Reduction of ethylene biosynthesis in sugarcane induces growth and investment in the non-enzymatic antioxidant apparatus.

KEY MESSAGE: Lower ethylene production in sugarcane results in plants with higher stature, expression of growth-promoting genes, higher photosynthetic rate, and increased antioxidant compounds. The hormone ethylene is involved in critical processes in sugarcane, such as the growth and accumulation of sucrose. The lack of mutants for ethylene biosynthesis or signaling genes makes it difficult to understand the role of this phytohormone throughout sugarcane development. This study aimed to evaluate the physiology and development of sugarcane plants with low ethylene production. To achieve this goal, we used RNA interference to silence three genes, ScACS1, ScACS2, and ScACS3, encoding 1-aminocyclopropane-1-carboxylic acid synthases (ACS), responsible for a limiting step of the ethylene biosynthesis pathway. Sugarcane plants with reduced ethylene levels presented increased growth, faster germination of lateral gems, and activation of non-enzymatic antioxidant mechanisms. We observed an augmentation in the expression of ScACO5, which encodes the final enzyme regulating ethylene biosynthesis, and ScERF1, encoding a transcription factor, linked to the ethylene response. The increase in plant height was correlated with higher expression of ScPIF3, ScPIF4, and ScPIF5, which encode for transcription factors related to growth induction. Interestingly, there was also an increase in the expression of the ScGAI gene, which encodes a DELLA protein, a growth repressor. The final content of sucrose in the stems was not affected by the low levels of ethylene, although the rate of CO2 assimilation was reduced. This study reports for the first time the impacts of low endogenous production of ethylene in sugarcane and provides helpful insights on the molecular mechanisms behind ethylene responses.

Amino Acid and Carbohydrate Metabolism Are Coordinated to Maintain Energetic Balance during Drought in Sugarcane.

The ability to expand crop plantations without irrigation is a major goal to increase agriculture sustainability. To achieve this end, we need to understand the mechanisms that govern plant growth responses under drought conditions. In this study, we combined physiological, transcriptomic, and genomic data to provide a comprehensive picture of drought and recovery responses in the leaves and roots of sugarcane. Transcriptomic profiling using oligoarrays and RNA-seq identified 2898 (out of 21,902) and 46,062 (out of 373,869) transcripts as differentially expressed, respectively. Co-expression analysis revealed modules enriched in photosynthesis, small molecule metabolism, alpha-amino acid metabolism, trehalose biosynthesis, serine family amino acid metabolism, and carbohydrate transport. Together, our findings reveal that carbohydrate metabolism is coordinated with the degradation of amino acids to provide carbon skeletons to the tricarboxylic acid cycle. This coordination may help to maintain energetic balance during drought stress adaptation, facilitating recovery after the stress is alleviated. Our results shed light on candidate regulatory elements and pave the way to biotechnology strategies towards the development of drought-tolerant sugarcane plants.

Bringing to light the molecular evolution of GUX genes in plants.

Hemicellulose and cellulose are essential polysaccharides for plant development and major components of cell wall. They are also an important energy source for the production of ethanol from plant biomass, but their conversion to fermentable sugars is hindered by the complex structure of cell walls. The glucuronic acid substitution of xylan (GUX) enzymes attach glucuronic acid to xylan, a major component of hemicellulose, decreasing the efficiency of enzymes used for ethanol production. Since loss-of-function gux mutants of Arabidopsis thaliana enhance enzyme accessibility and cell wall digestion without adverse phenotypes, GUX genes are potential targets for genetically improving energy crops. However, comprehensive identification of GUX in important species and their evolutionary history are largely lacking. Here, we identified putative GUX proteins using hidden Markov model searches with the GT8 domain and a GUX-specific motif, and inferred the phylogenetic relationship of 18 species with Maximum likelihood and Bayesian approaches. Each species presented a variable number of GUX, and their evolution can be explained by a mixture of divergent, concerted and birth-and-death evolutionary models. This is the first broad insight into the evolution of GUX gene family in plants and will potentially guide genetic and functional studies in species used for biofuel production.

