Is nitric oxide a critical key factor in ABA-induced stomatal closure?

The role of nitric oxide (NO) in abscisic acid (ABA)-induced stomatal closure is a matter of debate. We conducted experiments in Vicia faba leaves using NO gas and sodium nitroprusside (SNP), a NO-donor compound, and compared their effects to those of ABA. In epidermal strips, stomatal closure was induced by ABA but not by NO, casting doubt on the role of NO in ABA-mediated stomatal closure. Leaf discs and intact leaves showed a dual dose response to NO: stomatal aperture widened at low dosage and narrowed at high dosage. Overcoming stomatal resistance by means of high CO2 concentration ([CO2]) restored photosynthesis in ABA-treated leaf discs but not in those exposed to NO. NO inhibited photosynthesis immediately, causing an instantaneous increase in intercellular [CO2] (Ci), followed by stomatal closure. However, lowering Ci by using low ambient [CO2] showed that it was not the main factor in NO-induced stomatal closure. In intact leaves, the rate of stomatal closure in response to NO was about one order of magnitude less than after ABA application. Because of the different kinetics of photosynthesis and stomatal closure that were observed, we conclude that NO is not likely to be the key factor in ABA-induced rapid stomatal closure, but that it fine-tunes stomatal aperture via different pathways.

An ultra-dense integrated linkage map for hexaploid chrysanthemum enables multi-allelic QTL analysis.

KEY MESSAGE: We constructed the first integrated genetic linkage map in a polysomic hexaploid. This enabled us to estimate inheritance of parental haplotypes in the offspring and detect multi-allelic QTL. Construction and use of linkage maps are challenging in hexaploids with polysomic inheritance. Full map integration requires calculations of recombination frequency between markers with complex segregation types. In addition, detection of QTL in hexaploids requires information on all six alleles at one locus for each individual. We describe a method that we used to construct a fully integrated linkage map for chrysanthemum (Chrysanthemum × morifolium, 2n = 6x = 54). A bi-parental F1 population of 406 individuals was genotyped with an 183,000 SNP genotyping array. The resulting linkage map consisted of 30,312 segregating SNP markers of all possible marker dosage types, representing nine chromosomal linkage groups and 107 out of 108 expected homologues. Synteny with lettuce (Lactuca sativa) showed local colinearity. Overall, it was high enough to number the chrysanthemum chromosomal linkage groups according to those in lettuce. We used the integrated and phased linkage map to reconstruct inheritance of parental haplotypes in the F1 population. Estimated probabilities for the parental haplotypes were used for multi-allelic QTL analyses on four traits with different underlying genetic architectures. This resulted in the identification of major QTL that were affected by multiple alleles having a differential effect on the phenotype. The presented linkage map sets a standard for future genetic mapping analyses in chrysanthemum and closely related species. Moreover, the described methods are a major step forward for linkage mapping and QTL analysis in hexaploids.

Natural variation in stomatal response to closing stimuli among Arabidopsis thaliana accessions after exposure to low VPD as a tool to recognize the mechanism of disturbed stomatal functioning.

Stomatal responses to closing stimuli are disturbed after long-term exposure of plants to low vapour pressure deficit (VPD). The mechanism behind this disturbance is not fully understood. Genetic variation between naturally occurring ecotypes can be helpful to elucidate the mechanism controlling stomatal movements in different environments. We characterized the stomatal responses of 41 natural accessions of Arabidopsis thaliana to closing stimuli (ABA and desiccation) after they had been exposed for 4 days to moderate VPD (1.17 kPa) or low VPD (0.23 kPa). A fast screening system was used to test stomatal response to ABA using chlorophyll fluorescence imaging under low O2 concentrations of leaf discs floating on ABA solutions. In all accessions stomatal conductance (gs) was increased after prior exposure to low VPD. After exposure to low VPD, stomata of 39 out of 41 of the accessions showed a diminished ABA closing response; only stomata of low VPD-exposed Map-42 and C24 were as responsive to ABA as moderate VPD-exposed plants. In response to desiccation, most of the accessions showed a normal stomata closing response following low VPD exposure. Only low VPD-exposed Cvi-0 and Rrs-7 showed significantly less stomatal closure compared with moderate VPD-exposed plants. Using principle component analysis (PCA), accessions could be categorized to very sensitive, moderately sensitive, and less sensitive to closing stimuli. In conclusion, we present evidence for different stomatal responses to closing stimuli after long-term exposure to low VPD across Arabidopsis accessions. The variation can be a useful tool for finding the mechanism of stomatal malfunctioning.

