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The CRISPR/Cas9 system has been used for genome editing in several organisms, including higher plants. This system induces site-specific mutations in the genome based on the nucleotide sequence of engineered guide RNAs. The complex genomes of C4 grasses makes genome editing a challenge in key grass crops like maize (Zea mays), sorghum (Sorghum bicolor), Brachiaria spp., switchgrass (Panicum virgatum), and sugarcane (Saccharum spp.). Setaria viridis is a diploid C4 grass widely used as a model for these C4 crop plants. Here, an optimized CRISPR/Cas9 binary vector that exploits the non-homologous end joining (NHEJ) system was used to knockout a green fluorescent protein (gfp) transgene in S. viridis accession A10.1. Transformation of embryogenic callus by A. tumefaciens generated ten glufosinate-ammonium resistant transgenic events. In the T0 generation, 60% of the events were biallelic mutants in the gfp transgene with no detectable accumulation of GFP protein and without insertions or deletions in predicted off-target sites. The gfp mutations generated by CRISPR/Cas9 were stable and displayed Mendelian segregation in the T1 generation. Altogether, the system described here is a highly efficient genome editing system for S. viridis, an important model plant for functional genomics studies in C4 grasses. Also, this system is a potential tool for improvement of agronomic traits in C4 crop plants with complex genomes.

Water deficit is a major constraint for crops of economic importance in almost all agricultural regions. However, plants have an active defense system to adapt to these adverse conditions, acting in the reprogramming of gene expression responsible for encoding microRNAs (miRNAs). These miRNAs promote the regulation to the target gene expression by the post-transcriptional (PTGS) and transcriptional gene silencing (TGS), modulating several pathways including defense response to water deficit. The broader knowledge of the miRNA expression profile and its regulatory networks in response to water deficit can provide evidence for the development of new biotechnological tools for genetic improvement of several important crops. In this study, we usedThe online version contains supplementary material available at 10.1007/s12298-022-01226-z.

The objective of this work was to evaluate different traits of four corn (Zea mays) genotypes with contrasting responses to drought and to determine the main traits associated to such responses. The experiment was carried out in a greenhouse. The plants were grown in pots subjected to full irrigation. Drought was imposed to plants at 54 days after sowing and kept constant for 12 consecutive days; however, a group of plants remained under full irrigation. Traits related to leaf gas exchange, photochemical apparatus, growth, and yield were assessed, and data were subjected to hierarchical agglomerative clustering and principal component analysis. DKB 390 distinguishes from the other genotypes for growth and yield traits, while 2B-707 and DKB 390 discriminate from 'BRS 1030' and 'BRS 1010' for physiological traits. Ear length, kernel number per ear, above-ground dry matter, shoot dry matter, and plant height are the most important growth and yield traits to discriminate genotype-dependent drought tolerance. Among the physiological traits, the most important are: chlorophyll content, absorptivity, leaf temperature, maximum fluorescence in the dark-adapted state, minimum fluorescence in the dark-adapted state, water-use efficiency, and intercellular CO2 concentration. Resumo: O objetivo deste trabalho foi avaliar diferentes características de quatro genótipos de milho (Zea mays) com respostas contrastantes à seca e determinar as principais características associadas a tais respostas. O experimento foi realizado em casa de vegetação. As plantas foram cultivadas em vasos submetidos à irrigação plena. A seca foi imposta às plantas aos 54 dias após a semeadura e mantida constante por 12 dias consecutivos; no entanto, um grupo de plantas permaneceu sob irrigação plena. Avaliaram-se as características relacionadas às trocas gasosas foliares, ao aparato fotoquímico, ao crescimento e à produção. Os dados foram submetidos a agrupamento hierárquico e análise de componentes principais. DKB 390 distingue-se dos demais genótipos quanto às características de crescimento e produção, enquanto 2B-707 e DKB 390 distinguem-se dos genótipos 'BRS 1030' e 'BRS 1010' quanto às características fisiológicas. O comprimento da espiga, o número de grãos por espiga, a matéria seca da parte aérea e do caule e a altura de planta são as características de crescimento e produção mais importantes para discriminar os genótipos de milho quanto à tolerância à seca. Entre as características fisiológicas, as mais importantes são: conteúdo de clorofila, absortividade, temperatura da folha, fluorescência máxima no escuro, fluorescência mínima no escuro, eficiência no uso de água e concentração intercelular de CO2.

