Characterization Of Mechanism Involved In Trichome Formation And Embryo Development Regulated By Wo In Tomato

Plant trichomes, which are found in nearly all terrestrial plants, originate from epidermal cells. They contain two types, unicellular and multicellular, and serve as an excellent model to study cell fate determination. Trichomes in Arabidopsis are typical unicellular type, the regulatory pathway of which has been well characterized. A WD-bHLH-MYB complex triggers trichome formation in Arabidopsis by enhancing the expression of its downstream genes. Cotton fibres, resembling Arabidopsis trichomes, are controlled by a similar mechanism. However, the regulatory pathway of multicellular trichomes in snapdragon, petunia, tobacco and tomato is different from that in Arabidopsis and cotton. The Woolly gene (Wo), a spontaneous mutation in tomato, is responsible for trichome formation and embryo development. Therefore, cloning and characterization of this gene is very important for unfolding the mechanism of multicellular trichome formation and embryo development in tomato.We cloned and characterized Wo in this study by map-based cloning, and analyzed its expression pattern, subcellular localization and downstream genes. We inferred a regulatory model of trichome formation and embryo development in tomato. The main results are as follows:1. By comparing the genetic map (Tanksley,1992) with the published IL map, we inferred that Wo located in the mid-region of the long arm of chromosome 2, under the RFLP marker CT38. F2 populations segregated for the woolly phenotype were obtained by crossing IL2-3 and IL2-5 with LA3186. We determined Wo in the genetic interval between W124 and C2_At5g64670 using F2 nonwoolly individuals from the cross IL2-5×LA3186. We further carried out fine mapping based on the F2 progenies from the cross IL2-3×LA3186 using newly exploited markers between W124 and C2_At5g64670 according to the published BAC sequences and RFLP probe sequences, and delimited Wo in the region between STS64 and STS62, which were both closely linked to it with just one recombination event identified, and STS4 cosegregated with it.2. We tried to align with the sequences of C02HBa0006L05 and C02HBa0204D01, in which STS64 and STS62 located respectively. These two BAC clones formed a～200 kb contig with an 15328 bp overlapping end. We thus confirmed the location of Wo in this-200 kb physical region. Gene prediction of this～200 kb sequence indicated that this fragment contains 19 ORFs as automatically predicted by FGENESH and GENESCAN. One ORF (ORF-4) encodes a polypeptide of 730 amino acids, containing three conserved domains, HD box, bZip motif and START domain, which showed 73% sequence identities with a shoot epidermal cell differentiation related gene, PDF2. We thus considered this ORF as a good candidate for Wo.3. We amplified and sequenced the gDNA and cDNA sequences of this candidate gene from LA3186 and Ailsa Craig, and identified two alleles. One allele is identical to the sequence from Ailsa Craig, while the other contains a missense mutation (C-1904 to G) and results in an amino acid change of Pro to Arg.4. We constructed over-expression constructs of these two alleles using pMV2 vector and RNAi suppression construct using the pHELLSGATE2 vector. We introduced these vectors into nonwoolly plants by stable transformation and obtained 7 transformants exhibiting increased density of trichomes to various degrees. Two of these plants showed typical woolly phenotype. But these two plants showed dwarfism, no lateral root formation, failed to produce flowers and slow growth rate. RNAi transgenic plants showed ’no trichome’. We thus concluded that ORF-4 is Wo. Cosegregation analysis further demonstrated that our candidate is correct.5. RT-PCR analysis with total RNA extracted from young stems, functional leaves, shoot apexes, flowers, green fruits, and roots from LA3186 and nonwoolly plants revealed that Wo is expressed constitutively, with the shoot apexes being the highest. RNA in situ hybridization indicated that the transcripts of Wo were mainly localized in the shoot apical meristem, leaf primordia and epidermis of the young stem. No obvious difference in expression patterns between LA3186 and its nonwoolly segregants was observed. Promoter analysis demonstrated that GUS activity was detected in several tissues, including shoot apexes, axillary meristems, stems, and leaves, with higher activities at