Plasma membrane aquaporins regulate root hydraulic conductivity in the model plant Setaria viridis.

The high rate of productivity observed in panicoid crops is in part due to their extensive root system. Recently, green foxtail (Setaria viridis) has emerged as a genetic model system for panicoid grasses. Natural accessions of S. viridis originating from different parts of the world, with differential leaf physiological behavior, have been identified. This work focused on understanding the physiological and molecular mechanisms controlling root hydraulic conductivity and root-to-shoot gas exchange signaling in S. viridis. We identified 2 accessions, SHA and ZHA, with contrasting behavior at the leaf, root, and whole-plant levels. Our results indicated a role for root aquaporin (AQP) plasma membrane (PM) intrinsic proteins in the differential behavior of SHA and ZHA. Moreover, a different root hydraulic response to low levels of abscisic acid between SHA and ZHA was observed, which was associated with root AQPs. Using cell imaging, biochemical, and reverse genetic approaches, we identified PM intrinsic protein 1;6 (PIP1;6) as a possible PIP1 candidate that regulates radial root hydraulics and root-to-shoot signaling of gas exchange in S. viridis. In heterologous systems, PIP1;6 localized in the endoplasmic reticulum, and upon interaction with PIP2s, relocalization to the PM was observed. PIP1;6 was predominantly expressed at the root endodermis. Generation of knockout PIP1;6 plants (KO-PIP1;6) in S. viridis showed altered root hydraulic conductivity, altered gas exchange, and alteration of root transcriptional patterns. Our results indicate that PIPs are essential in regulating whole-plant water homeostasis in S. viridis. We conclude that root hydraulic conductivity and gas exchange are positively associated and are regulated by AQPs.

Suppression of a BAHD acyltransferase decreases p-coumaroyl on arabinoxylan and improves biomass digestibility in the model grass Setaria viridis.

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.

MicroRNAs expression profiles in early responses to different levels of water deficit in Setaria viridis.

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 used Setaria viridis accession A10.1 as a C4 model plant to widely investigate the miRNA expression profile in early responses to different levels of water deficit. Ecophysiological studies in Setaria viridis under water deficit and after rewatering demonstrated a drought tolerant accession, capable of a rapid recovery from the stress. Deep small RNA sequencing and degradome studies were performed in plants submitted to drought to identify differentially expressed miRNA genes and their predicted targets, using in silico analysis. Our findings showed that several miRNAs were differentially modulated in response to distinctive levels of water deficit and after rewatering. The predicted mRNA targets mainly corresponded to genes related to cell wall remodeling, antioxidant system and drought-related transcription factors, indicating that these genes are rapidly regulated in early responses to drought stress. The implications of these modulations are extensively discussed, and higher-effect miRNAs are suggested as major players for potential use in genetic engineering to improve drought tolerance in economically important crops, such as sugarcane, maize, and sorghum. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01226-z.

Enhanced aluminum tolerance in sugarcane: evaluation of SbMATE overexpression and genome-wide identification of ALMTs in Saccharum spp.

BACKGROUND: A 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 is one of 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. RESULTS: In 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. CONCLUSIONS: Our 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.

Salt tolerance of two perennial grass Brachypodium sylvaticum accessions.

KEY MESSAGE: We studied the salt stress tolerance of two accessions isolated from different areas of the world (Norway and Tunisia) and characterized the mechanism(s) regulating salt stress in Brachypodium sylvaticum Osl1 and Ain1. Perennial grasses are widely grown in different parts of the world as an important feedstock for renewable energy. Their perennial nature that reduces management practices and use of energy and agrochemicals give these biomass crops advantages when dealing with modern agriculture challenges such as soil erosion, increase in salinized marginal lands and the runoff of nutrients. Brachypodium sylvaticum is a perennial grass that was recently suggested as a suitable model for the study of biomass plant production and renewable energy. However, its plasticity to abiotic stress is not yet clear. We studied the salt stress tolerance of two accessions isolated from different areas of the world and characterized the mechanism(s) regulating salt stress in B. sylvaticum Osl1, originated from Oslo, Norway and Ain1, originated from Ain-Durham, Tunisia. Osl1 limited sodium transport from root to shoot, maintaining a better K/Na homeostasis and preventing toxicity damage in the shoot. This was accompanied by higher expression of HKT8 and SOS1 transporters in Osl1 as compared to Ain1. In addition, Osl1 salt tolerance was accompanied by higher abundance of the vacuolar proton pump pyrophosphatase and Na+/H+ antiporters (NHXs) leading to a better vacuolar pH homeostasis, efficient compartmentation of Na+ in the root vacuoles and salt tolerance. Although preliminary, our results further support previous results highlighting the role of Na+ transport systems in plant salt tolerance. The identification of salt tolerant and sensitive B. sylvaticum accessions can provide an experimental system for the study of the mechanisms and regulatory networks associated with stress tolerance in perennials grass.

