tobacco plants. (A) Seedlings 17 days after germination. (B) Root system of soil-grown plants at the flowering stage. (C) Root length, number of lateral roots (LR), and adventitious roots (AR) on day 10 after germination. (D) Dose-response curve of root growth inhibition by exogenous cytokinin. Seeds were sown on MS medium containing 3% sucrose and the indicated concentration of iPR. The length of primary roots was determined after 10 days of cultivation in the dark on vertically positioned plates. Bars indicate ± SD; n = 30.

Figure 4

Histology of shoot meristems, leaves, and root meristems. (A) Longitudinal median section through the vegetative SAM. P, leaf primordia. (B) Vascular tissue in second order veins of leaves. X, xylem, PH, a phloem bundle. (C) Cross sections of fully developed leaves. (D) Scanning electron microscopy of the upper leaf epidermis. (E) Root apices stained with 4′,6-diamidino-2-phenylindole. RM, root meristem. (F) Longitudinal median sections of root meristems 10 days after germination. RC, root cap; PM, promeristem. (G) Transverse root sections 10 mm from the apex. E, epidermis, C1–C4, cortical cell layer; X, xylem; PH, phloem. The material for the analysis of the SAM and the mature fully expanded leaves was from 38- and 100-day-old plants (clone AtCKX1-50), respectively, which were cultivated in a green house. Root analysis was performed with primary roots of seedlings 10 days after germination. Bars, 100 μm.