Influence of Sugarcane Variety on Rhizosphere Microbiota Under Irrigated and Water-Limiting Conditions.

Drought is one of the main problems linked to climate change that is faced by agriculture, affecting various globally important crops, including sugarcane. Environmentally sustainable strategies have been sought to mitigate the effects of climate change on crops. Among them, the use of beneficial microorganisms offers a promising approach. However, it is still necessary to understand the mechanisms that regulate plant-microorganism interactions, in normal situations and under stress. In this work, the rhizosphere metagenomes of two sugarcane varieties, one resistant and the other susceptible to drought, were compared under normal conditions and under water-limiting conditions. The results showed that for the drought-resistant sugarcane variety, bacteria belonging to the order Sphingomonadales and the family Xanthomonadaceae presented increased activities in terms of mobility, colonization, and cell growth. In contrast, the rhizosphere associated with the drought-sensitive variety exhibited increases of bacteria belonging to the family Polyangiaceae, and the genus Streptomyces, with modifications in DNA metabolism and ribosome binding proteins. The results pointed to variation in the rhizosphere microbiota that was modulated by the host plant genotype, revealing potential bacterial candidates that could be recruited to assist plants during water-limiting conditions.

Analysis of the PEBP gene family and identification of a novel FLOWERING LOCUS T orthologue in sugarcane.

Sugarcane (Saccharum spp.) is an important economic crop for both sugar and biomass, the yields of which are negatively affected by flowering. The molecular mechanisms controlling flowering in sugarcane are nevertheless poorly understood. RNA-seq data analysis and database searches have enabled a comprehensive description of the PEBP gene family in sugarcane. It is shown to consist of at least 13 FLOWERING LOCUS T (FT)-like genes, two MOTHER OF FT AND TFL (MFT)-like genes, and four TERMINAL FLOWER (TFL)-like genes. As expected, these genes all show very high homology to their corresponding genes in Sorghum, and also to FT-like, MFT-like, and TFL-like genes in maize, rice, and Arabidopsis. Functional analysis in Arabidopsis showed that the sugarcane ScFT3 gene can rescue the late flowering phenotype of the Arabidopsis ft-10 mutant, whereas ScFT5 cannot. High expression levels of ScFT3 in leaves of short day-induced sugarcane plants coincided with initial stages of floral induction in the shoot apical meristem as shown by histological analysis of meristem dissections. This suggests that ScFT3 is likely to play a role in floral induction in sugarcane; however, other sugarcane FT-like genes may also be involved in the flowering process.

Transcriptomic Analysis of Changes in Gene Expression During Flowering Induction in Sugarcane Under Controlled Photoperiodic Conditions.

Flowering is of utmost relevance for the agricultural productivity of the sugarcane bioeconomy, but data and knowledge of the genetic mechanisms underlying its photoperiodic induction are still scarce. An understanding of the molecular mechanisms that regulate the transition from vegetative to reproductive growth in sugarcane could provide better control of flowering for breeding. This study aimed to investigate the transcriptome of +1 mature leaves of a sugarcane cultivar subjected to florally inductive and non-inductive photoperiodic treatments to identify gene expression patterns and molecular regulatory modules. We identified 7,083 differentially expressed (DE) genes, of which 5,623 showed significant identity to other plant genes. Functional group analysis showed differential regulation of important metabolic pathways involved in plant development, such as plant hormones (i.e., cytokinin, gibberellin, and abscisic acid), light reactions, and photorespiration. Gene ontology enrichment analysis revealed evidence of upregulated processes and functions related to the response to abiotic stress, photoprotection, photosynthesis, light harvesting, and pigment biosynthesis, whereas important categories related to growth and vegetative development of plants, such as plant organ morphogenesis, shoot system development, macromolecule metabolic process, and lignin biosynthesis, were downregulated. Also, out of 76 sugarcane transcripts considered putative orthologs to flowering genes from other plants (such as Arabidopsis thaliana, Oryza sativa, and Sorghum bicolor), 21 transcripts were DE. Nine DE genes related to flowering and response to photoperiod were analyzed either at mature or spindle leaves at two development stages corresponding to the early stage of induction and inflorescence primordia formation. Finally, we report a set of flowering-induced long non-coding RNAs and describe their level of conservation to other crops, many of which showed expression patterns correlated against those in the functionally grouped gene network.

