Disrupting the cinnamyl alcohol dehydrogenase 1 gene (BdCAD1) leads to altered lignification and improved saccharification in Brachypodium distachyon

Brachypodium distachyon (Brachypodium) has been proposed as a model for grasses, but there is limited knowledge regarding its lignins and no data on lignin‐related mutants. The cinnamyl alcohol dehydrogenase (CAD) genes involved in lignification are promising targets to improve the cellulose‐to‐ethanol conversion process. Down‐regulation of CAD often induces a reddish coloration of lignified tissues. Based on this observation, we screened a chemically induced population of Brachypodium mutants (Bd21–3 background) for red culm coloration. We identified two mutants (Bd4179 and Bd7591), with mutations in the BdCAD1 gene. The mature stems of these mutants displayed reduced CAD activity and lower lignin content. Their lignins were enriched in 8–O–4‐ and 4–O–5‐coupled sinapaldehyde units, as well as resistant inter‐unit bonds and free phenolic groups. By contrast, there was no increase in coniferaldehyde end groups. Moreover, the amount of sinapic acid ester‐linked to cell walls was measured for the first time in a lignin‐related CAD grass mutant. Functional complementation of the Bd4179 mutant with the wild‐type BdCAD1 allele restored the wild‐type phenotype and lignification. Saccharification assays revealed that Bd4179 and Bd7591 lines were more susceptible to enzymatic hydrolysis than wild‐type plants. Here, we have demonstrated that BdCAD1 is involved in lignification of Brachypodium. We have shown that a single nucleotide change in BdCAD1 reduces the lignin level and increases the degree of branching of lignins through incorporation of sinapaldehyde. These changes make saccharification of cells walls pre‐treated with alkaline easier without compromising plant growth.     

短柄草柄锈菌在感病寄主二穗短柄草上发育过程的组织学研究

本研究采用荧光染色和荧光显微镜技术,系统研究了短柄草柄锈菌Puccinia brachypodii (分离系F-Co和K-Ki)在感病二穗短柄草Brachypodium distachyon自交系Bd21-3叶片中的发育过程。荧光显微镜观察结果表明,接种12-18 h时,病原菌通过芽管侵入短柄草叶表皮气孔后形成气孔下囊,产生初生菌丝并形成吸器母细胞,进而侵入植物细胞形成吸器;接种24-48 h后,病原菌初生菌丝分支并形成次生菌丝;接种72 h后,短柄草锈菌开始形成菌落。F-Co的发育过程快于K-Ki,在120-216 h时,F-Co的菌落扩展面积明显高于K-Ki。本研究证实F-Co、K-Ki和二穗短柄草均为亲和互作。     

Roles of BdUNICULME4 and BdLAXATUM‐A in the non‐domesticated grass Brachypodium distachyon

In cultivated grasses, tillering, spike architecture and seed shattering represent major agronomical traits. In barley, maize and rice, the NOOT‐BOP‐COCH‐LIKE (NBCL) genes play important roles in development, especially in ligule development, tillering and flower identity. However, compared with dicots, the role of grass NBCL genes is underinvestigated. To better understand the role of grass NBCLs and to overcome any effects of domestication that might conceal their original functions, we studied TILLING nbcl mutants in the non‐domesticated grass Brachypodium distachyon. In B. distachyon, the NBCL genes BdUNICULME4 (CUL4) and BdLAXATUM‐A (LAXA) are orthologous, respectively, to the barley HvUniculme4 and HvLaxatum‐a, to the maize Zmtassels replace upper ears1 and Zmtassels replace upper ears2 and to the rice OsBLADE‐ON‐PETIOLE1 and OsBLADE‐ON‐PETIOLE2/3. In B. distachyon, our reverse genetics study shows that CUL4 is not essential for the establishment of the blade–sheath boundary but is necessary for the development of the ligule and auricles. We report that CUL4 also exerts a positive role in tillering and a negative role in spikelet meristem activity. On the other hand, we demonstrate that LAXA plays a negative role in tillering, positively participates in spikelet development and contributes to the control of floral organ number and identity. In this work, we functionally characterized two new NBCL genes in a context of non‐domesticated grass and highlighted original roles for grass NBCL genes that are related to important agronomical traits.     

