A SEARCH OF BRASSICA SI-INVOLVED ORTHOLOGS IN BUCKWHEAT LEADS TO NOVEL BUCKWHEAT SEQUENCE IDENTIFICATION: MLPK POSSIBLY INVOLVED IN SI RESPONSE

- Self-incompatibility (SI) systems, gamethophytic (GSI) and sporophytic (SSI), prevent self-pollination in angiosperms. Buckwheat displays heteromorphic SSI, with pollination allowed only between different flower morphs - thrum and pin. The physiology of thrum and pin morph SI responses are entirely different, resembling homomorphic Brassica SSI and Prunus GSI responses, respectively. Considering angiosperm species may share ancestral SI genes , we examined the presence of Brassica and Prunus SI-involved gene orthologs in the buckwheat genome. We did not find evidence of SRK , SLG and SP11 Brassica or S-RNase and SFB Prunus orthologs in the buckwheat genome, but we found a Brassica MLPK ortholog. We report the partial nucleotide sequence of the buckwheat MLPK and discuss the possible implications of this finding.


INTRODUCTION
Self-incompatibility (SI) systems are widely distributed among angiosperms (in approximately 60% of all angiosperm species; Hiscock and Kues, 1999).They enable the discrimination between "self" and "non-self" pollen in flowering plants, forming intra-and interspecies reproductive barriers and preserving the genetic variability of species.Basic classification of SI systems to gametophytic (GSI) or sporophytic (SSI) is made according to pollen SI phenotype determinationin GSI systems the pollen self-incompatibility phenotype is determined by its own haploid genotype and in SSI systems it is determined by the diploid genotype of the mother plant.Also, GSI displaying species are always homomorphic (one type of flower per species), while SSI plant species may be homomorphic or heteromorphic (more than one type of flower per species).Today, GSI systems and homomorphic SSI systems are far better studied than heteromorphic SSI, with research into the Primula (McCubbin, 2008) and Fagopyrum (Miljuš-Djukić et al., 1998;Matsui et al., 2004;Matsui et al., 2007).
Common buckwheat (Fagopyrum esculentum Moench.) is of interest as an important nutritive crop in Asia, Australia, the USA and Western Europe.Buckwheat displays a heteromorphic SSI system -it is a distylous plant with two flower types (one flower type per plant), pin (long styles, short stamens) and thrum (short styles, long stamens), equally distributed among the population.Legitimate pollination is possible only between different flower morphs.In the case of illegitimate pollination in thrum flower morph, self-pollen tubes are stopped at the junction between the stigma and the style, while in pin flower morph self-pollen tubes grow to 2/3 of the style length (Miljuš-Djukić et al., 1998) before termination.
Although the physiological data about the SI response upon incompatible pollination in buckwheat are abundant (Miljuš-Djukić et al., 1998;Matsui et al., 2004;Matsui et al., 2007), the data concerning the SI reaction at a molecular level are still very scarce.Considering the different SI responses in two buckwheat morphs upon selfpollination, it is to be expected that different genes and different mechanisms underlying the SI reactions in those morphs will be found.
In light of the fact that the SI response in buckwheat thrum and pin pistils physiologically resembles the SI response in Brassica and Solanaceae, respectively, that similar biochemical processes underlay different SI systems (Miljuš-Djukić For the homomorphic SSI system in Brassica and S-RNase based GSI in Prunus, most of the SI-involved molecular components are well known.The SI reaction in Brassica includes S-locus receptor kinase (SRK), Slocus glycoprotein (SLG) and S-locus cysteine rich (SCR) protein (SP11 protein), with a myristoylated membrane bound kinase (MLPK) as SI signal transducer (Nasrallah et al., 1988;Stein et al., 1991;Goring et al., 1993;Nasrallah et al., 1994;Suzuki et al., 1999;Schopfer et al., 1999;Murase et al., 2004).In Prunus the SI response involves S-locus RNase (S-RNase) and S-locus F-box protein (SFB) (McClure et al., 1989;Lee et al., 1994;Ushijima et al., 2003).In contrast to the rapid SI response in Brassica with the immediate inhibition of self-pollen tube growth at the stigma surface, the S-RNase based GSI system allows self-pollen tube growth to 2/3 of the style's length before its termination.
In this paper we started with the identification of buckwheat SI-involved genes through a search for SI-involved orthologs.We investigated the presence of SRK, SLG, SP11 and MLPK Brassica orthologs as well as S-RNase and SFB Prunus orthologs in the buckwheat genome, using PCR primers designed from conserved regions of Brassica and Prunus genes.Also, for additional S-RNase identification we separated the styles' protein extracts by IEF and stained IEF gel specifically for RNases.The implications of the results are discussed.

