Abscisic acid regulation of heterophylly in Marsilea quadrifolia L.: effects of R-(-) and S-(+) isomers

The plant hormone abscisic acid (ABA) induces a developmental switch in the aquatic fern Marsilea quadrifolia, causing the formation of aerial type characteristics, including the elongation of petioles and roots, a change in leaf morphology, the expansion of leaf surface area, and the shortening of the internodes. A number of ABA-responsive heterophylly (ABRH) genes are induced early during the transition. Using optically pure isomers of ABA, it was found that both the natural S-(+)-ABA and the unnatural R-(-)-ABA are capable of inducing a heterophyllous switch and regulating ABRH gene expression. When dose responses are compared, the unnatural ABA gives stronger morphogenic effects than the natural ABA at the same concentration, it is effective at lower concentrations, and its optimal concentration is also lower compared with the natural ABA. Deuterium-labelled ABA enantiomers were used to trace the fate of applied ABA and to distinguish the applied compound and its metabolites from the endogenous counterparts. In tissues, the supplied (+)-ABA was metabolized principally to dihydrophaseic acid, while the supplied (-)-ABA was converted at a slower rate to 7'-hydroxy abscisic acid. Treatment with either enantiomer resulted in increased biosynthesis of ABA, as reflected in the accumulation of endogenous dihydrophaseic acid. Taken together, these results suggest two distinct mechanisms of action for (-)-ABA: either (-)-ABA is intrinsically active, or its activity is due to the stimulation of ABA biosynthesis.     

Heterophylly in Ranunculus flabellaris: The Effect of Abscisic Acid on Leaf Anatomy

Ranunculus flabellaris Raf., the yellow water crowfoot, exhibitsstriking heterophylly between submerged and terrestrial leaves.Leaves produced under water are highly divided with numerousnarrow lobes and deep sinuses, whereas terrestrial leaves havefew broad lobes and shallow sinuses. When plants are submergedin a 25 µM solution of ABA, the typical transition fromterrestrial to submerged leaves is completely suppressed and,instead, terrestrial-like leaves are produced. Image analysistechniques show that, in addition to this modification of leafmorphology, leaves produced under ABA treatment possess surfaceand internal features characteristic of terrestrial leaf anatomy.This study provides evidence that the environmental factorsthat influence the morphological and anatomical expression ofheterophylly may act through endogenous ABA. Ranunculus flabellaris, yellow water crowfoot, ABA, heterophylly, leaf anatomy     

Effects of exogenous ABA and cytokinin on leaf photosynthesis and grain protein accumulation in wheat ears cultured <Emphasis Type="Italic">in vitro</Emphasis>

A detached culture system and steady-state ¹⁵N labeling technique were used to study the effects of exogenous ABA and ZR on photosynthetic characteristics, nitrogen remobilization and the activities of key enzymes for nitrogen metabolism in detached wheat parts during grain protein accumulation. The differences in net photosynthetic rate, chlorophyll content (SPAD value) and soluble protein content in the flag leaves of detached culture system between the treatments of ABA and ZR showed that ABA facilitates the post-anthesis senescence course compared to the ZR treatment. The differences in the changes of ¹⁵N amount in different organs in the detached culture system between the ABA and ZR treatments showed that nitrogen remobilization from vegetative organs to the grain is accelerated by the ABA treatment but is delayed by ZR. The activities of GS and GPT in grains treated with ABA were significantly higher than those with the control treatment at 5 DAC, but reduced significantly compared with control at 11 DAC. The two enzyme activities in grains were reduced significantly by ZR at 5 DAC and increased significantly at 11 DAC, compared with those treated with ABA. The above changes of enzyme activity showed that the ABA treatment hastens amino acid conversion into grains and protein accumulation in grains, whereas the ZR treatment delays these processes. A significant reduction in grain weight with ABA treatment is associated with the reduction of net photosynthesis, chlorophyll content, and soluble protein content in flag leaves. Compared with the control and ZR treatments, a significant increase in grain protein content with the ABA treatment may result from the accelerating effects of ABA on N remobilization, amino acid conversion into grains and protein accumulation in grains.     

