Time-Series Transcriptomics Reveals That AGAMOUS-LIKE22 Affects Primary Metabolism and Developmental Processes in Drought-Stressed Arabidopsis.

In Arabidopsis thaliana, changes in metabolism and gene expression drive increased drought tolerance and initiate diverse drought avoidance and escape responses. To address regulatory processes that link these responses, we set out to identify genes that govern early responses to drought. To do this, a high-resolution time series transcriptomics data set was produced, coupled with detailed physiological and metabolic analyses of plants subjected to a slow transition from well-watered to drought conditions. A total of 1815 drought-responsive differentially expressed genes were identified. The early changes in gene expression coincided with a drop in carbon assimilation, and only in the late stages with an increase in foliar abscisic acid content. To identify gene regulatory networks (GRNs) mediating the transition between the early and late stages of drought, we used Bayesian network modeling of differentially expressed transcription factor (TF) genes. This approach identified AGAMOUS-LIKE22 (AGL22), as key hub gene in a TF GRN. It has previously been shown that AGL22 is involved in the transition from vegetative state to flowering but here we show that AGL22 expression influences steady state photosynthetic rates and lifetime water use. This suggests that AGL22 uniquely regulates a transcriptional network during drought stress, linking changes in primary metabolism and the initiation of stress responses.

Effects of kinetics of light-induced stomatal responses on photosynthesis and water-use efficiency.

Both photosynthesis (A) and stomatal conductance (gs ) respond to changing irradiance, yet stomatal responses are an order of magnitude slower than photosynthesis, resulting in noncoordination between A and gs in dynamic light environments. Infrared gas exchange analysis was used to examine the temporal responses and coordination of A and gs to a step increase and decrease in light in a range of different species, and the impact on intrinsic water use efficiency was evaluated. The temporal responses revealed a large range of strategies to save water or maximize photosynthesis in the different species used in this study but also displayed an uncoupling of A and gs in most of the species. The shape of the guard cells influenced the rapidity of response and the overall gs values achieved, with different impacts on A and Wi . The rapidity of gs in dumbbell-shaped guard cells could be attributed to size, whilst in elliptical-shaped guard cells features other than anatomy were more important for kinetics. Our findings suggest significant variation in the rapidity of stomatal responses amongst species, providing a novel target for improving photosynthesis and water use.

C3 photosynthesis in the desert plant Rhazya stricta is fully functional at high temperatures and light intensities.

The C3 plant Rhazya stricta is native to arid desert environment zones, where it experiences daily extremes of heat, light intensity (PAR) and high vapour pressure deficit (VPD). We measured the photosynthetic parameters in R. stricta in its native environment to assess the mechanisms that permit it to survive in these extreme conditions. Infrared gas exchange analysis examined diel changes in assimilation (A), stomatal conductance (gs ) and transpiration (E) on mature leaves of R. stricta. A/ci analysis was used to determine the effect of temperature on carboxylation capacity (Vc,max ) and the light- and CO2 -saturated rate of photosynthesis (Amax ). Combined chlorophyll fluorescence and gas exchange light response curve analysis at ambient and low oxygen showed that both carboxylation and oxygenation of Rubisco acted as the major sinks for the end products of electron transport. Physiological analysis in conjunction with gene expression analysis suggested that there are two isoforms of Rubisco activase which may provide an explanation for the ability of R. stricta to maintain Rubisco function at high temperatures. The potential to exploit this ability to cope with extreme temperatures is discussed in the context of future crop improvement.

Arabidopsis HEAT SHOCK TRANSCRIPTION FACTORA1b overexpression enhances water productivity, resistance to drought, and infection.

Heat-stressed crops suffer dehydration, depressed growth, and a consequent decline in water productivity, which is the yield of harvestable product as a function of lifetime water consumption and is a trait associated with plant growth and development. Heat shock transcription factor (HSF) genes have been implicated not only in thermotolerance but also in plant growth and development, and therefore could influence water productivity. Here it is demonstrated that Arabidopsis thaliana plants with increased HSFA1b expression showed increased water productivity and harvest index under water-replete and water-limiting conditions. In non-stressed HSFA1b-overexpressing (HSFA1bOx) plants, 509 genes showed altered expression, and these genes were not over-represented for development-associated genes but were for response to biotic stress. This confirmed an additional role for HSFA1b in maintaining basal disease resistance, which was stress hormone independent but involved H2O2 signalling. Fifty-five of the 509 genes harbour a variant of the heat shock element (HSE) in their promoters, here named HSE1b. Chromatin immunoprecipitation-PCR confirmed binding of HSFA1b to HSE1b in vivo, including in seven transcription factor genes. One of these is MULTIPROTEIN BRIDGING FACTOR1c (MBF1c). Plants overexpressing MBF1c showed enhanced basal resistance but not water productivity, thus partially phenocopying HSFA1bOx plants. A comparison of genes responsive to HSFA1b and MBF1c overexpression revealed a common group, none of which harbours a HSE1b motif. From this example, it is suggested that HSFA1b directly regulates 55 HSE1b-containing genes, which control the remaining 454 genes, collectively accounting for the stress defence and developmental phenotypes of HSFA1bOx.