Overexpression of an evolutionarily conserved drought-responsive sugarcane gene enhances salinity and drought resilience.

BACKGROUND AND AIMS: Improving drought adaptation is more pressing for crops such as sugarcane, rice, wheat and maize, given the high dependence of these crops on irrigation. One option for enhancing adaptation to water limitation in plants is by transgenic approaches. An increasing number of genes that are associated with mechanisms used by plants to cope with water scarcity have been discovered. Genes encoding proteins with unknown functions comprise a relevant fraction of the genes that are modulated by drought. We characterized a gene in response to environmental stresses to gain insight into the unknown fraction of the sugarcane genome. Scdr2 (Sugarcane drought-responsive 2) encodes a small protein and shares highly conserved sequences within monocots, dicots, algae and fungi. METHODS: Plants overexpressing the Scdr2 sugarcane gene were examined in response to salinity and drought. Measurements of the gas exchange parameters, germination rate, water content, dry mass and oxidative damage were performed. Seeds as well as juvenile plants were used to explore the resilience level of the transgenic plants when compared with wild-type plants. KEY RESULTS: Overexpression of Scdr2 enhanced germination rates in tobacco seeds under drought and salinity conditions. Juvenile transgenic plants overexpressing Scdr2 and subjected to drought and salinity stresses showed higher photosynthesis levels, internal CO2 concentration and stomatal conductance, reduced accumulation of hydrogen peroxide in the leaves, no penalty for photosystem II and faster recovery after submission to both stress conditions. Respiration was not strongly affected by both stresses in the Scdr2 transgenic plants, whereas wild-type plants exhibited increased respiration rates. CONCLUSIONS: Scdr2 is involved in the response mechanism to abiotic stresses. Higher levels of Scdr2 enhanced resilience to salinity and drought, and this protection correlated with reduced oxidative damage. Scdr2 confers, at the physiological level, advantages to climate limitations. Therefore, Scdr2 is a potential target for improving sugarcane resilience to abiotic stress.

Brassinosteroids, the Sixth Class of Phytohormones: A Molecular View from the Discovery to Hormonal Interactions in Plant Development and Stress Adaptation.

Phytohormones are natural chemical messengers that play critical roles in the regulation of plant growth and development as well as responses to biotic and abiotic stress factors, maintaining plant homeostasis, and allowing adaptation to environmental changes. The discovery of a new class of phytohormones, the brassinosteroids (BRs), almost 40 years ago opened a new era for the studies of plant growth and development and introduced new perspectives in the regulation of agronomic traits through their use in agriculture. BRs are a group of hormones with significant growth regulatory activity that act independently and in conjunction with other phytohormones to control different BR-regulated activities. Genetic and molecular research has increased our understanding of how BRs and their cross-talk with other phytohormones control several physiological and developmental processes. The present article provides an overview of BRs' discovery as well as recent findings on their interactions with other phytohormones at the transcriptional and post-transcriptional levels, in addition to clarifying how their network works to modulate plant growth, development, and responses to biotic and abiotic stresses.

ScGAI is a key regulator of culm development in sugarcane.

Sugarcane contributes more than 70% of sugar production and is the second largest feedstock for ethanol production globally. Since sugar accumulates in sugarcane culms, culm biomass and sucrose content are the most commercially important traits. Despite extensive breeding, progress in both cane yield and sugar content remains very slow in most countries. We hypothesize that manipulating the genetic elements controlling culm growth will alter source-sink regulation and help break down the yield barriers. In this study, we investigate the role of sugarcane ScGAI, an ortholog of SLR1/D8/RHT1/GAI, on culm development and source-sink regulation through a combination of molecular techniques and transgenic strategies. We show that ScGAI is a key molecular regulator of culm growth and development. Changing ScGAI activity created substantial culm growth and carbon allocation changes for structural molecules and storage. ScGAI regulates spatio-temporal growth of sugarcane culm and leaf by interacting with ScPIF3/PIF4 and ethylene signaling elements ScEIN3/ScEIL1, and its action appears to be regulated by SUMOylation in leaf but not in the culm. Collectively, the remarkable culm growth variation observed suggests that ScGAI could be used as an effective molecular breeding target for breaking the slow yield gain in sugarcane.

