Light Response Curves in Land Plants.

Light is the driving force for photosynthesis. Two techniques are commonly employed to help characterize the relationship between the light environment and photosynthesis in plants.Chlorophyll a fluorescence analysis is used to examine both the capacity for and the efficiency of the conversion of absorbed light into energy for photosynthesis. Additionally, gas exchange analysis is used to assess the utilization of that energy for carbon fixation. These techniques are used either in isolation or in combination to acquire light response curves that measure the response of the plant to sequential changes in irradiance. Light response curves can help users understand photosynthetic mechanisms, evaluate how plants respond to light conditions, or assess the extent of physiological plasticity within plants. In this chapter, we provide a generalized method for acquiring light response curves suitable for both chlorophyll a fluorescence and gas exchange techniques using commercially available apparatus. Depending on the equipment available, these methods can be applied individually or combined to acquire data simultaneously. The methods are broadly applicable to most land plants but are ideally suited to help those that are unfamiliar with these techniques.

Targeted knockdown of ribulose-1, 5-bisphosphate carboxylase-oxygenase in rice mesophyll cells.

We generated antisense constructs targeting two of the five Rubisco small subunit genes (OsRBCS2 and 4) which account for between 30-40 % of the RBCS transcript abundance in leaf blades. The constructs were driven by a maize phosphoenolpyruvate carboxylase (PEPC) promoter known to have enriched expression in mesophyll cells (MCs). In the resulting lines leaf, Rubisco protein content was reduced by between 30-50 % and CO2 assimilation rate was limited under photorespiratory and non-photorespiratory conditions. A relationship between Rubisco protein content and CO2 assimilation rate was found. This was associated with a significant reduction in dry biomass accumulation and grain yield of between 37-70%. In addition to serving as a resource for reducing Rubisco accumulation in a cell-preferential manner, these lines allow us to characterize gene function and isoform specific suppression on photosynthesis and growth. Our results suggest that the knockdown of multiple genes is required to completely reduce Rubisco accumulation in MCs.

A low CO2-responsive mutant of Setaria viridis reveals that reduced carbonic anhydrase limits C4 photosynthesis.

In C4 species, ß-carbonic anhydrase (CA), localized to the cytosol of the mesophyll cells, accelerates the interconversion of CO2 to HCO3-, the substrate used by phosphoenolpyruvate carboxylase (PEPC) in the first step of C4 photosynthesis. Here we describe the identification and characterization of low CO2-responsive mutant 1 (lcr1) isolated from an N-nitroso-N-methylurea- (NMU) treated Setaria viridis mutant population. Forward genetic investigation revealed that the mutated gene Sevir.5G247800 of lcr1 possessed a single nucleotide transition from cytosine to thymine in a ß-CA gene causing an amino acid change from leucine to phenylalanine. This resulted in severe reduction in growth and photosynthesis in the mutant. Both the CO2 compensation point and carbon isotope discrimination values of the mutant were significantly increased. Growth of the mutants was stunted when grown under ambient pCO2 but recovered at elevated pCO2. Further bioinformatics analyses revealed that the mutation has led to functional changes in one of the conserved residues of the protein, situated near the catalytic site. CA transcript accumulation in the mutant was 80% lower, CA protein accumulation 30% lower, and CA activity ~98% lower compared with the wild type. Changes in the abundance of other primary C4 pathway enzymes were observed; accumulation of PEPC protein was significantly increased and accumulation of malate dehydrogenase and malic enzyme decreased. The reduction of CA protein activity and abundance in lcr1 restricts the supply of bicarbonate to PEPC, limiting C4 photosynthesis and growth. This study establishes Sevir.5G247800 as the major CA allele in Setaria for C4 photosynthesis and provides important insights into the function of CA in C4 photosynthesis that would be required to generate a rice plant with a functional C4 biochemical pathway.

Immunolocalization Analysis of C4 Proteins in the Leaf Tissue of Rice.

