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.

Click processes orthogonal to CuAAC and SuFEx forge selectively modifiable fluorescent linkers.

The appeal of catalytic click chemistry is largely due to the copper-catalysed azide-alkyne cycloaddition (CuAAC) process, which is orthogonal to the more recently introduced sulfur-fluoride exchange (SuFEx). However, the triazole rings generated by CuAAC are not readily modifiable, and SuFEx connectors cannot be selectively functionalized, attributes that would be attractive in a click process. Here we introduce bisphosphine-copper-catalysed phenoxydiazaborinine formation (CuPDF), a link-and-in situ modify strategy for merging a nitrile, an allene, a diborane and a hydrazine. We also present copper- and palladium-catalysed quinoline formation (Cu/PdQNF), which is applicable in aqueous media, involving an aniline as the modifier. CuPDF and Cu/PdQNF are easy to perform and deliver robust, alterable and tunable fluorescent hubs. CuPDF and Cu/PdQNF are orthogonal to SuFEx and CuAAC, despite the latter and CuPDF also being catalysed by an organocopper species. These advantages were applied to protecting group-free syntheses of sequence-defined branched oligomers, a chemoselectively amendable polymer, three drug conjugates and a two-drug conjugate.

The cold-induced factor CBF3 mediates root stem cell activity, regeneration, and developmental responses to cold.

Plant growth and development involve the specification and regeneration of stem cell niches (SCNs). Although plants are exposed to disparate environmental conditions, how environmental cues affect developmental programs and stem cells is not well understood. Root stem cells are accommodated in meristems in SCNs around the quiescent center (QC), which maintains their activity. Using a combination of genetics and confocal microscopy to trace morphological defects and correlate them with changes in gene expression and protein levels, we show that the cold-induced transcription factor (TF) C-REPEAT BINDING FACTOR 3 (CBF3), which has previously been associated with cold acclimation, regulates root development, stem cell activity, and regeneration. CBF3 is integrated into the SHORT-ROOT (SHR) regulatory network, forming a feedback loop that maintains SHR expression. CBF3 is primarily expressed in the root endodermis, whereas the CBF3 protein is localized to other meristematic tissues, including root SCNs. Complementation of cbf3-1 using a wild-type CBF3 gene and a CBF3 fusion with reduced mobility show that CBF3 movement capacity is required for SCN patterning and regulates root growth. Notably, cold induces CBF3, affecting QC activity. Furthermore, exposure to moderate cold around 10°C-12°C promotes root regeneration and QC respecification in a CBF3-dependent manner during the recuperation period. By contrast, CBF3 does not appear to regulate stem cell survival, which has been associated with recuperation from more acute cold (∼4°C). We propose a role for CBF3 in mediating the molecular interrelationships among the cold response, stem cell activity, and development.

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.

Diastereo- and enantioselective synthesis of compounds with a trifluoromethyl- and fluoro-substituted carbon centre.

Molecules that contain one or more fluorine atoms are crucial to drug discovery. There are protocols available for the selective synthesis of different organofluorine compounds, including those with a fluoro-substituted or a trifluoromethyl-substituted stereogenic carbon centre. However, approaches for synthesizing compounds with a trifluoromethyl- and fluoro-substituent stereogenic carbon centre are far less common. This potentially impactful set of molecules thus remains severely underdeveloped. Here we introduce a catalytic regio-, diastereo- and enantioselective strategy for the preparation of homoallylic alcohols bearing a stereogenic carbon centre bound to a trifluoromethyl group and a fluorine atom. The process, which involves a polyfluoroallyl boronate and is catalysed by an in situ-formed organozinc complex, can be used for diastereodivergent preparation of tetrafluoro-monosaccharides, including ribose core analogues of the antiviral drug sofosbuvir (Sovaldi). Unexpected reactivity/selectivity profiles, probably originating from the trifluoromethyl- and fluoro-substituted carbon site, are discovered, foreshadowing other unique chemistries that remain unknown.

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.

Gastric ischemia: cross-sectional imaging findings via a case series.

Gastric ischemia is an uncommon, serious, and potentially fatal disease caused by diffuse or focal gastric vascular insufficiency. It can be caused by states of systemic hypotension or disseminated vasculitis or thrombosis, even though the stomach possesses collateral circulation that prevents it from developing to a certain extent. Computed tomography (CT) is the technique of choice to assess the extent of the disease, as it detects parietal hypoenhancement and gastric and/or portal pneumatosis. The treatment required depends on the etiology and ranges from surgery to resuscitation measures. This article presents the imaging findings from a series of three cases of gastric ischemia seen at our hospital.

