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Zm40S-S24 and Zm40S-S27: TWO MAIZE GENES CODING FOR RIBOSOMAL PROTEINS EXPRESSED IN THE ROOT APEX AND YOUNG SHOOTS Gaëlle C. Stukart 1, Virginia Stiefel.

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Presentation on theme: "Zm40S-S24 and Zm40S-S27: TWO MAIZE GENES CODING FOR RIBOSOMAL PROTEINS EXPRESSED IN THE ROOT APEX AND YOUNG SHOOTS Gaëlle C. Stukart 1, Virginia Stiefel."— Presentation transcript:

1 Zm40S-S24 and Zm40S-S27: TWO MAIZE GENES CODING FOR RIBOSOMAL PROTEINS EXPRESSED IN THE ROOT APEX AND YOUNG SHOOTS Gaëlle C. Stukart 1, Virginia Stiefel 3, Pere Puigdomènech 3, Paulo Arruda 1,2 and Marcelo Menossi 1,2 1. Centro de Biologia Molecular e Engenharia Genética, Universidade Estadual de Campinas, 2. Departamento de Genética e Evolução, Instituto de Biologia, Universidade Estadual de Campinas. Campinas, SP, Brazil 3. Departament de Genética Molecular, Institut de Biología Molecular, Barcelona, Espanha. Phone: , Fax: European Community Supported by: INTRODUCTION Aluminum (Al) is one of the main limiting factors of root growth in acid soils. These soils occupy about 84% of the Brazilian territory. The root apex is the major site of the Al toxicity. This project aimed to isolate genes expressed mainly in the root apex of Al-tolerant maize plants, widening the knowledge of this part of the plant. These genes and their promoters will be also useful to create transgenic Al-tolerant plants, improving the productivity. Two ribosomal-proteins cDNAs were isolated. They were named Zm40S-S24 and Zm40S-S27, due to their homology with ribosomal proteins. Plant material was obtained from the Germplasm Bank from the University of Campinas. Two maize lines, Cat (Al-tolerant) and S (Al-sensitive) were used. The mRNA Differential Display (D.D.) was used to compare different parts of the seedling: the root apex, the rest of the root and the shoot. This technique uses a T 12 VN primer (where V=A, C or G and N=A, C, G or T), that hibridizes to the polyadenylate tail of the mRNA, and arbitrary primers, that anneals at different positions in the sequence. After reverse transcription the cDNA from different parts was amplified and resolved in a DNA sequencing gel to detect the differential expression. The cDNAs were isolated and their differential expression was confirmed by Northern Blot using RNA from seedlings and adult leaves. Northern Blot was also used to evaluate the expression in response to Al, Lanthanum (La) and Sodium Chloride (NaCl). 5´RACE-PCR (Rapid Amplification of cDNA Ends) was used to characterize the complete sequence of the cDNA. To evaluate the genomic organization, a Southern Blot was made using probes corresponding to the 3´ region of the cDNA and the codifying region. The characterization of the spatial expression of the Zm40S-S24 gene was conducted by in situ hybridization using a DIG-labelled probe. MATERIALS AND METHODS Figure 1: Isolation of two cDNA, Zm40S-S24 and Zm40S-S27, using mRNA Differential Display. a, cDNAs from the root apex (A), rest of the root (R) and shoot (S) of Cat lines were resolved in a DNA sequencing gel. The two differentially expressed bands are indicated by arrows, the first one indicates the Zm40S-S27 and the second one Zm40S-S24. These bands were eluted from the gel, reamplified and cloned. b, steady state of Zm40S-S24 and Zm40S-S27 transcript accumulation in different parts of the plant. Zm40S-S24 and Zm40S-S27 probes were hybridized to total RNA from root apex (A), rest of the root (R), shoot (S) and adult leaf (L). Transcript levels of Zm40S-S24 show that its expression is higher in the root apex, but also present in the shoot. Transcript levels of Zm40S-S27 show that its expression is higher in the root apex, also high in the shoot and present in the rest of the root and in the leaves. Figure 3: Steady state of Zm40S-S24 and Zm40S-S27 transcript accumulation in response to Al, La and NaCl. To evaluate the expression of these genes in response to environmental stresses, maize seedlings from Cat100-6 (Al-tolerant) and S (Al-sensitive) were exposed to toxic concentrations of Al, La and NaCl for 24 and 48 hours. Total RNA from root tips was isolated and used in Northern blot experiments probed with DNA fragments corresponding to the 3 end of Zm40S-S24 (a) and Zm40S-S27 (b). NaCl stress represses the expression of both genes in both maize