Improving vanadium stress tolerance of watermelon by grafting onto bottle gourd and pumpkin rootstock

Vanadium (V) is a transition metal found in the Earth crust. V adversely affects plant growth and development. Besides several other management practices, grafting of scion cultivars onto appropriate rootstock provides a suitable solution. Grafting is an important agro-technical procedure utilized to enhance the capacity of plants to tolerate biotic and abiotic stresses. In this study, watermelon was grafted onto bottle gourd and pumpkin rootstock, and self-grafted watermelon plants were utilized as a control. V was applied at the rate of 50 mg/L under hydroponic conditions. The result showed that V application substantially reduces the growth of watermelon plants, however, grafting of watermelon onto bottle gourd and pumpkin rootstock improves V stress tolerance of watermelon by reducing the V concentration in leaf tissues, improving the relative chlorophyll content (SPAD index) and photosynthetic assimilation, up-regulating the expression of SOD (Cla008698, Cla0012125, Cla009820 and Cla001158), glutathione S-transferase (Cla013224) and glutathione peroxidase (Cla021039) genes in the leaves, and enhancing the activities of antioxidant enzymes (SOD, CAT). The scanning electron microscopy (SEM) of the root tips showed that minimal damage of roots was observed for pumpkin roots compared with the roots of watermelon and bottle gourd under V stress conditions. So far as we know, these results are the first evidence that grafting mitigates V stress in plants.     

Modest calcium increase in tomatoes expressing a variant of <Emphasis Type="Italic">Arabidopsis</Emphasis> cation/H<Superscript>+</Superscript> antiporter

The over-expression of Arabidopsis CAX1 and CAX2 causes transgenic tomato plants to reveal severe Ca²⁺ deficiency-like symptoms such as tip-burn and/or blossom end rot, despite there being sufficient Ca²⁺ in each plant part. To correct the symptoms and to moderately enhance the calcium level, a worldwide vegetable tomato was genetically engineered using a modified Arabidopsis cation/H⁺ antiporter sCAX2A, a mutant form of Arabidopsis CAX2. Compared with the wild-type, the sCAX2A-expressing tomato plants demonstrated elevated Ca²⁺ levels in the fruits with almost no changes in the levels of Mn²⁺, Cu²⁺, and Fe²⁺. Moreover, expression of sCAX2A in tomato plants did not show any significant alterations in their morphological phenotypes. Unlike 35S::sCAX1 construct, sCAX2A antiporter gene driven by 35S promoter can be a valuable tool for enriching Ca²⁺ contents in the tomato fruit without additional accumulation of the undesirable cations.     

Ectopic expression of Arabidopsis H+-pyrophosphatase AVP1 enhances drought resistance in bottle gourd (Lagenaria siceraria Standl.)

Bottle gourd (Lagenaria sicerar ia Standl.) has been used as a source of rootstock for grafting watermelon to improve its fruit quality. We report here the development of a bottle gourd with resistance to drought by ectopic expression of the Arabidopsis AVP1 gene that encodes a vacuolar H⁺-pyrophosphatase. The drought resistance of AVP1-expressing and wild-type plants was assessed by growing plants under drought conditions. After 12 days of water deprivation, both AVP1-expressing and wild-type plants demonstrated reduced growth. After 10 days of re-watering, wild-type plants showed minimal growth while the AVP1-expressing plants resumed rapid growth. Further, AVP1-expressing plants displayed longer primary roots and more robust root systems than wild-type plants.     

Expression of an Arabidopsis CAX2 variant in potato tubers increases calcium levels with no accumulation of manganese

Previously, we made a chimeric Arabidopsis thaliana vacuolar transporter CAX2B [a variant of N-terminus truncated form of CAX2 (sCAX2) containing the “B” domain from CAX1] that has enhanced calcium (Ca²⁺) substrate specificity and lost the manganese (Mn²⁺) transport capability of sCAX2. Here, we demonstrate that potato (Solanum tuberosum L.) tubers expressing the CAX2B contain 50–65% more calcium (Ca²⁺) than wild-type tubers. Moreover, expression of CAX2B in potatoes did not show any significant increase of the four metals tested, particularly manganese (Mn²⁺). The CAX2B-expressing potatoes have normally undergone the tuber/plant/tuber cycle for three generations; the trait appeared stable through the successive generations and showed no deleterious alternations on plant growth and development. These results demonstrate the enhanced substrate specificity of CAX2B in potato. Therefore, CAX2B can be a valuable tool for Ca²⁺ nutrient enrichment of potatoes with reduced accumulation of undesirable metals.     