Sequencing and functional validation of the JGI Brachypodium distachyon T‐DNA collection

Due to a large and growing collection of genomic and experimental resources, Brachypodium distachyon has emerged as a powerful experimental model for the grasses. To add to these resources we sequenced 21 165 T‐DNA lines, 15 569 of which were produced in this study. This increased the number of unique insertion sites in the T‐DNA collection by 21 078, bringing the overall total to 26 112. Thirty‐seven per cent (9754) of these insertion sites are within genes (including untranslated regions and introns) and 28% (7217) are within 500 bp of a gene. Approximately 31% of the genes in the v.2.1 annotation have been tagged in this population. To demonstrate the utility of this collection, we phenotypically characterized six T‐DNA lines with insertions in genes previously shown in other systems to be involved in cellulose biosynthesis, hemicellulose biosynthesis, secondary cell wall development, DNA damage repair, wax biosynthesis and chloroplast synthesis. In all cases, the phenotypes observed supported previous studies, demonstrating the utility of this collection for plant functional genomics. The Brachypodium T‐DNA collection can be accessed at http://jgi.doe.gov/our-science/science-programs/plant-genomics/brachypodium/brachypodium-t-dna-collection/ .     

p‐Coumaroyl‐CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon

Grass lignins contain substantial amounts of p‐coumarate (pCA) that acylate the side‐chains of the phenylpropanoid polymer backbone. An acyltransferase, named p‐coumaroyl‐CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol‐pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p‐coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide‐generated Bdpmt‐1 missense mutant had no (<0.5%) residual pCA on lignin, and BdPMT RNAi plants had levels as low as 10% of wild‐type, whereas the amounts of pCA acylating arabinosyl units on arabinoxylans in these PMT mutant plants remained unchanged. pCA acylation of lignin from BdPMT‐overexpressing plants was found to be more than three‐fold higher than that of wild‐type, but again the level on arabinosyl units remained unchanged. Taken together, these data are consistent with a defined role for grass PMT genes in encoding BAHD (BEAT, AHCT, HCBT, and DAT) acyltransferases that specifically acylate monolignols with pCA and produce monolignol p‐coumarate conjugates that are used for lignification in planta.     

In the grass species Brachypodium distachyon,the production of mixed‐linkage (1,3;1,4)‐β‐glucan (MLG) occurs in the Golgi apparatus

Mixed‐linkage (1,3;1,4)‐β‐glucan (MLG) is a glucose polymer with beneficial effects on human health and high potential for the agricultural industry. MLG is present predominantly in the cell wall of grasses and is synthesized by cellulose synthase‐like F or H families of proteins, with CSLF6 being the best‐characterized MLG synthase. Although the function of this enzyme in MLG production has been established, the site of MLG synthesis in the cell is debated. It has been proposed that MLG is synthesized at the plasma membrane, as occurs for cellulose and callose; in contrast, it has also been proposed that MLG is synthesized in the Golgi apparatus, as occurs for other matrix polysaccharides of the cell wall. Testing these conflicting possibilities is fundamentally important in the general understanding of the biosynthesis of the plant cell wall. Using immuno‐localization analyses with MLG‐specific antibody in Brachypodium and in barley, we found MLG present in the Golgi, in post‐Golgi structures and in the cell wall. Accordingly, analyses of a functional fluorescent protein fusion of CSLF6 stably expressed in Brachypodium demonstrated that the enzyme is localized in the Golgi. We also established that overproduction of MLG causes developmental and growth defects in Brachypodium as also occur in barley. Our results indicated that MLG production occurs in the Golgi similarly to other cell wall matrix polysaccharides, and supports the broadly applicable model in grasses that tight mechanisms control optimal MLG accumulation in the cell wall during development and growth. This work addresses the fundamental question of where mixed linkage (1,3;1,4)‐β‐glucan (MLG) is synthesized in plant cells. By analyzing the subcellular localization of MLG and MLG synthase in an endogenous system, we demonstrated that MLG synthesis occurs at the Golgi in Brachypodium and barley. A growth inhibition due to overproduced MLG in Brachypodium supports the general applicability of the model that a tight control of the cell wall polysaccharides accumulation is needed to maintain growth homeostasis during development.     

