Jorge Ernesto de Araujo Mariath

New Insights into Aluminum Tolerance in Rice: The ASR5 Protein Binds the STAR1 Promoter and Other Aluminum-Responsive Genes

Aluminum (Al) toxicity in plants is one of the primary constraints in crop production. Al³⁺, the most toxic form of Al, is released into soil under acidic conditions and causes extensive damage to plants, especially in the roots. In rice, Al tolerance requires the ASR5 gene, but the molecular function of ASR5 has remained unknown. Here, we perform genome-wide analyses to identify ASR5-dependent Al-responsive genes in rice. Based on ASR5_RNAi silencing in plants, a global transcriptome analysis identified a total of 961 genes that were responsive to Al treatment in wild-type rice roots. Of these genes, 909 did not respond to Al in the ASR5_RNAi plants, indicating a central role for ASR5 in Al-responsive gene expression. Under normal conditions, without Al treatment, the ASR5_RNAi plants expressed 1.756 genes differentially compared to the wild-type plants, and 446 of these genes responded to Al treatment in the wild-type plants. Chromatin immunoprecipitation followed by deep sequencing identified 104 putative target genes that were directly regulated by ASR5 binding to their promoters, including the STAR1 gene, which encodes an ABC transporter required for Al tolerance. Motif analysis of the binding peak sequences revealed the binding motif for ASR5, which was confirmed via in vitro DNA-binding assays using the STAR1 promoter. These results demonstrate that ASR5 acts as a key transcription factor that is essential for Al-responsive gene expression and Al tolerance in rice.

The mitochondrial glutathione peroxidase GPX3 is essential for H2O2 homeostasis and root and shoot development in rice

Glutathione (GSH) peroxidases (GPXs: EC 1.11.1.9 and EC1.11.1.12) are non-heme thiol peroxidases that catalyze the reduction of H2O2 or organic hydroperoxides to water, and they have been identified in almost all kingdoms of life. The rice glutathione peroxidase (OsGPX) gene family is comprised of 5 members spread throughout a range of sub cellular compartments. The OsGPX gene family is induced in response to exogenous H2O2 and cold stress. In contrast, they are down regulated in response to drought and UV-B light treatments. Transgenic rice plants have been generated that lack mitochondrial OsGPX3. These GPX3s plants showed shorter roots and shoots compared to non-transformed (NT) plants, and higher amounts of H2O2 mitochondrial release were observed in the roots of these plants cultivated under normal conditions. This accumulation of H2O2 is positively associated with shorter root length in GPX3s plants compared to NT ones. Moreover, GPX3 promoter analysis indicated that it is mainly expressed in root tissue. These results suggest that silencing the mitochondrial OsGPX3 gene impairs normal plant development and leads to a stress-induced morphogenic response via H2O2 accumulation.

Identification and expression analysis of castor bean (Ricinus communis) genes encoding enzymes from the triacylglycerol biosynthesis pathway

Castor bean (Ricinus communis) oil contains ricinoleic acid-rich triacylglycerols (TAGs). As a result of its physical and chemical properties, castor oil and its derivatives are used for numerous bio-based products. In this study, we survey the Castor Bean Genome Database to report the identification of TAG biosynthesis genes. A set of 26 genes encoding six distinct classes of enzymes involved in TAGs biosynthesis were identified. In silico characterization and sequence analysis allowed the identification of plastidic isoforms of glycerol-3-phosphate acyltransferase and lysophosphatidate acyltransferase enzyme families, involved in the prokaryotic lipid biosynthesis pathway, that form a cluster apart from the cytoplasmic isoforms, involved in the eukaryotic pathway. In addition, two distinct membrane bound diacylglycerol acyltransferase enzymes were identified. Quantitative expression pattern analyses demonstrated variations in gene expressions during castor seed development. A tendency of maximum expression level at the middle of seed development was observed. Our results represent snapshots of global transcriptional activities of genes encompassing six enzyme families involved in castor bean TAG biosynthesis that are present during seed development. These genes represent potential targets for biotechnological approaches to produce nutritionally and industrially desirable oils.

Cytological analysis of early microspore divisions and embryo formation in cultured soybean anthers

Young inflorescences of two Brazilian soybean cultivars (IAS 5 and RS 7) were subjected to 4 °C pretreatment for 0, 5, and 10 days. Cytological examinations of the t in vitro anthers were done during the first four weeks of culture. The cold pretreatment had no clear effect on the frequencies of symmetrical-binucleate microspores or multinucleate grains. The multinucleate grains might originate either by symmetrical or assymmetrical division. The best medium for callus and embryo induction was B5 long containing 2.0 mg l−1 2,4-dichlorophenoxy acetic acid and 0.5 mg l−1 benzyladenine. The frequency of anthers/calli with embryos was about 2% in all cultivars. Histological analysis of such embryos showed that they were similar to zygotic embryos.

