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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 CO

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.

RNA polymerase II is an essential multiprotein complex that transcribes thousands of genes, being a fundamental component of the transcription initiation complex. In eukaryotes, RNA polymerase II is formed by a 10-multisubunit conserved core complex, and two additional peripheral subunits, Rpb4 and Rpb7, form the Rpb4/7 subcomplex. Although transcription is vital for cell and organismal viability, little is known about the transcription initiation complex in sugarcane. An initial characterization of the sugarcane RNA polymerase subunit IV (ScRpb4) was performed. Our results demonstrate that ScRpb4 is evolutionarily conserved across kingdoms. At the molecular level, ScRpb4 expression was found in vegetative and reproductive tissues. Furthermore, the expression of ScRpb4 remained stable under various stress conditions, most likely to ensure a proper transcriptional response. Optimal conditions to express ScRpb4 in vitro for further studies were also identified. In this study, an initial characterization of the sugarcane polymerase II subunit IV is presented. Our results open the window to more specific experiments to study ScRpb4 function, for instance, crystal structure determination and pull-down assays as well as their function under biotic and abiotic stresses.

The number of areas subject to drought is likely to increase in response to climate change and will affect the yields of globally important crops such as sugarcane. In this respect, the aim of the present study was to assess the drought tolerance of sugarcane varieties in different phenophases and identify the physiological and biochemical characteristics associated with the lowest yield loss under drought conditions. Six of the most widely cultivated varieties in the region were studied, namely SP79-1011, RB855113, RB92579, RB867515, RB72454 and RB855536. Plant physiological characteristics were assessed during the dry season in the tillering, intense growth and ripening stages, and yield was quantified after 12 months of cultivation. This study shows that different sugarcane varieties are more affected by drought in the intense growth phenophase, since plants showed a greater decline in water and osmotic potential at midday during this period. The varieties most affected by drought in this phenophase were RB855536 and RB855113, which exhibited greater reductions in water and osmotic potential. They also had larger osmoregulator accumulation as a response to drought, but not enough to prevent dehydration, which likely contributed to the lower yield. The RB867515 and RB92579 varieties underwent no significant change in osmotic potential at midday during drought stress in the intense growth phase, despite of low leaf water potential, and little variation in the osmoregulators contents. For these reasons, these two varieties showed greater drought acclimation potentials and were more productive as submitted to drought conditions.

Nowadays, water deficit is one of the major environmental stress issues affecting sugarcane crops around the world. It has been causing significant production decrease due to the increased mortality rates generated by water stress in sugarcane plantations. The aim of the current study is to assess sugarcane morphological and yield responses during different phenophases of sugarcane varieties when plants are subjected to drought stress in the fields of a tropical region. Six sugarcane varieties, namely: SP79-1011, RB855113, RB92579, RB867515, RB72454, and RB855536, were subjected to water stress. The herein adopted water regimes were (i) irrigated crop and (ii) crop subjected to natural drought in the field. Plants were cultivated under water stress imposition throughout three phenological stages (i) tillering, three months after planting; (ii) intense growth, seven months after planting; and (iii) ripening, eleven months after planting. The irrigation treatment consisted of supplementing the crop with 50 mm of water per month during the dry season, whereas non-irrigated plants were naturally grown under water stress conditions throughout the experiment. Water stress mostly affected the sugarcane crop during the intense growth phase. Varieties RB72454 and RB855536 presented smaller green-leaf number, as well as narrower leaf width and smaller leaf area under water stress; besides, they showed low productive potential and high stress susceptibility index (SSI). Stalk height maintenance and the larger number of tillers in RB92579 plants subjected to water stress helped balancing yield rates. This variety also showed the best drought tolerance (DTI) and yield/tolerance indices (YTI); moreover, the principal component analysis evidenced that leaf area, plant height and yield were important factors to distinguish the most tolerant varieties. Such results show that RB92579 is more drought tolerant and has better physiological acclimation potential than the other five varieties; therefore, it can be recommended for crops subjected to drought periods.

Modulation of gibberellic acid signaling through ScGAI regulates culm development including change in phytomer production and source–sink regulation 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.

