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 188.8.131.52) 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 in 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 Ser419 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.
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.
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.
Plants are challenged by a large number of environmental stresses that reduce productivity and even cause death. Both chloroplasts and mitochondria produce reactive oxygen species under normal conditions; however, stress causes an imbalance in these species that leads to deviations from normal cellular conditions and a variety of toxic effects. Mitochondria have uncoupling proteins (UCPs) that uncouple electron transport from ATP synthesis. There is evidence that UCPs play a role in alleviating stress caused by reactive oxygen species overproduction. However, direct evidence that UCPs protect plants from abiotic stress is lacking.
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.
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.
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.
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.
The present article reports on the characterization of ScBAK1, a leucine-rich repeat receptor-like kinase from sugarcane (Saccharum spp.), expressed predominantly in bundle-sheath cells of the mature leaf and potentially involved in cellular signaling cascades mediated by high levels of sugar in this organ. In this report, it was shown that the ScBAK1 sequence was similar to the brassinosteroid insensitive1-associated receptor kinase1 (BAK1). The putative cytoplasmatic domain of ScBAK1 contains all the amino acids characteristic of protein kinases, and the extracellular domain contains five leucine-rich repeats and a putative leucine zipper. Transcripts of ScBAK1 were almost undetectable in sugarcane roots or in any other sink tissue, but accumulated abundantly in the mature leaves. The ScBAK1 expression was higher in the higher sugar content individuals from a population segregating for sugar content throughout the growing season. In situ hybridization in sugarcane leaves showed that the ScBAK1 mRNA accumulated at much higher levels in bundle-sheath cells than in mesophyll cells. In addition, using biolistic bombardment of onion epidermal cells, it was shown that ScBAK1-GFP fusions were localized in the plasma membrane as predicted for a receptor kinase. All together, the present data indicate that ScBAK1 might be a receptor involved in the regulation of specific processes in bundle-sheath cells and in sucrose synthesis in mature sugarcane leaves.
Sucrose content is a highly desirable trait in sugarcane as the worldwide demand for cost-effective biofuels surges. Sugarcane cultivars differ in their capacity to accumulate sucrose and breeding programs routinely perform crosses to identify genotypes able to produce more sucrose. Sucrose content in the mature internodes reach around 20% of the culms dry weight. Genotypes in the populations reflect their genetic program and may display contrasting growth, development, and physiology, all of which affect carbohydrate metabolism. Few studies have profiled gene expression related to sugarcanes sugar content. The identification of signal transduction components and transcription factors that might regulate sugar accumulation is highly desirable if we are to improve this characteristic of sugarcane plants.
The general transcription factor TFIIH is a multiprotein complex with different enzymatic activities such as helicase, protein kinase and DNA repair. MAT1 (ménage à trois 1) is one of the TFIIH subunits that has kinase activity and it is the third subunit of the cyclin-dependent kinase (CDK)-activating kinase (CAK), CDK7- cyclin H. The main objective of this work was to characterize ScMAT1, a sugarcane gene encoding a MAT1 homolog. Northern blots and in situ hybridization results showed that ScMAT1 was expressed in sugarcane mature leaf, leaf roll and inflorescence, and it was not differentially expressed in any of the other tissues analyzed such us bud and roots. In addition, ScMAT1 was not differentially expressed during different stress conditions and treatment with hormones. In situ hybridization analyses also showed that ScMAT1 was expressed in different cell types during leaf development. In order to identify proteins that interact with ScMAT1, a yeast two hybrid assay with ScMAT1 as bait was used to screen a sugarcane leaf cDNA library. The screening of yeast two hybrids yielded 14 positive clones. One of them is a cytochrome p450 family protein involved in oxidative degradation of toxic compounds. Other clones isolated are also related to plant responses to stress. To determine the subcellular localization of ScMAT1, a ScMAT1-GFP fusion was assayed in onion epidermal cell and the fluorescence was localized to the nucleus, in agreement with the putative role of ScMAT1 as a basal transcription factor.
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.
Sugarcane (Saccharum spp.) is one of the most important crops in the world. Drought stress is a major abiotic stress factor that significantly reduces sugarcane yields. However the gene network that mediates plant responses to water stress remains largely unknown in several crop species. Although several microRNAs that mediate post-transcriptional regulation during water stress have been described in other species, the role of the sugarcane microRNAs during drought stress has not been studied. The objective of this work was to identify sugarcane miRNAs that are differentially expressed under drought stress and to correlate this expression with the behavior of two sugarcane cultivars with different drought tolerances. The sugarcane cultivars RB867515 (higher drought tolerance) and RB855536 (lower drought tolerance) were cultivated in a greenhouse for three months and then subjected to drought for 2, 4, 6 or 8 days. By deep sequencing of small RNAs, we were able to identify 18 miRNA families. Among all of the miRNAs thus identified, seven were differentially expressed during drought. Six of these miRNAs were differentially expressed at two days of stress, and five miRNAs were differentially expressed at four days. The expression levels of five miRNAs (ssp-miR164, ssp-miR394, ssp-miR397, ssp-miR399-seq 1 and miR528) were validated by RT-qPCR (quantitative reverse transcriptase PCR). Six precursors and the targets of the differentially expressed miRNA were predicted using an in silico approach and validated by RT-qPCR; many of these targets may play important roles in drought tolerance. These findings constitute a significant increase in the number of identified miRNAs in sugarcane and contribute to the elucidation of the complex regulatory network that is activated by drought stress.
Drought is a major abiotic stress that affects crop productivity worldwide. Sugarcane can withstand periods of water scarcity during the final stage of culm maturation, during which sucrose accumulation occurs. Meanwhile, prolonged periods of drought can cause severe plant losses.
The presence of aluminum (Al) is one of the main factors limiting crop yield in Brazil and worldwide. Plant responses to Al are complex, and the use of techniques such as microarrays can facilitate their comprehension. In a previous work, we evaluated the transcriptome of two maize lines, Cat100-6 and S1587-17, after growing the plants for 1 or 3 days in acid soil (pH 4.1) or alkaline soil with Ca(OH)? (pH 5.5), and we identified genes that likely contribute to Al tolerance. The mapping of these genes to the chromosomes allowed the identification of the genes that are localized in maize QTLs previously reported in the literature as associated with the tolerant phenotype. We were able to map genes encoding proteins possibly involved with acid soil tolerance, such as the ones encoding an RNA binding protein, a protease inhibitor, replication factors, xyloglucan endotransglycosylase and cyclins, inside QTLs known to be important for the Al-tolerant phenotype.
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