Overexpression of a Sugarcane BAHD Acyltransferase Alters Hydroxycinnamate Content in Maize Cell Wall.

The purification of hydroxycinnamic acids [p-coumaric acid (pCA) and ferulic acid (FA)] from grass cell walls requires high-cost processes. Feedstocks with increased levels of one hydroxycinnamate in preference to the other are therefore highly desirable. We identified and conducted expression analysis for nine BAHD acyltransferase ScAts genes from sugarcane. The high conservation of AT10 proteins, together with their similar gene expression patterns, supported a similar role in distinct grasses. Overexpression of ScAT10 in maize resulted in up to 75% increase in total pCA content. Mild hydrolysis and derivatization followed by reductive cleavage (DFRC) analysis showed that pCA increase was restricted to the hemicellulosic portion of the cell wall. Furthermore, total FA content was reduced up to 88%, resulting in a 10-fold increase in the pCA/FA ratio. Thus, we functionally characterized a sugarcane gene involved in pCA content on hemicelluloses and generated a C4 plant that is promising for valorizing pCA production in biorefineries.

Selection and validation of reference genes by RT-qPCR under photoperiodic induction of flowering in sugarcane (Saccharum spp.).

Although reference genes have previously been used in the expression analysis of genes involved in sugarcane flowering they had not been experimentally validated for stability and consistency of expression between different samples over a wide range of experimental conditions. Here we report the analysis of candidate reference genes in different tissue types, at different temporal time-points, in both short and long day photoperiodic treatments. The stability of the candidate reference genes in all conditions was evaluated with NormFinder, BestKeeper, and RefFinder algorithms that complement each other for a more robust analysis. As the Normfinder algorithm was more appropriate for our experimental conditions, greater emphasis was placed on Normfinder when choosing the most stable genes. UBQ1 and TUB were shown to be the most stable reference genes to use for normalizing RT-qPCR gene expression data during floral induction, whilst 25SrRNA1 and GAPDH were the least stable. Their use as a reference gene pair was validated by analyzing the expression of two differentially expressed target genes (PIL5 and LHP1). The UBQ1/TUB reference genes combination was able to reveal small significant differences in gene expression of the two target genes that were not detectable when using the least stable reference gene combination. These results can be used to inform the choice of reference genes to use in the study of the sugarcane floral induction pathway. Our work also demonstrates that both PIL5 and LHP1 are significantly up-regulated in the initial stages of photoperiodic induction of flowering in sugarcane.

Genetic Differentiation of a New World Screwworm Fly Population from Uruguay Detected by SNPs, Mitochondrial DNA and Microsatellites in Two Consecutive Years.

The New World screwworm (NWS) fly, Cochliomyia hominivorax (Diptera: Calliphoridae), is an economically important ectoparasite currently distributed in South America and in the Caribbean basin. The successful eradication of this species in USA, Mexico and continental Central America was achieved by a control program based on the sterile insect technique (SIT). In order to implement a genetic control strategy over the NWS fly's current area of occurrence, first, it is necessary to understand the species dynamics and population structure. In order to address this objective, the spatial genetic structure of the NWS fly was previously reported in South America based on different genetic markers; however, to date, no study has investigated temporal changes in the genetic composition of its populations. In the current study, the temporal genetic structure of a NWS fly population from Uruguay was investigated through two consecutive samplings from the same locality over an interval of approximately 18 generations. The genetic structure was accessed with neutral and under selection SNPs obtained with genotyping-by-sequencing. The results gathered with these data were compared to estimates achieved with mitochondrial DNA sequences and eight microsatellite markers. Temporal changes in the genetic composition were revealed by all three molecular markers, which may be attributed to seasonal changes in the NWS fly's southern distribution. SNPs were employed for the first time for estimating the genetic structure in a NWS fly population; these results provide new clues and perspectives on its population genetic structure. This approach could have significant implications for the planning and implementation of management programs.