Identification of transcription factors that control the expression of the sugarcane ACS gene, which is likely involved in ethylene-controlled sucrose accumulation and the circadian regulation of ethylene biosynthesis., 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.

Resistance to drought stress is fundamental to plant survival and development. Abscisic acid (ABA) is one of the major hormones involved in different types of abiotic and biotic stress responses. ABA intracellular signaling has been extensively explored in Arabidopsis thaliana and occurs via a phosphorylation cascade mediated by three related protein kinases, denominated SnRK2s (SNF1-related protein kinases). However, the role of ABA signaling and the biochemistry of SnRK2 in crop plants remains underexplored. Considering the importance of the ABA hormone in abiotic stress tolerance, here we investigated the regulatory mechanism of sugarcane SnRK2s—known as stress/ABA-activated protein kinases (SAPKs). The crystal structure of ScSAPK10 revealed the characteristic SnRK2 family architecture, in which the regulatory SnRK2 box interacts with the kinase domain αC helix. To study sugarcane SnRK2 regulation, we produced a series of mutants for the protein regulatory domains SnRK2 box and ABA box. Mutations in ScSAPK8 SnRK2 box aimed at perturbing its interaction with the protein kinase domain reduced protein kinase activity in vitro. On the other hand, mutations to ScSAPK ABA box did not impact protein kinase activity but did alter the protein autophosphorylation pattern. Taken together, our results demonstrate that both SnRK2 and ABA boxes might play a role in sugarcane SnRK2 function.

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.

Sugarcane is a unique crop with the ability to accumulate high levels of sugar and is a commercially viable source of biomass for bioelectricity and second-generation bioethanol. Water deficit is the single largest abiotic stress affecting sugarcane productivity and the development of water use efficient and drought tolerant cultivars is an imperative for all major sugarcane producing countries.. This review summarizes the physiological and molecular studies on water deficit stress in sugarcane, with the aim to help formulate more effective research strategies for advancing our knowledge on genes and mechanisms underpinning plant response to water stress. We also overview transgenic studies in sugarcane, with an emphasis on the potential strategies to develop superior sugarcane varieties that improve crop productivity in drought-prone environments.

The sensitivity of root cells and root processes to toxic aluminium ions (Al3+) varies along the root axis. This study was established to assess the sensitivity of nutrient uptake to Al along the main root axis of maize genotypes that differ in resistance to Al and to test whether citrate, an Al-complexing compound that is unevenly released along the root axis, can play a role in protecting the root from Al-impaired nutrient uptake. A divided-root-chamber technique was used to measure net fluxes of calcium (Ca2+), magnesium (Mg2+), and potassium (K+) along intact roots of two maize genotypes differing in resistance to Al. The accumulation of Al along their main root axis was also measured in short-term experiments. Results of these experiments were compared with those of a previous study, where citrate exudation had been measured along identical maize root axes. Aluminium affected nutrient uptake widely along the root with strong effects in the apical region, reducing total Ca2+ and Mg2+ uptake, but not K+ uptake. The negative effects of Al3+ were more pronounced in the Al sensitive genotype than in the resistant one. The former also accumulated more Al in its roots than the latter, but this differential accumulation was observed only in the apical part of the root. The spatial pattern of nutrient uptake, irrespective of Al treatment, did not match that of Al-stimulated citrate exudation. Based on the differential sensitivity of the root axis of the two maize genotypes and especially on the extent of the root zones where these differences are expressed, it is suggested that the less Al-disturbed nutrient uptake of a genotype is associated with its resistance to Al.

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)₂ (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.

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.

Due to the strategic importance of sugarcane to Brazil, FAPESP, the main Sao Paulo state research funding agency, launched in 2008 the FAPESP Bioenergy Research Program (BIOEN, http://bioenfapesp.org). BIOEN aims to generate new knowledge and human resources for the improvement of the sugarcane and ethanol industry. As part of the BIOEN program, a Workshop on Sugarcane Improvement was held on March 18th and 19th 2009 in Sao Paulo, Brazil. The aim of the workshop was to explore present and future challenges for sugarcane improvement and its use as a sustainable bioenergy and biomaterial feedstock. The workshop was divided in four sections that represent important challenges for sugarcane improvement: a) gene discovery and sugarcane genomics, b) transgenics and controlled transgene expression, c) sugarcane physiology (photosynthesis, sucrose metabolism, and drought) and d) breeding and statistical genetics. This report summarizes the roadmap for the improvement of sugarcane.