Background: Sugarcane (Saccharum spp.) covers vast areas of land (around 25 million ha worldwide), and its processing is already linked into infrastructure for producing bioethanol in many countries. This makes it an ideal candidate for improving composition of its residues (mostly cell walls), making them more suitable for cellulosic ethanol production. In this paper, we report an approach to improving saccharification of sugarcane straw by RNAi silencing of the recently discovered BAHD01 gene responsible for feruloylation of grass cell walls. Results: We identified six BAHD genes in the sugarcane genome (SacBAHDs) and generated five lines with substantially decreased SacBAHD01 expression. To find optimal conditions for determining saccharification of sugarcane straw, we tried multiple combinations of solvent and temperature pretreatment conditions, devising a predictive model for finding their effects on glucose release. Under optimal conditions, demonstrated by Organosolv pretreatment using 30% ethanol for 240 min, transgenic lines showed increases in saccharification efficiency of up to 24%. The three lines with improved saccharification efficiency had lower cell-wall ferulate content but unchanged monosaccharide and lignin compositions. Conclusions: The silencing of SacBAHD01 gene and subsequent decrease of cell-wall ferulate contents indicate a promising novel biotechnological approach for improving the suitability of sugarcane residues for cellulosic ethanol production. In addition, the Organosolv pretreatment of the genetically modified biomass and the optimal conditions for the enzymatic hydrolysis presented here might be incorporated in the sugarcane industry for bioethanol production. Keywords: Sugarcane, Made available in DSpace on 2019-06-22T00:35:25Z (GMT). No. of bitstreams: 1 SilencingofaBAHDacyltransferase.pdf: 1991226 bytes, checksum: 2c98f01c74be1c72d0b268e90455a2cb (MD5) Previous issue date: 2019

The feasibility of thermography as a technique for plant screening aiming at drought-tolerance has been proven by its relationship with gas exchange, biomass, and yield. In this study, unlike most of the previous, thermography was applied for phenotyping contrasting maize genotypes whose classification for drought tolerance had already been established in the field. Our objective was to determine whether thermography-based classification would discriminate the maize genotypes in a similar way as the field selection in which just grain yield was taken into account as a criterion. We evaluated gas exchange, daily water consumption, leaf relative water content, aboveground biomass, and grain yield. Indeed, the screening of maize genotypes based on canopy temperature showed similar results to traditional methods. Nevertheless, canopy temperature only partially reflected gas exchange rates and daily water consumption in plants under drought. Part of the explanation may lie in the changes that drought had caused in plant leaves and canopy structure, altering absorption and dissipation of energy, photosynthesis, transpiration, and partitioning rates. Accordingly, although there was a negative relationship between grain yield and plant canopy temperature, it does not necessarily mean that plants whose canopies were maintained cooler under drought achieved the highest yield. Made available in DSpace on 2019-06-22T00:34:48Z (GMT). No. of bitstreams: 1 SEG05.12.12.001.00.05HUGO3ijms2002273v2.pdf: 6315240 bytes, checksum: ff562dd27405293d1747a55605057985 (MD5) Previous issue date: 2019

Summary MicroRNAs (miRNAs) modulate the abundance and spatial–temporal accumulation of target mRNAs and indirectly regulate several plant processes. Transcriptional regulation of the genes encoding miRNAs (MIR genes) can be activated by numerous transcription factors, which themselves are regulated by other miRNAs. Fine‐tuning of MIR genes or miRNAs is a powerful biotechnological strategy to improve tolerance to abiotic or biotic stresses in crops of economic importance. Current approaches for miRNA fine‐tuning are based on the down‐ or up‐regulation of MIR gene transcription and the use of genetic engineering tools to manipulate the final concentration of these miRNAs in the cytoplasm. Transgenesis, cisgenesis, intragenesis, artificial MIR genes, endogenous and artificial target mimicry, MIR genes editing using Meganucleases, ZNF proteins, TALENs and CRISPR/Cas9 or CRISPR/Cpf1, CRISPR/dCas9 or dCpf1, CRISPR13a, topical delivery of miRNAs and epigenetic memory have been successfully explored to MIR gene or miRNA modulation and improve agronomic traits in several model or crop plants. However, advantages and drawbacks of each of these new biotechnological tools (NBTs) are still not well understood. In this review, we provide a brief overview of the biogenesis and role of miRNAs in response to abiotic or biotic stresses, we present critically the main NBTs used for the manipulation of MIR genes and miRNAs, we show current efforts and findings with the MIR genes and miRNAs modulation in plants, and we summarize the advantages and drawbacks of these NBTs and provide some alternatives to overcome. Finally, challenges and future perspectives to miRNA modulating in important crops are also discussed.

