NUA positively regulates plant immunity by coordination with ESD4 to deSUMOylate TPR1 in Arabidopsis.

Nuclear pore complex (NPC) is composed of multiple nucleoporins (Nups). A plethora of studies have highlighted the significance of NPC in plant immunity. However, the specific roles of individual Nups are poorly understood. NUCLEAR PORE ANCHOR (NUA) is a component of NPC. Loss of NUA leads to an increase in SUMO conjugates and pleiotropic developmental defects in Arabidopsis thaliana. Herein, we revealed that NUA is required for plant defense against multiple pathogens. NUCLEAR PORE ANCHOR associates with the transcriptional corepressor TOPLESS-RELATED1 (TPR1) and contributes to TPR1 deSUMOylation. Significantly, NUA-interacting protein EARLY IN SHORT DAYS 4 (ESD4), a SUMO protease, specifically deSUMOylates TPR1. It has been previously established that the SUMO E3 ligase SAP AND MIZ1 DOMAIN-CONTAINING LIGASE 1 (SIZ1)-mediated SUMOylation of TPR1 represses the immune-related function of TPR1. Consistent with this notion, the hyper-SUMOylated TPR1 in nua-3 leads to upregulated expression of TPR1 target genes and compromised TPR1-mediated disease resistance. Taken together, our work uncovers a mechanism by which NUA positively regulates plant defense responses by coordination with ESD4 to deSUMOylate TPR1. Our findings, together with previous studies, reveal a regulatory module in which SIZ1 and NUA/ESD4 control the homeostasis of TPR1 SUMOylation to maintain proper immune output.

Predicting an individual's cerebellar activity from functional connectivity fingerprints.

The cerebellum is gaining scientific attention as a key neural substrate of cognitive function; however, individual differences in the cerebellar organization have not yet been well studied. Individual differences in functional brain organization can be closely tied to individual differences in brain connectivity. 'Connectome Fingerprinting' is a modeling approach that predicts an individual's brain activity from their connectome. Here, we extend 'Connectome Fingerprinting' (CF) to the cerebellum. We examined functional MRI data from 160 subjects (98 females) of the Human Connectome Project young adult dataset. For each of seven cognitive task paradigms, we constructed CF models from task activation maps and resting-state cortico-cerebellar functional connectomes, using a set of training subjects. For each model, we then predicted task activation in novel individual subjects, using their resting-state functional connectomes. In each cognitive paradigm, the CF models predicted individual subject cerebellar activity patterns with significantly greater precision than did predictions from the group average task activation. Examination of the CF models revealed that the cortico-cerebellar connections that carried the most information were those made with the non-motor portions of the cerebral cortex. These results demonstrate that the fine-scale functional connectivity between the cerebral cortex and cerebellum carries important information about individual differences in cerebellar functional organization. Additionally, CF modeling may be useful in the examination of patients with cerebellar dysfunction, since model predictions require only resting-state fMRI data which is more easily obtained than task fMRI.

Functional network reconfiguration supporting memory-guided attention.

Studies have identified several brain regions whose activations facilitate attentional deployment via long-term memories. We analyzed task-based functional connectivity at the network and node-specific level to characterize large-scale communication between brain regions underlying long-term memory guided attention. We predicted default mode, cognitive control, and dorsal attention subnetworks would contribute differentially to long-term memory guided attention, such that network-level connectivity would shift based on attentional demands, requiring contribution of memory-specific nodes within default mode and cognitive control subnetworks. We expected that these nodes would increase connectivity with one another and with dorsal attention subnetworks during long-term memory guided attention. Additionally, we hypothesized connectivity between cognitive control and dorsal attention subnetworks facilitating external attentional demands. Our results identified both network-based and node-specific interactions that facilitate different components of LTM-guided attention, suggesting a crucial role across the posterior precuneus and restrosplenial cortex, acting independently from the divisions of default mode and cognitive control subnetworks. We found a gradient of precuneus connectivity, with dorsal precuneus connecting to cognitive control and dorsal attention regions, and ventral precuneus connecting across all subnetworks. Additionally, retrosplenial cortex showed increased connectivity across subnetworks. We suggest that connectivity from dorsal posterior midline regions is critical for the integration of external information with internal memory that facilitates long-term memory guided attention.

