Decreased eggshell strength caused by impairment of uterine calcium transport coincide with higher bone minerals and quality in aged laying hens

Background Deteriorations in eggshell and bone quality are major challenges in aged laying hens.This study compared the differences of eggshell quality,bone parameters and their correlations as well as uterine physiologi-cal characteristics and the bone remodeling processes of hens laying eggs of different eggshell breaking strength to explore the mechanism of eggshell and bone quality reduction and their interaction.A total of 24074-week-old Hy-line Brown laying hens were selected and allocated to a high(HBS,44.83±1.31 N)or low(LBS,24.43±0.57 N)eggshell breaking strength group.Results A decreased thickness,weight and weight ratio of eggshells were observed in the LBS,accompanied with ultrastructural deterioration and total Ca reduction.Bone quality was negatively correlated with eggshell quality,marked with enhanced structures and increased components in the LBS.In the LBS,the mammillary knobs and effective layer grew slowly.At the initiation stage of eggshell calcification,a total of 130 differentially expressed genes(DEGs,122 upregulated and 8 downregulated)were identified in the uterus of hens in the LBS relative to those in the HBS.These DEGs were relevant to apoptosis due to the cellular Ca overload.Higher values of p62 protein level,caspase-8 activity,Bax protein expression and lower values of Bcl protein expression and Bcl/Bax ratio were seen in the LBS.TUNEL assay and hematoxylin-eosin staining showed a significant increase in TUNEL-positive cells and tissue damages in the uterus of the LBS.Although few DEGs were identified at the growth stage,similar uterine tissue damages were also observed in the LBS.The expressions of runt-related transcription factor 2 and osteocal-cin were upregulated in humeri of the LBS.Enlarged diameter and more structural damages of endocortical bones and decreased ash were observed in femurs of the HBS.Conclusion The lower eggshell breaking strength may be attributed to a declined Ca transport due to uterine tissue damages,which could affect eggshell calcification and lead to a weak ultrastructure.Impaired uterine Ca transport may result in reduced femoral bone resorption and increased humeral bone formation to maintain a higher mineral and bone quality in the LBS.

Genome-Wide Comparative in silico Analysis of Calcium Transporters of Rice and Sorghum

The mechanism of calcium uptake, translocation and accumulation in Poaceae has not yet been fully understood. To address this issue, we conducted genome-wide comparative in silico analysis of the calcium （Ca2＋） transporter gene family of two crop species, rice and sorghum. Gene annotation, identification of upstream cis-acting ele- ments, phylogenetic tree construction and syntenic mapping of the gene family were performed using several bio- informatics tools. A total of 31 Ca2＋ transporters, distributed on 9 out of 12 chromosomes, were predicted from rice genome, while 28 Ca2＋ transporters predicted from sorghum are distributed on all the chromosomes except chromosome 10 （Chr 10）. Interestingly, most of the genes on Chr 1 and Chr 3 show an inverse syntenic relation- ship between rice and sorghum. Multiple sequence alignment and motif analysis of these transporter proteins re- vealed high conservation between the two species. Phylogenetic tree could very well identify the subclasses of channels, ATPases and exchangers among the gene family. The in silico c/s-regulatory element analysis suggested diverse functions associated with light, stress and hormone responsiveness as well as endosperm- and meris- tem-specific gene expression. Further experiments are warranted to validate the in silico analysis of the predicted transporter gene family and elucidate the functions of Ca2＋ transporters in various biological processes.

Transcriptome-based insights into the calcium transport mechanism of chick chorioallantoic membrane

Chorioallantoic membrane(CAM)is responsible for respiratory gas exchange,eggshell calcium transport,embryonic acid-base equilibrium,allantoic ion,and water reabsorption during avian embryonic development.To further understand the timing of CAM gene expression during chick embryonic development,especially the calcium absorption mechanism,transcriptome quantitative comparative analysis was conducted on chick CAM during the embryonic period(E)of 9,13,17,and 20 days.A total of 6378 differentially expressed genes(DEGs)were identified.Functional enrichment analysis of DEGs showed that CAM DEGs were mainly involved in biological processes such as"ion transport regulation","immune response"and"cell cycle".Time series analysis of the differential genes showed that the functional cells of CAM began to proliferate and differentiate at E9 and the calcium content of egg embryo increased significantly at E13.Simultaneously,the observation of the ultrastructure of the eggshell showed that the interstice of the fiber layer was enlarged at E13,and the mastoid layer was partly exposed.Therefore,it is preliminarily inferred that CAM calcium transport starts at E13,and genes such as TRPV6,S100 A10,and RANKL cooperate to regulate calcium release and transport.