Immunolocalization analysis is a principal tool to study protein expression and subcellular distribution in plant cells or tissues. In this chapter, we present the method of the preparation of lightly fixed fresh rice leaf tissue for immunolocalization analysis and detection of the protein of interest using fluorescent probes by fluorescent microscopy. This method especially does not need the process of embedding plant materials that saves time and prevents alterations of cellular compounds and structure during sample preparation. Using this method, the C4 rice project compared the expressions of the proteins of interest among C4 model plants, wild-type rice, and transgenic or mutant plants and successfully selected the transgenic plants with the correct location of each protein to create a C4 rice prototype.

Overexpression of the chloroplastic 2-oxoglutarate/malate transporter disturbs carbon and nitrogen homeostasis in rice.

The chloroplastic 2-oxaloacetate (OAA)/malate transporter (OMT1 or DiT1) takes part in the malate valve that protects chloroplasts from excessive redox poise through export of malate and import of OAA. Together with the glutamate/malate transporter (DCT1 or DiT2), it connects carbon with nitrogen assimilation, by providing 2-oxoglutarate for the GS/GOGAT (glutamine synthetase/glutamate synthase) reaction and exporting glutamate to the cytoplasm. OMT1 further plays a prominent role in C4 photosynthesis: OAA resulting from phosphoenolpyruvate carboxylation is imported into the chloroplast, reduced to malate by plastidic NADP-malate dehydrogenase, and then exported for transport to bundle sheath cells. Both transport steps are catalyzed by OMT1, at the rate of net carbon assimilation. To engineer C4 photosynthesis into C3 crops, OMT1 must be expressed in high amounts on top of core C4 metabolic enzymes. We report here high-level expression of ZmOMT1 from maize in rice (Oryza sativa ssp. indica IR64). Increased activity of the transporter in transgenic rice was confirmed by reconstitution of transporter activity into proteoliposomes. Unexpectedly, overexpression of ZmOMT1 in rice negatively affected growth, CO2 assimilation rate, total free amino acid content, tricarboxylic acid cycle metabolites, as well as sucrose and starch contents. Accumulation of high amounts of aspartate and the impaired growth phenotype of OMT1 rice lines could be suppressed by simultaneous overexpression of ZmDiT2. Implications for engineering C4 rice are discussed.

Exploring Effective Sensory Experience in the Environmental Design of Sustainable Cafés.

The aim of this study was to explore and construct spatial indicators suitable for green café ambience. The indicators were further empirically verified. A three-round questionnaire survey, based on the Delphi method, was conducted with 15 experts, including university professors (food and beverage services management and interior environmental design), café operators, and personnel from government agencies. Data were collected, and the results on the characteristics of the repeated feedback from the experts were convergent. Thirty-six indicators suitable for the design of green café ambience were extracted, of which 17 were verified by actual cafés as highly operable. The five-sense indicators of sustainable green ambience design obtained in this study can facilitate positive customer experiences and enhance the appeal of maintaining sustainable green trends for cafés. These indicators can also provide references for café operators in business planning and green café ambience design.

The Relations Among Physical Indicators, Cognitive Status, Community Participation, and Depression of the Frail Male Elderly in Taiwan.

This study explored the basic attributes, physiological indices, cognitive states, and community participation of older male outpatients with frailty for predicting depression. Questionnaires were collected using purposive sampling from a medical clinic in a teaching hospital in northern Taiwan. One hundred and ninety frail men enrolled as participants. The results revealed that older male adults with frailty, the age, residence, income, self-reported health status, alcohol consumption, total instrumental activities of daily living (IADL) scores in physiological indices, IADL grouping, cognitive state score, each Mini-Mental State Examination category, and involvement and dedication scores exhibited statistical differences from depression scores. Depression determinants, such as an excellent and normal self-reported health status and IADL total score, could predict the depression status of male older adults with frailty. Nursing personnel should assess the self-reported health status and self-care ability of male older adults with frailty early to prevent or delay geriatric depression.

The Relationship Between Depression and Frailty in Community-Dwelling Older People: A Systematic Review and Meta-Analysis of 84,351 Older Adults.

