Effects of fungicide treatments on mycorrhizal communities and carbon acquisition in the mixotrophic Pyrola japonica (Ericaceae).

Pyrola japonica, a member of the family Ericaceae, is a mixotroph that grows on forest floors and obtains carbon (C) from both its photosynthesis and its mycorrhizal fungi. Its mycorrhizal community is dominated by Russulaceae. However, the mechanism of its C acquisition and its flexibility are not well understood. Our aim was to assess the impact of disturbance of the mycorrhizal fungal communities on C acquisition by P. japonica. We repeatedly applied a fungicide (Benomyl) to soils around P. japonica plants in a broad-leaved forest of central Japan, in order to disturb fungal associates near roots. After fungicide treatment, P. japonica roots were collected and subjected to barcoding by next-generation sequencing, focusing on the ITS2 region. The rate of mycorrhizal formation and α-diversity did not significantly change upon fungicide treatments. Irrespective of the treatments, Russulaceae represented more than 80% of the taxa. Leaves and seeds of the plants were analysed for 13C stable isotope ratios that reflect fungal C gain. Leaf and seed δ13C values with the fungicide treatment were significantly lower than those with the other treatments. Thus the fungicide did not affect mycorrhizal communities in the roots, but disturbed mycorrhizal fungal pathways via extraradical hyphae, and resulted in a more photosynthetic behaviour of P. japonica for leaves and seeds.

Water uptake patterns of tropical canopy trees in Borneo: species-specific and temporal variation and relationships with aboveground traits.

Root water uptake depth and its temporal variation are important determinants of tree mortality, resource partitioning and drought resistance; however, their effects on tropical trees remain poorly understood. In this study, we investigated interspecific differences in water uptake depth and its temporal variation using stable isotope analysis and examined the relationships between water uptake depth and aboveground traits in a humid aseasonal tropical rainforest in Borneo. Species-specific differences in water uptake depth were examined for six dominant dipterocarp species. Temporal variation in water uptake depth for various canopy trees was assessed in three periods with different soil moisture conditions. We then examined the relationships between water uptake depth and aboveground traits including wood density, maximum tree height, flowering frequency and growth rate. Dipterocarpus globosus appeared to be more reliant on deep water resources than the other dipterocarp species. Water uptake from the soil layers varied among the three sampling periods. Trees generally utilized deeper soil water during the second driest sampling period, when temperatures were lowest. During the driest and wettest sampling periods, species with higher flowering frequencies tended to preferentially uptake deep soil water. These results suggest that low temperature and soil moisture promote increased deep soil water uptake in the study region. Dynamic relationships between water uptake patterns and aboveground tree traits may be related to resource partitioning among co-existing species.

Regulation of the plastochron by three many-noded dwarf genes in barley.

The plastochron, the time interval between the formation of two successive leaves, is an important determinant of plant architecture. We genetically and phenotypically investigated many-noded dwarf (mnd) mutants in barley. The mnd mutants exhibited a shortened plastochron and a decreased leaf blade length, and resembled previously reported plastochron1 (pla1), pla2, and pla3 mutants in rice. In addition, the maturation of mnd leaves was accelerated, similar to pla mutants in rice. Several barley mnd alleles were derived from three genes-MND1, MND4, and MND8. Although MND4 coincided with a cytochrome P450 family gene that is a homolog of rice PLA1, we clarified that MND1 and MND8 encode an N-acetyltransferase-like protein and a MATE transporter-family protein, which are respectively orthologs of rice GW6a and maize BIGE1 and unrelated to PLA2 or PLA3. Expression analyses of the three MND genes revealed that MND1 and MND4 were expressed in limited regions of the shoot apical meristem and leaf primordia, but MND8 did not exhibit a specific expression pattern around the shoot apex. In addition, the expression levels of the three genes were interdependent among the various mutant backgrounds. Genetic analyses using the double mutants mnd4mnd8 and mnd1mnd8 indicated that MND1 and MND4 regulate the plastochron independently of MND8, suggesting that the plastochron in barley is controlled by multiple genetic pathways involving MND1, MND4, and MND8. Correlation analysis between leaf number and leaf blade length indicated that both traits exhibited a strong negative association among different genetic backgrounds but not in the same genetic background. We propose that MND genes function in the regulation of the plastochron and leaf growth and revealed conserved and diverse aspects of plastochron regulation via comparative analysis of barley and rice.