Prospecting Plant Growth-Promoting Bacteria Isolated from the Rhizosphere of Sugarcane Under Drought Stress.

In the rhizosphere, the soil bacteria and the plants are closely related, with the plant-associated microbiota playing an important role in promoting plant growth under both normal and stress conditions. In this study, the cultivable bacteria in the sugarcane rhizosphere under different levels of drought stress were characterized and screened for plant growth activities. The results suggested that the microbial community associated with the sugarcane rhizosphere was strongly affected by drought, but some important genera of bacteria such as Arthrobacter, Pseudomonas, Microbacterium, and Bacillus remained present during the entire experiment, indicating the adaptability of these organisms and their importance in the rhizosphere community. Many isolates exhibited positive results for one or more plant growth activity, and they were also capable of growing under simulated drought stress, suggesting that the microorganisms isolated from the sugarcane rhizosphere could be explored for uses such as biofertilizers or biocontrol agents in agriculture.

Gene Duplication in the Sugarcane Genome: A Case Study of Allele Interactions and Evolutionary Patterns in Two Genic Regions.

Sugarcane (Saccharum spp.) is highly polyploid and aneuploid. Modern cultivars are derived from hybridization between S. officinarum and S. spontaneum. This combination results in a genome exhibiting variable ploidy among different loci, a huge genome size (~10 Gb) and a high content of repetitive regions. An approach using genomic, transcriptomic, and genetic mapping can improve our knowledge of the behavior of genetics in sugarcane. The hypothetical HP600 and Centromere Protein C (CENP-C) genes from sugarcane were used to elucidate the allelic expression and genomic and genetic behaviors of this complex polyploid. The physically linked side-by-side genes HP600 and CENP-C were found in two different homeologous chromosome groups with ploidies of eight and ten. The first region (Region01) was a Sorghum bicolor ortholog region with all haplotypes of HP600 and CENP-C expressed, but HP600 exhibited an unbalanced haplotype expression. The second region (Region02) was a scrambled sugarcane sequence formed from different noncollinear genes containing partial duplications of HP600 and CENP-C (paralogs). This duplication resulted in a non-expressed HP600 pseudogene and a recombined fusion version of CENP-C and the orthologous gene Sobic.003G299500 with at least two chimeric gene haplotypes expressed. It was also determined that it occurred before Saccharum genus formation and after the separation of sorghum and sugarcane. A linkage map was constructed using markers from nonduplicated Region01 and for the duplication (Region01 and Region02). We compare the physical and linkage maps, demonstrating the possibility of mapping markers located in duplicated regions with markers in nonduplicated region. Our results contribute directly to the improvement of linkage mapping in complex polyploids and improve the integration of physical and genetic data for sugarcane breeding programs. Thus, we describe the complexity involved in sugarcane genetics and genomics and allelic dynamics, which can be useful for understanding complex polyploid genomes.

Diel oscillations in cell wall components and soluble sugars as a response to short-day in sugarcane (Saccharum sp.).