The FBH family of bHLH transcription factors controls ACC synthase expression in sugarcane.

Ethylene is a phytohormone involved in the regulation of several aspects of plant development and in responses to biotic and abiotic stress. The effects of exogenous application of ethylene to sugarcane plants are well characterized as growth inhibition of immature internodes and stimulation of sucrose accumulation. However, the molecular network underlying the control of ethylene biosynthesis in sugarcane remains largely unknown. The chemical reaction catalyzed by 1-aminocyclopropane-1-carboxylic acid synthase (ACS) is an important rate-limiting step that regulates ethylene production in plants. In this work, using a yeast one-hybrid approach, we identified three basic helix-loop-helix (bHLH) transcription factors, homologs of Arabidopsis FBH (FLOWERING BHLH), that bind to the promoter of ScACS2 (Sugarcane ACS2), a sugarcane type 3 ACS isozyme gene. Protein-protein interaction assays showed that sugarcane FBH1 (ScFBH1), ScFBH2, and ScFBH3 form homo- and heterodimers in the nucleus. Gene expression analysis revealed that ScFBHs and ScACS2 transcripts are more abundant in maturing internodes during afternoon and night. In addition, Arabidopsis functional analysis demonstrated that FBH controls ethylene production by regulating transcript levels of ACS7, a homolog of ScACS2. These results indicate that ScFBHs transcriptionally regulate ethylene biosynthesis in maturing internodes of sugarcane.

pGVG: a new Gateway-compatible vector for transformation of sugarcane and other monocot crops.

The successful development of genetically engineered monocots using Agrobacterium-mediated transformation has created an increasing demand for compatible vectors. We have developed a new expression vector, pGVG, for efficient transformation and expression of different constructs for gene overexpression and silencing in sugarcane. The pCAMBIA2300 binary vector was modified by adding Gateway recombination sites for fast gene transfer between vectors and the maize polyubiquitin promoter Ubi-1 (ZmUbi1), which is known to drive high gene expression levels in monocots. Transformation efficiency using the pGVG vector reached up to 14 transgenic events per gram of transformed callus. Transgenic plants expressing the ß-glucuronidase (GUS) reporter gene from pGVG showed high levels of GUS activity. qRT-PCR evaluations demonstrated success for both overexpression and hairpin-based silencing cassettes. Therefore, pGVG is suitable for plant transformation and subsequent applications for high-throughput production of stable transgenic sugarcane. The use of an expression cassette based on the ZmUbi1 promoter opens the possibility of using pGVG in other monocot species.

Oligomerization, membrane association, and in vivo phosphorylation of sugarcane UDP-glucose pyrophosphorylase.

Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H2O2 strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.

Physiological and transcriptional analyses of developmental stages along sugarcane leaf.

