Geraldo Magela de Almeida Cançado

Novel Properties of the Wheat Aluminum Tolerance Organic Acid Transporter (TaALMT1) Revealed by Electrophysiological Characterization in Xenopus Oocytes: Functional and Structural Implications

Many plant species avoid the phytotoxic effects of aluminum (Al) by exuding dicarboxylic and tricarboxylic acids that chelate and immobilize Al3+ at the root surface, thus preventing it from entering root cells. Several novel genes that encode membrane transporters from the ALMT and MATE families recently were cloned and implicated in mediating the organic acid transport underlying this Al tolerance response. Given our limited understanding of the functional properties of ALMTs, in this study a detailed characterization of the transport properties of TaALMT1 (formerly named ALMT1) from wheat (Triticum aestivum) expressed in Xenopus laevis oocytes was conducted. The electrophysiological findings are as follows. Although the activity of TaALMT1 is highly dependent on the presence of extracellular Al3+ (Km1/2 of approximately 5 μm Al3+ activity), TaALMT1 is functionally active and can mediate ion transport in the absence of extracellular Al3+. The lack of change in the reversal potential (Erev) upon exposure to Al3+ suggests that the “enhancement” of TaALMT1 malate transport by Al is not due to alteration in the transporters selectivity properties but is solely due to increases in its anion permeability. The consistent shift in the direction of the Erev as the intracellular malate activity increases indicates that TaALMT1 is selective for the transport of malate over other anions. The estimated permeability ratio between malate and chloride varied between 1 and 30. However, the complex behavior of the Erev as the extracellular Cl− activity was varied indicates that this estimate can only be used as a general guide to understanding the relative affinity of TaALMT1 for malate, representing only an approximation of those expected under physiologically relevant ionic conditions. TaALMT1 can also mediate a large anion influx (i.e. outward currents). TaALMT1 is permeable not only to malate but also to other physiologically relevant anions such as Cl−, NO3−, and SO42− (to a lesser degree).

Glutathione S-transferase and aluminum toxicity in maize

Aluminum (Al) toxicity induces changes in the expression of several genes, some of which are involved in plant responses to oxidative stress. Using mRNA differential display, we identified a maize Al-inducible cDNA encoding a glutathione S-transferase (GST). The gene was named GST27.2 owing to its homology to the maize gene GST27, which is known to be induced by xenobiotics. GST27.2 is present in the maize genome as a single copy and analysis of its expression pattern revealed that the gene is expressed mainly in the root tip. Expression was up-regulated in response to various Al and Cd concentrations in both Al-tolerant and Al-sensitive maize lines. Consistent with its role in plants, phylogenetic analysis of theta-type GSTs revealed that GST27.2 belongs to a group of proteins that respond to different stresses. Finally, structural analysis of the polypeptide chain indicates that the two amino acids that differ between GST27.2 and GST27 (E102K and P123L) could be responsible for alterations in activity and / or specificity. Together, these results suggest that GST27.2 may play an important part in plant defenses against Al toxicity.

Identification of genes responsive to the application of ethanol on sugarcane leaves

The control of gene expression in precise time and space is a desirable attribute of chemically inducible systems. Ethanol is a chemical inducer with favourable features, such as being inexpensive and easy to apply. The aim of this study was to identify ethanol-responsive genes in sugarcane. The cDNA macroarray technique was adopted to identify transcript changes in sugarcane leaves (Saccharum spp. cv SP80-3280) exposed to ethanol. The expression profiles of sugarcane genes were analysed using nylon filters containing 3,575 cDNA clones from the leaf roll library of the SUCEST project. Seventy expressed sequence tags (ESTs) presented altered expression patterns, including ESTs corresponding to genes related to transcriptional and translational processes, abiotic stress and others. Several genes of unknown function were also identified. Among the 48 ESTs up-regulated by ethanol, an abiotic stress-responsive protein and an unknown function gene presented rapid induction by ethanol. The macroarray data of selected ethanol-responsive EST were confirmed by RNA-blot hybridisation. The expression profile of the 48 up-regulated genes was compared in two other cultivars: SP89-1115 and SP90-3414. Surprisingly, no gene showed a similar expression profile in the three cultivars. This result suggests that sugarcane plants have a high diversity in their responses to ethanol.

