An ABC transporter is involved in the silicon-induced formation of casparian bands in the exodermis of rice

Silicon (Si) promotes the formation of Casparian bands (CB) in rice and reduces radial oxygen loss (ROL). Further transcriptomic approaches revealed several candidate genes involved in the Si-induced formation of CB such as ATP binding cassette (ABC) transporter, Class III peroxidases, ligases and transferases. Investigation of these genes by means of overexpression (OE) and knockout (KO) mutants revealed the contribution of the ABC transporter (OsABCG25) to CB formation in the exodermis, which was also reflected in the expression of other OsABCG25 in the Si-promoted formation of CB genes related to the phenylpropanoid pathway, such as phenylalanine-ammonia-lyase, diacylglycerol O-acyltransferase and 4-coumarate-CoA ligase. Differential CB development in mutants and Si supply also affected the barrier function of the exodermis. OE of the ABC transporter and Si supply reduced the ROL from roots and Fe uptake. No effect on ROL and Fe uptake could be observed for the KO mutant. The presented research confirms the impact of the OsABCG25 in the Si-promoted formation of CB and its barrier functions. © 2017 Hinrichs, Fleck, Biedermann, Ngo, Schreiber and Schenk

Silicon Promotes Exodermal Casparian Band Formation in Si-Accumulating and Si-Excluding Species by Forming Phenol Complexes

We studied the effect of Silicon (Si) on Casparian band (CB) development, chemical composition of the exodermal CB and Si deposition across the root in the Si accumulators rice and maize and the Si non-accumulator onion. Plants were cultivated in nutrient solution with and without Si supply. The CB development was determined in stained root cross-sections. The outer part of the roots containing the exodermis was isolated after enzymatic treatment. The exodermal suberin was transesterified with MeOH/BF3 and the chemical composition was measured using gas chromatography-mass spectroscopy (GC-MS) and flame ionization detector (GC-FID). Laser ablation-inductively coupled plasma-mass spectroscopy (LA-ICP-MS) was used to determine the Si deposition across root cross sections. Si promoted CB formation in the roots of Si-accumulator and Si non-accumulator species. The exodermal suberin was decreased in rice and maize due to decreased amounts of aromatic suberin fractions. Si did not affect the concentration of lignin and lignin-like polymers in the outer part of rice, maize and onion roots. The highest Si depositions were found in the tissues containing CB. These data along with literature were used to suggest a mechanism how Si promotes the CB development by forming complexes with phenols.DFG/SCHR 506/12-

Molecular Background of Pi Deficiency-Induced Root Hair Growth in Brassica carinata – A Fasciclin-Like Arabinogalactan Protein Is Involved

Formation of longer root hairs under limiting phosphate (P) conditions can increase the inorganic P (Pi) uptake. Here, regulatory candidate genes for Pi deficiency-induced root hair growth were identified by comparison of massive analysis of cDNA ends (MACE) provided expression profiles of two Brassica carinata cultivars (cv.) differing in their root hair response to Pi deficiency: cv. Bale develops longer root hairs under Pi deficiency, but not cv. Bacho. A split-root experiment was conducted for the differentiation between locally and systemically regulated genes. Furthermore, plants were exposed to nitrogen and potassium deficiency to identify P-specific reacting genes. The latter were knocked out by CRISPR/Cas9 and the effect on the root hair length was determined. About 500 genes were differentially expressed under Pi deficiency in cv. Bale, while these genes did not respond to the low P supply in cv. Bacho. Thirty-three candidate genes with a potential regulatory role were selected and the transcriptional regulation of 30 genes was confirmed by quantitative PCR. Only five candidate genes seemed to be either exclusively regulated locally (two) or systemically (three), whereas 25 genes seemed to be involved in both local and systemic signaling pathways. Potassium deficiency affected neither the root hair length nor the expression of the 30 candidate genes. By contrast, both P and nitrogen deficiency increased the root hair length, and both affected the transcript levels in 26 cases. However, four genes reacted specifically to Pi starvation. These genes and, additionally, INORGANIC PHOSPHATE TRANSPORTER 1 (BcPHT1) were targeted by CRISPR/Cas9. However, even if the transcript levels of five of these genes were clearly decreased, FASCICLIN-LIKE ARABINOGALACTAN PROTEIN 1 (BcFLA1) was the only gene whose downregulation reduced the root hair length in transgenic hairy roots under Pi-deficient conditions. To the best of our knowledge, this is the first study describing a fasciclin-like arabinogalactan protein with a predicted role in the Pi deficiency-induced root hair elongation

