Histopathology of the Plasmodiophora brassicae-Chinese Cabbage Interaction in Hosts Carrying Different Sources of Resistance

Clubroot is a serious soil-borne disease of crucifers caused by the obligate parasite Plasmodiophora brassicae. The genetic basis and histopathology of clubroot resistance in two Chinese cabbage (Brassica rapa ssp. pekinensis) inbred lines Bap055 and Bap246, challenged with pathotype 4 of P. brassicae, was evaluated. The Chinese cabbage cultivar “Juxin” served as a susceptible check. The resistance in Bap055 was found to be controlled by the CRa gene, while resistance in Bap246 fit a model of control by unknown recessive gene. Infection of the roots by P. brassicae was examined by inverted microscopy. Despite their resistance, primary and secondary infection were observed to occur in Bap055 and Bap246. Primary infection was detected at 2 days post-inoculation (DPI) in “Juxin,” at 4 DPI in Bap055, and at 6 DPI in Bap246. Infection occurred most quickly on “Juxin,” with 60% of the root hairs infected at 10 DPI, followed by Bap055 (31% of the root hairs infected at 12 DPI) and Bap246 (20% of the root hairs infected at 14 DPI). Secondary infection of “Juxin” was first observed at 8 DPI, while in Bap055 and Bap246, secondary infection was first observed at 10 DPI. At 14 DPI, the percentage of cortical infection in “Juxin,” Bap055 and Bap246 was 93.3, 20.0, and 11.1%, respectively. Although cortical infection was more widespread in Bap055 than in Bap246, secondary infection in both of these hosts was restricted relative to the susceptible check, and the vascular system remained intact. A large number of binucleate secondary plasmodia were observed in “Juxin” and the vascular system was disrupted at 16 DPI; in Bap055 and Bap246, only a few secondary plasmodia were visible, with no binucleate secondary plasmodia. The defense mechanisms and expression of resistance appears to differ between Chinese cabbage cultivars carrying different sources of resistance.

COE2 Is Required for the Root Foraging Response to Nitrogen Limitation

There are numerous exchanges of signals and materials between leaves and roots, including nitrogen, which is one of the essential nutrients for plant growth and development. In this study we identified and characterized the Chlorophyll A/B-Binding Protein (CAB) (named coe2 for CAB overexpression 2) mutant, which is defective in the development of chloroplasts and roots under normal growth conditions. The phenotype of coe2 is caused by a mutation in the Nitric Oxide Associated (NOA1) gene that is implicated in a wide range of chloroplast functions including the regulation of metabolism and signaling of nitric oxide (NO). A transcriptome analysis reveals that expression of genes involved in metabolism and lateral root development are strongly altered in coe2 seedlings compared with WT. COE2 is expressed in hypocotyls, roots, root hairs, and root caps. Both the accumulation of NO and the growth of lateral roots are enhanced in WT but not in coe2 under nitrogen limitation. These new findings suggest that COE2-dependent signaling not only coordinates gene expression but also promotes chloroplast development and function by modulating root development and absorption of nitrogen compounds.

Cell surface receptor kinase FERONIA linked to nutrient sensor TORC1 signaling controls root hair growth at low temperature in Arabidopsis thaliana

Root hairs (RH) are excellent model systems for studying cell size regulation since they elongate several hundred-fold their original size. Their growth is determined both by intrinsic and environmental signals. Although nutrients availability in the soil are key factors for a sustained plant growth, the molecular mechanisms underlying their perception and downstream signaling pathways remains unclear. Here, we identified that a low temperature triggers a strong RH cell elongation response involving the cell surface receptor kinase FERONIA (FER) and nutrient sensor TORC1 pathway. We found that FER is required to perceive limited nutrients availability caused by low temperature, to interacts with and activate TORC1-downstream components to trigger RH growth. Nitrates perceived and transported by NRT1.1 were found to mimic this growth response at low temperature. Our findings reveal a new molecular mechanism by which a central hub composed by FER-TORC1 controls RH cell elongation under low temperature.

Efficient Editing of CSLD2 Orthologue by CRISPR/Cas9 Affects Cell Morphogenesis of Root Hair in Spinach

CRISPR/Cas9 is a valuable tool and has been extensively employed to perform gene editing in plants. However, CRISPR/Cas9 has not been successfully used in spinach, an important leafy vegetable crop. Here, we precisely edited Spo23361 and Spo10340, two cellulose synthase-like D (CSLD) genes involved in root hair formation of spinach hairy roots, using CRISPR/Cas9 system. Four mutation types (i.e., replacement, insertion, deletion, and combined mutations) were observed, among which the deletion accounted for the vast majority (about 64.1%). Mutation rate differed largely among different targets. Seven homozygous/bi-allelic and eight heterozygous/chimeric mutated lines of Spo23361 were obtained from 15 independent transgenic hairy root lines. All of the seven homozygous/bi-allelic lines displayed bulking and short root hairs, which exhibited the characteristics of Arabidopsis csld2 mutants. Thirteen heterozygous/chimeric mutated lines, but no homozygous/bi-allelic lines, of Spo10340 were obtained from 15 independent transgenic hairy root lines, all of which showed similar phenotype of root hair with normal hairy roots. The transcriptomic analysis further revealed that multiple gene expressions for cell wall modulation and membrane trafficking were disturbed, which might result in the inhibition of root hair growth in Spo23361 mutants. Our results indicate that Agrobacterium rhizogenes-mediated transformation using CRISPR/Cas9 is a simple and efficient genome editing tool in spinach. It lays a solid foundation for large-scale genome editing in spinach in future.

