Cloning a putative self-incompatibility gene from the pollen of the grass Phalaris coerulescens.

In Phalaris coerulescens, gametophytic self-incompatibility is controlled by two unlinked genes: S and Z. A probable S gene has now been isolated and sequenced. This represents a novel self-incompatibility gene isolated from pollen in the multilocus system of a monocotyledonous plant. The gene is approximately 3 kb long, split by five introns, and exclusively expressed in the mature pollen. The deduced amino acid sequences from the S1, S2, and part of the S4 alleles showed that the protein has a variable N terminus and a conserved C terminus. The sequence of a complete mutant at the S locus indicated that mutations in the conserved C terminus, a thioredoxin-like region, led to loss of function. We propose that the gene has two distinct sections, a variable N terminus determining allele specificity and a conserved C terminus with the catalytic function. The gene structure and its deduced protein sequences strongly suggest that this monocotyledon has developed a self-incompatibility system entirely different from those operating in the dicotyledons. The possible interactions between S and Z genes in both pollen and stigma are discussed.     

S-protein mutants indicate a functional role for SBP in the self-incompatibility reaction of Papaver rhoeas

The self-incompatibility response involves S-allele specific recognition between stigmatic S proteins and incompatible pollen, resulting in S-specific pollen inhibition. In Papaver rhoeas, the pollen S gene product is predicted to be a receptor that interacts with the stigmatic S protein in an S specific manner. We recently identified an S protein binding protein (SBP) in pollen that binds stigmatic S proteins, although apparently not in an S-allele-specific manner. In order to investigate the functional significance of the interaction between S proteins and SBP, we constructed mutant derivatives of the S1 protein and tested their SBP-binding activity and their biological activity. Here we present an evaluation of nine mutant derivatives of the S1 protein. Western ligand blotting was used to show that mutations to amino acid residues in predicted loops 2 and 6 of the S1 protein cause significant reductions in their SBP-binding activity. These same mutants show a concomitant reduction in their ability to inhibit incompatible pollen. This establishes a direct link between SBP binding and inhibition of incompatible pollen and implicates SBP as a pollen component playing a key role in the self-incompatibility reaction. We discuss the possible nature of the contribution of SBP in the S-specific rejection of incompatible pollen.     

Ribonuclease activity of Petunia inflata S proteins is essential for rejection of self-pollen.

S proteins, pistil-specific ribonucleases that cosegregate with S alleles, have previously been shown to control rejection of self-pollen in Petunia inflata and Nicotiana alata, two solanaceous species that display gametophytic self-incompatibility. The ribonuclease activity of S proteins was thought to degrade RNA of self-pollen tubes, resulting in the arrest of their growth in the style. However, to date no direct evidence has been obtained. Here, the ribonuclease activity of S3 protein of P. inflata was abolished, and the effect on the pistil's ability to reject S3 pollen was examined. The S3 gene was mutagenized by replacing the codon for His-93, which has been implicated in ribonuclease activity, with a codon for asparagine, and the mutant S3 gene was introduced into P. inflata plants of S1S2 genotype. Two transgenic plants produced a level of mutant S3 protein comparable to that of the S3 protein produced in self-incompatible S1S3 and S2S3 plants, yet they failed to reject S3 pollen. The mutant S3 protein produced in these two transgenic plants did not exhibit any detectable ribonuclease activity. We have previously shown that transgenic plants (S1S2 plants transformed with the wild-type S3 gene) producing a normal level of wild-type S3 protein acquired the ability to reject S3 pollen completely. Thus, the results reported here provide direct evidence that the biochemical mechanism of gametophytic self-incompatibility in P. inflata involves the ribonuclease activity of S proteins.     