Chlorophyll fluorescence: a probe of photosynthesis in vivo.

The use of chlorophyll fluorescence to monitor photosynthetic performance in algae and plants is now widespread. This review examines how fluorescence parameters can be used to evaluate changes in photosystem II (PSII) photochemistry, linear electron flux, and CO(2) assimilation in vivo, and outlines the theoretical bases for the use of specific fluorescence parameters. Although fluorescence parameters can be measured easily, many potential problems may arise when they are applied to predict changes in photosynthetic performance. In particular, consideration is given to problems associated with accurate estimation of the PSII operating efficiency measured by fluorescence and its relationship with the rates of linear electron flux and CO(2) assimilation. The roles of photochemical and nonphotochemical quenching in the determination of changes in PSII operating efficiency are examined. Finally, applications of fluorescence imaging to studies of photosynthetic heterogeneity and the rapid screening of large numbers of plants for perturbations in photosynthesis and associated metabolism are considered.

The water-water cycle in leaves is not a major alternative electron sink for dissipation of excess excitation energy when CO(2) assimilation is restricted.

Electron flux from water via photosystem II (PSII) and PSI to oxygen (water-water cycle) may provide a mechanism for dissipation of excess excitation energy in leaves when CO(2) assimilation is restricted. Mass spectrometry was used to measure O(2) uptake and evolution together with CO(2) uptake in leaves of French bean and maize at CO(2) concentrations saturating for photosynthesis and the CO(2) compensation point. In French bean at high CO(2) and low O(2) concentrations no significant water-water cycle activity was observed. At the CO(2) compensation point and 3% O(2) a low rate of water-water cycle activity was observed, which accounted for 30% of the linear electron flux from water. In maize leaves negligible water-water cycle activity was detected at the compensation point. During induction of photosynthesis in maize linear electron flux was considerably greater than CO(2) assimilation, but no significant water-water cycle activity was detected. Miscanthus × giganteus grown at chilling temperature also exhibited rates of linear electron transport considerably in excess of CO(2) assimilation; however, no significant water-water cycle activity was detected. Clearly the water-water cycle can operate in leaves under some conditions, but it does not act as a major sink for excess excitation energy when CO(2) assimilation is restricted.

Imaging of reactive oxygen species in vivo.

Reactive oxygen species (ROS) are involved in many signalling pathways and numerous stress responses in plants. Consequently, it is important to be able to identify and localize ROS in vivo to evaluate their roles in signalling. A number of probes that have a high affinity for specific ROS and that are effectively taken up by cells and tissues are commercially available. Applications to intact leaves of singlet oxygen sensor green (SOSG), nitroblue tetrazolium (NBT), di-amino benzidine (DAB) and Amplex Red to detect singlet oxygen, superoxide and hydrogen peroxide are described. Imaging of the probes in the cells and tissues of leaves allows sites of ROS production to be identified.

Reductions in mesophyll and guard cell photosynthesis impact on the control of stomatal responses to light and CO2.

Transgenic antisense tobacco plants with a range of reductions in sedoheptulose-1,7-bisphosphatase (SBPase) activity were used to investigate the role of photosynthesis in stomatal opening responses. High resolution chlorophyll a fluorescence imaging showed that the quantum efficiency of photosystem II electron transport (F(q)(')/F(m)(')) was decreased similarly in both guard and mesophyll cells of the SBPase antisense plants compared to the wild-type plants. This demonstrated for the first time that photosynthetic operating efficiency in the guard cells responds to changes in the regeneration capacity of the Calvin cycle. The rate of stomatal opening in response to a 30 min, 10-fold step increase in red photon flux density in the leaves from the SBPase antisense plants was significantly greater than wild-type plants. Final stomatal conductance under red and mixed blue/red irradiance was greater in the antisense plants than in the wild-type control plants despite lower CO(2) assimilation rates and higher internal CO(2) concentrations. Increasing CO(2) concentration resulted in a similar stomatal closing response in wild-type and antisense plants when measured in red light. However, in the antisense plants with small reductions in SBPase activity greater stomatal conductances were observed at all C(i) levels. Together, these data suggest that the primary light-induced opening or CO(2)-dependent closing response of stomata is not dependent upon guard or mesophyll cell photosynthetic capacity, but that photosynthetic electron transport, or its end-products, regulate the control of stomatal responses to light and CO(2).