Commercial scale genetic transformation of mature seed embryo explants in maize.

A novel, efficient maize genetic transformation system was developed using Agrobacterium-mediated transformation of embryo explants from mature seeds. Seeds from field grown plants were sterilized and crushed to isolate embryo explants consisting of the coleoptile, leaf primordia, and shoot apical meristem which were then purified from the ground seed bulk preparation. The infection of relevant tissues of seed embryo explants (SEEs) by Agrobacterium was improved by the centrifugation of the explants. Transgenic plants were obtained by multiple bud induction on high cytokinin media, followed by plant regeneration on hormone-free medium. Three different selectable markers (cp4 epsps, aadA, and nptII) were successfully used for producing transgenic plants. Stable integration of transgenes in the maize genome was demonstrated by molecular analyses and germline transmission of the inserted transgenes to the next generation was confirmed by pollen segregation and progeny analysis. Phenotypic evidence for chimeric transgenic tissue was frequently observed in initial experiments but was significantly reduced by including a second bud induction step with optimized cytokinin concentration. Additional improvements, including culturing explants at an elevated temperature during bud induction led to the development of a revolutionary system for efficient transgenic plant production and genome editing. To our knowledge, this is the first report of successful transgenic plant regeneration through Agrobacterium-mediated transformation of maize mature SEEs. This system starts with mature seed that can be produced in large volumes and the SEEs explants are storable. It has significant advantages in terms of scalability and flexibility over methods that rely on immature explants.

The Na+/H+ antiporter SALT OVERLY SENSITIVE 1 regulates salt compensation of circadian rhythms by stabilizing GIGANTEA in Arabidopsis.

The circadian clock is a timekeeping, homeostatic system that temporally coordinates all major cellular processes. The function of the circadian clock is compensated in the face of variable environmental conditions ranging from normal to stress-inducing conditions. Salinity is a critical environmental factor affecting plant growth, and plants have evolved the SALT OVERLY SENSITIVE (SOS) pathway to acquire halotolerance. However, the regulatory systems for clock compensation under salinity are unclear. Here, we show that the plasma membrane Na+/H+ antiporter SOS1 specifically functions as a salt-specific circadian clock regulator via GIGANTEA (GI) in Arabidopsis thaliana. SOS1 directly interacts with GI in a salt-dependent manner and stabilizes this protein to sustain a proper clock period under salinity conditions. SOS1 function in circadian clock regulation requires the salt-mediated secondary messengers cytosolic free calcium and reactive oxygen species, pointing to a distinct regulatory role for SOS1 in addition to its function as a transporter to maintain Na+ homeostasis. Our results demonstrate that SOS1 maintains homeostasis of the salt response under high or daily fluctuating salt levels. These findings highlight the genetic capacity of the circadian clock to maintain timekeeping activity over a broad range of salinity levels.

Modelling of plant circadian clock for characterizing hypocotyl growth under different light quality conditions.

To meet the ever-increasing global food demand, the food production rate needs to be increased significantly in the near future. Speed breeding is considered as a promising agricultural technology solution to achieve the zero-hunger vision as specified in the United Nations Sustainable Development Goal 2. In speed breeding, the photoperiod of the artificial light has been manipulated to enhance crop productivity. In particular, regulating the photoperiod of different light qualities rather than solely white light can further improve speed breading. However, identifying the optimal light quality and the associated photoperiod simultaneously remains a challenging open problem due to complex interactions between multiple photoreceptors and proteins controlling plant growth. To tackle this, we develop a first comprehensive model describing the profound effect of multiple light qualities with different photoperiods on plant growth (i.e. hypocotyl growth). The model predicts that hypocotyls elongated more under red light compared to both red and blue light. Drawing similar findings from previous related studies, we propose that this might result from the competitive binding of red and blue light receptors, primarily Phytochrome B (phyB) and Cryptochrome 1 (cry1) for the core photomorphogenic regulator, CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1). This prediction is validated through an experimental study on Arabidopsis thaliana. Our work proposes a potential molecular mechanism underlying plant growth under different light qualities and ultimately suggests an optimal breeding protocol that takes into account light quality.