Ketamine effect on intracellular and mitochondrial calcium mobilization

The suppressive effects of ketamine on intracellular calcium has been reported in a variety of cells although the mechanisms involved are not well understood.The aim of this work was to evaluate the ketamine effect on the mitochondrial Ca^(2+)accumulation and the cellular Ca^(2+)mobilization using FLUO4-AM and flow cytometry.The results showed that mitochondria from ketamine injected animals presented a lower ability to retain calcium at concentrations higher than 20μM,as compared with controls(saline injected animals).In ad-dition,ketamine showed a significant decreased KCl-induced intracellular calcium concentration.KCl increased calcium influx through cellular depolarization.According to the data presented herein,ketamine presents a clear inhibitory effect on cytosolic Ca2+transport mechanisms,independently from their action on the calcium channel associated NMDA receptor.

Dietary supplementation with calcitriol or quercetin improved eggshell and bone quality by modulating calcium metabolism

This study was aimed to investigate the effects of dietary calcitriol or quercetin supplementation on eggshell and bone quality of laying hens.In trial 1,72 Hy-Line Brown layers(80-week-old)with weak-shelled strength(25 to 30 N)were assigned into 4 dietary treatments with 6 replicates of 3 birds and fed a basal diet(4%calcium level)or basal diets supplemented with 0.5%calcium,5μg/kg calcitriol or 500 mg/kg quercetin for 4 weeks.In trial 2,360 Hy-Line Brown layers(60-week-old)were divided into 3 groups with 8 replicates of 15 birds:control group(basal diet),calcitriol group(basal diet+5μg/kg calcitriol),and quercetin group(basal diet+500 mg/kg quercetin).This trial lasted for 12 weeks.The results showed that dietary calcitriol or quercetin improved eggshell quality in both trials(P<0.05).In trial 2,compared with the control group,both calcitriol and quercetin supplementations improved femoral bone quality,calcium retention of hens and calcium content in uterine fluid at 18.5 h post-oviposition(PO)(P<0.05),along with enhancing uterine morphology.Compared to the control group,supplemental calcitriol or quercetin up-regulated the relative mRNA expression levels of uterine transient receptor potential cation channel,subfamily V,member 6(TRPV6)at 8.5 h PO and plasma membrane calcium-ATPase(PMCA),vitamin D receptor(VDR),estrogen receptor alpha(ERα)at 18.5 h PO(P<0.05),but down-regulated the uterine caspase 3(CASP3)relative mRNA expression level at 8.5 h PO(P<0.05).Meanwhile,the femoral relative mRNA expression levels of tartrate-resistant acid phosphatase(TRAP)(up-regulated at 8.5 and 18.5 h PO)and alkaline phosphatase(ALP)(up-regulated at 8.5 h PO but down-regulated at 18.5 h PO)were also affected by calcitriol or quercetin supplementation(P<0.05).Compared to the calcitriol,quercetin increased hen-day egg production and femoral medullary bone volume/bone tissue volume but reduced femoral stiffness(P<0.05),which were accompanied by increased relative mRNA expression levels of uterine TRPV6,estrogen receptor beta(ERβ)at 18.5 h PO(P<0.05).Overall,both dietary calcitriol and quercetin could improve eggshell and bone quality by modulating calcium metabolism of aged layers.Compared to calcitriol,dietary quercetin up-regulated the expression of uterine calcium transporters,without affecting eggshell quality.

