Daniel P. Matton

Hypervariable Domains of Self-incompatibility RNases Mediate Allele-Specific Pollen Recognition

Self-incompatibility (SI) in angiosperms is a genetic mechanism that promotes outcrossing through rejection of self-pollen. In the Solanaceae, SI is determined by a multiallelic S locus whose only known product is an S RNase. S RNases show a characteristic pattern of five conserved and two hypervariable regions. These are thought to be involved in the catalytic function and in allelic specificity, respectively. When the Solanum chacoense S12S14 genotype is transformed with an S11 RNase, the styles of plants expressing significant levels of the transgene reject S11 pollen. A previously characterized S RNase, S13, differs from the S11 RNase by only 10 amino acids, four of which are located in the hypervariable regions. When S12S14 plants were transformed with a chimeric S11 gene in which these four residues were substituted with those present in the S13 RNase, the transgenic plants acquired the S13 phenotype. This result demonstrates that the S RNase hypervariable regions control allelic specificity.

Production of an S RNase with Dual Specificity Suggests a Novel Hypothesis for the Generation of New S Alleles

Gametophytic self-incompatibility in plants involves rejection of pollen when pistil and pollen share the same allele at the S locus. This locus is highly multiallelic, but the mechanism by which new functional S alleles are generated in nature has not been determined and remains one of the most intriguing conceptual barriers to a full understanding of selfincompatibility. The S11 and S13 RNases of Solanum chacoense differ by only 10 amino acids, but they are phenotypically distinct (i.e., they reject either S11 or S13 pollen, respectively). These RNases are thus ideally suited for a dissection of the elements involved in recognition specificity. We have previously found that the modification of four amino acid residues in the S11 RNase to match those in the S13 RNase was sufficient to completely replace the S11 phenotype with the S13 phenotype. We now show that an S11 RNase in which only three amino acid residues were modified to match those in the S13 RNase displays the unprecedented property of dual specificity (i.e., the simultaneous rejection of both S11 and S13 pollen). Thus, S12S14 plants expressing this hybrid S RNase rejected S11, S12, S13, and S14 pollen yet allowed S15 pollen to pass freely. Surprisingly, only a single base pair differs between the dual-specific S allele and a monospecific S13 allele. Dual-specific S RNases represent a previously unsuspected category of S alleles. We propose that dualspecific alleles play a critical role in establishing novel S alleles, because the plants harboring them could maintain their old recognition phenotype while acquiring a new one.

The Activation of the Potato PR-10a Gene Requires the Phosphorylation of the Nuclear Factor PBF-1.

The pathogenesis-related gene PR-10a (formerly STH[middot]2) is induced in various organs of potato after wounding, elicitor treatment, or infection by Phytophthora infestans. Deletion analysis of the promoter of the PR-10a gene enabled us to identify a 50-bp region, located between positions -155 and -105, necessary for the elicitor responsiveness of the [beta]-glucuronidase reporter gene in transgenic potato plants. Within this region, a 30-bp sequence, located between positions -135 and -105, was necessary for the activation of the promoter by the elicitor. However, strong promoter activity after elicitor treatment required the presence of a 20-bp sequence located between positions -155 and -135. The region between -135 and -105 was specifically recognized by two nuclear factors, PBF-1 (PR-10a Binding Factor 1) and PBF-2, and binding of PBF-1 was coordinated with the accumulation of the PR-10a mRNA. Gel shift assays using nuclear extracts pretreated with sodium deoxycholate or alkaline phosphatase suggested that PBF-1 is a multimeric factor in which at least one of the constituent proteins can be phosphorylated. Treatment with alkaline phosphatase also indicated that binding of PBF-1 is positively regulated by phosphorylation and that it is phosphorylated only in tissues in which PR-10a is expressed. The use of protein phosphatase and kinase inhibitors in vivo provided additional evidence that wounding and elicitor treatment induce the phosphorylation of PBF-1 and that this phosphorylation is associated with gene activation.