Extended Frontal Networks for Visual and Auditory Working Memory.

Working memory (WM) supports the persistent representation of transient sensory information. Visual and auditory stimuli place different demands on WM and recruit different brain networks. Separate auditory- and visual-biased WM networks extend into the frontal lobes, but several challenges confront attempts to parcellate human frontal cortex, including fine-grained organization and between-subject variability. Here, we use differential intrinsic functional connectivity from 2 visual-biased and 2 auditory-biased frontal structures to identify additional candidate sensory-biased regions in frontal cortex. We then examine direct contrasts of task functional magnetic resonance imaging during visual versus auditory 2-back WM to validate those candidate regions. Three visual-biased and 5 auditory-biased regions are robustly activated bilaterally in the frontal lobes of individual subjects (N = 14, 7 women). These regions exhibit a sensory preference during passive exposure to task stimuli, and that preference is stronger during WM. Hierarchical clustering analysis of intrinsic connectivity among novel and previously identified bilateral sensory-biased regions confirms that they functionally segregate into visual and auditory networks, even though the networks are anatomically interdigitated. We also observe that the frontotemporal auditory WM network is highly selective and exhibits strong functional connectivity to structures serving non-WM functions, while the frontoparietal visual WM network hierarchically merges into the multiple-demand cognitive system.

Frequency, composition and mobility of Escherichia coli-derived transposable elements in holdings of plasmid repositories.

By providing the scientific community with uniform and standardized resources of consistent quality, plasmid repositories play an important role in enabling scientific reproducibility. Plasmids containing insertion sequence elements (IS elements) represent a challenge from this perspective, as they can change the plasmid structure and function. In this study, we conducted a systematic analysis of a subset of plasmid stocks distributed by plasmid repositories (The Arabidopsis Biological Resource Center and Addgene) which carry unintended integrations of bacterial mobile genetic elements. The integration of insertion sequences was most often found in, but not limited to, pBR322-derived vectors, and did not affect the function of the specific plasmids. In certain cases, the entire stock was affected, but the majority of the stocks tested contained a mixture of the wild-type and the mutated plasmids, suggesting that the acquisition of IS elements likely occurred after the plasmids were acquired by the repositories. However, comparison of the sequencing results of the original samples revealed that some plasmids already carried insertion mutations at the time of donation. While an extensive BLAST analysis of 47 877 plasmids sequenced from the Addgene repository uncovered IS elements in only 1.12%, suggesting that IS contamination is not widespread, further tests showed that plasmid integration of IS elements can propagate in conventional Escherichia coli hosts over a few tens of generations. Use of IS-free E. coli hosts prevented the emergence of IS insertions as well as that of small indels, suggesting that the use of IS-free hosts by donors and repositories could help limit unexpected and unwanted IS integrations into plasmids.

TOC1 clock protein phosphorylation controls complex formation with NF-YB/C to repress hypocotyl growth.

Plant photoperiodic growth is coordinated by interactions between circadian clock and light signaling networks. How post-translational modifications of clock proteins affect these interactions to mediate rhythmic growth remains unclear. Here, we identify five phosphorylation sites in the Arabidopsis core clock protein TIMING OF CAB EXPRESSION 1 (TOC1) which when mutated to alanine eliminate detectable phosphorylation. The TOC1 phospho-mutant fails to fully rescue the clock, growth, and flowering phenotypes of the toc1 mutant. Further, the TOC1 phospho-mutant shows advanced phase, a faster degradation rate, reduced interactions with PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) and HISTONE DEACETYLASE 15 (HDA15), and poor binding at pre-dawn hypocotyl growth-related genes (PHGs), leading to a net de-repression of hypocotyl growth. NUCLEAR FACTOR Y subunits B and C (NF-YB/C) stabilize TOC1 at target promoters, and this novel trimeric complex (NF-TOC1) acts as a transcriptional co-repressor with HDA15 to inhibit PIF-mediated hypocotyl elongation. Collectively, we identify a molecular mechanism suggesting how phosphorylation of TOC1 alters its phase, stability, and physical interactions with co-regulators to precisely phase PHG expression to control photoperiodic hypocotyl growth.

