A FLORICAULA/LEAFY gene homolog is preferentially expressed in developing female cones of the tropical pine Pinus caribaea var. caribaea

Marcelo Carnier Dornelas; Adriana Pinheiro Martinelli Rodriguez

Universidade de São Paulo, Centro de Energia Nuclear na Agricultura, Laboratório de Biotecnologia Vegetal, Piracicaba, SP, Brazil

Correspondence

ABSTRACT

In angiosperms, flower formation is controlled by meristem identity genes, one of which, FLORICAULA (FLO)/LEAFY (LFY), plays a central role. It is not known if the formation of reproductive organs of pre-angiosperm species is similarly regulated. Here, we report the cloning of a conifer (Pinus caribaea var. caribaea) FLO/LFY homolog, named PcLFY. This gene has a large C-terminal region of high similarity to angiosperm FLO/LFY orthologs and shorter regions of local similarity. In contrast to angiosperms, conifers have two divergent genes resembling LFY. Gymnosperm FLO/LFY proteins constitute a separate clade, that can be divided into two divergent groups. Phylogenetic analysis of deduced protein sequences has shown that PcLFY belongs to the LFY-like clade. Northern hybridization analysis has revealed that PcLFY is preferentially expressed in developing female cones but not in developing male cones. This expression pattern was confirmed by in situ hybridization and is consistent with the hypothesis of PcLFY being involved in the determination of the female cone identity. Additionally, mutant complementation experiments have shown that the expression of the PcLFY coding region, driven by the Arabidopsis LFY promoter, can confer the wild-type phenotype to lfy-26 transgenic mutants, suggesting that both gymnosperm and angiosperm LFY homologs share the same biological role.

Key words: LEAFY, plant reproduction, development, gene expression, flowering.

Introduction

The molecular mechanisms underlying both intrinsic and extrinsic controls of pine shoot development are largely unknown. For both practical and scientific reasons, we are interested in mechanisms that bring about the formation of reproductive organs rather than vegetative shoots. The practical goal is to control flowering in this commercially important tropical forestry species. The knowledge of fundamental aspects of the reproductive development will allow us to genetically modify this process and to produce trees that flower earlier or are sterile. Genetic engineering of tropical pine, including regeneration of transgenic plants, has already been achieved (Walter et al., 1998). A further scientific goal is to uncover the regulation of reproduction in pre-angiosperm species and gain understanding of the evolution of this process in plants.

Several genes involved in the vegetative-to-reproductive transition have been cloned in angiosperm annual species (Levy and Dean, 1998; Lohmann and Weigel, 2002; Izawa et al., 2003). These genes can be loosely classified into one of the following three groups: (i) flowering-time genes that are involved in the vegetative-to-reproductive phase change by acting globally or locally in the shoot apical meristem; (ii) locally acting meristem identity genes that regulate the establishment of the floral meristem identity; and (iii) locally acting floral organ identity genes that regulate the identity of floral organs. These genes and their functions appear to be conserved in angiosperms, including monocotyledons (An et al., 1994; Pnuelli et al., 1994; Colombo et al., 1996; Mena et al., 1996). In contrast, there is very little information concerning pre-angiosperm plant species, in which only a few corresponding genes have been cloned (Picea abies, Tandre et al., 1995; Gnetum gnemon, Münster et al., 1997; Pinus radiata, Mouradov et al., 1998; Pinus radiata, Mellerowicz et al., 1998). The LEAFY (LFY) gene of Arabidopsis, or its Antirrhinum ortholog FLORICAULA (FLO), is one of the key regulatory genes involved in both the control of the vegetative-to-reproductive phase change (Blázquez et al., 1997) and the acquisition by axillary meristems of the floral meristem identity (Huala and Sussex, 1992; Weigel et al., 1992; Weigel and Nilsson, 1995). Plants overexpressing LFY flower early, produce flowers in the place of lateral vegetative shoots and convert their otherwise indeterminate terminal shoot meristems to flowers (Weigel and Nilsson, 1995; Blázquez et al., 1997). These effects can also be observed in heterologous angiosperm species (Nilsson and Weigel, 1997), indicating that the function of LFY is largely conserved. Mutations in FLO/LFY cause the conversion of flowers into shoots. In Arabidopsis, male-sterile flowers are occasionally produced even in mutants homozygous for the strongest lfy alleles, indicating that a parallel pathway may specify floral meristem identity. In contrast, in Antirrhinum, mutations in FLO abolish flowering (Coen et al., 1990). Thus, the activity of the parallel pathway specifying floral meristem identity can vary among species. This paper reports cloning and expression studies of a Pinus caribaea var. caribaea FLO/LFY homolog named PcLFY. In Pinus, genes NEEDLY (NLY) and PRFLL were reported, representing two divergent Pinus radiata LFY homologs (Mouradov et al., 1998; Mellerowicz et al., 1998). Sequence comparisons indicate that PRFLL and NLY represent divergent proteins, and that conifers, in contrast to angiosperms, have two LFY-like paralogs. PRFLL is largely expressed in buds and male cones but not in female cones or other somatic tissues, while NLY is preferentially expressed in female cones (Mouradov et al., 1998; Mellerowicz et al., 1998). We performed a detailed spatial and temporal analysis of the P. caribaea PcLFY gene, using Northern and in situ hybridization analysis, and our results were consistent with PcLFY being involved in female cone identity determination. Additionally, our experiments demonstrated that PcLFY can complement the lfy-26 Arabidopsis mutant, which shows the strongest mutant phenotype, suggesting that both pine and Arabidopsis LFY homologs may share the same biological role.

