Punctual mutations in lipoxygenase sequence expressed in black soybean seed coat 1

Lipoxygenase enzymes are relevant in several characteristics of soybean seeds, such as germination performance, storage capacity and tolerance to infection by microorganisms. In this sense, the study of allelic variations of the genes related to that enzyme may be promising for the development of molecular markers associated with phenotypic characteristics which are relevant to seed physiology. This study aimed to identify mutations in lipoxygenase sequence expressed in black soybean seed coat. After isolation of the sequence of interest by the cDNA-AFLP technique, such sequence was compared with sequences of nucleotides and proteins of the lipoxygenase present in yellow soybean seed coat. Analysis of multiple sequence alignment has shown an expressive number of changes in the sequence of nucleotides, which have resulted in the modification of 13 amino acids in the translated protein. The identification of such mutations widens the possibility for further studies involving the association of these allelic variations with phenotypic differences of interest to seeds physiology.


Introduction
The lipoxygenase enzyme catalyzes the hydroperoxidation of polyunsaturated fatty acids (Vick and Zimmerman, 1981).Soybean seeds contain three lipoxygenases isozymes called Lipoxygenase 1 (L-1), Lipoxygenase 2 (L-2) and Lipoxygenase 3 (L-3) (Axelrod et al., 1981).It was suggested that products of the lipoxygenases can be metabolized and produce compounds with distinct functions during germination, plant growth and development, as well as in the mechanisms of tolerance to pathogen infections (Dias et al., 2002;Akram et al., 2008).
Innumerous scientific studies have been performed aiming at investigating the effect of lipoxygenases in soybean seed performance.Negative effect of lipoxygenase was observed during soybean seed storage; however, in studies performed by Lima et al. (2010), it was observed that soybean seed deterioration was more influenced by the linolenic acid content than by the lipoxygenase enzyme, since the vigor of the genotypes with low linolenic acid content was higher during storage.
In contrast, Oliveira et al. (2006) showed that the presence of lipoxygenase contributes to favor emergence speed of soybean seedlings.In germinating soybean seeds, lipoxygenases were found not to be involved in lipid mobilization (Wang et al., 1999).The lack of substantial oxygenation of polyunsaturated fatty acids during the germination process supports the idea that soybean seed lipoxygenases might have been recruited to function as storage proteins despite their intact but obsolete enzymatic capacity (Siedow, 1991;Wang et al., 1999;Lenis et al., 2010).The presence of lipoxygenase in the germinative process was also detected in studies carried out with pea seeds (Pisum sativum L), in which there is a shift of lipoxygenase activity from radicles to shoots that accompanies the transition from seed lipoxygenases to vegetative lipoxygenases (Mo and Koster, 2006).
Secondary metabolites, resulting from lipoxygenase activity, also play an important role in the defense against pathogens, even in seeds.In studies conducted by Gao et al. (2009), some maize mutant seeds, without lipoxygenase, presented higher susceptibility to different species of Aspergillus spp.
Among soybean genotypes, genetic variability indeed occurs for some seed characteristics, including those related to coat color (Mertz et al., 2010), which may be used in studies aiming at identifying allelic variations of interest genes, thus facilitating comprehension of the role of enzymes or proteins of importance to seed physiology, as lipoxygenase.
Thus, this study aimed to identify mutations in the lipoxygenase sequence expressed in soybean genotypes with black coat coloration.

Material and Methods
The lipoxygenase sequence from black soybean seed coat was obtained from the AFLP Starter Primer Kit (Invitrogen) primer combination EcoRI-AGC/MseI-CTC which generated 12 polymorfi c fragments of cDNA -AFLP, differentially expressed between soybean seeds coats (Genotype TP) of black coat coloration and (cv.CD 202) of yellow coloration, as described by Mertz et al. (2010).One of the polymorphic fragments was aligned and showed high similarity with the lipoxygenase sequence.Afterwards, the same sequence was compared by means of global alignment with the lipoxygenase sequences deposited in data bank NCBI (Nacional Center for Biotechnology Information -http:// www.ncbi.nlm.nih.gov/) in order to detect the occurrence of preserved blocks and the occurrence of mutation of the insertion/ deletion type (INDELS) or of simple nucleotide polymorphism (SNPs).This was done by associating software programs ClustalW (Thompson et al., 1994) and BioEdit 7.0.0(Hall, 1999).After identifying the presence of mutations, the sequences were then translated into amino acids by using the software ExPASy -Translate tool (Gasteiger et al., 2003) in (www.expasy.ch/tools/dna.html) and aligned again by means of software programs ClustalW (Thompson et al., 1994) and BioEdit 7.0.0(Hall, 1999).This was performed to verify if the changes in the nucleotides resulted in changes in the sequence of amino acids of the protein.

Soybean Soybean Soybean T T TA A A TA T T TA TA TA T T TA T T T TGG GG GG
From the 13 modifi ed amino acids, eight were substituted by amino acids belonging to the same group; two amino acids were substitutions from the neutral polar group to the basic polar group, or vice-versa (Ser-Lys and His-Thr); two amino acids of the non-polar group were substituted by amino acids from the neutral polar group, or vice-versa (Ala-Thr and Gln-Ala); and one polar amino acid was substituted by another from the neutral polar group (Glu-Gly) (Table 1).

E E E E E E YC BC
Table 1.Changes in amino acid composition of the lipoxygenase protein expressed in the soybean seeds coats of black coloration.
The different colors represent the groups sorted as a function of the radical: red (non-polar); blue (polar neutral); black (polar acid); and green (polar basic).Such modifi cations may cause changes in the tridimensional structure of the protein and, consequently, its catalytic function (Skrzypczak-Jankun et al., 1997).
The phenotypic effects of these mutations are as much or more drastic, the bigger the difference in the chemical nature of the lateral chains of the amino acid residues; for example, the substitution of one hydrophilic amino acid (polar) by one amino acid with lateral chain of hydrophobic residue (nonpolar).This occurred when the substitution of Glutamine by Alanine (Gln -Ala), or yet when the substitution resulted in the inversion of the electric charge of the residue.In this study, the substitution of glutamic acid by glycine (Glu -Gly) occurred, i.e., of one amino acid with negative charge by one neutral amino acid, that is, without electric charge.
SNP variations (Single nucleotide polymorphism) were identifi ed in genes of the alpha amylase enzyme in different ecotypes of rice, aiming at associating these mutations with characteristics related to seed germination (Malone et al., 2008).
The role of LOXs in plants is ambiguous; some reports suggested that LOX and its hydroperoxide products might directly participate in senescence; however, data obtained by (Wang et al., 1990) suggested that LOX, especially LOX 2, may play an important physiological role in maintaining viability rather than promoting senescence of soybean seeds.
In addition, it was suggested that the lipoxygenases enzymes and their products may play a significant role in the seed germination process and in seed physiological quality (Dias et al., 2002;Oliveira et al., 2006).So, the identification of these expressed mutations in the sequence of lipoxygenases obtained in this study suggests the feasibility of further studies involving associations of these allelic variations with phenotypical difference for characteristics of interest to seed physiology, aiming at the development of molecular markers based on SNP.

Conclusion
Punctual mutations were identified in lipoxygenase sequences from black soybean seed coat.

Figure 1 .
Figure 1.Global alignment between lipoxygenase nucleotides sequences expressed in black soybean seed coat (BC) and lipoxygenase sequence of soybean genotypes with yellow seed coat (YC) (Lipoxygenase (U26457.1)-Glycine max).Black arrows indicate changes of nucleotides that result in amino acid substitution; red arrows indicate silent mutations, and blue arrows indicate mutations that occur after codon determination.