Advances in the diagnosis of multiorgan involvement in systemic sclerosis: a focus on MRI.

PURPOSE OF REVIEW: Systemic sclerosis (SSc) is a rare chronic multisystem autoimmune disease characterized by endothelial dysfunction, tissue hypoxia, and diffuse organ fibrosis. MRI provides a radiation free approach to noninvasively assess the key manifestations of SSc in multiple organs. The purpose of this review is to summarize recent advances in MRI techniques to provide diagnostic and prognostic information in patients with SSc. RECENT FINDINGS: MRI can probe processes that play a key role in the development of SSc-related complications, including neointima proliferation, fibrosis, and hypoxia. Feature tracking and parametric mapping MRI can detect cardiac involvement at the subclinical level. Contrast-free MRI angiography with Digital Artery Volume Index (DAVIX) assessment allow comprehensive assessment of hand involvement. T1 mapping and BOLD imaging can assess SSc effects on skeletal muscle, and lung MRI is becoming a key method for imaging of interstitial lung disease. As a new exciting application, the sodium content of the skin can be quantified by 23Na MRI reflective of glycosaminoglycan content. SUMMARY: Recent advances in MRI provide a unique opportunity to study the key pathophysiologic processes and clinical manifestations of SSc in multiple organs noninvasively, which can pave the way for the development of effective therapies.

RETINOBLASTOMA-RELATED Has Both Canonical and Noncanonical Regulatory Functions During Thermo-Morphogenic Responses in Arabidopsis Seedlings.

Warm temperatures accelerate plant growth, but the underlying molecular mechanism is not fully understood. Here, we show that increasing the temperature from 22°C to 28°C rapidly activates proliferation in the apical shoot and root meristems of wild-type Arabidopsis seedlings. We found that one of the central regulators of cell proliferation, the cell cycle inhibitor RETINOBLASTOMA-RELATED (RBR), is suppressed by warm temperatures. RBR became hyper-phosphorylated at a conserved CYCLIN-DEPENDENT KINASE (CDK) site in young seedlings growing at 28°C, in parallel with the stimulation of the expressions of the regulatory CYCLIN D/A subunits of CDK(s). Interestingly, while under warm temperatures ectopic RBR slowed down the acceleration of cell proliferation, it triggered elongation growth of post-mitotic cells in the hypocotyl. In agreement, the central regulatory genes of thermomorphogenic response, including PIF4 and PIF7, as well as their downstream auxin biosynthetic YUCCA genes (YUC1-2 and YUC8-9) were all up-regulated in the ectopic RBR expressing line but down-regulated in a mutant line with reduced RBR level. We suggest that RBR has both canonical and non-canonical functions under warm temperatures to control proliferative and elongation growth, respectively.

CT attenuation correction improves quantitative risk prediction by cardiac SPECT in obese patients.

PURPOSE: This study aimed to compare the predictive value of CT attenuation-corrected stress total perfusion deficit (AC-sTPD) and non-corrected stress TPD (NC-sTPD) for major adverse cardiac events (MACE) in obese patients undergoing cadmium zinc telluride (CZT) SPECT myocardial perfusion imaging (MPI). METHODS: The study included 4,585 patients who underwent CZT SPECT/CT MPI for clinical indications (chest pain: 56%, shortness of breath: 13%, other: 32%) at Yale New Haven Hospital (age: 64 ± 12 years, 45% female, body mass index [BMI]: 30.0 ± 6.3 kg/m2, prior coronary artery disease: 18%). The association between AC-sTPD or NC-sTPD and MACE defined as the composite end point of mortality, nonfatal myocardial infarction or late coronary revascularization (> 90 days after SPECT) was evaluated with survival analysis. RESULTS: During a median follow-up of 25 months, 453 patients (10%) experienced MACE. In patients with BMI ≥ 35 kg/m2 (n = 931), those with AC-sTPD ≥ 3% had worse MACE-free survival than those with AC-sTPD < 3% (HR: 2.23, 95% CI: 1.40 - 3.55, p = 0.002) with no difference in MACE-free survival between patients with NC-sTPD ≥ 3% and NC-sTPD < 3% (HR:1.06, 95% CI:0.67 - 1.68, p = 0.78). AC-sTPD had higher AUC than NC-sTPD for the detection of 2-year MACE in patients with BMI ≥ 35 kg/m2 (0.631 versus 0.541, p = 0.01). In the overall cohort AC-sTPD had a higher ROC area under the curve (AUC, 0.641) than NC-sTPD (0.608; P = 0.01) for detection of 2-year MACE. In patients with BMI ≥ 35 kg/m2 AC sTPD provided significant incremental prognostic value beyond NC sTPD (net reclassification index: 0.14 [95% CI: 0.20 - 0.28]). CONCLUSIONS: AC sTPD outperformed NC sTPD in predicting MACE in patients undergoing SPECT MPI with BMI ≥ 35 kg/m2. These findings highlight the superior prognostic value of AC-sTPD in this patient population and underscore the importance of CT attenuation correction.