Silicon enhances suberization and lignification in roots of rice (Oryza sativa)

The beneficial element silicon (Si) may affect radial oxygen loss (ROL) of rice roots depending on suberization of the exodermis and lignification of sclerenchyma. Thus, the effect of Si nutrition on the oxidation power of rice roots, suberization and lignification was examined. In addition, Si-induced alterations of the transcript levels of 265 genes related to suberin and lignin synthesis were studied by custom-made microarray and quantitative Real Time-PCR. Without Si supply, the oxidation zone of 12 cm long adventitious roots extended along the entire root length but with Si supply the oxidation zone was restricted to 5 cm behind the root tip. This pattern coincided with enhanced suberization of the exodermis and lignification of sclerenchyma by Si supply. Suberization of the exodermis started, with and without Si supply, at 4–5 cm and 8–9 cm distance from the root tip (drt), respectively. Si significantly increased transcript abundance of 12 genes, while two genes had a reduced transcript level. A gene coding for a leucine-rich repeat protein exhibited a 25-fold higher transcript level with Si nutrition. Physiological, histochemical, and molecular-biological data showing that Si has an active impact on rice root anatomy and gene transcription is presented here

HVEM Signalling Promotes Colitis

Background Tumor necrosis factor super family (TNFSF) members regulate important processes involved in cell proliferation, survival and differentiation and are therefore crucial for the balance between homeostasis and inflammatory responses. Several members of the TNFSF are closely associated with inflammatory bowel disease (IBD). Thus, they represent interesting new targets for therapeutic treatment of IBD. Methodology/Principal Findings We have used mice deficient in TNFSF member HVEM in experimental models of IBD to investigate its role in the disease process. Two models of IBD were employed: i) chemical-induced colitis primarily mediated by innate immune cells; and ii) colitis initiated by CD4+CD45RBhigh T cells following their transfer into immuno-deficient RAG1-/- hosts. In both models of disease the absence of HVEM resulted in a significant reduction in colitis and inflammatory cytokine production. Conclusions These data show that HVEM stimulatory signals promote experimental colitis driven by innate or adaptive immune cells

The Expression of P-Responsive Genes is Related to Root Hair Growth

Phosphorus deficiency results in the formation of longer root hairs in Brassica carinata cv. Bale to exhaust a greater soil volume. Recently, suppression subtractive hybridization revealed genes that were regulated in P-starved roots in Brassica carinata. Sequencing identified genes sharing homologies to a leucine-rich repeat protein kinase (LRR), a transcription factor of the 14-3-3 family, and a proline rich family protein (PRP) in Arabidopsis thaliana. This study aims at characterizing the expression pattern of these putative P-responsive genes by specifying the response to several nutritional stress factors and by inducing or discontinuing phosphorus deprivation. Root hair length was enhanced with phosphorus and nitrogen deprivation but not with potassium depletion. Likewise, the transcription level of LRR and PRP was increased in -P and -N conditions whereas there was no clear response for 14-3-3. One day after varying P supply, phenotypically the root hair length was changed. Correspondingly, the expression of the root-specific LRR and PRP was induced by P-deficiency and inhibited by the addition of P. The response to P starvation of 14-3-3 followed no clear trend. P-resupply increased the gene expression as well as P removal with a short term repression after 3 hours. In summary, the expression of a LRR receptor-like protein kinase and a proline rich protein suggests a function of these genes in inducing root hair growth. The putative transcription factor 14-3-3 may be of importance in rapid but unspecific stress response