Suppression of a single BAHD gene in Setaria viridis causes large, stable decreases in cell wall feruloylation and increases biomass digestibility.

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.

Selective separation of Cu, Ni and Ag from printed circuit board waste using an environmentally safe technique.

Printed circuit boards (PCBs) make up a large part of e-waste and include high concentrations of high-value metals. Therefore, the recovery of these metals is interesting from both the environmental and economic points of view. Here, the extraction/separation of copper, nickel and silver from PCB leachate was studied using an aqueous two-phase system (ATPS) formed by triblock copolymers with an electrolyte and water, which is in compliance with the principles of green chemistry. The best conditions for the selective extraction consisted of 1-(2-pyridylazo)-2-naphthol (3.5 mmol kg-1) at pH = 6.0 in 6 sequential steps for the Cu(II), dimethylglyoxime (5.00 mmol kg-1) at pH = 9.0 for the Ni(II) and thiocyanate (5.20 mmol kg-1) at pH = 9.0 for the Ag(I). These conditions were applied sequentially for extraction of Cu, Ni and Ag from the PCB leachate, obtaining high separation factor (S) values between the analyte and the metallic concomitants (SCu,Ni = 1,460, SCu,Fe = 15,500, SCu,Ag = 15,900, SNi,Fe = 32,700, SNi,Ag = 34,700 and SAg,Fe = 4800). The maximum extraction percentages (%E) for Cu, Ni and Ag were 99.9%, 99.9% and 99.8%, respectively. After the extraction, a single step stripping process was performed, resulting in more than 82% of the ion available in a clean lower phase. For the first time, an ATPS has been used for sequential extraction of several metal analytes from a real sample.

The influence of Aspergillus niger transcription factors AraR and XlnR in the gene expression during growth in D-xylose, L-arabinose and steam-exploded sugarcane bagasse.

The interest in the conversion of plant biomass to renewable fuels such as bioethanol has led to an increased investigation into the processes regulating biomass saccharification. The filamentous fungus Aspergillus niger is an important microorganism capable of producing a wide variety of plant biomass degrading enzymes. In A. niger the transcriptional activator XlnR and its close homolog, AraR, controls the main (hemi-)cellulolytic system responsible for plant polysaccharide degradation. Sugarcane is used worldwide as a feedstock for sugar and ethanol production, while the lignocellulosic residual bagasse can be used in different industrial applications, including ethanol production. The use of pentose sugars from hemicelluloses represents an opportunity to further increase production efficiencies. In the present study, we describe a global gene expression analysis of A. niger XlnR- and AraR-deficient mutant strains, grown on a D-xylose/L-arabinose monosaccharide mixture and steam-exploded sugarcane bagasse. Different gene sets of CAZy enzymes and sugar transporters were shown to be individually or dually regulated by XlnR and AraR, with XlnR appearing to be the major regulator on complex polysaccharides. Our study contributes to understanding of the complex regulatory mechanisms responsible for plant polysaccharide-degrading gene expression, and opens new possibilities for the engineering of fungi able to produce more efficient enzymatic cocktails to be used in biofuel production.

A new species and new records of microcaddisflies (Trichoptera: Hydroptilidae) from northeastern Brazil.

The Hydroptilidae constitute the most diverse and widespread family of Trichoptera in the world. About 90 species of microcaddisflies have been recorded from Brazil, but the northeastern region is one of the least-sampled. In this work we describe a new species of Metrichia from Pernambuco State. Furthermore, we present the first records of Flintiella andreae Angrisano 1995 and Oxyethira bettyae Thomson & Holzenthal 2012 from Brazil, and four other new records from the northeastern region.

Corrigendum: Modification of plant cell walls with hydroxycinnamic acids by BAHD acyltransferases.

[This corrects the article DOI: 10.3389/fpls.2022.1088879.].

Modification of plant cell walls with hydroxycinnamic acids by BAHD acyltransferases.

In the last decade it has become clear that enzymes in the "BAHD" family of acyl-CoA transferases play important roles in the addition of phenolic acids to form ester-linked moieties on cell wall polymers. We focus here on the addition of two such phenolics-the hydroxycinnamates, ferulate and p-coumarate-to two cell wall polymers, glucuronoarabinoxylan and to lignin. The resulting ester-linked feruloyl and p-coumaroyl moities are key features of the cell walls of grasses and other commelinid monocots. The capacity of ferulate to participate in radical oxidative coupling means that its addition to glucuronoarabinoxylan or to lignin has profound implications for the properties of the cell wall - allowing respectively oxidative crosslinking to glucuronoarabinoxylan chains or introducing ester bonds into lignin polymers. A subclade of ~10 BAHD genes in grasses is now known to (1) contain genes strongly implicated in addition of p-coumarate or ferulate to glucuronoarabinoxylan (2) encode enzymes that add p-coumarate or ferulate to lignin precursors. Here, we review the evidence for functions of these genes and the biotechnological applications of manipulating them, discuss our understanding of mechanisms involved, and highlight outstanding questions for future research.

