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Flooding is a frequent environmental stress that reduces soybean ( Glycine max ) growth and grain yield in many producing areas in the world, such as, e.g., in the United States, Southeast Asia and Southern Brazil. In these regions, soybean is frequently cultivated in lowland areas by rotating with rice ( Oryza sativa ), which provides numerous technical, economic and environmental benefits. Given these realities, this work aimed to characterize physiological responses, identify genes differentially expressed under flooding stress in Brazilian soybean genotypes with contrasting flooding tolerance, and select SNPs with potential use for marker-assisted selection. Soybean cultivars TECIRGA 6070 (flooding tolerant) and FUNDACEP 62 (flooding sensitive) were grown up to the V6 growth stage and then flooding stress was imposed. Total RNA was extracted from leaves 24 h after the stress was imposed and sequenced. In total, 421 induced and 291 repressed genes were identified in both genotypes. TECIRGA 6070 presented 284 and 460 genes up- and down-regulated, respectively, under flooding conditions. Of those, 100 and 148 genes were exclusively up- and down-regulated, respectively, in the tolerant genotype. Based on the RNA sequencing data, SNPs in differentially expressed genes in response to flooding stress were identified. Finally, 38 SNPs, located in genes with functional annotation for response to abiotic stresses, were found in TECIRGA 6070 and absent in FUNDACEP 62. To validate them, 22 SNPs were selected for designing KASP assays that were used to genotype a panel of 11 contrasting genotypes with known phenotypes. In addition, the phenotypic and grain yield impacts were analyzed in four field experiments using a panel of 166 Brazilian soybean genotypes. Five SNPs possibly related to flooding tolerance in Brazilian soybean genotypes were identified. The information generated from this research will be useful to develop soybean genotypes adapted to poorly drained soils or areas subject to flooding.

Osmotins are multifunctional proteins belonging to the thaumatin-like family related to plant stress responses. To better understand the functions of soybean osmotins in drought stress response, the current study presents the characterisation of four previously described proteins and a novel putative soybean osmotin (GmOLPa-like). Gene and protein structure as well as gene expression analyses were conducted on different tissues and developmental stages of two soybean cultivars with varying dehydration sensitivities (BR16 and EMB48 are highly and slightly sensitive, respectively). The analysed osmotin sequences share the conserved amino acid signature and 3D structure of the thaumatin-like family. Some differences were observed in the conserved regions of protein sequences and in the electrostatic surface potential. P21-like present the most similar electrostatic potential to osmotins previously characterised as promoters of drought tolerance in Nicotiana tabacum and Solanum nigrum . Gene expression analysis indicated that soybean osmotins were differentially expressed in different organs (leaves and roots), developmental stages (R1 and V3), and cultivars in response to dehydration. In addition, under dehydration conditions, the highest level of gene expression was detected for GmOLPa-like and P21-like osmotins in the leaves and roots, respectively, of the less drought sensitive cultivar. Altogether, the results suggest an involvement of these genes in drought stress tolerance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Faillace, Caruso, Timmers, Favero, Guzman, Rechenmacher, de Oliveira-Busatto, de Souza, Bredemeier and Bodanese-Zanettini.)

Osmotin is an important multifunctional protein related to plant stress responses and is classified into the thaumatin-like protein (TLP) family. Using genome-wide and phylogenetic approaches, we investigated osmotin origin and diversification across plant TLP evolution. Genomic and protein in silico analysis tools were also accessed and considered for the study conclusions. Phylogenetic analysis including a total of 722 sequences from 32 Viridiplantae species allowed the identification of an osmotin group that includes all previously characterized osmotins. Based on the phylogenetic tree results, it is evident that the osmotin group emerged from spermatophytes. Phylogenetic separation and gene expansion could be accounted for by an exclusive motif composition and organization that emerged and was maintained following tandem and block duplications as well as natural selection. The TLP family conserved residues and structures that were also identified in the sequences of the osmotin group, thus suggesting their maintenance for defense responses. The gene expression of Arabidopsis and rice putative osmotins reinforces its roles during stress response.

