Involvement of Vacuolar Processing Enzyme CgVPE1 in Vacuole Rupture in the Programmed Cell Death during the Development of the Secretory Cavity in Citrus grandis 'Tomentosa' Fruits.

Vacuolar processing enzymes (VPEs) with caspase-1-like activity are closely associated with vacuole rupture. The destruction of vacuoles is one of the characteristics of programmed cell death (PCD) in plants. However, whether VPE is involved in the vacuole destruction of cells during secretory cavity formation in Citrus plants remains unclear. This research identified a CgVPE1 gene that encoded the VPE and utilized cytology and molecular biology techniques to explore its temporal and spatial expression characteristics during the PCD process of secretory cavity cells in the Citrus grandis 'Tomentosa' fruit. The results showed that CgVPE1 is an enzyme with VPE and caspase-1-like activity that can self-cleave into a mature enzyme in an acidic environment. CgVPE1 is specifically expressed in the epithelial cells of secretory cavities. In addition, it mainly accumulates in vacuoles before it is ruptured in the secretory cavity cells. The spatial and temporal immunolocalization of CgVPE1 showed a strong relationship with the change in vacuole structure during PCD in secretory cavity cells. In addition, the change in the two types of VPE proteins from proenzymes to mature enzymes was closely related to the change in CgVPE1 localization. Our results indicate that CgVPE1 plays a vital role in PCD, causing vacuole rupture in cells during the development of the secretory cavity in C. grandis 'Tomentosa' fruits.

Transcriptome and Metabolome Analyses Provide Insights into the Flavonoid Accumulation in Peels of Citrus reticulata 'Chachi'.

The quality of Chinese medicinal materials depends on the content of bioactive components, which are affected by the environmental factors of different planting regions. In this research, integrated analysis of the transcriptome and metabolome of C. reticulata 'Chachi' was performed in two regions, and three orchards were included in the analysis. In total, only 192 compounds were found in fresh peels, and among 18 differentially accumulated flavonoid metabolites, 15 flavonoids were enriched in peels from the Xinhui planting region. In total, 1228 genes were up-regulated in peels from Xinhui, including the CHS and GST genes, which are involved in the salt stress response. Overall, based on the correlation analysis of flavonoid content and gene expression in peels of C. reticulata 'Chachi', we concluded that the authenticity of the GCRP from Xinhui may be closely related to the higher content of naringin and narirutin, and the increase in the content of these may be due to the highly saline environment of the Xinhui region.

The expansion of mesophyll cells is coordinated with the division of chloroplasts in diploid and tetraploid Arabidopsis thaliana.

MAIN CONCLUSION: Cell expression is coordinated with chloroplast division in diploid and tetraploid Arabidopsis thaliana, polyploidy promoted the expansion of mesophyll cells and chloroplast division in A. thaliana. Cell development and differentiation are always accompanied by cell expansion and chloroplast division in plants, but the relationship between them is still relatively unknown. To confirm the relationship between cell expansion and chloroplast division during the leaf development process of diploid and tetraploid Arabidopsis thaliana, we systematically analyzed the expansion of mesophyll cells and the division of chloroplasts through cytological observation and gene-expression characteristics. As a result, in diploid and tetraploid A. thaliana, there were two peaks in both mesophyll cell expansion and chloroplast division during the leaf development process. Tetraploid A. thaliana mesophyll cells were larger and contained more chloroplasts than diploid A. thaliana mesophyll cells, which indicated that cell division and cell expansion were coordinated with chloroplast division in A. thaliana and that polyploidy further promoted mesophyll cell expansion and chloroplast division.

Ca2+-dependent nuclease is involved in DNA degradation during the formation of the secretory cavity by programmed cell death in fruit of Citrus grandis 'Tomentosa'.

