RESUMO
Potassium (K+) is a critical determinant of salinity tolerance, and H2O2 has been recognized as an important signaling molecule that mediates many physiological responses. However, the details of how H2O2 signaling regulates K+ uptake in the root under salt stress remain elusive. In this study, salt-sensitive cucumber and salt-tolerant pumpkin which belong to the same family, Cucurbitaceae, were used to answer the above question. We show that higher salt tolerance in pumpkin was related to its superior ability for K+ uptake and higher H2O2 accumulation in the root apex. Transcriptome analysis showed that salinity induced 5816 (3005 up- and 2811 down-) and 4679 (3965 up- and 714 down-) differentially expressed genes (DEGs) in cucumber and pumpkin, respectively. DEGs encoding NADPH oxidase (respiratory burst oxidase homolog D; RBOHD), 14-3-3 protein (GRF12), plasma membrane H+-ATPase (AHA1), and potassium transporter (HAK5) showed higher expression in pumpkin than in cucumber under salinity stress. Treatment with the NADPH oxidase inhibitor diphenylene iodonium resulted in lower RBOHD, GRF12, AHA1, and HAK5 expression, reduced plasma membrane H+-ATPase activity, and lower K+ uptake, leading to a loss of the salinity tolerance trait in pumpkin. The opposite results were obtained when the plants were pre-treated with exogenous H2O2. Knocking out of RBOHD in pumpkin by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9] editing of coding sequences resulted in lower root apex H2O2 and K+ content and GRF12, AHA1, and HAK5 expression, ultimately resulting in a salt-sensitive phenotype. However, ectopic expression of pumpkin RBOHD in Arabidopsis led to the opposite effect. Taken together, this study shows that RBOHD-dependent H2O2 signaling in the root apex is important for pumpkin salt tolerance and suggests a novel mechanism that confers this trait, namely RBOHD-mediated transcriptional and post-translational activation of plasma membrane H+-ATPase operating upstream of HAK5 K+ uptake transporters.
Assuntos
Membrana Celular/metabolismo , Cucurbitaceae/metabolismo , Potássio/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Membrana Celular/efeitos dos fármacos , Cucurbita/efeitos dos fármacos , Cucurbita/metabolismo , Cucurbitaceae/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/metabolismo , Oniocompostos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Tolerância ao Sal/genética , Tolerância ao Sal/fisiologiaRESUMO
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.
Assuntos
Cucumis sativus/fisiologia , Cucurbita/fisiologia , Peróxido de Hidrogênio/metabolismo , NADPH Oxidases/genética , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Tolerância ao Sal , Cucumis sativus/enzimologia , Cucumis sativus/genética , Cucurbita/enzimologia , Cucurbita/genética , NADPH Oxidases/metabolismo , Proteínas de Plantas/metabolismoRESUMO
Tissue tolerance to salinity stress is a complex physiological trait composed of multiple 'sub-traits' such as Na+ compartmentalization, K+ retention, and osmotic tolerance. Previous studies have shown that some Cucurbita species employ tissue tolerance to combat salinity and we aimed to identify the physiological and molecular mechanisms involved. Five C. maxima (salt-tolerant) and five C. moschata (salt-sensitive) genotypes were comprehensively assessed for their salt tolerance mechanisms and the results showed that tissue-specific transport characteristics enabled the more tolerant lines to deal with the salt load. This mechanism was associated with the ability of the tolerant species to accumulate more Na+ in the leaf vein and to retain more K+ in the leaf mesophyll. In addition, C. maxima more efficiently retained K+ in the roots when exposed to transient NaCl stress and it was also able to store more Na+ in the xylem parenchyma and cortex in the leaf vein. Compared with C. moschata, C. maxima was also able to rapidly close stomata at early stages of salt stress, thus avoiding water loss; this difference was attributed to higher accumulation of ABA in the leaf. Transcriptome and qRT-PCR analyses revealed critical roles of high-affinity potassium (HKT1) and intracellular Na+/H+ (NHX4/6) transporters as components of the mechanism enabling Na+ exclusion from the leaf mesophyll and Na+ sequestration in the leaf vein. Also essential was a higher expression of NCED3s (encoding 9-cis-epoxycarotenoid dioxygenase, a key rate-limiting enzyme in ABA biosynthesis), which resulted in greater ABA accumulation in the mesophyll and earlier stomata closure in C. maxima.
Assuntos
Ácido Abscísico/metabolismo , Cucurbita/fisiologia , Estômatos de Plantas/fisiologia , Potássio/metabolismo , Sódio/metabolismo , Células do Mesofilo/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/metabolismo , Tolerância ao Sal , Especificidade da EspécieRESUMO
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⺠transport from the pumpkin rootstock to the cucumber scion. High-affinity potassium transporters (HKTs) are crucial for the long distance transport of Na⺠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 Naâº-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⺠accumulation in the plants shoots. Finally, the CmHKT1;1 transgenic line as the rootstock decreased the Na⺠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⺠transport from the rootstock to the scion and can further be useful for engineering salt tolerance in cucurbit crops.
