Herbivore-induced plant volatiles (HIPVs) are widely recognized as an ecologically important defensive response of plants against herbivory. Although the induction of this 'cry for help' has been well documented, only a few studies have investigated the inhibition of HIPVs by herbivores and little is known about whether herbivores have evolved mechanisms to inhibit the release of HIPVs. To examine the role of herbivore effectors in modulating HIPVs and stomatal dynamics, we conducted series of experiments combining pharmacological, surgical, genetic (CRISPR-Cas9) and chemical (GC-MS analysis) approaches. We show that the salivary enzyme, glucose oxidase (GOX), secreted by the caterpillar Helicoverpa zea on leaves, causes stomatal closure in tomato (Solanum lycopersicum) within 5 min, and in both tomato and soybean (Glycine max) for at least 48 h. GOX also inhibits the emission of several HIPVs during feeding by H. zea, including (Z)-3-hexenol, (Z)-jasmone and (Z)-3-hexenyl acetate, which are important airborne signals in plant defenses. Our findings highlight a potential adaptive strategy where an insect herbivore inhibits plant airborne defenses during feeding by exploiting the association between stomatal dynamics and HIPV emission.
Vertical variation in leaf gas exchange characteristics of trees grown in a lowland dipterocarp forest in Peninsular Malaysia was investigated. Maximum net photosynthetic rate, stomatal conductance, and electron transport rate of leaves at the upper canopy, lower canopy, and forest floor were studied in situ with saturated condition photosynthetic photon flux density. The dark respiration rate of leaves at the various heights was also studied. Relationships among gas exchange characteristics, and also with nitrogen content per unit leaf area and leaf dry matter per area were clearly detected, forming general equations representing the vertical profile of several important parameters related to gas exchange. Numerical analysis revealed that the vertical distribution of gas exchange parameters was well determined showing both larger carbon gain for the whole canopy and at the same time positive carbon gain for the leaves of the lowest layer. For correct estimation of gas exchange at both leaf and canopy scales using multi-layer models, it is essential to consider the vertical distribution of gas exchange parameters with proper scaling coefficients.
The vertical structure of a tropical rain forest is complex and multilayered, with strong variation of micro-environment with height up to the canopy. We investigated the relation between morphological traits of leaf surfaces and tree ecological characteristics in a Malaysian tropical rain forest. The shapes and densities of stomata and trichomes on the abaxial leaf surfaces and their relation with leaf characteristics such as leaf area and leaf mass per area (LMA) were studied in 136 tree species in 35 families with different growth forms in the tropical moist forest. Leaf physiological properties were also measured in 50 canopy and emergent species. Most tree species had flat type (40.4 %) or mound type (39.7 %) stomata. In addition, 84 species (61.76 %) in 22 families had trichomes, including those with glandular (17.65 %) and non-glandular trichomes (44.11 %). Most leaf characteristics significantly varied among the growth form types: species in canopy and emergent layers and canopy gap conditions had higher stomatal density, stomatal pore index (SPI), trichome density and LMA than species in understory and subcanopy layers, though the relation of phylogenetically independent contrasts to each characteristic was not statistically significant, except for leaf stomatal density, SPI and LMA. Intrinsic water use efficiency in canopy and emergent tree species with higher trichome densities was greater than in species with lower trichome densities. These results suggest that tree species in tropical rain forests adapt to a spatial difference in their growth forms, which are considerably affected by phylogenetic context, by having different stomatal and trichome shapes and/or densities.
In this study, we demonstrated the occurrence of stomatal patchiness and its spatial scale in leaves from various sizes of trees grown in a lowland dipterocarp forest in Peninsular Malaysia. To evaluate the patterns of stomatal behavior, we used three techniques simultaneously to analyze heterobaric or homobaric leaves from five tree species ranging from 0.6 to 31 m in height: (i) diurnal changes in chlorophyll fluorescence imaging, (ii) observation and simulation of leaf gas-exchange rates and (iii) a pressure-infiltration method. Measurements were performed in situ with 1000 or 500 μmol m(-2) s(-1) photosynthetic photon flux density. Diurnal patterns in the spatial distribution of photosynthetic electron transport rate (J) mapped from chlorophyll fluorescence images, a comparison of observed and simulated leaf gas-exchange rates, and the spatial distribution of stomatal apertures obtained from the acid-fuchsin-infiltrated area showed that patchy stomatal closure coupled with severe midday depression of photosynthesis occurred in Neobalanocarpus heimii (King) Ashton, a higher canopy tree with heterobaric leaves due to the higher leaf temperature and vapor pressure deficit. However, subcanopy or understory trees showed uniform stomatal behavior throughout the day, although they also have heterobaric leaves. These results suggest that the occurrence of stomatal patchiness is determined by tree size and/or environmental conditions. The analysis of spatial scale by chlorophyll fluorescence imaging showed that several adjacent anatomical patches (lamina areas bounded by bundle-sheath extensions within the lamina) may co-operate for the distributed patterns of J and stomatal apertures.
