Using Macroarrays Containing Sugarcane ESTs to Identify Aluminum-induced Genes in Maize
Felix, J.M., 1) Duarte, R.D.,1) Jorge, R.A. 2), Arruda, P., 1) and Menossi, M. 1)
1) Centro de Biologia Molecular e Engenharia Genética, Departamento de Genética e Evolução, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), CP 6010, Campinas, SP, 13083-870, Brazil.
2) Departamento de Físico-Química, Instituto de Química, UNICAMP, Campinas, SP, Brazil.
Key words: aluminum, acid soils, cDNA arrays, cross-species hybridization, maize, sugarcane
Aluminum (Al) toxicity affects the productivity of several crops in acid soils, which comprise up to 40% of arable land in the world. The first and most dramatic symptom of Al toxicity is the inhibition of root growth. Our group has been involved in the Sugarcane Expressed Sequence Tag (SUCEST) project which has generated around 50,000 genes from several tissues. Based on the high level of nucleotide identity found between sugarcane and maize genes, radiolabeled cDNA probes from maize would be expected to hybridize with the sugarcane ESTs on the arrays. In this work, we have confirmed this hypothesis by showing that sugarcane macroarrays can be used to identify Al-induced genes in maize.
Aluminum (Al) is the most abundant metal in the earth’s crust. Since many plant species are sensitive to micromolar concentrations of Al, the potential for soils to be Al toxic is considerable (Delhaize and Ryan, 1995). In acid soils, the solubilization of Al is enhanced and Al toxicity is a major factor limiting plant production. The primary effect of Al in plants is the inhibition of root growth, which causes a decrease in water and nutrient uptake. Because Al exerts a strong selective force, plants have developed strategies to overcome Al toxicity.
The cloning of genes induced in response to this ion is a key step to clarifying the mechanisms of Al toxicity and tolerance in plants. In the past five years, the hybridization of DNA arrays has become a powerful tool for evaluating the expression of thousands of genes in parallel (Scheena et al, 1995).
The use of heterologous probes to screen cDNA and genomic libraries is common practice, especially for studying closely related species. As our lab has been working in the sugarcane EST project (sucest.lbi.ic.unicamp.br), we decided to evaluate whether DNA arrays containing sugarcane ESTs from root libraries could be used to identify Al-induced genes from maize.
Materials and methods
Plasmids containing sugarcane ESTs from root libraries of the SUCEST project were denatured with 0.2 M NaOH in 96-well microtitre plates and deposited on Hybond N nylon filters (12 cm x 8 cm) using a 96-pin manual replicator (V&P Scientific, USA), to give a total of 384 ESTs in duplicate. DNA was fixed to the filters by baking at 80?C.
Zea mays seedlings from the inbred lines Cat100-6 (Al-tolerant) and S1585-17 (Al-sensitive) were grown in nutrient solution containing different Al concentrations (Moon et al, 1997). Total RNA from root apex was extracted accoding to Logemann et al. (1987). 32Alpha-P-labeled cDNA probes were obtained from 30 ;g of total RNA as described by Schummer et al. o(1999). DNA array filters were hybridized for 16 h at 42C using high stringency hybridization conditions and then
exposed to X-ray films (Sambrook et al, 1989). Autoradiographies were inspected to detect differences in the signal from control and treated cDNA probes. Northern blot hybridizations were done as described by Sambrook et al. (1989), using high stringency hybridization conditions.
Results 32Two replicate membranes containing 384 sugarcane ESTs were obtained. The alpha-P-labeled cDNA probes
were synthesized from total RNA extracted from the root tips of Cat100-6 seedlings which had or had not been exposed to Al. After hybridization to the DNA arrays, three ESTs corresponding to Al-induced genes were identified (indicated by the arrows in Figure 1A). The deduced protein of these clones showed identity to a root-specific protein ZRP3, an S-adenosyl methionine synthetase and a water channel protein (not shown). The corresponding maize genes were identified in the maize genome database (gremlin3.zool.iastate.edu). There was a good correlation between the pattern detected in the arrays and the results observed in northern blots containing RNAs from the root apex of both maize lines exposed to increasing concentrations of Al (Figure 1 B; only the result for the ZRP3 gene is shown).
Root specific protein (ZRP3) -maizeRoot specific protein (ZRP3) -maize
151515000555505050-6-6-6(x 10(x 10(x 10)activity)activity)activity858585Cat100-6Cat100-6
S 1587-17S 1587-17
Figure 1: Identification of Al-induced genes in maize using arrays containing sugarcane ESTs. (A) Two replicate
membranes containing 384 sugarcane ESTs were hybridized with probes from Cat100-6 seedlings which had (Aluminum) or had not (Control) been exposed to 50 ;M of Al activity. The arrows indicate the ESTs corresponding to Al-induced genes. (B) Accumulation of the ZRP3 homolog probe was evaluated by northern blot using RNA from the root apex of both lines exposed to increasing Al activities. The total RNA stained with ethidium bromide before being transferred to the membrane is shown under each lane.
The high homology between the sugarcane and maize genomes allowed the use of cross-species hybridizations to evaluate gene expression. With this method, we identified three maize genes induced by Al treatment. There are no previous reports on the induction of these genes by Al.
S-Adenosylmethionine synthetase (SAM-S) catalyzes the biosynthesis of S-adenosylmethionine (Adomet) from Met and ATP. The expression of SAM-S is significantly altered in response to salt (Espartero et al.,1994) and water (Mayne et al, 1996) stress. Since SAM is involved in several biological process, we cannot speculate on its role in the response to Al toxicity.
The water channel protein belongs to a family of membrane integral proteins (MIPs) found in the tonoplast and plasma membrane. These proteins are involved in the transmembrane transport of water during plant growth and development and in response to stress. The water conductivity (Lp) of roots varies in response to several environmental factors, including water and salt availability, low temperatures and nutritional deficiency (Maurel, 1997). Since Al affects membrane permeability (Deleers et al, 1986), it will probably change root Lp. Increased levels of this protein could help regulate water transport through membranes, thereby preventing major changes in root Lp.
We are currently quantifying the hybridization signals with a phosphorimager. The data obtained so far have allowed the identification of several other genes that were clustered according to their expression pattern.
In conclusion, cross-species hybridizations is providing a greater understanding of the defenses activated by maize plants in response to Al stress.
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