Plant material

Material for genomic DNA and RNA extraction and for microscopy of vegetative and reproductive tissues of Pinus caribaea var. caribaea was collected in the fields of Escola Superior de Agricultura Luiz de Queiroz, of the University of São Paulo (Piracicaba, SP, Brazil). Young expanding needles were collected for the extraction of genomic DNA. RNA-blot, in situ hybridization and SEM analyses were performed on plant tissues collected and fixed at different developmental stages during two growing seasons corresponding to the years 2000 to 2003.

DNA and RNA extraction, library construction and gene cloning

Total RNA for cDNA library construction and Northern experiments was isolated from young pine leaves (needles), vegetative apices, developing male and female cones at different developmental stages and roots of recently germinated seeds (germinated on wet paper in the dark for 4 days), using the RNeasy plant Minikit (QIAGEN) according to the manufacturer's instructions. Genomic DNA for PCR amplification, Southern analysis and construction of genomic libraries was isolated by the traditional CTAB-based method (Sambrook et al., 1989).

The genomic clones of PcLFY were isolated by screening 300,000 plaques from a P. caribaea genomic library (76 x 10^-6 pfu) constructed with partially Sau3A-digested genomic DNA, using the Packagene Lambda Packing Systems (Promega). This screening was performed with a biotin-labeled probe (North2South chemiluminescent system, Pierce), using the entire Arabidopsis LFY cDNA from plasmid pDW124 (Weigel et al., 1992) as template. Two adjacent BamHI fragments (P45B with 3.35 kbp, and P65B with 6.4 kbp) containing the genomic PcLFY sequence were subcloned into pBluescriptKS (Clontech). Subclones were prepared by nested deletions (as described by Zhu and Clark, 1995) and sequenced on an ABI Prism 377 automated sequencer (Perkin-Elmer/Applied Biosystems), using the DYEnamic ET terminator Cycle Sequencing Kit (Amersham/ Pharmacia Biotech, USA) coupled with M13 reverse and forward primers according to the manufacturer's instructions. The complete PcLFY genomic sequence was deposited in the GenBank under the accession number AY640315.

A cDNA library was constructed using total RNA from a mix of male and female cones at different developmental stages. The poly-A fraction RNA was isolated (Sussman et al., 2000), and the first strand of cDNA was synthesized using the SuperScript cloning system (Life Technologies). The cDNA library screening was performed using a PCR-based strategy (Sussman et al., 2000) and the LFY-specific degenerated primers L1: 5'-CGGAY ATIAAYAARCCIAARATGMGICAYTA-3'and L4: 5'-C GGATCCGTGICK-IARIYKIGTIGGIACRTA-3' (Frohlich and Meyerowitz, 1997). The insert sizes of the positive clones were determined by PCR using the M13 forward and reverse primers, and the five positive clones were sequenced on both strands. The longest PcLFY cDNA sequence was deposited in the GenBank under the accession number AY640316.

