Characterization of a novel Brassica napus kinase, BNK1.

A novel plant protein kinase, designated Brassica napus kinase 1 (BNK1), was isolated from a lambda-pistil cDNA library. The deduced BNK1 protein contains all eleven conserved subdomains of a kinase and encodes a functional serine/threonine protein kinase. Phylogenetic analysis of several plant protein kinase subfamilies showed that BNK1 is most closely related to the NAK subfamily of protein kinases. Genomic Southern blot analysis revealed that BNK1 is a single copy gene in the B. napus genome and does not appear to be a member of a multigene family. Expression studies revealed that the BNK1 transcript was ubiquitously expressed throughout the plant, with highest levels in stem and pistil tissues.

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.

Further analysis of the interactions between the Brassica S receptor kinase and three interacting proteins (ARC1, THL1 and THL2) in the yeast two-hybrid system.

The yeast two-hybrid system was used to further characterize the interactions between the Brassica S receptor kinase (SRK) and three putative substrates, ARC1 and the two thioredoxin h proteins, THL1 and THL2. Interactions were generally detectable with kinase domains of both Class I and Class II SRKs. Chimeric constructs were made between the SRK910 kinase domain and the non-interacting Arabidopsis RLK5 kinase domain. Only one chimeric construct, SRR2, interacted with THL1 and THL2, while none of the chimeras were able to interact with ARC1. SRR2 is largely made up of RLK5 kinase domain with the N-terminal end being derived from the SRK910 kinase domain and was the only chimeric construct that retained kinase activity. Deletion or substitution of a conserved cysteine at the N-terminal end of the SRK910 kinase domain resulted in loss of interaction with THL1 and THL2, while the addition of this cysteine to a related receptor kinase, SFR1, conferred the ability to interact with the thioredoxin h proteins. In addition, substitution of the cysteines in the THL1 active site abolished the interaction. Lastly, the two Arabidopsis thioredoxin h clones most closely related to THL1 and THL2 were found to interact with the SRK kinase domains. Thus, the nature of the interaction of the thioredoxin h clones with SRK involves the reducing activity of these proteins and is restricted to the class of thioredoxin h proteins which have the variant CPPC active site.

Mechanisms of self-incompatibility in flowering plants.

Self-incompatibility is a widespread mechanism in flowering plants that prevents inbreeding and promotes outcrossing. The self-incompatibility response is genetically controlled by one or more multi-allelic loci, and relies on a series of complex cellular interactions between the self-incompatible pollen and pistil. Although self-incompatibility functions ultimately to prevent self-fertilization, flowering plants have evolved several unique mechanisms for rejecting the self-incompatible pollen. The self-incompatibility system in the Solanaceae makes use of a multi-allelic RNase in the pistil to block incompatible pollen tube growth. In contrast, the Papaveraceae system appears to have complex cellular responses such as calcium fluxes, actin rearrangements, and programmed cell death occurring in the incompatible pollen tube. Finally, the Brassicaceae system has a receptor kinase signalling pathway activated in the pistil leading to pollen rejection. This review highlights the recent advances made towards understanding the cellular mechanisms involved in these self-incompatibility systems and discusses the striking differences between these systems.

Transformation of Arabidopsis with a Brassica SLG/SRK region and ARC1 gene is not sufficient to transfer the self-incompatibility phenotype.