Pollen-expressed RLCKs control pollen tube burst

In angiosperms,the pollen tube enters the receptive synergid cell,where it ruptures to release its cytoplasm along with two sperm cells.This interaction is complex,and the exact signal transducers that trigger the bursting of pollen tubes are not well understood.In this study,we identify three homologous receptor-like cytoplasmic kinases(RLCKs)expressed in pollen tubes of Arabidopsis,Delayed Burst 1/2/3(DEB1/2/3),which play a crucial role in this process.These genes produce proteins localized on the plasma membrane,and their knockout causes delayed pollen tube burst and entrance of additional pollen tubes into the embryo sac due to fertilization recovery.We show that DEBs interact with the Ca^(2+)pump ACA9,influencing the dynamics of cytoplasmic Ca^(2+)in pollen tubes through phosphorylation.These results highlight the importance of DEBs as key signal transducers and the critical function of the DEB-ACA9 axis in timely pollen tube burst in synergids.

Calcium Signaling in Plant Endosymbiotic Organelles： Mechanism and Role in Physiology

Recent studies have demonstrated that chloroplasts and mitochondria evoke specific Ca2＋ signals in response to biotic and abiotic stresses in a stress-dependent manner. The identification of Ca2＋ transporters and Ca2＋signaling mol- ecules in chloroplasts and mitochondria implies that they play roles in controlling not only intra-organellar functions, but also extra-organellar processes such as plant immunity and stress responses. It appears that organellar Ca2＋ signaling might be more important to plant cell functions than previously thought. This review briefly summarizes what is known about the molecular basis of Ca2＋ signaling in plant mitochondria and chloroplasts.

Calcium-Dependent Protein Kinase CPK21 Functions in Abiotic Stress Response in Arabidopsis thaliana

Calcium-dependent protein kinases （CDPKs） comprise a family of plant serine/threonine protein kinases in which the calcium sensing domain and the kinase effector domain are combined within one molecule. So far, a biological function in abiotic stress signaling has only been reported for few CDPK isoforms, whereas the underlying biochemical mechanism for these CDPKs is still mainly unknown. Here, we show that CPK21 from Arabidopsis thaliana is biochemically activated in vivo in response to hyperosmotic stress. Loss-of-function seedlings of cpk21 are more tolerant to hyperosmotic stress and mutant plants show increased stress responses with respect to marker gene expression and metabolite accumulation. In transgenic Arabidopsis complementation lines in the cpk21 mutant background, in which either CPK21 wildtype, or a full-length enzyme variant carrying an amino-acid substitution were stably expressed, stress responsitivity was restored by CPK21 but not with the kinase inactive variant. The biochemical characterization of in planta synthesized and purified CPK21 protein revealed that within the calcium-binding domain, N-terminal EF1- and EF2-motifs compared to C-terminal EF3- and EF4-motifs differ in their contribution to calcium-regulated kinase activity, suggesting a crucial role for the N-terminal EF-hand pair. Our data provide evidence for CPK21 contributing in abiotic stress signaling and suggest that the N-terminal EF-hand pair is a calcium-sensing determinant controlling specificity of CPK21 function.

Calcium and Calmodulin-Mediated Regulation of Gene Expression in Plants

Sessile plants have developed a very delicate system to sense diverse kinds of endogenous developmental cues and exogenous environmental stimuli by using a simple Ca^2＋ ion. Calmodulin （CAM） is the predominant Ca^2＋ sensor and plays a crucial role in decoding the Ca^2＋ signatures into proper cellular responses in various cellular compartments in eukaryotes. A growing body of evidence points to the importance of Ca^2＋ and CaM in the regulation of the transcriptional process during plant responses to endogenous and exogenous stimuli. Here, we review recent progress in the identification of transcriptional regulators modulated by Ca^2＋ and CaM and in the assessment of their functional significance during plant signal transduction in response to biotic and abiotic stresses and developmental cues.

