Genome-wide identification and expression analysis of the cryptochromes reveal the CsCRY1 role under low-light-stress in cucumber.

Introduction: Low-light-stress is a common meteorological disaster that can result in slender seedlings. The photoreceptors play a crucial role in perceiving and regulating plants' tolerance to low-light-stress. However, the low-light-stress tolerance of cucumber has not been effectively evaluated, and the functions of these photoreceptor genes in cucumber, particularly under low-light-stress conditions, are not clear. Methods: Herein, we evaluated the growth characteristics of cucumber seedlings under various LED light treatment. The low-light-stress tolerant cucumber CR and intolerant cucumber CR were used as plant materials for gene expression analysis, and then the function of CsCRY1 was analyzed. Results: The results revealed that light treatment below 40 µmol m-2 s-1 can quickly and effectively induce low-light-stress response. Then, cucumber CR exhibited remarkable tolerance to low-light-stress was screened. Moreover, a total of 11 photoreceptor genes were identified and evaluated. Among them, the cryptochrome 1 (CRY1) had the highest expression level and was only induced in the low-light sensitive cucumber CS. The transcript CsaV3_3G047490.1 is predicted to encode a previously unknown CsCRY1 protein, which lacks 70 amino acids at its C-terminus due to alternative 5' splice sites within the final intron of the CsCRY1 gene. Discussion: CRY1 is a crucial photoreceptor that plays pivotal roles in regulating plants' tolerance to low-light stress. In this study, we discovered that alternative splicing of CsCRY1 generates multiple transcripts encoding distinct CsCRY1 protein variants, providing valuable insights for future exploration and utilization of CsCRY1 in cucumber.

CmoNAC1 in pumpkin rootstocks improves salt tolerance of grafted cucumbers by binding to the promoters of CmoRBOHD1, CmoNCED6, CmoAKT1;2 and CmoHKT1;1 to regulate H2O2, ABA signaling and K+/Na+ homeostasis.

The NAC transcription factor is a type of plant-specific transcription factor that can regulate plant salt tolerance, but the underlying mechanism is unclear in grafted vegetables. H2O2 and ABA in pumpkin rootstocks can be transported to cucumber scion leaves, promoting stomatal closure to improve salt tolerance of grafted cucumbers. Despite these observations, the regulatory mechanism is unknown. Here, our research revealed that CmoNAC1 is a key transcription factor that regulates H2O2 and ABA signaling in pumpkin roots under salt stress. The function of CmoNAC1 was analyzed using root transformation and RNA-seq, and we found that pumpkin CmoNAC1 promoted the production of H2O2 and ABA via CmoRBOHD1 and CmoNCED6, respectively, and regulated K+/Na+ homeostasis via CmoAKT1;2, CmoHKT1;1, and CmoSOS1 to improve salt tolerance of grafted cucumbers. Root knockout of CmoNAC1 resulted in a significant decrease in H2O2 (52.9% and 32.1%) and ABA (21.8% and 42.7%) content and K+/Na+ ratio (81.5% and 56.3%) in leaf and roots of grafted cucumber, respectively, while overexpression showed the opposite effect. The root transformation experiment showed that CmoNCED6 could improve salt tolerance of grafted cucumbers by regulating ABA production and K+/Na+ homeostasis under salt stress. Finally, we found that CmoNAC1 bound to the promoters of CmoRBOHD1, CmoNCED6, CmoAKT1;2, and CmoHKT1;1 using yeast one-hybrid, luciferase, and electrophoretic mobility shift assays. In conclusion, pumpkin CmoNAC1 not only binds to the promoters of CmoRBOHD1 and CmoNCED6 to regulate the production of H2O2 and ABA signals in roots, but also binds to the promoters of CmoAKT1;2 and CmoHKT1;1 to increase the K+/Na+ ratio, thus improving salt tolerance of grafted cucumbers.

Transcriptomic and functional characterization reveals CsHAK5;3 as a key player in K+ homeostasis in grafted cucumbers under saline conditions.

