No abstract

Self-incompatibility in the Brassicaceae is controlled by multiple haplotypes encoding the pollen ligand (S-locus protein 11, SP11, also known as S-locus cysteine-rich protein, SCR) and its stigmatic receptor (S-receptor kinase, SRK). A haplotype-specific interaction between SP11/SCR and SRK triggers the self-incompatibility response that leads to self-pollen rejection, but the signalling pathway remains largely unknown. Here we show that Ca(2+) influx into stigma papilla cells mediates self-incompatibility signalling. Using self-incompatible Arabidopsis thaliana expressing SP11/SCR and SRK, we found that self-pollination specifically induced an increase in cytoplasmic Ca(2+) ([Ca(2+)]cyt) in papilla cells. Direct application of SP11/SCR to the papilla cell protoplasts induced Ca(2+) increase, which was inhibited by D-(-)-2-amino-5-phosphonopentanoic acid (AP-5), a glutamate receptor channel blocker. An artificial increase in [Ca(2+)]cyt in papilla cells arrested wild-type (WT) pollen hydration. Treatment of papilla cells with AP-5 interfered with self-incompatibility, and Ca(2+) increase on the self-incompatibility response was reduced in the glutamate receptor-like channel (GLR) gene mutants. These results suggest that Ca(2+) influx mediated by GLR is the essential self-incompatibility response leading to self-pollen rejection.

In the Brassicaceae, intraspecific non-self pollen (compatible pollen) can germinate and grow into stigmatic papilla cells, while self-pollen or interspecific pollen is rejected at this stage. However, the mechanisms underlying this selective acceptance of compatible pollen remain unclear. Here, using a cell-impermeant calcium indicator, we showed that the compatible pollen coat contains signaling molecules that stimulate Ca 2+ export from the papilla cells. Transcriptome analyses of stigmas suggested that autoinhibited Ca 2+ -ATPase13 (ACA13) was induced after both compatible pollination and compatible pollen coat treatment. A complementation test using a yeast Saccharomyces cerevisiae strain lacking major Ca 2+ transport systems suggested that ACA13 indeed functions as an autoinhibited Ca 2+ transporter. ACA13 transcription increased in papilla cells and in transmitting tracts after pollination. ACA13 protein localized to the plasma membrane and to vesicles near the Golgi body and accumulated at the pollen tube penetration site after pollination. The stigma of a T-DNA insertion line of ACA13 exhibited reduced Ca 2+ export, as well as defects in compatible pollen germination and seed production. These findings suggest that stigmatic ACA13 functions in the export of Ca 2+ to the compatible pollen tube, which promotes successful fertilization.

Caffeine (1,3,7-trimethylxanthine) is derived from xanthosine through three successive transfers of methyl groups and a single ribose removal in coffee plants. The methyl group transfer is catalyzed by N-zmethyltransferases, xanthosine methyltransferase (XMT), 7-methylxanthine methyltransferase (MXMT) and 3,7-dimethylxanthine methyltransferase (DXMT). We previously cloned three genes encoding each of these N-methyltransferases from coffee plants, and reconstituted the final sequence of the caffeine synthetic pathway in vitro. In the present study, we simultaneously expressed these coffee genes in tobacco plants (Nicotiana tabacum), using a multiple-gene transfer method, and confirmed successful caffeine production up to 5 microg g(-1) fresh weight in leaves of the resulting transgenic plants. Their effects on feeding behavior of tobacco cutworms (Spodoptera litura), which damage a wide range of crops, were then examined. Leaf disc choice test showed that caterpillars selectively fed on the wild-type control materials, or positively avoided the transgenic materials. The results suggest a novel approach to confer self-defense by producing caffeine in planta. A second generation of transgenic crops containing caffeine may save labor and agricultural costs and also mitigate the environmental load of pesticides in future.

TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetic method that can be employed to generate allelic series of induced mutations in targeted genes for functional analyses. To date, TILLING resources in Arabidopsis thaliana are only available in accessions Columbia and Landsberg erecta. Here, we extended the Arabidopsis TILLING resources by developing a new population of ethyl methanesulfonate (EMS)-induced mutant lines in another commonly used A. thaliana accession C24. A permanent collection of 3,509 independent EMS mutagenized M2 lines was developed in A. thaliana accession C24, and designated C24TILL. Using the TILLING method to search C24TILL for mutations in four selected genes identified a total of 73 mutations, comprising 69.6% missense, 29.0% sense, and 1.4% nonsense mutations. Consistent with the propensity of EMS to induce guanine alkylation, 98.4% of the observed mutations were G/C to A/T transitions. Based on the mutations identified in the four target genes, the overall mutation density in the C24TILL collection was estimated to be 1/345 kb. TILLING the DUO POLLEN 1 (DUO1) gene from the C24TILL collection identified a truncation mutation leading to a deficiency in sperm cell differentiation. Taken together, a new TILLING resource, the C24TILL collection, was generated for A. thaliana accession C24. The C24TILL collection provides an allelic series of induced point mutations that will serve as a useful alternative reverse genetic resource for functional genetic studies in A. thaliana.