Grafting Cucumber Onto Pumpkin Induced Early Stomatal Closure by Increasing ABA Sensitivity Under Salinity Conditions.
ABSTRACT: During early periods of salt stress, reduced stomatal opening can prevent water loss and wilting. Abscisic acid (ABA) signal plays an important role in this process. Here, we show that cucumber grafted onto pumpkin exhibits rapid stomatal closure, which helps plants to adapt to osmotic stress caused by salinity. Increased ABA contents in the roots, xylem sap, and leaves were evaluated in two grafting combinations (self-grafted cucumber and cucumber grafted onto pumpkin rootstock). The expression levels of ABA biosynthetic or signaling related genes NCED2 (9-cis-epoxycarotenoid dioxygenase gene 2), ABCG22 (ATP-binding cassette transporter genes 22), PP2C (type-2C protein phosphatases), and SnRK2.1 (sucrose non-fermenting 1-related protein kinases 2) were investigated. Results showed that a root-sourced ABA signal led to decreased stomatal opening and transpiration in the plants grafted onto pumpkin. Furthermore, plants grafted onto pumpkin had increased sensitivity to ABA, compared with self-grafted cucumbers. The inhibition of ABA biosynthesis with fluridon in roots increased the transpiration rate (Tr) and stomatal conductance (Gs) in the leaves. Our study demonstrated that the roots of pumpkin increases the sensitivity of the scion to ABA delivered from the roots to the shoots, and enhances osmotic tolerance under NaCl stress. Such a mechanism can be greatly exploited to benefit vegetable production particularly in semiarid saline regions.
Project description:Balancing stomata-dependent CO2 assimilation and transpiration is a key challenge for increasing crop productivity and water use efficiency under drought stress for sustainable crop production worldwide. Here, we show that cucumber and luffa plants with luffa as rootstock have intrinsically increased water use efficiency, decreased transpiration rate and less affected CO2 assimilation capacity following drought stress over those with cucumber as rootstock. Drought accelerated abscisic acid (ABA) accumulation in roots, xylem sap and leaves, and induced the transcript of ABA signaling genes, leading to a decreased stomatal aperture and transpiration in the plants grafted onto luffa roots as compared to plants grafted onto cucumber roots. Furthermore, stomatal movement in the plants grafted onto luffa roots had an increased sensitivity to ABA. Inhibition of ABA biosynthesis in luffa roots decreased the drought tolerance in cucumber and luffa plants. Our study demonstrates that the roots of luffa have developed an enhanced ability to sense the changes in root-zone moisture and could eventually deliver modest level of ABA from roots to shoots that enhances water use efficiency under drought stress. Such a mechanism could be greatly exploited to benefit the agricultural production especially in arid and semi-arid areas.
Project description:Plant salt tolerance can be improved by grafting onto salt-tolerant rootstocks. However, the underlying signaling mechanisms behind this phenomenon remain largely unknown. To address this issue, we used a range of physiological and molecular techniques to study responses of self-grafted and pumpkin-grafted cucumber plants exposed to 75 mM NaCl stress. Pumpkin grafting significantly increased the salt tolerance of cucumber plants, as revealed by higher plant dry weight, chlorophyll content and photochemical efficiency (Fv/Fm), and lower leaf Na+ content. Salinity stress resulted in a sharp increase in H2O2 production, reaching a peak 3 h after salt treatment in the pumpkin-grafted cucumber. This enhancement was accompanied by elevated relative expression of respiratory burst oxidase homologue (RBOH) genes RbohD and RbohF and a higher NADPH oxidase activity. However, this increase was much delayed in the self-grafted plants, and the difference between the two grafting combinations disappeared after 24 h. The decreased leaf Na+ content of pumpkin-grafted plants was achieved by higher Na+ exclusion in roots, which was driven by the Na+/H+ antiporter energized by the plasma membrane H+-ATPase, as evidenced by the higher plasma membrane H+-ATPase activity and higher transcript levels for PMA and SOS1. In addition, early stomatal closure was also observed in the pumpkin-grafted cucumber plants, reducing water loss and maintaining the plant's hydration status. When pumpkin-grafted plants were pretreated with an NADPH oxidase inhibitor, diphenylene iodonium (DPI), the H2O2 level decreased significantly, to the level found in self-grafted plants, resulting in the loss of the salt tolerance. Inhibition of the NADPH oxidase-mediated H2O2 signaling in the root also abolished a rapid stomatal closure in the pumpkin-grafted plants. We concluded that the pumpkin-grafted cucumber plants increase their salt tolerance via a mechanism involving the root-sourced respiratory burst oxidase homologue-dependent H2O2 production, which enhances Na+ exclusion from the root and promotes an early stomatal closure.