PLAC8 is a cysteine-rich protein described as a central mediator of tumor evolution in mammals; as such, it represents a promising candidate for diagnostic and therapeutic targeting. The human PLAC8 gene is also involved in contact hypersensitivity response and presents a role in psoriatic skin. In plants, PLAC8 motif-containing proteins are involved in the determination of organ size and growth, response to infection, Ca 2+ influx, Cd resistance, and zinc detoxification. In general, PLAC8 motif-containing proteins present the conserved CCXXXXCPC or CLXXXXCPC region. However, there is no devised nomenclature for the PLAC8 motif-containing proteins. Here, through the analysis of 445 sequences, we show that PLAC8 motif-containing proteins constitute a unique gene family, and we propose a unified nomenclature. This is the first report indicating the existence of different groups of PLAC8 proteins, which we have called types I, II, and III. Type I genes are found in mammals, fungi, plants, and algae, and types II and III are exclusive to plants. Our study describes for the first time PLAC8 type III proteins. Whether these sequences maintain their known functional role or possess distinct functions of types I and II genes remains unclear.

Lipopolysaccharide-induced tumor necrosis factor-α (LITAF) is a membrane protein that is highly dependent on correct location to exert transcription factor activity and protein quality control. In humans, LITAF, PIG7 (p53-inducible gene 7), and SIMPLE (small integral membrane protein of the lysosome/late endosome) refer to the same gene, which acts as a tumor suppressor. Several studies have shown that the transcription factor activity and nuclear translocation of LITAF protein are critical for the induction of several immune cells via classical pathways. In plants, LITAF protein corresponds to the plasma membrane protein AtGILP (Arabidopsis thaliana GSH-induced LITAF domain protein). The conservation of LITAF proteins across species and their putative role is still unclear. In this study, we investigate the LITAF-containing proteins, which we call GILP proteins, in Viridiplantae. We identified a total of 59 genes in 46 species, whose gene copies range from one to three. Phylogenetic analysis showed that multiple copies were originated via block duplication posteriorly to monocot and eudicot separation. Analysis of the LITAF domain of GILP proteins allowed the identification of a putative domain signature in Viridiplantae, containing a CXXCX41HXCPXC motif. The subcellular location for the majority of GILP proteins was predicted to be in the plasma membrane, based on a transmembrane domain positioned within the LITAF domain. In silico analysis showed that the GILP genes are neither tissue-specific nor ubiquitously expressed, being responsive to stress conditions. Finally, investigation of the GILP protein network resulted in the identification of genes whose families are known to be involved with biotic and/or abiotic stress responses. Together, the expression modulation of GILP genes associated with their plasma membrane location suggests that they could act in the signaling of biotic/abiotic stress response in plants.

Urease catalyzes the hydrolysis of urea to ammonia and carbon dioxide. The ammonia (nitrogen (N) product of urease activity) is incorporated into organic compounds. Thus, urease is involved in N remobilization, as well as in primary N assimilation. Two urease isoforms have been described for soybean: the embryo-specific, encoded by the Eu1 gene, and the ubiquitous urease, encoded by Eu4. A third urease-encoding gene was recently identified, designated Eu5, which encodes the putative protein product SBU-III. The present study aimed to evaluate the contribution of soybean ureases to seed germination and plant development. Analyses were performed using Eu1/Eu4/Eu5-co-suppressed transgenic plants and mutants of the Eu1 and Eu4 urease structural genes, as well as a urease-null mutant (eu3-a) that activates neither the ubiquitous nor embryo-specific ureases. The co-suppressed plants presented a developmental delay during the first month after germination; shoots and roots were significantly smaller and lighter. Slower development was observed for the double eu1-a/eu4-a mutant and the eu3-a single mutant. The N content in transgenic plants was significantly lower than in non-transgenic plants. Among the mutants, eu3-a presented the lowest and eu1-a the highest N content. Altogether, these results indicate that increased ureolytic activity plays an important role in plant development.