Contrast-enhanced computed tomography for ex vivo assessment of human kidneys: A proof-of-concept study.

BACKGROUND: Ex vivo perfusion of transplant-declined human organs has emerged as a promising platform to study the response of an organ to novel therapeutic strategies. However, to fully realize the capability of this platform for performing translational research in human organ pathophysiology, there is a need for robust assays to assess organ function and disease. State-of-the-art research methods rely on analyses of biopsies taken during perfusion, which both damages the organ and only provides localized information. Developing non-invasive, whole organ methods of assessment is critical to the further development of this research platform. METHODS: We use ex vivo cold infusion scanning (EXCIS) with contrast-enhanced computed tomography (CT) to quantify perfusion in kidneys preserved ex vivo. EXCIS-CT computes three complementary metrics for whole organ assessment: a dynamic assessment of contrast filling, a measure of vascular network anatomical structure, and a static assessment of perfusion heterogeneity. RESULTS: These metrics were applied to a series of six transplant-declined human kidneys, which demonstrated a range of anatomies and perfusion. Lastly, two transplant-declined human kidneys were imaged before and after a 1-h period of ex vivo normothermic perfusion (NMP). We found variable responses to NMP, with one kidney maintaining the vascular network and hemodynamics and the other showing significant changes in vessel size and spatial perfusion profile. CONCLUSIONS: EXCIS-CT provides metrics that can be used to characterize whole organ perfusion and vascular function.

The REPLUMLESS Transcription Factor Controls the Expression of the RECEPTOR-LIKE CYTOPLASMIC KINASE VI_A2 Gene Involved in Shoot and Fruit Patterning of Arabidopsis thaliana.

The promoter of the RECEPTOR-LIKE CYTOPLASMIC KINASE VI_A2 (RLCK VI_A2) gene contains nine binding sites for the REPLUMLESS (RPL) transcription factor. In agreement, the expression of the kinase gene was strongly downregulated in the rpl-4 mutant. Comparing phenotypes of loss-of-function mutants, it was revealed that both genes are involved in stem growth, phyllotaxis, organization of the vascular tissues, and the replum, highlighting potential functional interactions. The expression of the RLCKVI_A2 gene from the constitutive 35S promoter could not complement the rpl-4 phenotypes but exhibited a dominant positive effect on stem growth and affected vascular differentiation and organization. The results also indicated that the number of vascular bundles is regulated independently from stem thickness. Although our study cannot demonstrate a direct link between the RPL and RLVKVI_A2 genes, it highlights the significance of the proper developmental regulation of the RLCKVI_A2 promoter for balanced stem development.

Integration of coronary artery calcium scoring from CT attenuation scans by machine learning improves prediction of adverse cardiovascular events in patients undergoing SPECT/CT myocardial perfusion imaging.