Project description:With the aim to clarifying the role of the ABA/H2O2 signaling cascade in the regulating the antioxidant capacity of grafted cucumber plants in response to Ca(NO3)2 stress, we investigated the relationship between ABA-mediated H2O2 production and the activities of antioxidant enzymes in the leaves of pumpkin-grafted cucumber seedlings. The results showed that both ABA and H2O2 were detected in pumpkin-grafted cucumber seedlings in response to Ca(NO3)2 treatment within 0.5 h in the leaves and peaked at 3 and 6 h after Ca(NO3)2 treatment, respectively, compared to the levels under control conditions. The activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and peroxidase (POD) in pumpkin-grafted cucumber leaves gradually increased over time and peaked at 12 h of Ca(NO3)2 stress. Furthermore, in the leaves of pumpkin-grafted cucumber seedlings, the H2O2 generation, the antioxidant enzyme activities and the expression of SOD, POD and cAPX were strongly blocked by an inhibitor of ABA under Ca(NO3)2 stress, but this effect was eliminated by the addition of exogenous ABA. Moreover, the activities and gene expressions of these antioxidant enzymes in pumpkin-grafted leaves were almost inhibited under Ca(NO3)2 stress by pretreatment with ROS scavengers. These results suggest that the pumpkin grafting-induced ABA accumulation mediated H2O2 generation, resulting in the induction of antioxidant defense systems in leaves exposed to Ca(NO3)2 stress in the ABA/H2O2 signaling pathway.
Project description:Background:Quantitative real-time PCR (qRT-PCR) is a commonly used high-throughput technique to measure mRNA transcript levels. The accuracy of this evaluation of gene expression depends on the use of optimal reference genes. Cucumber-pumpkin grafted plants, made by grafting a cucumber scion onto pumpkin rootstock, are superior to either parent plant, as grafting conveys many advantages. However, although many reliable reference genes have been identified in both cucumber and pumpkin, none have been obtained for cucumber-pumpkin grafted plants. Methods:In this work, 12 candidate reference genes, including eight traditional genes and four novel genes identified from our transcriptome data, were selected to assess their expression stability. Their expression levels in 25 samples, including three cucumber and three pumpkin samples from different organs, and 19 cucumber-pumpkin grafted samples from different organs, conditions, and varieties, were analyzed by qRT-PCR, and the stability of their expression was assessed by the comparative ?Ct method, geNorm, NormFinder, BestKeeper, and RefFinder. Results:The results showed that the most suitable reference gene varied dependent on the organs, conditions, and varieties. CACS and 40SRPS8 were the most stable reference genes for all samples in our research. TIP41 and CACS showed the most stable expression in different cucumber organs, TIP41 and PP2A were the optimal reference genes in pumpkin organs, and CACS and 40SRPS8 were the most stable genes in all grafted cucumber samples. However, the optimal reference gene varied under different conditions. CACS and 40SRPS8 were the best combination of genes in different organs of cucumber-pumpkin grafted plants, TUA and RPL36Aa were the most stable in the graft union under cold stress, LEA26 and ARF showed the most stable expression in the graft union during the healing process, and TIP41 and PP2A were the most stable across different varieties of cucumber-pumpkin grafted plants. The use of LEA26, ARF and LEA26+ARF as reference genes were further verified by analyzing the expression levels of csaCYCD3;1, csaRUL, cmoRUL, and cmoPIN in the graft union at different time points after grafting. Discussion:This work is the first report of appropriate reference genes in grafted cucumber plants and provides useful information for the study of gene expression and molecular mechanisms in cucumber-pumpkin grafted plants.
