Cell wall-mediated root development is targeted by a soil-borne bacterial pathogen to promote infection.

Plant pathogens manipulate host development, facilitating colonization and proliferation. Ralstonia solanacearum is a soil-borne bacterial pathogen that penetrates roots and colonizes plants through the vascular system, causing wilting and death. Here, we find that RipAC, an effector protein from R. solanacearum, alters root development in Arabidopsis, promoting the formation of lateral roots and root hairs. RipAC interacts with CELLULOSE SYNTHASE (CESA)-INTERACTIVE PROTEIN 1 (CSI1), which regulates the activity of CESA complexes at the plasma membrane. RipAC disrupts CESA-CSI1 interaction, leading to a reduction in cellulose content, root developmental alterations, and a promotion of bacterial pathogenicity. We find that CSI1 also associates with the receptor kinase FERONIA, forming a complex that negatively regulates immunity in roots; this interaction, however, is not affected by RipAC. Our work reveals a bacterial virulence strategy that selectively affects the activities of a host target, promoting anatomical alterations that facilitate infection without causing activation of immunity.

Synthetically derived BiAux modulates auxin co-receptor activity to stimulate lateral root formation.

The root system plays an essential role in plant growth and adaptation to the surrounding environment. The root clock periodically specifies lateral root prebranch sites (PBS), where a group of pericycle founder cells (FC) is primed to become lateral root founder cells and eventually give rise to lateral root primordia or lateral roots (LRs). This clock-driven organ formation process is tightly controlled by modulation of auxin content and signaling. Auxin perception entails the physical interaction of TRANSPORT INHIBITOR RESPONSE 1 (TIR1) or AUXIN SIGNALING F-BOX (AFBs) proteins with AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) repressors to form a co-receptor system. Despite the apparent simplicity, the understanding of how specific auxin co-receptors are assembled remains unclear. We identified the compound bis-methyl auxin conjugated with N-glucoside, or BiAux, in Arabidopsis (Arabidopsis thaliana) that specifically induces the formation of PBS and the emergence of LR, with a slight effect on root elongation. Docking analyses indicated that BiAux binds to F-box proteins, and we showed that BiAux function depends on TIR1 and AFB2 F-box proteins and AUXIN RESPONSE FACTOR 7 activity, which is involved in FC specification and LR formation. Finally, using a yeast (Saccharomyces cerevisiae) heterologous expression system, we showed that BiAux favors the assemblage of specific co-receptors subunits involved in LR formation and enhances AUXIN/INDOLE-3-ACETIC ACID 28 protein degradation. These results indicate that BiAux acts as an allosteric modulator of specific auxin co-receptors. Therefore, BiAux exerts a fine-tune regulation of auxin signaling aimed to the specific formation of LR among the many development processes regulated by auxin.

Temperature changes in the root ecosystem affect plant functionality.

Climate change is increasing the frequency of extreme heat events that aggravate its negative impact on plant development and agricultural yield. Most experiments designed to study plant adaption to heat stress apply homogeneous high temperatures to both shoot and root. However, this treatment does not mimic the conditions in natural fields, where roots grow in a dark environment with a descending temperature gradient. Excessively high temperatures severely decrease cell division in the root meristem, compromising root growth, while increasing the division of quiescent center cells, likely in an attempt to maintain the stem cell niche under such harsh conditions. Here, we engineered the TGRooZ, a device that generates a temperature gradient for in vitro or greenhouse growth assays. The root systems of plants exposed to high shoot temperatures but cultivated in the TGRooZ grow efficiently and maintain their functionality to sustain proper shoot growth and development. Furthermore, gene expression and rhizosphere or root microbiome composition are significantly less affected in TGRooZ-grown roots than in high-temperature-grown roots, correlating with higher root functionality. Our data indicate that use of the TGRooZ in heat-stress studies can improve our knowledge of plant response to high temperatures, demonstrating its applicability from laboratory studies to the field.

Novel SRY-box transcription factor 9 variant in campomelic dysplasia and the location of missense and nonsense variants along the protein domains: A case report.