BackgroundA major limiting factor for plant growth is the aluminum (Al) toxicity in acidic soils, especially in tropical regions. The exclusion of Al from the root apex through root exudation of organic acids such as malate and citrate are both the most ubiquitous tolerance mechanisms in the plant kingdom. Two families of anion channels that confer Al tolerance are well described in the literature, ALMT and MATE family. ResultsIn this study, sugarcane plants constitutively overexpressing the Sorghum bicolor MATE gene (SbMATE) showed improved tolerance to Al when compared to non-transgenic (NT) plants, characterized by sustained root growth and exclusion of aluminum from the root apex based on the result obtained with hematoxylin staining. In addition, genome-wide analysis of the recently released sugarcane genome identified 11 ALMT genes and molecular studies showed potential new targets for aluminum tolerance. ConclusionsOur results indicate that the transgenic plants overexpressing the Sorghum bicolor MATE has an improved tolerance to Al. The expression profile of ALMT genes revels potential candidate genes to be used has an alternative for agricultural expansion in Brazil and other areas with aluminum toxicity in poor and acid soils.

Grass cell walls have hydroxycinnamic acids attached to arabinosyl residues of arabinoxylan (AX), and certain BAHD acyltransferases are involved in their addition. In this study, we characterized one of these BAHD genes in the cell wall of the model grass Setaria viridis. RNAi silenced lines of S. viridis (SvBAHD05) presented a decrease of up to 42% of ester-linked p-coumarate (pCA) and 50% of pCA-arabinofuranosyl, across three generations. Biomass from SvBAHD05 silenced plants exhibited up to 32% increase in biomass saccharification after acid pre-treatment, with no change in total lignin. Molecular dynamics simulations suggested that SvBAHD05 is a p-coumaroyl coenzyme A transferase (PAT) mainly involved in the addition of pCA to the arabinofuranosyl residues of AX in Setaria. Thus, our results provide evidence of p-coumaroylation of AX promoted by SvBAHD05 acyltransferase in the cell wall of the model grass S. viridis. Furthermore, SvBAHD05 is a promising biotechnological target to engineer crops for improved biomass digestibility for biofuels, biorefineries and animal feeding.

The objective of this work was to evaluate physiological processes in contrasting physic nut (Jatropha curcas) genotypes during the flowering and fruiting stages. Gas exchange parameters were measured using an infrared gas analyzer, and morphological traits were evaluated during each stage under natural conditions, in a randomized complete block design with five replicates. Differences in yield between J. curcas accessions were not related to photosynthetic performance, but rather to the number of inflorescences and female flowers, which are genetically determined. Moreover, the net CO2 assimilation was equivalent in both genotypes, although they produced different amount of fruit. The genotypes differed consistently in terms of carboxylation efficiency and ribulose bisphosphate regeneration. Finally, J. curcas branch growth is not impaired by the increased sink strength during fruiting, and fruit may have been actually exerting a positive effect on the net CO2 assimilation, which may have enabled plants to maintain growth while producing flowers and fruits. Resumo: O objetivo deste trabalho foi avaliar processos fisiológicos em genótipos contrastantes de pinhão-manso (Jatropha curcas), durante as fases de florescimento e frutificação. As trocas gasosas foram mensuradas com analisador de gás por infravermelho, e as características morfológicas foram avaliadas em cada fase sob condições naturais, em delineamento de blocos ao acaso, com cinco repetições. As diferenças no rendimento entre os acessos de J. curcas não estiveram relacionadas ao desempenho fotossintético, mas sim ao número de inflorescências e flores femininas, que são geneticamente determinadas. Além disso, a assimilação líquida de CO2 foi similar em ambos os genótipos, apesar de eles terem diferido na produção de frutos. Os genótipos diferiram consistentemente quanto à eficiência de carboxilação e à regeneração da ribulose bifosfato. Finalmente, o crescimento de ramo de J. curcas não é prejudicado pelo aumento no dreno durante a frutificação, e os frutos possivelmente estavam exercendo efeito positivo na assimilação líquida de CO2, o que pode ter permitido que as plantas mantivessem o crescimento ao produzir flores e frutos.

O objetivo deste estudo foi avaliar os impactos econômicos e ambientais ex ante da cultivar de cana-de-açúcar ‘Cana Flex’, bem como o retorno do investimento em pesquisa e desenvolvimento. A ‘Cana Flex’ é voltada ao melhor aproveitamento da biomassa na produção de etanol de segunda geração (E2G). Consideraram-se cenários de adoção, para estimar ganhos econômicos na produção de E2G e na comercialização de créditos de descarbonização (CBios), no âmbito da Política Nacional de Biocombustíveis (RenovaBio). Os resultados indicam que, se 10% do bagaço de cana produzido na safra 2018/2019 fosse destinado à produção de E2G, o diferencial da ‘Cana Flex’, em comparação a uma variedade não modificada, implicaria em aumento de 393.263,6 m3 em E2G, em redução de emissão de 748.983,06 t CO2eq, e em ganhos de R$ 37,4 milhões na comercialização de CBios. Diante dos ganhos com CBios, o investimento em pesquisa e desenvolvimento retornaria a taxa interna de retorno (TIR) de 47% e o valor presente líquido (VPL) de R$ 87,9 milhões, em 20 anos. A ‘Cana Flex’ tem grande potencial para contribuir para a viabilidade econômica do E2G no Brasil, elevando a produção e gerando ganhos econômicos ao setor produtivo, além de contribuir para o alcance das metas brasileiras de redução das emissões de gases de efeito estufa.