Cecal bascule with secondary gastric outlet obstruction in addition to a large bowel closed obstruction: A case report.

We present a case of cecal bascule in a 60-year-old woman with abdominal pain and vomiting. Imaging tests revealed a cecal bascule causing mechanic obstruction of the stomach. Besides a small bowel dilatation was not seen, the distended cecum was extrinsically obstructing the antrum and therefore, the gastric outlet. Cecal bascule is a form of cecal volvulus without the axial twisted component. The cecum folds anterior or anteromedially on itself and, because of a valve mechanism, becomes distended. The pressure increases and it begins to compromise arterial blood flow so life-threatening complications, such as bowel ischemia and infarction can occur. Prompt diagnosis is therefore crucial and plain radiographs and computed tomography are the preferred imaging techniques for this purpose.

An auxin-regulable oscillatory circuit drives the root clock in Arabidopsis.

In Arabidopsis, the root clock regulates the spacing of lateral organs along the primary root through oscillating gene expression. The core molecular mechanism that drives the root clock periodicity and how it is modified by exogenous cues such as auxin and gravity remain unknown. We identified the key elements of the oscillator (AUXIN RESPONSE FACTOR 7, its auxin-sensitive inhibitor IAA18/POTENT, and auxin) that form a negative regulatory loop circuit in the oscillation zone. Through multilevel computer modeling fitted to experimental data, we explain how gene expression oscillations coordinate with cell division and growth to create the periodic pattern of organ spacing. Furthermore, gravistimulation experiments based on the model predictions show that external auxin stimuli can lead to entrainment of the root clock. Our work demonstrates the mechanism underlying a robust biological clock and how it can respond to external stimuli.

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.

Root-knot nematodes induce gall formation by recruiting developmental pathways of post-embryonic organogenesis and regeneration to promote transient pluripotency.

Root-knot nematodes (RKNs; Meloidogyne spp.) induce new post-embryogenic organs within the roots (galls) where they stablish and differentiate nematode feeding cells, giant cells (GCs). The developmental programmes and functional genes involved remain poorly defined. Arabidopsis root apical meristem (RAM), lateral root (LR) and callus marker lines, SHORT-ROOT/SHR, SCARECROW/SCR, SCHIZORIZA/SCZ, WUSCHEL-RELATED-HOMEOBOX-5/WOX5, AUXIN-RESPONSIVE-FACTOR-5/ARF5, ARABIDOPSIS-HISTIDINE PHOSPHOTRANSFER-PROTEIN-6/AHP6, GATA-TRANSCRIPTION FACTOR-23/GATA23 and S-PHASE-KINASE-ASSOCIATED-PROTEIN2B/SKP2B, were analysed for nematode-dependent expression. Their corresponding loss-of-function lines, including those for LR upstream regulators, SOLITARY ROOT/SLR/IAA14, BONDELOS/BDL/IAA12 and INDOLE-3-ACETIC-ACID-INDUCIBLE-28/IAA28, were tested for RKN resistance/tolerance. LR genes, for example ARF5 (key factor for root stem-cell niche regeneration), GATA23 (which specifies pluripotent founder cells) and AHP6 (cytokinin-signalling-inhibitor regulating pericycle cell-divisions orientation), show a crucial function during gall formation. RKNs do not compromise the number of founder cells or LR primordia but locally induce gall formation possibly by tuning the auxin/cytokinin balance in which AHP6 might be necessary. Key RAM marker genes were induced and functional in galls. Therefore, the activation of plant developmental programmes promoting transient-pluripotency/stemness leads to the generation of quiescent-centre and meristematic-like cell identities within the vascular cylinder of galls. Nematodes enlist developmental pathways of new organogenesis and/or root regeneration in the vascular cells of galls. This should determine meristematic cell identities with sufficient transient pluripotency for gall organogenesis.

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.

Catalytic Enantioselective Boryl and Silyl Substitution with Trifluoromethyl Alkenes: Scope, Utility, and Mechanistic Nuances of Cu-F ß-Elimination.