Neural correlates associated with impaired global motion perception in cerebral visual impairment (CVI).

Cerebral visual impairment (CVI) is associated with a wide range of visual perceptual deficits including global motion processing. However, the underlying neurophysiological basis for these impairments remain poorly understood. We investigated global motion processing abilities in individuals with CVI compared to neurotypical controls using a combined behavioral and multi-modal neuroimaging approach. We found that CVI participants had a significantly higher mean motion coherence threshold (determined using a random dot kinematogram pattern simulating optic flow motion) compared to controls. Using functional magnetic resonance imaging (fMRI), we investigated activation response profiles in functionally defined early (i.e. primary visual cortex; area V1) and higher order (i.e. middle temporal cortex; area hMT+) stages of motion processing. In area V1, responses to increasing motion coherence were similar in both groups. However, in the CVI group, activation in area hMT+ was significantly reduced compared to controls, and consistent with a surround facilitation (rather than suppression) response profile. White matter tract reconstruction obtained from high angular resolution diffusion imaging (HARDI) revealed evidence of increased mean, axial, and radial diffusivities within cortico-cortical (i.e. V1-hMT+), but not thalamo-hMT+ connections. Overall, our results suggest that global motion processing deficits in CVI may be associated with impaired signal integration and segregation mechanisms, as well as white matter integrity at the level of area hMT+.

Cost-effective circadian mechanism: rhythmic degradation of circadian proteins spontaneously emerges without rhythmic post-translational regulation.

Circadian protein oscillations are maintained by the lifelong repetition of protein production and degradation in daily balance. It comes at the cost of ever-replayed, futile protein synthesis each day. This biosynthetic cost with a given oscillatory protein profile is relievable by a rhythmic, not constant, degradation rate that selectively peaks at the right time of day but remains low elsewhere, saving much of the gross protein loss and of the replenishing protein synthesis. Here, our mathematical modeling reveals that the rhythmic degradation rate of proteins with circadian production spontaneously emerges under steady and limited activity of proteolytic mediators and does not necessarily require rhythmic post-translational regulation of previous focus. Additional (yet steady) post-translational modifications in a proteolytic pathway can further facilitate the degradation's rhythmicity in favor of the biosynthetic cost saving. Our work is supported by animal and plant circadian data, offering a generic mechanism for potentially widespread, time-dependent protein turnover.

Individual Subject Approaches to Mapping Sensory-Biased and Multiple-Demand Regions in Human Frontal Cortex.

Sensory modality, widely accepted as a key factor in the functional organization of posterior cortical areas, also shapes the organization of human frontal lobes. 'Deep imaging,' or the practice of collecting a sizable amount of data on individual subjects, offers significant advantages in revealing fine-scale aspects of functional organization of the human brain. Here, we review deep imaging approaches to mapping multiple sensory-biased and multiple-demand regions within human lateral frontal cortex. In addition, we discuss how deep imaging methods can be transferred to large public data sets to further extend functional mapping at the group level. We also review how 'connectome fingerprinting' approaches, combined with deep imaging, can be used to localize fine-grained functional organization in individual subjects using resting-state data. Finally, we summarize current 'best practices' for deep imaging.

Attention and Default Mode Network Assessments of Meditation Experience during Active Cognition and Rest.

Meditation experience has previously been shown to improve performance on behavioral assessments of attention, but the neural bases of this improvement are unknown. Two prominent, strongly competing networks exist in the human cortex: a dorsal attention network, that is activated during focused attention, and a default mode network, that is suppressed during attentionally demanding tasks. Prior studies suggest that strong anti-correlations between these networks indicate good brain health. In addition, a third network, a ventral attention network, serves as a "circuit-breaker" that transiently disrupts and redirects focused attention to permit salient stimuli to capture attention. Here, we used functional magnetic resonance imaging to contrast cortical network activation between experienced focused attention Vipassana meditators and matched controls. Participants performed two attention tasks during scanning: a sustained attention task and an attention-capture task. Meditators demonstrated increased magnitude of differential activation in the dorsal attention vs. default mode network in a sustained attention task, relative to controls. In contrast, there were no evident attention network differences between meditators and controls in an attentional reorienting paradigm. A resting state functional connectivity analysis revealed a greater magnitude of anticorrelation between dorsal attention and default mode networks in the meditators as compared to both our local control group and a n = 168 Human Connectome Project dataset. These results demonstrate, with both task- and rest-based fMRI data, increased stability in sustained attention processes without an associated attentional capture cost in meditators. Task and resting-state results, which revealed stronger anticorrelations between dorsal attention and default mode networks in experienced mediators than in controls, are consistent with a brain health benefit of long-term meditation practice.