The secretory cavity is a typical structure in Citrus fruit and is formed by schizolysigeny. Previous reports have indicated that programmed cell death (PCD) is involved in the degradation of secretory cavity cells in the fruit, and that the spatio-temporal location of calcium is closely related to nuclear DNA degradation in this process; however, the molecular mechanisms underlying this Ca2+ regulation remain largely unknown. Here, we identified CgCaN that encodes a Ca2+-dependent DNase in the fruit of Citrus grandis 'Tomentosa', the function of which was studied using calcium ion localization, DNase activity assays, in situ hybridization, and protein immunolocalization. The results suggested that the full-length cDNA of CgCaN contains an ORF of 1011 bp that encodes a protein 336 amino acids in length with a SNase-like functional domain. CgCaN digests dsDNA at neutral pH in a Ca2+-dependent manner. In situ hybridization signals of CgCaN were particularly distributed in the secretory cavity cells. Ca2+ and Ca2+-dependent DNases were mainly observed in the condensed chromatin and in the nucleolus. In addition, spatio-temporal expression patterns of CgCaN and its protein coincided with the time-points that corresponded to chromatin degradation and nuclear rupture during the PCD in the development of the fruit secretory cavity. Taken together, our results suggest that Ca2+-dependent DNases play direct roles in nuclear DNA degradation during the PCD of secretory cavity cells during Citrus fruit development. Given the consistency of the expression patterns of genes regulated by calmodulin (CaM) and calcium-dependent protein kinases (CDPK) and the dynamics of calcium accumulation, we speculate that CaM and CDPK proteins might be involved in Ca2+ transport from the extracellular walls through the cytoplasm and into the nucleus to activate CgCaN for DNA degradation.

CELLULASE6 and MANNANASE7 Affect Cell Differentiation and Silique Dehiscence.

Cellulases, hemicellulases, and pectinases play important roles in fruit development and maturation. Although mutants with defects in these processes have not been reported for cellulase or hemicellulase genes, the pectinases ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE1 (ADPG1) and ADPG2 were previously shown to be essential for silique dehiscence in Arabidopsis (Arabidopsis thaliana). Here, we demonstrate that the cellulase gene CELLULASE6 (CEL6) and the hemicellulase gene MANNANASE7 (MAN7) function in the development and dehiscence of Arabidopsis siliques. We found that these genes were expressed in both vegetative and reproductive organs and that their expression in the silique partially depended on the INDEHISCENT and ALCATRAZ transcription factors. Cell differentiation was delayed in the dehiscence zone of cel6 and man7 mutant siliques at early flower development stage 17, and a comparison of the spatio-temporal patterns of CEL6 and MAN7 expression with the locations of delayed cell differentiation in the cel6 and man7 mutants revealed that CEL6 and MAN7 likely indirectly affect the timing of cell differentiation in the silique valve at this stage. CEL6 and MAN7 were also found to promote cell degeneration in the separation layer in nearly mature siliques, as cells in this layer remained intact in the cel6 and man7 mutants and the cel6-1 man7-3 double mutant, whereas they degenerated in the wild-type control. Phenotypic studies of single, double, triple, and quadruple mutants revealed that higher-order mutant combinations of cel6-1, man7-3, and adpg1-1 and adpg2-1 produced more severe silique indehiscent phenotypes than the corresponding lower-order mutant combinations, except for some combinations involving cel6-1, man7-3, and adpg2-1 Our results demonstrate that the ability of the silique to dehisce can be manipulated to different degrees by altering the activities of various cell wall-modifying enzymes.

Ca2+- and Zn2+-dependent nucleases co-participate in nuclear DNA degradation during programmed cell death in secretory cavity development in Citrus fruits.

Calcium (Ca2+)- and zinc Zn2+-dependent nucleases play pivotal roles in plant nuclear DNA degradation in programmed cell death (PCD). However, the mechanisms by which these two nucleases co-participate in PCD-associated nuclear DNA degradation remain unclear. Here, the spatiotemporal expression patterns of two nucleases (CrCAN and CrENDO1) were analyzed qualitatively and quantitatively during PCD in secretory cavity formation in Citrus reticulata 'Chachi' fruits. Results show that the middle and late initial cell stages and lumen-forming stages are key stages for nuclear degradation during the secretory cavity development. CAN and ENDO1 exhibited potent in vitro DNA degradation activity at pH 8.0 and pH 5.5, respectively. Quantitative real-time reverse-transcription polymerase chain reaction, in situ hybridization assays, the subcellular localization of Ca2+ and Zn2+, and immunocytochemical localization showed that CrCAN was activated at the middle and late initial cell stages, while CrENDO1 was activated at the late initial cell and lumen-forming stages. Furthermore, we used immunocytochemical double-labelling to simultaneously locate CrCAN and CrENDO1. The DNA degradation activity of the two nucleases was verified by simulating the change of intracellular pH in vitro. Our results also showed that CrCAN and CrENDO1 worked respectively and co-participated in nuclear DNA degradation during PCD of secretory cavity cells. In conclusion, we propose the model for the synergistic effect of Ca2+- and Zn2+-dependent nucleases (CrCAN and CrENDO1) in co-participating in nuclear DNA degradation during secretory cavity cell PCD in Citrus fruits. Our findings provide direct experimental evidence for exploring different ion-dependent nucleases involved in nuclear degradation during plant PCD.