Sugarcane (Saccharum spp.) is a monocotyledonous semi-perennial C4 grass of the Poaceae family. Its capacity to accumulate high content of sucrose and biomass makes it one of the most important crops for sugar and biofuel production. Conventional methods of sugarcane breeding have shown several limitations due to its complex polyploid and aneuploid genome. However, improvement by biotechnological engineering is currently the most promising alternative to introduce economically important traits. In this work, we present an improved protocol for Agrobacterium tumefaciens-mediated transformation of commercial sugarcane hybrids using immature top stalk-derived embryogenic callus cultures. The callus cultures are transformed with preconditioned A. tumefaciens carrying a binary vector that encodes expression cassettes for a gene of interest and the bialaphos resistance gene (bar confers resistance to glufosinate-ammonium herbicide). This protocol has been used to successfully transform a commercial sugarcane cultivar, SP80-3280, highlighting: (i) reduced recalcitrance and oxidation; (ii) high yield of embryogenic callus; (iii) improved selection; and (iv) shoot regeneration and rooting of the transformed plants. Altogether, these improvements generated a transformation efficiency of 2.2%. This protocol provides a reliable tool for a routine procedure for sugarcane improvement by genetic engineering. © 2017 by John Wiley & Sons, Inc.

As técnicas tradicionais de fenotipagem de plantas usam características facilmente mensuráveis, com base em medidas relacionadas, por exemplo, ao peso e ao tamanho das plantas. Tais medidas podem ser realizadas em qualquer horário, sem protocolo, preparação prévia da amostra ou conhecimentos profundos para a obtenção e interpretação dos dados. Por isso, são as mais usadas na experimentação a campo, embora envolvam métodos destrutivos e sejam altamente demandadoras de tempo, recursos financeiros e mão-de-obra. Em razão dos desafios atuais relacionados à competitividade do mercado de cultivares, à sustentabilidade do agronegócio e às mudanças climáticas globais, os cientistas de plantas têm buscado agregar modernas técnicas de fenotipagem ao processo de desenvolvimento de novas cultivares. Tais técnicas devem permitir uma análise ampla, detalhada e precisa da manifestação das informações genéticas do indivíduo e sua interação com o meio ambiente, aumentando assim a eficiência e eficácia do processo de seleção. Consequentemente, as novas técnicas de fenotipagem têm avançado em escala, precisão, reprodutibilidade e complexidade em relação às técnicas tradicionais, principalmente pelo uso de imagens geradas em plataformas automatizadas. Tais plataformas são constituídas de estações para a geração de imagens que utilizam diferentes técnicas espectroscópicas, para cobrir diferentes regiões do espectro eletromagnético. As estações mais comuns capturam imagens digitais RGB, de fluorescência da clorofila, UV/VIS, termográficas e na região do NIR, as quais possibilitam a avaliação do crescimento das plantas, do aparato fotoquímico, dos teores de pigmentos, da temperatura da copa e de diferentes metabólitos encontrado nas plantas. Traditional techniques of plant phenotyping use easily measurable traits, based on measures related for instance to plant weight and size. Such measurements can be made at any time, without protocol, prior sample preparation or deep knowledge for obtaining and interpreting the data. However, they require field experimentation, financial resources and manpower, use destructive methods and are time-consuming. Due to the current challenges related to the competitiveness of the cultivars market, the sustainability of agribusiness and global climate change, plant scientists have sought more specific and dynamics features, which are used to assist in explaining the responses of plants to the environment. To evaluate these characteristics, plant phenotyping has advanced in scale, precision, reproducibility and complexity compared to traditional techniques, especially by using automated platforms images. Such platforms consist of automated imaging stations that use different techniques to cover different regions of the electromagnetic spectrum. The most common imaging stations capture digital RGB, chlorophyll fluorescence, UV/VIS, thermographic and NIR images, which enable the evaluation of plant growth, photochemical apparatus, pigments content, canopy temperature and several metabolites found in plants. Keywords: plant evaluation, spectroscopy, phenotyping platforms, sensors