Analysis of the Ca2+ domain in the Arabidopsis H+/Ca2+ antiporters CAX1 and CAX3

Ca²⁺ levels in plants are controlled in part by H⁺/Ca²⁺ exchangers. Structure/function analysis of the Arabidopsis H⁺/cation exchanger, CAX1, revealed that a nine amino acid region (87–95) is involved in CAX1-mediated Ca²⁺ specificity. CAX3 is 77% identical (93% similar) to CAX1, and when expressed in yeast, localizes to the vacuole but does not suppress yeast mutants defective in vacuolar Ca²⁺ transport. Transgenic tobacco plants expressing CAX3 containing the 9 amino acid Ca²⁺ domain (Cad) from CAX1 (CAX3-9) displayed altered stress sensitivities similar to CAX1-expressing plants, whereas CAX3-9-expressing plants did not have any altered stress sensitivities. A single leucine-to-isoleucine change at position 87 (CAX3-I) within the Cad of CAX3 allows this protein to weakly transport Ca²⁺ in yeast (less than 10% of CAX1). Site-directed mutagenesis of the leucine in the CAX3 Cad demonstrated that no amino acid change tested could confer more activity than CAX3-I. Transport studies in yeast demonstrated that the first three amino acids of the CAX1 Cad could confer twice the Ca²⁺ transport capability compared to CAX3-I. The entire Cad of CAX3 (87–95) inserted into CAX1 abolishes CAX1-mediated Ca²⁺ transport. However, single, double, or triple amino acid replacements within the native CAX1 Cad did not block CAX1 mediated Ca²⁺ transport. Together these findings suggest that other domains within CAX1 and CAX3 influence Ca²⁺ transport. This study has implications for the ability to engineer CAX-mediated transport in plants by manipulating Cad residues.     

Improving magnesium uptake,photosynthesis and antioxidant enzyme activities of watermelon by grafting onto pumpkin rootstock under low magnesium

Background and aims

Magnesium (Mg) is an essential macronutrient that plays an important role in numerous physiological and biochemical processes of plant. However, Mg deficiency commonly occurs worldwide. Watermelon is an important crop that often suffers from Mg deficiency. This study aims to test whether watermelon performance can be improved by grafting onto rootstocks under low Mg and to clarify the underlying physiological mechanism.

Methods

Self-grafted, bottle gourd (Jingxinzhen No.1) and pumpkin (Jingxinzhen No.4) rootstock-grafted plants were treated with three Mg concentrations: 2.0 mM (normal condition), 0.4 mM (moderate stress), and 0.04 mM (severe stress) for 16 days under hydroponic conditions. Ungrafted watermelon and pumpkin were treated with 2.0 mM and 0.04 mM for 12 days.

Results

The growth of the plants was not affected by 0.4 mM Mg; however, plant growth decreased under 0.04 mM Mg in all graft combinations compared with control (2.0 mM Mg). Pumpkin rootstock grafting significantly increased watermelon growth under low Mg stress (0.04 mM Mg), compared with self-grafted and bottle gourd-grafted plants. The Mg²⁺ uptake of watermelon plants was increased by grafting onto pumpkin rootstocks, however, root-to-shoot transport capacity of Mg²⁺ was similar compared with self-grafted plants under 0.04 mM Mg. Gene expression analysis showed that magnesium transporter genes MGT1, MGT3, MGT4, and MGT5 may play an important role in higher Mg²⁺ uptake of pumpkin root. The photosynthetic parameters and activities of superoxide dismutase, peroxidase and catalase were significantly higher, but malonaldehyde (MDA) content were lower in the pumpkin rootstock grafted plants compared with other graft combinations under 0.04 mM Mg.

Conclusion

Our results provide strong evidence that pumpkin rootstock ‘Jinxinzhen No. 4’ grafting can improve watermelon performance under low Mg stress. The enhanced plant performance is attributed to higher root Mg²⁺ uptake and the improvement of photosynthesis and antioxidant enzyme activities.