Characterization of a WRKY transcription factor expressed in late torpedo-stage embryos of Solanum chacoense

A novel WRKY-like transcription factor was isolated from a screen for weakly expressed mRNAs in ovules in the self-incompatible wild potato species Solanum chacoense Bitt. This protein, named ScWRKY1, consisted of 525 amino acids and can be classified as a WRKY group-I member, having two WRKY domains. It is expressed at low levels in stems, roots, and petals, and expressed at much higher levels in leaves. Interestingly, although barely detectable in developing seeds, it is strongly and transiently expressed in fertilized ovules bearing late torpedo-staged embryos, suggesting a specific role during embryogenesis.

Identification of cis-acting elements involved in the regulation of the pathogenesis-related gene STH-2 in potato

We have characterized a genomic clone containing the potato pathogenesis-related genes STH-2 and STH-21. The two genes are found 4 kb apart on the same chromosome and their sequences are highly similar. They present the same transcriptional orientation and are both interrupted by a single intron. A chimaeric gene consisting of 1015 bp of 5′-flanking sequence and part of the first exon of STH-2 fused to the bacterial β-glucuronidase gene was highly-expressed in tubers of transgenic potato plants after wounding and elicitor treatments. The levels of activity observed in these transgenic plants parallel those observed for the accumulation of STH-2 mRNAs under similar conditions. This indicates that cis-acting elements necessary for the proper activation of the gene are present within 1 kb of 5′-flanking sequences. Functional analysis of 5′ deletions of the STH-2/GUS constructs by transient expression in leaf protoplasts revealed the presence of an upstream regulatory sequence between -135 and -52 which contains a TGAC motif, and a possible negative regulatory region between -52 and -28. A factor present in nuclear extracts of wounded potato tubers was found to bind specifically to nucleotides located between -135 to -105, suggesting that this region contains important cis-regulatory elements.

Alcohol dehydrogenase gene expression in potato following elicitor and stress treatment.

A cDNA clone corresponding to a mRNA that rapidly accumulates during the hypersensitive-like response induced by elicitor treatment of potato (Solanum tuberosum L.) tuber was characterized. The clone encodes a polypeptide (Mr=41097) having 83%–85% amino acid identity with known plant alcohol dehydrogenase sequences (ADH; EC 1.1.1.1). The identity of the clone was confirmed by measuring the ADH enzyme activity in extracts of Escherichia coli transformed with the cDNA clone. In potato tuber disks, a wide range of stresses, including treatment with fatty acid elicitors, salicylic acid, UV light and anaerobiosis, was shown to induce accumulation of Adh transcripts. In stems, a high constitutive level of Adh transcripts could be detected in 4-week old plants, but not in 8-week old plants. However, the mRNA could be induced to accumulate in stems of 8-week old plants by treatment with arachidonic acid elicitor or by anaerobiosis. Induction in leaves was also obtained during anaerobiosis and after treatment with a Phytophthora infestans mycelial homogenate.

Characterization of five RALF-like genes from Solanum chacoense provides support for a developmental role in plants

Five RALF (rapid alkalinization factor)-like genes, named ScRALF1 to 5, were isolated from fertilized ovule and ovary cDNA libraries of Solanum chacoense. They showed high sequence similarities with the RALF protein sequence from Nicotiana tabacum, and exhibited the characteristic architecture of RALF polypeptides. All ScRALFs were moderately to highly expressed at some stage of fruit maturation. ScRALF1 and ScRALF3 were predominantly expressed in ovaries and larger fruits, while ScRALF2, ScRALF4, and ScRALF5 were also expressed in other tissues, indicating that while some RALFs may be involved in fruit maturation, others could be involved in other developmental processes. Wounding or treatment of plants with growth regulators involved in plant defense responses had no significant impact on the mRNA level of any of these genes. These results suggest and support previous data showing that RALF peptides are more likely to act as a small peptide involved in plant development than in defense responses.

Pollination, wounding and jasmonate treatments induce the expression of a developmentally regulated pistil dioxygenase at a distance, in the ovary, in the wild potato Solanum chacoense Bitt.