Defense-Related Calcium Signaling Mutants Uncovered via a Quantitative High-Throughput Screen in Arabidopsis thaliana

Calcium acts as a second messenger for signaling to a variety of stimuli including MAMPs （Microbe-Associated Molecular Patterns）, such as fig22 and elf18 that are derived from bacterial flagellin and elongation factor Tu, respectively. Here, Arabidopsis thaliana mutants with changed calcium elevation （cce） in response to fig22 treatment were isolated and characterized. Besides novel mutant alleles of the fig22 receptor, FLS2 （Flagellin-Sensitive 2）, and the receptor-associated kinase, BAK1 （Brassinosteroid receptor 1-Associated Kinase 1）, the new cce mutants can be categorized into two main groups--those with a reduced or an enhanced calcium elevation. Moreover, cce mutants from both groups show differ- ential phenotypes to different sets of MAMPs. Thus, these mutants will facilitate the discovery of novel components in early MAMP signaling and bridge the gaps in current knowledge of calcium signaling during plant-microbe interactions. Last but not least, the screening method is optimized for speed （covering 384 plants in 3 or 10 h） and can be adapted to genetically dissect any other stimuli that induce a change in calcium levels.

Distinctive characteristics and functions of multiple mitochondrial Ca^(2+) influx mechanisms

Intracellular Ca2+ is vital for cell physiology.Disruption of Ca2+ homeostasis contributes to human diseases such as heart failure,neuron-degeneration,and diabetes.To ensure an effective intracellular Ca2+ dynamics,various Ca2+ transport proteins localized in different cellular regions have to work in coordination.The central role of mitochondrial Ca2+ transport mechanisms in responding to physiological Ca2+ pulses in cytosol is to take up Ca2+ for regulating energy production and shaping the amplitude and duration of Ca2+ transients in various micro-domains.Since the discovery that isolated mitochondria can take up large quantities of Ca2+ approximately 5 decades ago,extensive studies have been focused on the functional characterization and implication of ion channels that dictate Ca2+ transport across the inner mitochondrial membrane.The mitochondrial Ca2+ uptake sensitive to non-specific inhibitors ruthenium red and Ru360 has long been considered as the activity of mitochondrial Ca2+ uniporter(MCU) .The general consensus is that MCU is dominantly or exclusively responsible for the mitochondrial Ca2+ influx.Since multiple Ca2+ influx mechanisms(e.g.L-,T-,and N-type Ca2+ channel) have their unique functions in the plasma membrane,it is plausible that mitochondrial inner membrane has more than just MCU to decode complex intracellular Ca2+ signaling in various cell types.During the last decade,four molecular identities related to mitochondrial Ca2+ influx mechanisms have been identified.These are mitochondrial ryanodine receptor,mitochondrial uncoupling proteins,LETM1(Ca2+ /H+ exchanger) ,and MCU and its Ca2+ sensing regulatory subunit MICU1.Here,we briefly review recent progress in these and other reported mitochondrial Ca2+ influx pathways and their differences in kinetics,Ca2+ dependence,and pharmacological characteristics.Their potential physiological and pathological implications are also discussed.

Cytosolic Ca^2＋ Signals Enhance the Vacuolar Ion Conductivity of Bulging Arabidopsis Root Hair Cells

Plant cell expansion depends on the uptake of solutes across the plasma membrane and their storage within the vacuole. In contrast to the well-studied plasma membrane, little is known about the regulation of ion transport at the vacuolar membrane. We therefore established an experimental approach to study vacuolar ion transport in intact Arabidopsis root cells, with multi-barreled microelectrodes. The subcellular position of electrodes was detected by imaging current-injected fluorescent dyes. Comparison of measurements with electrodes in the cytosol and vacuole revealed an average vacuolar membrane potential of -31 inV. Voltage clamp recordings of single vacuoles resolved the activity of voltage-independent and slowly deactivating channels. In bulging root hairs that express the Ca2＋ sensor R-GECO1, rapid elevation of the cytosolic Ca^2＋ concentration was observed, after impalement with microelectrodes, or injection of the Ca^2＋ chelator BAPTA. Elevation of the cytosolic Ca^2＋ level stimulated the activity of voltage- independent channels in the vacuolar membrane. Likewise, the vacuolar ion conductance was enhanced during a sudden increase of the cytosolic Ca^2＋ level in cells injected with fluorescent Ca^2＋ indicator FURA-2. These data thus show that cytosolic Ca^2＋ signals can rapidly activate vacuolar ion channels, which may prevent rupture of the vacuolar membrane, when facing mechanical forces.