Sucrose is the major product of photosynthesis in many higher plants. It is transported from the source tissue through the phloem to various sink tissues to support plant growth, development and reproduction. Knowledge on the signal transduction pathways involved in sucrose synthesis in mature leaves is limited. Using a microarray approach, we analyzed the expression profiles of 1920 sugarcane genes encoding signal transduction elements, transcription factors and stress-related proteins. We used individuals from a population segregating for sugar content and gene expression profiles were obtained from seven individuals with highest and seven with lowest sugar content. Surprisingly, from the 24 differentially expressed genes, 19 were more expressed in plants containing low-sugar content. Three of these genes encoded 14-3-3 like proteins, which have been found to reduce sucrose phosphate synthase (SPS) activity. Another encoded an SNF1-related protein similar to a protein kinase that phosphorylates SPS in vitro making it a target for the interaction with 14-3-3 proteins. The up-regulation of eight stress related genes in the lower sugar content plants supports a view that sugar levels modulate a complex signal transduction network that seems to involve responses that are related to stress. Evidence that hormone signaling is related to the sucrose content was also found. These data reinforced the usefulness of genomic approaches to uncover how sucrose metabolism can be regulated in sugarcane.

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.

Summary • Aluminum (Al) toxicity is a major factor limiting crop yields on acid soils. There is considerable genotypic variation for Al tolerance in most common plant species. In maize (Zea mays), Al tolerance is a complex phenomenon involving multiple genes and physiological mechanisms yet uncharacterized. • To begin elucidating the molecular basis of maize Al toxicity and tolerance, a detailed temporal analysis of root gene expression under Al stress was performed using microarrays with Al-tolerant and Al-sensitive genotypes. • Al altered the expression of significantly more genes in the Al-sensitive genotype, presumably as a result of more severe Al toxicity. Nevertheless, several Al-regulated genes exhibited higher expression in the Al-tolerant genotype. Cell wall-related genes, as well as low phosphate-responsive genes, were found to be regulated by Al. In addition, the expression patterns of genes related to Al-activated citrate release indicated that in maize this mechanism is probably regulated by the expression level and/or function of the citrate transporter. • This study is the first comprehensive survey of global transcriptional regulation under Al stress. The results described here will help to further our understanding of how mechanisms of Al toxicity and tolerance in maize are regulated at the transcriptional level.

The phytotoxic effects of aluminum (Al) on root systems of crop plants constitute a major agricultural problem in many areas of the world. Root exudation of Al-chelating molecules such as low-molecular-weight organic acids has been shown to be an important mechanism of plant Al tolerance/resistance. Differences observed in the physiology and electrophysiology of root function for two maize genotypes with contrasting Al tolerance revealed an association between rates of Al-activated root organic acid release and Al tolerance. Using these genotypes, we cloned ZmALMT1, a maize gene homologous to the wheat ALMT1 and Arabidopsis AtALMT1 genes that have recently been described as encoding functional, Al-activated transporters that play a role in tolerance by mediating Al-activated organic acid exudation in roots. The ZmALMT1 cDNA encodes a 451 amino acid protein containing six transmembrane helices. Transient expression of a ZmALMT1::GFP chimera confirmed that the protein is targeted to the plant cell plasma membrane. We addressed whether ZmALMT1 might underlie the Al-resistance response (i.e. Al-activated citrate exudation) observed in the roots of the Al-tolerant genotype. The physiological, gene expression and functional data from this study confirm that ZmALMT1 is a plasma membrane transporter that is capable of mediating elective anion efflux and influx. However, gene expression data as well as biophysical transport characteristics obtained from Xenopus oocytes expressing ZmALMT1 indicate that this transporter is implicated in the selective transport of anions involved in mineral nutrition and ion homeostasis processes, rather than mediating a specific Al-activated citrate exudation response at the rhizosphere of maize roots.