Southern and Northern hybridization

Southern blotting was performed as described in Sambrook et al. (1989), using genomic DNA digested with XhoI and PstI and blotted on a Hybond-N Plus membrane (Amersham). Northern experiments were performed using 10 mg of total RNA extracted from male and female developing cones (a mix of different developmental stages), vegetative apices and young needles, separated in a denaturating agarose gel (Sambrook et al., 1989) and hybridized to a PcLFY probe.

The PcLFY probe used in both Southern and Northern experiments was a 235bp PCR product obtained from the 3' transcribed region of the gene, using the primers P13: 5'-CTCCAAGTGACAGAGCTGACG-3' and P25: 5'-CT GCTGGATGTGCAACAT-3', and a PcLFY cDNA clone as template. PCR reactions were performed in a final volume of 25 mL with an initial 3 min denaturation cycle at 96 °C, followed by 40 cycles of 96 °C for 40 s; 45 °C for 30 s, and 72 °C for 2 min. The PCR product was purified using the CONCERT Rapid PCR Purification System (Gibco-Life Sciences). The probe was labeled with fluorescein, using the DCP-Star GeneImage System (Pharmacia-Amersham). Hybridization conditions, washing stringencies and detection were those suggested by the kit manufacturer. As a control for gel loading in Northern experiments, the stripped membrane was re-hybridized with a heterologous probe for a constitutively expressed gene, under low stringency, using cDNA for an Arabidopsis ubiquitin as template (GenBank accession AB5432).

Preparation of slides, digoxigenin-labeling of RNA probes, and hybridization were performed as described elsewhere (Dornelas et al., 1999; 2000). A high-stringency hybridization condition was achieved using 50% formamide in the hybridization solution and washes with up to 0.1 % SSC at 55 °C. The template for the PcLFY digoxigenin-labeled probe was the 1,498 bp cDNA fragment containing the complete coding region, cloned in pGEM-T vector. The hybridized sections were viewed immediately and photographed under a Zeiss Axiovert 35 microscope.

Microscopy

All plant material collected for microscopy was immediately fixed in 4% paraformaldehyde under vacuum for 24 h and dehydrated to absolute ethanol, where it was stored at 4 °C until needed. For light microscopy, the dehydrated samples were embedded in Historesin (Leica, 2-hydroxyethyl-methacrylate). Resin polymerization was carried out at room temperature for 48 h. After polymerization, serial sections of 5-8 µm were obtained and stained with 0.05% toluidine blue (Dornelas et al., 1992). The histological sections were observed and photographed under a Zeiss Axiovert 35 microscope.

Alternatively, the plant material was initially dissected in absolute ethanol under an Olympus dissecting microscope. The resultant material was critical point-dried with CO₂ in a Balzer's drier and further dissected, when necessary. The samples were mounted in metallic stubs with carbon conductive adhesive tape, coated with colloidal gold and observed at 10-20 kV using a ZEISS DSM 940 A or a LEO 435 VP scanning electron microscope.

Sequence comparisons

The partial PcLFY sequences obtained were manipulated in a standard word processor and aligned using ClustalW (Thompson et al., 1994), before being checked for similarity with sequences already deposited in public databases, using BLASTX (Altschul et al., 1997). The complete nucleotide and protein sequences of different LFY homologs were retrieved from GenBank (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi) and aligned with the entire nucleotide and deduced protein sequences of PcLFY, using ClustalW (Thompson et al., 1994). Distance matrixes were obtained from the alignments, and comparative trees were built using TreeView (Page, 2000). Alternatively, parsimony analysis was performed using PAUP (Swofford, 1998).