Loss of pollen-S function in two self-compatible selections of Prunus avium is associated with deletion/mutation of an S haplotype-specific F-box gene

Recently, an S haplotype-specific F-box (SFB) gene has been proposed as a candidate for the pollen-S specificity gene of RNase-mediated gametophytic self-incompatibility in Prunus (Rosaceae). We have examined two pollen-part mutant haplotypes of sweet cherry (Prunus avium). Both were found to retain the S-RNase, which determines stylar specificity, but one (S3' in JI 2434) has a deletion including the haplotype-specific SFB gene, and the other (S4' in JI 2420) has a frame-shift mutation of the haplotype-specific SFB gene, causing amino acid substitutions and premature termination of the protein. The loss or significant alteration of this highly polymorphic gene and the concomitant loss of pollen self-incompatibility function provides compelling evidence that the SFB gene encodes the pollen specificity component of self-incompatibility in Prunus. These loss-of-function mutations are inconsistent with SFB being the inactivator of non-self S-RNases and indicate the presence of a general inactivation mechanism, with SFB conferring specificity by protecting self S-RNases from inactivation.     

Thioredoxin activity in the C terminus of Phalaris S protein

Self-incompatibility in the grass Phalaris coerulescens is controlled by two genes S and Z . Isolation and sequencing of two S alleles showed that they encode proteins that are highly conserved at the C terminus with significant homology to thioredoxin H proteins. In particular, the residues in and around the active site of thioredoxin, Trp-Cys-Gly-Pro-Cys, are perfectly conserved. The C terminus of the S protein has been expressed in Eschericia coli and purified to homogeneity on Ni-NTA resin. Functional assays showed that the protein has thioredoxin activity; it can act as a substrate for E. coli thioredoxin reductase and also catalyse the reduction of insulin by dithiothreitol. The possible role of thioredoxin-like activity of the S protein in mediating the incompatibility reaction in Phalaris is discussed.     

Identification of residues in a hydrophilic loop of the Papaver rhoeas S protein that play a crucial role in recognition of incompatible pollen.

The self-incompatibility response involves S allele-specific recognition between stigmatic S proteins and incompatible pollen. This response results in pollen inhibition. Defining the amino acid residues within the stigmatic S proteins that participate in S allele-specific inhibition of incompatible pollen is essential for the elucidation of the molecular basis of the self-incompatibility response. We have constructed mutant derivatives of the S1 protein from Papaver rhoeas by using site-directed mutagenesis and have tested their biological activity. This has enabled us to identify amino acid residues in the stigmatic S proteins of P. rhoeas that are required for S-specific inhibition of incompatible pollen. We report here the identification of several amino acid residues in the predicted hydrophilic loop 6 of the P. rhoeas stigmatic S1 protein that are involved in the inhibition of S1 pollen. Mutation of the only hypervariable amino acid, which is situated in this loop, resulted in the complete loss of ability of the S protein to inhibit S1 pollen. This clearly demonstrates that this residue plays a crucial role in pollen recognition and may also participate in defining allelic specificity. We have also established the importance of highly conserved amino acids adjacent to this hypervariable site. Our studies demonstrate that both variable and conserved amino acids in the region of the S protein corresponding to surface loop 6 are key elements that play a role in the recognition and inhibition of incompatible pollen in the pollen-pistil self-incompatibility reaction.     

An F-box gene linked to the self-incompatibility (S) locus of Antirrhinum is expressed specifically in pollen and tapetum

In many flowering plants, self-fertilization is prevented by an intraspecific reproductive barrier known as self-incompatibility (SI), that, in most cases, is controlled by a single multiallelic S locus. So far, the only known S locus product in self-incompatible species from the Solanaceae, Scrophulariaceae and Rosaceae is a class of ribonucleases called S RNases. Molecular and transgenic analyses have shown that S RNases are responsible for pollen rejection by the pistil but have no role in pollen expression of SI, which appears to be mediated by a gene called the pollen self-incompatibility or Sp gene. To identify possible candidates for this gene, we investigated the genomic structure of the S locus in Antirrhinum, a member of the Scrophulariaceae. A novel F-box gene, AhSLF-S2, encoded by the S2 allele, with the expected features of the Sp gene was identified. AhSLF-S2 is located 9 kb downstream of S2 RNase gene and encodes a polypeptide of 376 amino acids with a conserved F-box domain in its amino-terminal part. Hypothetical genes homologous to AhSLF-S2 are apparent in the sequenced genomic DNA of Arabidopsis and rice. Together, they define a large gene family, named SLF (S locus F-box) family. AhSLF-S2 is highly polymorphic and is specifically expressed in tapetum, microspores and pollen grains in an allele-specific manner. The possibility that Sp encodes an F-box protein and the implications of this for the operation of self-incompatibility are discussed.     

