Zm40S-S24 and Zm40S-S27: TWO MAIZE GENES CODING FOR RIBOSOMAL PROTEINS EXPRESSED IN THE ROOT APEX AND

YOUNG SHOOTSGaëlle C. Stukart1, Virginia Stiefel3, Pere Puigdomènech3, Paulo Arruda1,2 and Marcelo Menossi1 ,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, Brazil3. Departament de Genética Molecular, Institut de Biología Molecular, Barcelona, Espanha.e-mail:stukart@unicamp.brPhone: 19-788-1143, Fax: 19-788-1089

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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 100-6 (Al-tolerant) and S1587-17 (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 T12VN 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 100-6 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 S1587-17 (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 S1587-17, 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 S1587-17. This

is in accordance with the higher tolerance to both ions observed in

Cat100-6 when compared to S2587-17 (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 100-6 and S 1587-

17 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 100-6 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 100-6 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 gtggtcttca361 agttttgctc tatgccagat actagcgtca tgaactgcct ttagctaaga ttttccataa421 gttgttctcg agcagctatg tgttgcatgt tggctacttc caaacttata tctggtttgg481 tttggattgt gatgaactaa tggctctttg aatttatatt atgatgttga cgatatttgg541 cgacatctgg tttcttggtg aaaaaaaaaa