Assuntos
Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Cucumis sativus/crescimento & desenvolvimento , Cucurbita/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sódio/metabolismo , Simportadores/genética , Simportadores/metabolismo , Membrana Celular/metabolismo , Cucumis sativus/genética , Cucurbita/genética , Cucurbita/metabolismo , Regulação da Expressão Gênica de Plantas , Folhas de Planta/metabolismo , Brotos de Planta/genética , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Tolerância ao Sal , Regulação para CimaRESUMO
Potassium (K) is one of the essential nutrients for crops, and K⺠deficiency highly restricts crop yield and quality. Watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] is an economically important crop that often suffers from K⺠deficiency. To elucidate the underlying tolerance mechanism of watermelon to K⺠deficiency and to improve K efficiency of watermelon and other crops in the future, two watermelon genotypes, namely, YS and 8424, that exhibit contrasting K efficiencies were studied to compare their response mechanisms to K⺠deficiency. YS was more tolerant of K⺠deficiency and displayed less inhibited root growth than 8424. Roots of YS and 8424 seedlings with or without K⺠supply were harvested at 6 and 120 h after treatment (HAT), and their transcriptomes were analyzed by Illumina RNA sequencing. Different regulation mechanisms of the root Kâº-uptake genes for short- and long-term stress were observed. Genes involved in jasmonic acid and reactive oxygen species production; Ca²âº and receptor-like kinase signaling; lignin biosynthesis; and other stress-related genes were repressed in YS, whereas a large number of such stress-related genes were induced in 8424 at 120 HAT. These results suggested that repressed defense and stress response can save energy for better root growth in YS, which can facilitate K⺠uptake and increase K efficiency and tolerance to K⺠deficiency. This study presents the first global root transcriptome in watermelon and provides new insights into the molecular mechanisms underlying tolerance to K⺠deficiency of K-efficient watermelon genotypes.
Assuntos
Citrullus/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana Transportadoras/genética , Raízes de Plantas/genética , Potássio/metabolismo , Transcriptoma , Biomassa , Cátions/análise , Cátions/metabolismo , Citrullus/classificação , Citrullus/fisiologia , Regulação para Baixo , Perfilação da Expressão Gênica , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Modelos Biológicos , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/fisiologia , Potássio/análise , Canais de Potássio Corretores do Fluxo de Internalização/genética , Análise de Sequência de RNA , Estresse Fisiológico , Regulação para CimaRESUMO
Grafting onto salt-tolerant pumpkin rootstock can increase cucumber salt tolerance. Previous studies have suggested that this can be attributed to pumpkin roots with higher capacity to limit the transport of Na(+) to the shoot than cucumber roots. However, the mechanism remains unclear. This study investigated the transport of Na(+) in salt-tolerant pumpkin and salt-sensitive cucumber plants under high (200 mM) or moderate (90 mM) NaCl stress. Scanning ion-selective electrode technique showed that pumpkin roots exhibited a higher capacity to extrude Na(+), and a correspondingly increased H(+) influx under 200 or 90 mM NaCl stress. The 200 mM NaCl induced Na(+)/H(+) exchange in the root was inhibited by amiloride (a Na(+)/H(+) antiporter inhibitor) or vanadate [a plasma membrane (PM) H(+) -ATPase inhibitor], indicating that Na(+) exclusion in salt stressed pumpkin and cucumber roots was the result of an active Na(+)/H(+) antiporter across the PM, and the Na(+)/H(+) antiporter system in salt stressed pumpkin roots was sufficient to exclude Na(+) X-ray microanalysis showed higher Na(+) in the cortex, but lower Na(+) in the stele of pumpkin roots than that in cucumber roots under 90 mM NaCl stress, suggesting that the highly vacuolated root cortical cells of pumpkin roots could sequester more Na(+), limit the radial transport of Na(+) to the stele and thus restrict the transport of Na(+) to the shoot. These results provide direct evidence for pumpkin roots with higher capacity to limit the transport of Na(+) to the shoot than cucumber roots.
Assuntos
Cucumis sativus/fisiologia , Cucurbita/fisiologia , Microanálise por Sonda Eletrônica/métodos , Proteínas de Plantas/metabolismo , Sódio/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Cucumis sativus/efeitos dos fármacos , Cucurbita/efeitos dos fármacos , Eletrodos Seletivos de Íons , Epiderme Vegetal/efeitos dos fármacos , Epiderme Vegetal/fisiologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/fisiologia , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/fisiologia , Potássio/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Tolerância ao Sal , Cloreto de Sódio/farmacologia , Trocadores de Sódio-Hidrogênio/metabolismo , Estresse FisiológicoRESUMO
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.