Isolation of buckwheat genomic DNA
The fresh leaves of greenhouse-grown buckwheat (Fagopyrum esculentum Moench) were collected, frozen in liquid nitrogen, ground into a fine powder and used for genomic DNA isolation (DNeasy Plant Mini Kit, Qiagen).

PCR identification of target genes
Degenerate forward and reverse primers were designed according to published sequences of SRK, SLG, SP11 and MLPK genes in the genus Brassica (http://www.ncbi.nlm.nih.gov) and adjusted according to buckwheat codone usage.All primer sequences and annealing temperatures used for the search of Brassica orthologs are given in Table 1.The PCR conditions were: 1. 94ºC 2 min; 2. 94ºC 1 min, Tann 1 min, 72ºC 1 min; repeated 34 times; 3. 72ºC 10 min.The study of Prunus orthologs was conducted using specific primers and PCR conditions as stated in Banović et al., (2009).
The PCR product of the targetted MLPK gene was purified (PCR Purification Kit, Qiagen), ligated with deoxyadenine and cloned into the pGEM-T Easy Vector System (Promega).Plasmids with inserts of an appropriate size were selected by PCR amplification using MLPK specific primers and by restriction digestion with EcoRI and KpnI (New England BioLabs, UK).
The nucleotide sequence was obtained using an ABI3730XL DNA Analyzer (Applied Biosystems) (commercially done by Macrogen company).

Isoelectric focusing of stylar protein extracts and staining for RNase activity
Whole protein stylar extracts were prepared, separated by isoelectric focusing (IEF) and the IEF gel was stained for ribonuclease activity as described by Bošković and Tobutt (1996).The conditions for electrophoresis were as in Banović et al., (2009).

Computer-assisted analysis
The obtained partial MLPK nucleotide sequence published in this paper was deposited at the NCBI data base under accession number FJ858190.The deduced amino acid sequence was compared to other protein sequences in the NCBI data base using the BLASTP search program (www.ncbi.nlm.nih.govWeb server).

RESULTS AND DISCUSSION
A search for Brassica and Prunus SI-involved orthologs in buckwheat genome gave further results.We did not find evidence for SRK, SLG or SP11 Brassica orthologs in the buckwheat genome -all PCR amplifications using gene specific primers (Table 1) were without amplification product.However, PCR amplification using MLPK specific primers (Table 1) gave a specific amplification product.
We obtained partial a MLPK nucleotide sequence that is 728 nucleotides long, contains 4 exons and corresponds to the kinase region of MLPK.It shows a high similarity at the amino acid level to the protein kinases of other plant species ranging from 81% (Trifolium pratense) to 89% (Populus trichocarpa).
MLPKs being orthologs and sharing a general function in the mediation of signaling processes does not exclude the possibility that those signaling processes may be involved in a spectrum of different roles, only one of which is SI response.With respect to the similar manifestation of the SI response in the thrum pistils of buckwheat and the SI response in Brassica, there is a reasonable possibility that buckwheat MLPK may be involved in SI response as well.
Further, buckwheat's partial MLPK sequence showed a 80% similarity to the MLPKf2 of Brassica rapa (AB121973) and a 80% similarity to the APK1A of Arabidopsis thaliana (AT1G07570) at the amino acid level (Figure 1).According to Murase et al., (2004) molecular mechanisms that produce two alternative MLPK (or APK1) transcripts and regulate their expression patterns are conserved in the genera of Brassica and Arabidopsis.It is probable that these mechanisms are conserved in other plant species sharing SIinvolved MLPK orthologs.Therefore, in the forthcoming period we are going to obtain a full MLPK sequence and if present in isoforms, to investigate their expression pattern.The next step will be to deduce the possible involvement of MLPK in the SI cascade underlying incompatible pollinations in the thrum flower morph of buckwheat.
Regarding the Prunus orthologs S-RNase and SFB, we find no evidence of their presence in the buckwheat genome.PCR amplifications using gene specific primers gave no amplification product.Also, IEF protein separation of the buckwheat style protein extracts specifically stained for RNases revealed no presence of basic S-RNases in either of the unpollinated buckwheat styles or in the self and non-self pollinated styles of both morphs.Therefore, in the buckwheat pin morph flower that shares a similar SI response physiology with Prunus, the SI response is not based on S-RNases and SFB.It remains to uncover the molecules that are SI-involved in the pin morph as well as the thrum, through S-locus mapping and 2D-PAGE style protein extracts' separation and identification.

Table 1 .
et al., 2003)and that phylogenetically distant flowering plant species may share ancestral genes, we decided to investigate if the SI responses in two buckwheat morphs involve similar SI components as those identified in Brassica and Prunus.