BACKGROUND: Sugarcane is a tropical crop that can accumulate high concentration of sucrose in the stem as a storage carbohydrate. For that reason, sugarcane accounts for approximately 75% of all the sugar produced in the world and has become the main sugar source to produce first-generation bioethanol in Brazil. Daily rhythms cause plants to adapt and coordinate their metabolism to achieve maximum photosynthesis and carbohydrate production throughout the day. Circadian rhythms arise from the interaction of an internal oscillator and external stimuli, whereas diel rhythms occur in response to a light-dark cycle. Diel signalling contributes to synchronizing circadian rhythms to photoperiods, and levels of carbohydrates oscillate in a diel fashion. Under regular photoperiods, they are synthesized during the daytime and consumed throughout the night as an energy reserve. However, short days can induce higher rates of synthesis during daytime and lower rates of consumption in the dark. Cell wall carbohydrates are also diurnally regulated, and it has been shown that celluloses, hemicelluloses and pectin are deposited/degraded at different times of the day. To assess the diel carbohydrate profile in young sugarcane plants, we measured soluble sugars and cell wall components along a time course in plants subjected either to a regular day or short day. RESULTS: Short-day influenced sucrose synthesis and cell wall components. In short-day a 44% increase in sucrose concentration was detected in the dark, but was stable during the day. Cellulose, hemicellulose and pectin also fluctuate within a 24 h interval when subjected to a short day. A 38% increase in leaf sheath cellulose was observed from the middle of the day to the first hour of the night. Leaf sheath pectin and hemicellulose also increased from the day to the night, while it decreased in leaves. CONCLUSIONS: The presented data show diurnal patterns of soluble sugar metabolism together with temporal regulation of cell wall metabolism for a short day, suggesting that diel signalling has a role in how sugarcane manages sugar accumulation and partitioning. Understanding cell wall synthesis/degradation dynamics may help to improve the yield of sugarcane.

Selection and validation of reference genes for gene expression studies in Klebsiella pneumoniae using Reverse Transcription Quantitative real-time PCR.

For reliable results, Reverse Transcription Quantitative real-time Polymerase Chain Reaction (RT-qPCR) analyses depend on stably expressed reference genes for data normalization purposes. Klebsiella pneumoniae is an opportunistic Gram-negative bacterium that has become a serious threat worldwide. Unfortunately, there is no consensus for an ideal reference gene for RT-qPCR data normalization on K. pneumoniae. In this study, the expression profile of eleven candidate reference genes was assessed in K. pneumoniae cells submitted to various experimental conditions, and the expression stability of these candidate genes was evaluated using statistical algorithms BestKeeper, NormFinder, geNorm, Delta CT and RefFinder. The statistical analyses ranked recA, rho, proC and rpoD as the most suitable reference genes for accurate RT-qPCR data normalization in K. pneumoniae. The reliability of the proposed reference genes was validated by normalizing the relative expression of iron-regulated genes in K. pneumoniae cells submitted to iron-replete and iron-limited conditions. This work emphasizes that the stable expression of any potential reference candidate gene must be validated in each physiological condition or experimental treatment under study.

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.

Entre sanatório e terreiros: Ulysses Pernambucano, René Ribeiro e o projeto reformista da psiquiatria social de Recife (1919-1949)

Entre as décadas de 1920 e 1930 o estado de Pernambuco passou por duas reformas psiquiátricas lideradas pelo médico Ulysses Pernambucano. A primeira reforma (1924-1926) combateu os métodos coercitivos tradicionalmente utilizados no tratamento dos “loucos agitados”, tais como o uso de celas, camisas de força e sedativos. Na segunda reforma (1931-1935), utilizando-se o discurso da Higiene Mental, retirou-se a centralidade do hospício no tratamento psiquiátrico por meio da criação de uma colônia agrícola, de serviços ambulatoriais, de um hospital aberto e de um serviço de profilaxia (higiene) das doenças mentais. O combate àquilo que se entendia como sendo as causas sociais da loucura, fez com que um grupo de psiquiatras do Recife empreendesse uma série de pesquisas urbanas visando futuras transformações sociais. Assim, iniciaram estudos pioneiros em diversos campos, como a religiosidade afro-brasileira, bem como buscaram canais de diálogo com outros campos do saber, tais como a Sociologia, a Psicologia, o Serviço Social e a Antropologia. O objetivo deste trabalho é analisar as trajetórias de Ulysses Pernambucano e de seu aluno, René Ribeiro, para que, por meio delas, se possa evidenciar os debates, os projetos, os conflitos políticos e a proposta interdisciplinar que estavam sendo colocados no contexto psiquiátrico da época –entre as décadas de 1910 e 1940. Buscou-se evidenciar como o discurso da Higiene Mental foi utilizado para articular e legitimar uma agenda de reformas que eram, a um só tempo, científicas e sociais.(AU)

Identification, classification and transcriptional profiles of dirigent domain-containing proteins in sugarcane.