Background: Campomelic dysplasia (CD) is a rare disorder that involves the skeletal and genital systems. This condition has been associated with a diverse set of mutations in the SRY-box transcription factor 9 (SOX9) gene. Case presentation: We herein report a case involving a 4-year-old female patient with CD, female sex reversal, type 1 Arnold-Chiari malformation, and bilateral conductive hearing loss and investigate the causal mutation. Whole-exome sequencing analysis detected a novel Trp115X* variant in the SOX9 gene. We performed a literature review of the reported cases and demonstrated that the missense variants were located only in the self-dimerization domain (DIM) and high-mobility group box domains. We also reported that variants in the DIM domain do not cause sex reversal and identified that the amino acid sequences that were mutated in the patients with campomelic dysplasia are evolutionarily conserved among primates. Conclusions: We suggest that missense variants cannot be located in the K2, PQA, and PQS given that these domains function critically for transcriptional activation or repression of target genes and evolve under purifying selection.

Effects of Combined Abiotic Stresses Related to Climate Change on Root Growth in Crops.

Climate change is a major threat to crop productivity that negatively affects food security worldwide. Increase in global temperatures are usually accompanied by drought, flooding and changes in soil nutrients composition that dramatically reduced crop yields. Against the backdrop of climate change, human population increase and subsequent rise in food demand, finding new solutions for crop adaptation to environmental stresses is essential. The effects of single abiotic stress on crops have been widely studied, but in the field abiotic stresses tend to occur in combination rather than individually. Physiological, metabolic and molecular responses of crops to combined abiotic stresses seem to be significantly different to individual stresses. Although in recent years an increasing number of studies have addressed the effects of abiotic stress combinations, the information related to the root system response is still scarce. Roots are the underground organs that directly contact with the soil and sense many of these abiotic stresses. Understanding the effects of abiotic stress combinations in the root system would help to find new breeding tools to develop more resilient crops. This review will summarize the current knowledge regarding the effects of combined abiotic stress in the root system in crops. First, we will provide a general overview of root responses to particular abiotic stresses. Then, we will describe how these root responses are integrated when crops are challenged to the combination of different abiotic stress. We will focus on the main changes on root system architecture (RSA) and physiology influencing crop productivity and yield and convey the latest information on the key molecular, hormonal and genetic regulatory pathways underlying root responses to these combinatorial stresses. Finally, we will discuss possible directions for future research and the main challenges needed to be tackled to translate this knowledge into useful tools to enhance crop tolerance.

The co-chaperone HOP participates in TIR1 stabilisation and in auxin response in plants.

HOP (HSP70-HSP90 organising protein) is a conserved family of co-chaperones well known in mammals for its role in the folding of signalling proteins associated with development. In plants, HOP proteins have been involved in the response to multiple stresses, but their role in plant development remains elusive. Herein, we describe that the members of the HOP family participate in different aspects of plant development as well as in the response to warm temperatures through the regulation of auxin signalling. Arabidopsis hop1 hop2 hop3 triple mutant shows different auxin-related phenotypes and a reduced auxin sensitivity. HOP interacts with TIR1 auxin coreceptor in vivo. Furthermore, TIR1 accumulation and auxin transcriptional response are reduced in the hop1 hop2 hop3 triple mutant, suggesting that HOP's function in auxin signalling is related, at least, to TIR1 interaction and stabilisation. Interestingly, HOP proteins form part of the same complexes as SGT1b (a different HSP90 co-chaperone) and these co-chaperones synergistically cooperate in auxin signalling. This study provides relevant data about the role of HOP in auxin regulation in plants and uncovers that both co-chaperones, SGT1b and HOP, cooperate in the stabilisation of common targets involved in plant development.

May the dark be with roots: a perspective on how root illumination may bias in vitro research on plant-environment interactions.