New insights on the role of HCN in root hair elongation through single cell proteomics

Root hairs are specialized structures involved in water and nutrient uptake by plants. They elongate from epidermal cells following a complex developmental program. β-cyanoalanine synthase (CAS), which is mainly involved in hydrogen cyanide (HCN) detoxification in Arabidopsis thaliana, plays a role in root hair elongation, as evidenced by the fact that cas-c1 mutants show a severe defect in root hair shape. In addition to root hairs, CAS C1 is expressed in the quiescent center and meristem. However, the cas-c1 mutation has no visible effect on either tissue, in both control and nutrient-deprivation conditions. To identify its role in root hair formation, we conducted single cell proteomics analysis by isolating root hair cells using Fluorescence-Activated Cell Sorting (FACS) from wild type and cas-c1 mutants. We also analyzed the presence of S-cyanylation, a protein post-translational modification (PTM) mediated by HCN and affecting cysteine residues and protein activity, in proteins of wild type and cas-c1 mutants. We found that several proteins involved in root hair development, related to the receptor kinase FERONIA signaling and to DNA methylation, are modified by this new post-translational modification.

Survival of Plants During Short-Term BOA-OH Exposure: ROS Related Gene Expression and Detoxification Reactions Are Accompanied With Fast Membrane Lipid Repair in Root Tips

For the characterization of BOA-OH insensitive plants, we studied the time-dependent effects of the benzoxazolinone-4/5/6/7-OH isomers on maize roots. Exposure of Zea mays seedlings to 0.5 mM BOA-OH elicits root zone-specific reactions by the formation of dark rings and spots in the zone of lateral roots, high catalase activity on root hairs, and no visible defense reaction at the root tip. We studied BOA-6-OH- short-term effects on membrane lipids and fatty acids in maize root tips in comparison to the benzoxazinone-free species Abutilon theophrasti Medik. Decreased contents of phosphatidylinositol in A. theophrasti and phosphatidylcholine in maize were found after 10–30 min. In the youngest tissue, α-linoleic acid (18:2), decreased considerably in both species and recovered within one hr. Disturbances in membrane phospholipid contents were balanced in both species within 30–60 min. Triacylglycerols (TAGs) were also affected, but levels of maize diacylglycerols (DAGs) were almost unchanged, suggesting a release of fatty acids for membrane lipid regeneration from TAGs while resulting DAGs are buildings blocks for phospholipid reconstitution, concomitant with BOA-6-OH glucosylation. Expression of superoxide dismutase (SOD2) and of ER-bound oleoyl desaturase (FAD2-2) genes were contemporaneously up regulated in contrast to the catalase CAT1, while CAT3 was arguably involved at a later stage of the detoxification process. Immuno-responses were not elicited in short-terms, since the expression of NPR1, POX12 were barely affected, PR4 after 6 h with BOA-4/7-OH and PR1 after 24 h with BOA-5/6-OH. The rapid membrane recovery, reactive oxygen species, and allelochemical detoxification may be characteristic for BOA-OH insensitive plants.

Proteomics and lipidomics integrated analysis of outer membrane vesicles indicates that glycerophospholipid metabolism contributes to the early symbiosis between Sinorhizobium fredii HH103 and soybean

Gram-negative bacteria can produce outer membrane vesicles (OMVs), and most functional studies of OMVs have been focused on mammalian-bacterial interactions. However, research on the OMVs of rhizobia is still limited so far. In this work, we isolated and purified OMVs from Sinorhizobium fredii HH103 under free-living conditions that was set as control (C-OMVs) and symbiosis-mimicking conditions that was induced by genistein (G-OMVs). The soybean roots treated with G-OMVs displayed significant deformation of root hairs. G-OMVs significantly induced the expression of nodulation genes related to early symbiosis, while inhibited that of the defense genes of soybean. Proteomics analysis identified a total of 93 differential proteins between C-OMVs and G-OMVs, which are mainly associated with ribosome synthesis, flagellar assembly, two-component system, ABC transporters, oxidative phosphorylation, nitrogen metabolism, quorum sensing, glycerophospholipid metabolism and peptidoglycan biosynthesis. A total of 45 differential lipids were identified in lipidomics analysis. Correlation analysis of OMV proteome and lipidome data revealed that glycerophospholipid metabolism is the enriched KEGG metabolic pathway, and the expression of phosphatidylserine decarboxylase was significantly up-regulated in G-OMVs. The changes in three lipids related to symbiosis in the glycerophospholipid metabolism pathway were verified by ELISA. Our results indicate that glycerophospholipid metabolism contributes to rhizobia-soybean symbiosis via OMVs.