Suppression by ABA of salicylic acid and lignin accumulation and the expression of multiple genes,in <Emphasis Type="Italic">Arabidopsis</Emphasis> infected with <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tomato</Emphasis>

Abscisic acid (ABA) has been implicated in determining the outcome of interactions between many plants and their pathogens. We had previously shown that increased concentrations of ABA within leaves of Arabidopsis induced susceptibility towards an avirulent strain of Pseudomonas syringae pathovar (pv.) tomato. We now show that ABA induces susceptibility via suppression of the accumulation of components crucial for a resistance response. Lignin and salicylic acid concentrations in leaves were increased during a resistant interaction but reduced when plants were treated with ABA. The reduction in lignin and salicylic acid production was independent of the development of the hypersensitive response (HR), indicating that, in this host-pathogen system, HR is not required for resistance. Genome-wide gene expression analysis using microarrays showed that treatment with ABA suppressed the expression of many defence-related genes, including those important for phenylpropanoid biosynthesis and those encoding resistance-related proteins. Together, these results show that resistance induction in Arabidopsis to an avirulent strain of P. syringae pv. tomato is regulated by ABA. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Leaves may function as temperature sensors in the heterophylly of Rorippa aquatica (Brassicaceae)

Many plants show heterophylly, which is variation in leaf form within a plant owing to environmental change. The molecular mechanisms underlying heterophylly have recently been investigated in several plant species. However, little is known about how plants exhibiting heterophylly sense environmental cues. Here, we used Rorippa aquatica (Brassicaceae), which shows heterophylly, to investigate whether a single leaf can sense and transit changes in ambient temperature. The morphology of newly developed leaves after single-leaf warming treatment was significantly different from that of mock-treated control leaves, suggesting that leaves are sensing organs that mediate the responses to changes in ambient temperature in R. aquatica.     

Regulation of the KNOX-GA Gene Module Induces Heterophyllic Alteration in North American Lake Cress

Plants show leaf form alteration in response to changes in the surrounding environment, and this phenomenon is called heterophylly. Although heterophylly is seen across plant species, the regulatory mechanisms involved are largely unknown. Here, we investigated the mechanism underlying heterophylly in Rorippa aquatica (Brassicaceae), also known as North American lake cress. R. aquatica develops pinnately dissected leaves in submerged conditions, whereas it forms simple leaves with serrated margins in terrestrial conditions. We found that the expression levels of KNOTTED1-LIKE HOMEOBOX (KNOX1) orthologs changed in response to changes in the surrounding environment (e.g., change of ambient temperature; below or above water) and that the accumulation of gibberellin (GA), which is thought to be regulated by KNOX1 genes, also changed in the leaf primordia. We further demonstrated that exogenous GA affects the complexity of leaf form in this species. Moreover, RNA-seq revealed a relationship between light intensity and leaf form. These results suggest that regulation of GA level via KNOX1 genes is involved in regulating heterophylly in R. aquatica. The mechanism responsible for morphological diversification of leaf form among species may also govern the variation of leaf form within a species in response to environmental changes.     

Foliar sprays with ABA promote growth of Ilex paraguariensis by alleviating diurnal water stress

Stomatal closure, relative water content (RWC) and vegetative growth were monitored in Ilex paraguariensis plants grown under well-watered conditions with a photosynthetic photon flux density (PPFD) varying from 100% to 1.5%, and sprayed weekly with either distilled water (control) or 1.89 mM abscisic acid (ABA). ABA treatments caused stomatal closure, ranging from 62% to 73%. These treatments also increased RWC in the early evening from 82% to 92% and 88% to 94% in mature and immature leaves, respectively. Such alleviation of the water stress was correlated with increases in leaf area, leaf dry weight (DW), shoot length and shoot DW. On day 35 from the beginning of the experiment, the increases in DW of both leaves and shoots were 1.5-fold at the 1.5% PPFD and 3-fold (for leaves) and 4.5-fold (for shoots) under 100% PPFD. In water-sprayed control plants grown under 1.5% PPFD shoot length also increased significantly, although these shoots contained more ABA (assessed by capillary gas chromatography–mass spectrometry) than those of plants grown under 100% PPFD. These results show that ABA sprayed on to leaves promotes growth in I. paraguariensis plants by alleviating diurnal water stress.     

