Local auxin transport regulation in the nascent nodule - an overview in nodulating plants and an investigation into the cytokinin receptor, cre1 - mediated control of auxin transport in medicago truncatula

Legumes form a symbiotic relationship with a group of bacteria, collectively known as rhizobia. The bacterial symbiont fixes atmospheric nitrogen within root nodules, thus providing the host with an assimilative nitrogen source. Nodule formation involves a complex signalling pathway within the legume host. The plant hormone auxin is involved in nodule organogenesis, but how auxin regulates nodulation is still poorly described. Several studies have found increased auxin signalling in nodule primordia, but so far auxin metabolites have never been quantified during the early stages of nodulation. Therefore, the first aim of this thesis was to establish methods for auxin quantification in legume roots. The presumed build-up of auxin in nodule primordia has been predicted to be due to inhibition of auxin export from cells at the nodule initiation site, but the regulation of auxin transport has not been tested systematically in different legumes. Therefore, the second aim was to compare auxin concentrations and auxin transport changes during nodulation in different legumes. Third, the regulation of auxin transport and auxin accumulation was placed into the known signalling pathway of nodulation in the model legume, Medicago truncatula. Auxins are naturally present in low quantities in the root. We developed an LC-MS/MS method for the accurate and sensitive quantification of auxins in root tissues. The method was validated and produced sensitive limits of detection / quantification and correlation coefficients. To compare the role of auxin between indeterminate and determinate nodule types, we measured auxin transport and auxin content in M. truncatula (forming indeterminate nodules) and Lotus japonicus (forming determinate nodules). In addition to acropetal auxin transport, basipetal auxin transport was regulated in response to rhizobia inoculation in both legumes. Different auxins with distinct levels of abundance were detected in separate legumes, with some unique to the nodule tissues. Auxin concentrations increased at the early stages of nodule formation in M. truncatula, but not Lotus japonicus. The inhibition of acropetal polar auxin transport by rhizobia occurred only in indeterminate nodule-forming legumes and correlated with the ability of synthetic auxin transport inhibitors to induce pseudonodules in those legumes. Finally, we investigated the role of the cytokinin receptor CRE1 in modulating auxin transport during nodulation in M. truncatula. We found that cytokinin signalling through CRE1 is necessary for inhibition of acropetal auxin transport, increased auxin concentration and auxin signalling in response to rhizobia. The CRE1 receptor was also required for the correct induction of several flavonoids, which could act as endogenous auxin transport inhibitors. External application of those flavonoids rescued nodulation in the cre1 nodulation-deficient mutant. In conclusion, we demonstrated that the auxin transport machinery is a crucial component in the host legume that is regulated in response to rhizobia. Auxin transport changes could explain measured changes in auxin concentrations during nodule initiation of M. truncatula, but not L. japonicus. Auxin transport control is mediated by endogenous flavonoids, and both flavonoid induction and auxin transport control are regulated by cytokinin signalling in M. truncatula

Auxin transport, metabolism, and signalling during nodule initiation: indeterminate and determinate nodules

Most legumes can form a unique type of lateral organ on their roots: root nodules. These structures host symbiotic nitrogen-fixing bacteria called rhizobia. Several different types of nodules can be found in nature, but the two best-studied types are called indeterminate and determinate nodules. These two types differ with respect to the presence or absence of a persistent nodule meristem, which consistently correlates with the cortical cell layers giving rise to the nodule primordia. Similar to other plant developmental processes, auxin signalling overlaps with the site of organ initiation and meristem activity. Here, we review how auxin contributes to early nodule development. We focus on changes in auxin transport, signalling, and metabolism during nodule initiation, describing both experimental evidence and computer modelling. We discuss how indeterminate and determinate nodules may differ in their mechanisms for generating localized auxin response maxima and highlight outstanding questions for future research

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula

Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation

Flavonoids and auxin transport Inhibitors rescue symbiotic nodulation in the <em>medicago truncatula</em> cytokinin perception mutant <em>cre1</em>

Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro-and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation

Molecular signals controlling the inhibition of nodulation by nitrate in Medicago truncatula

The presence of nitrogen inhibits legume nodule formation, but the mechanism of this inhibition is poorly understood. We found that 2.5 mM nitrate and above significantly inhibited nodule initiation but not root hair curling in Medicago trunatula. We analyzed protein abundance in M. truncatula roots after treatment with either 0 or 2.5 mM nitrate in the presence or absence of its symbiont Sinorhizobium meliloti after 1, 2 and 5 days following inoculation. Two-dimensional gel electrophoresis combined with mass spectrometry was used to identify 106 differentially accumulated proteins responding to nitrate addition, inoculation or time point. While flavonoid-related proteins were less abundant in the presence of nitrate, addition of Nod gene-inducing flavonoids to the Sinorhizobium culture did not rescue nodulation. Accumulation of auxin in response to rhizobia, which is also controlled by flavonoids, still occurred in the presence of nitrate, but did not localize to a nodule initiation site. Several of the changes included defense- and redox-related proteins, and visualization of reactive oxygen species indicated that their induction in root hairs following Sinorhizobium inoculation was inhibited by nitrate. In summary, the presence of nitrate appears to inhibit nodulation via multiple pathways, including changes to flavonoid metabolism, defense responses and redox changes.This work was supported by funding from the Australian Research Council (ARC) through the ARC Centre of Excellence for Integrative Legume Research (CE0348212), a Future Fellowship to UM (FT100100669), and by an ARC Discovery Grant (DP120102970)

A snapshot of the root phenotyping landscape in 2021

Root phenotyping describes methods for measuring root properties, or traits. While root phenotyping can be challenging, it is advancing quickly. In order for the field to move forward, it is essential to understand the current state and challenges of root phenotyping, as well as the pressing needs of the root biology community. In this letter, we present and discuss the results of a survey that was created and disseminated by members of the Graduate Student and Postdoc Ambassador Program at the 11th symposium of the International Society of Root Research. This survey aimed to (1) provide an overview of the objectives, biological models and methodological approaches used in root phenotyping studies, and (2) identify the main limitations currently faced by plant scientists with regard to root phenotyping. Our survey highlighted that (1) monocotyledonous crops dominate the root phenotyping landscape, (2) root phenotyping is mainly used to quantify morphological and architectural root traits, (3) 2D root scanning/imaging is the most widely used root phenotyping technique, (4) time-consuming tasks are an important barrier to root phenotyping, (5) there is a need for standardised, high-throughput methods to sample and phenotype roots, particularly under field conditions, and to improve our understanding of trait-function relationships

An integrated command and control architecture concept for unmanned systems in the year 2030

U.S. Forces require an integrated Command and Control Architecture that enables operations of a dynamic mix of manned and unmanned systems. The level of autonomous behavior correlates to: 1) the amount of trust with the reporting vehicles, and 2) the multi-spectral perspective of the observations. The intent to illuminate the architectural issues for force protection in 2030 was based on a multi-phased analytical model of High Value Unit (HVU) defense. The results showed that autonomous unmanned aerial vehicles are required to defeat high-speed incoming missiles. To evaluate the level of autonomous behavior required for an integrated combat architecture, geometric distributions were modeled to determine force positioning, based on a scenario driven Detect-to-Engage timeline. Discrete event simulation was used to schedule operations, and a datalink budget assessment of communications to determine the critical failure paths in the the integrated combat architecture. The command and control principles used in the integrated combat architecture were based on Boyd's OODA (Obseve, Orient, Decide, and Act) Loop. A conservative fleet size estimate, given the uncertainties of the coverage overlap and radar detection range, a fleet size of 35 should be anticipated given an UAV detection range of 20km and radar coverage overlap of 4 seconds.http://archive.org/details/anintegratedcomm109455244US Navy (USN) authorsApproved for public release; distribution is unlimited