OBJECTIVES: In this study we investigated the correlation between depression and frailty in older adults. Additionally, correlations among study designs (prospective vs. cross-sectional), regions, depression indices, frailty indices, covariance corrections, and sexes were explored to support the analysis. METHODS: A systematic literature review and meta-analysis were conducted. A total of 84,351 older adults, all 65 years of age or older, were analyzed. Both authors independently extracted and examined retrieved articles. Searched keywords included "depression" or "depressive"; "frailty" or "frail"; and "older people," "elderly," "geriatric," or "senior." Articles published between January 2000 and December 2016 were searched. A literature quality assessment was conducted in accordance with the guidelines of the Preferred Reporting Items for Systematic REVIEWS AND META-ANALYSES: Systematic literature searches were conducted on the Embase, PubMed, MEDLINE, Cumulative Index to Nursing and Allied Health Literature, and Cochrane Library databases, and collected studies were analyzed using a random effects model. RESULTS: Fourteen studies on people 65 years of age or older were collected, and a correlation analysis was conducted for depression and frailty. According to the meta-analysis, the risk for frailty due to depression was nonsignificant among the subgroups for study design (p for heterogeneity = .149), region (p = .429), depression criteria (p = .934), covariate adjustment (p = .702), and frailty criteria (p = .661). Notably, the risk for frailty due to depression was significantly higher in men than in women (pooled odds ratios for men and women: 4.76 and 2.25, respectively; Qbetween χ2 = 9.93, p = .002). CONCLUSION: Older adults with depression are more prone to frailty than are those without depression. Regardless of study design, region, depression index, frailty index, and covariance corrections, no significant differences were observed in the results of studies on depression and frailty in older adults. The only factor that had a significant influence was sex; older men with depression were at a higher risk for frailty than were older women with depression. CLINICAL RELEVANCE: Depression and frailty are pertinent health concerns related to geriatric syndromes. Because older adults with depression have a high risk for frailty, nursing personnel should use a depression index as early as possible to screen for depression and further reduce the occurrence of frailty in older adults. Furthermore, based on the aforementioned differences between the sexes, special attention should be paid to older men with depression to reduce their risk for frailty.

Transgenic maize phosphoenolpyruvate carboxylase alters leaf-atmosphere CO2 and 13CO2 exchanges in Oryza sativa.

The engineering process of C4 photosynthesis into C3 plants requires an increased activity of phosphoenolpyruvate carboxylase (PEPC) in the cytosol of leaf mesophyll cells. The literature varies on the physiological effect of transgenic maize (Zea mays) PEPC (ZmPEPC) leaf expression in Oryza sativa (rice). Therefore, to address this issue, leaf-atmosphere CO2 and 13CO2 exchanges were measured, both in the light (at atmospheric O2 partial pressure of 1.84 kPa and at different CO2 levels) and in the dark, in transgenic rice expressing ZmPEPC and wild-type (WT) plants. The in vitro PEPC activity was 25 times higher in the PEPC overexpressing (PEPC-OE) plants (~20% of maize) compared to the negligible activity in WT. In the PEPC-OE plants, the estimated fraction of carboxylation by PEPC (ß) was ~6% and leaf net biochemical discrimination against 13CO2[Formula: see text] was ~ 2‰ lower than in WT. However, there were no differences in leaf net CO2 assimilation rates (A) between genotypes, while the leaf dark respiration rates (Rd) over three hours after light-dark transition were enhanced (~ 30%) and with a higher 13C composition [Formula: see text] in the PEPC-OE plants compared to WT. These data indicate that ZmPEPC in the PEPC-OE rice plants contributes to leaf carbon metabolism in both the light and in the dark. However, there are some factors, potentially posttranslational regulation and PEP availability, which reduce ZmPEPC activity in vivo.

Knockdown of glycine decarboxylase complex alters photorespiratory carbon isotope fractionation in Oryza sativa leaves.