BACKGROUND: Machine learning (ML) has been previously applied for prognostication in patients undergoing SPECT myocardial perfusion imaging (MPI). We evaluated whether including attenuation CT coronary artery calcification (CAC) scoring improves ML prediction of major adverse cardiovascular events (MACE) in patients undergoing SPECT/CT MPI. METHODS: From the REFINE SPECT Registry 4770 patients with SPECT/CT performed at a single center were included (age: 64 ± 12 years, 45% female). ML algorithm (XGBoost) inputs were clinical risk factors, stress variables, SPECT imaging parameters, and expert-observer CAC scoring using CT attenuation correction scans performed to obtain CT attenuation maps. The ML model was trained and validated using tenfold hold-out validation. Receiver Operator Characteristics (ROC) curves were analyzed for prediction of MACE. MACE-free survival was evaluated with standard survival analyses. RESULTS: During a median follow-up of 24.1 months, 475 patients (10%) experienced MACE. Higher area under the ROC curve for MACE was observed with ML when CAC scoring was included (CAC-ML score, 0.77, 95% confidence interval [CI] 0.75-0.79) compared to ML without CAC (ML score, 0.75, 95% CI 0.73-0.77, P = .005) and when compared to CAC score alone (0.71, 95% CI 0.68-0.73, P < .001). Among clinical, imaging, and stress parameters, CAC score had highest variable importance for ML. On survival analysis patients with high CAC-ML score (> 0.091) had higher event rate when compared to patients with low CAC-ML score (hazard ratio 5.3, 95% CI 4.3-6.5, P < .001). CONCLUSION: Integration of attenuation CT CAC scoring improves the predictive value of ML risk score for MACE prediction in patients undergoing SPECT MPI.

Impaired left-ventricular global longitudinal strain by feature-tracking cardiac MRI predicts mortality in systemic sclerosis.

Impaired left-ventricular (LV) and right-ventricular (RV) cardiac magnetic resonance (CMR) strain has been documented in systemic sclerosis (SSc). However, it is unknown whether the CMR strain is predictive of adverse outcomes in SSc. Therefore, we set out to investigate the prognostic value of CMR strain in SSc. Patients with SSc who underwent CMR for clinical indications between 11/2010 and 07/2020 were retrospectively studied. LV and RV strain was evaluated by feature tracking. The association between strain, late gadolinium enhancement (LGE), and survival was evaluated with time to event and Cox-regression analyses. During the study period, 42 patients with SSc (age: 57 ± 14 years, 83% female, 57% limited cutaneous SSc, SSc duration: 7 ± 8 years) underwent CMR. During the median follow-up of 3.6 years, 11 patients died (26%). Compared to surviving patients, patients who died had significantly worse LV GLS (- 8.2 ± 6.2% versus - 12.1 ± 2.9%, p = 0.03), but no difference in LV global radial, circumferential, or RV strain values. Patients within the quartile of most impaired LV GLS (≥ - 12.8%, n = 10) had worse survival when compared to patients with preserved LV GLS (< - 12.8%, n = 32, log-rank p = 0.02), which persisted after controlling for LV cardiac output, LV cardiac index, reduced LV ejection fraction, or presence of LGE. In addition, patients who had both impaired LV GLS and LGE (n = 5) had worse survival than patients with LGE or impaired GLS alone (n = 14) and compared to those without any of these features (n = 17, p = 0.003). In our retrospective cohort of patients with SSc undergoing CMR for clinical indications, LV GLS and LGE were found to be predictive of overall survival.

A Common Molecular Signature Indicates the Pre-Meristematic State of Plant Calli.