There is a growing demand for renewable energy, and sugarcane is a promising bioenergy crop. In Brazil, the largest sugarcane producer in the world, sugarcane plantations are expanding into areas where severe droughts are common. Recent evidence has highlighted the role of miRNAs in regulating drought responses in several species, including sugarcane. This review summarizes the data from miRNA expression profiles observed in a wide array of experimental conditions using different sugarcane cultivars that differ in their tolerance to drought. We uncovered a complex regulation of sugarcane miRNAs in response to drought and discussed these data with the miRNA profiles observed in other plant species. The predicted miRNA targets revealed different transcription factors, proteins involved in tolerance to oxidative stress, cell modification, as well as hormone signaling. Some of these proteins might regulate sugarcane responses to drought, such as reduction of internode growth and shoot branching and increased leaf senescence. A better understanding on the regulatory network from miRNAs and their targets under drought stress has a great potential to contribute to sugarcane improvement, either as molecular markers as well as by using biotechnological approaches.

Coffea arabica, one of the most important breeding species in the world, is susceptible to the leaf miner Leucoptera coffeella, which causes severe damage to coffee plantations. A closely related coffee species (Coffea racemosa) resistant to this insect was crossed with C. arabica and resulted in segregating progenies with resistance or susceptibility to coffee leaf miner. The aim of this work was to isolate and characterize the genes involved in coffee resistance to this pest. Subtracted cDNA libraries enriched in genes preferentially expressed in coffee plants resistant to L. coffeella were constructed. Approximately 1500 clones were spotted on nylon membranes and hybridized to cDNA probes derived from RNA samples from infestation experiments. Several genes were differentially expressed. We selected expressed ESTs with the most interesting expression profiles and confirmed the up-regulation of five of them (Class III Chitinase PR-8, signal peptidase complex subunit SPC25, photosystem gene psaH, a putative calcium exchanger similar to CAX9 and a homeotic gene BEL) by RNA blot. The possible functions of these genes in coffee resistance and coffee development, and a hypothetical defense mechanism against L. coffeela are discussed.

The basic leucine zipper (bZIP) proteins form a large family of transcriptional factors in plants and other eukaryotes. Plant bZIP transcriptional factors are divided into subfamilies and are involved in regulating a large range of physiological processes, from plant development to responses to biotic and abiotic stimuli. In this work, we cloned a novel bZIP of sugarcane into the pET3c vector and expressed the recombinant SCbZIP1 (66-170) protein in Escherichia coli BL21 (DE3) plysS. The recombinant protein was purified by heat-treatment and reversed phase chromatography. Northern blot analysis showed that SCbZIP1 was expressed early in development on day 4, but was not induced by abscisic acid (ABA) or exposure to cold. The identity of the recombinant protein was confirmed by mass spectrometry and CD spectroscopy showed an alpha-helical content of 33%. Electrophoretic mobility assays showed that SCbZIP1 (66-170) bound strongly to G-box, Hex and C-box DNA motifs. SCbZIP1 (66-170) was phosphorylated in vitro by a series of protein kinases and its DNA-binding affinity was strongly decreased after phosphorylation by CKII. SCbZIP1 (66-170) also underwent homo- and heterodimerization with truncated forms of the bZIP transcription factor Opaque 2 from Coix.

Tropical and subtropical plants are generally sensitive to cold and can show appreciable variation in their response to cold stress when exposed to low positive temperatures. Using nylon filter arrays, we analyzed the expression profile of 1,536 expressed sequence tags (ESTs) of sugarcane (Saccharum sp. cv SP80-3280) exposed to cold for 3 to 48 h. Thirty-four cold-inducible ESTs were identified, of which 20 were novel cold-responsive genes that had not previously been reported as being cold inducible, including cellulose synthase, ABI3-interacting protein 2, a negative transcription regulator, phosphate transporter, and others, as well as several unknown genes. In addition, 25 ESTs were identified as being down-regulated during cold exposure. Using a database of cold-regulated proteins reported for other plants, we searched for homologs in the sugarcane EST project database (SUCEST), which contains 263,000 ESTs. Thirty-three homologous putative cold-regulated proteins were identified in the SUCEST database. On the basis of the expression profiles of the cold-inducible genes and the data-mining results, we propose a molecular model for the sugarcane response to low temperature.