The influence of reduced glycine decarboxylase complex (GDC) activity on leaf atmosphere CO2 and 13CO2 exchange was tested in transgenic Oryza sativa with the GDC H-subunit knocked down in leaf mesophyll cells. Leaf measurements on transgenic gdch knockdown and wild-type plants were carried out in the light under photorespiratory and low photorespiratory conditions (i.e. 18.4 kPa and 1.84 kPa atmospheric O2 partial pressure, respectively), and in the dark. Under approximately current ambient O2 partial pressure (18.4 kPa pO2), the gdch knockdown plants showed an expected photorespiratory-deficient phenotype, with lower leaf net CO2 assimilation rates (A) than the wild-type. Additionally, under these conditions, the gdch knockdown plants had greater leaf net discrimination against 13CO2 (Δo) than the wild-type. This difference in Δo was in part due to lower 13C photorespiratory fractionation (f) ascribed to alternative decarboxylation of photorespiratory intermediates. Furthermore, the leaf dark respiration rate (Rd) was enhanced and the 13CO2 composition of respired CO2 (δ13CRd) showed a tendency to be more depleted in the gdch knockdown plants. These changes in Rd and δ13CRd were due to the amount and carbon isotopic composition of substrates available for dark respiration. These results demonstrate that impairment of the photorespiratory pathway affects leaf 13CO2 exchange, particularly the 13C decarboxylation fractionation associated with photorespiration.

Frail Phenotype and Disability Prediction in Community-Dwelling Older People: A Systematic Review and Meta-Analysis of Prospective Cohort Studies.

BACKGROUND: The World Health Organization has identified frailty as a crucial factor affecting successful aging. Systematic literature reviews have yet to focus on the relationship between stages of frailty and disability in community-dwelling older adults. PURPOSE: The purpose of this study was to investigate the relationships between various frailty types and disability in community-dwelling older adults and to explore how various frailty criteria have been used to assess disability in this population. METHODS: A systematic literature review and meta-analysis were conducted on articles from the following databases: Cochrane Library, CINAHL, PubMed, and Ovid. Database search criteria included articles that were published between January 2001 and July 2017 and study samples that included community-dwelling adults aged 60 years and older. We excluded studies that were conducted in institutions or hospitals and experimental studies on frailty. Two reviewers independently assessed eligibility and extracted data. A random-effects model was used to analyze the literature and to calculate the pooled disability of frailty. RESULTS: In total, nine studies with a total sample of 32,998 participants that recorded 8,666 disabilities and a mean follow-up time of 30.4 months (SD = 29.26) were pooled for the meta-analysis. Using various indicators to predict the risk of disability compared with robust older adults, those with frailty faced a higher risk of disability, followed by older adults at risk of frailty. CONCLUSIONS/IMPLICATIONS FOR PRACTICE: Frailty is a crucial health consideration among older adults. Those who are frail have the highest relative risk of disability, followed by those at risk of frailty. Early assessment of frailty may effectively prevent the occurrence of frailty-induced disability in older adults.

The Relationship of Frailty and Hospitalization Among Older People: Evidence From a Meta-Analysis.

OBJECTIVES: This research explored the relationship between the stages of frailty and risk for hospitalization in older adults and evaluated the risk for hospitalization among the elderly in relation to various frailty assessment indexes. METHODS: A systematic literature review and meta-analysis were carried out. A total of 32,998 older people, 8,666 of whom were hospitalized, were included in this study. Two of the researchers independently collected and reviewed the literature. The key search terms used were "frailty" or "frail," "hospitalization," and "older people" or "older" or "geriatric" or "senior." Data were recorded from January 2001 to July 2016. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were adopted for quality assessment. A systematic search was carried out using Embase and Scopus to analyze the collected literature. FINDINGS: A meta-analysis was conducted on eight studies that discussed the relationship between frailty and hospitalization risk in older adults 65 years of age or older. The results showed that frail older people exhibited the highest risk for hospitalization, following by prefrail and robust older people. Next, different frailty assessment indicators were used to predict the risk for hospitalization among older people. All of these indexes also showed that older persons with frailty had the highest risk for hospitalization, followed by prefrail older people. CONCLUSIONS: Frailty is a vital issue in geriatric care and is a crucial factor in the hospitalization of older people. Frail older people were at the highest risk for hospitalization, following by prefrail and robust older people. Assessing frailty as early as possible can reduce the hospitalization risk among older people. CLINICAL RELEVANCE: Professional nursing staff should use frailty indicators in a timely fashion to assess the status of frailty in older people and should effectively develop frailty prevention strategies to decrease the risk for hospitalization and to enhance quality of life for older adults.

Candidate regulators of Early Leaf Development in Maize Perturb Hormone Signalling and Secondary Cell Wall Formation When Constitutively Expressed in Rice.