Transcriptional regulations of pollen tube reception are associated with the fertility of the ginger species Zingiber zerumbet and Zingiber corallinum.

Zingiber zerumbet and Zingiber corallinum are economically valuable species in the genus Zingiber. While Z. corallinum is sexually active, Z. zerumbet adopts clonal propagation, although it has the potential for sexual reproduction. It is unclear so far at which step during the sexual reproduction of Z. zerumbet inhibition occurs, and what are the regulatory mechanisms underlying this inhibition. Here, by comparing with the fertile species Z. corallinum using microscopy-based methods, we show that rare differences were observed in Z. zerumbet up to the point when the pollen tubes invaded the ovules. However, a significantly higher percentage of ovules still contained intact pollen tubes 24 h after pollination, suggesting pollen tube rupture was impaired in this species. Further RNA-seq analysis generated accordant results, showing that the transcription of ANX and FER, as well as genes for the partners in the same complexes (e.g., BUPS and LRE, respectively), and those putative peptide signals (e.g., RALF34), were timely activated in Z. corallinum, which ensured the pollen tubes being able to grow, reorient to ovules, and receipt by embryo sacs. In Z. zerumbet, genes for these complexes were cooperatively suppressed, which would result in the maintenance of PT integrity due to the disruption of RALF34-ANX/BUPS signaling in PT and the failure of PT reception by an active synergid due to the insufficiency of the synergid-harbored FER/LRE complex. Taking the results from the cytological and RNA-seq studies together, a model is proposed to illustrate the possible regulation mechanisms in Z. zerumbet and Z. corallinum, in which the regulations for pollen tube rupture and reception are proposed as the barrier for sexual reproduction in Z. zerumbet.

Soil conditions and the plant microbiome boost the accumulation of monoterpenes in the fruit of Citrus reticulata 'Chachi'.

BACKGROUND: The medicinal material quality of Citrus reticulata 'Chachi' differs depending on the bioactive components influenced by the planting area. Environmental factors, such as soil nutrients, the plant-associated microbiome and climatic conditions, play important roles in the accumulation of bioactive components in citrus. However, how these environmental factors mediate the production of bioactive components of medicinal plants remains understudied. RESULTS: Here, a multi-omics approach was used to clarify the role of environmental factors such as soil nutrients and the root-associated microbiome on the accumulation of monoterpenes in the peel of C. reticulata 'Chachi' procured from core (geo-authentic product region) and non-core (non-geo-authentic product region) geographical regions. The soil environment (high salinity, Mg, Mn and K) enhanced the monoterpene content by promoting the expression of salt stress-responsive genes and terpene backbone synthase in the host plants from the core region. The microbial effects on the monoterpene accumulation of citrus from the core region were further verified by synthetic community (SynCom) experiments. Rhizosphere microorganisms activated terpene synthesis and promoted monoterpene accumulation through interactions with the host immune system. Endophyte microorganisms derived from soil with the potential for terpene synthesis might enhance monoterpene accumulation in citrus by providing precursors of monoterpenes. CONCLUSIONS: Overall, this study demonstrated that both soil properties and the soil microbiome impacted monoterpene production in citrus peel, thus providing an essential basis for increasing fruit quality via reasonable fertilization and precision microbiota management. Video Abstract.

Individualized pharmaceutical care for a patient after pancreaticoduodenectomy with trypsin replacement nutritional therapy: A case report.