Reduced susceptibility to Fusarium head blight in Brachypodium distachyon through priming with the Fusarium mycotoxin deoxynivalenol

The fungal cereal pathogen Fusarium graminearum produces deoxynivalenol (DON) during infection. The mycotoxin DON is associated with Fusarium head blight (FHB), a disease that can cause vast grain losses. Whilst investigating the suitability of Brachypodium distachyon as a model for spreading resistance to F. graminearum, we unexpectedly discovered that DON pretreatment of spikelets could reduce susceptibility to FHB in this model grass. We started to analyse the cell wall changes in spikelets after infection with F. graminearum wild‐type and defined mutants: the DON‐deficient Δtri5 mutant and the DON‐producing lipase disruption mutant Δfgl1, both infecting only directly inoculated florets, and the mitogen‐activated protein (MAP) kinase disruption mutant Δgpmk1, with strongly decreased virulence but intact DON production. At 14 days post‐inoculation, the glucose amounts in the non‐cellulosic cell wall fraction were only increased in spikelets infected with the DON‐producing strains wild‐type, Δfgl1 and Δgpmk1. Hence, we tested for DON‐induced cell wall changes in B. distachyon, which were most prominent at DON concentrations ranging from 1 to 100 ppb. To test the involvement of DON in defence priming, we pretreated spikelets with DON at a concentration of 1 ppm prior to F. graminearum wild‐type infection, which significantly reduced FHB disease symptoms. The analysis of cell wall composition and plant defence‐related gene expression after DON pretreatment and fungal infection suggested that DON‐induced priming of the spikelet tissue contributed to the reduced susceptibility to FHB.     

Drought‐inducible changes in the histone modification H3K9ac are associated with drought‐responsive gene expression in Brachypodium distachyon

• H3K9ac, an epigenetic marker, is widely distributed in plant genomes. H3K9ac enhances gene expression, which is highly conserved in eukaryotes. However, genome‐wide studies of H3K9ac in monocot species are limited, and the changes in H3K9ac under drought stress for individual genes are still not clear.
• We analysed changes in the H3K9ac level of Brachypodium distachyon under 20% PEG‐6000‐simulated drought stress conditions. We also performed chromatin immunoprecipitation, followed by next generation sequencing (ChIP‐seq) on H3K9ac to reveal changes in H3K9ac for individual genes at the genome‐wide level.
• Our study showed that H3K9ac was mainly enriched in gene exon regions. Drought increased or decreased the H3K9ac level at specific genomic loci. We identified 40 genes associated with increased H3K9ac levels and 36 genes associated with decreased H3K9ac levels under drought stress. Further, RT‐qPCR analyses showed that H3K9ac was positively associated with gene expression of those drought‐responsive genes.
• We conclude that H3K9ac enhances the expression level of a large number of drought‐responsive genes under drought stress in B. distachyon. The data presented here will help to reveal the correlation of some specific drought‐responsive genes and their enriched H3K9ac levels in the model plant B. distachyon.

     

An essential role of caffeoyl shikimate esterase in monolignol biosynthesis in Medicago truncatula

Biochemical and genetic analyses have previously identified caffeoyl shikimate esterase (CSE) as an enzyme in the monolignol biosynthesis pathway in Arabidopsis thaliana, although the generality of this finding has been questioned. Here we show the presence of CSE genes and associated enzyme activity in barrel medic (Medicago truncatula, dicot, Leguminosae), poplar (Populus deltoides, dicot, Salicaceae), and switchgrass (Panicum virgatum, monocot, Poaceae). Loss of function of CSE in transposon insertion lines of M. truncatula results in severe dwarfing, altered development, reduction in lignin content, and preferential accumulation of hydroxyphenyl units in lignin, indicating that the CSE enzyme is critical for normal lignification in this species. However, the model grass Brachypodium distachyon and corn (Zea mays) do not possess orthologs of the currently characterized CSE genes, and crude protein extracts from stems of these species exhibit only a weak esterase activity with caffeoyl shikimate. Our results suggest that the reaction catalyzed by CSE may not be essential for lignification in all plant species.     