     

Increased calcium in carrots by expression of an Arabidopsis H+/Ca2+ transporter

We demonstrate that carrots expressing the Arabidopsis H⁺/Ca²⁺ transporter CAX1 (Cation Exchanger 1) contained up to 50% more calcium (Ca) than plants transformed with control vectors. The CAX1-expressing carrots were fertile, and robust plant growth was seen in the majority of the transgenic plants. CAX1-expressing carrots were crossed to a commercial carrot variety to confirm that the increased Ca accumulation was mediated by CAX1-expression, and the increased Ca content was clearly correlated with the transgene. This study suggests that modulation of ion transporters could be an important means of increasing the Ca content of agriculturally important crops. To our knowledge, this study represents the first attempts to use biotechnology to increase the Ca content of an agriculturally important crop.     

Functional Studies of Split Arabidopsis Ca2+/H+ Exchangers

In plants, high capacity tonoplast cation/H⁺ antiport is mediated in part by a family of cation exchanger (CAX) transporters. Functional association between CAX1 and CAX3 has previously been shown. In this study we further examine the interactions between CAX protein domains through the use of nonfunctional halves of CAX transporters. We demonstrate that a protein coding for an N-terminal half of an activated variant of CAX1 (sCAX1) can associate with the C-terminal half of either CAX1 or CAX3 to form a functional transporter that may exhibit unique transport properties. Using yeast split ubiquitin, in planta bimolecular fluorescence complementation, and gel shift experiments, we demonstrate a physical interaction among the half proteins. Moreover, the half-proteins both independently localized to the same yeast endomembrane. Co-expressing variants of N- and C-terminal halves of CAX1 and CAX3 in yeast suggested that the N-terminal region mediates Ca²⁺ transport, whereas the C-terminal half defines salt tolerance phenotypes. Furthermore, in yeast assays, auto-inhibited CAX1 could be differentially activated by CAX split proteins. The N-terminal half of CAX1 when co-expressed with CAX1 activated Ca²⁺ transport, whereas co-expressing C-terminal halves of CAX variants with CAX1 conferred salt tolerance but no apparent Ca²⁺ transport. These findings demonstrate plasticity through hetero-CAX complex formation as well as a novel means to engineer CAX transport.     

Identification and functional characterization of <Emphasis Type="Italic">APRR2</Emphasis> controlling green immature fruit color in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Nitrogen (N) is a macronutrient essential for plant growth and development. Meanwhile, grafting is a method used to alleviate stress tolerance of various biotic and abiotic factors. This study aims to investigate how pumpkin grafting (PG) improves N use efficiency of watermelon. A commercial watermelon cultivar “Zaojia 8424” [Citrullus lanatus (Thunb.) Matsum. and Nakai.] was self-grafted and then grafted onto pumpkin (Cucurbita maxima?×C. moschata) rootstock cv. Qingyan Zhenmu No. 1. The grafted plants were exposed to two levels of N (9 and 0.2 mM) under hydroponic conditions. The grafted plants were harvested at days 11 and 22 after low N (0.2 mM) treatment. PG improved the N use efficiency of watermelon scion through the vigorous root system of pumpkin rootstock that enhanced the uptake and accumulation of N, P, K, Ca, Mg, B, and Mn in watermelon. Gene expressions of nitrate reductase (Cla002787, Cla002791, and Cla023145) and nitrite reductase (Cla013062) genes were increased, promoting N assimilation. Mesophyll thickness and SPAD index (relative chlorophyll measurement) were also improved. Furthermore, pumpkin rootstock also enhanced the supply of zeatine riboside (ZR) and isopentenyl adenosine (iPA) in the leaves, promoting shoot growth. All these lead to improved plant growth and nitrogen use efficiency of pumpkin rootstock-grafted watermelon plants.     

