SRK, the stigma-specific S locus receptor kinase of Brassica, is targeted to the plasma membrane in transgenic tobacco.

The S locus receptor kinase (SRK) gene is one of two S locus genes required for the self-incompatibility response in Brassica. We have identified the product of the SRK6 gene in B. oleracea stigmas and have shown that it has characteristics of an integral membrane protein. When expressed in transgenic tobacco, SRK6 is glycosylated and targeted to the plasma membrane. These results provide definitive biochemical evidence for the existence in plants of a plasma membrane-localized transmembrane protein kinase with a known cell-cell recognition function. The timing of SRK expression in stigmas follows a time course similar to that previously described for another S locus-linked gene, the S locus glycoprotein (SLG) gene, and correlates with the ability of stigmas to mount a self-incompatibility response. Based on SRK6 promoter studies, the site of gene expression overlaps with that of SLG and exhibits predominant expression in the stigmatic papillar cells. Although reporter gene studies indicated that the SRK promoter was active in pollen, SRK protein was not detected in pollen, suggesting that SRK functions as a cell surface receptor exclusively in the papillar cells of the stigma.     

Promoters derived from banana bunchy top virus-associated components S1 and S2 drive transgene expression in both tobacco and banana

Potential promoter regions of the Banana bunchy top virus (BBTV)-associated DNA components S1 and S2 were fused to the #-glucuronidase reporter gene and assessed for activity in both tobacco (Nicotiana tabacum cv. Xanthi) and banana (Musa spp. cv. Bluggoe). Transient assays indicated that all the S1- and S2-derived promoters were active and had greater expression in tobacco than banana. In stably transformed tobacco and banana, the S1- and S2-derived promoters directed expression in root meristems and trichomes. The S1 promoter was also expressed in the vascular tissue of leaves and roots, while both the S1 and S2 promoters were active in tobacco leaf trichomes and pollen. In banana, expression was significantly enhanced by the inclusion of the maize polyubiquitin intron 3' to the promoter. Interestingly, there was some evidence to indicate that S1 promoter fragments containing part of the open reading frame at the 5' end of the promoter had enhanced activity, suggesting that promoter elements may not be confined to the non-coding region.     

Genomic organization of the S locus: Identification and characterization of genes in SLG/SRK region of S(9) haplotype of Brassica campestris (syn. rapa).

In Brassica, two self-incompatibility genes, encoding SLG (S locus glycoprotein) and SRK (S-receptor kinase), are located at the S locus and expressed in the stigma. Recent molecular analysis has revealed that the S locus is highly polymorphic and contains several genes, i.e., SLG, SRK, the as-yet-unidentified pollen S gene(s), and other linked genes. In the present study, we searched for expressed sequences in a 76-kb SLG/SRK region of the S(9) haplotype of Brassica campestris (syn. rapa) and identified 10 genes in addition to the four previously identified (SLG(9), SRK(9), SAE1, and SLL2) in this haplotype. This gene density (1 gene/5.4 kb) suggests that the S locus is embedded in a gene-rich region of the genome. The average G + C content in this region is 32.6%. An En/Spm-type transposon-like element was found downstream of SLG(9). Among the genes we identified that had not previously been found to be linked to the S locus were genes encoding a small cysteine-rich protein, a J-domain protein, and an antisilencing protein (ASF1) homologue. The small cysteine-rich protein was similar to a pollen coat protein, named PCP-A1, which had previously been shown to bind SLG.     

Coevolution of the S-locus genes SRK,SLG and SP11/SCR in Brassica oleracea and B. rapa

Brassica self-incompatibility (SI) is controlled by SLG and SRK expressed in the stigma and by SP11/SCR expressed in the anther. We determined the sequences of the S domains of 36 SRK alleles, 13 SLG alleles, and 14 SP11 alleles from Brassica oleracea and B. rapa. We found three S haplotypes lacking SLG genes in B. rapa, confirming that SLG is not essential for the SI recognition system. Together with reported sequences, the nucleotide diversities per synonymous and nonsynonymous site (pi(S) and pi(N)) at the SRK, SLG, and SP11 loci within B. oleracea were computed. The ratios of pi(N):pi(S) for SP11 and the hypervariable region of SRK were significantly >1, suggesting operation of diversifying selection to maintain the diversity of these regions. In the phylogenetic trees of 12 SP11 sequences and their linked SRK alleles, the tree topology was not significantly different between SP11 and SRK, suggesting a tight linkage of male and female SI determinants during the evolutionary course of these haplotypes. Genetic exchanges between SLG and SRK seem to be frequent; three such recent exchanges were detected. The evolution of S haplotypes and the effect of gene conversion on self-incompatibility are discussed.     