Measurement of O2 Uptake and Evolution in Leaves In Vivo Using Stable Isotopes and Membrane Inlet Mass Spectrometry.

Oxygen is both product and substrate of photosynthesis and metabolism in plants, by oxygen evolution through water splitting and uptake by photorespiration and respiration. It is important to investigate these processes simultaneously in leaves, especially in response to environmental variables, such as light and temperature. To distinguish between processes that evolve or take up O2 in leaves in the light, in vivo gas exchange of stable isotopes of oxygen and membrane inlet mass spectrometry is used. A closed-cuvette system for gas exchange of leaf disks is described, using the stable isotopes 16O2 and 18O2, with a semipermeable membrane gas inlet and isotope mass separation and detection by mass spectrometry. Measurement of evolution and uptake, as well as CO2 uptake, at a range of light levels allows composition of a light-response curve, here described for French bean and maize leaf disks.

GIANT CHLOROPLAST 1 is essential for correct plastid division in Arabidopsis.

Plastids are vital plant organelles involved in many essential biological processes. Plastids are not created de novo but divide by binary fission mediated by nuclear-encoded proteins of both prokaryotic and eukaryotic origin. Although several plastid division proteins have been identified in plants, limited information exists regarding possible division control mechanisms. Here, we describe the identification of GIANT CHLOROPLAST 1 (GC1), a new nuclear-encoded protein essential for correct plastid division in Arabidopsis. GC1 is plastid-localized and is anchored to the stromal surface of the chloroplast inner envelope by a C-terminal amphipathic helix. In Arabidopsis, GC1 deficiency results in mesophyll cells harbouring one to two giant chloroplasts, whilst GC1 overexpression has no effect on division. GC1 can form homodimers but does not show any interaction with the Arabidopsis plastid division proteins AtFtsZ1-1, AtFtsZ2-1, AtMinD1, or AtMinE1. Analysis reveals that GC1-deficient giant chloroplasts contain densely packed wild-type-like thylakoid membranes and that GC1-deficient leaves exhibit lower rates of CO(2) assimilation compared to wild-type. Although GC1 shows similarity to a putative cyanobacterial SulA cell division inhibitor, our findings suggest that GC1 does not act as a plastid division inhibitor but, rather, as a positive factor at an early stage of the division process.

A photoprotective role for O(2) as an alternative electron sink in photosynthesis?

Photoprotection of the photosynthetic apparatus has two essential elements: first, the thermal dissipation of excess excitation energy in the photosystem II antennae (i.e. non-photochemical quenching), and second, the ability of photosystem II to transfer electrons to acceptors within the chloroplast (i.e. photochemical quenching). Recent studies indicate that the proportion of absorbed photons that are thermally dissipated through the non-photochemical pathway often reaches a maximum well before saturating irradiances are reached. Hence, photochemical quenching is crucial for photoprotection at saturating light intensities. When plants are exposed to environmental stresses and the availability of CO(2) within the leaf is restricted, the reduction of oxygen by both the photorespiratory and the Mehler ascorbate peroxidase pathways appears to play a critical photoprotective role, substituting for CO(2) in sustaining electron flow. Induction of high activity of the Mehler ascorbate peroxidase pathway may be associated with acclimation to environmental stress.

Treatment of chronic painful diabetic neuropathy with isosorbide dinitrate spray: a double-blind placebo-controlled cross-over study.

OBJECTIVE: Considerable evidence implicates impaired nitric oxide (NO) generation in the pathogenesis of diabetic neuropathic pain. We therefore conducted a pilot study to examine the effects of isosorbide dinitrate (ISDN), a NO donor with local vasodilating properties, in spray form in the management of chronic neuropathic pain. RESEARCH DESIGN AND METHODS: The study was of double-blind, randomized, placebo-controlled, and two-period cross-over design. After a 2-week run-in period, 22 diabetic patients (13 men, 20 with type 2 diabetes, age [mean +/- SE] 63.7 +/- 1.8 years, duration of diabetes 9.1 +/- 1.5 years, duration of painful neuropathy 2.6 +/- 0.4 years) were randomized to receive ISDN or placebo sprays for 4 weeks, exchanging their treatment for a further 4 weeks after a 2-week wash-out period. The patients administered the spray to both feet before bedtime. Biweekly pain and other sensory symptoms were assessed using a visual analog scale (VAS) and the Lickert scale, respectively. RESULTS: ISDN spray reduced overall neuropathic pain (P = 0.02) and burning sensation (P = 0.006). No treatment difference was observed with other sensory modalities (hot/cold sensation, tingling, numbness, hyperesthesia, and jabbing-like sensation). At study completion, 11 patients (50%) reported benefit and wished to continue using the ISDN spray, 4 (18%) preferred the placebo spray, and the remaining 7 (32%) were undecided. CONCLUSIONS: ISDN spray offers an alternative and effective pharmacological option in relieving overall pain and burning sensation in the management of painful diabetic neuropathy. The potential of ISDN spray in alleviating other specific sensory symptoms associated with diabetic peripheral neuropathy merits further study.