Project description:Soil salinity adversely affects the growth and yield of crops, including cucumber, one of the most important vegetables in the world. Grafting with salt-tolerant pumpkin as the rootstock effectively improves the growth of cucumber under different salt conditions by limiting Na? transport from the pumpkin rootstock to the cucumber scion. High-affinity potassium transporters (HKTs) are crucial for the long distance transport of Na? in plants, but the function of pumpkin HKTs in this process of grafted cucumber plants remains unclear. In this work, we have characterized CmHKT1;1 as a member of the HKT gene family in Cucurbita moschata and observed an obvious upregulation of CmHKT1;1 in roots under NaCl stress conditions. Heterologous expression analyses in yeast mutants indicated that CmHKT1;1 is a Na?-selective transporter. The transient expression in tobacco epidermal cells and in situ hybridization showed CmHKT1;1 localization at plasma membrane, and preferential expression in root stele. Moreover, ectopic expression of CmHKT1;1 in cucumber decreased the Na? accumulation in the plants shoots. Finally, the CmHKT1;1 transgenic line as the rootstock decreased the Na? content in the wild type shoots. These findings suggest that CmHKT1;1 plays a key role in the salt tolerance of grafted cucumber by limiting Na? transport from the rootstock to the scion and can further be useful for engineering salt tolerance in cucurbit crops.
Project description:Leaf abscisic acid concentration ([ABA]) during growth influences morpho-physiological traits associated with the plant's ability to cope with stress. A dose-response curve between [ABA] during growth and the leaf's ability to regulate water loss during desiccation or rehydrate upon re-watering was obtained. Rosa hybrida plants were grown at two relative air humidities (RHs, 60% or 90%) under different soil water potentials (-0.01, -0.06, or -0.08MPa) or upon grafting onto the rootstock of a cultivar sustaining [ABA] at elevated RH. Measurements included [ABA], stomatal anatomical features, stomatal responsiveness to desiccation, and the ability of leaves, desiccated to varying degrees, to recover their weight (rehydrate) following re-watering. Transpiration efficiency (plant mass per transpired water) was also determined. Soil water deficit resulted in a lower transpiration rate and higher transpiration efficiency at both RHs. The lowest [ABA] was observed in well-watered plants grown at high RH. [ABA] was increased by soil water deficit or grafting, at both RHs. The growth environment-induced changes in stomatal size were mediated by [ABA]. When [ABA] was increased from the level of (well-watered) high RH-grown plants to the value of (well-watered) plants grown at moderate RH, stomatal responsiveness was proportionally improved. A further increase in [ABA] did not affect stomatal responsiveness to desiccation. [ABA] was positively related to the ability of dehydrated leaves to rehydrate. The data indicate a growth [ABA]-related threshold for stomatal sensitivity to desiccation, which was not apparent either for stomatal size or for recovery (rehydration) upon re-watering.
Project description:Pericarp wax of cucumber is an important economic trait, determining sales and marketing. Grafting of cucumber onto pumpkin rootstock (Cucurbita moschata) is an effective way to produce glossy cucumber fruits. However, the molecular regulation mechanism of this phenomenon remains largely unknown. In the present study, transcriptome analyses, genome-wide DNA methylation sequencing, and wax metabolite analysis were performed on the pericarp of self-rooted versus grafted cucumber. We identified the AP2/ERF-type transcription factor CsWIN1 as methylated and significantly upregulated in grafted cucumber compared to self-rooted cucumber. The increased expression of CsWIN1 was also positively correlated with several key wax biosynthesis genes, including CsCER1, CsCER1-1, CsCER4, CsKCS1, and the wax transporter gene CsABC. The transcriptome expression level of these genes was validated through qRT-PCR profiles. Furthermore, wax metabolite analysis showed that more wax ester (C20 fatty acid composition), but fewer alkanes (C29 and C31) were deposited in grafted cucumber pericarp. The higher expression of CsWIN1 and wax biosynthesis genes was reflected in the glossier appearance of grafted pericarp, possibly the result of higher wax ester content and higher integration of small trichomes in the pericarp. This study demonstrates that grafting can affect the content and composition of pericarp wax in cucumber grafted on pumpkin, and a unique regulation model of CsWIN1 for wax biosynthesis may exist in cucumber.
