SRD1 is involved in the auxin-mediated initial thickening growth of storage root by enhancing proliferation of metaxylem and cambium cells in sweetpotato (Ipomoea batatas)

Abstract

A sweetpotato (Ipomoea batatas cv. 'Jinhongmi') MADS-box protein cDNA (SRD1) has been isolated from an early stage storage root cDNA library. The role of the SRD1 gene in the formation of the storage root in sweetpotato was investigated by an expression pattern analysis and characterization of SRD1-overexpressing (ox) transgenic sweetpotato plants. Transcripts of SRD1 were detected only in root tissues, with the fibrous root having low levels of the transcript and the young storage root showing relatively higher transcript levels. SRD1 mRNA was mainly found in the actively dividing cells, including the vascular and cambium cells of the young storage root. The transcript level of SRD1 in the fibrous roots increased in response to 1000 muM indole-3-acetic acid (IAA) applied exogenously. During the early stage of storage root development, the endogenous IAA content and SRD1 transcript level increased concomitantly, suggesting an involvement of SRD1 during the early stage of the auxin-dependent development of the storage root. SRD1-ox sweetpotato plants cultured in vitro produced thicker and shorter fibrous roots than wild-type plants. The metaxylem and cambium cells of the fibrous roots of SRD1-ox plants showed markedly enhanced proliferation, resulting in the fibrous roots of these plants showing an earlier thickening growth than those of wild-type plants. Taken together, these results demonstrate that SRD1 plays a role in the formation of storage roots by activating the proliferation of cambium and metaxylem cells to induce the initial thickening growth of storage roots in an auxin-dependent manner.

Fig. 1.

Expression pattern of SRD1. (A) Developmental stages of sweetpotato. (B) RNA gel blot analysis of SRD1. Full-length SRD1 cDNA was used as a probe. The ethidium bromide-stained rRNA is shown as a loading control (lower panel). FR, fibrous root (diameter <0.2 cm); YSR, young storage root (diameter 0.5–1.0 cm); MSR, mature storage root (diameter >5 cm); FR-MSR, fibrous root from mature storage root stage. (This figure is available in colour at JXB online.)

Fig. 2.

Subcellular localization of SRD1 in onion epidermal cells. (A) The control GFP. (B–E) SRD1::GFP fusion protein. (A, B, D) Fluorescent images. (C, E) Visible light images. (D) Onion cells were stained with the DNA binding dye DAPI. (E) Merged image of GFP with DAPI signal. GFP, green fluorescent protein, DAPI, 4′,6-diamidino-2-phenylindole. (This figure is available in colour at JXB online.)

Fig. 3.

Localization of SRD1 transcripts in young storage root of sweetpotato. Young storage root (diameter 5 mm) and digoxigenin-labelled sense and antisense SRD1 probes were used in in situ hybridization. (A–C) Cross-section of a young storage root. (D–F) Cross-section hybridized with the sense riboprobe. (G– I) Cross-section hybridized with the antisense riboprobe. PC, primary cambium; SC, secondary cambium; PH, primary phloem. (This figure is available in colour at JXB online.)

Fig. 4.

Effect of auxin on the expression of SRD1. Total RNA was extracted from the fibrous roots treated with various concentrations of indole-3-acetic acid (IAA). Real-time RT-PCR data were normalized to those for the endogenous β-tubulin gene. Error bars indicate the standard deviation between three technical replicates measured on fibrous roots collected from at least three different sweetpotato plantlets and subsequently pooled for analysis. Semi-quantitative RT-PCR analyses were conducted with each designated gene-specific primer. The sweetpotato β-tubulin gene was used as an equal loading internal control. (A) Transcript levels of SRD1 in response to treatment with various concentrations of exogenous IAA for 3 h. (B) Time course of the expression of SRD1 and three sweetpotato AUX/IAA genes (BU690901, BU691583, and BU691819) in response to IAA (500 μM) for various time periods (0–24 h).

Fig. 5.

Endogenous IAA concentration and SRD1 transcript levels during early-stage storage root development. (A) Endogenous IAA levels in the unpigmented (diameter 1.0 mm) and pigmented (diameter 1.5 mm) fibrous root, thick root (diameter 3.0 mm), and young storage root (diameter 5.0 mm). Each sample represents pools of roots with a designated maximum diameter from at least three different sweetpotato plants. (B) Transcript levels of SRD1 and three AUX/IAA genes (BU690901, BU691583, and BU691819) in the roots with designated maximum diameters. Real-time RT-PCR data were normalized to those for the endogenous β-tubulin gene. Error bars indicate the standard deviation between three technical replicates measured on roots with a designated maximum diameter collected from at least three different sweetpotato plants and subsequently pooled for analysis.

Fig. 6.

Anatomical characteristics of three different types of sweetpotato roots. Cross-sections of fibrous, thick, and storage roots were prepared from sweetpotato [I. batatas (L.) Lam. cv. ‘Jinhongmi’] roots. The number in parentheses indicates the maximum diameter of each root. PH, phloem; MXY, metaxylem; PXY, protoxylem; PC, primary cambium; SC, secondary cambium; SXY, secondary xylem element. (This figure is available in colour at JXB online.)

Fig. 7.

Characterization of SRD1-ox sweetpotato plants. (A) SRD1 transcript levels in SRD1-ox plants. Total RNA was isolated from fibrous root tissue of the sweetpotato plants cultured in vitro. Semi-quantitative RT-PCR analysis was conducted with SRD1-specific primers. Numbers 1–5 represent SRD1-ox sweetpotato lines 1–5. Sweetpotato β-tubulin was used as an equal loading internal control. WT, wild-type. (B) Morphology of the fibrous roots in SRD1-ox plants. Pictures were taken from the bottom of the culture plate for a better view of the root morphology. (C) Microscopic observation of the transverse section of fibrous root in the area marked with a box in B. The lower panel is an enlarged image of the upper panel. MPH, metaphloem; MX, metaxylem; PC, primary cambium; SC, secondary cambium; PXY, protoxylem. (D) Quantitative characterization of fibrous root development in SRD1-ox lines 1 and 3. Data were collected from the sweetpotato plants cultured in vitro at 14 d after planting and are the means±SD from three separate measurements of three individual plants. Root diameter and cell numbers were measured on transverse sections of fibrous roots. Root diameter was determined by measuring the largest diameter on the circle enclosed by epidermal cells. Numbers of metaxylem cells include numbers of mature and immature metaxylem cells. For determination of cell numbers and root diameters, three cell files from three individual plants were used. WT, wild-type. (This figure is available in colour at JXB online.)