芸薹属植物自交不亲和性的分子机制

芸薹属植物自交不亲和性受单一位点的复等位基因控制,此位点命名为S位点,它决定柱头表面花粉识别的专一性,S位点糖蛋白基因（SLG）和S受体激酶基因（SRK)是控制芸薹属植物花柱自交不亲和性的两个关键因子,本文介绍了编码自产不亲和性的S位点的SLG,SRK和花粉S基因的鉴定,结构及功能,并对其信号传导途径的可能机制做了简要概述。     

芸苔属植物自交不亲和性的分子机制

芸薹属植物自交不亲和性受单一位点的复等位基因控制,此位点命名为S位点。它决定柱头表面花粉识别的专一性。S位点糖蛋白基因（SLG）和S受体激酶基因（SRK）是控制芸薹属植物花柱自交不亲和性的两个关键因子。本文介绍了编码自交不亲和性的S位点的SLG、SRK和花粉S基因的鉴定、结构及功能,并对其信号传导途径的可能机制做了简要概述。     

Analysis of a maize α-tubulin gene promoter by transient expression and in transgenic tobacco plants

The pattern of expression directed by the promoter of the maize Tub α 1 gene was investigated by analysis of chloramphenicol acetyl transferase (CAT) and β-glucuronidase (GUS) activities in transient expression experiments of maize and tobacco protoplasts. The same promoter was also investigated by histochemical GUS analysis in transgenic tobacco plants containing promoter gene fusions. As determined by histochemical tests, the Tub α 1 promoter gene preferentially directs GUS expression in regenerating root tip meristems and pollen. This pattern corresponds to the distinctive features of natural expression of the gene in maize as determined by Northern analysis. However, no expression is observed in other meristematic tissues of the transgenic tobacco plants, as in shoot apex or in coleoptiles, which is weakly detected in maize. Analysis of the regulatory properties of 5' promoter deletions showed that the proximal region of the promoter, from positions −1410 or −449 to 15 bp upstream of the ATG, is sufficient to establish the qualitative pattern of expression in transgenic tobacco plants. Deletions to positions −352 or −117 abolished the expression in roots, but not in pollen, suggesting that upstream of these positions there are elements responsible for the pattern in root. Further deletions abolished all the promoter activity, suggesting that this promoter region contains the elements essential for expression in pollen. The different patterns and levels of transient and stable expression are discussed.     

Creation and analysis of a novel chimeric promoter for the complete containment of pollen- and seed-mediated gene flow

Effective containment of gene flow in transgenic plants requires a promoter that is highly specific for male and female gametes or tissues. Here, we report the creation of a novel pollen-, stigma- and carpel-specific (PSC) promoter through the fusion of the pollen-specific LAT52 and carpel-specific AGL5 enhancers to a stigma-specific SLG promoter. Gene expression analysis showed that fusion of the LAT52 enhancer to the SLG promoter enables the latter to gain pollen-specific activity while the acquirement of carpel-specific activity requires the correct orientation of the inserted AGL5 enhancer in the PSC promoter, and only a forward- but not a reverse-oriented one is functional. The resulting fPSC promoter, when fused to DT-A, generated at least three aberrant gynoecium phenotypes. Type I plants exhibited shortened stigmatic tissues, resembling plants containing the DT-A gene controlled by the SLG promoter. However, type II and III plants displayed partial or complete ablation of gynoecia, and were unable to support the reproductive process. Type II and III plants also produced severely perturbed anthers and pollen in comparison to type I or SLG::DT-A plants, and transgenic pollen grains were unable, when out-crossed with control plants, to pass the transgene to the next generation in all plants examined, indicating that they are selectively eliminated. This tissue-specific ablation or perturbation is highly specific, and does not compromise vegetative growth. Evidently, the fPSC promoter faithfully acquires tissue specificity from the incorporated enhancers and promoter, and should have a practical application for transgene containment in non-fruit and -grain producing plant crops.     

Structural and transcriptional comparative analysis of the S locus regions in two self-incompatible Brassica napus lines