Transport of mRNA molecules coding NAC domain protein in grafted pear and transgenic tobacco

Grafting is an important cultivation method and recent research on the mechanism of interactions between rootstock nad scion is focused on the long-distance transport of mRNA and small RNAs in the phloem. Among these transportable molecules, NACP gene coding NAM, ATAF1/2, CUC2 (NAC) domain protein might be involved in apical meristem development. Here, we report the transport of NACP mRNA between Chinese pear (Pyrus bretschneideri) cv. Yali (scion) and the wild Pyrus betulaefolia Bunge (rootstock). Our results indicated that NACP mRNA can be transported in both directions from the 3^rd to 10^th day after micro-grafting. It can also be transported to the shoot apex 30 to 70 cm away from graft-union in 2-year-old grafted trees. For further investigation, transgenic tobaccos with 35S: P. betulaefolia-NACP construct were grafted on wild-type tobaccos (Nicotiana tabacum L. cv. Samsun). The sustainable transport of Pyrus-NACP mRNA through the graft-union occurred from the 15^th day after grafting.     

Kinetic mechanism of the Ca2+-dependent switch-on and switch-off of cardiac troponin in myofibrils

The kinetics of Ca²⁺-dependent conformational changes of human cardiac troponin (cTn) were studied on isolated cTn and within the sarcomeric environment of myofibrils. Human cTnC was selectively labeled on cysteine 84 with N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole and reconstituted with cTnI and cTnT to the cTn complex, which was incorporated into guinea pig cardiac myofibrils. These exchanged myofibrils, or the isolated cTn, were rapidly mixed in a stopped-flow apparatus with different [Ca²⁺] or the Ca²⁺-buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid to determine the kinetics of the switch-on or switch-off, respectively, of cTn. Activation of myofibrils with high [Ca²⁺] (pCa 4.6) induced a biphasic fluorescence increase with rate constants of >2000 s⁻¹ and ∼330 s⁻¹, respectively. At low [Ca²⁺] (pCa 6.6), the slower rate was reduced to ∼25 s⁻¹, but was still ∼50-fold higher than the rate constant of Ca²⁺-induced myofibrillar force development measured in a mechanical setup. Decreasing [Ca²⁺] from pCa 5.0-7.9 induced a fluorescence decay with a rate constant of 39 s⁻¹, which was approximately fivefold faster than force relaxation. Modeling the data indicates two sequentially coupled conformational changes of cTnC in myofibrils: 1), rapid Ca²⁺-binding (k_B ≈ 120 μM⁻¹ s⁻¹) and dissociation (k_D ≈ 550 s⁻¹); and 2), slower switch-on (k_on = 390s⁻¹) and switch-off (k_off = 36s⁻¹) kinetics. At high [Ca²⁺], ∼90% of cTnC is switched on. Both switch-on and switch-off kinetics of incorporated cTn were around fourfold faster than those of isolated cTn. In conclusion, the switch kinetics of cTn are sensitively changed by its structural integration in the sarcomere and directly rate-limit neither cardiac myofibrillar contraction nor relaxation.     

Ectopic expression of a maize calreticulin mitigates calcium deficiency-like disorders in sCAX1-expressing tobacco and tomato

Deregulated expression of an Arabidopsis H⁺/Ca²⁺ antiporter (sCAX1) in agricultural crops increases total calcium (Ca²⁺) but may result in yield losses due to Ca²⁺ deficiency-like symptoms. Here we demonstrate that co-expression of a maize calreticulin (CRT, a Ca²⁺ binding protein located at endoplasmic reticulum) in sCAX1-expressing tobacco and tomato plants mitigated these adverse effects while maintaining enhanced Ca²⁺ content. Co-expression of CRT and sCAX1 could alleviate the hypersensitivity to ion imbalance in tobacco plants. Furthermore, blossom-end rot (BER) in tomato may be linked to changes in CAX activity and enhanced CRT expression mitigated BER in sCAX1 expressing lines. These findings suggest that co-expressing Ca²⁺ transporters and binding proteins at different intracellular compartments can alter the content and distribution of Ca²⁺ within the plant matrix.     