A novel NADPH thioredoxin reductase, localized in the chloroplast, which deficiency causes hypersensitivity to abiotic stress in Arabidopsis thaliana

Plants contain three thioredoxin systems. Chloroplast thioredoxins are reduced by ferredoxin-thioredoxin reductase, whereas the cytosolic and mitochondrial thioredoxins are reduced by NADPH thioredoxin reductase (NTR). There is high similarity among NTRs from plants, lower eukaryotes, and bacteria, which are different from mammal NTR. Here we describe the OsNTRC gene from rice encoding a novel NTR with a thioredoxin-like domain at the C terminus, hence, a putative NTR/thioredoxin system in a single polypeptide. Orthologous genes were found in other plants and cyanobacteria, but not in bacteria, yeast, or mammals. Full-length OsNTRC and constructs with truncated NTR and thioredoxin domains were expressed in Escherichia coli as His-tagged polypeptides, and a polyclonal antibody specifically cross-reacting with the OsNTRC enzyme was raised. An in vitro activity assay showed that OsNTRC is a bifunctional enzyme with both NTR and thioredoxin activity but is not an NTR/thioredoxin system. Although the OsNTRC gene was expressed in roots and shoots of rice seedlings, the protein was exclusively found in shoots and mature leaves. Moreover, fractionation experiments showed that OsNTRC is localized to the chloroplast. An Arabidopsis NTRC knock-out mutant showed growth inhibition and hypersensitivity to methyl viologen, drought, and salt stress. These results suggest that the NTRC gene is involved in plant protection against oxidative stress.     

NaTrxh is an essential protein for pollen rejection in Nicotiana by increasing S‐RNase activity

In self‐incompatible Solanaceae, the pistil protein S‐RNase contributes to S‐specific pollen rejection in conspecific crosses, as well as to rejecting pollen from foreign species or whole clades. However, S‐RNase alone is not sufficient for either type of pollen rejection. We describe a thioredoxin (Trx) type h from Nicotiana alata, NaTrxh, which interacts with and reduces S‐RNase in vitro. Here, we show that expressing a redox‐inactive mutant, NaTrxh_SS, suppresses both S‐specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia. Biochemical experiments provide evidence that NaTrxh specifically reduces the Cys₁₅₅‐Cys₁₈₅ disulphide bond of S_C10‐Rnase, resulting in a significant increase of its ribonuclease activity. This reduction and increase in S‐RNase activity by NaTrxh helps to explain why S‐RNase alone could be insufficient for pollen rejection.     

The C-terminal residues of bacteriophage T7 gene 4 helicase-primase coordinate helicase and DNA polymerase activities

The gene 4 protein of bacteriophage T7 plays a central role in DNA replication by providing both helicase and primase activities. The C-terminal helicase domain is not only responsible for DNA-dependent dTTP hydrolysis, translocation, and DNA unwinding, but it also interacts with T7 DNA polymerase to coordinate helicase and polymerase activities. The C-terminal 17 residues of gene 4 protein are critical for its interaction with the T7 DNA polymerase/thioredoxin complex. This C terminus is highly acidic; replacement of these residues with uncharged residues leads to a loss of interaction with T7 DNA polymerase/thioredoxin and an increase in oligomerization of the gene 4 protein. Such an alteration on the C terminus results in a reduced efficiency in strand displacement DNA synthesis catalyzed by gene 4 protein and T7 DNA polymerase/thioredoxin. Replacement of the C-terminal amino acid, phenylalanine, with non-aromatic residues also leads to a loss of interaction of gene 4 protein with T7 DNA polymerase/thioredoxin. However, neither of these modifications of the C terminus affects helicase and primase activities. A chimeric gene 4 protein containing the acidic C terminus of the T7 gene 2.5 single-stranded DNA-binding protein is more active in strand displacement synthesis. Gene 4 hexamers containing even one subunit of a defective C terminus are defective in their interaction with T7 DNA polymerase.     

Site-directed mutagenesis reveals the involvement of an additional thioredoxin-dependent regulatory site in the activation of recombinant sorghum leaf NADP-malate dehydrogenase.