Abscisic acid does not mediate NaCl-induced accumulation of 70-kDa subunit tonoplast H+ -ATPase message in tomato

There is an increased accumulation of message for the catalytic (70-kDa) subunit of the tonoplast H⁺-ATPase in leaves of tomato (Lycopersicon esculentum L.) plants responding to NaCl. To determine if abscisic acid (ABA) mediates this response, message accumulation was examined in treatments designed to separate exposure to NaCl from increases in endogenous ABA. Under three different experimental conditions, salt-induced changes in the accumulation of 70-kDa message were unrelated to any change in endogenous ABA. The results were as follows: (i) under drought stress, plants accumulated levels of ABA similar to those measured in salt-treated plants; however, no increase in 70-kDa subunit message was observed; (ii) the ABA-deficient mutant sitiens exhibited an increased accumulation of message despite the absence of NaCl-induced accumulation of ABA; and (iii) the inhibitor of general isoprenoid biosynthesis, Lovastatin, blocked NaCl-induced accumulation of ABA but did not alter NaCl-induced accumulation of message. In addition to these three experimental responses, application of exogenous ABA increased endogenous ABA levels without any comparable increase in message accumulation. Based on these results, it is concluded that ABA does not mediate the NaCl-induced accumulation of 70-kDa subunit tonoplast H⁺ -ATPase message accumulation in tomato.     

Seasonal changes in endogenous ABA and IAA and the influence of applied ABA and auxin in relation to shoot growth and abscission in Valencia Orange (Citrus sinensis (L.) Osbeck)

Seasonal changes in endogenous IAA and ABA were measured by gas chromatography/mass-spectrometry. Highest concentrations of ABA occurred in leaves. There was a major ABA peak in early spring (up to 1360 ng g^–1 dw). Levels were low in summer (90 ng g^–1 dw). There was a minor ABA peak in autumn. Endogenous IAA in leaves was highest in winter/spring (up to 76 ng g^–1 dw). Applied ABA promoted abscission of leaves and shoots while applied NAA delayed abscission. The main peak in leaf-ABA content was followed by extensive shoot abscission. The involvement of ABA and IAA in regulation of flush growth was not clear.This paper is dedicated to Michael G. Mullins, who died on 13 November 1990, for his outstanding contribution to horticulture.     

ABA Initiates Anthocyanin Production in Grape Cell Cultures

Abscisic acid (ABA) has a well-known positive impact on grape ripening, especially color development, but its role in the initiation of anthocyanin synthesis remains unclear. To elucidate this point, ABA treatment was applied to a simple Vitis vinifera model, consisting of Cabernet Sauvignon cell suspensions that do not spontaneously produce anthocyanins under laboratory conditions. Endogenous ABA levels, the expression of some genes in the upstream part of the anthocyanin pathway, and anthocyanin content were determined. Exogenous ABA treatment sharply increased cell ABA content and induced both structural and regulatory genes involved in anthocyanin production. These changes were promptly detected, as early as 6 h after ABA treatment, whereas anthocyanin production was observed only after 4 days in culture. These results demonstrate that ABA promotes anthocyanin synthesis in grape cell culture.     

