In Situ Hybridization of RcSCR1

In an attempt to examine the expression site of RcSCR1, digoxigenin labelled sense and anti-sense probes were synthesized. Two different methods were used to synthesize these probes, (1) the full-length probe was synthesized and then partially hydrolyzed to about 200 bases and (2) a new plasmid was constructed, which contains the 3’-NTR of the RcSCR1.

To construct the new plasmid, a PCR was performed to amplify the RcSCR1 3’-NTR fragment. Afterwards the PCR product was checked in an agarose gel and then the DNA fragment of RcSCR1 3’-NTR was ligated to pGEM-T vector. The pSCR1NTR (Figure 30), which contains the RcSCR1 3’-NTR, was cut by NotI or NcoI. The sense probe and the anti-sense probe were obtained by in vitro transcription using SP6 RNA polymerase or T7 RNA polymerase. Because the sense and anti-sense probes were short enough, they need not be hydrolyzed. Then the in situ hybridization was performed with different probes.

200 ng of probe were added to 1 ml hybridization solution.

An optimal sucrose transport rate was found in 5-day-old Ricinus seedlings (Komor, 1977).

Using tissues sections of 5-day-old seedlings to perform in situ hybridization and strong hybridization signal should be found in cotyledons. The results indicated that no hybridization signal was detected in the sections, which were hybridized with the sense probe. But a strong hybridization signal was detected in the sections, which were hybridized with the anti-sense probe (Figure 31).

pSCR1NTR

Figure 30: The restriction map of pSCR1NTR. The 3´-NTR of RcSCR1 was cloned to pGEM-T vector.

This plasmid was used to generate RNA probe encoded to 3´-NTR of RcSCR1.

Using hydrolyzed probes to perform in situ hybridization led to the similar results compared to using non-hydrolyzed digoxigenin-labelled probes. To examine the expression of RcSCR1 during the development of the seedling, in situ hybridization was performed with the endosperm and the cotyledons of 2 - 6 days old seedlings. No hybridization signals were detected in all sections of day 2 to day 6 samples that were incubated with the hybridization buffer and sense probe (Figure 32, A, D, G, J and M).

Day 2 cotyledons showed only very weak hybridization signals with the anti-sense probe(Figure 32, B and C). In the day 3 - 5 samples, the signals in the cotyledons were relatively stronger and were detected mainly in the lower epidermis (Figure 32, E, F, H, I, K and L). In the day 6 sample, the highest expression of RcSCR1 was foound in the palisade parenchyma cell layer (Figure 32, N and O). Under low magnification, some dark staining was seen in the endosperm sections of day 4, 5 and 6 samples, but the hybridization signal was hardly seen under high magnification, where it was clearly detected in the cells of the cotyledons. No hybridization signal was found in the phloem tissue of the cotyledons at any age of the seedling.

Different tissue sections of 6 days old seedlings were used for RcSCR1 in situ hybridization (Figure 33). The results indicated that no hybridization signal was detected in the sections, which were hybridized with the sense probe (Figure 33, A, C). But a strong hybridization signal was detected in the sections, which were hybridized with the anti-sense probe (Figure 33, B, D). In these sections, the hybridization signals were localized in the cotyledons and the hypocotyl but not in the endosperm (Figure 33, B). In the cotyledons, the RcSCR1 transcript was detected mainly in the palisade parenchyma cell layer (Figure 33, B), in the hypocotyl, it was detected in the phloem area (Figure 33, D).

Figure 31 : In situ hybridization of RcSCR1. Cross sections of cotyledons of day 5 seedling were used. They were hybridized with digoxigenin -labelled anti-sense probe or sense probes. RcSCR1 mRNA could be found in the lower epidermis of the cotyledons. The arrows indicate the hybridization signal. Bar = 50 µm.

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Figure 32: (next page) In situ hybridization of RcSCR1. All sections (10 µm thick) were hybridized with digoxigenin-labelled anti-sense or sense probe. (A), (D), (G), (J) and (M) are negative controls.

They were hybridized with the sense probe. (B), (C), (E), (F), (H), (I), (K), (L), (N) and (O) are samples.

They were treated with the anti-sense probe. (A), (B), (D), (E), (G), (H), (J), (K), (M) and (N) were observed under low magnification. The arrows indicate the localization of RcSCR1 mRNA. (A), (B) and (C) : cotyledons and endosperm sections of day 2; (D), (E) and (F) : cotyledons and endosperm of day 3; (G), (H) and (I) : cotyledons and endosperm sections of day 4; (J), (K) and (L) : cotyledons and endosperm sections of day 5; (M), (N) and (O) : cotyledons and endosperm sections of day 6. Bar in (A), (B), (D), (E), (G), (H), (J), (K), (L) and (M) = 1 mm; bar in (C), (F), (I), (L) and (O) = 100 µm.

Sense Anti-sense Anti-sense

d a y 2

d a y 3

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Expression of RcSCR1 was not only found in the cotyledons, but also in other tissues.

RcSCR1 was found in most of the cells of the young developing leaves of the seedling (Figure 34, A). The RcSCR1 was also found in the phloem of the hypocotyl and the petiole

(Figure 34, B and C). In the young developing seed, RcSCR1 was also found in the seed coat (Figure 35, A and B). The results indicated that RcSCR1 expression is not restricted to the germinating seedling, but is also found in d eveloping seeds and other tissues.

Sense probe Anti-sense probe

Figure 33: In situ hybridization of RcSCR1. (A) and (C) are negative controls. They were hybridized with the sense probe. (B) and (D) are samples. They were hybridized with the anti-sense probe. The arrows indicate the hybridization signal. Cross section of (A) and (B): cotyledons of day 6; (C) and (D): hypocotyl cross sections of day 6; Ph, phloem; Xy, xylem. Bar in (A) to (B) = 200 µm, bar in (C), (D) = 50 µm.

Figure 34: RcSCR1 in situ hybridization of different tissues of Ricinus communis. The sections were hybridized with anti-sense probe. (A) young developing leaf, (B) petiole. The arrows indicate the hybridization signals of RcSCR1.

Anti-sense

Sense

Figure 35 : RcSCR1 in situ hybridization of developing seeds of Ricinus communis. The RcSCR1 mRNA was found in the seed coat of developing seeds. The arrows indicate the RcSCR1 mRNA in the cross section of developing seeds. (A) and (B) were hybridized with anti-sense probe. (C) and (D) were hybridized with sense probe.

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C D

A B