Stomatal malfunctioning under low VPD conditions: induced by alterations in stomatal morphology and leaf anatomy or in the ABA signaling?

Exposing plants to low VPD reduces leaf capacity to maintain adequate water status thereafter. To find the impact of VPD on functioning of stomata, stomatal morphology and leaf anatomy, fava bean plants were grown at low (L, 0.23 kPa) or moderate (M, 1.17 kPa) VPDs and some plants that developed their leaves at moderate VPD were then transferred for 4 days to low VPD (M→L). Part of the M→L-plants were sprayed with ABA (abscisic acid) during exposure to L. L-plants showed bigger stomata, larger pore area, thinner leaves and less spongy cells compared with M-plants. Stomatal morphology (except aperture) and leaf anatomy of the M→L-plants were almost similar to the M-plants, while their transpiration rate and stomatal conductance were identical to that of L-plants. The stomatal response to ABA was lost in L-plants, but also after 1-day exposure of M-plants to low VPD. The level of foliar ABA sharply decreased within 1-day exposure to L, while the level of ABA-GE (ABA-glucose ester) was not affected. Spraying ABA during the exposure to L prevented loss of stomatal closing response thereafter. The effect of low VPD was largely depending on exposure time: the stomatal responsiveness to ABA was lost after 1-day exposure to low VPD, while the responsiveness to desiccation was gradually lost during 4-day exposure to low VPD. Leaf anatomical and stomatal morphological alterations due to low VPD were not the main cause of loss of stomatal closure response to closing stimuli.

Can prolonged exposure to low VPD disturb the ABA signalling in stomatal guard cells?

The response of stomata to many environmental factors is well documented. Multiple signalling pathways for abscisic acid (ABA)-induced stomatal closure have been proposed over the last decades. However, it seems that exposure of a leaf for a long time (several days) to some environmental conditions generates a sort of memory in the guard cells that results in the loss of suitable responses of the stomata to closing stimuli, such as desiccation and ABA. In this review paper we discuss changes in the normal pattern of signal transduction that could account for disruption of guard cell signalling after long-term exposure to some environmental conditions, with special emphasis on long-term low vapour pressure deficit (VPD).

Dynamics of adaptation of stomatal behaviour to moderate or high relative air humidity in Tradescantia virginiana.

Chlorophyll fluorescence imaging was used to measure stomatal closure in response to desiccation of Tradescantia virginiana leaves grown under high (90%) and moderate (55%) relative humidities (RHs), or transferred between these humidities. Stomata in leaves grown at high RH were less responsive to desiccation than those of leaves grown at moderate RH. Stomata of plants transferred from moderate RH conditions to high RH showed the same diminished closure in response to desiccation as did stomata that developed at high RH. This response was found both when the leaves were fully expanded and when still actively expanding during the moderate RH pre-treatment. Four days of exposure to high RH was the minimal exposure time to induce the diminished closure response. When leaves were grown in high RH prior to a 10 d moderate RH treatment, the reduced stomatal closure response to desiccation was only reversed in leaves (regions) which were actively expanding during moderate RH treatment. This indicates that with respect to stomatal responses to desiccation, high RH leaf regions have a limited capacity to adapt to moderate RH conditions. The decrease in responsiveness to desiccation of the stomata, induced by long-term exposure to high RH, was not due to osmotic adjustment in the leaves. Within 1 d after transferring moderate RH-grown plants to a high RH, the abscisic acid (ABA) concentration of their leaves decreased to the low level of ABA found in high RH-grown leaves. The closure response in leaves exposed to high RH for 5 d, however, could not be fully restored by the application of ABA. Transferring plants from high to moderate RH resulted in increased ABA levels within 2 d without a recovery of the stomatal closing response. It is discussed that the diminished stomatal closure in plants exposed to high RH could be due to changes in the signalling pathway for ABA-related closure of stomata or to an increased sequestration of ABA by mesophyll tissue or the symplast in the epidermis, induced by a longer period (several days) of a low ABA level.

The role of abscisic acid in disturbed stomatal response characteristics of Tradescantia virginiana during growth at high relative air humidity.

