Down-regulation of the IbEXP1 gene enhanced storage root development in sweetpotato

Abstract

The role of an expansin gene (IbEXP1) in the formation of the storage root (SR) was investigated by expression pattern analysis and characterization of IbEXP1-antisense sweetpotato (Ipomoea batatas cv. Yulmi) plants in an attempt to elucidate the molecular mechanism underlying SR development in sweetpotato. The transcript level of IbEXP1 was high in the fibrous root (FR) and petiole at the FR stage, but decreased significantly at the young storage root (YSR) stage. IbEXP1-antisense plants cultured in vitro produced FRs which were both thicker and shorter than those of wild-type (WT) plants. Elongation growth of the epidermal cells was significantly reduced, and metaxylem and cambium cell proliferation was markedly enhanced in the FRs of IbEXP1-antisense plants, resulting in an earlier thickening growth in these plants relative to WT plants. There was a marked reduction in the lignification of the central stele of the FRs of the IbEXP1-antisense plants, suggesting that the FRs of the mutant plants possessed a higher potential than those of WT plants to develop into SRs. IbEXP1-antisense plants cultured in soil produced a larger number of SRs and, consequently, total SR weight per IbEXP1-antisense plant was greater than that per WT plant. These results demonstrate that SR development was accelerated in IbEXP1-antisense plants and suggest that IbEXP1 plays a negative role in the formation of SR by suppressing the proliferation of metaxylem and cambium cells to inhibit the initial thickening growth of SRs. IbEXP1 is the first sweetpotato gene whose role in SR development has been directly identified in soil-grown transgenic sweetpotato plants.

Fig. 1.

Expression pattern of IbEXP1. (A) Developmental stages of sweetpotato. (B) RNA gel blot analysis of IbEXP1. Full-length IbEXP1 cDNA was used as a probe. The ethidium bromide-stained rRNA is shown as a loading control (lower panel). FR, fibrous root (diameter <0.2cm); YSR, young storage root (diameter 0.5–1.0cm); MSR, mature storage root (diameter >5cm); FR-MSR, fibrous root from mature storage root stage. (This figure is available in colour at JXB online.)

Fig. 2.

Effect of various hormones on the expression of IbEXP1. Transcript levels of IbEXP1 in response to treatment with various concentrations of exogenous IAA, JA, or BA for 3h. Total RNA was extracted from the fibrous roots treated with the three hormones. Real-time RT-PCR data were normalized to those for the endogenous β-tubulin gene. Error bars indicate the standard deviation (SD) between three technical replicates measured on fibrous roots collected from at least three different sweetpotato plantlets and subsequently pooled for analysis.

Fig. 3.

Characterization of IbEXP1-antisense sweetpotato plants. (A) Transcript levels of IbEXP1 in IbEXP1-antisense plants. Real-time RT-PCR analysis was carried out with total RNAs extracted from fibrous roots grown in vitro. Data were normalized to those for the endogenous β-tubulin gene. Error bars indicate the standard deviation (SD) between three technical replicates measured on fibrous roots collected from at least three different sweetpotato plantlets. (B) Morphology of the IbEXP1-antisense plants. Pictures were taken at 10 d after planting. (C) Root length of IbEXP1-antisense plants. Root length was measured with the three longest roots of each plant. (D) Root diameter of IbEXP1-antisense plants. Root diameter was measured with a dial caliper by measuring the thickest root of each plant. (C and D) Data were collected from sweetpotato plants cultured in vitro for 10 d after planting and are the means ±SD from three separate measurements of three individual plants. Different letters above the bars indicate significantly different means (P < 0.05) as analysed by Duncan’s multiple range test using the SAS statistical program (SAS Institute, Cary, NC, USA). (A–D) Numbers #1-#3 represent IbEXP1-antisense sweetpotato lines #1-#3, respectively. WT, wild type. (This figure is available in colour at JXB online.)

Fig. 4.

Effect of down-regulation of IbEXP1 on the growth of epidermal cells in fibrous roots. (A) Longitudinal sections of epidermis were prepared from fibrous roots of sweetpotato plants cultured in vitro for 10 d after planting. The scale bar represents 50 µm. (B) The length of epidermal cells of the fibrous root. (C) The width of epidermal cells of the fibrous root. (B and C) Between 15 and 30 cells of at least three individual plants were measured. Data are the means ±standard deviation (SD) from three separate experiments. (A–C) Numbers #1 and #2 represent IbEXP1-antisense sweetpotato lines #1 and #2, respectively. WT, wild type. (This figure is available in colour at JXB online.)

Fig. 5.

Effect of down-regulation of IbEXP1 on cell proliferation in fibrous roots. Transverse sections of fibrous roots were prepared from in vitro cultured sweetpotato plants at 10 d after planting. The right panel is an enlarged image of the left panel. Numbers #1 and #2 represent IbEXP1-antisense sweetpotato lines #1 and #2, respectively. Scale bars: 200 µm. MPH, metaphloem; MX, metaxylem; PC, primary cambium; WT, wild type. (This figure is available in colour at JXB online.)

Fig. 6.

Altered lignification in the fibrous roots from IbEXP1-antisense sweetpotato plants. (A) Histochemical analysis of lignin deposition in the fibrous roots from IbEXP1-antisense plants. Transverse hand-prepared sections of the fibrous roots from the wild-type (WT) and IbEXP1-antisense plants (#1 and #2) cultured in vitro for 3 weeks were stained with phloroglucinol-HCl. The stele areas of the FRs in the left panel are enlarged in the right panel. Lignified material is stained violet. EP, epidermis; CMX, central metaxylem; MX, metaxylem. (B) Transcript levels of the cinnamyl alcohol dehydrogenase (CAD) gene in IbEXP1-antisense plants. Total RNAs were extracted from fibrous roots of WT and IbEXP1-antisense plants (#1 and #2) cultured in vitro for 3 weeks. Real-time RT-PCR data were normalized to those for the endogenous β-tubulin gene. Error bars indicate the standard deviation (SD) between three technical replicates measured on fibrous roots collected from at least three different sweetpotato plants and subsequently pooled for analysis. (This figure is available in colour at JXB online.)

Fig. 7.

Phenotypic alteration in IbEXP1-antisense sweetpotato plants grown in soil. Sweetpotato plants were grown in soil and harvested at 5 months after planting. Numbers #1–#3 represent IbEXP1-antisense sweetpotato lines #1–#3, respectively. WT, wild type. (This figure is available in colour at JXB online.)

Fig. 8.

Storage root development in IbEXP1-antisense sweetpotato plants. (A) Site of storage root development. Red arrows represent the FRSS [fibrous root between storage root and underground stem]. (B) Length of the FRSS. Data were collected from the sweetpotato plants grown in soil at 5 months after planting and are the means ±standard deviation (SD) from three separate measurements of three individual plants. (A and B) Numbers #1–#3 represent IbEXP1-antisense sweetpotato lines #1–#3, respectively. WT, wild type. (This figure is available in colour at JXB online.)