A sepal-expressed ADP-glucose pyrophosphorylase gene (NtAGP) is required for petal expansion growth in 'Xanthi' tobacco

Abstract

In this study, a tobacco (Nicotiana tabacum 'Xanthi') ADP-glucose pyrophosphorylase cDNA (NtAGP) was isolated from a flower bud cDNA library and the role of NtAGP in the growth of the floral organ was characterized. The expression of NtAGP was high in the sepal, moderate in the carpel and stamen, and low in the petal tissues. NtAGP-antisense plants produced flowers with abnormal petal limbs due to the early termination of the expansion growth of the petal limbs between the corolla lobes. Microscopic observation of the limb region revealed that cell expansion was limited in NtAGP-antisense plants but that cell numbers remained unchanged. mRNA levels of NtAGP, ADP-glucose pyrophosphorylase activity, and starch content in the sepal tissues of NtAGP-antisense plants were reduced, resulting in significantly lower levels of sugars (sucrose, glucose, and fructose) in the petal limbs. The feeding of these sugars to flower buds of the NtAGP-antisense plants restored the expansion growth in the limb area between the corolla lobes. Expansion growth of the petal limb between the corolla lobes was severely arrested in 'Xanthi' flowers from which sepals were removed, indicating that sepal carbohydrates are essential for petal limb expansion growth. These results demonstrate that NtAGP plays a crucial role in the morphogenesis of petal limbs in 'Xanthi' through the synthesis of starch, which is the main carbohydrate source for expansion growth of petal limbs, in sepal tissues.

Figure 1.

Genomic Southern-blot analysis of NtAGP. Genomic DNA gel blots were hybridized with full-length NtAGP cDNA or the 205-bp NtAGP-specific DNA fragment. Each lane represents 50 μg of genomic DNA digested with the indicated restriction enzymes. The DNA size markers are indicated in kilobases (kb).

Figure 2.

Expression pattern of NtAGP mRNA. Each lane represents 30 μg of total RNA prepared from the designated tissues. Equal loading of RNA in each lane was verified by the amount of ethidium bromide-stained rRNA shown in the bottom panel. The 205-bp NtAGP-specific DNA fragment was used as a probe. A, Tissue-specific expression of NtAGP. B, Expression of NtAGP mRNA during flower development. The five developmental stages are defined by bud size: <1 cm, bud stage 1; 1 to 2 cm, bud stage 2; 2 to 3 cm, bud stage 3; 3 to 4 cm, bud stage 4; open flower, stage 5. C, Flower organ-specific expression of NtAGP. Total RNA was isolated from the designated organs at open flower stage.

Figure 3.

Phenotypical alteration in the NtAGP-antisense plants. A, Petal parts of the tobacco flowers are illustrated. B, The growth of petal limbs was compared between NtAGP-antisense plant and the ‘Xanthi’ plant. Each developmental stage was classified according to the morphological markers described in Table I. NtAGP, NtAGP-antisense line. [See online article for color version of this figure.]

Figure 4.

NtAGP transcript levels in NtAGP-antisense tobacco plants. Total RNA was extracted from the sepal, anther, and ovary from flowers at stage 9 and from the fully expanded leaf of the flowering plant. Each sample represents pools of material collected from at least five different tobacco plants. Semiquantitative RT-PCR analyses were conducted with NtAGP-specific primers. Tobacco β-tubulin was used as an equal loading internal control. A, Semiquantitative RT-PCR analysis of NtAGP transcript levels in the sepal, leaf, anther, and ovary. Twenty-two cycles of PCR amplification (described in the “Materials and Methods” section) were employed with the sepal, anther, and ovary RNA and 26 cycles with leaf RNA. B, Relative NtAGP transcript levels in the sepal and leaf. Quantification of the NtAGP PCR products was performed using a scanning densitometer. Relative transcript levels are expressed as percentages of those in the ‘Xanthi’.

Figure 5.

Morphological characterization of the petal limb in NtAGP-antisense plants. A, Comparison of flower morphology of antisense lines 3, 4, and 5 at stage 13. B, Comparison of limb length of antisense lines 3, 4, and 5 at stage 13. f, Limb length at the middle of the corolla lobe; g, limb length in the area between the corolla lobes. Measurements were taken on 20 flowers from at least five individual plants of each genotype at stage 13. [See online article for color version of this figure.]

Figure 6.

Effects of NtAGP down-regulation on cell morphology. A, Microscopic observations of the petal limbs at stage 13 in the area marked with a box. B and D, Tangential sections of adaxial epidermis. C and E, Longitudinal cross sections of the petal limb. F and G, Horizontal cross sections of the petal limb. [See online article for color version of this figure.]

Figure 7.

Effects of NtAGP down-regulation on carbohydrate content. Each measurement was performed with pools of sepals or petals from at least five individual plants. Values are the means ± sd of three separate measurements. A, AGPase activity in the sepal from 2-cm-long flower buds. B, Starch content in the sepal from 2-cm-long flower buds. C, Iodine staining of the sepal from 2-cm-long flower buds. [See online article for color version of this figure.]

Figure 8.

Phenotype rescue by sugar feeding. Flower buds of NtAGP-antisense line 3 at stage 9 were collected and incubated in distilled water supplemented with 0 or 200 mm Suc, Glc (Glu in B), or Fru. Three different flower buds were used for each sugar and each sugar concentration. The rescue test was repeated three times. A, Morphology of the petal limbs fed the three sugars; pictures were obtained 72 h after incubation initiation. B, Comparison of the length of the petal limbs fed various sugars. Limb length was measured at 72 h after incubation initiation. The petal lengths described in Figure 5 are represented as f and g. NT, Nontreated. [See online article for color version of this figure.]

Figure 9.

The role of sepal in petal limb growth. Sepals were removed from two different developmental stages of ‘Xanthi’ flower buds, and expansion growth of petal limbs was observed at 72 h after sepal elimination. Five independent floral buds were employed for each developmental stage. The sepal elimination test was repeated three times. A, Developmental stages of floral buds when sepals were removed. 1, Two-centimeter-long floral bud; 2, 4-cm-long floral bud. B, Petal limb morphology observed at 72 h after sepal elimination. Numbers indicate each developmental stage of floral buds when sepals were removed. Control, Petal limbs from flower buds with sepals. [See online article for color version of this figure.]