Drought is today, and perhaps even more in the future, the main challenge for grain crops, resulting in a drastic yield reduction. Thus, it is of great interest to obtain soybean genotypes tolerant to water deficit. The drought tolerance trait is difficult to obtain through classical breeding due to its polygenic basis. In this context, genetic engineering is presented as a way to achieve this attribute. The ability to modulate the expression of many genes placed the transcription factors as promising biotechnological targets to develop stress tolerant cultivars. The WRKY proteins form a large family of transcription factors that are involved in important physiological and biochemical processes in plants, including the response to water deficit. In this study, the expression pattern determined by qPCR showed that, GmWRKY6, GmWRKY46, GmWRKY56, GmWRKY106 and GmWRKY149 genes are differentially expressed between a drought tolerant and a susceptible soybean genotype in water stress conditions. The in silico promoter and coexpression analysis indicate that these genes act in a stress physiological background., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Drought limits wheat production in the Brazilian Cerrado biome. In order to search for candidate genes associated to the response to water deficit, we analyzed the gene expression profiles, under severe drought stress, in roots and leaves of the cultivar MGS1 Aliança, a well-adapted cultivar to the Cerrado. A set of 4,422 candidate genes was found in roots and leaves. The number of down-regulated transcripts in roots was higher than the up-regulated transcripts, while the opposite occurred in leaves. The number of common transcripts between the two tissues was 1,249, while 2,124 were specific to roots and 1,049 specific to leaves. Quantitative RT-PCR analysis revealed a 0.78 correlation with the expression data. The candidate genes were distributed across all chromosomes and component genomes, but a greater number was mapped on the B genome, particularly on chromosomes 3B, 5B and 2B. When considering both tissues, 116 different pathways were induced. One common pathway, among the top three activated pathways in both tissues, was starch and sucrose metabolism. These results pave the way for future marker development and selection of important genes and are useful for understanding the metabolic pathways involved in wheat drought response.

In plants, ureases have been related to urea degradation, to defense against pathogenic fungi and phytophagous insects, and to the soybean-Bradyrhizobium japonicum symbiosis. Two urease isoforms have been described for soybean: the embryo-specific, encoded by Eu1 gene, and the ubiquitous urease, encoded by Eu4. A third urease-encoding locus exists in the completed soybean genome. The gene was designated Eu5 and the putative product of its ORF as SBU-III. Phylogenetic analysis shows that 41 plant, moss and algal ureases have diverged from a common ancestor protein, but ureases from monocots, eudicots and ancient species have evolved independently. Genomes of ancient organisms present a single urease-encoding gene and urease-encoding gene duplication has occurred independently along the evolution of some eudicot species. SBU-III has a shorter amino acid sequence, since many gaps are found when compared to other sequences. A mutation in a highly conserved amino acid residue suggests absence of ureolytic activity, but the overall protein architecture remains very similar to the other ureases. The expression profile of urease-encoding genes in different organs and developmental stages was determined by RT-qPCR. Eu5 transcripts were detected in seeds one day after dormancy break, roots of young plants and embryos of developing seeds. Eu1 and Eu4 transcripts were found in all analyzed organs, but Eu4 expression was more prominent in seeds one day after dormancy break whereas Eu1 predominated in developing seeds. The evidence suggests that SBU-III may not be involved in nitrogen availability to plants, but it could be involved in other biological role(s)., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

The Lesion Simulating Disease (LSD) genes encode a family of zinc finger proteins that play a role in programmed cell death (PCD) and other biological processes, such as plant growth and photosynthesis. In the present study, we report the reconstruction of the evolutionary history of the LSD gene family in Viridiplantae. Phylogenetic analysis revealed that the monocot and eudicot genes were distributed along the phylogeny, indicating that the expansion of the family occurred prior to the diversification between these clades. Sequences encoding proteins that present one, two, or three LSD domains formed separate groups. The secondary structure of these different LSD proteins presented a similar composition, with the β-sheets being their main component. The evolution by gene duplication was identified only to the genes that contain three LSD domains, which generated proteins with equal structure. Moreover, genes encoding proteins with one or two LSD domains evolved as single-copy genes and did not result from loss or gain in LSD domains. These results were corroborated by synteny analysis among regions containing paralogous/orthologous genes in Glycine max and Populus trichocarpa. The Ka/Ks ratio between paralogous/orthologous genes revealed that a subfunctionalization process possibly could be occurring with the LSD genes, explaining the involvement of LSD members in different biological processes, in addition to the negative regulation of PCD. This study presents important novelty in the evolutionary history of the LSD family and provides a basis for future research on individual LSD genes and their involvement in important pathway networks in plants.

