Auxin and cytokinin have opposite effects on amyloplast development and the expression of starch synthesis genes in cultured bright yellow-2 tobacco cells

Abstract

In cultured Bright Yellow-2 (BY-2) tobacco (Nicotiana tabacum) cells, the depletion of auxin (2,4-dichlorophenoxyacetic acid) in the culture medium induces the accumulation of starch. This is accelerated by the addition of cytokinin (benzyladenine). Light and electron microscopic observations revealed that this amyloplast formation involves drastic changes in plastid morphology. The effects of auxin and cytokinin on amyloplast development were investigated by adding auxin or cytokinin to cells grown in a hormone-free culture. Auxin repressed amyloplast development, whereas cytokinin accelerated starch accumulation regardless of the timing of hormone addition. RNA gel-blot analysis revealed that the accumulation of the ADP-glucose pyrophosphorylase small subunit gene (AgpS), granule-bound starch synthase, and starch branching enzyme transcripts were also affected by hormonal conditions. High levels of AgpS, granule-bound starch synthase, and starch branching enzyme transcripts accumulated in amyloplast-developing cells grown in auxin-depleted conditions. Furthermore, the addition of auxin to the cells cultured in hormone-free medium reduced the level of AgpS transcripts, whereas the addition of cytokinin increased it, irrespective of the timing of hormone addition. These results suggest that auxin and cytokinin exert opposite effects on amyloplast development by regulating the expression of the genes required for starch biosynthesis.

Figure 1

Effect of hormonal conditions on BY-2 cells. Changes in cell number (top), and starch content per cell (bottom) during culture in D medium (▪), F medium (○), and B medium (▴) are shown. Data are the means of three independent experiments. Vertical bars represent the se.

Figure 2

Frequency histogram of the fluorescence intensities of cell nuclei measured using a video-intensified photon-counting microscope system and expressed in C values, where the fluorescence intensity emitted from prophase cell nuclei was defined as 4C. BY-2 cells were cultured in D, F, or B medium for 48 h and harvested. After fixation, cell nuclei were stained with DAPI and their fluorescence intensities were quantified. Arrows indicate peaks corresponding to C values of the G1 and G2 phase. Note that these histograms do not contain data for M-phase nuclei. Mitotic indices of 9.4%, 2.1%, and 1.5% were observed in nuclei cultured in D, F, and B medium, respectively.

Figure 3

Photomicrographs of amyloplasts in BY-2 cultured cells stained with iodine. Stationary-phase (a–d), 2-d-old cells cultured in D medium (e–h), F medium (i–l), or B medium (m–p) are shown. a, c, e, g, i, k, m, and o, Cells observed with the condenser diaphragm closed; b, d, f, h, j, l, and p, cells observed with the condenser diaphragm open. a and b, c and d, e and f, g and h, i and j, k and l, m and n, and o and p show the same fields. a, b, e, f, i, j, m, and n and c, d, g, h, k, l, o, and p are at the same magnification. Arrows indicate starch granules. N, Cell nucleus. The bars in n and p represent 50 and 10 μm, respectively.

Figure 4

Electron micrographs of leucoplast-like plastids in stationary-phase cells (a), proplastids observed in cells cultured in D medium for 48 h (b), and differentiated amyloplasts in 2-d-old cells cultured in F medium (c) and B medium (d), which are filled with starch grains. M, Mitochondrion; P, plastid; S, starch granule. Scale bar represents 500 nm.

Figure 5

Effects of auxin application on growth and starch accumulation of BY-2 cells grown in hormone-free medium. The left panels show the change in cell number, while the right panels show the change in starch content during culture. Arrowheads indicate the time of auxin addition (final concentrations of 0.2 mg/L). Data are the means from three independent experiments. Vertical bars represent the se. ○, Control cells (without auxin addition); ▪, sample cells (auxin added during culture). BY-2 cells ceased to accumulate starch and started cell division whenever auxin was added to the culture.

Figure 6

Effects of cytokinin application on growth and starch accumulation of BY-2 cells grown in hormone-free medium. The left panels show the change in cell number, while the right panels show the change in starch content during culture. Arrowheads indicate the time of cytokinin addition (final concentration of 1 mg/L). The values are the means from three independent experiments. The vertical bars represent the se. ○, Control cells (without cytokinin addition); ▪, sample cells (cytokinin added during amyloplast development). Starch accumulation was accelerated and cell proliferation was repressed in response to cytokinin addition.

Figure 7

Comparison of AgpS transcript levels under various hormonal conditions. Left, Total RNA was extracted from BY-2 cells cultured for 0, 12, 24, 36, and 48 h in D, F, or B medium and subjected to quantitative RNA gel-blot analyses. Each lane was loaded with 10 μg of total RNA for detection of AgpS and EF-1α, 0.1 μg of total RNA for rRNA. Hybridization signals for transcripts of AgpS (top), elongation factor 1α (middle), and 26S rRNA (bottom) are shown. Right, Changes in the steady-state level of AgpS transcripts under various hormonal conditions. The hybridization signal densities were quantified using the Lane analyzer program. Minor loading differences were accounted for by quantifying the rRNA signals of the 1/100-fold diluted RNA used for the detection of AgpS. The relative accumulation of AgpS mRNA is expressed as a relative value, where the accumulation of stationary-phase cell transcripts is defined as 1. Data are the means from three independent experiments. Vertical bars represent the se. ▪, D medium; ○, F medium; and ▴, B medium.

Figure 8

Comparison of GBSS and SBE transcript levels under various hormonal conditions. Total RNA was extracted from BY-2 cells cultured for 0, 12, 24, 36, and 48 h in D, F, or B medium and subjected to quantitative RNA gel-blot analyses. Each lane was loaded with 20 μg of total RNA for detection. The same samples of RNA used for determining AgpS transcript levels were used for this blot.

Figure 9

Response of AgpS gene expression to auxin and cytokinin added during culture. A, RNA gel-blot analysis. Auxin (left) or cytokinin (right) was added to the culture 0, 12, 24, and 36 h after transferring the stationary-phase cells to hormone-free (F) medium. Total RNA was extracted from the cells at 12-h intervals and subjected to quantitative RNA gel-blot analysis using AgpS as a probe. Arrowheads indicate the time of hormone addition. B, Changes in AgpS transcript levels. The hybridization signal densities were quantified using the Lane analyzer program, and the AgpS mRNA levels were expressed as relative values, where the transcript level in stationary-phase cells was defined as 1. Minor loading differences were calibrated by quantification of the rRNA signal density (not shown). Arrowheads indicate the time of hormone addition. Data are the means from three independent experiments. Vertical bars represent the se. ○, Control cells (no hormone addition); ▪, plus 2,4-D; and ▴, plus BA.