BACKGROUND: Sugarcane is one of the major crops worldwide. It is cultivated in over 100 countries on 22 million ha. The complex genetic architecture and the lack of a complete genomic sequence in sugarcane hamper the adoption of molecular approaches to study its physiology and to develop new varieties. Investments on the development of new sugarcane varieties have been made to maximize sucrose yield, a trait dependent on photosynthetic capacity. However, detailed studies on sugarcane leaves are scarce. In this work, we report the first molecular and physiological characterization of events taking place along a leaf developmental gradient in sugarcane. RESULTS: Photosynthetic response to CO2 indicated divergence in photosynthetic capacity based on PEPcase activity, corroborated by activity quantification (both in vivo and in vitro) and distinct levels of carbon discrimination on different segments along leaf length. Additionally, leaf segments had contrasting amount of chlorophyll, nitrogen and sugars. RNA-Seq data indicated a plethora of biochemical pathways differentially expressed along the leaf. Some transcription factors families were enriched on each segment and their putative functions corroborate with the distinct developmental stages. Several genes with higher expression in the middle segment, the one with the highest photosynthetic rates, were identified and their role in sugarcane productivity is discussed. Interestingly, sugarcane leaf segments had a different transcriptional behavior compared to previously published data from maize. CONCLUSION: This is the first report of leaf developmental analysis in sugarcane. Our data on sugarcane is another source of information for further studies aiming to understand and/or improve C4 photosynthesis. The segments used in this work were distinct in their physiological status allowing deeper molecular analysis. Although limited in some aspects, the comparison to maize indicates that all data acquired on one C4 species cannot always be easily extrapolated to other species. However, our data indicates that some transcriptional factors were segment-specific and the sugarcane leaf undergoes through the process of suberizarion, photosynthesis establishment and senescence.

Transcriptome analysis highlights changes in the leaves of maize plants cultivated in acidic soil containing toxic levels of Al(3+).

Soil acidity limits crop yields worldwide and is a common result of aluminum (Al) phytotoxicity, which is known to inhibit root growth. Here, we compared the transcriptome of leaves from maize seedlings grown under control conditions (soil without free Al) and under acidic soil containing toxic levels of Al. This study reports, for the first time, the complex transcriptional changes that occur in the leaves of maize plants grown in acidic soil with phytotoxic levels of Al. Our data indicate that 668 genes were differentially expressed in the leaves of plants grown in acidic soil, which is significantly greater than that observed in our previous work with roots. Genes encoding TCA cycle enzymes were upregulated, although no specific transporter of organic acids was differentially expressed in leaves. We also provide evidence for positive roles for auxin and brassinosteroids in Al tolerance, whereas gibberellin and jasmonate may have negative roles. Our data indicate that plant responses to acidic soil with high Al content are not restricted to the root; tolerance mechanisms are also displayed in the aerial parts of the plant, thus indicating that the entire plant responds to stress.

Fine-Tuning of Arabidopsis thaliana Response to Endophytic Colonization by Gluconacetobacter diazotrophicus PAL5 Revealed by Transcriptomic Analysis.

Gluconacetobacter diazotrophicus is a diazotrophic endophytic bacterium that promotes the growth and development of several plant species. However, the molecular mechanisms activated during plant response to this bacterium remain unclear. Here, we used the RNA-seq approach to understand better the effect of G. diazotrophicus PAL5 on the transcriptome of shoot and root tissues of Arabidopsis thaliana. G. diazotrophicus colonized A. thaliana roots and promoted growth, increasing leaf area and biomass. The transcriptomic analysis revealed several differentially expressed genes (DEGs) between inoculated and non-inoculated plants in the shoot and root tissues. A higher number of DEGs were up-regulated in roots compared to shoots. Genes up-regulated in both shoot and root tissues were associated with nitrogen metabolism, production of glucosinolates and flavonoids, receptor kinases, and transcription factors. In contrast, the main groups of down-regulated genes were associated with pathogenesis-related proteins and heat-shock proteins in both shoot and root tissues. Genes encoding enzymes involved in cell wall biogenesis and modification were down-regulated in shoots and up-regulated in roots. In contrast, genes associated with ROS detoxification were up-regulated in shoots and down-regulated in roots. These results highlight the fine-tuning of the transcriptional regulation of A. thaliana in response to colonization by G. diazotrophicus PAL5.

Differentially delayed root proteome responses to salt stress in sugar cane varieties.