Self-incompatibility (SI) in Brassica is controlled by a single locus, termed the S locus. There is evidence that two of the S locus genes, SLG, which encodes a secreted glycoprotein, and SRK, which encodes a putative receptor kinase, are required for SI on the stigma side. The current model postulates that a pollen ligand recognizing the SLG/SRK receptors is encoded in the genomic region defined by the SLG and SRK genes. A fosmid contig of approximately 65 kb spanning the SLG-910 and SRK-910 genes was isolated from the Brassica napus W1 line. A new gene, SLL3, was identified using a novel approach combining cDNA subtraction and direct selection. This gene encodes a putative secreted small peptide and exists as multiple copies in the Brassica genome. Sequencing analysis of the 65-kb contig revealed seven additional genes and a transposon. None of these seven genes exhibited features expected of S genes on the pollen side. An approximately 88-kb contig of the A14 S region also was isolated from the B. napus T2 line and sequenced. Comparison of the two S regions revealed that (1) the gene organization downstream of SLG in both S haplotypes is highly colinear; (2) the distance between SLG-A14 and SRK-A14 genes is much larger than that between SLG-910 and SRK-910, with the intervening region filled with retroelements and haplotype-specific genes; and (3) the gene organization downstream of SRK in the two haplotypes is divergent. These observations lead us to propose that the SLG downstream region might be one border of the S locus and that the accumulation of heteromorphic sequences, such as retroelements as well as haplotype-unique genes, may act as a mechanism to suppress recombination between SLG and SRK.     

Molecular characterization of mature pollen-specific genes encoding novel small cysteine-rich proteins in rice (Oryza sativa L.)

In our previous cDNA microarray analysis, we identified 53 mature anther-specific genes, whose function was unknown, in rice. We reanalyzed these genes from the viewpoint of the specific amino acid motif. Out of 53 genes, three genes, Os-26, Os-32, and Os-169 (renamed as OsSCP1, OsSCP2, and OsSCP3), encoded cysteine-rich motif (Cys-X₃-Cys-X₁₃-Cys-X₃-Cys), indicating that they were novel small cysteine-rich proteins. From the search of specific elements in promoter regions, several pollen-specific elements were found. In order to determine whether three promoters were functional in pollen or not, the gene constructs with promoter regions fused to the β-glucuronidase gene were transformed into tobacco. Histochemical analysis showed that these promoters were active in the mature pollen grains and pollen tubes. Furthermore, OsSCP1 and OsSCP3 formed a multigene family tandemly in the rice genome. From the results, OsSCPs might have important roles in mature pollen development and pollen tube growth.     

Deletion analysis of pollen-expressed promoters

Summary We have used microprojectile bombardment of tobacco pollen to study the DNA sequences involved in the expression of pollen-expressed genes. Promoter-reporter gene fusions constructed with the promoters of three different pollen-expressed genes from tomato (LAT52, LAT56, and LAT59) and either theβ-glucuronidase or luciferase reporter genes were assayed by bombarding hydrated tobacco pollen with the gene constructs precipitated onto tungsten microprojectiles. Reporter gene expression can be assayed within 30 min, with the maximal level of expression between 6 and 12 h after bombardment. By constructing and assaying promoter deletion derivatives, we have been able to delimit regions of the promoters that are necessary for high level expression in pollen. We also demonstrate that results with this transient expression system parallel the expression levels seen in pollen from stably transformed transgenic plants. The microprojectile bombardment assay can be used to rapidly test constructs for pollen expression beforeAgrobacterium-mediated plant transformation. Furthermore, it may be possible to adapt the microprojectile bombardment technique to achieve stable transformation of pollen. Presented in the Session-in-Depth Genetic Transformation and Genetic Analysis Using Microprojectile Bombardment at the 41st Annual Meeting of the Tissue Culture Association, Houston, Texas, June 10–13, 1990. This work was supported by the U.S. Department of Agriculture, Washington, DC, ARS CRIS 5335-22230-002-00D, and by the NSF Center for Plant Developmental Biology, UC-Berkeley, DIR-8719933.     

Expression of the S receptor kinase in self-compatible Brassica napus cv. Westar leads to the allele-specific rejection of self-incompatible Brassica napus pollen

Expression of an S receptor kinase (SRK910) transgene in the self-compatible Brassica napus cv. Westar conferred on the transgenic pistil the ability to reject pollen from the self-incompatible Brassica napus W1 line, which carries the S910 allele. In one of the SRK transgenic lines, 1C, virtually no seeds were produced when the transgenic pistils were pollinated with W1 pollen (Mean number of seeds per pod = 1.22). This response was specific to the W1 pollen since pollen from a different self-incompatible Brassica napus line (T2) and self-pollinations were fully compatible. Westar plants expressing an S locus glycoprotein transgene (SLG910) did not show any self-incompatibility response towards W1 pollen. Transgenic Westar plants resulting from crosses between the 1C SRK transgenic line and three SLG910 transgenic lines were also tested for rejection of W1 pollen. The additional expression of the SLG910 transgene in the SRK910 transgenic plants did not cause any significant further reduction in seed production (Mean seeds/pod = 1.04) or have any detectable effects on the number of pollen grains that adhered to the pistil. Thus, while the allele-specific SLG gene was previously reported to have an enhancing effect on the self-incompatibility response, no evidence for such a role was found in this study.