Communities of mycorrhizal fungi in different trophic types of Asiatic Pyrola japonica sensu lato (Ericaceae).

Mixotrophic plants obtain carbon by their own photosynthetic activity and from their root-associated mycorrhizal fungi. Mixotrophy is deemed a pre-adaptation for evolution of mycoheterotrophic nutrition, where plants fully depend on fungi and lose their photosynthetic activity. The aim of this study was to clarify mycorrhizal dependency and heterotrophy level in various phenotypes of mixotrophic Pyrola japonica (Ericaceae), encompassing green individuals, rare achlorophyllous variants (albinos) and a form with minute leaves, P. japonica f. subaphylla. These three phenotypes were collected in two Japanese forests. Phylogenetic analysis of both plants and mycorrhizal fungi was conducted based on DNA barcoding. Enrichment in 13C among organs (leaves, stems and roots) of the phenotypes with reference plants and fungal fruitbodies were compared by measuring stable carbon isotopic ratio. All plants were placed in the same clade, with f. subaphylla as a separate subclade. Leaf 13C abundances of albinos were congruent with a fully mycoheterotrophic nutrition, suggesting that green P. japonica leaves are 36.8% heterotrophic, while rhizomes are 74.0% heterotrophic. There were no significant differences in δ13C values among organs in both albino P. japonica and P. japonica f. subaphylla, suggesting full and high mycoheterotrophic nutrition, respectively. Among 55 molecular operational taxonomic units (OTUs) detected as symbionts, the genus Russula was the most abundant in each phenotype and its dominance was significantly higher in albino P. japonica and P. japonica f. subaphylla. Russula spp. detected in P. japonica f. subaphylla showed higher dissimilarity with other phenotypes. These results suggest that P. japonica sensu lato is prone to evolve mycoheterotrophic variants, in a process that changes its mycorrhizal preferences, especially towards the genus Russula for which this species has a marked preference.

Agreement among Goldmann applanation tonometer, iCare, and Icare PRO rebound tonometers; non-contact tonometer; and Tonopen XL in healthy elderly subjects.

PURPOSE: To evaluate the inter-device agreement among the Goldmann applanation tonometer (GAT), iCare and Icare PRO rebound tonometers, non-contact tonometer (NCT), and Tonopen XL tonometer. METHODS: Sixty healthy elderly subjects were enrolled. The intraocular pressure (IOP) in each subject's right eye was measured thrice using each of the five tonometers. Intra-device agreement was evaluated by calculating intraclass correlation coefficients (ICCs). Inter-device agreement was evaluated by ICC and Bland-Altman analyses. RESULTS: ICCs for intra-device agreement for each tonometer were >0.8. IOP as measured by iCare (mean ± SD, 11.6 ± 2.5 mmHg) was significantly lower (p < 0.05) than that measured by GAT (14.0 ± 2.8 mmHg), NCT (13.6 ± 2.5 mmHg), Tonopen XL (13.7 ± 4.1 mmHg), and Icare PRO (12.6 ± 2.2 mmHg; Bonferroni test). There was no significant difference in mean IOP among GAT, NCT, and Tonopen XL. Regarding inter-device agreement, ICC was lower between Tonopen XL and other tonometers (all ICCs < 0.4). However, ICCs of GAT, iCare, Icare PRO, and NCT showed good agreement (0.576-0.700). The Bland-Altman analysis revealed that the width of the 95% limits of agreement was larger between the Tonopen XL and the other tonometers ranged from 14.94 to 16.47 mmHg. Among the other tonometers, however, the widths of 95% limits of agreement ranged from 7.91 to 9.24 mmHg. CONCLUSION: There was good inter-device agreement among GAT, rebound tonometers, and NCT. Tonopen XL shows the worst agreement with the other tonometers; therefore, we should pay attention to its' respective IOP. CLINICAL TRIAL REGISTRATION: Japan Clinical Trials Register; number: UMIN000011544.

Comparison of semi-automated center-dot and fully automated endothelial cell analyses from specular microscopy images.