Soil salinity is a limiting factor to sugar cane crop development, although in general plants present variable mechanisms of tolerance to salinity stress. The molecular basis underlying these mechanisms can be inferred by using proteomic analysis. Thus, the objective of this work was to identify differentially expressed proteins in sugar cane plants submitted to salinity stress. For that, a greenhouse experiment was established with four sugar cane varieties and two salt conditions, 0 mM (control) and 200 mM NaCl. Physiological and proteomics analyses were performed after 2 and 72 h of stress induction by salt. Distinct physiological responses to salinity stress were observed in the varieties and linked to tolerance mechanisms. In proteomic analysis, the roots soluble protein fraction was extracted, quantified, and analyzed through bidimensional electrophoresis. Gel images analyses were done computationally, where in each contrast only one variable was considered (salinity condition or variety). Differential spots were excised, digested by trypsin, and identified via mass spectrometry. The tolerant variety RB867515 showed the highest accumulation of proteins involved in growth, development, carbohydrate and energy metabolism, reactive oxygen species metabolization, protein protection, and membrane stabilization after 2 h of stress. On the other hand, the presence of these proteins in the sensitive variety was verified only in stress treatment after 72 h. These data indicate that these stress responses pathways play a role in the tolerance to salinity in sugar cane, and their effectiveness for phenotypical tolerance depends on early stress detection and activation of the coding genes expression.

Effects of drought on the microtranscriptome of field-grown sugarcane plants.

Sugarcane (Saccharum spp.) is the most promising crop for renewable energy. Among the diverse stresses that affect plant productivity, drought stress frequently causes losses in sugarcane fields. Although several studies have addressed plant responses to drought using controlled environments, plant responses under field conditions are largely unknown. Recently, microRNA (miRNA)-mediated post-transcriptional regulation has been described as an important and decisive component in vegetal development and stress resistance modulation. The role of miRNAs in sugarcane responses to drought under field conditions is currently not known. Two sugarcane cultivars differing in drought tolerance were grown in the field with and without irrigation (rainfed) for 7 months. By using small RNA deep sequencing, we were able to identify 18 miRNA families comprising 30 mature miRNA sequences. Among these families, we found 13 mature miRNAs that were differentially expressed in drought-stressed plants. Seven miRNAs were differentially expressed in both cultivars. The target genes for many of the differentially expressed mature miRNAs were predicted, and some of them were validated by quantitative reverse transcription PCR. Among the targets, we found transcription factors, transporters, proteins associated with senescence, and proteins involved with flower development. All of these data increase our understanding of the role of miRNAs in the complex regulation of drought stress in field-grown sugarcane, providing valuable tools to develop new sugarcane cultivars tolerant to drought stress.

Molecular and Computational Strategies to Increase the Efficiency of CRISPR-Based Techniques.

The prokaryote-derived Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas mediated gene editing tools have revolutionized our ability to precisely manipulate specific genome sequences in plants and animals. The simplicity, precision, affordability, and robustness of this technology have allowed a myriad of genomes from a diverse group of plant species to be successfully edited. Even though CRISPR/Cas, base editing, and prime editing technologies have been rapidly adopted and implemented in plants, their editing efficiency rate and specificity varies greatly. In this review, we provide a critical overview of the recent advances in CRISPR/Cas9-derived technologies and their implications on enhancing editing efficiency. We highlight the major efforts of engineering Cas9, Cas12a, Cas12b, and Cas12f proteins aiming to improve their efficiencies. We also provide a perspective on the global future of agriculturally based products using DNA-free CRISPR/Cas techniques. The improvement of CRISPR-based technologies efficiency will enable the implementation of genome editing tools in a variety of crop plants, as well as accelerate progress in basic research and molecular breeding.

Molecular characterization of a miraculin-like gene differentially expressed during coffee development and coffee leaf miner infestation.