inbred lines. This repression is lower in S , in accordance with its higher tolerance to this salt (not shown). Al and La stress do not change the expression of Zm40S-S24 and Zm40S- S27 in Cat100-6, but they cause a slight decrease in S This is in accordance with the higher tolerance to both ions observed in Cat100-6 when compared to S (not shown). Figure 2: Complete cDNA sequence and deduced protein of Zm40S-S24 and Zm40S-S27, obtained by mRNA Differential Display and 5 RACE-PCR. The sequence found in the D.D. is shown in red. In each case three oligonucleotides were designed and used to amplify the missing 5 cDNA sequence by means of 5 RACE-PCR. The numbers on the left refer to the bases, and the numbers on the right refer to the aminoacids. Zm40S-S24 (a) and Zm40S-S27 (b) showed significant homology to other 40S proteins. Figure 4: Genomic organization of Zm40S-S24 and Zm40S-S27. Genomic DNA isolated from adult leaves of Cat and S lines was digested by Xba I (X), Hind III (H), Bam HI (B) and Eco RI (E). The sequence probe (a) showed that Zm40S-S24 and Zm40S-S27 have just one copy in genome, but the RACE sequence probe (b) has many copies. These results indicated that Zm40S-S24 and Zm40S-S27 are single copy genes that belong to a large family of genes coding for ribossomal proteins. Figure 5: In situ hybridization of Zm40S-S24 to maize roots and shoots. In order to identify the region within the root apex and the shoot where the Zm40S-S24 mRNA accumulates, in situ hybridization experiments were performed. The Zm40S-S24 antisense DIG-labeled probe (a) was hybridized to Cat root section. Results show that Zm40S-S24 mRNA is detected in meristematic cells. No signal could be detected using the sense probe (b). The Zm40S-S24 antisense DIG-labeled probe (c) was hybridized to Cat shoot section. Results show that Zm40S-S24 mRNA is detected in the leaf primordia but not in the coleoptile. No signal could be detected using the sense probe (d). Two genes coding 40S ribosomal subunit were identified. Our results regarding the expression of these genes are in accordance with the housekeeping role of these ribossomal proteins. However, we found that both genes, Zm40S-S24 and Zm40S-S27, are subjected to a spatial control, being expressed mainly in the root apex and young shoots. This control of gene expression was clearly for the Zm40S-S24 by in situ hybridization. As these genes are part of a multigenic protein family, we spectulate that other members could be specific for other parts of the plant. CONCLUSION a M T N R L L A R K Q F V L (13) 1 cttccgcact tngcaagttc atgaccaacc gcctgctggc ccgcaaacaa tttgtgcttg E V I H P G R A N V S K A E L K E R L A (33) 61 aggttatcca ccccggccgc gccaacgtct ccaaggcgga gttgaaggag aggcttgcca K M Y E V K D P N T I F V F K F R T H F (53) 121 agatgtacga ggtgaaggac cccaacacca tcttcgtctt caagttccgc acccacttcg G G G K S T G F G L I Y D N L E S A K K (73) 181 gtggaggcaa gtccactggc ttcggcctca tctacgataa cctcgagtct gccaagaagt F E P K Y R L I R N G L A T K V E K S R (93) 241 tcgagcccaa ataccgcctc atcaggaatg gtcttgctac taaggttgag aagtcccgca K Q M K E R K N R A K K I R G V K K T K (113) 301 agcaaatgaa agagcggaag aacagggcca agaagatccg tggtgtcaag aagaccaagg A G D A K K K (120) 361 ctggtgacgc caagaagaag taaacagtca tttatatttc tgtattactg tcctgaactc 421 tgggtggtcg tctagttgca atgtcataat tatggtcgtg ttaggtttcg ttccgcactc 481 agcagtaagt gaagtttgta anttcctgga tgtgaactta cccggattgg ctgtagcatg 541 ttgtgagatg agaaggctat tttgtcatgc agntnngtca aaaaaaaaaa aa b 1 tcccggctca gacttctgct gcagcgcctc cgccgccgct gccgcatcta acgcagttcc M V L Q N D I D L L N P P(13) 61 ctccgccgca gcccgaggca agatggttct ccagaacgac attgatctgc tcaacccgcc A E L E K L K H K K K R L V Q S P N S F(33) 121 ggcagaactc gagaagctca agcacaagaa aaagagactc gtccagtcgc ccaactcctt F M D V K C Q G C F S I T T V F S H S Q(53) 181 cttcatggat gttaagtgcc aggggtgctt cagcataacc actgttttca gccactccca T V V V C P G C Q T V L C Q P T G G K A(73) 241 gactgttgtt gtgtgccctg gctgccaaac tgtcctctgc caacccacag gaggaaaggc R L T E G C S F R R K G D(86) 301 aaggctcact gagggctgtt ctttccgcag gaagggagac tagagcggat gtggtcttca 361 agttttgctc tatgccagat actagcgtca tgaactgcct ttagctaaga ttttccataa 421 gttgttctcg agcagctatg tgttgcatgt tggctacttc caaacttata tctggtttgg 481 tttggattgt gatgaactaa tggctctttg aatttatatt atgatgttga cgatatttgg 541 cgacatctgg tttcttggtg aaaaaaaaaa


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