Programmed cell death (PCD) is an ordered cell suicide that removes unwanted or damaged cells, playing a role in defense to environmental stresses and pathogen invasion. PCD is component of the life cycle of plants, occurring throughout development from embryogenesis to the death. Metacaspases are cysteine proteases present in plants, fungi, and protists. In certain plant-pathogen interactions, the PCD seems to be mediated by metacaspases. We adopted a comparative genomic approach to identify genes coding for the metacaspases in Viridiplantae. We observed that the metacaspase was divided into types I and II, based on their protein structure. The type I has a metacaspase domain at the C-terminus region, presenting or not a zinc finger motif in the N-terminus region and a prodomain rich in proline. Metacaspase type II does not feature the prodomain and the zinc finger, but has a linker between caspase-like catalytic domains of 20 kDa (p20) and 10 kDa (p10). A high conservation was observed in the zinc finger domain (type I proteins) and in p20 and p10 subunits (types I and II proteins). The phylogeny showed that the metacaspases are divided into three principal groups: type I with and without zinc finger domain and type II metacaspases. The algae and moss are presented as outgroup, suggesting that these three classes of metacaspases originated in the early stages of Viridiplantae, being the absence of the zinc finger domain the ancient condition. The study of metacaspase can clarify their assignment and involvement in plant PCD mechanisms.

Key Message: The work describes an ASR knockdown transcriptomic analysis by deep sequencing of rice root seedlings and the transactivation of ASR cis-acting elements in the upstream region of a MIR gene. MicroRNAs are key regulators of gene expression that guide post-transcriptional control of plant development and responses to environmental stresses. ASR (ABA, Stress and Ripening) proteins are plant-specific transcription factors with key roles in different biological processes. In rice, ASR proteins have been suggested to participate in the regulation of stress response genes. This work describes the transcriptomic analysis by deep sequencing two libraries, comparing miRNA abundance from the roots of transgenic ASR5 knockdown rice seedlings with that of the roots of wild-type non-transformed rice seedlings. Members of 59 miRNA families were detected, and 276 mature miRNAs were identified. Our analysis detected 112 miRNAs that were differentially expressed between the two libraries. A predicted inverse correlation between miR167abc and its target gene (LOC_Os07g29820) was confirmed using RT-qPCR. Protoplast transactivation assays showed that ASR5 is able to recognize binding sites upstream of the MIR167a gene and drive its expression in vivo. Together, our data establish a comparative study of miRNAome profiles and is the first study to suggest the involvement of ASR proteins in miRNA gene regulation.

Background: Drought is by far the most important environmental factor contributing to yield losses in crops, including soybeans [Glycine max (L.) Merr.]. To address this problem, a gene that encodes an osmotin-like protein isolated from Solanum nigrum var. americanum (SnOLP) driven by the UBQ3 promoter from Arabidopsis thaliana was transferred into the soybean genome by particle bombardment., Results: Two independently transformed soybean lines expressing SnOLP were produced. Segregation analyses indicated single-locus insertions for both lines. qPCR analysis suggested a single insertion of SnOLP in the genomes of both transgenic lines, but one copy of the hpt gene was inserted in the first line and two in the second line. Transgenic plants exhibited no remarkable phenotypic alterations in the seven analyzed generations. When subjected to water deficit, transgenic plants performed better than the control ones. Leaf physiological measurements revealed that transgenic soybean plants maintained higher leaf water potential at predawn, higher net CO2 assimilation rate, higher stomatal conductance and higher transpiration rate than non-transgenic plants. Grain production and 100-grain weight were affected by water supply. Decrease in grain productivity and 100-grain weight were observed for both transgenic and non-transgenic plants under water deficit; however, it was more pronounced for non-transgenic plants. Moreover, transgenic lines showed significantly higher 100-grain weight than non-transgenic plants under water shortage., Conclusions: This is the first report showing that expression of SnOLP in transgenic soybeans improved physiological responses and yield components of plants when subjected to water deficit, highlighting the potential of this gene for biotechnological applications.