The Expression of P-Responsive Genes is Related to Root Hair Growth

Phosphorus deficiency results in the formation of longer root hairs in Brassica carinata cv. Bale to exhaust a greater soil volume. Recently, suppression subtractive hybridization revealed genes that were regulated in P-starved roots in Brassica carinata. Sequencing identified genes sharing homologies to a leucine-rich repeat protein kinase (LRR), a transcription factor of the 14-3-3 family, and a proline rich family protein (PRP) in Arabidopsis thaliana. This study aims at characterizing the expression pattern of these putative P-responsive genes by specifying the response to several nutritional stress factors and by inducing or discontinuing phosphorus deprivation. Root hair length was enhanced with phosphorus and nitrogen deprivation but not with potassium depletion. Likewise, the transcription level of LRR and PRP was increased in -P and -N conditions whereas there was no clear response for 14-3-3. One day after varying P supply, phenotypically the root hair length was changed. Correspondingly, the expression of the root-specific LRR and PRP was induced by P-deficiency and inhibited by the addition of P. The response to P starvation of 14-3-3 followed no clear trend. P-resupply increased the gene expression as well as P removal with a short term repression after 3 hours. In summary, the expression of a LRR receptor-like protein kinase and a proline rich protein suggests a function of these genes in inducing root hair growth. The putative transcription factor 14-3-3 may be of importance in rapid but unspecific stress response

Mechanisms of phosphorus efficiency in potato genotypes

The objective of this study was to evaluate potato genotypes for P efficiency and to identify the mechanisms of P efficiency. Two efficient and two inefficient genotypes were selected from screening experiments and grown in soil as well in nutrient solution at different P lev-els. Genotypes CGN 17903 and CIP 384321.3 were clearly superior to genotypes CGN 22367 and CGN 18233 in terms of relative growth rate (RGR) of shoot d.m.. P-efficient genotypes had about 15% longer root hairs compared to P-inefficient genotypes but this did not enhance P uptake rate per cm root length. However, P efficiency of genotype CGN 17903 was related to higher P utilization efficiency and that of CIP 384321.3 to both higher P uptake efficiency in terms of root-shoot ratio and intermediate utilization efficiency. The higher RGR of the efficient genotypes resulted from enhanced net assimilation rate (NAR) rather than from leaf area ratio (LAR). Net photosynthetic rate was similar for all geno-types. However for the P-inefficient genotype CGN 22367, the lower NAR could be ex-plained by increased leaf dark respiration. For P-inefficient genotype CGN 18233 it is speculated that increased carbon cost of root respiration or carbon exudation or both caused low NAR, since dark respiration of this genotype was similar to that of P-efficient geno-types. Keywords: dark respiration, net assimilation rate, net photosynthetic rate, P utilization effi-ciency, P uptake rate, root hair, root-shoot rati

Efficient generation of mutations mediated by CRISPR/Cas9 in the hairy root transformation system of Brassica carinata.

A protocol for the induction of site-directed deletions and insertions in the genome of Brassica carinata with CRISPR is described. The construct containing the Cas9 nuclease and the guide RNA (gRNA) was delivered by the hairy root transformation technique, and a successful transformation was monitored by GFP fluorescence. PAGE analysis of an amplified region, presumably containing the deletions and insertions, demonstrated up to seven different indels in one transgenic root and in all analyzed roots a wildtype allele of the modified gene was not detectable. Interestingly, many of these mutations consisted of relatively large indels with up to 112 bp. The exact size of the deletions was determined to allow an estimation whether the targeted gene was not functional due to a considerable deletion or a frame shift within the open reading frame. This allowed a direct phenotypic assessment of the previously characterized roots and, in fact, deletions in FASCICLIN-LIKE ARABINOGALACTAN PROTEIN 1 (BcFLA1)-a gene with an expression pattern consistent with a role in root hair architecture-resulted in shorter root hairs compared to control roots ectopically expressing an allele of the gene that cannot be targeted by the gRNA in parallel to the CRISPR construct. As an additional line of evidence, we monitored BcFLA1 expression with qPCR and detected a significant reduction of the transcript in roots with an active CRISPR construct compared to the control, although residual amounts of the transcript were detected, possibly due to inefficient nonsense-mediated mRNA decay. Additionally, the presence of deletions and insertions were verified by Sanger sequencing of the respective amplicons. In summary we demonstrate the successful application of CRISPR/Cas9 in hairy roots of B. carinata, the proof of its effectiveness and its effect on the root hair phenotype. This study paves the way for experimental strategies involving the phenotypic assessment of gene lesions by CRISPR which do not require germline transmission