Phosphate deficiency-dependent upregulation of UDP-glucose pyrophosphorylase genes is insensitive to ABA and ethylene status in Arabidopsis leaves

The effects of inorganic phosphate (Pi) deficiency and ABA/ethylene status on expression of UDP-glucose pyrophosphorylase (UGPase) genes (Ugp), involved in sucrose/polysaccharide metabolism, were investigated. Both wild-type (wt), aba and abi mutants (ABA-deficient and -in-sensitive), etr, ein and eto (ethylene resistant and overproducing) grown on Pi-deficient and complete nutrient solution, as well as phol (Pi-deficient) mutants of Arabidopsis thaliana were used for experiments. Generally, Pi-deficiency conditions (including mannose feeding to decrease cytosolic Pi pool) resulted in an increase of Ugp expression in the leaves, under all experimental conditions. Mutant backgrounds reflecting differences in ABA or ethylene status/ sensitivity had no effect on the level of Ugp up-regulation by Pi-stress. Furthermore, feeding ABA to the leaves of wt and pho1 plants had no effect on Ugp expression, regardless of the sucrose status in the leaves. The data suggest that Pi deficiency leading to up-regulation of Ugp acts independently of ABA and ethylene status.     

Transpiration, CO2 assimilation, WUE, and stomatal aperture in leaves of Viscum album (L.): Effect of abscisic acid (ABA) in the xylem sap of its host (Populus x euamericana).

Leaves of the mistletoe Viscum album (L.) show a high rate of transpiration, even when the host is under severe drought stress. The hypothesis that a strong control of ABA influx from the xylem sap of the host into the mistletoe prevents stomatal closure in mistletoe leaves was tested under the following conditions: sections of poplar twigs carrying a mistletoe were perfused with artificial xylem sap that contained different ABA concentrations and both transpiration and ABA levels were analysed in mistletoe leaves. Despite variation by a factor of 10(4), the ABA content of the host xylem did not affect ABA levels, leaf transpiration, CO(2) assimilation, WUE, or the degree of stomatal aperture in mistletoe leaves. These observations support the hypothesis of a strong control of ABA influx from the host of the xylem into the mistletoe, although degradation of ABA before it enters the mistletoe leaves cannot be excluded. This mechanism may ensure a water and nutritional status favourable for the mistletoe, even if the water status of the host is impaired. Despite the lack of short-term sensitivity of ABA levels in mistletoe leaves to even strong changes of ABA levels in the xylem sap of the host, ABA levels in mistletoe leaves were relatively high compared to ABA levels in the leaves of several tree species including poplar. Since significant transpiration of the mistletoe leaves was observed despite high ABA levels, a diminished sensitivity of the stomata of mistletoe leaves to ABA has to be concluded. The stomatal density of adaxial Viscum leaves of 89+/-23 stomata per mm is lower than those reported in a study performed at the end of the 19th century.     

Activation of abscisic acid biosynthesis in the leaves of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> in response to water deficit

It is well known that endogenous abscisic acid (ABA) levels increase rapidly in response to drought stress and that this induces stomatal closure. In Arabidopsis thaliana, ABA levels increased rapidly in the leaves and roots when intact wild-type whole plants were exposed to drought stress. However, if the leaves and roots were separated and exposed to drought independently, the ABA level increased only in the leaves. These results suggest that, under our experimental conditions, ABA is synthesized mainly in the leaves in response to drought stress and that some of the ABA accumulated in the leaves is transported to the roots. Tracer experiments using isotope-labeled ABA indicate that the movement of ABA from leaves to roots is activated by water deficit in the roots. We also demonstrate that the endogenous ABA level in the leaves increased only when the leaves themselves were exposed to drought stress, suggesting that leaves play a major role in the production of ABA in response to acute water shortage.     