All grass leaves are strap-shaped with a series of parallel veins running from base to tip, but the distance between each pair of veins, and the cell-types that develop between them, differs depending on whether the plant performs C3 or C4 photosynthesis. As part of a multinational effort to introduce C4 traits into rice to boost crop yield, candidate regulators of C4 leaf anatomy were previously identified through an analysis of maize leaf transcriptomes. Here we tested the potential of 60 of those candidate genes to alter leaf anatomy in rice. In each case, transgenic rice lines were generated in which the maize gene was constitutively expressed. Lines grouped into three phenotypic classes: (1) indistinguishable from wild-type; (2) aberrant shoot and/or root growth indicating possible perturbations to hormone homeostasis; and (3) altered secondary cell wall formation. One of the genes in class 3 defines a novel monocot-specific family. None of the genes were individually sufficient to induce C4-like vein patterning or cell-type differentiation in rice. A better understanding of gene function in C4 plants is now needed to inform more sophisticated engineering attempts to alter leaf anatomy in C3 plants.

CRISPR-Cas9 and CRISPR-Cpf1 mediated targeting of a stomatal developmental gene EPFL9 in rice.

KEY MESSAGE: CRISPR-Cas9/Cpf1 system with its unique gene targeting efficiency, could be an important tool for functional study of early developmental genes through the generation of successful knockout plants. The introduction and utilization of systems biology approaches have identified several genes that are involved in early development of a plant and with such knowledge a robust tool is required for the functional validation of putative candidate genes thus obtained. The development of the CRISPR-Cas9/Cpf1 genome editing system has provided a convenient tool for creating loss of function mutants for genes of interest. The present study utilized CRISPR/Cas9 and CRISPR-Cpf1 technology to knock out an early developmental gene EPFL9 (Epidermal Patterning Factor like-9, a positive regulator of stomatal development in Arabidopsis) orthologue in rice. Germ-line mutants that were generated showed edits that were carried forward into the T2 generation when Cas9-free homozygous mutants were obtained. The homozygous mutant plants showed more than an eightfold reduction in stomatal density on the abaxial leaf surface of the edited rice plants. Potential off-target analysis showed no significant off-target effects. This study also utilized the CRISPR-LbCpf1 (Lachnospiracae bacterium Cpf1) to target the same OsEPFL9 gene to test the activity of this class-2 CRISPR system in rice and found that Cpf1 is also capable of genome editing and edits get transmitted through generations with similar phenotypic changes seen with CRISPR-Cas9. This study demonstrates the application of CRISPR-Cas9/Cpf1 to precisely target genomic locations and develop transgene-free homozygous heritable gene edits and confirms that the loss of function analysis of the candidate genes emerging from different systems biology based approaches, could be performed, and therefore, this system adds value in the validation of gene function studies.

Overexpression of OsSAP16 Regulates Photosynthesis and the Expression of a Broad Range of Stress Response Genes in Rice (Oryza sativa L.).

This study set out to identify and characterize transcription factors regulating photosynthesis in rice. Screening populations of rice T-DNA activation lines led to the identification of a T-DNA mutant with an increase in intrinsic water use efficiency (iWUE) under well-watered conditions. Flanking sequence analysis showed that the T-DNA construct was located upstream of LOC_Os07g38240 (OsSAP16) encoding for a stress-associated protein (SAP). A second mutant identified with activation in the same gene exhibited the same phenotype; expression of OsSAP16 was shown to be enhanced in both lines. There were no differences in stomatal development or morphology in either of these mutants, although overexpression of OsSAP16 reduced stomatal conductance. This phenotype limited CO2 uptake and the rate of photosynthesis, which resulted in the accumulation of less biomass in the two mutants. Whole transcriptome analysis showed that overexpression of OsSAP16 led to global changes in gene expression consistent with the function of zinc-finger transcription factors. These results show that the gene is involved in modulating the response of rice to drought stress through regulation of the expression of a set of stress-associated genes.

High-level ß1-integrin expression in a subpopulation of highly tumorigenic oral cancer cells.