Drought regimes can be characterized by the variability in the quantity of rainfall and the duration of rainless periods. However, most research on plant response to drought has ignored the impacts of rainfall variation, especially with regard to the influence of nonstructural carbohydrates (NSCs) in promoting drought resistance. To test the hypothesis that these components of drought differentially affect NSC dynamics and seedling resistance, we tracked NSC in plant tissues of tropical tree seedlings in response to manipulations of the volume and frequency of water applied. NSC concentrations decreased in woody tissues under infrequent-high watering but increased under no watering. A faster decline of growth relative to stomatal conductance in the no watering treatment was consistent with NSC accumulation as a result of an uncoupling of growth and photosynthesis, while usage of stored NSCs in woody tissues to maintain function may account for the NSC decline under infrequent-high watering. NSCs, and specifically stem NSCs, contributed to drought resistance under severe water deficits, while NSCs had a less clear role in drought resistance to variability in water availability. The contrasting response of NSCs to water variability and deficit indicates that unique processes support seedling resistance to these components of drought.
To date, the exact route and mode of transmission of Helicobacter pylori remains elusive. The detection of H. pylori in food using molecular approaches has led us to postulate that the gastric pathogen may survive in the extragastric environment for an extended period. In this study, we show that H. pylori prolongs its survival by forming biofilm and micro-colonies on vegetables. The biofilm forming capability of H. pylori is both strain and vegetable dependent. H. pylori strains were classified into high and low biofilm formers based on their highest relative biofilm units (BU). High biofilm formers survived longer on vegetables compared to low biofilm formers. The bacteria survived better on cabbage compared to other vegetables tested. In addition, images captured on scanning electron and confocal laser scanning microscopes revealed that the bacteria were able to form biofilm and reside as micro-colonies on vegetable surfaces, strengthening the notion of possible survival of H. pylori on vegetables for an extended period of time. Taken together, the ability of H. pylori to form biofilm on vegetables (a common food source for human) potentially plays an important role in its survival, serving as a mode of transmission of H. pylori in the extragastric environment.
We investigated effects of heterogeneous stomatal behavior on diurnal patterns of leaf gas exchange in 10 tree species. Observations were made in middle and upper canopy layers of potted tropical rainforest trees in a nursery at the Forest Research Institute Malaysia. Measurements were taken from 29 January to 3 February 2010. We measured in situ diurnal changes in net photosynthetic rate and stomatal conductance in three leaves of each species under natural light. In both top-canopy and sub-canopy species, midday depression of net assimilation rate occurred in late morning. Numerical analysis showed that patchy bimodal stomatal behavior occurred only during midday depression, suggesting that the distribution pattern of stomatal apertures (either uniform or non-uniform stomatal behavior) varies flexibly within single days. Direct observation of stomatal aperture using Suzuki's Universal Micro-Printing (SUMP) method demonstrated midday patchy stomatal closure that fits a bimodal pattern in Shorea leprosula Miq., Shorea macrantha Brandis. and Dipterocarpus tempehes V.Sl. Inhibition of net assimilation rate and stomatal conductance appears to be a response to changes in vapor pressure deficit (VPD). Variable stomatal closure with increasing VPD is a mechanism used by a range of species to prevent excess water loss from leaves through evapotranspiration (viz., inhibition of midday leaf gas exchange). Bimodal stomatal closure may occur among adjacent stomata within a single patch, rather than among patches on a single leaf. Our results suggest the occurrence of patches at several scales within single leaves. Further analysis should consider variable spatial scales in heterogeneous stomatal behavior between and within patches and within single leaves.
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.