RATIONALE: Optimal nutritional therapy for pancreaticoduodenectomy (PD) has been debated; however, little is known about key points of pancreatin enteric-coated capsule administration, a critical component of the PD treatment regimen. Patients often report elevations in tablet platoon and steatorrhea, and steatorrhea may adversely affect nutritional therapy for PD. Herein, we report a case of individualized pharmaceutical care for a patient after PD with trypsin replacement nutritional therapy. PATIENT CONCERNS AND DIAGNOSIS: After PD with trypsin replacement nutritional therapy, the patient developed acute steatorrhea. INTERVENTION: Individualized pharmaceutical care was provided by clinical pharmacists to address intolerance to pancreatin enteric-coated capsules following PD. OUTCOMES: The clinical pharmacist's integration into the patient's treatment plan enhanced pharmacotherapy optimization, especially through pharmacokinetic monitoring and interventions related to nutritional therapy. LESSON: Pharmaceutical care by clinical pharmacists aids in ensuring the safety and efficacy of drugs and nutritional treatment. Clinical pharmacists should be members of the nutrition support team.

Effects of individualized administration of folic acid on prothrombotic state and vascular endothelial function with H-type hypertension: A double-blinded, randomized clinical cohort study.

BACKGROUND: Hypertension and hyperhomocysteinemia (HHcy) have long been associated with adverse cardiovascular and cerebrovascular health outcomes. This study evaluated the effect of individualized administration of folic acid (FA) on homocysteine (Hcy) levels, prothrombotic state, and blood pressure (BP) in patients with H-type hypertension (combination of HHcy and hypertension). METHODS: In this double-blinded, randomized clinical cohort study, 126 patients with H-type hypertension who were treated at our hospital were randomly divided into treatment and control groups (n = 55 each). The control group was treated with oral levamlodipine besylate tablets 2.5 mg and placebo, once a day (in the morning). The treatment group was first treated with oral levamlodipine besylate 2.5 mg and FA tablets 0.8 mg, once a day (in the morning), for 12 weeks. Then, in a second 12-week phase, the FA dose was adjusted using the methylene tetrahydrofolate reductase C677 polymorphism genotype. The levels of Hcy and coagulation factors, prothrombotic state parameters, BP, and adverse drug reactions were compared between the 2 groups. RESULTS: Pretreatment general patient characteristics, including Hcy levels, were similar between the 2 groups (P > .05). BP and prothrombotic status did not differ before and after the first phase of treatment (P > .05). However, Hcy and endothelin-1 (ET-1) levels decreased, while nitric oxide levels increased significantly in the intervention group (P < .05). In the second phase, after 3 months' treatment with an FA dose adjusted according to methylene tetrahydrofolate reductase C677T genotype, the Hcy and ET-1/NO levels were significantly decreased in the intervention group and were lower than those after the first treatment phase and lower than in the control group (P < .01). BP, D-dimer levels, and fibrinogen scores were significantly lower after the second treatment phase (P < .01). There was no significant difference in the incidence of adverse drug reactions between the 2 groups (P > .05). CONCLUSIONS: Individualized administration of FA tablets can effectively reduce BP, and Hcy and coagulation factor levels, and significantly improve prothrombotic status in patients with H-type hypertension.

Correlation of the temporal and spatial expression patterns of HQT with the biosynthesis and accumulation of chlorogenic acid in Lonicera japonica flowers.

Hydroxycinnamoyl-CoA quinate transferase (HQT) is one of the key enzymes in the biosynthesis of chlorogenic acid (CGA) in the flowers of Lonicera japonica. However, the spatiotemporal expression patterns of HQT and its relationship to the dynamics of CGA biosynthesis, transport, and storage remain largely unknown. In this study, we collected L. japonica flower samples at different growth stages (S1-S6) and examined the spatiotemporal expression pattern of HQT and the dynamic accumulation patterns of CGA using a combination of molecular and cytological techniques. Our results suggest that the spatiotemporal expression pattern of HQT is directly correlated with dynamic changes in CGA accumulation and distribution in L. japonica flowers. We further show that CGA is synthesized primarily in the cytoplasm and chloroplasts. CGA synthesized in the cytoplasm first accumulates in specialized vesicles and is then transferred to large central vacuoles for storage by fusion of CGA-containing vesicles with vacuoles. Furthermore, CGA synthesized in the chloroplasts appears to be transferred into the vacuoles for storage by direct membrane fusion between the tonoplast and the disrupted chloroplast membranes. Collectively, our results suggest that CGA is synthesized in chloroplasts and cytoplasm and finally transferred to the vacuole for long-term storage.