The chloroplastic enzyme NADP-malate dehydrogenase is activated by a reversible thiol/disulfide interchange with reduced thioredoxin. Its target disulfide bridge is considered to be located at the amino terminus. To further substantiate the regulatory role of this disulfide, site-directed mutagenesis has been used to replace each or both of the amino-terminal cysteines of the sorghum leaf NADP-malate dehydrogenase, expressed in Escherichia coli, by serines. A truncation mutant lacking the amino terminus has also been produced. Surprisingly, the mutant proteins still required activation by reduced thioredoxin. However, their activation was almost instantaneous, whereas the native enzyme reached full activity after a 10-20 min preincubation. The 8 1/2 for reduced thioredoxin was decreased 2-fold in the mutants, but their Km values for NADPH and oxaloacetate did not change significantly. The inhibition of activation by NADP and inhibition of activity by thiol-derivatizing agents were also retained. These results are interpreted as an indication that two thioredoxin-dependent reduction steps are involved in NADP-dependent malate dehydrogenase light activation, hence that two disulfides per monomer participate in the process. The overall activation rate would depend on a conformational change following the reduction of the amino-terminal disulfide bridge. The amino terminus also plays a role in the dimerization of the protein.     

A Mutant S3 RNase of Petunia inflata Lacking RNase Activity Has an Allele-Specific Dominant Negative Effect on Self-Incompatibility Interactions

Gametophytic self-incompatibility in the Solanaceae is controlled by a multiallelic locus called the S locus. Growth of pollen tubes in the pistil is inhibited when the pollen has one of the two S alleles carried by the pistil. The products of a number of pistil S alleles[mdash]S proteins or S RNases[mdash]have been identified, and their role in controlling the pistil's ability to reject self-pollen has been positively established. In contrast, the existence of pollen S allele products has so far been inferred entirely from genetic evidence. Here, we introduced a modified S3 gene of Petunia inflata encoding an S3 RNase lacking RNase activity into P. inflata plants of the S2S3 genotype to determine whether the production of the mutant protein, designated S3(H93R), would have any effect on the ability of the transgenic plants to reject S2 and S3 pollen. Analysis of the self-incompatibility behavior of 49 primary transgenic plants and the progeny of three plants (H30, H37, and H40) that produced S3(H93R) in addition to producing wild-type levels of endogenous S2 and S3 RNases revealed that S3(H93R) had a dominant negative effect on the function of the S3 RNase in rejecting self-pollen; however, it had no effect on the function of the S2 RNase. One likely explanation of the results is that S3(H93R) competes with the S3 RNase for binding to a common molecule, which is presumably the product of the pollen S3 allele.     

黑曲霉硫氧还蛋白的克隆表达纯化及其酶活位点特征分析

首次从黑曲霉Aspergillus niger全基因组中克隆出黑曲霉硫氧还原蛋白基因AnTrx,并对其编码蛋白的第33-37位保守区的活性位点实施定点突变C34S、C37S及C34S-C37S,获得相应的3个定点突变基因。将野生型AnTrx及其突变子分别在大肠杆菌Escherichia coli中诱导表达,比浊法测定纯化的各表达产物还原牛胰岛素α与β链之间二硫键的活性。结果表明,AnTrx的3个突变体都不表现明显催化活性。当突变型与野生型AnTrx等量混合后,发现突变型AnTrx-C34S可显著提高野生型AnTrx的催化效率,而突变型AnTrx-C37S却无此功能。由此证明,AnTrx活性结构域的第37位Cys残基上的巯基能参与攻击硫氧还蛋白和底物蛋白所形成的二硫键而释放被还原的底物蛋白,而第34位Cys残基同其他微生物的同一活性域一样参与硫氧化还蛋白与底物的结合。这一结果有助于认识真菌硫氧还蛋白第37位活性位点的作用。     

Interaction of mutant thioredoxins of Escherichia coli with the gene 5 protein of phage T7. The redox capacity of thioredoxin is not required for stimulation of DNA polymerase activity