PURPOSE: To evaluate two specular microscopy analysis methods across different endothelial cell densities (ECDs). METHODS: Endothelial images of one eye from each of 45 patients were taken by using three different specular microscopes (three replicates each). To determine the consistency of the center-dot method, we compared SP-6000 and SP-2000P images. CME-530 and SP-6000 images were compared to assess the consistency of the fully automated method. The SP-6000 images from the two methods were compared. Intraclass correlation coefficients (ICCs) for the three measurements were calculated, and parametric multiple comparisons tests and Bland-Altman analysis were performed. RESULTS: The ECD mean value was 2425 ± 883 (range 516-3707) cells/mm2. ICC values were > 0.9 for all three microscopes for ECD, but the coefficients of variation (CVs) were 0.3-0.6. For ECD measurements, Bland-Altman analysis revealed that the mean difference was 42 cells/mm2 between the SP-2000P and SP-6000 for the center-dot method; 57 cells/mm2 between the SP-6000 measurements from both methods; and -5 cells/mm2 between the SP-6000 and CME-530 for the fully automated method (95% limits of agreement: - 201 to 284 cell/mm2, - 410 to 522 cells/mm2, and - 327 to 318 cells/mm2, respectively). For CV measurements, the mean differences were - 3, - 12, and 13% (95% limits of agreement - 18 to 11, - 26 to 2, and - 5 to 32%, respectively). CONCLUSIONS: Despite using three replicate measurements, the precision of the center-dot method with the SP-2000P and SP-6000 software was only ± 10% for ECD data and was even worse for the fully automated method. CLINICAL TRIAL REGISTRATION: Japan Clinical Trials Register ( http://www.umin.ac.jp/ctr/index/htm9 ) number UMIN 000015236.

Evapotranspiration and water source of a tropical rainforest in peninsular Malaysia.

To evaluate water use and the supporting water source of a tropical rainforest, a 4-year assessment of evapotranspiration (ET) was conducted in Pasoh Forest Reserve, a lowland dipterocarp forest in Peninsular Malaysia. The eddy covariance method and isotope signals of rain, plant, soil, and stream waters were used to determine forest water sources under different moisture conditions. Four sampling events were conducted to collect soil and plant twig samples in wet, moderate, dry, and very dry conditions for the identification of isotopic signals. Annual ET from 2012 to 2015 was quite stable with an average of 1,182 ± 26 mm, and a substantial daily ET was observed even during drought periods, although some decline was observed, corresponding with volumetric soil water content. During the wet period, water for ET was supplied from the surface soil layer between 0 and 0.5 m, whereas in the dry period, approximately 50% to 90% was supplied from the deeper soil layer below 0.5-m depth, originating from water precipitated several months previously at this forest. Isotope signatures demonstrated that the water sources of the plants, soil, and stream were all different. Water in plants was often different from soil water, probably because plant water came from a different source than water that was strongly bound to the soil particles. Plants showed no preference for soil depth with their size, whereas the existence of storage water in the xylem was suggested. The evapotranspiration at this forest is balanced and maintained using most of the available water sources except for a proportion of rapid response run-off.

Prostaglandin-associated periorbitopathy in latanoprost users.

PURPOSE: We investigated the incidence of prostaglandin-associated periorbitopathy (PAP) in subjects with glaucoma treated with latanoprost ophthalmic solution. SUBJECTS AND METHODS: One eye and the forehead in 22 subjects were evaluated. All patients had used latanoprost for more than 1 year (range, 12 to 45 months; mean, 26.0 months) and were prostaglandin F2α analogue treatment-naïve. Digital photographs of the subjects obtained before latanoprost therapy and at the last examination were compared retrospectively. Four signs of PAP (deepening of the upper eyelid sulcus (DUES), upper eyelid ptosis, flattening of the lower eyelid bags, and inferior scleral show) and supplemental side effects around the eyelids (eyelash growth, poliosis, and eyelid pigmentation) were judged to be negative or positive by three independent observers. If the observers unanimously rated a sign as positive, the result was defined as positive. RESULTS: Twelve subjects (54.5%) had no apparent signs. Three subjects were judged to have DUES (13.6%), and two subjects each were judged to have flattening of the lower eyelid bags and eyelid pigmentation (9.0%). The other signs were judged as positive in only one subject each, respectively (4.5%). A univariate logistic regression analysis showed no significant associations between any of the signs and age, sex, or the duration of therapy. CONCLUSION: Latanoprost induced DUES, upper eyelid ptosis, flattening of the lower eyelid bags, inferior scleral show, and supplemental side effects around the eyelids; however, the rates of such occurrence might be relatively low.