Dirigent (DIR) proteins, encoded by DIR genes, are referred to as "dirigent" because they direct the outcome of the coupling of the monolignol coniferyl alcohol into (+) or (-) pinoresinol, the first intermediates in the enantiocomplementary pathways for lignan biosynthesis. DIR domain-containing or DIR-like proteins are, thus, termed for not having a clear characterization. A transcriptome- and genome-wide survey of DIR domain-containing proteins in sugarcane was carried out, in addition to phylogenetic, physicochemical and transcriptional analyses. A total of 120 non-redundant sequences containing the DIR domain were identified and classified into 64 groups according to phylogenetic and sequence alignment analyses. In silico analysis of transcript abundance showed that these sequences are expressed at low levels in leaves and genes in the same phylogenetic clade have similar expression patterns. Expression analysis of ShDIR1-like transcripts in the culm internodes of sugarcane demonstrates their abundance in mature internodes, their induction by nitrogen fertilization and their predominant expression in cells that have a lignified secondary cell wall, such as vascular bundles of young internodes and parenchymal cells of the pith of mature internodes. Due to the lack of information about the functional role of DIR in plants, a possible relationship is discussed between the ShDIR1-like transcriptional profile and cell wall development in parenchyma cells of sugarcane culm, which typically accumulates large amounts of sucrose. The number of genes encoding the DIR domain-containing proteins in sugarcane is intriguing and is an indication per se that these proteins may have an important metabolic role and thus deserve to be better studied.

Genome-Wide Survey of Genes Under Positive Selection in Avian Pathogenic Escherichia coli Strains.

The ability to obtain bacterial genomes from the same host has allowed for comparative studies that help in the understanding of the molecular evolution of specific pathotypes. Avian pathogenic Escherichia coli (APEC) is a group of extraintestinal strains responsible for causing colibacillosis in birds. APEC is also suggested to possess a role as a zoonotic agent. Despite its importance, APEC pathogenesis still has several cryptic pathogenic processes that need to be better understood. In this work, a genome-wide survey of eight APEC strains for genes with evidence of recombination revealed that ∼14% of the homologous groups evaluated present signs of recombination. Enrichment analyses revealed that nine Gene Ontology (GO) terms were significantly more represented in recombinant genes. Among these GO terms, several were noted to be ATP-related categories. The search for positive selection in these APEC genomes revealed 32 groups of homologous genes with evidence of positive selection. Among these groups, we found several related to cell metabolism, as well as several uncharacterized genes, beyond the well-known virulence factors ompC, lamB, waaW, waaL, and fliC. A GO term enrichment test showed a prevalence of terms related to bacterial cell contact with the external environment (e.g., viral entry into host cell, detection of virus, pore complex, bacterial-type flagellum filament C, and porin activity). Finally, the genes with evidence of positive selection were retrieved from genomes of non-APEC strains and tested as were done for APEC strains. The result revealed that none of the groups of genes presented evidence of positive selection, confirming that the analysis was effective in inferring positive selection for APEC and not for E. coli in general, which means that the study of the genes with evidence of positive selection identified in this study can contribute for the better understanding of APEC pathogenesis processes.

Ethylene-induced transcriptional and hormonal responses at the onset of sugarcane ripening.