In response to different degrees of mechanical injury, certain plant cells re-enter the division cycle to provide cells for tissue replenishment, tissue rejoining, de novo organ formation, and/or wound healing. The intermediate tissue formed by the dividing cells is called a callus. Callus formation can also be induced artificially in vitro by wounding and/or hormone (auxin and cytokinin) treatments. The callus tissue can be maintained in culture, providing starting material for de novo organ or embryo regeneration and thus serving as the basis for many plant biotechnology applications. Due to the biotechnological importance of callus cultures and the scientific interest in the developmental flexibility of somatic plant cells, the initial molecular steps of callus formation have been studied in detail. It was revealed that callus initiation can follow various ways, depending on the organ from which it develops and the inducer, but they converge on a seemingly identical tissue. It is not known, however, if callus is indeed a special tissue with a defined gene expression signature, whether it is a malformed meristem, or a mass of so-called "undifferentiated" cells, as is mostly believed. In this paper, I review the various mechanisms of plant regeneration that may converge on callus initiation. I discuss the role of plant hormones in the detour of callus formation from normal development. Finally, I compare various Arabidopsis gene expression datasets obtained a few days, two weeks, or several years after callus induction and identify 21 genes, including genes of key transcription factors controlling cell division and differentiation in meristematic regions, which were upregulated in all investigated callus samples. I summarize the information available on all 21 genes that point to the pre-meristematic nature of callus tissues underlying their wide regeneration potential.

Feasibility study of PET dynamic imaging of [18F]DHMT for quantification of reactive oxygen species in the myocardium of large animals.

OBJECTIVES: We aimed to develop a dynamic imaging technique for a novel PET superoxide tracer, [18F]DHMT, to allow for absolute quantification of myocardial reactive oxygen species (ROS) production in a large animal model. METHODS: Six beagle dogs underwent a single baseline dynamic [18F]DHMT PET study, whereas one animal underwent three serial dynamic studies over the course of chronic doxorubicin administration (1 mg·kg-1·week-1 for 15 weeks). During the scans, sequential arterial blood samples were obtained for plasma metabolite correction. The optimal compartment model and graphical analysis method were identified for kinetic modeling. Values for the left ventricular (LV) net influx rate, Ki, were reported for all the studies and compared with the LV standard uptake values (SUVs) and the LV-to-blood pool SUV ratios from the 60 to 90 minute static images. Parametric images were also generated. RESULTS: [18F]DHMT followed irreversible kinetics once oxidized within the myocardium in the presence of superoxide, as evidenced by the fitting generated by the irreversible two-tissue (2Ti) compartment model and the linearity of Patlak analysis. Myocardial Ki values showed a weak correlation with LV SUV (R2 = 0.27), but a strong correlation with LV-to-blood pool SUV ratio (R2 = 0.92). Generation of high-quality parametric images showed superior myocardial to blood contrast compared to static images. CONCLUSIONS: A dynamic PET imaging technique for [18F]DHMT was developed with full and simplified kinetic modeling for absolute quantification of myocardial superoxide production in a large animal model.

PET Myocardial Blood Flow for Post-transplant Surveillance and Cardiac Allograft Vasculopathy in Heart Transplant Recipients.

PURPOSE OF REVIEW: Cardiac allograft vasculopathy (CAV) is a late-occurring complication of heart transplantation significantly limiting overall graft survival. In the last few years, evidence has been growing about the use of positron emission tomography (PET) myocardial perfusion imaging with integrated myocardial blood flow (MBF) quantification in heart transplant recipients. RECENT FINDINGS: Multiple studies have demonstrated that PET MBF assessment can be utilized to establish the diagnosis of CAV noninvasively and can be employed for prognostication. PET MBF quantification has also helped to define the link between transplant rejection and CAV. In addition, limited data suggests that PET MBF quantification can be used in heart transplant patients for serial monitoring of CAV. PET myocardial perfusion imaging integrating MBF quantification shows great promise for the evaluation of CAV with good diagnostic and prognostic performance.

Plants in Microgravity: Molecular and Technological Perspectives.

Plants are vital components of our ecosystem for a balanced life here on Earth, as a source of both food and oxygen for survival. Recent space exploration has extended the field of plant biology, allowing for future studies on life support farming on distant planets. This exploration will utilize life support technologies for long-term human space flights and settlements. Such longer space missions will depend on the supply of clean air, food, and proper waste management. The ubiquitous force of gravity is known to impact plant growth and development. Despite this, we still have limited knowledge about how plants can sense and adapt to microgravity in space. Thus, the ability of plants to survive in microgravity in space settings becomes an intriguing topic to be investigated in detail. The new knowledge could be applied to provide food for astronaut missions to space and could also teach us more about how plants can adapt to unique environments. Here, we briefly review and discuss the current knowledge about plant gravity-sensing mechanisms and the experimental possibilities to research microgravity-effects on plants either on the Earth or in orbit.