Grafting can improve the salt tolerance of many crops. However, critical genes in scions responsive to rootstock under salt stress remain a mystery. We found that pumpkin rootstock decreased the content of Na+ by 70.24 %, increased the content of K+ by 25.9 %, and increased the K+/Na+ ratio by 366.0 % in cucumber scion leaves. RNA-seq analysis showed that ion transport-related genes were the key genes involved in salt stress tolerance in grafted cucumber. The identification and analysis of the expression of K+ transporter proteins in cucumber and pumpkin revealed six and five HAK5 members, respectively. The expression of CsHAK5;3 in cucumber was elevated in different graft combinations under salt stress and most notably in cucumber scion/pumpkin rootstock. CsHAK5;3 was localized to the plasma membrane, and a yeast complementation assay revealed that it can transport K+. CsHAK5;3 knockout in hairy root mutants decreased the K+ content of leaves (45.6 %) and roots (50.3 %), increased the Na+ content of leaves (29.3 %) and roots (34.8 %), and decreased the K+/Na+ ratio of the leaves (57.9 %) and roots (62.9 %) in cucumber. However, CsHAK5;3 overexpression in hairy roots increased the K+ content of the leaves (31.2 %) and roots (38.3 %), decreased the Na+ content of leaves (17.2 %) and roots (14.3 %), and increased the K+/Na+ ratio of leaves (58.9 %) and roots (61.6 %) in cucumber. In conclusion, CsHAK5;3 in cucumber can mediate K+ transport and is one of the key target pumpkin genes that enhance salt tolerance of cucumber grafted.

An efficient root transformation system for CRISPR/Cas9-based analyses of shoot-root communication in cucurbit crops.

Cucurbit crops are suitable models for studying long-distance signaling in horticultural plants. Although thousands of substances are graft transmissible in cucurbits, functional studies have been hampered by the lack of efficient genetic transformation systems. Here, we report a convenient and efficient root transformation method for several cucurbit crops that will facilitate studies of functional genes and shoot-root crosstalk. We obtained healthy plants with completely transformed roots and non-transgenic shoots within 6 weeks. Furthermore, we combined this root transformation method with grafting, which allowed for gene manipulation in the rootstock. We validated our system by exploring salt tolerance mechanisms using a cucumber (Cucumis sativus)/pumpkin (Cucurbita moschata Duch.) (scion/rootstock) graft in which the sodium transporter gene High-affinity K+ transporter1 (CmoHKT1;1) was edited in the pumpkin rootstock, and by overexpressing the pumpkin tonoplast Na+/H+ antiporter gene Sodium hydrogen exchanger4 (CmoNHX4) in cucumber roots.

Overexpression of Melon Tonoplast Sugar Transporter CmTST1 Improved Root Growth under High Sugar Content.

Sugar allocation is based on the source-to-sink and intracellular transport between different organelles, and sugar transporters are usually involved in these processes. Tonoplast sugar transporters (TST) are responsible for transporting sugar into vacuoles; however, the role of TSTs in root growth and the response to abiotic stress is poorly studied. Here, RNA analysis and promoter-ß-glucuronidase staining revealed that a melon TST1 gene (CmTST1) is highly expressed in the roots. The sugar feeding experiment results showed that the expression of CmTST1 in the roots was induced by a relatively high level of sucrose (6%), glucose (3%), and fructose (3%). The ectopic overexpression of CmTST1 in Arabidopsis improved the root and shoot growth of seedlings under high exogenous sugar stress. Furthermore, the ectopic expression of CmTST1 promoted the expression of plasma membrane-located sugar transporters. We proposed that CmTST1 plays a key role in importing sugar transport into the vacuoles of roots in response to metabolic demands to maintain cytosolic sugar homeostasis.

Grafting Cucumber Onto Pumpkin Induced Early Stomatal Closure by Increasing ABA Sensitivity Under Salinity Conditions.

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.

Tissue-specific respiratory burst oxidase homolog-dependent H2O2 signaling to the plasma membrane H+-ATPase confers potassium uptake and salinity tolerance in Cucurbitaceae.

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.

iTRAQ-based quantitative proteomics analysis of cold stress-induced mechanisms in grafted watermelon seedlings.

Rootstock grafting can improve the cold tolerance of watermelon. However, the molecular mechanisms underlying this process remain unknown. Herein, we used an isobaric tag for relative and absolute quantification (iTRAQ)-based quantitative proteomics approach for the comparative analysis of protein abundances in self-grafted (SG) and pumpkin rootstock-grafted (RG) watermelon seedlings in response to cold stress. A total of 4796 distinct proteins were identified, and 752 proteins were significantly differentially accumulated in grafted watermelon seedling leaves after 48 h cold stress. Based on bioinformatics analysis, the cold tolerance of RG watermelon seedlings might be related to more energy produced through photosynthesis, carbon metabolism, and oxidative phosphorylation, compared with that of SG watermelon seedlings. RG watermelon seedlings could cope with cold stress by improving the scavenging capacity of ROS and arginine biosynthesis. Posttranscriptional regulation and protein homeostasis also play important roles for grafted watermelon seedlings to adapt to cold stress. Several protein kinases involved in signal transduction may act as positive regulators in RG watermelon seedling leaves suffering from cold stress. In addition, iTRAQ data were confirmed to be reliable by the assays of physiological indicators and relative transcript levels of eight genes. BIOLOGICAL SIGNIFICANCE: Rootstock grafting is regarded as an effective method to enhance the cold tolerance of watermelon seedlings. To elucidate the cold tolerance mechanism of grafted watermelon, an iTRAQ-based quantitative proteomics approach combined with bioinformatics analysis was employed to identify differentially accumulated proteins in SG and RG watermelon seedlings between cold stress and control conditions. This study provided additional insight into the molecular basis of the grafted watermelon seedlings in response to cold stress.