Self-incompatibility (SI) promotes outbreeding in flowering plants, and in Brassica SI is genetically controlled by the S locus. Self-incompatible Brassica and self-fertile Arabidopsis belong to the same crucifer family. In addition, a comparative analysis reveals a high degree of microsynteny between the B. campestris S locus and its homologous region in Arabidopsis--with the notable exception that the Brassica SI genes, SLG and SRK, are missing. Brassica ARC1 encodes a component of the SRK signal transduction pathway leading to self-pollen rejection, and no closely related ARC1 homolog has been identified in Arabidopsis. The purpose of the research reported here was to introduce Brassica SI components into Arabidopsis in an attempt to compensate for the missing genes and to investigate whether the SI phenotype can be transferred. Inserts of approximately 40 kb from the fosmid clones F20 and F22, which span the B. napus W1 SLG-SRK region, were cloned into the plant transformation vector pBIBAC2. Transgenic plants were generated that expressed the Brassica SI genes in the flower buds. In addition, the endogenous, SLG-like, gene AtS1 was not co-suppressed by the Brassica SLG transgene. No SI phenotype was observed among the T1 BIBAC2-F20 and BIBAC2-F22 transgenic plants. When the ARC1 gene was transformed into BIBAC2-F20 or BIBAC2-F22 plants, the resulting BIBAC2-F20-ARC1 and BIBAC2-F22-ARC1 plants still set seeds normally, and no rejection response was observed when self-incompatible B. napus W1 pollen was placed on BIBAC2-F20-ARC1 or BIBAC2-F22-ARC1 Arabidopsis stigmas. Taken together, our results suggest that complementing Arabidopsis genome with Brassica SLG, SRK and ARC1 genes is unlikely to be sufficient to transfer the SI phenotype.

A breakdown of Brassica self-incompatibility in ARC1 antisense transgenic plants.

Self-incompatibility, the rejection of self pollen, is the most widespread mechanism by which flowering plants prevent inbreeding. In Brassica, the S receptor kinase (SRK) has been implicated in the self-incompatibility response, but the molecular mechanisms involving SRK are unknown. One putative downstream effector for SRK is ARC1, a protein that binds to the SRK kinase domain. Here it is shown that suppression of ARC1 messenger RNA levels in the self-incompatible Brassica napus W1 line is correlated with a partial breakdown of self-incompatibility, resulting in seed production. This provides strong evidence that ARC1 is a positive effector of the Brassica self-incompatibility response.

Neither compatible nor self-incompatible pollinations of Brassica napus involve reorganization of the papillar cytoskeleton.

Compatible pollination of Brassica napus necessitates pollen hydration, pollen germination and growth of the pollen tube through the loosened walls of stigmatic papillar cells, whereas self-incompatible (SI) pollinations fail at one of these stages. Analyses of the early stages of pollination show that at high (but not low) relative humidities, both compatible and SI pollen hydrates, but SI germination is reduced and the rare pollen tubes generally fail to penetrate the papillar walls, although there is some wall loosening. Inside the papillae, both compatible and SI interactions may induce the formation of callose, but there is no evidence for a major accumulation of cytoplasm or secretory vesicles in the vicinity of the pollen tubes and neither microtubule nor F-actin patterns re-arrange in this zone. These observations indicate that the source of the wall-loosening enzymes is probably the pollen tube or pollen coat, and that the common cellular responses of plants to attempted invasions have become suppressed in the papilla-pollen tube interaction.

Binding of an arm repeat protein to the kinase domain of the S-locus receptor kinase.

Screening of a yeast two-hybrid library for proteins that interact with the kinase domain of an S-locus receptor kinase (SRK) resulted in the isolation of a plant protein called ARC1 (Arm Repeat Containing). This interaction was mediated by the C-terminal region of ARC1 in which five arm repeat units were identified. Using the yeast two-hybrid system and in vitro binding assays, ARC1 was found to interact specifically with the kinase domains from SRK-910 and SRK-A14 but failed to interact with kinase domains from two different Arabidopsis receptor-like kinases. In addition, treatment with a protein phosphatase or the use of a kinase-inactive mutant reduced or abolished the binding of ARC1 to the SRK-910 kinase domain, indicating that the interaction was phosphorylation dependent. Lastly, RNA blot analysis revealed that the expression of ARC1 is restricted to the stigma, the site of the self-incompatibility response.

Interrelationships between cytoplasmic Ca2+ peaks, pollen hydration and plasma membrane conductances during compatible and incompatible pollinations of Brassica napus papillae.