Crosstalk between the Arabidopsis Glutathione Peroxidase-Like 5 Isoenzyme (AtGPXL5) and Ethylene.

Glutathione peroxidases (GPXs) are important antioxidant enzymes in animals. Plants contain GPX-like (GPXL) enzymes, which-in contrast to GPXs-contain cysteine in their active site instead of selenocysteine. Although several studies proved their importance in development and stress responses, their interaction with ethylene (ET) signalling is not known. Our aim was to investigate the involvement of AtGPXL5 in ET biosynthesis and/or signalling using Atgpxl5 mutant and AtGPXL5 cDNA-overexpressing (OX-AtGPXL5) lines. Four-day-old dark-grown Atgpxl5 seedlings had shorter hypocotyls and primary roots, while OX-AtGPXL5 seedlings exhibited a similar phenotype as wild type under normal conditions. Six-week-old OX-AtGPXL5 plants contained less H2O2 and malondialdehyde, but higher polyamine and similar ascorbate- and glutathione contents and redox potential (EGSH) than the Col-0. One-day treatment with the ET-precursor 1-aminocyclopropane-1-carboxylic acid (ACC) induced the activity of glutathione- and thioredoxin peroxidases and some other ROS-processing enzymes. In the Atgpxl5 mutants, the EGSH became more oxidised; parallelly, it produced more ethylene after the ACC treatment than other genotypes. Although the enhanced ET evolution measured in the Atgpxl5 mutant can be the result of the increased ROS level, the altered expression pattern of ET-related genes both in the Atgpxl5 and OX-AtGPXL5 plants suggests the interplay between AtGPXL5 and ethylene signalling.

First-in-Human Experience With Peritoneal Direct Sodium Removal Using a Zero-Sodium Solution: A New Candidate Therapy for Volume Overload.

BACKGROUND: Loop diuretics have well-described toxicities, and loss of response to these agents is common. Alternative strategies are needed for the maintenance of euvolemia in heart failure (HF). Nonrenal removal of sodium directly across the peritoneal membrane (direct sodium removal [DSR]) with a sodium-free osmotic solution should result in extraction of large quantities of sodium with limited off-target solute removal. METHODS: This article describes the preclinical development and first-in-human proof of concept for DSR. Sodium-free 10% dextrose was used as the DSR solution. Porcine experiments were conducted to investigate the optimal dwell time, safety, and scalability and to determine the effect of experimental heart failure. In the human study, participants with end-stage renal disease on peritoneal dialysis (PD) underwent randomization and crossover to either a 2-hour dwell with 1 L DSR solution or standard PD solution (Dianeal 4.25% dextrose, Baxter). The primary end point was completion of the 2-hour dwell without significant discomfort or adverse events, and the secondary end point was difference in sodium removal between DSR and standard PD solution. RESULTS: Porcine experiments revealed that 1 L DSR solution removed 4.1±0.4 g sodium in 2 hours with negligible off-target solute removal and overall stable serum electrolytes. Increasing the volume of DSR solution cycled across the peritoneum increased sodium removal and substantially decreased plasma volume (P=0.005). In the setting of experimental heart failure with elevated right atrial pressure, sodium removal was ≈4 times greater than in healthy animals (P<0.001). In the human proof-of-concept study, DSR solution was well tolerated and not associated with significant discomfort or adverse events. Plasma electrolyte concentrations were stable, and off-target solute removal was negligible. Sodium removal was substantially higher with DSR (4.5±0.4 g) compared with standard PD solution (1.0±0.3 g; P<0.0001). CONCLUSIONS: DSR was well tolerated in both animals and human subjects and produced substantially greater sodium removal than standard PD solution. Additional research evaluating the use of DSR as a method to prevent and treat hypervolemia in heart failure is warranted. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03801226.