Background: Many previous studies have shown that soybean WRKY transcription factors are involved in the plant response to biotic and abiotic stresses. Phakopsora pachyrhizi is the causal agent of Asian Soybean Rust, one of the most important soybean diseases. There are evidences that WRKYs are involved in the resistance of some soybean genotypes against that fungus. The number of WRKY genes already annotated in soybean genome was underrepresented. In the present study, a genome-wide annotation of the soybean WRKY family was carried out and members involved in the response to P. pachyrhizi were identified., Results: As a result of a soybean genomic databases search, 182 WRKY-encoding genes were annotated and 33 putative pseudogenes identified. Genes involved in the response to P. pachyrhizi infection were identified using superSAGE, RNA-Seq of microdissected lesions and microarray experiments. Seventy-five genes were differentially expressed during fungal infection. The expression of eight WRKY genes was validated by RT-qPCR. The expression of these genes in a resistant genotype was earlier and/or stronger compared with a susceptible genotype in response to P. pachyrhizi infection. Soybean somatic embryos were transformed in order to overexpress or silence WRKY genes. Embryos overexpressing a WRKY gene were obtained, but they were unable to convert into plants. When infected with P. pachyrhizi, the leaves of the silenced transgenic line showed a higher number of lesions than the wild-type plants., Conclusions: The present study reports a genome-wide annotation of soybean WRKY family. The participation of some members in response to P. pachyrhizi infection was demonstrated. The results contribute to the elucidation of gene function and suggest the manipulation of WRKYs as a strategy to increase fungal resistance in soybean plants.

Water deficiency decreases plant growth and productivity. Several mechanisms are activated in response to dehydration that allows plants to cope with stress, including factors controlling stomatal aperture and ramified root system development. In addition, ABA metabolism is also implicated in the regulation of drought responses. The basic helix-loop-helix (bHLH) proteins, a large family of conserved transcription factors that regulates many cellular processes in eukaryotic organisms, are also involved in several responses that are important for plants to cope with drought stress. This review discusses distinct mechanisms related to drought-adaptive responses, especially the possible involvement of the bHLH transcription factors such as MUTE, implicated in stomatal development; RD22, [corrected] an ABA-responsive gene; EGL3 and GL3, involved in thichome and root hair development; and SPT, which play roles in repressing leaf expansion. Transcription factors are potential targets for new strategies to increase the tolerance of cultivars to drought stress. Recognition of gene regulatory networks in crops is challenging, and the manipulation of bHLH genes as well as components that mediate bHLH transcription factor responses in different pathways could be essential to achieve abiotic stress tolerance in plants through genetic manipulation., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The Lesion Simulating Disease (LSD) genes encode a family of zinc finger proteins that are reported to play an important role in the hypersensitive response and programmed cell death (PCD) that are caused by biotic and abiotic stresses. In the present study, 117 putative LSD family members were identified in Viridiplantae. Genes with one, two, or three conserved LSD domains were identified. Proteins with three LSD domains were highly represented in the species analyzed and were present in basal organisms. Proteins with two LSD domains were identified only in the Embryophyte clade, and proteins possessing one LSD domain were highly represented in grass species. Expression analyses of Glycine max LSD (GmLSD) genes were performed by real-time quantitative polymerase chain reaction. The results indicated that GmLSD genes are not ubiquitously expressed in soybean organs and that their expression patterns are instead organ-dependent. The expression of the majority of GmLSD genes is modulated in soybean during Phakopsora pachyrhizi infection. In addition, the expression of some GmLSD genes is modulated in plants under dehydration stress. These results suggest the involvement of GmLSD genes in the response of soybean to both biotic and abiotic stresses.