Gene expression profiling in maize roots under aluminum stress

To investigate the molecular mechanisms of Al toxicity, cross-species cDNA array approach was employed to identify expressed sequence tags (ESTs) regulated by Al stress in root tips of Al-tolerant maize (Zea mays) genotype Cat100-6 and Al-sensitive genotype S1587-17. Due to the high degree of conservation observed between sugarcane and maize, we have analyzed the expression profiling of maize genes using 2 304 sugarcane (ESTs) obtained from different libraries. We have identified 85 ESTs in Al stressed maize root tips with significantly altered expression. Among the up-regulated ESTs, we have found genes encoding previously identified proteins induced by Al stress, such as phenyl ammonia-lyase, chitinase, Bowman-Birk proteinase inhibitor, and wali7. In addition, several novel genes up-and downregulated by Al stress were identified in both genotypes.

Overexpression of BdMATE Gene Improves Aluminum Tolerance in Setaria viridis

Acidic soils are distributed worldwide, predominantly in tropical and subtropical areas, reaching around 50% of the arable soil. This type of soil strongly reduces crop production, mainly because of the presence of aluminum, which has its solubility increased at low pH levels. A well-known physiological mechanism used by plants to cope with Al stress involves activation of membrane transporters responsible for organic acid anions secretion from the root apex to the rhizosphere, which chelate Al, preventing its absorption by roots. In sorghum, a membrane transporter gene belonging to multidrug and toxic compound extrusion (MATE) family was identified and characterized as an aluminum-activated citrate transporter gene responsible for Al tolerance in this crop. Setaria viridis is an emerging model for C4 species and it is an important model to validate some genes for further C4 crops transformation, such as sugarcane, maize, and wheat. In the present work, Setaria viridis was used as a model plant to overexpress a newly identified MATE gene from Brachypodium distachyon (BdMATE), closely related to SbMATE, for aluminum tolerance assays. Transgenic S. viridis plants overexpressing a BdMATE presented an improved Al tolerance phenotype, characterized by sustained root growth and exclusion of aluminum from the root apex in transgenic plants, as confirmed by hematoxylin assay. In addition, transgenic plants showed higher root citrate exudation into the rhizosphere, suggesting that Al tolerance improvement in these plants could be related to the chelation of the metal by the organic acid anion. These results suggest that BdMATE gene can be used to transform C4 crops of economic importance with improved aluminum tolerance.

Population structure of jatropha and its implication for the breeding program.

Jatropha (Jatropha curcas L.) has potential as an oilseed crop that requires the development of technology for its exploitation. The objective of this study was to assess the population structure and the genetic diversity in jatropha accessions at a global level using simple sequence repeat (SSR) molecular markers. Jatropha accessions (N = 109) from 10 countries were genotyped using 10 SSR markers. The results showed a low level of genetic diversity among 92 accessions originating from India, Mozambique, Ethiopia, Tanzania, Brazil, Honduras, and Indonesia, which were grouped in one cluster. In contrast, accessions from Mexico and Costa Rica showed high level of genetic variability. These accessions may be used to increase the genetic diversity of jatropha in the breeding populations. The study also showed the need of collecting activity from the center of diversity (Mexico and Costa Rica) to aggregate the genetic diversity in the international collections of jatropha.