We have investigated Ca(2+)-involving cell signaling, plasma membrane potentials and conductances and callose formation during early stages of pollination of papillae of Brassica napus. Using fluorescence imaging of calcium green-1, we found that application of a range of pollen types and controls all rapidly produced small localized peaks in papillar cytoplasmic [Ca2+]. This response was more frequent in compatible than incompatible interactions and was correlated with subsequent hydration of the applied pollen grains, indicating that it may be a differential prerequisite of the compatible signaling pathway leading to successful pollinations. In contrast, a slight trend to increased plasma membrane conductance (but with no indications of action potential-like responses) and also callose deposition in papillae adjacent to pollen grains followed pollination in both SC and SI interactions, indicating that alterations in plasma membrane permeability and callose deposition during early phases of pollination are not primary determinants of the fate of attempted pollinations.

Molecular characterization of the S locus in two self-incompatible Brassica napus lines.

In Brassica species, self-incompatibility has been mapped genetically to a single chromosomal location. In this region, there are two closely linked genes coding for the S locus glycoprotein (SLG) and S locus receptor kinase (SRK). They appear to comprise the pistil component of the self-incompatibility reaction. SLG and SRK are thought to recognize an unknown pollen component on the incompatible pollen, and the gene encoding this pollen component must also be linked to the SLG and SRK genes. To further our understanding of self-incompatibility, the chromosomal region carrying the SLG and SRK genes has been studied. The physical region between the SLG-910 and the SRK-910 genes in the Brassica napus W1 line was cloned, and a search for genes expressed in the anther revealed two additional S locus genes located downstream of the SLG-910 gene. Because these two genes are novel and are conserved at other S alleles, we designated them as SLL1 and SLL2 (for S locus-linked genes 1 and 2, respectively). The SLL1 gene is S locus specific, whereas the SLL2 gene is not only present at the S locus but is also present in other parts of the genomes in both self-incompatible and self-compatible Brassica ssp lines. Expression of the SLL1 gene is only detectable in anthers of self-incompatible plants and is developmentally regulated during anther development, whereas the SLL2 gene is expressed in anthers and stigmas in both self-incompatible and self-compatible plants, with the highest levels of expression occurring in the stigmas. Although SLL1 and SLL2 are linked to the S locus region, it is not clear whether these genes function in self-incompatibility or serve some other cellular roles in pollen-pistil functions.

Two members of the thioredoxin-h family interact with the kinase domain of a Brassica S locus receptor kinase.

To determine potential targets of the S locus receptor kinase (SRK) during the Brassica self-incompatibility response, a yeast two-hybrid library was screened with the SRK-910 protein kinase domain. Two thioredoxin-h-like clones, THL-1 and THL-2, were found to interact specifically with the SRK-910 protein kinase domain and not to interact with the protein kinase domains from the Arabidopsis receptor-like protein kinases (RLK) RLK4 and RLK5. The interaction between THL-1 and the SRK-910 protein kinase domain was confirmed using coimmunoprecipitation experiments with fusion proteins produced in Escherichia coli. THL-1 has thioredoxin activity based on an insulin reduction assay, and THL-1 is weakly phosphorylated by the SRK-910 protein kinase domain. THL-1 and THL-2 are both expressed in a variety of tissues but show some differences in steady state mRNA levels, with THL-2 being preferentially expressed in floral tissues. This indicates a more general biological function for these thioredoxins in addition to a potential role as effector molecules in the self-incompatibility signal cascade.

Features of the extracellular domain of the S-locus receptor kinase from Brassica.

An S-receptor kinase (SRK) gene associated with self-incompatibility in a Brassica napus subsp. oleifera line has been characterized. The SRK-A14 cDNA shows the highest levels of homology in the 5' end to the SLG-A14 cDNA present at the same locus. RNA blot analysis shows that the SRK-A14 gene is expressed predominantly in the pistil, and at lower levels in the anthers. The predicted amino acid sequences from the extracellular domain of the SRK-A14 gene and three other SRK genes were compared. The different SRK extracellular domains were for the most part very similar, with the exception of two variable regions containing a high level of amino acid alterations. These extracellular domains also contain a region of similarity to the immunoglobulin domains present in members of the immunoglobulin superfamily. These findings may define regions of the SRK protein that are necessary for interactions between SRK and other proteins.