The relation between Al-toxicity and oxidative stress was studied for two inbred lines of maize (Zea mays L.), Cat100-6 (Al-tolerant) and S1587-17 (Al-sensitive). Peroxidase (PX), catalase (CAT) and superoxide dismutase (SOD) activities were determined in root tips of both lines, exposed to different Al(3+) concentrations and times of exposure. No increases were observed in CAT activities in either line, although SOD and PX were found to be 1.7 and 2.0 times greater than initial levels, respectively, in sensitive maize treated with 36 microM of Al(3+) for 48 h. The results indicate that Al(3+) induces the dose- and time dependent formation of reactive oxygen species (ROS) and subsequent protein oxidation in S1587-17, although not in Cat100-6. After exposure to 36 microM of Al(3+) for 48 h, the formation of 20+/-2 nmol of carbonyls per mg of protein was observed in S1587-17. The onset of protein oxidation took place after the drop of the relative root growth observed in the sensitive line, indicating that oxidative stress is not the primary cause of root growth inhibition. The presence of Al(3+) did not induce lipid peroxidation in either lines, contrasting with the observations in other species. These results, in conjunction with the data presented in the literature, indicate that oxidative stress caused by Al may harm several components of the cell, depending on the plant species. Moreover, Al(3+) treatment and oxidative stress in the sensitive maize line induced cell death in root tip cells, an event revealed by the high chromatin fragmentation detected by TUNEL analysis.

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.

The role of calmodulin on Al toxicity was studied in two maize ( Zea mays L.) inbred lines, Cat 100-6 (Al-tolerant) and S 1587-17 (Al-sensitive). Increasing levels of Al induced the release of malate at similar rate by roots of both genotypes, while the exudation of citrate, a stronger Al-binding compound, was 3.5 times higher in Cat 100-6 seedlings exposed to 16.2×10 −6 Al 3+ activity. The calmodulin inhibitor trifluoperazine significantly reduced the root growth in both genotypes, mimicking the main effect of Al. However, when Cat 100-6 and S 1587-17 seedlings were challenged with Al in conjunction with trifluoperazine, no further reduction in root growth or any other effect of Al toxicity was observed. The rate of Al-induced citrate exudation by both genotypes was not affected by treatment with trifluoperazine or calmidazolium, another calmodulin inhibitor. The Al 3+ interaction with cytoplasmic CaM was estimated using models for the binding of Al 3+ and Mg 2+ with CaM and physiological concentrations of citrate, CaM, InsP 3 , ATP, ADP, Al 3+ and Mg 2+ . In this simulation, Al 3+ associated with citrate and InsP 3 , but not with CaM. We conclude that calmodulin is not relevant to the physiological processes leading to the Al tolerance in maize, nor is it a primary target for Al toxicity.

The translational construct, containing 719 bp of promoter and 5′-untranslated region and the first 16 bp of coding region, of the maize gene Hrgp encoding a hydroxyproline-rich glycoprotein fused to a glucuronidase reporter cassette, has been tested for activity in different maize tissues by microprojectile bombardment. The promoter has been found to be very active in the tissues of the plant, such as meristems or young shoots, with high cell wall formation activity where a high expression has also been shown for the endogenous gene. The promoter was also shown to be very active in cell types with a protection role such as in pericarp or styles and in cell types where the reinforcement of the cell wall is needed, as styles, auricles and cortical cells in the root tip. The promoter activity is developmentally regulated in the endosperm, being highest simultaneously with active cell division at the early-mid stages of development. In the presence of ethylene, the promoter shows an increased activity in accordance with the increment of mRNA accumulation observed in the plant upon ethylene treatment. It is concluded that the promoter fragment starting at −719 bp (numbering related to the ATG) of the Hrgp gene keeps the essential cis-DNA elements necessary for spatial, temporal and hormonal gene expression in maize.

A protocol is described for simultaneous histochemical detection of LacZ and Gus activity in plant tissues after microprojectile bombardment. The suitability of two different Gus substrates (Salmon-glcA, Magenta-β-d-glcA) is compared. This detection system is used to assay the number of cells expressing either or both reporter gene. This technique is used as a qualitative assay to demonstrate the tissue specificity of a Hrgp promoter in maize embryos, and to measure ABA responsiveness of a Lea promoter in Arabidopsis. The promoter to be studied is linked to the gus reporter gene and the lacZ reporter gene linked to the CaMV 35S promoter is used as a constitutive internal control. The use of an internal control drastically reduces the data variation inherent to microprojectile bombardment.

