45. Isolation of genes related to sexual reproduction in rice by simplified differential display

K. T. YOSHIDA and T. KUBOYAMA

Graduate School of Agricultural and Life Science, University of Tokyo, Tokyo, 113 Japan

It has long been suggested that many cell-cell interactive events between pollen and pistil during pollen-tube elongation are necessary for successful sexual reproduction in flowering plants. Many molecular markers that express specifically during the reproductive phase are necessary in order to understand the molecular basis of sexual reproduction. We have developed a simplified differential display technique that is effective in detecting and cloning of differentially expressed transcripts (Yoshida et al. 1994). In this study, we used the simplified differential display technique to isolate cDNAs that preferentially accumulate in rice pistils during pollen-tube elongation through fertilization.

In preliminary experiments, the time course of pollen-tube elongation in a rice pistil was examined in a japonica rice cultivar Kamenoo. A rice flower remains open for about 1 hour. Pollen tubes reach the bottom of the ovary during this flowering time. About 90 minutes after the beginning of flowering, a pollen tube enters the micropyle of the ovule. Fertilization is supposed to occur within 2 to 4 hours after flowering. To isolate sexual reproduction-associated genes, we compared mRNA populations from pistils at flowering and at 2 to 4 hours after flowering with those from pistils at 1 day before flowering and ovaries at 1 day after flowering. The cDNAs were prepared from these four mRNA populations using random primers. After removal of the random primers, simplified differential display was used to isolate genes that are preferentially expressed in pistils at flowering and/or 2 to 4 hours after flowering. The RT-PCR products were separated using 1.6% agarose gels and visualized by ethidium bromide staining. Of the 32 different primers tested, 13 primers produced bands that are expected for sexual reproduction-associated cDNA fragments. Of 19 candidates for genes preferentially expressed in pistils after flowering, 11cDNA fragments were recovered from the gel and cloned into vectors for further analysis.

Nucleotide sequences of both ends of the cDNA inserts were determined. To investigate the possible identities of these clones, the sequences were used to search the DNA and protein databases using the BLAST program. As shown in Table 1, two of the clones, RFF07 and RFF09, out of 11 were identified as phospholipase D (PLD) and methylmalonate semialdehyde dehydrogenase, respectively. Methylmalonate semialdehyde dehydrogenase is related to the metabolism of valine and thymine (Kedishvili et al. 1992). PLD generates phosphatidic acid from structural lipids and is related to signal transduction mediated by lipid-derived second messengers (Liscovitch 1992: Nishizuka 1995). Recently, it was demonstrated that PLD is activated through G proteins and it was suggested that signal transduction via PLD activation exists in plants (Munnik et al. 1995), as in animals. PLD in flowering rice pistils might therefore have a potential role in cell to cell communication during sexual reproduction. Further analysis focused on this clone, RFF07.

Because the genes identified using simplified differential display are expected to be expressed only at very low levels and are not easily detected by Northern blot analysis, RT-PCR analysis was performed in order to understand the expression pattern of RFF07. An internal primer set of 18 bases was designed for amplification of the RFF07 gene fragment. The expression level of the gene corresponding to RFF07 dramatically increased at flowering and rapidly decreased afterwards (Fig. 1). The expression pattern of RFF07 was consistent with the pattern observed in the differential display. It is noteworthy that the expression level of the transcript was much lower in pistils at flowering after emasculation with hot water than in normal pistils. The specific expression of the RFF07 may therefore be associated with sexual reproduction (pollen-tube invasion into a stigma or elongation through a style) and not with flowering itself. Lower level of expression was also observed in the embryos at 14 days after flowering. This may indicate that the RFF07 gene has another role in embryo development.

Table 1. Summaries of cDNA clones of rice that are expressed after flowering in nucleotide match with known genes

Clone
Length of
inserts
Length
compared
Putatively
identified genes
RFF01
543
543
none
RFF02
1000
1000
none
RFF03
512
512
none
RFF04
338
338
none
RFF05
1200
827
none
RFF06
598
598
none
RFF07
257 
257
Phospholipase D 
RFF08
1250
879
None
RFF09
411 
411 
Methylmalonate semialdehyde
dehydrogenase 
RFF10
322
322
none
RFF11
169
169
none

Fig. 1 . RT-PCR analysis of RFF07 with cDNA templates prepared from pistils at 1 day before flowering (BFP), pistils at flowering that were emasculated with hot water (FEP), pistils at flowering (FP), pistils at 2 to 4 hours after flowering (AFP), and ovaries at 1 day after flowering, embryos at 14 days after flowering (14E).

References

Kedishvili, N.Y., K.M. Popov, P.M. Rougraff, Y. Zhao, D.W. Crabb and R.A. Harris, 1992. CoA-dependent methylmalonate-semialdehyde dehydrogenase, a unique member of the aldehyde dehydrogenase superfamily. J. Biol. Chem. 267: 19724-19729.

Liscovitch, M., 1992. Crosstalk among multiple signal-activated phospholipases. Trends Biochem. Sci. 17: 393-399.

Munnik, T., S.A. Arisz, T. de Vrije and A. Musgrave, 1995. G protein activation stimulates phospholipase Dsignaling in plants. Plant Cell 7: 2197-2210.

Nishizuka, Y., 1995. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J. 9: 484-496.

Yoshida, K.T., S. Naito and G. Takeda, 1994. cDNA cloning of regeneration-specific genes in rice by differential screening of randomly amplified cDNAs using RAPD primers. Plant Cell Physiol. 35: 1003-1009.