Cross-sectional study of the association between a deepening of the upper eyelid sulcus-like appearance and wide-open eyes.

BACKGROUND: Deepening of the upper eyelid sulcus (DUES) is a common complication of prostaglandin F2α analog treatment, which causes cosmetic problems. However, identifying this condition using photographs is difficult due to such problems as the camera flash effects, blepharoptosis or wide-open eyes. PURPOSE: We investigated the association between a DUES-like appearance and wide-open eyes regarding the presence of wide-open eyes as a cause for overestimating the incidence of DUES. SUBJECTS AND METHODS: One eye and the forehead in 100 subjects (31 younger subjects, 30 older subjects and 39 patients with blepharoptosis) were evaluated in the present study. Digital photographs of the subjects with natural open and wide-open eyes were taken with a flash. Five signs (a puffy eyelid, the presence/absence of the upper eyelid sulcus (UES), wrinkles on the forehead with natural open eyes and an increase in the number of wrinkles on the forehead and a DUES-like appearance with wide-open eyes) were judged to be negative or positive by three independent observers. Univariate and multivariate logistic regression analyses were performed to determine the independent predictor(s) of a DUES-like appearance with wide-open eyes. RESULTS: Fourteen subjects (four young, three old and seven subjects with blepharoptosis) were judged to have a DUES-like appearance with wide-open eyes (14%). The only predictive factor was the presence of UES in the patients with natural open eyes (odds ratio = 17.244, 95% confidence interval: 3.447-86.270, P<0.001). Among the 12 UES-positive subjects, six (50%) exhibited a DUES-like appearance with wide-open eyes. CONCLUSIONS: The presence of wide-open eyes can thus cause a DUES-like appearance. Blepharoptosis itself is not a predictive factor; however, care should be taken not to overestimate the incidence of DUES, especially in patients with UES with natural open eyes, as a DUES-like appearance can be caused by wide-open eyes, even in treatment-naïve patients. TRIAL REGISTRATION: UMIN000010500.

Midday depression of leaf CO2 exchange within the crown of Dipterocarpus sublamellatus in a lowland dipterocarp forest in Peninsular Malaysia.

We observed diurnal and seasonal patterns of leaf-scale gas exchange within the crown of a Dipterocarpus sublamellatus Foxw. tree growing in a lowland dipterocarp forest at Pasoh, Peninsular Malaysia. Observations were carried out nine times over 6 years, from September 2002 to December 2007. Observation periods included both wet and mild-dry periods, and natural and saturated photosynthetic photon flux density (PPFD) light conditions. In situ measurements of the diurnal change in net photosynthetic rate and in stomatal conductance were carried out on canopy leaves of a 40-m-tall D. sublamellatus tree, which was accessed from a canopy corridor. A diurnal change in electron transport rate was observed under saturated PPFD conditions. The maximum net assimilation rate was approximately 10 micromol m(-2) s(-1). There was a clear inhibition of the net assimilation rate coupled with stomatal closure after late morning and this inhibition occurred year-round. Although the electron transport rate decreased alongside this inhibition, it sometimes followed on. Numerical analysis showed that the main factor in the inhibition of the net assimilation rate was patchy bimodal stomatal closure, which occurred in both mild-dry and wet periods. The midday depression occurred year-round, though there are fluctuations in soil moisture during the mild-dry and wet periods. The magnitude of the inhibition was not related to soil water content but was related to vapor pressure deficit (VPD): that is, whether the days were sunny and hot or cloudy and cool. On cloudy, cool days in the wet period, the net photosynthesis was only moderately inhibited, but it still decreased in the afternoon and was coupled with patchy stomatal closure, even in quite moderate VPD, leaf temperature and PPFD conditions. Our results suggest that patchy stomatal closure signaled by the increase in VPD, in transpiration and by circadian rhythms, was the key factor in constraining midday leaf gas exchange of the D. sublamellatus canopy leaves.

Environmental effects on oxygen isotope enrichment of leaf water in cotton leaves.