Uptake, Movement, Activity, and Persistence of an Abscisic Acid Analog (8′ Acetylene ABA Methyl Ester) in Marigold and Tomato

The abscisic acid (ABA) analog 8′ acetylene ABA methyl ester (PBI 429) was evaluated for its potential to alter the growth and moisture use of bedding plants during nursery production. Treating seedlings with the ABA analog as a root-dip slowed moisture use and growth of tomato seedlings under greenhouse conditions. In marigolds, comparable ABA analog treatments had no effect on growth and limited effects on plant moisture use. To determine whether these differences in response to treatment with the ABA analog were associated with differences in absorption of the analog and/or its persistence, the ABA analog was applied either as a foliar spray or root-dip, and the resulting concentrations of the ABA analog were monitored over a 10-day interval in both the roots and the leaves. In both crops, the ABA analog was detected in both leaf and root tissues irrespective of the mode of application, suggesting systemic movement of the analog. Tissue concentrations of the ABA analog were consistently lower in the foliar treatment than in the root-dip. The uptake and the retention of the ABA analog over time was similar in leaves of the two test crops, but less of the ABA analog was absorbed and retained in the roots of marigold plants than in the tomatoes. This suggests that the observed differences in responses of these two plant species to application of ABA analogs may be related to differences in retention or accumulation of ABA in the roots rather than to differences in the total amount of ABA analog absorbed or its movement and retention in the plant system. Levels of endogenous ABA were not significantly altered by application of the ABA analog.     

Genetic association mapping identifies single nucleotide polymorphisms in genes that affect abscisic acid levels in maize floral tissues during drought

In maize, water stress at flowering causes loss of kernel set and productivity. While changes in the levels of sugars and abscisic acid (ABA) are thought to play a role in this stress response, the mechanistic basis and genes involved are not known. A candidate gene approach was used with association mapping to identify loci involved in accumulation of carbohydrates and ABA metabolites during stress. A panel of single nucleotide polymorphisms (SNPs) in genes from these metabolic pathways and in genes for reproductive development and stress response was used to genotype 350 tropical and subtropical maize inbred lines that were well watered or water stressed at flowering. Pre-pollination ears, silks, and leaves were analysed for sugars, starch, proline, ABA, ABA-glucose ester, and phaseic acid. ABA and sugar levels in silks and ears were negatively correlated with their growth. Association mapping with 1229 SNPs in 540 candidate genes identified an SNP in the maize homologue of the Arabidopsis MADS-box gene, PISTILLATA, which was significantly associated with phaseic acid in ears of well-watered plants, and an SNP in pyruvate dehydrogenase kinase, a key regulator of carbon flux into respiration, that was associated with silk sugar concentration. An SNP in an aldehyde oxidase gene was significantly associated with ABA levels in silks of water-stressed plants. Given the short range over which decay of linkage disequilibrium occurs in maize, the results indicate that allelic variation in these genes affects ABA and carbohydrate metabolism in floral tissues during drought.     

Heterophylly in Ranunculus flabellaris: The Effect of Abscisic Acid on Leaf Ultrastructure

Ranunculus flabellaris Rafin., an aquatic buttercup, exhibitsheterophylly at the level of cellular ultrastructure. Comparedto terrestrial leaves, underwater leaves have thinner epidermalcell walls and more numerous paramural bodies per epidermaland mesophyll cell cross-section. The number of chloroplastsand mitochondria in cell cross-sections also contrasts betweenthe two leaf types. Despite within-and between-leaf variations,different patterns of organelle distribution for the two leafforms were found using principal coordinates analysis. In addition,underwater leaf chloroplasts are smaller, have fewer grana,a greater number of thylakoids/granum, and less starch comparedto chloroplasts from terrestrial leaves. At the ultrastructurallevel, submergence in ABA solution does not produce a leaf withas many characteristics of the terrestrial environment, as shownin previous studies of leaf morphology and anatomy. While numberand distribution of organelles in ABA-treated leaves are similarto terrestrial leaves, some features of chloroplast internalstructure and paramural body number and distribution resembleunderwater leaves. It is postulated that ABA acts as a morphogeninvolved in guiding the irreversible processes of leaf development,but certain subcellular characteristics may be determined directlyby the physical environment. Difficulties encountered in quantitativeanalyses of cellular ultrastructure are discussed. Ranunculus flabellaris, ABA, heterophylly, leaf ultrastructure, principal coordinates analysis