Pumpkin CmHKT1;1 Controls Shoot Na⁺ Accumulation via Limiting Na⁺ Transport from Rootstock to Scion in Grafted Cucumber.

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.

An early ABA-induced stomatal closure, Na+ sequestration in leaf vein and K+ retention in mesophyll confer salt tissue tolerance in Cucurbita species.

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.

Boron: Functions and Approaches to Enhance Its Availability in Plants for Sustainable Agriculture.

Boron (B) is an essential trace element required for the physiological functioning of higher plants. B deficiency is considered as a nutritional disorder that adversely affects the metabolism and growth of plants. B is involved in the structural and functional integrity of the cell wall and membranes, ion fluxes (H⁺, K⁺, PO43−, Rb⁺, Ca2+) across the membranes, cell division and elongation, nitrogen and carbohydrate metabolism, sugar transport, cytoskeletal proteins, and plasmalemma-bound enzymes, nucleic acid, indoleacetic acid, polyamines, ascorbic acid, and phenol metabolism and transport. This review critically examines the functions of B in plants, deficiency symptoms, and the mechanism of B uptake and transport under limited B conditions. B deficiency can be mitigated by inorganic fertilizer supplementation, but the deleterious impact of frequent fertilizer application disrupts soil fertility and creates environmental pollution. Considering this, we have summarized the available information regarding alternative approaches, such as root structural modification, grafting, application of biostimulators (mycorrhizal fungi (MF) and rhizobacteria), and nanotechnology, that can be effectively utilized for B acquisition, leading to resource conservation. Additionally, we have discussed several new aspects, such as the combination of grafting or MF with nanotechnology, combined inoculation of arbuscular MF and rhizobacteria, melatonin application, and the use of natural and synthetic chelators, that possibly play a role in B uptake and translocation under B stress conditions.

Wheat Intercropping Enhances the Resistance of Watermelon to Fusarium Wilt.

A fungus Fusarium oxysporum F. sp. niveum (FON) is the causal organism of Fusarium wilt in watermelon. In this study, we evaluated the effect of wheat intercropping on the Fusarium wilt of watermelon. Our results showed that wheat intercropping decreases the incidence of Fusarium wilt of watermelon, likely due to the secretion of coumaric acid from the roots of wheat that dramatically inhibits FON spore germination, sporulation, and growth. The secretion of p-hydroxybenzoic acid, ferulic acid, and cinnamic acid from the roots of watermelon stimulates FON spore germination, sporulation, and growth. The secretion of phenolic acids and organic acids from the roots of watermelon is also promoted by FON infection. However, secretion of phenolic acids and organic acids from the roots of watermelon is substantially reduced under wheat intercropping systems. FON infection increases the accumulation of free and conjugated salicylic acid (SA) in watermelon grown under wheat intercropping systems through isochorismate (ICS) and phenylalanine ammonia-lyase (PAL) pathways. Furthermore, wheat intercropping up-regulates the expression of disease-and defense-responsive genes and improves the activities of corresponding pathogenesis-related (PR) enzymes in the roots of watermelon. In conclusion, the secretion of coumaric acid from the roots of wheat and changes in the composition of phenolic acid and organic acid secretion from the roots of watermelon suppress Fusarium wilt of watermelon under wheat intercropping system. Meanwhile, wheat intercropping also enhanced the resistance of watermelon to FON by up-regulating the expression of disease-and defense-responsive genes in watermelon.

Root respiratory burst oxidase homologue-dependent H2O2 production confers salt tolerance on a grafted cucumber by controlling Na+ exclusion and stomatal closure.

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.

Ectopic Expression of Pumpkin NAC Transcription Factor CmNAC1 Improves Multiple Abiotic Stress Tolerance in Arabidopsis.

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.