A T-DNA mutation in the RNA helicase eIF4A confers a dose-dependent dwarfing phenotype in Brachypodium distachyon

In a survey of the BrachyTAG mutant population of Brachypodium distachyon, we identified a line carrying a T-DNA insertion in one of the two eukaryotic initiation factor 4A (eIF4A) genes present in the nuclear genome. The eif4a homozygous mutant plants were slow-growing, and exhibited reduced final plant stature due to a decrease in both cell number and cell size, consistent with roles for eIF4A in both cell division and cell growth. Hemizygous plants displayed a semi-dwarfing phenotype, in which stem length was reduced but leaf length was normal. Linkage between the insertion site and phenotype was confirmed, and we show that the level of eIF4A protein is strongly reduced in the mutant. Transformation of the Brachypodium homozygous mutant with a genomic copy of the Arabidopsis eIF4A-1 gene partially complemented the growth phenotype, indicating that gene function is conserved between mono- and dicotyledonous species. This study identifies eIF4A as a novel dose-dependent regulator of stem elongation, and demonstrates the utility of Brachypodium as a model for grass and cereals research.     

Host‐induced gene silencing of an important pathogenicity factor PsCPK1 in Puccinia striiformis f. sp. tritici enhances resistance of wheat to stripe rust

Rust fungi are devastating plant pathogens and cause a large economic impact on wheat production worldwide. To overcome this rapid loss of resistance in varieties, we generated stable transgenic wheat plants expressing short interfering RNAs (siRNAs) targeting potentially vital genes of Puccinia striiformis f. sp. tritici (Pst). Protein kinase A (PKA) has been proved to play important roles in regulating the virulence of phytopathogenic fungi. PsCPK1, a PKA catalytic subunit gene from Pst, is highly induced at the early infection stage of Pst. The instantaneous silencing of PsCPK1 by barley stripe mosaic virus (BSMV)‐mediated host‐induced gene silencing (HIGS) results in a significant reduction in the length of infection hyphae and disease phenotype. These results indicate that PsCPK1 is an important pathogenicity factor by regulating Pst growth and development. Two transgenic lines expressing the RNA interference (RNAi) construct in a normally susceptible wheat cultivar displayed high levels of stable and consistent resistance to Pst throughout the T₃ to T₄ generations. The presence of the interfering RNAs in transgenic wheat plants was confirmed by northern blotting, and these RNAs were found to efficiently down‐regulate PsCPK1 expression in wheat. This study addresses important aspects for the development of fungal‐derived resistance through the expression of silencing constructs in host plants as a powerful strategy to control cereal rust diseases.     

Cell wall proteomics contributes to explore the functional proteins of Brachypodium distachyon grains

The plant cell wall is the first barrier in response to external stimuli and cell wall proteins (CWPs) can play an important role in the modulation of plant growth and development. In the past 10 years, the plant cell wall proteomics has increasingly become a very active research filed, which provides a broader understanding of CWPs for people. The cell wall proteome of Arabidopsis, rice, and other model plants has begun to take shape, and proteomic technology has become an effective way to identify the candidate functional CWPs in large scale. The challenging work of Francin‐Allami et al. (Proteomics 2015, 15, 2296–2306) is a vital step toward building the most extensive cell wall proteome of a monocot species. They identified 299 cell wall proteins in Brachypodium distachyon grains, and also compared the grain cell wall proteome with those of B. distachyon culms and leaves, which provides a new perspective for further explaining the plant cell wall structures and remodeling mechanism.     