Complementation of the Arabidopsis lfy-26 mutant

The XbaI–SmaI PcLFY fragment, carrying the coding region of PcLFY, with its endogenous start and stop codons, was obtained from plasmid pPCLFY and blunt-ended using DNA polymerase I (Klenow fragment). An intermediate pDW132P vector was prepared by cloning the polished fragment described above into the SmaI site of pDW132, containing the Arabidopsis LFY promoter (a gift from D. Weigel, Salk Institute, LA Jolla CA, USA). The correct orientation of the cloning process was checked by endonuclease digestion. The PstI-SpeI fragment from the resultant pDW132P (LFY::PcLFY) vector was blunt-ended with Klenow and cloned into the plant transformation vector pSKI015 (a gift from D. Weigel, Salk Institute, LA Jolla CA, USA), that contains the bar gene, allowing selection with the herbicide Basta (Sylvet), constituting the pSKI015P vector. Arabidopsis plants (Columbia ecotype) transgenic for pSKI015E T-DNA were generated by using Agrobacterium tumefaciens-mediated in planta transformation, as described by Bechtold and Pelletier (1998). Putatively transformed seeds were selected upon germination on sand wetted with a Basta (Sylvet) solution at 500 µL.mL^-1. Homozygous (Basta-resistant) lines were created by selfing. The resistant:sensitive segregation ratio was used to estimate the number of transformed T-DNA loci. T2 lines, homozygous for the LFY::PcLFY T-DNA loci, were identified by sowing 200-300 T2 seeds, derived from different T1 plants under selective conditions. Transgenic and non-transgenic plants were grown in growth chambers at 23 °C under illumination with fluorescent light: long-day (LD) conditions (16 h of light/8 h of darkness) or short-day (SD) conditions (8 h of light/16 h of darkness). Finally, LFY::PcLFY transformants in the Columbia ecotype were crossed to the strong lfy-26 mutant allele in the Landsberg erecta background (wild-type and mutant Arabidopsis seeds were obtained from the ABRC seed stock at the Ohio State University facility at Columbus, Ohio, USA). To genotype F2 plants at the LFY locus, CAPS (Cleared Amplified Polymorphic Sequences; Konieczny and Ausubel, 1993) markers that distinguished between Columbia and Landsberg were used (URL: http://www.salk.eduyLABSypbio-wycaps.html). Transgenic and non-transgenic Arabidopsis flowers and inflorescences at different developmental stages were photographed under a stereomicroscope or analyzed by SEM.

Shoot and female cone development in Pinus caribaea var. caribaea

Vegetative and reproductive development in Pinus species is regulated by an integrated system of ontogenetic, positional and environmental factors. These factors determine the rate of formation, the type and the differentiation of primordia produced at the flanks of the apical meristem and in axils (Doak, 1935). In the juvenile stage, the meristem continuously produces primary needle primordia that elongate and form photosynthetic organs (Figure 1A). Some of the primary needle primordia are accompanied by axillary meristems that form either long or short shoot primordia (Riding, 1972). Long shoot primordia give rise to branches, whereas short shoot primordia form secondary needles while their apical meristems cease to function. As pine seedlings pass into the adult stage, the primary needles shorten and develop into scale-like cataphylls (Doak, 1935). Some cataphylls become bud scales, enclosing both apical and axillary meristems together with associated primordia and forming closed buds (Figure 1B and E). Bud growth becomes cyclic, with sequential formation of various types of primordia within each cycle. The number of cycles produced annually and bud morphology vary among pine species (Doak, 1935). The apical meristem produces bud scales and bracts subtending axillary short and long shoot primordia (Figure 1B and E). In addition, two new kinds of axillary primordia are formed: the male and female cone primordia. Male cone primordia usually develop in buds on subordinate branches located within the lower crown. These buds produce only one growth cycle during a year. Female cone primordia develop in buds on dominant branches located within the upper crown. These buds produce up to five growth cycles each year (Doak, 1935). Typically, female cones (Figure 1D and F) are found in the first two cycles initiated in the summer (Bollman, 1983). During each cycle, the meristem produces primordia in a defined sequence (Doak, 1935). Male and female cone primordia are not present in all buds, indicating that the formation of reproductive primordia in mature plants is regulated by some other factors, in addition to the ontogenetic stage and position. These factors probably include environment and gibberellins (Cecich et al., 1994).