OBJECTIVES: The ß1 integrin (CD29) is a putative marker for cancerous epithelial stem cells. Cancer stem cells are essential to drive tumor growth, recurrence, and metastasis. We investigated the role of ß1-integrin expression in the development of malignant phenotypes of oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: Immunostaining was used to analyze the expression levels of ß1 integrins in different types of cell colonies and tumor spheres. The results of cell viability and migration assays with and without siRNA knockdown of ß1-integrin expression were compared. Cells expressing ß1 integrins were evaluated for their tumorigenicity in mice. The expression of ß1 integrins in human specimens of oral cancers at different clinical stages was semiquantified based on immunohistochemical staining of the ß1-integrin protein. RESULTS: The expression level of ß1 integrins in Meng-1 oral epidermoid carcinoma cells (OECM-1) cells was significantly higher in holoclonal colonies and tumor spheres compared to control cells. The knockdown of ß1-integrin expression in OECM-1 cells reduced cell proliferation, migration, and tumor sphere formation. Beta-1 integrin (+) cells were more tumorigenic in the mouse xenograft model than ß1 integrin (-) cells. In the human specimens, the expression level of the ß1-integrin protein positively correlated with the clinical stage. CONCLUSION: The expression of ß1 integrin in OECM-1 cells is involved in the development of malignant phenotypes of OSCC. CLINICAL RELEVANCE: Inhibitors for ß1-integrin signaling may be suitable to become target-specific therapies for OSCC.

Isolating stromal stem cells from periodontal granulation tissues.

Stem cell therapy is a promising area in regenerative medicine. Periodontal granulation tissues are often discarded during conventional surgery. If stromal stem cells can be isolated from these tissues, they can be used for subsequent surgery on the same patient. Fifteen human periodontal granulation tissue samples were obtained from intrabony defects during surgery. Immunohistochemistry (IHC) was carried out on five of the samples to identify STRO-1, a marker of mesenchymal stem cells. Five samples underwent flow cytometry analysis for the same marker. The remaining five samples were characterized by "colony formation unit-fibroblast" (CFU-f) assay and selected for proliferation assay, flow cytometry of stem cell markers, immunocytochemistry (ICC), multipotent differentiation assays, and repairing critical-size defects in mice. The ratio of STRO-1(+) cells detected by IHC was 5.91 ± 1.50%. The analysis of flow cytometry for STRO-1 was 6.70 ± 0.81%. Approximately two thirds of the CFU-f colonies had a strong reaction to STRO-1 in ICC staining. The cells were multipotent both in vitro and in vivo. Mice given bone grafts and stem cells showed significantly better bone healing than those without stem cells. Multipotent stromal stem cells can be isolated from human periodontal granulation tissues. These cells improve new bone formation when transplanted in mouse calvarial defects. Isolating stem cells from relatively accessible sites without extra procedures is clinically advantageous. This study demonstrated that human periodontal granulation tissues contain isolatable multipotent stem cells. The cells may be a good source for autotransplantation in subsequent treatment.

Transcriptome analysis during somatic embryogenesis of the tropical monocot Elaeis guineensis: evidence for conserved gene functions in early development.

With the aim of understanding the molecular mechanisms underlying somatic embryogenesis (SE) in oil palm, we examined transcriptome changes that occur when embryogenic suspension cells are initiated to develop somatic embryos. Two reciprocal suppression subtractive hybridization (SSH) libraries were constructed from oil palm embryogenic cell suspensions: one in which embryo development was blocked by the presence of the synthetic auxin analogue 2,4-dichlorophenoxyacetic acid (2,4-D: ) in the medium (proliferation library); and another in which cells were stimulated to form embryos by the removal of 2,4-D: from the medium (initiation library). A total of 1867 Expressed Sequence Tags (ESTs) consisting of 1567 potential unigenes were assembled from the two libraries. Functional annotation indicated that 928 of the ESTs correspond to proteins that have either no similarity to sequences in public databases or are of unknown function. Gene Ontology (GO) terms assigned to the two EST populations give clues to the underlying molecular functions, biological processes and cellular components involved in the initiation of embryo development. Macroarrays were used for transcript profiling the ESTs during SE. Hierarchical cluster analysis of differential transcript accumulation revealed 4 distinct profiles containing a total of 192 statistically significant developmentally regulated transcripts. Similarities and differences between the global results obtained with in vitro systems from dicots, monocots and gymnosperms will be discussed.