A Novel Signaling Pathway Required for Arabidopsis Endodermal Root Organization Shapes the Rhizosphere Microbiome.

The Casparian strip (CS) constitutes a physical diffusion barrier to water and nutrients in plant roots, which is formed by the polar deposition of lignin polymer in the endodermis tissue. The precise pattern of lignin deposition is determined by the scaffolding activity of membrane-bound Casparian Strip domain proteins (CASPs), but little is known of the mechanism(s) directing this process. Here, we demonstrate that Endodermis-specific Receptor-like Kinase 1 (ERK1) and, to a lesser extent, ROP Binding Kinase1 (RBK1) are also involved in regulating CS formation, with the former playing an essential role in lignin deposition as well as in the localization of CASP1. We show that ERK1 is localized to the cytoplasm and nucleus of the endodermis and that together with the circadian clock regulator, Time for Coffee (TIC), forms part of a novel signaling pathway necessary for correct CS organization and suberization of the endodermis, with their single or combined loss of function resulting in altered root microbiome composition. In addition, we found that other mutants displaying defects in suberin deposition at the CS also display altered root exudates and microbiome composition. Thus, our work reveals a complex network of signaling factors operating within the root endodermis that establish both the CS diffusion barrier and influence the microbial composition of the rhizosphere.

Evaluation of cardiac allograft vasculopathy by positron emission tomography.

Cardiac allograft vasculopathy (CAV) remains one of the most important late occurring complications in heart transplant (HT) recipients significantly effecting graft survival. Recently, there has been tremendous focus on the development of effective and safe non-invasive diagnostic strategies for the diagnosis of CAV employing a wide range of imaging technologies. During the past decade multiple studies have been published using positron emission tomography (PET) myocardial perfusion imaging, establishing the value of PET myocardial blood flow quantification for the evaluation of CAV. These independent investigations demonstrate that PET can be successfully used to establish the diagnosis of CAV, can be utilized for prognostication and may be used for serial monitoring of HT recipients. In addition, molecular imaging techniques have started to emerge as new tools to enhance our knowledge to better understand the pathophysiology of CAV.

The AtCRK5 Protein Kinase Is Required to Maintain the ROS NO Balance Affecting the PIN2-Mediated Root Gravitropic Response in Arabidopsis.

The Arabidopsis AtCRK5 protein kinase is involved in the establishment of the proper auxin gradient in many developmental processes. Among others, the Atcrk5-1 mutant was reported to exhibit a delayed gravitropic response via compromised PIN2-mediated auxin transport at the root tip. Here, we report that this phenotype correlates with lower superoxide anion (O2•-) and hydrogen peroxide (H2O2) levels but a higher nitric oxide (NO) content in the mutant root tips in comparison to the wild type (AtCol-0). The oxidative stress inducer paraquat (PQ) triggering formation of O2•- (and consequently, H2O2) was able to rescue the gravitropic response of Atcrk5-1 roots. The direct application of H2O2 had the same effect. Under gravistimulation, correct auxin distribution was restored (at least partially) by PQ or H2O2 treatment in the mutant root tips. In agreement, the redistribution of the PIN2 auxin efflux carrier was similar in the gravistimulated PQ-treated mutant and untreated wild type roots. It was also found that PQ-treatment decreased the endogenous NO level at the root tip to normal levels. Furthermore, the mutant phenotype could be reverted by direct manipulation of the endogenous NO level using an NO scavenger (cPTIO). The potential involvement of AtCRK5 protein kinase in the control of auxin-ROS-NO-PIN2-auxin regulatory loop is discussed.

The Arabidopsis RLCK VI_A2 Kinase Controls Seedling and Plant Growth in Parallel with Gibberellin.