BackgroundDrought 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.Methodology/principal findingsIn a previous study, we evaluated the transcriptome of drought-stressed plants to better understand sugarcane responses to drought. Among the up-regulated genes was Scdr1 (sugarcane drought-responsive 1). The aim of the research reported here was to characterize this gene. Scdr1 encodes a putative protein containing 248 amino acids with a large number of proline (19%) and cysteine (13%) residues. Phylogenetic analysis showed that ScDR1is in a clade with homologs from other monocotyledonous plants, separate from those of dicotyledonous plants. The expression of Scdr1 in different varieties of sugarcane plants has not shown a clear association with drought tolerance.Conclusions/significanceThe overexpression of Scdr1 in transgenic tobacco plants increased their tolerance to drought, salinity and oxidative stress, as demonstrated by increased photosynthesis, water content, biomass, germination rate, chlorophyll content and reduced accumulation of ROS. Physiological parameters, such as transpiration rate (E), net photosynthesis (A), stomatal conductance (gs) and internal leaf CO(2) concentration, were less affected by abiotic stresses in transgenic Scdr1 plants compared with wild-type plants. Overall, our results indicated that Scdr1 conferred tolerance to multiple abiotic stresses, highlighting the potential of this gene for biotechnological applications.

Made available in DSpace on 2013-08-28T14:11:36Z (GMT). No. of bitstreams: 1 WOS000294678300009.pdf: 677555 bytes, checksum: ad618b66656cc8525114d2f7e9aaa25d (MD5) Made available in DSpace on 2013-09-30T18:37:55Z (GMT). No. of bitstreams: 2 WOS000294678300009.pdf: 677555 bytes, checksum: ad618b66656cc8525114d2f7e9aaa25d (MD5) WOS000294678300009.pdf.txt: 49587 bytes, checksum: 573fd52d922ca0fd3a6f37a99178f29e (MD5) Previous issue date: 2011-08-30 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:50:29Z No. of bitstreams: 2 WOS000294678300009.pdf: 677555 bytes, checksum: ad618b66656cc8525114d2f7e9aaa25d (MD5) WOS000294678300009.pdf.txt: 49587 bytes, checksum: 573fd52d922ca0fd3a6f37a99178f29e (MD5) Made available in DSpace on 2014-05-20T13:50:29Z (GMT). No. of bitstreams: 2 WOS000294678300009.pdf: 677555 bytes, checksum: ad618b66656cc8525114d2f7e9aaa25d (MD5) WOS000294678300009.pdf.txt: 49587 bytes, checksum: 573fd52d922ca0fd3a6f37a99178f29e (MD5) Previous issue date: 2011-08-30 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Financiadora de Estudos e Projetos (FINEP) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Background: 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.Methodology/Principal Findings: Tolerances to salt and water deficit were analyzed in transgenic tobacco plants that overexpress a UCP (AtUCP1) from Arabidopsis thaliana. Seeds of AtUCP1 transgenic lines germinated faster, and adult plants showed better responses to drought and salt stress than wild-type (WT) plants. These phenotypes correlated with increased water retention and higher gas exchange parameters in transgenic plants that overexpress AtUCP1. WT plants exhibited increased respiration under stress, while transgenic plants were only slightly affected. Furthermore, the transgenic plants showed reduced accumulation of hydrogen peroxide in stressed leaves compared with WT plants.Conclusions/Significance: Higher levels of AtUCP1 improved tolerance to multiple abiotic stresses, and this protection was correlated with lower oxidative stress. Our data support previous assumptions that UCPs reduce the imbalance of reactive oxygen species. Our data also suggest that UCPs may play a role in stomatal closure, which agrees with other evidence of a direct relationship between these proteins and photosynthesis. Manipulation of the UCP protein expression in mitochondria is a new avenue for crop improvement and may lead to crops with greater tolerance for challenging environmental conditions. Univ Estadual Campinas, Inst Biol, Lab Genoma Func, Dept Genet Evolucao & Bioagentes, Campinas, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Genet, Botucatu, SP, Brazil Univ Estadual Campinas, Inst Biol, Dept Biol Vegetal, Campinas, Brazil Univ Estadual Paulista, Inst Biociencias, Dept Genet, Botucatu, SP, Brazil

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.