Cloning and sequence analysis

Evolutionary relationships

A maximum parsimony consensus tree was constructed to access the phylogenetic relationships of PcLFY with its other gymnosperm and angiosperm counterparts (Figure 3). PcLFY is closely related to P. radiata PRFLL, but the distance between PcLFY and NLY exceeded the distance between most divergent angiosperm LFY orthologs. PcLFY was slightly closer to angiosperm proteins than NLY (distances 60-69% vs. 64-72%). When we considered only the gymnosperm LFY-like homologs, it became apparent that the gymnosperm sequences formed two groups: one containing NLY, and the other containing PcLFY and PRFLL. The phylogenetic relations of PcLFY with other plant LFY-related proteins revealed three facts. First, the gymnosperm sequences formed a group distinct from angiosperms. Second, the sequences of PcLFY and NLY were more divergent than any two angiosperm LFY orthologs. Third, the gymnosperm sequences formed two groups, named LFY-like and NLY-like. The mostly male theory of the origin of angiosperm flowers (Frohlich, 2003) assumes that the co-expression of genes responsible for the identity of male and female organs in a same structure would be sufficient to produce bisexual flowers in the angiosperm ancestor. Thus, the presence of two homologs of LFY in gymnosperms and of only one in most angiosperms (probably due to the loss of the NLY-like lineage) would be a key observation to corroborate the mostly male theory (Frohlich and Parker, 2000; Frohlich, 2003)

Gene copy number determination and expression analyses

To establish whether the PcLFY gene is a single-copy gene or part of a multigene family, we performed Southern Blot hybridization using digested P. caribaea genomic DNA. The hybridization pattern obtained using low stringency washes (2xSSC, 42 °C) suggested that an additional PcLFY-like gene may exist in the P. caribaea genome (Figure 4A). However, using more stringent washes (i.e., 0.1 x SSC, 65 °C), a single band was detected in each lane (data not shown). We also used the higher stringent conditions (i.e., 0.1 x SSC, 65 °C) to perform a Northern Blot analysis (Figure 4B), aiming to determine the steady-state PcLFY mRNA level in vegetative and reproductive tissues of P. caribaea. A single band corresponding to the transcript size of about 1.5 kb was observed, predominantly in developing female cones. This size corresponded to the cloned cDNA size and the transcript size of angiosperm LFY orthologs (Coen et al., 1990; Weigel et al., 1992; Kelly et al., 1995; Anthony et al., 1993).

The ontogenetic pattern of PcLFY expression was further studied by in situ hybridization, in longitudinal sections of shoot apices of adult (15 years-old) plants (Figure 5). Low levels of the PcLFY transcript were found in all types of meristems, but the hybridization signals were higher in developing female cones (Figure 5). With the Northern hybridization analysis, where the level of steady-state RNA was much lower in developing male cones and vegetative buds than in the developing female cones, some degree of expression was observed in vegetative tissues and male cones by in situ hybridization. The reason for that was probably a dilution of the peripheral bud tissues by very large pith and vascular tissue in the dominant mature buds, or maybe the probe and the stringency used in the hybridizations were not specific enough. No PcLFY transcript was detected in differentiating bracts or needles.

Complementation of Arabidopsis lfy mutants by the expression of PcLFY

The coding region of the PcLFY cDNA was fused downstream to the Arabidopsis LFY promoter and used to transform Arabidopsis plants that were crossed to the strong-phenotype lfy-26 Arabidopsis mutant. Upon identification of homozygous transgenic mutant plants (verified by CAPs genotyping, data not shown, Konieczny and Ausubel, 1993), their phenotype was analyzed. Complete restoration of the wild-type phenotype was observed (Figure 6). Early-arising (basal) flowers were replaced by bracts with secondary inflorescence shoots in the Arabidopsis lfy-26 mutants, whereas later arising flowers were replaced by small bracts, in whose axils abnormal flowers developed (Figure 6B and C; Weigel et al., 1992). These abnormal flowers contained sepals and carpels but no petals or stamens, these later being usually homeotically substituted by more sepals and carpels, respectively (Figure 6C; Weigel et al., 1992). In contrast, wild-type flowers typically contain four sepals, four petals, six stamens and two carpels. The lfy-26 floral phenotype was largely complemented by the LFY::PcLFY transgene. The main shoot of these plants developed flowers in both basal and apical positions, and most of these contained all four floral organ types (Figure 6D).