Outcrossing rate in olive assessed by microsatellite and inter simple sequence repeat (ISSR) markers

Olive is known to be an allogamous species. The aim of this study was to estimate the magnitude of cross-pollination rate using microsatellite simple sequence repeats (SSR) and inter simple sequence repeats (ISSR) molecular markers in olive genotypes. The DNA from maternal plants and 23 progenies of two different accessions, Ascolano USA and MGS GRAP541 were extracted and screened with two microsatellite and ten ISSR markers. The outcrossing rate and other related parameters were analyzed using the MLTR application. The set of estimates, individually and collectively, support the hypothesis of frequent allogamy in both olive genotypes evaluated, with high rates of outcrossing in both markers. Keywords: Olea europaea L., outcrossing rate, zygotic embryos, mating system, molecular markers

Myrciaria dubia, an Amazonian fruit: population structure and its implications for germplasm conservation and genetic improvement

Myrciaria dubia (camu-camu) is an Amazon tree that produces a tart fruit with high vitamin C content. It is probably the fruit with the highest vitamin C content among all Brazilian fruit crops and it can be used to supplement daily vitamin C dose. This property has attracted the attention of consumers and, consequently, encouraged fruit farmers to produce it. In order to identify and select potential accessions for commercial exploitation and breeding programs, M. dubia has received considerable research attention. The identification and characterization of genetic diversity, as well as identification of the population structure of accessions preserved in germplasm banks are fundamental for the success of any breeding program. The objective of this study was to evaluate the genetic variability of 10 M. dubia populations obtained from the shores of Reis Lake, located in the municipality of Caracaraí, Roraima, Brazil. Fourteen polymorphic inter simple sequence repeat (ISSR) markers were used to study the population genetic diversity, which resulted in 108 identified alleles. Among the 14 primers, GCV, UBC810, and UBC827 produced the highest number of alleles. The study illustrated the suitability and efficiency of ISSR markers to study the genetic diversity of M. dubia accessions. We also revealed the existence of high genetic variability among both accessions and populations that can be exploited in future breeding programs and conservation activities of this species.

Molecular characterization and population structure study of cambuci: strategy for conservation and genetic improvement.

Cambuci (Campomanesia phaea) belongs to the Myrtaceae family and is native to the Atlantic Forest of Brazil. It has ecological and social appeal but is exposed to problems associated with environmental degradation and expansion of agricultural activities in the region. Comprehensive studies on this species are rare, making its conservation and genetic improvement difficult. Thus, it is important to develop research activities to understand the current situation of the species as well as to make recommendations for its conservation and use. This study was performed to characterize the cambuci accessions found in the germplasm bank of Coordenadoria de Assistência Técnica Integral using inter-simple sequence repeat markers, with the goal of understanding the plants population structure. The results showed the existence of some level of genetic diversity among the cambuci accessions that could be exploited for the genetic improvement of the species. Principal coordinate analysis and discriminant analysis clustered the 80 accessions into three groups, whereas Bayesian model-based clustering analysis clustered them into two groups. The formation of two cluster groups and the high membership coefficients within the groups pointed out the importance of further collection to cover more areas and more genetic variability within the species. The study also showed the lack of conservation activities; therefore, more attention from the appropriate organizations is needed to plan and implement natural and ex situ conservation activities.

Effect of coconut water on growth of olive embryos cultured in vitro

The experiment was carried out to determine the appropriate dose of coconut water as supplement for in vitro cultivation of zygotic embryos from 19 olive genotypes. The isolated embryos of the olive seeds were immersed on culture medium containing 0 (control), 25, 50, and 100mL L-1 of fresh and sterile coconut water and kept for 45 days under controlled environment. The percentage of germination, shoot length, number of roots, number of leaves and number of internodes were measured for all 19 olive genotypes. The ANOVA of the parameters evaluated showed significant genotypes x doses of coconut water interaction for shoot length, number of leaves and number of internodes and the dose of 100mL L-1 produced the best results overall as indicated by the means of measured parameters. However, the study showed the importance of determining the appropriate dose of coconut water for each genotype under consideration as shown by significant genotype x dose of coconut water interaction effect.