Populational Diversity on Leaf Morphology of Maté (Ilex paraguariensis A. St.-Hil., Aquifoliaceae)

Mate (Ilex paraguariensis A. St.-Hil.) is a native plant from southern Brazil and a raw material for beverages. To research on population variability of the species, we compared leaf morphology of three populations from Mato Grosso do Sul (MS), Parana (PR) and Santa Catarina (SC) States. Lamina measurements included area (A), perimeter (P), length (l), maximum width (w), distance between maximum width and lamina base (dw), base angle (BÂ), biomass (m), and the indexes: relative distance of maximum width (dw/l), ratio length/width (l/w), relative perimeter (RP= P / (l + w), rectangular area index (RAI = A / (l x w)) and leaf mass/area (m/A). We verified differences among populations on A (MS SC) and m/A (MS>PR, MS=SC, PR=SC). The lowest RAI of SC could be explained, at least partially, by a positive correlation between RAI and BÂ (r= 0.6043, p<0.01).

Somatic embryogenesis from immature cotyledons of soybean (Glycine max (L.) Merr.): ontogeny of somatic embryos

Soybean somatic embryos obtained from immature zygotic embryos were histologically analyzed in order to determine possible ontogenetic routes followed by these embryos. The proembryo stage and globular, heart-shaped, torpedo and cotyledonary embryo stages were found, closely resembling the ontogeny of zygotic embryos. However, the absence of a characteristic suspensor as well as the delay in the establishment of inner organization were the main differences between these two embryogenic processes.

Ovule ontogeny in Rubiaceae (Juss.): Chomelia obtusa (Cinchonoideae–Guettardeae) and Ixora coccinea (Ixoroideae–Ixoreae)

SummaryOvule development and gynosporogenesis (megasporogenesis) of two Rubiaceae, Chomelia obtusa (Cinchonoideae–Guettardeae) and Ixora coccinea (Ixoroideae–Ixoreae) were studied. Based on the new results it is proposed that the Ixora type’ of ovule development is established because of the convex nucellus and the presence of several archesporial cells. This result supports the close relationships of Cinchonoideae and Ixoroideae established by molecular systematic studies.

Histology of embryogenic responses in soybean anther culture

In order to clarify the embryogenic responses in soybean anther culture, anthers of four cultivars were cultured under known conditions to trigger androgenic response. A histological study was performed with anthers in vivo and with approximately 100 explants sampled after 9, 12, 15, 18, 21, 30 and 45 days of culture. In vitro culture triggered the frequent accumulation of phenolic compounds on the locular and anther surfaces, and also caused the destruction of cells and tissues in complex structure such as the tapetum, microspores and pollen grains. Somatic embryogenesis of unicellular origin was observed from the epidermis and the middle layer, and of multicellular origin from connective calluses. No androgenic response could be observed in the anthers of these four soybean genotypes, in the medium and conditions indicated. We point out to the need of changing the approach to the study of androgenesis in soybean, either by using culture conditions unfavourable to the proliferation of diploid tissues, or by culturing isolated microspores.

Cold tolerance in rice germinating seeds revealed by deep RNAseq analysis of contrasting indica genotypes

Rice productivity is largely affected by low temperature, which can be harmful throughout plant development, from germination to grain filling. Germination of indica rice cultivars under cold is slow and not uniform, resulting in irregular emergence and small plant population. To identify and characterize novel genes involved in cold tolerance during the germination stage, two indica rice genotypes (sister lines previously identified as cold-tolerant and cold-sensitive) were used in parallel transcriptomic analysis (RNAseq) under cold treatment (seeds germinating at 13 °C for 7 days). We detected 1,361 differentially expressed transcripts. Differences in gene expression found by RNAseq were confirmed for 11 selected genes using RT-qPCR. Biological processes enhanced in the cold-tolerant seedlings include: cell division and expansion (confirmed by anatomical sections of germinating seeds), cell wall integrity and extensibility, water uptake and membrane transport capacity, sucrose synthesis, generation of simple sugars, unsaturation of membrane fatty acids, wax biosynthesis, antioxidant capacity (confirmed by histochemical staining of H2O2), and hormone and Ca(2+)-signaling. The cold-sensitive seedlings respond to low temperature stress increasing synthesis of HSPs and dehydrins, along with enhanced ubiquitin/proteasome protein degradation pathway and polyamine biosynthesis. Our findings can be useful in future biotechnological approaches aiming to cold tolerance in indica rice.

Comparative transcriptional analysis provides new insights into the molecular basis of adventitious rooting recalcitrance in Eucalyptus

Adventitious rooting (AR) is essential in clonal propagation. Eucalyptus globulus is relevant for the cellulose industry due to its low lignin content. However, several useful clones are recalcitrant to AR, often requiring exogenous auxin, adding cost to clonal garden operations. In contrast, E. grandis is an easy-to-root species widely used in clonal forestry. Aiming at contributing to the elucidation of recalcitrance causes in E. globulus, we conducted a comparative analysis with these two species differing in rooting competence, combining gene expression and anatomical techniques. Recalcitrance in E. globulus is reversed by exposure to exogenous indole-3-acetic acid (IAA), which promotes important gene expression modifications in both species. The endogenous content of IAA was significantly higher in E. grandis than in E. globulus. The cambium zone was identified as an active area during AR, concentrating the first cell divisions. Immunolocalization assay showed auxin accumulation in cambium cells, further indicating the importance of this region for rooting. We then performed a cambium zone-specific gene expression analysis during AR using laser microdissection. The results indicated that the auxin-related genes TOPLESS and IAA12/BODENLOS and the cytokinin-related gene ARR1may act as negative regulators of AR, possibly contributing to the hard-to-root phenotype of E. globulus.