Expansins refer to a family of closely related non-enzymatic proteins found in the plant cell wall that are involved in the cell wall loosening. In addition, expansins appear to be involved in different physiological and environmental responses in plants such as leaf and stem initiation and growth, stomata opening and closing, reproduction, ripening and stress tolerance. Sugarcane (Saccharum spp.) is one of the main crops grown worldwide. Lignocellulosic biomass from sugarcane is one of the most promising raw materials for the ethanol industry. However, the efficient use of lignocellulosic biomass requires the optimization of several steps, including the access of some enzymes to the hemicellulosic matrix. The addition of expansins in an enzymatic cocktail or their genetic manipulation could drastically improve the saccharification process of feedstock biomass by weakening the hydrogen bonds between polysaccharides present in plant cell walls. In this study, the expansin gene family in sugarcane was identified and characterized by in silico analysis. Ninety two putative expansins in sugarcane (SacEXPs) were categorized in three subfamilies after phylogenetic analysis. The expression profile of some expansin genes in leaves of sugarcane in different developmental stages was also investigated. This study intended to provide suitable expansin targets for genetic manipulation of sugarcane aiming at biomass and yield improvement.

© 2018 Santiago et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Expansins refer to a family of closely related non-enzymatic proteins found in the plant cell wall that are involved in the cell wall loosening. In addition, expansins appear to be involved in different physiological and environmental responses in plants such as leaf and stem initiation and growth, stomata opening and closing, reproduction, ripening and stress tolerance. Sugarcane (Saccharum spp.) is one of the main crops grown worldwide. Lignocellulosic biomass from sugarcane is one of the most promising raw materials for the ethanol industry. However, the efficient use of lignocellulosic biomass requires the optimization of several steps, including the access of some enzymes to the hemicellulosic matrix. The addition of expansins in an enzymatic cocktail or their genetic manipulation could drastically improve the saccharification process of feedstock biomass by weakening the hydrogen bonds between polysaccharides present in plant cell walls. In this study, the expansin gene family in sugarcane was identified and characterized by in silico analysis. Ninety two putative expansins in sugarcane (SacEXPs) were categorized in three subfamilies after phylogenetic analysis. The expression profile of some expansin genes in leaves of sugarcane in different developmental stages was also investigated. This study intended to provide suitable expansin targets for genetic manipulation of sugarcane aiming at biomass and yield improvement.

Feruloylation of arabinoxylan (AX) in grass cell walls is a key determinant of recalcitrance to enzyme attack, making it a target for improvement of grass crops, and of interest in grass evolution. Definitive evidence on the genes responsible is lacking so we studied a candidate gene that we identified within the BAHD acyl-CoA transferase family. We used RNA interference (RNAi) silencing of orthologs in the model grasses Setaria viridis (SvBAHD01) and Brachypodium distachyon (BdBAHD01) and determined effects on AX feruloylation. Silencing of SvBAHD01 in Setaria resulted in a c. 60% decrease in AX feruloylation in stems consistently across four generations. Silencing of BdBAHD01 in Brachypodium stems decreased feruloylation much less, possibly due to higher expression of functionally redundant genes. Setaria SvBAHD01 RNAi plants showed: no decrease in total lignin, approximately doubled arabinose acylated by p-coumarate, changes in two-dimensional NMR spectra of unfractionated cell walls consistent with biochemical estimates, no effect on total biomass production and an increase in biomass saccharification efficiency of 40-60%. We provide the first strong evidence for a key role of the BAHD01 gene in AX feruloylation and demonstrate that it is a promising target for improvement of grass crops for biofuel, biorefining and animal nutrition applications.

Drought is one of the most challenging agricultural issues limiting sustainable sugarcane production and, in some cases, yield losses caused by drought are nearly 50%. DREB proteins play vital regulatory roles in abiotic stress responses in plants. The transcription factor DREB2A interacts with a cis-acting DRE sequence to activate the expression of downstream genes that are involved in drought-, salt- and heat-stress response in Arabidopsis thaliana. In the present study, we evaluated the effects of stress-inducible over-expression of AtDREB2A CA on gene expression, leaf water potential (ΨL), relative water content (RWC), sucrose content and gas exchanges of sugarcane plants submitted to a four-days water deficit treatment in a rhizotron-grown root system. The plants were also phenotyped by scanning the roots and measuring morphological parameters of the shoot. The stress-inducible expression of AtDREB2A CA in transgenic sugarcane led to the up-regulation of genes involved in plant response to drought stress. The transgenic plants maintained higher RWC and ΨL over 4 days after withholding water and had higher photosynthetic rates until the 3rd day of water-deficit. Induced expression of AtDREB2A CA in sugarcane increased sucrose levels and improved bud sprouting of the transgenic plants. Our results indicate that induced expression of AtDREB2A CA in sugarcane enhanced its drought tolerance without biomass penalty.

After the publication of the article [1], it was brought to our attention that the GenBank Accession number for the EF1 is missing in Methods section. Please find the accession number in the erratum below.