Towards Neuroscience of the Everyday World (NEW) using functional Near-Infrared Spectroscopy.

Functional Near-Infrared Spectroscopy (fNIRS) assesses human brain activity by noninvasively measuring changes of cerebral hemoglobin concentrations caused by modulation of neuronal activity. Recent progress in signal processing and advances in system design, such as miniaturization, wearability and system sensitivity, have strengthened fNIRS as a viable and cost-effective complement to functional Magnetic Resonance Imaging (fMRI), expanding the repertoire of experimental studies that can be performed by the neuroscience community. The availability of fNIRS and Electroencephalography (EEG) for routine, increasingly unconstrained, and mobile brain imaging is leading towards a new domain that we term "Neuroscience of the Everyday World" (NEW). In this light, we review recent advances in hardware, study design and signal processing, and discuss challenges and future directions towards achieving NEW.

Post-Translational Mechanisms of Plant Circadian Regulation.

The molecular components of the circadian system possess the interesting feature of acting together to create a self-sustaining oscillator, while at the same time acting individually, and in complexes, to confer phase-specific circadian control over a wide range of physiological and developmental outputs. This means that many circadian oscillator proteins are simultaneously also part of the circadian output pathway. Most studies have focused on transcriptional control of circadian rhythms, but work in plants and metazoans has shown the importance of post-transcriptional and post-translational processes within the circadian system. Here we highlight recent work describing post-translational mechanisms that impact both the function of the oscillator and the clock-controlled outputs.

Stimulus-Specific Visual Working Memory Representations in Human Cerebellar Lobule VIIb/VIIIa.

fMRI research has revealed that cerebellar lobule VIIb/VIIIa exhibits load-dependent activity that increases with the number of items held in visual working memory (VWM). However, it remains unclear whether these cerebellar responses reflect processes specific to VWM or more general visual attentional mechanisms. To investigate this question, we examined whether cerebellar activity during the delay period of a VWM task is selective for stimuli held in working memory. A sample of male and female human subjects performed a VWM continuous report task in which they were retroactively cued to remember the direction of motion of moving dot stimuli. Cerebellar lobule VIIb/VIIIa delay-period activation accurately decoded the direction of the remembered stimulus, as did frontal and parietal regions of the dorsal attention network. Arguing against a motor explanation, no other cerebellar area exhibited stimulus specificity, including the oculomotor vermis, a key area associated with eye movement control. Finer-scale analysis revealed that the medial portion of lobule VIIb and to a lesser degree the lateral most portion of lobules VIIb and VIIIa, which exhibit robust resting state connectivity with frontal and parietal regions of the dorsal attention network, encoded the identity of the remembered stimulus, while intermediate portions of lobule VIIb/VIIIa did not. These findings of stimulus-specific coding of VWM within lobule VIIb/VIIIa indicate for the first time that the distributed network responsible for the encoding and maintenance of mnemonic representations extends to the cerebellum.SIGNIFICANCE STATEMENT There is considerable debate concerning where in the brain the contents of visual working memory (VWM) are stored. To date, this literature has primarily focused on the role of regions located within cerebral cortex. There is growing evidence for cerebellar involvement in higher-order cognitive functions including working memory. While the cerebellum has been previously shown to be recruited by VWM paradigms, it is unclear whether any portion of cerebellum actively encodes and maintains mnemonic representations. The present study demonstrates that cerebellar lobule VIIb/VIIIa activity patterns are selective for remembered stimuli and that this selectivity persists in the absence of perceptual input. These findings provide novel evidence for the participation of cerebellar structures in the persistent storage of visual information.