Discussion

The LFY homologs have been consistently reported to be involved in flower formation in angiosperms (Lohmann and Weigel, 2002). The existence of its homologs in pre-angiosperm plant species that do not form flowers was confirmed by cloning of cDNAs from some gymnosperm species (Mouradov et al., 1998; Mellerowicz et al., 1998; Frohlich and Parker, 2000). Phylogenetic analysis (Figure 3) has demonstrated that gymnosperm sequences form a separate clade from angiosperms and that gymnosperms have, in contrast to angiosperms (Kelly et al., 1995), two paralogous genes resembling FLO/LFY (see also Frohlich and Parker, 2000). These two genes, represented by PcLFY and NLY, had rather divergent sequences, yet they shared some features that distinguished them from their angiosperm counterparts. The existence of two divergent LFY-related gene lineages in gymnosperms and the presumptive loss of one of these lineages in angiosperms may be one of the causative forces of the origin of bisexual flowers in angiosperms (Frohlich, 2003). Proline-rich and acidic domains in LFY and FLO (Coen et al., 1990; Weigel et al., 1992), whose presence indicates that these proteins are transcription factors, were not evident in conifers (Figure 2). In contrast, the C-terminal part of the protein, whose function has not been elucidated so far, was highly conserved between angiosperms and conifers. The significance of differences between angiosperms and conifers will become clear when the LFY protein is functionally analyzed by deletion and domain-swamping experiments.

The biological role of the expression of PcLFY or angiosperm LFY orthologs in vegetative buds is still unclear. Whereas neither lesions in LFY or FLO (Coen et al., 1990; Huala and Sussex, 1992; Weigel et al., 1992) nor ectopic over-expression of LFY (Weigel and Nilsson, 1995; Nilsson and Weigel, 1997) affected vegetative development in several angiosperms, it might not be the case in more primitive species. If the flower is homologous to an entire shoot of a cordaite-like progenitor (Hickey and Taylor, 1996), LFY function might have evolved from a gene that controls some aspects of shoot development, for example cell determinacy (Kelly et al., 1995; Hofer et al., 1997). Regardless of the ground level of LFY expression, floral stimulus, be it photoperiod, far-red light treatments, or gibberellin application, was reported to rapidly up-regulate the expression of this gene in diverse angiosperms (Coen et al., 1990; Weigel et al., 1992; Carpenter et al., 1995; Anthony et al., 1993; Blázquez et al., 1997; Hempel et al., 1997). The up-regulation preceded the attainment of developmental commitment to flower (Hempel et al., 1997), and the level of LFY activity determined how rapidly plants started producing flowers (Blázquez et al., 1997). These data are consistent with LFY being a necessary and quantitative, but not always sufficient, factor of floral determination. Our observations of high PcLFY expression in buds with undifferentiated, and therefore possibly not determined, female cone primordia is consistent with PcLFY playing an analogous role in the determination of the female cone primordium identity. In contrast, PcLFY does complement the defects observed in male reproductive development in Arabidopsis lfy-26 mutants (Figure 6). It is thus tempting to speculate that two LFY-like conifer paralogs represented by PcLFY and NLY have separate roles in the reproductive determination of separate male and female reproductive organs. Unlike unisexual angiosperm flowers that have a perfect flower ancestry, conifer cones evolved as truly unisexual organs. It is conceivable that separate meristem identity genes were involved in the determination of male and female organs, and that the function of female organ determination became redundant when the perfect flower evolved. Future comparative in situ-expression studies, analysis of transgenic plants, and studies of a broad range of pre-angiosperm species are likely to resolve these questions.

Acknowledgments

The authors wish to thank Weber A. Neves do Amaral (ESALQ/USP, Forestry Department) for great support on early stages of this work; F.C.A. Tavares and G. Bandel (ESALQ/USP, Genetics Department) for providing an excellent research environment; E.W. Kitajima for maintaining the scanning electron microscope facility at NAP/ MEPA (University of São Paulo, ESALQ, Piracicaba, Brazil); TAIR and the Ohio State University for the Arabidopsis seed stock maintenance; D. Weigel (Salk Institute, La Jolla, USA) for the generous gifts of plasmids pDW124, pDW132 and pSKI015. MCD is grateful to the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for financial support. APMR acknowledges the support of CNPq by means of a research fellowship.

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Correspondence to
M.C. Dornelas
Universidade de São Paulo, Centro de Energia Nuclear na Agricultura
Laboratório de Biotecnologia Vegetal
Av. Centenário 303
13400-970 Piracicaba, SP, Brazil
E-mail: mcdornel@cena.usp.br

Received: May 31, 2004; Accepted: February 15, 2005.

Associate Editor: Everaldo Gonçalves de Barros