Background Photosynthesis can be roughly separated into biochemical and photochemical processes. Both are affected by drought and can be assessed by non-invasive standard methods. Gas exchange, which mainly assesses the first process, has well-defined protocols. It is considered a standard method for evaluation of plant responses to drought. Under such stress, assessment of photochemical apparatus by chlorophyll fluorescence needs improvement to become faster and reproducible, especially in growing plants under field conditions. For this, we developed a protocol based on chlorophyll fluorescence imaging, using a rapid light curve approach. Results Almost all parameters obtained by rapid light curves have shown statistical differences between control and drought stressed maize plants. However, most of them were affected by induction processes, relaxation rate, and/or differences in chlorophyll content; while they all were influenced by actinic light intensity on each light step of light curve. Only the normalized parameters related to photochemical and non-photochemical quenching were strongly correlated with data obtained by gas exchange, but only from the light step in which the linear electron flow reached saturation. Conclusions The procedure developed in this study for discrimination of plant responses to water deficit stress proved to be as fast, efficient and reliable as the standard technique of gas exchange in order to discriminate the responses of maize genotypes to drought. However, unlike that, there is no need to perform daily and time consuming calibration routines. Moreover, plant acclimation to the dark is not required. The protocol can be applied to plants growing in both controlled conditions and full sunlight in the field. In addition, it generates parameters in a fast and accurate measurement process, which enables evaluating several plants in a short period of time. Electronic supplementary material The online version of this article (doi:10.1186/s13007-017-0209-z) contains supplementary material, which is available to authorized users.

Background Second-generation ethanol (2G-bioethanol) uses lignocellulosic feedstocks for ethanol production. Sugarcane is one among the most suitable crops for biofuel production. Its juice is extracted for sugar production, while sugarcane bagasse, straw, and senescing leaves are considered industrial waste. Senescence is the age-dependent deterioration of plant cells, ultimately leading to cell death and completion of the plant life cycle. Because senescing leaves may also be used for biofuel production, understanding the process of natural senescence, including remobilization of nutrients and its effect on cell walls can provide useful information for 2G-bioethanol production from sugarcane leaves. Results The natural senescence process in leaves of the commercial sugarcane cultivar RB867515 was investigated. Senescence was characterized by strong reduction in photosynthetic pigments content, remobilization of the nutrients N, P, K, B, Cu, Fe, and Zn, and accumulation of Ca, S, Mg, B, Mn, and Al. No significant changes in the cell-wall composition occurred, and only small changes in the expression of cell wall-related genes were observed, suggesting that cell walls are preserved during senescence. Senescence-marker genes, such as SAG12-like and XET-like genes, were also identified in sugarcane and found to be highly expressed. Conclusions Our study on nutrient remobilization under senescence in a vigorous sugarcane cultivar can contribute to the understanding on how nutrient balance in a high-yielding crop is achieved. In general, neutral monosaccharide profile did not change significantly with leaf senescence, suggesting that senescing leaves of sugarcane can be as a feedstock for biofuel production using pretreatments established for non-senescing leaves without additional efforts. Based on our findings, the potential biotechnological applications for the improvement of sugarcane cultivars are discussed. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0568-0) contains supplementary material, which is available to authorized users.

Real-time PCR (RT-qPCR) expression analysis is a powerful analytical technique, but reliable results depend on the use of stable reference genes for proper normalization. This study proposed to test the expression stability of 13 candidate reference genes in Setaria viridis, a monocot species recently proposed as a new C4 model plant. Gene expression stability of these genes was assayed across different tissues and developmental stages of Setaria and under drought or aluminum stress. In general, our results showed Protein Kinase, RNA Binding Protein and SDH as the most stable genes. Moreover, pairwise analysis showed that two reference genes were sufficient to normalize the gene expression data under each condition. By contrast, GAPDH and ACT were the least stably expressed genes tested. Validation of suitable reference genes was carried out to profile the expression of P5CS and GolS during abiotic stress. In addition, normalization of gene expression of SuSy, involved in sugar metabolism, was assayed in the developmental dataset. This study provides a list of reliable reference genes for transcript normalization in S. viridis in different tissues and stages of development and under abiotic stresses, which will facilitate genetic studies in this monocot model plant.

