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.

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.

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.

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.

Arsenite toxicity and uptake rate of rice (Oryza sativa L.) in vivo.

Toxicity threshold of arsenite on intact rice seedlings was determined and arsenite uptake characteristics were investigated using non-toxic concentrations of arsenite. The arsenite toxicity threshold was 2.4 µM arsenite which reduced growth by 10% (EC(10)). The two highest arsenite levels induced wilting of seedlings and reduced both, transpiration rate and net photosynthetic rate. Arsenic content in plant tissue increased up to 10.7 µM arsenite and then declined with increasing arsenite concentration in the treatment solution. The contents of Si, P, K, and of micronutrients Cu, Fe, Mn and Zn in shoot d.m. were reduced by arsenite levels ≥ 5.3 µM. In the non-toxic range, arsenite uptake rate was linearly related to arsenite concentration. High arsenite levels reduced growth without being taken up which might be due to increasing binding of arsenite to proteins at the outer side of the plasmalemma.

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.

Evaluation of soil characteristics potentially affecting arsenic concentration in paddy rice (Oryza sativa L.).

Paddy rice may contribute considerably to the human intake of As. The knowledge of soil characteristics affecting the As content of the rice plant enables the development of agricultural measures for controlling As uptake. During field surveys in 2004 and 2006, plant samples from 68 fields (Italy, Po-area) revealed markedly differing As concentration in polished rice. The soil factors total As(aqua regia), pH, grain size fractions, total C, plant available P(CAL), poorly crystalline Fe(oxal.) and plant available Si(Na-acetate) content that potentially affect As content of rice were determined. A multiple linear regression analysis showed a significant positive influence of the total As(aqua regia) and plant available P(CAL) content and a negative influence of the poorly crystalline Fe(oxal.) content of the soil on the As content in polished rice and rice straw. Si concentration in rice straw varied widely and was negatively related to As content in straw and polished rice.

Arsenic in rice (Oryza sativa L.) related to dynamics of arsenic and silicic acid in paddy soils.

Paddy rice is a global staple food which in some circumstances can contain high levels of the toxic element arsenic (As). In order to elucidate factors influencing As dissolution in the soil solution during paddy rice cultivation, rice (Oryza sativa L. "Selenio") was cultivated to maturity in six paddy soils in the greenhouse in 2005 and 2006. Concentrations of Mn, Fe, As, P, and silicic acid in soil solution and As concentrations in rice straw and polished rice grain were determined. There was a close relationship between Fe and As concentrations in the soil solution, suggesting that the major part of dissolved As originated from reduced iron-(hydr)oxide. However, in addition to the factors causing As dissolution in the soil, other factors influenced the uptake of As by rice. The inhibitory effect of indigenous silicic acid in the soil solution on As uptake was clearly shown. This implied that soils with high plant available Si contents resulted in low plant As contents and that Si application to soils may decrease the As content of rice.