The plant-specific receptor-like cytoplasmic kinases (RLCKs) form a large, poorly characterized family. Members of the RLCK VI_A class of dicots have a unique characteristic: their activity is regulated by Rho-of-plants (ROP) GTPases. The biological function of one of these kinases was investigated using a T-DNA insertion mutant and RNA interference. Loss of RLCK VI_A2 function resulted in restricted cell expansion and seedling growth. Although these phenotypes could be rescued by exogenous gibberellin, the mutant did not exhibit lower levels of active gibberellins nor decreased gibberellin sensitivity. Transcriptome analysis confirmed that gibberellin is not the direct target of the kinase; its absence rather affected the metabolism and signalling of other hormones such as auxin. It is hypothesized that gibberellins and the RLCK VI_A2 kinase act in parallel to regulate cell expansion and plant growth. Gene expression studies also indicated that the kinase might have an overlapping role with the transcription factor circuit (PIF4-BZR1-ARF6) controlling skotomorphogenesis-related hypocotyl/cotyledon elongation. Furthermore, the transcriptomic changes revealed that the loss of RLCK VI_A2 function alters cellular processes that are associated with cell membranes, take place at the cell periphery or in the apoplast, and are related to cellular transport and/or cell wall reorganisation.

AtCRK5 Protein Kinase Exhibits a Regulatory Role in Hypocotyl Hook Development during Skotomorphogenesis.

Seedling establishment following germination requires the fine tuning of plant hormone levels including that of auxin. Directional movement of auxin has a central role in the associated processes, among others, in hypocotyl hook development. Regulated auxin transport is ensured by several transporters (PINs, AUX1, ABCB) and their tight cooperation. Here we describe the regulatory role of the Arabidopsis thaliana CRK5 protein kinase during hypocotyl hook formation/opening influencing auxin transport and the auxin-ethylene-GA hormonal crosstalk. It was found that the Atcrk5-1 mutant exhibits an impaired hypocotyl hook establishment phenotype resulting only in limited bending in the dark. The Atcrk5-1 mutant proved to be deficient in the maintenance of local auxin accumulation at the concave side of the hypocotyl hook as demonstrated by decreased fluorescence of the auxin sensor DR5::GFP. Abundance of the polar auxin transport (PAT) proteins PIN3, PIN7, and AUX1 were also decreased in the Atcrk5-1 hypocotyl hook. The AtCRK5 protein kinase was reported to regulate PIN2 protein activity by phosphorylation during the root gravitropic response. Here it is shown that AtCRK5 can also phosphorylate in vitro the hydrophilic loops of PIN3. We propose that AtCRK5 may regulate hypocotyl hook formation in Arabidopsis thaliana through the phosphorylation of polar auxin transport (PAT) proteins, the fine tuning of auxin transport, and consequently the coordination of auxin-ethylene-GA levels.

CRK5 Protein Kinase Contributes to the Progression of Embryogenesis of Arabidopsis thaliana.

The fine tuning of hormone (e.g., auxin and gibberellin) levels and hormone signaling is required for maintaining normal embryogenesis. Embryo polarity, for example, is ensured by the directional movement of auxin that is controlled by various types of auxin transporters. Here, we present pieces of evidence for the auxin-gibberellic acid (GA) hormonal crosstalk during embryo development and the regulatory role of the Arabidopsis thaliana Calcium-Dependent Protein Kinase-Related Kinase 5 (AtCRK5) in this regard. It is pointed out that the embryogenesis of the Atcrk5-1 mutant is delayed in comparison to the wild type. This delay is accompanied with a decrease in the levels of GA and auxin, as well as the abundance of the polar auxin transport (PAT) proteins PIN1, PIN4, and PIN7 in the mutant embryos. We have previously showed that AtCRK5 can regulate the PIN2 and PIN3 proteins either directly by phosphorylation or indirectly affecting the GA level during the root gravitropic and hypocotyl hook bending responses. In this manuscript, we provide evidence that the AtCRK5 protein kinase can in vitro phosphorylate the hydrophilic loops of additional PIN proteins that are important for embryogenesis. We propose that AtCRK5 can govern embryo development in Arabidopsis through the fine tuning of auxin-GA level and the accumulation of certain polar auxin transport proteins.