Roots anchor plants to the soil, providing them with nutrients and water while creating a defence network and facilitating beneficial interactions with a multitude of living organisms and climatological conditions. To facilitate morphological and molecular studies, root research has been conducted using in vitro systems. However, under natural conditions, roots grow in the dark, mainly in the absence of illumination, except for the relatively low illumination of the upper soil surface, and this has been largely ignored. Here, we discuss the results found over the last decade on how experimental exposure of roots to light may bias root development and responses through the alteration of hormonal signalling, cytoskeleton organization, reactive oxygen species or the accumulation of flavonoids, among other factors. Illumination alters the uptake of nutrients or water, and also affects the response of the roots to abiotic stresses and root interactions with the microbiota. Furthermore, we review in vitro systems created to maintain roots in darkness, and provide a comparative analysis of root transcriptomes obtained with these devices. Finally, we identify other experimental variables that should be considered to better mimic soil conditions, whose improvement would benefit studies using in vitro cultivation or enclosed ecosystems.

Differential proteome between ejaculate and epididymal sperm represents a key factor for sperm freezability in wild small ruminants.

Epididymal sperm shows higher cryoresistance than ejaculated sperm. Although the sperm proteome seems to affect cell cryoresistance, studies aiming at identifying proteins involved in sperm freezing-tolerance are scarce. The aims of this study were to investigate differences of sperm freezability and proteome between epididymal and ejaculated sperm in three mountain ungulates: Iberian ibex, Mouflon and Chamois. Sperm samples were cryopreserved in straws by slow freezing. Tandem mass tag-labeled peptides from sperm samples were analyzed by high performance liquid chromatography coupled to a mass spectrometer in three technical replicates. The statistical analysis was done using the moderated t-test of the R package limma. Differences of freezability between both types of sperm were associated with differences of the proteome. Overall, epididymal sperm showed higher freezability than ejaculated sperm. Between 1490 and 1883 proteins were quantified in each species and type of sperm sample. Cross species comparisons revealed a total of 76 proteins that were more abundant in epididymal than in ejaculated sperm in the three species of study whereas 3 proteins were more abundant in ejaculated than epididymal sperm in the three species of study (adjusted P < 0.05; |log2| fold-change > 0.5). Many of the proteins that were associated with higher cryoresistance are involved in stress response and redox homeostasis. In conclusion, marked changes of sperm proteome were detected between epididymal and ejaculated sperm. This work contributes to update the sperm proteome of small ruminants and to identify candidate markers of sperm freezability.

Fluorescence-Activated Cell Sorting Using the D-Root Device and Optimization for Scarce and/or Non-Accessible Root Cell Populations.

Fluorescence-activated cell sorting (FACS) is a technique used to isolate specific cell populations based on characteristics detected by flow cytometry. FACS has been broadly used in transcriptomic analyses of individual cell types during development or under different environmental conditions. Different protoplast extraction protocols are available for plant roots; however, they were designed for accessible cell populations, which normally were grown in the presence of light, a non-natural and stressful environment for roots. Here, we report a protocol using FACS to isolate root protoplasts from Arabidopsis green fluorescent protein (GFP)-marked lines using the minimum number of enzymes necessary for an optimal yield, and with the root system grown in darkness in the D-Root device. This device mimics natural conditions as the shoot grows in the presence of light while the roots grow in darkness. In addition, we optimized this protocol for specific patterns of scarce cell types inside more differentiated tissues using the mCherry fluorescent protein. We provide detailed experimental protocols for effective protoplasting, subsequent purification through FACS, and RNA extraction. Using this RNA, we generated cDNA and sequencing libraries, proving that our methods can be used for genome-wide transcriptomic analyses of any cell-type from roots grown in darkness.

Alternative Polyadenylation and Salicylic Acid Modulate Root Responses to Low Nitrogen Availability.

Nitrogen (N) is probably the most important macronutrient and its scarcity limits plant growth, development and fitness. N starvation response has been largely studied by transcriptomic analyses, but little is known about the role of alternative polyadenylation (APA) in such response. In this work, we show that N starvation modifies poly(A) usage in a large number of transcripts, some of them mediated by FIP1, a component of the polyadenylation machinery. Interestingly, the number of mRNAs isoforms with poly(A) tags located in protein-coding regions or 5'-UTRs significantly increases in response to N starvation. The set of genes affected by APA in response to N deficiency is enriched in N-metabolism, oxidation-reduction processes, response to stresses, and hormone responses, among others. A hormone profile analysis shows that the levels of salicylic acid (SA), a phytohormone that reduces nitrate accumulation and root growth, increase significantly upon N starvation. Meta-analyses of APA-affected and fip1-2-deregulated genes indicate a connection between the nitrogen starvation response and salicylic acid (SA) signaling. Genetic analyses show that SA may be important for preventing the overgrowth of the root system in low N environments. This work provides new insights on how plants interconnect different pathways, such as defense-related hormonal signaling and the regulation of genomic information by APA, to fine-tune the response to low N availability.