Characterization of Rubus fruticosus mitochondria and salicylic acid inhibition of reactive oxygen species generation at Complex III/Q cycle: potential implications for hypersensitive response in plants.

In addition to adenosine triphosphate (ATP) production, mitochondria have been implicated in the regulation of several physiological responses in plants, such as programmed cell death (PCD) activation. Salicylic acid (SA) and reactive oxygen species (ROS) are essential signaling molecules involved in such physiological responses; however, the mechanisms by which they act remain unknown. In non-photosynthesizing tissues, mitochondria appear to serve as the main source of ROS generation. Evidence suggests that SA and ROS could regulate plant PCD through a synergistic mechanism that involves mitochondria. Herein, we isolate and characterize the mitochondria from non-photosynthesizing cell suspension cultures of Rubus fruticosus. Furthermore, we assess the primary site of ROS generation and the effects of SA on isolated organelles. Mitochondrial Complex III was found to be the major source of ROS generation in this model. In addition, we discovered that SA inhibits the electron transport chain by inactivating the semiquinone radical during the Q cycle. Computational analyses confirmed the experimental data, and a mechanism for this action is proposed.

Efficient genome editing and gene knockout in Setaria viridis with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway.

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.

Updated checklist of caddisflies (Insecta: Trichoptera) from the state of Piauí, Northeast Brazil, including a new species and new geographical records.

Some regions, such as the Northeast Region of Brazil, are still severely understudied and the trichopteran fauna of this region is not entirely known, mainly in areas from Cerrado and Caatinga biomes. Currently, 25 species have been reported from Piauí State, but most of these records are concentrated from one locality. This study aimed to update the knowledge of Trichoptera diversity in this state, including the description of a new species and new geographic records for the Northeast region and Brazil. The samples were collected in seven municipalities, using various traps. Specimens were also received from the municipality of Bom Jesus. Thirty-two species were added for Trichoptera fauna of Piauí, 14 of these represent new records for Northeast Brazil; furthermore, Neotrichia palma Flint 1982 is a new record for Brazil. Additionally, a new species, Cernotina longa sp. nov., is describe and illustrate, being the third nominal record of the genus for the Caatinga biome. Thus, with the contribution of this study, 57 species, 20 genera and seven families of caddisflies are known for Piauí State.

Identification and characterization of core abscisic acid (ABA) signaling components and their gene expression profile in response to abiotic stresses in Setaria viridis.

Abscisic acid (ABA) is an essential phytohormone that regulates growth, development and adaptation of plants to environmental stresses. In Arabidopsis and other higher plants, ABA signal transduction involves three core components namely PYR/PYL/RCAR ABA receptors (PYLs), type 2C protein phosphatases (PP2Cs) and class III SNF-1-related protein kinase 2 (SnRK2s). In the present study, we reported the identification and characterization of the core ABA signaling components in Setaria viridis, an emerging model plant for cereals and feedstock crops presenting C4 metabolism, leading to the identification of eight PYL (SvPYL1 to 8), twelve PP2C (SvPP2C1 to 12) and eleven SnRK2 (SvSnRK2.1 through SvSnRK2.11) genes. In order to study the expression profiles of these genes, two different S. viridis accessions (A10.1 and Ast-1) were submitted to drought, salinity and cold stresses, in addition to application of exogenous ABA. Differential gene expression profiles were observed in each treatment and plant genotype, demonstrating variations of ABA stress responses within the same species. These differential responses to stresses were also assessed by physiological measurements such as photosynthesis, stomatal conductance and transpiration rate. This study allows a detailed analysis of gene expression of the core ABA signaling components in Setaria viridis submitted to different treatments and provides suitable targets for genetic engineering of C4 plants aiming tolerance to abiotic stresses.

Silencing of a BAHD acyltransferase in sugarcane increases biomass digestibility.

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.

Correction to: Silencing of a BAHD acyltransferase in sugarcane increases biomass digestibility.

[This corrects the article DOI: 10.1186/s13068-019-1450-7.].

Genome-wide identification, characterization and expression profile analysis of expansins gene family in sugarcane (Saccharum spp.).

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.

Correction: Genome-wide identification, characterization and expression profile analysis of expansins gene family in sugarcane (Saccharum spp.).

[This corrects the article DOI: 10.1371/journal.pone.0191081.].