Handling Arabidopsis and Other Brassicaceae: Growth, Preservation of Seeds, Transformation, and Genetic Crosses.

Achieving optimal plant growth is essential for the advancement of Arabidopsis thaliana (Arabidopsis) research. Over the last 20 years, the Arabidopsis Biological Resource Center (ABRC) has collected and developed a series of best-practice protocols, some of which are presented in this chapter. Arabidopsis can be grown in a variety of locations, growth media, and environmental conditions. Some mutant genotypes, natural accessions, and Arabidopsis relatives require strictly controlled growth conditions best provided by growth rooms, chambers, or incubators. Other lines can be grown in less-controlled greenhouse settings. Although the majority of lines can be grown in soil, certain experimental purposes require utilization of sterile solid or liquid growth media. These include the selection of primary transformants, identification of homozygous lethal individuals in a segregating population, or bulking of a large amount of plant material. The importance of controlling, observing, and recording growth conditions is emphasized and appropriate equipment for monitoring these conditions is listed. Proper conditions for seed harvest and preservation, as well as seed quality control procedures, are also described. In addition, plant transformation and genetic crosses, two of the methods that revolutionized Arabidopsis genetics, are discussed.

Gradients of functional organization in posterior parietal cortex revealed by visual attention, visual short-term memory, and intrinsic functional connectivity.

Visual attention and visual working memory tasks recruit a common network of lateral frontal cortical (LFC) and posterior parietal cortical (PPC) regions. Here, we examine finer-scale organization of this frontoparietal network. Three LFC regions recruited by visual cognition tasks, superior precentral sulcus (sPCS), inferior precentral sulcus (iPCS), and mid inferior frontal sulcus (midIFS) exhibit differential patterns of resting-state functional connectivity to PPC. A broad dorsomedial to ventrolateral gradient is observed, with sPCS connectivity dominating in the dorsomedial PPC band, iPCS dominating in the middle band, and midIFS dominating in the ventrolateral band. These connectivity-defined subregions of PPC capture differential task activation between a pair of visual attention and working memory tasks. The relative functional connectivity of sPCS and iPCS also varies along the rostral-caudal axis of the retinotopic regions of PPC. iPCS connectivity is relatively stronger near the IPS0/IPS1 and IPS2/IPS3 borders, especially on the lateral portions of these borders, which each preferentially encode central visual field representations. In contrast, sPCS connectivity is relatively stronger elsewhere in retinotopic IPS regions which preferentially encode peripheral visual field representations. These findings reveal fine-scale gradients in functional connectivity within the frontoparietal visual network that capture a high-degree of specificity in PPC functional organization.

Compensatory Mutations in GI and ZTL May Modulate Temperature Compensation in the Circadian Clock.

Circadian systems share the three properties of entrainment, free-running period, and temperature compensation (TC). TC ensures nearly the same period over a broad range of physiologically relevant temperatures; however, the mechanisms behind TC remain poorly understood. Here, we identify single point mutations in two key elements of the Arabidopsis circadian clock, GIGANTEA (GI) and ZEITLUPE (ZTL), which likely act as compensatory substitutions to establish a remarkably constant free-running period over a wide range of temperatures. Using near-isogenic lines generated from the introgression of the Cape Verde Islands (Cvi) alleles of GI and ZTL into the Landsberg erecta (Ler) background, we show how longer periods in the Cvi background at higher temperatures correlate with a difference in strength of the GI/ZTL interaction. Pairwise interaction testing of all GI/ZTL allelic combinations shows similar affinities for isogenic alleles at 22°C, but very poor interaction between GI (Cvi) and ZTL (Cvi) at higher temperature. In vivo, this would result in lower ZTL levels at high temperatures leading to longer periods in the Cvi background. Mismatched allelic combinations result in extremely strong or weak GI/ZTL interactions, indicating how the corresponding natural variants likely became fixed through epistatic selection. Additionally, molecular characterization of GI (Cvi) reveals a novel functional motif that can modulate the GI/ZTL interaction as well as nucleocytoplasmic partitioning. Taken together, these results identify a plausible temperature-dependent molecular mechanism, which contributes to the robustness of TC through natural variation in GI and ZTL alleles found on the Cape Verde Islands.