The last few years have seen a significant increase in the number of large-scale sequencing projects generating whole genome databases. These sequence databases can be surveyed (genome sequence survey) for tandem repeats as an alternative means to develop microsatellites for monitoring and selecting natural populations and cultivars of Jatropha curcas. A total of 100 tandem repeats were revealed from mining 368 genomic surveyed sequences available in the Kazusa DNA Research Institute database. Twenty microsatellite sequences were successfully amplified, resulting in repeatable and scorable polymerase chain reaction products. Genotyping of J. curcas accessions from the Guatemalan population revealed 18 polymorphic loci. The average number of alleles per locus was 6.9, and allelic sizes ranged from 94 to 299 bp. Expected and observed heterozygosities ranged from 0.118 to 0.906 and from 0.082 to 0.794, respectively. Polymorphic information content values ranged from 0.114 (JcSSR-34) to 0.886 (JcSSR-33) with an average of 0.627. Analysis with Micro-Checker indicated few null alleles for locus JcSSR-37 in Guatemalan populations, which may be a possible cause of its deviation from Hardy-Weinberg equilibrium, even after Bonferroni's correction. No loci showed significant linkage disequilibrium. These microsatellite loci are expected to be valuable molecular markers in J. curcas because they show high levels of polymorphism and heterozygosity.

Jatropha curcas L. is a perennial oilseed crop belonging to the Euphorbiaceae family, whose oil content in seeds varies from 33 to 38%, giving a yield potential of over 1200 kg of oil per hectare. However, it is a non-domesticated species and research is required for commercial exploration of this species for biodiesel production. The strategies of Embrapa’s jatropha breeding program aim at developing cultivars with high yield and oil content, non-toxic (absence of phorbol esters), resistant to biotic and abiotic stresses and adapted to the main producing regions of Brazil. The program activities started with the enrichment and characterization of the germplasm bank, currently with over 200 accessions from different regions of Brazil. Depending on the specific objectives of the program, different selection and breeding methods are employed. In order to understand the genetic control of specific traits and to generate segregating populations, experimental designs such as diallel crosses, which allow the estimation of heterosis, general combining ability and specific combining ability among genotypes, have been adopted. In addition, molecular markers such as SSR and SNPs are being developed and may help in early selection for characters such as the absence of toxicity in the grains. The program also includes the study on genotype × environment interaction with the evaluation of the progenies/improved clones in different regions of Brazil, which is essential for recommending cultivars for specific or broad climatic conditions. In conclusion, considering that J. curcas is a perennial species and still not domesticated, approximately 5-7 years will be required to obtain improved cultivars and evidence-based information on crop production systems to support commercial cultivation.

Stable transformation of Coffea canephora P. was obtained by particle bombardment of embryogenic tissue. Leaf explants were cultured on medium supplemented with 5 µM isopentenyl-adenosine to induce direct embryogenesis. Explants with somatic embryos were transferred to half strength MS medium with 9 µM 2,4 dichlorophenoxyacetic acid. After 2 weeks, the explants with somatic embryos and embryogenic tissue were bombarded with tungsten particles (M-25) carrying the plasmid pCambia3301 (containing the bar and uidA genes) using a high pressure helium microprojectile device. The bombarded explants were submitted to selection on medium containing 5 µM ammonium glufosinate herbicide as selective agent. After 6 months, putative transgenic embryos were transferred to a growth regulator-free medium for germination. The regenerated plantlets were β-glucuronidase (GUS) positive whereas no GUS activity was observed in non-transgenic controls. Incorporation of the bar gene into the genome was confirmed by PCR and Southern blot analysis of the regenerated transformed plants. Greenhouse grown transgenic coffee plants were found to withstand the recommended level of the herbicide Finale™ for weed control.

The phytohormone ethylene is involved in several physiological and developmental processes in higher plants, including ripening of fruits, abscission of organs and tissues, senescence, wound response as well as in other abiotic stresses. The enzyme 1-aminocyclopropane-1-carboxylate oxidase (ACO) catalyzes the last step of ethylene biosynthesis. The production of ethylene and the expression of a Coffea arabica ACO gene during the last stages of fruit maturation were investigated. A rapid increase of ethylene production at the green-yellow stage of fruit ripening, after the end of endosperm formation, and a decrease of ethylene production at the cherry stage indicates a climacteric phase during ripening. An ACC oxidase (Ca-ACO) from coffee fruit cDNA was cloned and characterized using primers previously reported. The cDNA is homologous to previously described ACC oxidase cDNA in Coffea. The nucleotide and amino acid deduced sequences of the clone showed high homology with ACO from climacteric fruits. Northern blots were performed to determine the Ca-ACO transcription pattern from different tissues and from fruits at different ripening stages. Coffee fruits at an early ripening stage (green) showed the lowest level of Ca-ACO transcript accumulation. The transcript levels of Ca-ACO did not change significantly during the later stages, suggesting the presence of post- transcriptional control mechanisms. These results, taken together, strongly suggest a climacteric nature of coffee fruit ripening.