RESUMO
Drought, cold and salinity are the major environmental stresses that limit agricultural productivity. NAC transcription factors regulate the stress response in plants. Pumpkin (Cucurbita moschata) is an important cucurbit vegetable crop and it has strong resistance to abiotic stress; however, the biological functions of stress-related NAC genes in this crop are largely unknown. This study reports the function of CmNAC1, a stress-responsive pumpkin NAC domain protein. The CmNAC1-GFP fusion protein was transiently expressed in tobacco leaves for subcellular localization analysis, and we found that CmNAC1 is localized in the nucleus. Transactivation assay in yeast cells revealed that CmNAC1 functions as a transcription activator, and its transactivation domain is located in the C-terminus. CmNAC1 was ubiquitously expressed in different organs, and its transcript was induced by salinity, cold, dehydration, H2O2, and abscisic acid (ABA) treatment. Furthermore, the ectopic expression (EE) of CmNAC1 in Arabidopsis led to ABA hypersensitivity and enhanced tolerance to salinity, drought and cold stress. In addition, five ABA-responsive elements were enriched in CmNAC1 promoter. The CmNAC1-EE plants exhibited different root architecture, leaf morphology, and significantly high concentration of ABA compared with WT Arabidopsis under normal conditions. Our results indicated that CmNAC1 is a critical factor in ABA signaling pathways and it can be utilized in transgenic breeding to improve the abiotic stress tolerance of crops.
RESUMO
[This corrects the article on p. 1230 in vol. 6, PMID: 26793210.].
RESUMO
MicroRNAs (miRNAs), a class of small non-coding RNAs, recognize their mRNA targets based on perfect sequence complementarity. MiRNAs lead to broader changes in gene expression after plants are exposed to stress. High-throughput sequencing is an effective method to identify and profile small RNA populations in non-model plants under salt stresses, significantly improving our knowledge regarding miRNA functions in salt tolerance. Cucurbits are sensitive to soil salinity, and the Cucurbita genus is used as the rootstock of other cucurbits to enhance salt tolerance. Several cucurbit crops have been used for miRNA sequencing but salt stress-related miRNAs in cucurbit species have not been reported. In this study, we subjected two Cucurbita germplasm, namely, N12 (Cucurbita. maxima Duch.) and N15 (Cucurbita. moschata Duch.), with different sodium accumulation patterns, to Illumina sequencing to determine small RNA populations in root tissues after 4 h of salt treatment and control. A total of 21,548,326 and 19,394,108 reads were generated from the control and salt-treated N12 root tissues, respectively. By contrast, 19,108,240 and 20,546,052 reads were obtained from the control and salt-treated N15 root tissues, respectively. Fifty-eight conserved miRNA families and 33 novel miRNAs were identified in the two Cucurbita germplasm. Seven miRNAs (six conserved miRNAs and one novel miRNAs) were up-regulated in salt-treated N12 and N15 samples. Most target genes of differentially expressed novel miRNAs were transcription factors and salt stress-responsive proteins, including dehydration-induced protein, cation/H+ antiporter 18, and CBL-interacting serine/threonine-protein kinase. The differential expression of miRNAs between the two Cucurbita germplasm under salt stress conditions and their target genes demonstrated that novel miRNAs play an important role in the response of the two Cucurbita germplasm to salt stress. The present study initially explored small RNAs in the response of pumpkin to salt stress, and provided valuable information on novel miRNAs and their target genes in Cucurbita.
Assuntos
Cucurbita , MicroRNAs , RNA de Plantas , Tolerância ao Sal/fisiologia , Estresse Fisiológico/fisiologia , Cucurbita/genética , Cucurbita/metabolismo , MicroRNAs/biossíntese , MicroRNAs/genética , RNA de Plantas/biossíntese , RNA de Plantas/genéticaRESUMO
Melatonin (N-acetyl-5-methoxytryptamine) is a ubiquitous molecule with pleiotropic actions in different organisms. It performs many important functions in human, animals, and plants; these range from regulating circadian rhythms in animals to controlling senescence in plants. In this review, we summarize the available information regarding the presence of melatonin in different plant species, along with highlighting its biosynthesis and mechanisms of action. We also collected the available information on the effects of melatonin application on commercially important crops to improve their growth and development. Additionally, we have identified many new aspects where melatonin may have possible roles in plants, for example, its function in improving the storage life and quality of fruits and vegetables, its role in vascular reconnection during the grafting process and nutrient uptake from roots by modifying root architecture. Another potentially important aspect is the production of melatonin-rich food crops (cereals, fruits, and vegetables) through combination of conventional and modern breeding approaches, to increase plant resistance against biotic and abiotic stress, leading to improved crop yields, and the nutraceutical value of produce to solve food security issues.