Rock Fragment Content in Soils Shift Root Foraging Behavior in Xerophytic Species

Aims Root traits associated with resource foraging, including fine-root branching intensity, root hair and mycorrhiza, may change in soils with various physical structure indicated by rock fragment content (RFC), while how these traits covariate at the level of individual root branching order is largely unknown.Methods We subjected two xerophytic species, Artemisia vestita (subshrub) and Bauhinia brachycarpa (shrub), to increasing RFC gradients (0%, 25%, 50% and 75%, v v-1) in an arid environment and measured fine-root traits related to resource foraging.Results Root hair density and mycorrhizal colonization of both species decreased with increasing root order, but increased in 3rd- and 4th-order roots at high RFCs (50% or 75%). The two species tend to produce more root hairs than mycorrhizas under the high RFCs. For both species, root hair density and mycorrhizal colonization intensity were negatively correlated with root length and root diameter. Rockiness reduced root branching intensity in both species comparing with rock-free soil. At the same level of RFC, A. vestita had thicker roots and lower branching intensity than B. brachycarpa, and tended to produce more root hairs.Conclusion Our results suggest the high RFC soil conditions stimulated greater foraging functions in higher root orders. We found evidence for a greater investment in root hairs and mycorrhizal symbioses as opposed to building an extensive root system in rocky soils. The subshrub and shrub species took different approaches to foraging in the rocky soil through distinctive trait syndromes of fine-root components.

Several Yeast Species Induce Iron Deficiency Responses in Cucumber Plants (Cucumis sativus L.)

Iron (Fe) deficiency is a first-order agronomic problem that causes a significant decrease in crop yield and quality. Paradoxically, Fe is very abundant in most soils, mainly in its oxidized form, but is poorly soluble and with low availability for plants. In order to alleviate this situation, plants develop different morphological and physiological Fe-deficiency responses, mainly in their roots, to facilitate Fe mobilization and acquisition. Even so, Fe fertilizers, mainly Fe chelates, are widely used in modern agriculture, causing environmental problems and increasing the costs of production, due to the high prices of these products. One of the most sustainable and promising alternatives to the use of agrochemicals is the better management of the rhizosphere and the beneficial microbial communities presented there. The main objective of this research has been to evaluate the ability of several yeast species, such as Debaryomyces hansenii, Saccharomyces cerevisiae and Hansenula polymorpha, to induce Fe-deficiency responses in cucumber plants. To date, there are no studies on the roles played by yeasts on the Fe nutrition of plants. Experiments were carried out with cucumber plants grown in a hydroponic growth system. The effects of the three yeast species on some of the most important Fe-deficiency responses developed by dicot (Strategy I) plants, such as enhanced ferric reductase activity and Fe2+ transport, acidification of the rhizosphere, and proliferation of subapical root hairs, were evaluated. The results obtained show the inductive character of the three yeast species, mainly of Debaryomyces hansenii and Hansenula polymorpha, on the Fe-deficiency responses evaluated in this study. This opens a promising line of study on the use of these microorganisms as Fe biofertilizers in a more sustainable and environmentally friendly agriculture.

EIN3 and RSL4 interfere with an MYB–bHLH–WD40 complex to mediate ethylene-induced ectopic root hair formation in Arabidopsis

The alternating cell specifications of root epidermis to form hair cells or nonhair cells in Arabidopsis are determined by the expression level of GL2, which is activated by an MYB–bHLH–WD40 (WER–GL3–TTG1) transcriptional complex. The phytohormone ethylene (ET) has a unique effect of inducing N-position epidermal cells to form root hairs. However, the molecular mechanisms underlying ET-induced ectopic root hair development remain enigmatic. Here, we show that ET promotes ectopic root hair formation through down-regulation of GL2 expression. ET-activated transcription factors EIN3 and its homolog EIL1 mediate this regulation. Molecular and biochemical analyses further revealed that EIN3 physically interacts with TTG1 and interferes with the interaction between TTG1 and GL3, resulting in reduced activation of GL2 by the WER–GL3–TTG1 complex. Furthermore, we found through genetic analysis that the master regulator of root hair elongation, RSL4, which is directly activated by EIN3, also participates in ET-induced ectopic root hair development. RSL4 negatively regulates the expression of GL2, likely through a mechanism similar to that of EIN3. Therefore, our work reveals that EIN3 may inhibit gene expression by affecting the formation of transcription-activating protein complexes and suggests an unexpected mutual inhibition between the hair elongation factor, RSL4, and the hair specification factor, GL2. Overall, this study provides a molecular framework for the integration of ET signaling and intrinsic root hair development pathway in modulating root epidermal cell specification.