An S receptor kinase gene in self-compatible Brassica napus has a 1-bp deletion.

S locus glycoprotein (SLG) and S locus receptor kinase (SRK) cDNAs were isolated from an S allele present in a number of self-compatible Brassica napus lines. This A10 allele did not segregate with self-incompatibility in crosses involving other self-incompatible B. napus lines. The SLG-A10 cDNA was found to contain an intact open reading frame and was predicted to encode an SLG protein with sequence similarities to those previously associated with phenotypically strong self-incompatibility reactions. SLG-A10 transcripts were detected in the developing stigma at steady state levels even higher than those detected for SLG alleles linked with self-incompatibility. Analysis of the corresponding SRK-A10 cDNA showed that it was very similar to other S locus receptor kinase genes and was expressed predominantly in the stigma. However, a 1-bp deletion was detected in the SRK gene toward the 3' end of the SLG homology domain. This deletion would lead to premature termination of translation and the production of a truncated SRK protein. The A10 allele was determined to represent a B. oleracea S allele based on its segregation pattern with the B. oleracea S24 allele when both these alleles were present in the same B. napus background. These results suggest that a functional SRK gene is required for Brassica self-incompatibility.

Developmental regulation and cell type-specific expression of the murine gamma F-crystallin gene is mediated through a lens-specific element containing the gamma F-1 binding site.

The mouse gamma F-crystallin gene, one of six differentially regulated members of the gamma-crystallin gene family, is expressed exclusively in central nuclear fiber cells of the adult lens. The expression of this gene is controlled through regulatory elements contained in two upstream enhancers and the proximal promoter. Here we show that while the upstream enhancers and the proximal promoter could each direct gene expression in fiber cells formed at early stages of lens growth and development, cooperation between these elements is required to achieve expression in fiber cells formed at later stages. Evidence is provided that cooperative interaction between these elements modulates gene expression by increasing promoter strength. We also show that sequences within the proximal promoter region that bind lens cell nuclear factor gamma F-1 are sufficient to elicit gene expression in central nuclear fiber cells of the adult lens.

Identification of an S-locus glycoprotein allele introgressed from B. napus ssp. rapifera to B. napus ssp. oleifera.

Self-incompatible Brassica napus ssp. oleifera lines were generated by introgressing the S-locus from the self-incompatible B. napus ssp. rapifera Z line into the self-compatible cultivars, Topas and Regent, resulting in T2 and R2, respectively. Screening of a cDNA library made from R2 stigma RNA produced several candidate SLG (S-locus glycoprotein) cDNAs. One of the cDNAs, A14, was found to be represented in only the R2, T2 and Z lines. In addition, the corresponding A14 gene was demonstrated to segregate with the T2 self-incompatibility phenotype in an F2 population derived from a cross between T2 and Topas, and to exhibit high mRNA levels in the stigmas prior to anthesis. Sequence analysis of the A14 cDNA revealed close homology to B. oleracea SLG alleles associated with a Class I high activity self-incompatibility phenotype.

The S-locus receptor kinase gene in a self-incompatible Brassica napus line encodes a functional serine/threonine kinase.

An S-receptor kinase (SRK) cDNA, SRK-910, from the active S-locus in a self-incompatible Brassica napus W1 line has been isolated and characterized. The SRK-910 gene is predominantly expressed in pistils and segregates with the W1 self-incompatibility phenotype in an F2 population derived from a cross between the self-incompatible W1 line and a self-compatible Westar line. Analysis of the predicted amino acid sequence demonstrated that the extracellular receptor domain is highly homologous to S-locus glycoproteins, whereas the cytoplasmic kinase domain contains conserved amino acids present in serine/threonine kinases. An SRK-910 kinase protein fusion was produced in Escherichia coli and found to contain kinase activity. Phosphoamino acid analysis confirmed that only serine and threonine residues were phosphorylated. Thus, the SRK-910 gene encodes a functional serine/threonine receptor kinase.

Use of the polymerase chain reaction to isolate an S-locus glycoprotein cDNA introgressed from Brassica campestris into B. napus ssp. oleifera.