Role of cis-zeatin in root responses to phosphate starvation.

Phosphate (Pi) is an essential nutrient for all organisms. Roots are underground organs, but the majority of the root biology studies have been done on root systems growing in the presence of light. Root illumination alters the Pi starvation response (PSR) at different intensities. Thus, we have analyzed morphological, transcriptional and physiological responses to Pi starvation in dark-grown roots. We have identified new genes and pathways regulated by Pi starvation that were not described previously. We also show that Pi-starved plants increase the cis-zeatin (cZ) : trans-zeatin (tZ) ratio. Transcriptomic analyses show that tZ preferentially represses cell cycle and PSR genes, whereas cZ induces genes involved in cell and root hair elongation and differentiation. In fact, cZ-treated seedlings show longer root system as well as longer root hairs compared with tZ-treated seedlings, increasing the total absorbing surface. Mutants with low cZ concentrations do not allocate free Pi in roots during Pi starvation. We propose that Pi-starved plants increase the cZ : tZ ratio to maintain basal cytokinin responses and allocate Pi in the root system to sustain its growth. Therefore, cZ acts as a PSR hormone that stimulates root and root hair elongation to enlarge the root absorbing surface and to increase Pi concentrations in roots.

mTOR Expression in Liver Transplant Candidates with Hepatocellular Carcinoma: Impact on Histological Features and Tumour Recurrence.

(1) Background: The mammalian target of rapamycin (mTOR) pathway activation is critical for hepatocellular carcinoma (HCC) progression. We aimed to evaluate the mTOR tissue expression in liver transplant (LT) patients and to analyse its influence on post-LT outcomes. (2) Methods: Prospective study including a cohort of HCC patients who underwent LT (2012⁻2015). MTOR pathway expression was evaluated in the explanted liver by using the "PathScan Intracellular Signalling Array Kit" (Cell Signalling). Kaplan-Meier and Cox regression analyses were performed to evaluate post-LT HCC recurrence. (3) Results: Forty-nine patients were included (average age 56.4 ± 6, 14.3% females). Phospho-mTOR (Ser2448) was over-expressed in peritumoral tissue as compared with tumoral tissue (ΔSignal 22.2%; p < 0.001). The mTOR activators were also increased in peritumoral tissue (phospho-Akt (Thr308) ΔSignal 18.2%, p = 0.004; phospho-AMPKa (Thr172) ΔSignal 56.3%, p < 0.001), as they were the downstream effectors responsible for cell growth/survival (phospho-p70S6K (Thr389) ΔSignal 33.3%, p < 0.001 and phospho-S6RP (Ser235/236) ΔSignal 54.6%, p < 0.001). MTOR expression was increased in patients with multinodular HCC (tumoral p = 0.01; peritumoral p = 0.001). Increased phospho-mTOR in tumoral tissue was associated with higher HCC recurrence rates after LT (23.8% vs. 5.9% at 24 months, p = 0.04). (4) Conclusion: mTOR pathway is over-expressed in patients with multinodular HCC and is it associated with increased post-LT tumour recurrence rates.

A light-sensitive mutation in Arabidopsis LEW3 reveals the important role of N-glycosylation in root growth and development.