Project description:To determine whether drought-induced root jasmonate [jasmonic acid (JA) and jasmonic acid-isoleucine (JA-Ile)] accumulation affected shoot responses to drying soil, near-isogenic wild-type (WT) tomato (Solanum lycopersicum cv. Castlemart) and the def-1 mutant (which fails to accumulate jasmonates during water deficit) were self- and reciprocally grafted. Rootstock hydraulic conductance was entirely rootstock dependent and significantly lower in def-1, yet def-1 scions maintained a higher leaf water potential as the soil dried due to their lower stomatal conductance (gs). Stomatal sensitivity to drying soil (the slope of gsversus soil water content) was low in def-1 self-grafts but was normalized by grafting onto WT rootstocks. Although soil drying increased 12-oxo-phytodienoic acid (OPDA; a JA precursor and putative antitranspirant) concentrations in def-1 scions, foliar JA accumulation was negligible and foliar ABA accumulation reduced compared with WT scions. A WT rootstock increased drought-induced ABA and JA accumulation in def-1 scions, but decreased OPDA accumulation. Xylem-borne jasmonates were biologically active, since supplying exogenous JA via the transpiration stream to detached leaves decreased transpiration of WT seedlings but had the opposite effect in def-1. Thus foliar accumulation of both ABA and JA at WT levels is required for both maximum (well-watered) gs and stomatal sensitivity to drying soil.
Project description:Plants are sessile organisms, and their growth and development is detrimentally affected by environmental stresses such as drought and high salinity. Defense mechanisms are tightly regulated and complex processes, which respond to changing environmental conditions; however, the precise mechanisms that function under adverse conditions remain unclear. Here, we report the identification and functional characterization of the CaOSR1 gene, which functions in the adaptive response to abiotic stress. We found that CaOSR1 gene expression in pepper leaves was up-regulated after exposure to abscisic acid (ABA), drought, and high salinity. In addition, we demonstrated that the fusion protein of CaOSR1 with green fluorescent protein (GFP) is localized in the nucleus. We used CaOSR1-silenced pepper plants and CaOSR1-OX-overexpressing (OX) transgenic Arabidopsis plants to show that the CaOSR1 protein regulates the osmotic stress response. CaOSR1-silenced pepper plants showed increased drought susceptibility, and this was accompanied by a high transpiration rate. CaOSR1-OX plants displayed phenotypes that were hypersensitive to ABA and hyposensitive to osmotic stress, during the seed germination and seedling growth stages; furthermore, these plants exhibited enhanced drought tolerance at the adult stage, and this was characterized by higher leaf temperatures and smaller stomatal apertures because of ABA hypersensitivity. Taken together, our data indicate that CaOSR1 positively regulates osmotic stress tolerance via ABA-mediated cell signaling. These findings suggest an involvement of a novel protein in ABA and osmotic stress signalings in plants.
Project description:End-of-season drought or "terminal drought," which occurs after flowering, is considered the most significant abiotic stress affecting crop yields. Wheat crop production in Mediterranean-type environments is often exposed to terminal drought due to decreasing rainfall and rapid increases in temperature and evapotranspiration during spring when wheat crops enter the reproductive stage. Under such conditions, every millimeter of extra soil water extracted by the roots benefits grain filling and yield and improves water use efficiency (WUE). When terminal drought develops, soil dries from the top, exposing the top part of the root system to dry soil while the bottom part is in contact with available soil water. Plant roots sense the drying soil and produce signals, which on transmission to shoots trigger stomatal closure to regulate crop water use through transpiration. However, transpiration is linked to crop growth and productivity and limiting transpiration may reduce potential yield. While an early and high degree of stomatal closure affects photosynthesis and hence biomass production, a late and low degree of stomatal closure exhausts available soil water rapidly which results in yield losses through a reduction in post-anthesis water use. The plant hormone abscisic acid (ABA) is considered the major chemical signal involved in stomatal regulation. Wheat genotypes differ in their ability to produce ABA under drought and also in their stomatal sensitivity to ABA. In this viewpoint article we discuss the possibilities of exploiting genotypic differences in ABA response to soil drying in regulating the use of water under terminal drought. Root density distribution in the upper drying layers of the soil profile is identified as a candidate trait that can affect ABA accumulation and subsequent stomatal closure. We also examine whether leaf ABA can be designated as a surrogate characteristic for improved WUE in wheat to sustain grain yield under terminal drought. Ease of collecting leaf samples to quantify ABA compared to extracting xylem sap will facilitate rapid screening of a large number of germplasm for drought tolerance.