shoot apexes, axillary meristems, young stems, and adaxial young leaves. In addition, we also observed high GUS activity in lateral root primordial, gland cells of typeⅦtrichomes and developing embryos.6. Transient expression of CaMV35S::Wo-EGFP fusion construct in onion epidermal cells showed that Wo localized in the nucleus and membrane.7. Cytological observation of developing embryos showed that no obvious abnormality in embryo was observed untill 7 DPA, embryos at 7 DPA were arrested at the globular stage and stopped further development, subsequently cell number increased, while normal embryos continue to differentiate into mature embryos.8. Observation of semi-thin sections indicated that initial patterns of trichome formation varies from each other, and basal cells of each trichome contain special character. SEM observation indicated that the density of type I trichomes in LA3186 was much higher than that of the nonwoolly plants; clusters of trichomes consisting of two or three type 1 trichomes also obviously increased in LA3186. While type I trichomes were nearly absent in the RNAi knockdown plants.9. Sequence alignment of allelic lines LA0053 (Wo-), LA1531 (Wo"), MF802 (unknown), LA0258 (Wom) and LA1908 (Womz) indicated that LA0053 and MF802 are identical at the Wo locus which, like that of LA3186, is heterozygous; LA1531 and LA0258 are identical and homozygous at the Wo locus, with a single allele containing a point mutation at nt 1700, causing an amino acid change of Arg to Leu; the Wo locus in LA 1908 is also homozygous, and this Wo allele contains a point mutation at nt 2075, resulting in an amino acid change of Ile to Thr.10. We analyzed the sequence similarity between Wo encoded by these alleles with eleven homologues of other species. They showed 46～79% sequence identity to these homologues. And the C-terminus of Wo where all the mutations are located is highly conserved among all these homologues.11. Microarray analysis between LA3186 and its nonwoolly segregants demonstrated that approximately 186 genes were found to be regulated in woolly plants in comparison with nonwoolly plants, and 159 of them are up-regulated (85.5%),27 of them are down-regulated (15.5%).12. Transcript level of U226950 in the woolly plants (LA3186, LA0258 and LA 1908) was much higher than that in nonwoolly plants. The full length of U226950 is 321 bp, encodes a protein with 106 amino acids, which showed 55% sequence identity with a hypothetical B-type cyclin gene in Arabidopsis, AT5G06270.1. We thus named this gene as SlCycB2. Ectopic expression of CYCLIN B1;2 in trichomes can induce mitotic divisions and result in the formation of trichome clusters and multicellular trichomes. However, there was no significant similarity between SlCycB2 and CYCLIN B1;2. As leaves of LA3186 have obviously increased number of trichome clusters and multicellular trichomes (typeⅠ), we speculated that SlCycB2 may function downstream of Wo and thereby regulate trichome formation in tomato.13. Expression pattern of SlCycB2 in LA3186 was similar to that of Wo. Importantly, SlCycB2 expression was significantly higher in Wo-overexpressing transgenic plants but lower in Wo-RNAi plants. These results further demonstrated that SlCycB2 expression was regulated by Wo.14. We down-regulated SlCycB2 by RNAi in LA3186 and found that the RNAi lines showed obvious decrease in trichome density. SEM observation showed that type I trichomes nearly disappeared, and type V-like trichomes increased, most of which have many branches.15. Promoter sequence analysis showed that an 11 bp DNA motif (CTAATTGTTTA) encompassed in the promoter region, which has been demonstrated as a cis-element responsible for direct binding with HD domain. Yeast one-hybrid experiment indicated that Wo had no binding activity with this element, suggesting that SlCycB2 may not be regulated by promoter binding with Wo. BiFC and yeast two-hybrid experiments demonstrated that Wo can physically interact with SlCycB2. These data indicated SlCycB2 was regulated by Wo through their direct protein interaction.16. B-type cyclins function at the G2/M transition during mitosis, we thus inferred that a dramatic increase in type I trichome number may be attributed to the enhancement of mitosis in the epidermal cells destined to become trichomes, in which SlCycB2 expression was up-regulated by Wo. This transcriptional regulatory pathway is distinct from that characterized in Arabidopsis and cotton.