In the present study, we have analyzed transformed plants of the citrus rootstock Carrizo citrange constitutively expressing a Δ1-pyrroline-5-carboxylate synthetase mutant gene (p5cs), which encodes the rate-limiting enzyme of the proline biosynthetic pathway. Transgenic plants grew normally in vivo and accumulated high levels of proline in the leaves. In well-watered conditions, the photosynthetic rate of Carrizo citrange transgenic plants was not significantly different from the non-transformed control. The transgenic plants presented superior osmotic adjustment and significantly higher photosynthetic rate than the control plants after 15 days without watering. Under water deficit, the leaf water potential of non-transformed plants was close to zero after 17 days, and the photosynthetic rate was almost null 2 days later. On the other hand, in transgenic plants similar measurements were only observed after 24 days of water stress. The use of citrus rootstocks with increased proline accumulation appears to be a promising approach to maintain the productivity of plants under drought stress condition.

Transgenic plants of the important citrus rootstock Swingle citrumelo (Citrus paradisi Macf .×Poncirus trifoliata L. Raf.) were obtained using Agrobacterium tumefaciens-mediated transformation of seedling epicotyl tissue. Thin sections were co-cultured with Agrobacterium strain EHA105 carrying pBI121, a plasmid that contains the nptII (pnos) gene and the uidA (p35S) gene for scorable marker β-glucuronidase (GUS). The use of thin sections and the application of increasing selection pressure (25 and 50 mg l−1 kanamycin) reduced the occurrence of escapes. With the procedure described here, a transformation efficiency of 8.6% was achieved, with less than 10% of escapes. Stable integration of the transgene sequence was confirmed by histochemical assay of β-glucuronidase, PCR and Southern hybridization.

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed “mesosynteny” is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors., PLoS Genetics, 7 (6), ISSN:1553-7390, ISSN:1553-7404

Studies were carried out to optimize the conditions for transient gene expression through particle bombardment on Carrizo citrange (Citrus sinensis x Poncirus trifoliata) thin epicotyl sections. The best conditions for transient GUS expression were: M-25 tungsten particles, 1550 psi helium pressure, 9 cm distance between specimen and DNA/particle holder and culture of explants in a high osmolarity medium (0.2 M mannitol + 0.2 M sorbitol) 4 h prior and 20 h after bombardment. Under these conditions, an average of 102 blue spots per bombardment (20 explants/plate) were achieved. This protocol is currently being used for transformation of Carrizo citrange and sweet orange (Citrus sinensis).Nosso objetivo é desenvolver um protocolo para transformação genética de Citrus através do bombardeamento de partículas usando cortes finos de epicótilo como explantes alvo. Neste trabalho foram otimizadas condições para a expressão transiente usando citrange carrizo (Citrus sinensis x Poncirus trifoliata) como modelo. Epicótilos de plântulas de citrange carrizo germinadas in vitro foram cortadas transversalmente em seções de 1 mm e cultivadas em meio MS com 5 miM no escuro. Depois de uma semana, explantes foram bombardeados com o plasmídio pE2113-GUS usando um aparelho de alta pressão de hélio (PDS 1000/He Bio-Rad). As melhores condições para a expressão transiente foram: partículas de tungstênio M-25, pressão de hélio de 1550 psi, distância de 9 cm entre os explantes e DNA/particle holder e cultivo dos explantes em meio com alta osmolaridade (0,2 M manitol + 0,2 M sorbitol) 4 h antes e 20 h depois do bombardeamento. Usando estes parâmetros, uma média de 102 pontos azuis por bombardeamento (20 explantes/placa) foi obtida. Este protocolo esta sendo usado atualmente para transformação de citrange Carrizo e adaptado para transformação de laranja doce (Citrus sinensis).

An efficient system for in vitro plant regeneration from thin transversal stem sections explants (1–2 mm) using mature tissues of sweet orange cv. Pera was developed. Explants were cultured in different media to evaluate the frequency of regeneration and size of buds. A high percentage of explants (54% with 3.1 buds/explant) producing large buds (1–4 mm) was observed when the explants were cultivated for 2 weeks on Murashige and Skoog medium and then transferred to Woody Plant medium (WPM). Both media were supplemented with 1.8 μM 6-benzylaminopurine and 0.7 μM gibberellic acid. Adventitious buds were regenerated into whole plants by in vitro shoot-tip grafting. Regenerated plants started to flower after 12 months in the greenhouse, confirming their mature nature.

A system for in vitro plant regeneration from thin transversal epicotyl sections of sweet orange (Citrus sinenis L Osbeck cvs Pera, Valencia and Folha Murcha) was developed. Thin section explants (1-2 mm) from seedlings produced adventitious shoot buds on MS medium supplemented with BA (0.5-5 μM). Optimal shoot induction was achieved on 2-5 μM BA. Explants with buds were transferred to elongation media in order to improve the recovery of normal shoots. Higher number of elongated shoots was obtained on medium with 0.5 μM BA. Elongated shoots were rooted on half strength MS medium without growth regulators and transferred to soil for acclimatization. The advantages of this system for transformation are discussed.