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.

The general transcription factor TFIIH is a multiprotein complex with different enzymatic activities such as helicase, protein kinase and DNA repair. MAT1 (menage a 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.

Because of the economical relevance of sugarcane and its high potential as a source of biofuel, it is important to understand how this crop will respond to the foreseen increase in atmospheric [CO(2)]. The effects of increased [CO(2)] on photosynthesis, development and carbohydrate metabolism were studied in sugarcane (Saccharum ssp.). Plants were grown at ambient (approximately 370 ppm) and elevated (approximately 720 ppm) [CO(2)] during 50 weeks in open-top chambers. The plants grown under elevated CO(2) showed, at the end of such period, an increase of about 30% in photosynthesis and 17% in height, and accumulated 40% more biomass in comparison with the plants grown at ambient [CO(2)]. These plants also had lower stomatal conductance and transpiration rates (-37 and -32%, respectively), and higher water-use efficiency (c.a. 62%). cDNA microarray analyses revealed a differential expression of 35 genes on the leaves (14 repressed and 22 induced) by elevated CO(2). The latter are mainly related to photosynthesis and development. Industrial productivity analysis showed an increase of about 29% in sucrose content. These data suggest that sugarcane crops increase productivity in higher [CO(2)], and that this might be related, as previously observed for maize and sorghum, to transient drought stress.

Sugarcane is a highly productive crop used for centuries as the main source of sugar and recently to produce ethanol, a renewable bio-fuel energy source. There is increased interest in this crop due to the impending need to decrease fossil fuel usage. Sugarcane has a highly polyploid genome. Expressed sequence tag (EST) sequencing has significantly contributed to gene discovery and expression studies used to associate function with sugarcane genes. A significant amount of data exists on regulatory events controlling responses to herbivory, drought, and phosphate deficiency, which cause important constraints on yield and on endophytic bacteria, which are highly beneficial. The means to reduce drought, phosphate deficiency, and herbivory by the sugarcane borer have a negative impact on the environment. Improved tolerance for these constraints is being sought. Sugarcane's ability to accumulate sucrose up to 16% of its culm dry weight is a challenge for genetic manipulation. Genome-based technology such as cDNA microarray data indicates genes associated with sugar content that may be used to develop new varieties improved for sucrose content or for traits that restrict the expansion of the cultivated land. The genes can also be used as molecular markers of agronomic traits in traditional breeding programs.

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.

Jasmonic acid (JA) and its ester methyl jasmonate (MeJA) are linolenic acid-derived signaling molecules involved in plant development and stress responses. MeJA regulates gene expression at transcription, RNA processing and translation. We investigated the changes in gene expression in sugarcane leaves exposed to MeJA using cDNA arrays. Total RNA isolated at 0, 0.5, 1, 3, 6, and 12 h following MeJA treatment was labeled with alpha-33P-dCTP and hybridized to nylon filters containing 1,536 cDNA clones. A significant increase in gene expression in response to MeJA was detected for both novel and well known stress-related genes, while genes participating in photosynthesis and carbohydrate assimilation were down-regulated. Searches for conserved domains in unknown proteins and digital mRNA expression profile analysis revealed putative new stress-related proteins up-regulated by MeJA and the tissues where the MeJA-regulated genes are preferably expressed. O ácido jasmônico (JA) e seu éster metil-jasmonato (MeJA) são moléculas sinalizadoras derivadas do ácido linolênico e estão envolvidas no desenvolvimento da planta e na resposta aos estresses. MeJA regula a expressão gênica ao nível transcricional, do processamento do RNA e da tradução. Investigamos as mudanças na expressão gênica em folhas de cana-de-açúcar expostas ao MeJA usando arranjos de cDNA. O RNA total isolado a 0, 0,5, 1, 3, 6 e 12 horas após o tratamento com MeJA foi utilizado para a síntese de sondas contendo alfa-33P-dCTP, as quais foram, posteriormente, hibridizadas em membranas de náilon contendo 1.536 clones de cDNA. Um aumento significativo na expressão gênica em resposta ao MeJA foi detectado em genes que respondem a estresses e também em genes com função desconhecida, enquanto os genes que participam da fotossíntese e da assimilação de carboidrato foram reprimidos. A busca por domínios conservados em proteínas desconhecidas e a análise digital do perfil de expressão de mRNA revelaram possíveis proteínas novas relacionadas a estresses induzidas por MeJA e os tecidos onde os genes regulados por MeJA são preferivelmente expressos.