The effects of ethephon as a sugarcane ripener are attributed to ethylene. However, the role of this phytohormone at the molecular level is unknown. We performed a transcriptome analysis combined with the evaluation of sucrose metabolism and hormone profiling of sugarcane plants sprayed with ethephon or aminoethoxyvinylglycine (AVG), an ethylene inhibitor, at the onset of ripening. The differential response between ethephon and AVG on sucrose level and sucrose synthase activity in internodes indicates ethylene as a potential regulator of sink strength. The correlation between hormone levels and transcriptional changes suggests ethylene as a trigger of multiple hormone signal cascades, with approximately 18% of differentially expressed genes involved in hormone biosynthesis, metabolism, signalling, and response. A defence response elicited in leaves favoured salicylic acid over the ethylene/jasmonic acid pathway, while the upper internode was prone to respond to ethylene with strong stimuli on ethylene biosynthesis and signalling genes. Besides, ethylene acted synergistically with abscisic acid, another ripening factor, and antagonistically with gibberellin and auxin. We identified potential ethylene target genes and characterized the hormonal status during ripening, providing insights into the action of ethylene at the site of sucrose accumulation. A molecular model of ethylene interplay with other hormones is proposed.

Analysis of Three Sugarcane Homo/Homeologous Regions Suggests Independent Polyploidization Events of Saccharum officinarum and Saccharum spontaneum.

Whole genome duplication has played an important role in plant evolution and diversification. Sugarcane is an important crop with a complex hybrid polyploid genome, for which the process of adaptation to polyploidy is still poorly understood. In order to improve our knowledge about sugarcane genome evolution and the homo/homeologous gene expression balance, we sequenced and analyzed 27 BACs (Bacterial Artificial Chromosome) of sugarcane R570 cultivar, containing the putative single-copy genes LFY (seven haplotypes), PHYC (four haplotypes), and TOR (seven haplotypes). Comparative genomic approaches showed that these sugarcane loci presented a high degree of conservation of gene content and collinearity (synteny) with sorghum and rice orthologous regions, but were invaded by transposable elements (TE). All the homo/homeologous haplotypes of LFY, PHYC, and TOR are likely to be functional, because they are all under purifying selection (dN/dS ≪ 1). However, they were found to participate in a nonequivalently manner to the overall expression of the corresponding gene. SNPs, indels, and amino acid substitutions allowed inferring the S. officinarum or S. spontaneum origin of the TOR haplotypes, which further led to the estimation that these two sugarcane ancestral species diverged between 2.5 and 3.5 Ma. In addition, analysis of shared TE insertions in TOR haplotypes suggested that two autopolyploidization may have occurred in the lineage that gave rise to S. officinarum, after its divergence from S. spontaneum.

Functional and genetic characterization of hydrocarbon biodegrader and exopolymer-producing clones from a petroleum reservoir metagenomic library.

Microbial degradation of petroleum is a worldwide issue, which causes physico-chemical changes in its compounds, diminishing its commercial value. Biosurfactants are chemically diverse molecules that can be produced by several microorganisms and can enable microbial access to hydrocarbons. In order to investigate both microbial activities, function-driven screening assays for biosurfactant production and hydrocarbon biodegradation were carried out from a metagenomic fosmid library. It was constructed from the total DNA extracted from aerobic and anaerobic enrichments from a Brazilian biodegraded petroleum sample. A sum of 10 clones were selected in order to evaluate their ability to produce exopolymers (EPS) with emulsifying activity, as well as to characterize the gene sequences, harbored by the fosmid clones, through 454 pyrosequencing. Functional analyses confirmed the ability of some clones to produce surfactant compounds. Regarding hydrocarbon as microbial carbon sources, n-alkane (in mixture or not) and naphthalene were preferentially consumed as substrates. Analysis of sequence data set revealed the presence of genes related to xenobiotics biodegradation and carbohydrate metabolism. These data were corroborated by the results of hydrocarbon biodegradation and biosurfactant production detected in the evaluated clones.

Genome-wide identification and characterization of tRNA-derived RNA fragments in land plants.