Are we underestimating diabetes-related lower-extremity amputation rates? Results and benefits of the first prospective study.

OBJECTIVE: The objective of this study was to accurately determine the incidence of lower-extremity amputation using prospective data collection and to compare the results with those obtained by retrospective methods. RESEARCH DESIGN AND METHODS: The study was carried out over a 3-year period in a large district general hospital covering a clearly defined and relatively static population. All diabetic inpatients with foot problems were identified and followed-up until discharge or death. The demographic and admission details, medical history, investigations, procedures, and history and etiology of the foot lesion were collected twice weekly by a specialist nurse and podiatrist from all relevant wards. Thus, all subjects who underwent amputation could be identified. For comparison, retrospective data were collected from the hospital coding activities database, operating theater log books, anesthetic database, and limb-fitting records. RESULTS: The total population of the region in 2000 was 337,859, of which 9,183 were known to have diabetes. The total number of amputations during the 3-year survey period was 79, of which 45 were major and 34 minor. In our local population, the mean incidence during the survey period (1997-2000) equates to 7.8/100,000 general population and 2.85/1,000 diabetic population for all amputations, 4.5/100,000 general population and 1.62/1,000 diabetic population for major amputations, and 3.3/100,000 general population and 1.23/1,000 diabetic population for minor amputations. The prospective survey detected all lower-extremity amputations identified by the various retrospective methods; however, for the reverse, this was not the case. All of the retrospective methods, including the most commonly used (ICD-9 and OPCS-4 coding), failed to detect all of the cases revealed by the prospective survey (error rate ranging from 4.2 to 90.6%), and between 4.5 and 17.4% of amputations were misclassified. CONCLUSIONS: This study demonstrates the advantages of prospective data collection as a means of determining the incidence of lower-extremity amputations and highlights the limitations of retrospective data collection methods, which underestimate the incidence. In particular, the operating theater records, which have been the gold standard for many surveys, were found to be unreliable. Moreover, we have shown a 47% reduction in the major amputations during the survey period. Thus, we recommend that a prospective audit be incorporated into the activities of the specialist foot care team as a means of assessing and improving clinical care.

Comparative roles of microvascular and nerve function in foot ulceration in type 2 diabetes.

OBJECTIVE: To determine the relative roles of different modalities of sensory nerve function (large and small fiber) and the role of microvascular dysfunction in foot ulceration in type 2 diabetic subjects. RESEARCH DESIGN AND METHODS: A total of 20 control subjects and 18 type 2 diabetic subjects with foot ulceration and 20 without were studied. None of the subjects had clinical features of peripheral vascular disease. The Computer-Aided Sensory Evaluator IV (CASE IV) was used to determine vibration detection threshold (VDT), cold detection threshold (CDT), warm detection threshold (WDT), and heat pain onset threshold (HPO). Vibration perception threshold (VPT) was also assessed by a neurothesiometer. Microvascular function (maximum hyperemia to skin heating to 44 degrees C) was assessed using laser Doppler flowmetry (mean maximum hyperemia using laser Doppler flowmeter [LDF(max)]), laser Doppler imaging (mean maximum hyperemia using laser Doppler imager [LDI(max)]), and skin oxygenation with transcutaneous oxygen tension (TcpO(2)). RESULTS: VPT, VDT, CDT, and HPO were all significantly higher in individuals with ulceration than in those without (VPT and VDT: P < 0.0001) (CDT and HPO: P = 0.01). LDF(max), LDI(max), and TcpO(2) were significantly lower in the two diabetic groups than in the control subjects, but there was no difference between individuals with and without ulceration. Univariate logistic regression analysis revealed similar odds ratios for foot ulceration for VDT, CDT, HPO, and VPT (OR 1.97 [95% CI 1.30-2.98], 1.58 [1.20-2.08], 2.30 [1.21-4.37], and 1.24 [1.08-1.42], respectively). None of the microvascular parameters yielded significant odds ratios for ulceration. CONCLUSIONS: This study found that there was no additional value in measuring small-fiber function with the CASE IV over measuring vibration by either CASE IV or the inexpensive neurothesiometer in discriminating between individuals with and without ulceration. Furthermore, none of the tests of microvascular function including the TcpO(2) were able to discriminate between individuals with and without ulceration, suggesting that such tests may not be of benefit in identifying subjects at greater risk of foot ulceration.