Light-harvesting complexes (LHCs) in photosystem II (PSII) regulate glutathione (GSH) functions in plants. To investigate whether LHCs control GSH biosynthesis that modifies guard cell abscisic acid (ABA) sensitivity, we evaluated GSH content, stomatal aperture, reactive oxygen species (ROS), weight loss and plant growth using a ch1-1 mutant that was defective of LHCs and compared this with wild-type (WT) Arabidopsis thaliana plants. Glutathione monoethyl ester (GSHmee) increased but 1-chloro-2,4 dinitrobenzene (CDNB) decreased the GSH content in the guard cells. The guard cells of the ch1-1 mutants accumulated significantly less GSH than the WT plants. The guard cells of the ch1-1 mutants also showed higher sensitivity to ABA than the WT plants. The CDNB treatment increased but the GSHmee treatment decreased the ABA sensitivity of the guard cells without affecting ABA-induced ROS production. Dark and light treatments altered the GSH content and stomatal aperture of the guard cells of ch1-1 and WT plants, irrespective of CDNB and GSHmee. The ch1-1 mutant contained fewer guard cells and displayed poor growth, late flowering and stumpy weight loss compared with the WT plants. This study suggests that defective LHCs reduced the GSH content in the guard cells and increased sensitivity to ABA, resulting in stomatal closure.
Stable carbon isotope (δ(13)C) series were developed from analysis of sequential radial wood increments from AD 1850 to AD 2009 for four mature primary rainforest trees from the Danum and Imbak areas of Sabah, Malaysia. The aseasonal equatorial climate meant that conventional dendrochronology was not possible as the tree species investigated do not exhibit clear annual rings or dateable growth bands. Chronology was established using radiocarbon dating to model age-growth relationships and date the carbon isotopic series from which the intrinsic water-use efficiency (IWUE) was calculated. The two Eusideroxylon zwageri trees from Imbak yielded ages of their pith/central wood (±1 sigma) of 670 ± 40 and 759 ± 40 years old; the less dense Shorea johorensis and Shorea superba trees at Danum yielded ages of 240 ± 40 and 330 ± 40 years, respectively. All trees studied exhibit an increase in the IWUE since AD 1960. This reflects, in part, a response of the forest to increasing atmospheric carbon dioxide concentration. Unlike studies of some northern European trees, no clear plateau in this response was observed. A change in the IWUE implies an associated modification of the local carbon and/or hydrological cycles. To resolve these uncertainties, a shift in emphasis away from high-resolution studies towards long, well-replicated time series is proposed to develop the environmental data essential for model evaluation. Identification of old (greater than 700 years) ringless trees demonstrates their potential in assessing the impacts of climatic and atmospheric change. It also shows the scientific and applied value of a conservation policy that ensures the survival of primary forest containing particularly old trees (as in Imbak Canyon and Danum).
Dehydration-responsive element binding (DREB) transcription factor plays an important role in controlling the expression of abiotic stress responsive genes. An intronless oil palm EgDREB1 was isolated and confirmed to be a nuclear localized protein. Electrophoretic mobility shift and yeast one-hybrid assays validated its ability to interact with DRE/CRT motif. Its close evolutionary relation to the dicot NtDREB2 suggests a universal regulatory role. In order to determine its involvement in abiotic stress response, functional characterization was performed in oil palm seedlings subjected to different levels of drought severity and in EgDREB1 transgenic tomato seedlings treated by abiotic stresses. Its expression in roots and leaves was compared with several antioxidant genes using quantitative real-time PCR. Early accumulation of EgDREB1 in oil palm roots under mild drought suggests possible involvement in the initiation of signaling communication from root to shoot. Ectopic expression of EgDREB1 in T1 transgenic tomato seedlings enhanced expression of DRE/CRT and non-DRE/CRT containing genes, including tomato peroxidase (LePOD), ascorbate peroxidase (LeAPX), catalase (LeCAT), superoxide dismutase (LeSOD), glutathione reductase (LeGR), glutathione peroxidase (LeGP), heat shock protein 70 (LeHSP70), late embryogenesis abundant (LeLEA), metallothionine type 2 (LeMET2), delta 1-pyrroline-5- carboxylate synthetase (LePCS), ABA-aldehyde oxidase (LeAAO) and 9-cis- Epoxycarotenoid dioxygenase (LeECD) under PEG treatment and cold stress (4 °C). Altogether, these findings suggest that EgDREB1 is a functional regulator in enhancing tolerance to drought and cold stress.