Catalytic enantioselective methods are introduced that allow access to a variety of allyl boronates and silanes that contain a difluoroalkene unit; the resulting products may be used for the preparation of organofluorine compounds in high enantiomeric purity. Furthermore, a number of key mechanistic aspects of the transformations have been investigated and analyzed. Thus, first, an NHC-Cu-catalyzed method for boryl substitution with F3C-substituted alkenes is introduced. These processes, unlike the previously reported strategies, are applicable to alkyl as well as aryl substituted substrates, afford allyl boronates bearing a difluoroalkene moiety (up to 98% yield and 95:5 er). Second, the corresponding silyl substitutions, the first reported cases of their kind, are presented (up to 94% yield and 97:3 er). Third, experimental and computational (DFT) investigations are described that shed light on key mechanistic aspects of the catalytic processes. Evidence (X-ray structures of Cu-alkyl intermediates and kinetic studies) is put forth illustrating that the initial Cu-boryl and Cu-silyl addition is significantly faster than the ensuing Cu-F elimination, and that the latter step can be facilitated by either a mild Lewis acid (e.g., a Li or Na cation) or a nucleophilic promoter (e.g., an alkoxide). These findings together with DFT studies demonstrate that Cu-F ß-elimination probably proceeds with anti-stereochemistry. Representative cases of ways through which the new mechanistic understanding may be used to rationalize previously disclosed findings, significantly improve a transformation, or develop new diastereo- and enantioselective catalytic methods are provided. For example, an explanation is provided regarding why bisphosphine-Cu complexes do not efficiently promote boryl substitutions with aryl-substituted substrates, but the corresponding silyl substitutions are facile, and how the size of a ligand can impact regioselectivity and efficiency.

Different Strategies for Designing Dual-Catalytic Enantioselective Processes: From Fully Cooperative to Non-cooperative Systems.

An emerging area of research in chemistry requires that we learn how to manage the characteristics of a pair of co-catalysts so that a transformation proceeds as we wish it to. These are processes during which one catalyst first generates a non-isolable intermediate, which then in situ undergoes a reaction that is promoted by a different catalyst. This scenario raises several design issues. Since co-catalysts often have overlapping functions, what if there is an exchange of ligands between two organometallic catalysts? How can we be certain that a co-catalyst reacts specifically with a particular intermediate? What if the less reactive co-catalyst must engage first, and the one that is more active needs to wait its turn? How might we orchestrate the proper sequence of events? While many dual-catalytic processes have been introduced and reviews are available, there are subtle yet crucial distinguishing attributes that remain unappreciated. While the terms "dual-catalysis" and "cooperative catalysis" are often used interchangeably, on many occasions the catalysts are not entirely cooperative. Here, we will discuss how chemists have been able to harmonize the opposing functions of catalysts to achieve high efficiency and/or stereoselectivity. We will show that the progress achieved thus far is likely the preamble to the future development of non-orthogonal multi-catalytic processes (i.e., transformations involving several catalysts that are not inherently cooperative) where the order with which each catalyst enters the fray will demand additional innovative strategies.

Copper-Hydride-Catalyzed Enantioselective Processes with Allenyl Boronates. Mechanistic Nuances, Scope, and Utility in Target-Oriented Synthesis.

Synthesis of complex bioactive molecules is substantially facilitated by transformations that efficiently and stereoselectively generate polyfunctional compounds. Designing such processes is hardly straightforward, however, especially when the desired route runs counter to the inherently favored reactivity profiles. Furthermore, in addition to being efficient and stereoselective, it is crucial that the products generated can be easily and stereodivergently modified. Here, we introduce a catalytic process that delivers versatile and otherwise difficult-to-access organoboron entities by combining an allenylboronate, a hydride, and an allylic phosphate. Two unique selectivity problems had to be solved: avoiding rapid side reaction of a Cu-H complex with an allylic phosphate, while promoting its addition to an allenylboronate as opposed to the commonly utilized boron-copper exchange. The utility of the approach is demonstrated by applications to concise preparation of the linear fragment of pumiliotoxin B (myotonic, cardiotonic) and enantioselective synthesis and structure confirmation of netamine C, a member of a family of anti-tumor and anti-malarial natural products. Completion of the latter routes required the following noteworthy developments: (1) a two-step all-catalytic sequence for conversion of a terminal alkene to a monosubstituted alkyne; (2) a catalytic SN2'- and enantioselective allylic substitution method involving a mild alkylzinc halide reagent; and (3) a diastereoselective [3+2]-cycloaddition to assemble the polycyclic structure of a guanidyl polycyclic natural product.