Transgenic plants represent an invaluable tool for molecular, genetic, biochemical and physiological studies by gene overexpression or silencing, transposon-based mutagenesis, protein sub-cellular localization and/or promoter characterization as well as a breakthrough for breeding programs, allowing the production of novel and genetically diverse genotypes. However, the stable transformation of soybean cannot yet be considered to be routine because it depends on the ability to combine efficient transformation and regeneration techniques. Two methods have been used with relative success to produce completely and stably transformed plants: particle bombardment and the Agrobacterium tumefaciens system. In addition, transformation by Agrobacterium rhizogenes has been used as a powerful tool for functional studies. Most available information on gene function is based on heterologous expression systems. However, as the activity of many promoters or proteins frequently depends on specific interactions that only occur in homologous backgrounds, a final confirmation based on a homologous expression system is desirable. With respect to soybean biotech improvement, transgenic lines with agronomical, nutritional and pharmaceutical traits have been obtained, including herbicide-tolerant soybeans, which represented the principal biotech crop in 2011, occupying 47% of the global biotech area.

Environmental stresses caused by either abiotic or biotic factors greatly affect agriculture. As for soybean [Glycine max (L.) Merril], one of the most important crop species in the world, the situation is not different. In order to deal with these stresses, plants have evolved a variety of sophisticated molecular mechanisms, to which the transcriptional regulation of target-genes by transcription factors is crucial. Even though the involvement of several transcription factor families has been widely reported in stress response, there still is a lot to be uncovered, especially in soybean. Therefore, the objective of this study was to investigate the role of bHLH and trihelix-GT transcription factors in soybean responses to environmental stresses. Gene annotation, data mining for stress response, and phylogenetic analysis of members from both families are presented herein. At least 45 bHLH (from subgroup 25) and 63 trihelix-GT putative genes reside in the soybean genome. Among them, at least 14 bHLH and 11 trihelix-GT seem to be involved in responses to abiotic/biotic stresses. Phylogenetic analysis successfully clustered these with members from other plant species. Nevertheless, bHLH and trihelix-GT genes encompass almost three times more members in soybean than in Arabidopsis or rice, with many of these grouping into new clades with no apparent near orthologs in the other analyzed species. Our results represent an important step towards unraveling the functional roles of plant bHLH and trihelix-GT transcription factors in response to environmental cues.

The soybean ubiquitous urease (encoded by GmEu4) is responsible for recycling metabolically derived urea. Additional biological roles have been demonstrated for plant ureases, notably in toxicity to other organisms. However, urease enzymatic activity is not related to its toxicity. The role of GmEu4 in soybean susceptibility to fungi was investigated in this study. A differential expression pattern of GmEu4 was observed in susceptible and resistant genotypes of soybeans over the course of a Phakopsora pachyrhizi infection, especially 24 h after infection. Twenty-nine adult, transgenic soybean plants, representing six independently transformed lines, were obtained. Although the initial aim of this study was to overexpress GmEu4, the transgenic plants exhibited GmEu4 co-suppression and decreased ureolytic activity. The growth of Rhizoctonia solani, Phomopsis sp., and Penicillium herguei in media containing a crude protein extract from either transgenic or non-transgenic leaves was evaluated. The fungal growth was higher in the protein extracts from transgenic urease-deprived plants than in extracts from non-transgenic controls. When infected by P. pachyrhizi uredospores, detached leaves of urease-deprived plants developed a significantly higher number of lesions, pustules and erupted pustules than leaves of non-transgenic plants containing normal levels of the enzyme. The results of the present work show that the soybean plants were more susceptible to fungi in the absence of urease. It was not possible to overexpress active GmEu4. For future work, overexpression of urease fungitoxic peptides could be attempted as an alternative approach.