KEY MESSAGE: The manuscript by Alves et al. entitled "Genome-wide identification and characterization of tRNA-derived RNA fragments in land plants" describes the identification and characterization of tRNAderived sRNA fragments in plants. By combining bioinformatic analysis and genetic and molecular approaches, we show that tRF biogenesis does not rely on canonical microRNA/siRNA processing machinery (i.e., independent of DICER-LIKE proteins). Moreover, we provide evidences that the Arabidopsis S-like Ribonuclease 1 (RNS1) might be involved in the biogenesis of tRFs. Detailed analyses showed that plant tRFs are sorted into different types of ARGONAUTE proteins and that they have potential target candidate genes. Our work advances the understanding of the tRF biology in plants by providing evidences that plant and animal tRFs shared common features and raising the hypothesis that an interplay between tRFs and other sRNAs might be important to fine-tune gene expression and protein biosynthesis in plant cells. Small RNA (sRNA) fragments derived from tRNAs (3'-loop, 5'-loop, anti-codon loop), named tRFs, have been reported in several organisms, including humans and plants. Although they may interfere with gene expression, their biogenesis and biological functions in plants remain poorly understood. Here, we capitalized on small RNA sequencing data from distinct species such as Arabidopsis thaliana, Oryza sativa, and Physcomitrella patens to examine the diversity of plant tRFs and provide insight into their properties. In silico analyzes of 19 to 25-nt tRFs derived from 5' (tRF-5s) and 3'CCA (tRF-3s) tRNA loops in these three evolutionary distant species showed that they are conserved and their abundance did not correlate with the number of genomic copies of the parental tRNAs. Moreover, tRF-5 is the most abundant variant in all three species. In silico and in vivo expression analyses unraveled differential accumulation of tRFs in Arabidopsis tissues/organs, suggesting that they are not byproducts of tRNA degradation. We also verified that the biogenesis of most Arabidopsis 19-25 nt tRF-5s and tRF-3s is not primarily dependent on DICER-LIKE proteins, though they seem to be associated with ARGONAUTE proteins and have few potential targets. Finally, we provide evidence that Arabidopsis ribonuclease RNS1 might be involved in the processing and/or degradation of tRFs. Our data support the notion that an interplay between tRFs and other sRNAs might be important to fine tune gene expression and protein biosynthesis in plant cells.

Sugarcane Serine Peptidase Inhibitors, Serine Peptidases, and Clp Protease System Subunits Associated with Sugarcane Borer (Diatraea saccharalis) Herbivory and Wounding.

Sugarcane's (Saccharum spp.) response to Diatraea saccharalis (F.) (Lepidoptera: (Crambidae) herbivory was investigated using a macroarray spotted with 248 sugarcane Expressed Sequence Tags (ESTs) encoding serine peptidase inhibitors, serine peptidases. and Clp protease system subunits. Our results showed that after nine hours of herbivory, 13 sugarcane genes were upregulated and nine were downregulated. Among the upregulated genes, nine were similar to serine peptidase inhibitors and four were similar to Bowman-Birk Inhibitors (BBIs). Phylogenetic analysis revealed that these sequences belong to a phylogenetic group of sugarcane BBIs that are potentially involved in plant defense against insect predation. The remaining four upregulated genes included serine peptidases and one homolog to the Arabidopsis AAA+ chaperone subunit ClpD, which is a member of the Clp protease system. Among the downregulated genes, five were homologous to serine peptidases and four were homologous to Arabidopsis Clp subunits (three homologous to Clp AAA+ chaperones and one to a ClpP-related ClpR subunit). Although the roles of serine peptidase inhibitors in plant defenses against herbivory have been extensively investigated, the roles of plant serine peptidases and the Clp protease system represent a new and underexplored field of study. The up- and downregulated D. saccharalis genes presented in this study may be candidate genes for the further investigation of the sugarcane response to herbivory.