In this study, the role of abscisic acid (ABA) in altered stomatal responses of Tradescantia virginiana leaves grown at high relative air humidity (RH) was investigated. A lower ABA concentration was found in leaves grown at high RH compared with leaves grown at moderate RH. As a result of a daily application of 20 microM ABA to leaves for 3 weeks during growth at high RH, the stomata of ABA-treated leaves grown at high RH showed the same behaviour as did the stomata of leaves grown at moderate RH. For example, they closed rapidly when exposed to desiccation. Providing a high RH around a single leaf of a plant during growth at moderate RH changed the stomatal responses of this leaf. The stomata in this leaf grown at high RH did not close completely in response to desiccation in contrast to the stomata of the other leaves from the same plant. The ABA concentration on a fresh weight basis, though not on a dry weight basis, of this leaf was significantly lower than that of the others. Moreover, less closure of stomata was found in the older leaves of plants grown at high RH in response to desiccation compared with younger leaves. This was correlated with a lower ABA concentration in these leaves on a fresh weight basis, though not on a dry weight basis. Stomata of leaves grown at moderate RH closed in response to short-term application of ABA or sodium nitroprusside (SNP), while for leaves grown at high RH there was a clear difference in stomatal responses between the leaf margins and main-vein areas. The stomatal aperture in response to short-term application of ABA or SNP at the leaf margins of leaves grown at high RH remained significantly wider than in the main-vein areas. It was concluded that: (i) a long-term low ABA concentration in well-watered plants during growth at high RH could be a reason for less or no stomatal closure under conditions of drought stress; and (ii) the long-term ABA concentration on a fresh weight basis rather than on a dry weight basis is likely to be responsible for structural or physiological changes in stomata during leaf growth.

Dynamics of spatial heterogeneity of stomatal closure in Tradescantia virginiana altered by growth at high relative air humidity.

The spatial heterogeneity of stomatal closure in response to rapid desiccation of excised well-watered Tradescantia virginiana leaves grown at moderate (55%) or high (90%) relative air humidity (RH) was studied using a chlorophyll fluorescence imaging system under non-photorespiratory conditions. Following rapid desiccation, excised leaves grown at high RH had both a greater heterogeneity and a higher average value of PSII efficiency (Phi(PSII)) compared with leaves grown at moderate RH. Larger decreases in relative water content resulted in smaller decreases in water potential and Phi(PSII) of high RH-grown leaves compared with moderate RH-grown leaves. Moreover, the Phi(PSII) of excised high RH-grown leaves decreased less with decreasing water potential, implying that the stomata of high RH-grown leaves are less sensitive to decreases in leaf water potential compared with moderate RH-grown leaves. After desiccation, some non-closing stomata were distributed around the main vein in high RH-grown leaves. Direct measurements of stomatal aperture showed 77% stomatal closure in the margins after 2 h desiccation compared with 40% closure of stomata in the main-vein areas in high RH-grown leaves. Faster closure of stomata in leaf margins compared with main-vein areas of leaves grown at high RH was related to substantially lower relative water content in these areas of the leaves.

Flower opening and closure: a review.

Flower opening and closure are traits of a reproductive syndrome, as it allows pollen removal and/or pollination. Various types of opening can be distinguished such as nocturnal and diurnal and single or repetitive. Opening is generally due to cell expansion. Osmotic solute levels increase by the conversion of polysaccharides (starch or fructan) to monosaccharides, and/or the uptake of sugars from the apoplast. Repeated opening and closure movements are often brought about by differential elongation. In tulip petals, for example, the upper and lower sides of the mesophyll exhibit a 10 degrees C difference in optimum temperature for elongation growth, resulting in opening in the morning and closure in the evening. Opening and closure in several other species is regulated by changes in light intensity and, in some species with nocturnal opening, by an increase in relative humidity. A minimum duration of darkness and light are usually required for opening and closure, respectively, in flowers that open during the day. Both phytochrome and a blue light receptor seem involved in light perception. In some species, opening and closure are regulated by an endogenous rhythm, which, in all cases investigated, can be reset by changes from dark to light and/or light to dark. So far, Arabidopsis mutants have not been used to investigate the timing of flower opening and closure. As its flowers open and close in a circadian fashion, several mutants that are involved in the circadian clock and its light input may help to provide an insight into this type of flower opening. The co-ordination of processes culminating in synchronized flower opening is, in many species, highly intricate. The complex control by endogenous and exogenous factors sets flower opening and closure apart from most other growth processes.