Metabolomic response of Calotropis procera growing in the desert to changes in water availability.

Water availability is a major limitation for agricultural productivity. Plants growing in severe arid climates such as deserts provide tools for studying plant growth and performance under extreme drought conditions. The perennial species Calotropis procera used in this study is a shrub growing in many arid areas which has an exceptional ability to adapt and be productive in severe arid conditions. We describe the results of studying the metabolomic response of wild C procera plants growing in the desert to a one time water supply. Leaves of C. procera plants were taken at three time points before and 1 hour, 6 hours and 12 hours after watering and subjected to a metabolomics and lipidomics analysis. Analysis of the data reveals that within one hour after watering C. procera has already responded on the metabolic level to the sudden water availability as evidenced by major changes such as increased levels of most amino acids, a decrease in sucrose, raffinose and maltitol, a decrease in storage lipids (triacylglycerols) and an increase in membrane lipids including photosynthetic membranes. These changes still prevail at the 6 hour time point after watering however 12 hours after watering the metabolomics data are essentially indistinguishable from the prewatering state thus demonstrating not only a rapid response to water availability but also a rapid response to loss of water. Taken together these data suggest that the ability of C. procera to survive under the very harsh drought conditions prevailing in the desert might be associated with its rapid adjustments to water availability and losses.

The high light response in Arabidopsis involves ABA signaling between vascular and bundle sheath cells.

Previously, it has been shown that Arabidopsis thaliana leaves exposed to high light accumulate hydrogen peroxide (H2O2) in bundle sheath cell (BSC) chloroplasts as part of a retrograde signaling network that induces ASCORBATE PEROXIDASE2 (APX2). Abscisic acid (ABA) signaling has been postulated to be involved in this network. To investigate the proposed role of ABA, a combination of physiological, pharmacological, bioinformatic, and molecular genetic approaches was used. ABA biosynthesis is initiated in vascular parenchyma and activates a signaling network in neighboring BSCs. This signaling network includes the Galpha subunit of the heterotrimeric G protein complex, the OPEN STOMATA1 protein kinase, and extracellular H2O2, which together coordinate with a redox-retrograde signal from BSC chloroplasts to activate APX2 expression. High light-responsive genes expressed in other leaf tissues are subject to a coordination of chloroplast retrograde signaling and transcellular signaling activated by ABA synthesized in vascular cells. ABA is necessary for the successful adjustment of the leaf to repeated episodes of high light. This process involves maintenance of photochemical quenching, which is required for dissipation of excess excitation energy.

PHOTOSYNTHESIS AND PRODUCTION OF HYDROGEN PEROXIDE BY SYMBIODINIUM (PYRRHOPHYTA) PHYLOTYPES WITH DIFFERENT THERMAL TOLERANCES(1).

Occurrences whereby cnidaria lose their symbiotic dinoflagellate microalgae (Symbiodinium spp.) are increasing in frequency and intensity. These so-called bleaching events are most often related to an increase in water temperature, which is thought to limit certain Symbiodinium phylotypes from effectively dissipating absorbed excitation energy that is otherwise used for photochemistry. Here, we examined photosynthetic characteristics and hydrogen peroxide (H2 O2 ) production, a possible signal involved in bleaching, from two Symbiodinium types (a thermally "tolerant" A1 and "sensitive" B1) representative of cnidaria-Symbiodinium symbioses of reef-building Caribbean corals. Under steady-state growth at 26°C, a higher efficiency of PSII photochemistry, rate of electron turnover, and rate of O2 production were observed for type A1 than for B1. The two types responded very differently to a period of elevated temperature (32°C): type A1 increased light-driven O2 consumption but not the amount of H2 O2 produced; in contrast, type B1 increased the amount of H2 O2 produced without an increase in light-driven O2 consumption. Therefore, our results are consistent with previous suggestions that the thermal tolerance of Symbiodinium is related to adaptive constraints associated with photosynthesis and that sensitive phylotypes are more prone to H2 O2 production. Understanding these adaptive differences in the genus Symbiodinium will be crucial if we are to interpret the response of symbiotic associations, including reef-building corals, to environmental change.