Two cDNA clones homologous to myrosinase-binding proteins (MBPs) were identified by differential display in Arabidopsis thaliana (L.) Heynh. The cDNAs (MBP1 and MBP2) correspond to two open-reading frames found in a gene cluster of seven putative MBP genes located on chromosome 1. The predicted proteins MBP1 and MBP2 are similar to lectins and plant aggregating factors. In addition, MBP2 contains a region of high content of proline and alanine residues, commonly found in arabinogalactan proteins and hydroxyproline-rich glycoproteins. Transcripts corresponding to MBP1 and MBP2 genes are exclusively and abundantly expressed in flowers but are not detected in male-sterile flowers of coi1 plants, insensitive to jasmonic acid. Northern analysis and in situ hybridization revealed that MBP mRNAs are present in higher levels in immature flowers and are localized in several floral organs, including the ovary, ovules, style, anthers and filament. Transcripts of the Arabidopsis myrosinase gene TGG1 show a pattern of expression similar to that observed for the MBP genes during flower development; however, they are also abundant in green tissues and are only partially affected by COI1. Crude preparations of soluble proteins from leaf and flower extracts of wild-type Arabidopsis showed myrosinase activity when sinigrin was used as substrate. In contrast, coi1 plants showed significantly reduced myrosinase activities in both leaves and flowers. The results show that COI1 controls MBP expression in flowers and significantly affects the expression and activity of myrosinase in Arabidopsis.

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.

Plant cells are highly organised, and many biological processes are associated with specialised subcellular structures. Subcellular localisation is a key feature of proteins, since it is related to biological function. The subcellular localisation of such proteins can be predicted, providing information that is particularly relevant to those proteins with unknown or putative function. We performed the first in silico genome-wide subcellular localisation analysis for the sugarcane transcriptome (with 11 882 predicted proteins) and found that most of the proteins were localised in four compartments: nucleus (44%), cytosol (19%), mitochondria (12%) and secretory destinations (11%). We also showed that ~19% of the proteins were localised in multiple compartments. Other results allowed identification of a potential set of sugarcane proteins that could show dual targeting by the use of N-truncated forms that started from the nearest downstream in-frame AUG codons. This study was a first step in increasing knowledge about the subcellular localisation of the sugarcane proteome.

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. Electronic supplementary material The online version of this article (doi:10.1007/s00425-012-1795-7) contains supplementary material, which is available to authorized users.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0694-z) contains supplementary material, which is available to authorized users.

Hydroxyproline-rich glycoproteins (HRGP) are structural components of the plant cell wall. Hrgp genes from maize and related species have a conserved 500 bp sequence in the 5'-flanking region, and all Hrgp genes from monocots have an intron located in the 3' untranslated region. To study the role of these conserved regions, several deletions of the Hrgp gene were fused to the beta-glucuronidase ( GUS) gene and used to transform maize tissues by particle bombardment. The overall pattern of GUS activity directed by sequential deletions of the Hrgp promoter was different in embryos and young shoots. In embryos, the activity of the full-length Hrgp promoter was in the same range as that of the p35SI promoter construct, based on the strong 35S promoter, whereas in the fast-growing young shoots it was 20 times higher. A putative silencer element specific for young shoots was found in the -1,076/-700 promoter region. Other major cis elements for Hrgp expression are probably located in the regions spanning -699/-510 and -297/-160. Sequences close to the initial ATG and mRNA leader were also important since deletion of the region -52/+16 caused a 75% reduction in promoter activity. The presence of the Hrgp intron in the 3' untranslated region changed the levels of GUS activity directed by the Hrgp and the 35S promoters. This pattern of activity was complex, and was dependent on the promoter and cell type analysed.