A bacterial strain designated SBR5(T) was isolated from the rhizosphere of Triticum aestivum. A phylogenetic analysis based on the 16S rRNA gene sequence placed the isolate within the genus Paenibacillus, being most closely related to Paenibacillus graminis RSA19(T) (98.1 % similarity). The isolate was a Gram-reaction-variable, motile, facultatively anaerobic bacterium, with spores in a terminal position in cells. Starch was utilized and dihydroxyacetone and catalase were produced. Strain SBR5(T) displayed plant-growth-promoting rhizobacteria characteristics: the ability to fix nitrogen and to produce siderophores and indole-3-acetic acid. The DNA G+C content was 55.1 mol%. Chemotaxonomic analysis of the isolated strain revealed that MK-7 was the predominant menaquinone, while the major fatty acid was anteiso-C(15 : 0). DNA-DNA hybridization values between strain SBR5(T) and P. graminis RSA19(T), Paenibacillus odorifer TOD45(T) and Paenibacillus borealis KK19(T) were 43, 35 and 28 %, respectively. These DNA relatedness data and the results of phylogenetic and phenotypic analyses showed that strain SBR5(T) should be considered as the nitrogen-fixing type strain of a novel species of the genus Paenibacillus, for which the name Paenibacillus riograndensis sp. nov. is proposed. The type strain is SBR5(T) (=CCGB 1313(T) =CECT 7330(T)).

The processes that have shaped the extraordinary species diversity in neotropical rainforests are poorly understood, and knowledge about the patterns of genetic diversity across species' ranges is scarce, in contrast to other regions of the globe. We have conducted a range-wide study of genetic diversity in a plant endemic to the Brazilian Atlantic Rainforest, Vriesea gigantea (Bromeliaceae), based on a combined data set of nuclear microsatellites and chloroplast (cp) DNA markers typed in 429 plants from 13 populations. The results indicate a strong negative correlation between genetic diversity and population latitude, consistent with historical forest expansion from the northern half of the present distribution range. A deep phylogeographic split exists between the Brazilian states of São Paulo and Rio de Janeiro at ca. 23 degrees S latitude, probably reflecting past population isolation within more than one glacial refuge during the climatic changes of the Pleistocene. A comparison of genetic structures at cpDNA and nuclear markers revealed a pollen/seed flow ratio of more than 3:1, thus indicating an important role of the pollinating animals (that is, bats) in shaping the population genetic structure of this species. Diversity was reduced for cpDNA markers in the island populations off the coast, and reduced diversity and increased differentiation were observed for both nuclear and cpDNA at the edges of the species' range. The link between patterns of genetic and species diversity supports the hypothesis that both were shaped by the same biogeographic processes, triggered by the climatic oscillations of the Pleistocene.

In this work, a total of 311 putative nitrogen-fixing bacilli were isolated from seven distinct wheat production zones of the Rio Grande do Sul State, Brazil. Strains belonging to several species were grouped into 40 different nifH-RFLP-PCR profiles. The genus Paenibacillus was the most prominent group in both the rhizosphere (77.8%) and soil (79%). Paenibacillus borealis was the most frequently identified species, followed by Paenibacillus graminis. The remainder of the isolated bacteria belonged to the genus Bacillus sp. Indolic compound production (indole 3-acetic acid (IAA), indolepyruvic acid (IPyA) and indoleacetamide (IAM)) was detected in 33.6% and 26% of the isolates from the rhizosphere and soil, respectively. Among the 311 isolates, nine were able to solubilize phosphate and 48 were able to produce siderophores. The isolates SBR5, CSR16 and EsR7, identified by the 16S rRNA gene sequence as strains of Paenibacillus sp., were chosen for in vivo experiments in a greenhouse and proved to be very efficient in promoting a significant increase in the shoot and dry matter of wheat plants. Those strains could be useful in formulation of new inoculants, improving the cropping systems into which they can be most profitably applied.