Pollination and fertilization trigger unique developmental programs leading to embryogenesis, ovary maturation and seed set. Pistil tissues are actively involved in pollen tube growth and respond to the presence of the growing pollen tubes by modulating the expression of specific genes. Using subtractive hybridization to isolate genes involved in pollen-pistil interactions and fertilization, we have isolated a pollination- and fertilization-induced dioxygenase which is predominantly expressed in the pistil. In situ hybridization analyses revealed that the SPP2 dioxygenase (Solanum pollinated pistil) from the self-incompatible wild potato Solanum chacoense Bitt. is also developmentally regulated, with mRNA levels gradually regressing from the tip of the style towards the ovary during pistil development. At maturity, the upper limit of SPP2 transcript distribution coincided with the abscission zone of the style and SPP2 dioxygenase expression in ovaries coincided with the fertilization receptivity period of the flower. Pollination, as well as wounding of the style, induced an increase in SPP2 mRNA steady-state levels at a distance, in the ovary. Treatments with stress hormones including methyl jasmonate, jasmonic acid and salicylic acid mimicked the wound response and also induced SPP2 transcripts in the ovary. The SPP2 dioxygenase could be involved in the biosynthesis of deterrent alkaloids in reproductive tissues or in generating chemical signals involved in pollen tube guidance.

Characterization of a fertilization-induced and developmentally regulated plasma-membrane aquaporin expressed in reproductive tissues, in the wild potato Solanum chacoense Bitt.

Abstract. Fertilization triggers a unique and complex developmental program leading to embryogenesis and seed set. Growth and differentiation are accompanied by rapid metabolic changes as well as massive cellular reorganization inside the fertilized ovule. Using differential display to isolate genes involved in pollen–pistil interactions and early fertilization events, we isolated from Solanum chacoense Bitt. a fertilization-induced plasma-membrane intrinsic protein of the PIP2 family that is predominantly expressed in pistil and anthers tissues. Major intrinsic proteins (MIPs) consist of a large family of highly conserved membrane-spanning proteins that are mainly represented in plants by the aquaporins. Aquaporins, mainly of the PIP and TIP type, have been shown in many species to serve as water channels. In S. chacoense the ScPIP2a mRNA is developmentally regulated during anthesis, with mRNA levels gradually decreasing as the pistil reaches maturity. In flowers, strongest expression was observed in elongating styles, in stamens, and transiently in ovules following fertilization. In styles, maximal expression levels correlated with phases of rapid style elongation and with the formation of epidermal papillae. ScPIP2a mRNA was also strongly expressed in developing fruit, consistent with a role in cell expansion during maturation and development.

Characterization of a cytosolic nucleoside diphosphate kinase associated with cell division and growth in potato

A cDNA encoding Solanum chacoense cytosolic NDPK (NDPK1, EC 2.7.4.6) was isolated. The open reading frame encoded a 148 amino acid protein that shares homology with other cytosolic NDPKs including a conserved N-terminal domain. S. chacoense NDPK1 was expressed in Escherichia coli as a 6×His-tagged protein and purified by affinity chromatography. The recombinant protein exhibited a pattern of abortive complex formation suggesting that the enzyme is strongly regulated by the NTP/NDP ratio. A polyclonal antibody generated against recombinant NDPK1 was specific for the cytosolic isoform in Solanum tuberosum as shown from immunoprecipitation experiments and immunoblot analysis of chloroplasts and mitochondria preparations. NDPK activity and NDPK1 protein were found at different levels in various vegetative and reproductive tissues. DEAE fractogel analyses of NDPK activity in root tips, leaves, tubers and cell cultures suggest that NDPK1 constitutes the bulk of extractable NDPK activity in all these organs. NDPK activity and NDPK1 protein levels raised during the exponential growth phase of potato cell cultures whereas no rise in activity or NDPK1 protein was observed when sucrose concentration in the culture was manipulated to limit growth. Activity measurements, immunoblot analysis as well as immunolocalization experiments performed on potato root tips and shoot apical buds demonstrated that NDPK1 was predominantly localized in the meristematic zones and provascular tissues of the apical regions. These data suggest that NDPK1 plays a specific role in the supply of UTP during early growth of plant meristematic and provascular tissues.