Plant roots have the potential capacity to grow almost indefinitely if meristematic and lateral branching is sustained. In a genetic screen we identified an Arabidopsis mutant showing limited root growth (lrg1) due to defects in cell division and elongation in the root meristem. Positional cloning determined that lrg1 affects an alpha-1,2-mannosyltransferase gene, LEW3, involved in protein N-glycosylation. The lrg1 mutation causes a synonymous substitution that alters the correct splicing of the fourth intron in LEW3, causing a mix of wild-type and truncated protein. LRG1 RNA missplicing in roots and short root phenotypes in lrg1 are light-intensity dependent. This mutation disrupts a GC-base pair in a three-base-pair stem with a four-nucleotide loop, which seems to be necessary for correct LEW3 RNA splicing. We found that the lrg1 short root phenotype correlates with high levels of reactive oxygen species and low pH in the apoplast. Proteomic analyses of N-glycosylated proteins identified GLU23/PYK10 and PRX34 as N-glycosylation targets of LRG1 activity. The lrg1 mutation reduces the positive interaction between Arabidopsis and Serendipita indica. A prx34 mutant showed a significant reduction in root growth, which is additive to lrg1. Taken together our work highlights the important role of N-glycosylation in root growth and development.

The transcription factor OBP4 controls root growth and promotes callus formation.

Plant growth and development require a continuous balance between cell division and differentiation. In root meristems, differentiated cells acquire specialized functions, losing their mitotic potential. Some plant cells, such as pericycle cells, have a remarkable plasticity to regenerate new organs. The molecular mechanisms underlying cell reprogramming are not completely known. In this work, a functional screening of transcription factors identified Arabidopsis OBP4 (OBF Binding Protein 4) as a novel regulator of root growth and cell elongation and differentiation. Overexpression of OBP4 regulates the levels of a large number of transcripts in roots, many involved in hormonal signaling and callus formation. OBP4 controls cell elongation and differentiation in root cells. OBP4 does not induce cell division in the root meristem, but promotes pericycle cell proliferation, forming callus-like structures at the root tip, as shown by the expression of stem cell markers. Callus formation is enhanced by ectopic expression of OBP4 in the wild-type or alf4-1, but is significantly reduced in roots that have lower levels of OBP4. Our data provide molecular insights into how differentiated root cells acquire the potential to generate callus, a pluripotent mass of cells that can regenerate fully functional plant organs.

Control of Arabidopsis lateral root primordium boundaries by MYB36.

Root branching in plants relies on the de novo formation of lateral roots. These are initiated from founder cells, triggering new formative divisions that generate lateral root primordia (LRP). The LRP size and shape depends on the balance between positive and negative signals that control cell proliferation. The mechanisms controlling proliferation potential of LRP cells remains poorly understood. We found that Arabidopsis thaliana MYB36, which have been previously shown to regulate genes required for Casparian strip formation and the transition from proliferation to differentiation in the primary root, plays a new role in controlling LRP development at later stages. We found that MYB36 is a novel component of LR development at later stages. MYB36 was expressed in the cells surrounding LRP where it controls a set of peroxidase genes, which maintain reactive oxygen species (ROS) balance. This was required to define the transition between proliferating and arrested cells inside the LRP, coinciding with the change from flat to dome-shaped primordia. Reducing the levels of hydrogen peroxide (H2 O2 ) in myb36-5 significantly rescues the mutant phenotype. Our results uncover a role for MYB36 outside the endodermis during LRP development through a mechanism analogous to regulating the proliferation/differentiation transition in the root meristem.

Characterisation of a flavonoid ligand of the fungal protein Alt a 1.

Spores of pathogenic fungi are virtually ubiquitous and cause human disease and severe losses in crops. The endophytic fungi Alternaria species produce host-selective phytotoxins. Alt a 1 is a strongly allergenic protein found in A. alternata that causes severe asthma. Despite the well-established pathogenicity of Alt a 1, the molecular mechanisms underlying its action and physiological function remain largely unknown. To gain insight into the role played by this protein in the pathogenicity of the fungus, we studied production of Alt a 1 and its activity in spores. We found that Alt a 1 accumulates inside spores and that its release with a ligand is pH-dependent, with optimum production in the 5.0-6.5 interval. The Alt a 1 ligand was identified as a methylated flavonoid that inhibits plant root growth and detoxifies reactive oxygen species. We also found that Alt a 1 changes its oligomerization state depending on the pH of the surrounding medium and that these changes facilitate the release of the ligand. Based on these results, we propose that release of Alt a 1 should be a pathogenic target in approaches used to block plant defenses and consequently to favor fungal entry into the plant.