The characterization of a coffee gene encoding a protein similar to miraculin-like proteins, which are members of the plant Kunitz serine trypsin inhibitor (STI) family of proteinase inhibitors (PIs), is described. PIs are important proteins in plant defence against insects and in the regulation of proteolysis during plant development. This gene has high identity with the Richadella dulcifica taste-modifying protein miraculin and with the tomato protein LeMir; and was named as CoMir (Coffea miraculin). Structural protein modelling indicated that CoMir had structural similarities with the Kunitz STI proteins, but suggested specific folding structures. CoMir was up-regulated after coffee leaf miner (Leucoptera coffella) oviposition in resistant plants of a progeny derived from crosses between C. racemosa (resistant) and C. arabica (susceptible). Interestingly, this gene was down-regulated during coffee leaf miner herbivory in susceptible plants. CoMir expression was up-regulated after abscisic acid application and wounding stress and was prominent during the early stages of flower and fruit development. In situ hybridization revealed that CoMir transcripts accumulated in the anther tissues that display programmed cell death (tapetum, endothecium and stomium) and in the metaxylem vessels of the petals, stigma and leaves. In addition, the recombinant protein CoMir shows inhibitory activity against trypsin. According to the present results CoMir may act in proteolytic regulation during coffee development and in the defence against L. coffeella. The similarity of CoMir with other Kunitz STI proteins and the role of CoMir in plant development and plant stress are discussed.

A transcriptomic analysis of gene expression in the venom gland of the snake Bothrops alternatus (urutu).

BACKGROUND: The genus Bothrops is widespread throughout Central and South America and is the principal cause of snakebite in these regions. Transcriptomic and proteomic studies have examined the venom composition of several species in this genus, but many others remain to be studied. In this work, we used a transcriptomic approach to examine the venom gland genes of Bothrops alternatus, a clinically important species found in southeastern and southern Brazil, Uruguay, northern Argentina and eastern Paraguay. RESULTS: A cDNA library of 5,350 expressed sequence tags (ESTs) was produced and assembled into 838 contigs and 4512 singletons. BLAST searches of relevant databases showed 30% hits and 70% no-hits, with toxin-related transcripts accounting for 23% and 78% of the total transcripts and hits, respectively. Gene ontology analysis identified non-toxin genes related to general metabolism, transcription and translation, processing and sorting, (polypeptide) degradation, structural functions and cell regulation. The major groups of toxin transcripts identified were metalloproteinases (81%), bradykinin-potentiating peptides/C-type natriuretic peptides (8.8%), phospholipases A2 (5.6%), serine proteinases (1.9%) and C-type lectins (1.5%). Metalloproteinases were almost exclusively type PIII proteins, with few type PII and no type PI proteins. Phospholipases A2 were essentially acidic; no basic PLA2 were detected. Minor toxin transcripts were related to L-amino acid oxidase, cysteine-rich secretory proteins, dipeptidylpeptidase IV, hyaluronidase, three-finger toxins and ohanin. Two non-toxic proteins, thioredoxin and double-specificity phosphatase Dusp6, showed high sequence identity to similar proteins from other snakes. In addition to the above features, single-nucleotide polymorphisms, microsatellites, transposable elements and inverted repeats that could contribute to toxin diversity were observed. CONCLUSIONS: Bothrops alternatus venom gland contains the major toxin classes described for other Bothrops venoms based on trancriptomic and proteomic studies. The predominance of type PIII metalloproteinases agrees with the well-known hemorrhagic activity of this venom, whereas the lower content of serine proteases and C-type lectins could contribute to less marked coagulopathy following envenoming by this species. The lack of basic PLA2 agrees with the lower myotoxicity of this venom compared to other Bothrops species with these toxins. Together, these results contribute to our understanding of the physiopathology of envenoming by this species.

Transcriptional profile of maize roots under acid soil growth.

