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.

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

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

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.

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.

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.

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.

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

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

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.

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.

Improved Genetic Transformation of Sugarcane (Saccharum spp.) Embryogenic Callus Mediated by Agrobacterium tumefaciens.

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.

Characterization of sugarcane (Saccharum spp.) leaf senescence: implications for biofuel production.

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.

Selection of reliable reference genes for RT-qPCR analysis during developmental stages and abiotic stress in Setaria viridis.

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 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.

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.