Flavonols Mediate Root Phototropism and Growth through Regulation of Proliferation-to-Differentiation Transition.

Roots normally grow in darkness, but they may be exposed to light. After perceiving light, roots bend to escape from light (root light avoidance) and reduce their growth. How root light avoidance responses are regulated is not well understood. Here, we show that illumination induces the accumulation of flavonols in Arabidopsis thaliana roots. During root illumination, flavonols rapidly accumulate at the side closer to light in the transition zone. This accumulation promotes asymmetrical cell elongation and causes differential growth between the two sides, leading to root bending. Furthermore, roots illuminated for a long period of time accumulate high levels of flavonols. This high flavonol content decreases both auxin signaling and PLETHORA gradient as well as superoxide radical content, resulting in reduction of cell proliferation. In addition, cytokinin and hydrogen peroxide, which promote root differentiation, induce flavonol accumulation in the root transition zone. As an outcome of prolonged light exposure and flavonol accumulation, root growth is reduced and a different root developmental zonation is established. Finally, we observed that these differentiation-related pathways are required for root light avoidance. We propose that flavonols function as positional signals, integrating hormonal and reactive oxygen species pathways to regulate root growth direction and rate in response to light.

Everolimus is safe within the first month after liver transplantation.

BACKGROUND & AIMS: Sirolimus should not be started within the first month after liver transplantation (LT) because of an increased risk of adverse outcomes. The evidence regarding everolimus is lacking but the manufacturer transposed the same warning. We aimed to evaluate the safety of everolimus started within the first month after LT. METHODS: A consecutive cohort 187 LT patients (2009-2013) with a tacrolimus-based immunosuppression was evaluated. Patients starting everolimus within the first month after LT (n = 33; 17.6%) were compared with those starting everolimus thereafter (n = 25; 13.4%) or not receiving everolimus (n = 129; 69%). The median follow-up after LT was 21 months (IQR 7-36). Prospective outcomes were evaluated by using Kaplan-Meier curves and Cox's regression. RESULTS: The incidence of hepatic artery thrombosis was not significantly different in patients early treated with everolimus when compared with the remaining cohort (0% vs 9.1%; p = 0.12). Other vascular complications occurred in 9.1% of patients with early everolimus vs 7.3% in the remaining cohort (p = 0.72). No wound healing complications were detected with early everolimus. There were similar rates of incisional hernia (p = 0.31), infections (p = 0.15), renal impairment (p = 0.37), and histologically-proven acute rejection (p = 0.24) between groups. The rates of hyperlipidemia were increased with early everolimus (29.9% vs 16.5% at 3 years; p = 0.018). Graft loss and mortality rates were similar between groups (p = 0.34 and p = 0.94 respectively), after adjusting for possible confounding factors. CONCLUSIONS: Everolimus combined with reduced tacrolimus proved to be safe within the first month after LT. Future trials may be allowed to implement everolimus early after LT.

D-Root: a system for cultivating plants with the roots in darkness or under different light conditions.

In nature roots grow in the dark and away from light (negative phototropism). However, most current research in root biology has been carried out with the root system grown in the presence of light. Here, we have engineered a device, called Dark-Root (D-Root), to grow plants in vitro with the aerial part exposed to the normal light/dark photoperiod while the roots are in the dark or exposed to specific wavelengths or light intensities. D-Root provides an efficient system for cultivating a large number of seedlings and easily characterizing root architecture in the dark. At the morphological level, root illumination shortens root length and promotes early emergence of lateral roots, therefore inducing expansion of the root system. Surprisingly, root illumination also affects shoot development, including flowering time. Our analyses also show that root illumination alters the proper response to hormones or abiotic stress (e.g. salt or osmotic stress) and nutrient starvation, enhancing inhibition of root growth. In conclusion, D-Root provides a growing system closer to the natural one for assaying Arabidopsis plants, and therefore its use will contribute to a better understanding of the mechanisms involved in root development, hormonal signaling and stress responses.