BACKGROUND: Aluminum (Al) toxicity is one of the most important yield-limiting factors of many crops worldwide. The primary symptom of Al toxicity syndrome is the inhibition of root growth leading to poor water and nutrient absorption. Al tolerance has been extensively studied using hydroponic experiments. However, unlike soil conditions, this method does not address all of the components that are necessary for proper root growth and development. In the present study, we grew two maize genotypes with contrasting tolerance to Al in soil containing toxic levels of Al and then compared their transcriptomic responses. RESULTS: When grown in acid soil containing toxic levels of Al, the Al-sensitive genotype (S1587-17) showed greater root growth inhibition, more Al accumulation and more callose deposition in root tips than did the tolerant genotype (Cat100-6). Transcriptome profiling showed a higher number of genes differentially expressed in S1587-17 grown in acid soil, probably due to secondary effects of Al toxicity. Genes involved in the biosynthesis of organic acids, which are frequently associated with an Al tolerance response, were not differentially regulated in both genotypes after acid soil exposure. However, genes related to the biosynthesis of auxin, ethylene and lignin were up-regulated in the Al-sensitive genotype, indicating that these pathways might be associated with root growth inhibition. By comparing the two maize lines, we were able to discover genes up-regulated only in the Al-tolerant line that also presented higher absolute levels than those observed in the Al-sensitive line. These genes encoded a lipase hydrolase, a retinol dehydrogenase, a glycine-rich protein, a member of the WRKY transcriptional family and two unknown proteins. CONCLUSIONS: This work provides the first characterization of the physiological and transcriptional responses of maize roots when grown in acid soil containing toxic levels of Al. The transcriptome profiles highlighted several pathways that are related to Al toxicity and tolerance during growth in acid soil. We found several genes that were not found in previous studies using hydroponic experiments, increasing our understanding of plant responses to acid soil. The use of two germplasms with markedly different Al tolerances allowed the identification of genes that are a valuable tool for assessing the mechanisms of Al tolerance in maize in acid soil.

Molecular characterization of ScTFIIAgamma, encoding the putative TFIIA small subunit from sugarcane.

Transcription mediated by RNA polymerase II depends on a set of different transcription factors to form the pre-initiation complex. TFIIA is involved in the construction of this complex and increases the affinity of TBP for the DNA union region in vitro. In this study, we characterized the ScTFIIAgamma gene, which encodes a homolog of the smaller subunit (gamma) of transcription factor TFIIA in sugarcane. RNA blot analysis showed that ScTFIIAgamma transcripts accumulate in all tissues evaluated, with higher levels in leaf roll and flowers. In situ hybridization showed that ScTFIIAgamma was expressed in different cells of the reproductive meristem. In sugarcane plantlets, methyl jasmonate and absicic acid treatments as well as phosphate starvation had no influence on ScTFIIAgamma transcript accumulation. The subcelullar localization assay demonstrates that ScTFIIAgamma protein is directed to the cell nucleus. The phylogenetic analysis, the expression in several tissues and under different treatments and the nuclear localization are in line with the putative role of ScTFIIAgamma as a subunit of basal transcription factor.

Plant 3' Regulatory Regions From mRNA-Encoding Genes and Their Uses to Modulate Expression.

Molecular biotechnology has made it possible to explore the potential of plants for different purposes. The 3' regulatory regions have a great diversity of cis-regulatory elements directly involved in polyadenylation, stability, transport and mRNA translation, essential to achieve the desired levels of gene expression. A complex interaction between the cleavage and polyadenylation molecular complex and cis-elements determine the polyadenylation site, which may result in the choice of non-canonical sites, resulting in alternative polyadenylation events, involved in the regulation of more than 80% of the genes expressed in plants. In addition, after transcription, a wide array of RNA-binding proteins interacts with cis-acting elements located mainly in the 3' untranslated region, determining the fate of mRNAs in eukaryotic cells. Although a small number of 3' regulatory regions have been identified and validated so far, many studies have shown that plant 3' regulatory regions have a higher potential to regulate gene expression in plants compared to widely used 3' regulatory regions, such as NOS and OCS from Agrobacterium tumefaciens and 35S from cauliflower mosaic virus. In this review, we discuss the role of 3' regulatory regions in gene expression, and the superior potential that plant 3' regulatory regions have compared to NOS, OCS and 35S 3' regulatory regions.