Rice OsPAD4 functions differently from Arabidopsis AtPAD4 in host‐pathogen interactions

The extensively studied Arabidopsis phytoalexin deficient 4 (AtPAD4) gene plays an important role in Arabidopsis disease resistance; however, the function of its sequence ortholog in rice is unknown. Here, we show that rice OsPAD4 appears not to be the functional ortholog of AtPAD4 in host‐pathogen interactions, and that the OsPAD4 encodes a plasma membrane protein but that AtPAD4 encodes a cytoplasmic and nuclear protein. Suppression of OsPAD4 by RNA interference (RNAi) increased rice susceptibility to the biotrophic pathogen Xanthomonas oryzae pv. oryzae (Xoo), which causes bacteria blight disease in local tissue. OsPAD4‐RNAi plants also show compromised wound‐induced systemic resistance to Xoo. The increased susceptibility to Xoo was associated with reduced accumulation of jasmonic acid (JA) and phytoalexin momilactone A (MOA). Exogenous application of JA complemented the phenotype of OsPAD4‐RNAi plants in response to Xoo. The following results suggest that OsPAD4 functions differently than AtPAD4 in response to pathogen infection. First, OsPAD4 plays an important role in wound‐induced systemic resistance, whereas AtPAD4 mediates systemic acquired resistance. Second, OsPAD4‐involved defense signaling against Xoo is JA‐dependent, but AtPAD4‐involved defense signaling against biotrophic pathogens is salicylic acid‐dependent. Finally, OsPAD4 is required for the accumulation of terpenoid‐type phytoalexin MOA in rice‐bacterium interactions, but AtPAD4‐mediated resistance is associated with the accumulation of indole‐type phytoalexin camalexin.     

Overexpression of the PtSOS2 gene improves tolerance to salt stress in transgenic poplar plants

In higher plants, the salt overly sensitive (SOS) signalling pathway plays a crucial role in maintaining ion homoeostasis and conferring salt tolerance under salinity condition. Previously, we functionally characterized the conserved SOS pathway in the woody plant Populus trichocarpa. In this study, we demonstrate that overexpression of the constitutively active form of PtSOS2 (PtSOS2TD), one of the key components of this pathway, significantly increased salt tolerance in aspen hybrid clone Shanxin Yang (Populus davidiana × Populus bolleana). Compared to the wild‐type control, transgenic plants constitutively expressing PtSOS2TD exhibited more vigorous growth and produced greater biomass in the presence of high concentrations of NaCl. The improved salt tolerance was associated with a decreased Na⁺ accumulation in the leaves of transgenic plants. Further analyses revealed that plasma membrane Na⁺/H⁺ exchange activity and Na⁺ efflux in transgenic plants were significantly higher than those in the wild‐type plants. Moreover, transgenic plants showed improved capacity in scavenging reactive oxygen species (ROS) generated by salt stress. Taken together, our results suggest that PtSOS2 could serve as an ideal target gene to genetically engineer salt‐tolerant trees.     

Enhanced resistance to soybean cyst nematode Heterodera glycines in transgenic soybean by silencing putative CLE receptors

CLE peptides are small extracellular proteins important in regulating plant meristematic activity through the CLE‐receptor kinase‐WOX signalling module. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem) and vascular cambium are controlled by CLE signalling pathways. Interestingly, plant‐parasitic cyst nematodes secrete CLE‐like effector proteins, which act as ligand mimics of plant CLE peptides and are required for successful parasitism. Recently, we demonstrated that Arabidopsis CLE receptors CLAVATA1 (CLV1), the CLAVATA2 (CLV2)/CORYNE (CRN) heterodimer receptor complex and RECEPTOR‐LIKE PROTEIN KINASE 2 (RPK2), which transmit the CLV3 signal in the SAM, are required for perception of beet cyst nematode Heterodera schachtii CLEs. Reduction in nematode infection was observed in clv1, clv2, crn, rpk2 and combined double and triple mutants. In an effort to develop nematode resistance in an agriculturally important crop, orthologues of Arabidopsis receptors including CLV1, CLV2, CRN and RPK2 were identified from soybean, a host for the soybean cyst nematode Heterodera glycines. For each of the receptors, there are at least two paralogues in the soybean genome. Localization studies showed that most receptors are expressed in the root, but vary in their level of expression and spatial expression patterns. Expression in nematode‐induced feeding cells was also confirmed. In vitro direct binding of the soybean receptors with the HgCLE peptide was analysed. Knock‐down of the receptors in soybean hairy roots showed enhanced resistance to SCN. Our findings suggest that targeted disruption of nematode CLE signalling may be a potential means to engineer nematode resistance in crop plants.