Paradoxical buffering of calcium by calsequestrin demonstrated for the calcium store of skeletal muscle

Contractile activation in striated muscles requires a Ca²⁺ reservoir of large capacity inside the sarcoplasmic reticulum (SR), presumably the protein calsequestrin. The buffering power of calsequestrin in vitro has a paradoxical dependence on [Ca²⁺] that should be valuable for function. Here, we demonstrate that this dependence is present in living cells. Ca²⁺ signals elicited by membrane depolarization under voltage clamp were compared in single skeletal fibers of wild-type (WT) and double (d) Casq-null mice, which lack both calsequestrin isoforms. In nulls, Ca²⁺ release started normally, but the store depleted much more rapidly than in the WT. This deficit was reflected in the evolution of SR evacuability, E, which is directly proportional to SR Ca²⁺ permeability and inversely to its Ca²⁺ buffering power, B. In WT mice E starts low and increases progressively as the SR is depleted. In dCasq-nulls, E started high and decreased upon Ca²⁺ depletion. An elevated E in nulls is consistent with the decrease in B expected upon deletion of calsequestrin. The different value and time course of E in cells without calsequestrin indicate that the normal evolution of E reflects loss of B upon SR Ca²⁺ depletion. Decrement of B upon SR depletion was supported further. When SR calcium was reduced by exposure to low extracellular [Ca²⁺], release kinetics in the WT became similar to that in the dCasq-null. E became much higher, similar to that of null cells. These results indicate that calsequestrin not only stores Ca²⁺, but also varies its affinity in ways that progressively increase the ability of the store to deliver Ca²⁺ as it becomes depleted, a novel feedback mechanism of potentially valuable functional implications. The study revealed a surprisingly modest loss of Ca²⁺ storage capacity in null cells, which may reflect concurrent changes, rather than detract from the physiological importance of calsequestrin.     

Cucurbit Grafting

Due to limited availability of arable land and high market demand for off-season vegetables, cucurbits (plants in the family Cucurbitaceae) are continuously cultivated under unfavorable conditions in some countries. These conditions include environments that are too cold, wet, or dry, or are cool low-light winter greenhouses. Successive cropping can increase salinity, the incidence of cucurbit pests, and soilborne diseases like fusarium wilt caused by Fusarium spp. These conditions cause various physiological and pathological disorders leading to severe crop loss. Chemical pest control is expensive, not always effective, and can harm the environment. Grafting can overcome many of these problems. In fact, in many parts of the world, grafting is a routine technique in continuous cropping systems. It was first commonly used in Japan during the late 1920s by grafting watermelon [Citrullus lanatus (Thunb.) Matsum. and Nakai] onto pumpkin [Cucurbita moschata Duchesne ex. Poir] rootstocks. Soon after, watermelons were grafted onto bottle gourd [Lagenaria siceraria (Molina) Standl.] rootstocks. This practice helped control declining yield due to soilborne diseases. China produces more than half the world's watermelons and cucumbers (Cucumis sativus L.), and approximately 20% of these are grafted. Use of rootstocks can enhance plant vigor through vigorous attainment of soil nutrients, avoidance of soil pathogens and tolerance of low soil temperatures, salinity, and wet-soil conditions. The type of rootstock affects cucurbit plant growth, yield, and fruit quality. Cucurbit grafting is rare in the United States, but with continued loss of quality disease-free farmland along with the phase-out of methyl bromide, the U.S. cucurbit industry sees grafting as an attractive option. Some seed companies now offer watermelon transplants grafted onto squash or bottle gourd rootstocks, and some transplant facilities offer grafting services. There have been thorough analyses of cucurbit grafting in other countries, but the literature in English is limited. This review summarizes the state of the cucurbit grafting industry on a global level, translating work published in many languages.     

Ca2+/H+ exchange by acidic organelles regulates cell migration in vivo

Increasing evidence implicates Ca²⁺ in the control of cell migration. However, the underlying mechanisms are incompletely understood. Acidic Ca²⁺ stores are fast emerging as signaling centers. But how Ca²⁺ is taken up by these organelles in metazoans and the physiological relevance for migration is unclear. Here, we identify a vertebrate Ca²⁺/H⁺ exchanger (CAX) as part of a widespread family of homologues in animals. CAX is expressed in neural crest cells and required for their migration in vivo. It localizes to acidic organelles, tempers evoked Ca²⁺ signals, and regulates cell-matrix adhesion during migration. Our data provide new molecular insight into how Ca²⁺ is handled by acidic organelles and link this to migration, thereby underscoring the role of noncanonical Ca²⁺ stores in the control of Ca²⁺-dependent function.