DNA polymerase activity in Escherichia coli cells infected with bacteriophage T7 resides in a protein complex consisting of the T7 gene 5 protein and E. coli thioredoxin in a 1 to 1 stoichiometry. We have analyzed nine mutant thioredoxins, both in vivo and in vitro, for their ability to interact with the T7 gene 5 protein and stimulate the DNA polymerase and exonuclease activities inherent in gene 5 protein. The efficiency of plating of T7 on E. coli thioredoxin mutants depends strongly on the copy number of the respective mutant thioredoxin allele. Plating efficiencies at a constant copy number correlate well with the affinity of the purified mutant proteins for T7 gene 5 protein. The observed dissociation constant, Kobs, is increased between 5 and several hundredfold at 42 degrees C compared to wild-type thioredoxin. The maximum polymerase activity of the reconstituted gene 5 protein-thioredoxin complex at saturating concentrations of mutant thioredoxins, however, is reduced by less than 20%. Consequently, none of the mutant thioredoxins acts as a competitive inhibitor of wild-type thioredoxin. The active-site disulfide of thioredoxin is not essential for the activities of the gene 5 protein-thioredoxin complex. Both cysteines can be replaced without significantly affecting the maximum polymerase or exonuclease activities. Substitution or alkylation of either cysteine, however, reduces the affinity for gene 5 protein drastically, indicating that the active site is part of the thioredoxin surface involved in the protein-protein interaction.     

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.     

Self-compatibility of two apricot selections is associated with two pollen-part mutations of different nature

Loss of pollen-S function in Prunus self-compatible mutants has recently been associated with deletions or insertions in S-haplotype-specific F-box (SFB) genes. We have studied two self-compatible cultivars of apricot (Prunus armeniaca), Currot (S(C)S(C)) and Canino (S(2)S(C)), sharing the naturally occurring self-compatible (S(C))-haplotype. Sequence analysis showed that whereas the S(C)-RNase is unaltered, a 358-bp insertion is found in the SFB(C) gene, resulting in the expression of a truncated protein. The alteration of this gene is associated with self-incompatibility (SI) breakdown, supporting previous evidence that points to SFB being the pollen-S gene of the Prunus SI S-locus. On the other hand, PCR analysis of progenies derived from Canino showed that pollen grains carrying the S(2)-haplotype were also able to overcome the incompatibility barrier. However, alterations in the SFB(2) gene or evidence of pollen-S duplications were not detected. A new class of F-box genes encoding a previously uncharacterized protein with high sequence similarity (approximately 62%) to Prunus SFB proteins was identified in this work, but the available data rules them out of producing S-heteroallelic pollen and thus the cause of the pollen-part mutation. These results suggest that cv Canino has an additional mutation, not linked to the S-locus, which causes a loss of pollen-S activity when present in pollen. As a whole, these findings support the proposal that the S-locus products besides other S-locus independent factors are required for gametophytic SI in Prunus.     

Carbohydrate moiety of the Petunia inflata S3 protein is not required for self-incompatibility interactions between pollen and pistil.

For Petunia inflata and Nicotiana alata, which display gametophytic self-incompatibility, S proteins (the products of the multiallelic S gene in the pistil) have been shown to control the pistil's ability to recognize and reject self-pollen. The biochemical mechanism for rejection of self-pollen by S proteins has been shown to involve their ribonuclease activity; however, the molecular basis for self/non-self recognition by S proteins is not yet understood. Here, we addressed whether the glycan chain of the S3 protein of P. inflata is involved in self/non-self recognition by producing a nonglycosylated S3 protein in transgenic plants and examining the effect of deglycosylation on the ability of the S3 protein to reject S3 pollen. The S3 gene was mutagenized by replacing the codon for Asn-29, which is the only potential N-glycosylation site of the S3 protein, with a codon for Asp, and the mutant S3 gene was introduced into P. inflata plants of the S1S2 genotype. Six transgenic plants that produced a normal level of the nonglycosylated S3 protein acquired the ability to reject S3 pollen completely. These results suggest that the carbohydrate moiety of the S3 protein does not play a role in recognition or rejection of self-pollen and that the S allele specificity determinant of the S3 protein and those S proteins that contain a single glycan chain at the same site as the S3 protein must reside in the amino acid sequence itself.