The oxygen isotope enrichment of bulk leaf water (Delta(b)) was measured in cotton (Gossypium hirsutum) leaves to test the Craig-Gordon and Farquhar-Gan models under different environmental conditions. Delta(b) increased with increasing leaf-to-air vapor pressure difference (VPd) as an overall result of the responses to the ratio of ambient to intercellular vapor pressures (e(a)/e(i)) and to stomatal conductance (g(s)). The oxygen isotope enrichment of lamina water relative to source water (Delta(1)), which increased with increasing VPd, was estimated by mass balance between less enriched water in primary veins and enriched water in the leaf. The Craig-Gordon model overestimated Delta(b) (and Delta(1)), as expected. Such discrepancies increased with increase in transpiration rate (E), supporting the Farquhar-Gan model, which gave reasonable predictions of Delta(b) and Delta(1) with an L of 7.9 mm, much less than the total radial effective length L(r) of 43 mm. The fitted values of L for Delta(1) of individual leaves showed little dependence on VPd and temperature, supporting the assumption that the Farquhar-Gan formulation is relevant and useful in describing leaf water isotopic enrichment.

Seasonal fluctuations and temperature dependence of leaf gas exchange parameters of co-occurring evergreen and deciduous trees in a temperate broad-leaved forest.

Seasonal fluctuations in leaf gas exchange parameters were investigated in three evergreen (Quercus glauca Thunb., Cinnamomum camphora Sieb. and Castanopsis cuspidata Schottky) and one deciduous (Quercus serrata Thunb.) co-occurring, dominant tree species in a temperate broad-leaved forest. Dark respiration rate (Rn), maximum carboxylation rate (Vcmax) and stomatal coefficient (m), the ratio of stomatal conductance to net assimilation rate after adjustment to the vapor pressure deficit and internal carbon dioxide (CO2) concentration, were derived inversely from instantaneous field gas exchange data (one-point method). The normalized values of Rn and Vcmax at the reference temperature of 25 degrees C (Rn25, Vcmax25) and their temperature dependencies (Delta Ha(Rn), Delta Ha(Vcmax)) were analyzed. Parameter Vcmax25 ranged from 24.0-40.3 micromol m(-2) s(-1) and Delta Ha(Vcmax) ranged from 29.1- 67.0 kJ mol(-1). Parameter Rn25 ranged from 0.6-1.4 micromol m(-2) s(-1) and Delta Ha(Rn) ranged from 47.4-95.4 kJ mol(-1). The stomatal coefficient ranged from 7.2-8.2. For the three evergreen trees, a single set of Vcmax25 and Rn25 parameters and temperature dependence curves produced satisfactory estimates of carbon uptake throughout the year, except during the period of simultaneous leaf fall and leaf expansion, which occurs in April and May. In the deciduous oak, declines in Vcmax25 were observed after summer, along with changes in Vcmax25 and Rn25 during the leaf expansion period. In all species, variation in m during periods of leaf expansion and drought should be considered in modeling studies. We conclude that the changes in normalized gas exchange parameters during periods of leaf expansion and drought need to be considered when modeling carbon uptake of evergreen broad-leaved species.

Coupling of calcium transport with ATP hydrolysis in scallop sarcoplasmic reticulum.

Previously, we showed that incubation of the scallop sarcoplasmic reticulum (SR) with EGTA at above 37 degrees C resulted in the uncoupling of ATP hydrolysis with Ca2+ transport [Nagata et al. (1996) J. Biochem. 119, 1100-1105]. We have extended this study by comparing the kinetic behavior of Ca2+ release and binding to the uncoupled SR with that of intact scallop or rabbit SR. The change in the Ca2+ concentration in the reaction medium, as determined as the absorption of APIII, was followed using a stopped flow system. Intact scallop SR was preincubated with Ca2+ in the presence of a Ca2+ ionophore, A23187, and then ATP was added to initiate the reaction. The Ca2+ level in the medium increased to the maximum level in several seconds, and then slowly decreased to the initial low level. The rising and subsequent slow decay phases could be related to the dissociation and reassociation of Ca2+ with the Ca-ATPase, respectively. When uncoupled scallop SR vesicles were preincubated with CaCl2 in the absence of A23187 and then the reaction was initiated by the addition of ATP, a remarkable amount of Ca2+ was released from the SR vesicles into the cytosolic solution, whereas, with intact scallop or rabbit SR, only a sharp decrease in the Ca2+ level was observed. Based on these findings, we concluded that the heat treatment of scallop SR in EGTA may alter the conformation of the Ca-ATPase, thereby causing Ca2+ to be released from the enzyme, during the catalytic cycle, at the cytoplasmic surface, but not at the lumenal surface of SR vesicles.