Plant fertility is a central subject of many questions in plant evolutionary and conservation biology. Pollen availability, abiotic resources, and flowering pattern can limit fruit and seed production. Open pollination and pollen supplementation studies are used to estimate any pollen limitation in natural populations. To study the impact of these factors on the reproductive success of Vriesea gigantea, an epiphytic bromeliad in the Atlantic Rainforest in Brazil, its fertility in four natural populations in Itapuã State Park was assessed by considering plant and inflorescence size, flower production, fruit and seed set, flower and fruit set pattern, and seed viability and germination rate. Supplemental pollination in adult plants was used to determine whether fruit production in V. gigantea is limited by reception of pollen. The results showed that V. gigantea has a high production of flowers, fruits, and seeds. Seeds are highly viable in all populations, presenting an average germination rate of 94% (SE ± 3.5). Plants of V. gigantea from Itapuã State Park are highly fertile. The high proportion of fruit and seed set after manual hand pollination indicates that the species is self-compatible. Pollination treatments showed evidence of pollinator limitation in the Itapuã State Park population.

Chromosome number, meiotic behavior, and pollen viability were analyzed in 15 species of two genera, Vriesea and Aechmea, native to Rio Grande do Sul, Brazil. This study is the first cytogenetic analysis of these taxa. The chromosome numbers are all n = 25, consistent with the proposed base number of x = 25 for Bromeliaceae. All examined taxa displayed regular bivalent pairing and chromosome segregation at meiosis. Observed meiotic abnormalities include univalents in metaphase I; missing or extra chromosomes and precocious division of centromeres in metaphase II; laggards in telophase I and anaphase II/telophase II. The high pollen viability (>88%) reflects a regular meiosis.

The effect of ethylene on somatic embryogenesis from cotyledons of soybean (Glycine max) cultivars `Bragg', `IAS-5', and `RS-7' was studied through the application of silver nitrate or aminoethoxyvinylglycine. The addition of these chemicals to the induction medium had no effect on embryo induction, in spite of aminoethoxyvinylglycine having decreased ethylene production and silver nitrate enhancing it. However, subsequent histodif-ferentiation and conversion capacity of somatic embryos was affected by treatments applied to the induction medium. The effects of ethylene on embryo histodifferentiation and conversion were genotype-specific. Cultivars `IAS-5' and `RS-7' produced high frequencies of dicotyledonous embryos and had high conversion rates. These were also the least affected by alterations in ethylene production. For `Bragg', which has a low regeneration capacity, the use of aminoethoxyvinylglycine led to a significant improvement in the frequency of normal embryo formation as well as in the frequency of conversion into plants. The results suggest that the use of ethylene inhibitors during the induction process may facilitate plant recovery from soybean genotypes, such as `Bragg', which have a low regeneration capacity.

Employing a different culture strategy, we obtained a greatly improved frequency of embryo rescue in intersubgeneric soybean hybrids. Successful crosses were obtained in 31 different genotype combinations between nine Brazilian soybean lines as the female parents and 12 accessions from Glycine canescens, G. microphylla, G. tabacina and G. tomentella. The hybrid pod retention rate dropped to about 10% during the first 8 days after pollination and stayed largely unchanged up to the 20th day. Immature harvested seeds fell into three size groups: Group 1, smaller than 1.3 mm (mostly empty seed coats); Group 2, 1.9-5.0 mm; Group 3, larger than 5 mm (from selfing). A total of 90 putative hybrid embryos were rescued using a highly enriched B5 medium to nourish the newly dissected embryos. The growing embryos were then placed in a high osmotic, modified B5 medium to induce maturation and dormancy. Schenk and Hildebrandt medium was used to germinate the dormant, partially dehydrated, physiologically mature embryos. Approximately 37% of the rescued embryos developed into plantlets in vitro, and approximately 8% grew into mature plants in the greenhouse. Morphological, cytological and isoenzyme patterns confirmed the hybrid status of all seven mature plants, all of which were generated using G. tomentella G 9943 as the paternal parent. It was observed that all soybean lines crossed with G 9943 were capable of producing mature hybrid plants. There was no correlation between the initial size of Group 2 seeds and plant survival rate. The hybrids were cloned by grafting and treated with colchicine. One of the treated plants displayed chromosome doubling.