A self-incompatible canola-quality Brassica napus ssp. oleifera line (W1) was generated by introgressing the S-locus from a self-incompatible B. campestris plant into the Westar cultivar. Using the polymerase chain reaction (PCR) with primers derived from conserved regions in S-locus glycoprotein (SLG) alleles, the central region of the active SLG gene (910) was obtained. The remaining portions of the cDNA for this 910 gene were subsequently cloned using the PCR-rapid amplification of cDNA ends (RACE) procedure. Sequence analysis revealed that the 910 cDNA show a high degree of sequence similarity to SLG alleles associated with Class I self-incompatible lines. The 910 gene was found to be absent in the original self-compatible cv. Westar (B. napus) and segregated with self-incompatibility in a mixed population generated from a cross between self-incompatible W1 and self-compatible Westar. RNA blot analysis indicated that high levels of 910 mRNAs were present in the stigma as buds approached anthesis. Thus, the SLG allele of W1 transferred from B. campestris via backcrosses to a line of cv. Westar has been identified.

Temporal regulation of six crystallin transcripts during mouse lens development.

Using the polymerase chain reaction (PCR) and RNA blot analysis, we have examined the differential expression patterns of the gamma-crystallins during lens development. Since only four of these genes had been previously characterized, the cDNAs for the remaining two genes, gamma C and gamma F, were isolated and sequenced. The steady-state mRNA profiles were then determined by RNA blot analysis of samples from embryonic stages to 180 days after birth, with gene-specific probes for gamma A, gamma B, gamma C, and gamma D, and a common probe for gamma E and gamma F. Due to the paucity of mismatches between the gamma E and gamma F-crystallin genes, the PCR technique was exploited to determine their relative abundance. The data showed that while all six gamma-crystallin genes were expressed in the embryonic lens, they were differentially regulated during development. At early stages, the levels of gamma B and gamma C mRNAs were found to be relatively low in comparison to those for gamma A, gamma D, gamma E and gamma F. After 30-40 days, however, the levels of gamma A, gamma E, and gamma F mRNAs declined rapidly, and the gamma B, gamma C and gamma D transcripts became the major gamma-crystallin mRNA species. The utility of the PCR technique in studying the relative abundance of steady-state gamma-crystallin mRNAs was also investigated.

Transformation of a partial nopaline synthase gene into tobacco suppresses the expression of a resident wild-type gene.

A portion of the nopaline synthase gene under the control of the cauliflower mosaic virus 35S promoter was used to transform a tobacco plant that had previously been transformed with a wild-type nopaline synthase (nos) gene. Unexpectedly, in all nine primary transformants tested the wild-type nos expression was virtually completely suppressed. In contrast, plants transformed with the control vector DNA, which differed only in the absence of the partial nos gene, did not show any inhibition of nos expression. Progeny plants were analyzed for the stability of the gene-silencing phenotype. All of the progeny that carried both the wild-type and partial nos genes had no detectable nopaline synthase activity. In addition, wild-type nos mRNA could not be detected in these plants. In most plants in which the wild-type gene was segregated away from the partial nos gene, wild-type levels of activity were detected. Although DNA methylation has been shown to be correlated with a decrease in promoter activity in plants, none of the progeny appeared to carry a methylated nos promoter. The underlying mechanism causing this gene suppression phenomenon is unclear at this time.

In situ detection of beta-galactosidase in lenses of transgenic mice with a gamma-crystallin/lacZ gene.

Transgenic mice carrying the gamma 2-crystallin promoter fused to the coding region of the bacterial lacZ gene were generated. The offspring of three founder mice expressed high levels of the enzyme solely in the central nuclear fiber cells of the lens as measured by an in situ assay for the detection of beta-galactosidase activity. These results suggest that gamma 2-crystallin sequences between -759 to +45 contain essential information required for appropriate tissue-specific and temporal regulation of the mouse gamma 2-crystallin gene. In a broader context, this study also demonstrates the utility of beta-galactosidase hybrid gene constructs for monitoring the activity of gene regulatory elements in transgenic mice.