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.     

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.

     

Transmission of RNA silencing signal through grafting confers virus resistance from transgenically silenced tobacco rootstocks to non-transgenic tomato and tobacco scions

We examined the transmission of RNA silencing signal in non-transgenic tomato and tobacco scions grafted onto the tobacco Sd1 rootstocks, which is silenced in both NtTOM1 and NtTOM3 required for tobamovirus multiplication. When the non-transgenic tomato scions were grafted onto the Sd1 rootstocks, RT-PCR analysis of the scions showed the reduced level of mRNA compared with that before grafting in both LeTH3 and LeTH1, tomato homologs of NtTOM1 and NtTOM3, respectively. siRNAs from both genes were detected in the scions after grafting but not before grafting. Further tomato scions were inoculated with Tomato mosaic virus (ToMV) and used for virus infection. They showed very low level of virus accumulation. Necrotic responding tobacco to tobamovirus was grafted onto the rootstock of Sdl. RT-PCR analysis showed low level expression of both NtTOM1 and NtTOM3 in the scions but siRNA was detected after grafting. When the leaves of scions were inoculated with ToMV or Tobacco mosaic virus, they produced very few local necrotic lesions (LNLs) while the control scions did many LNLs. These results suggest that RNA silencing was transmitted to non-transgenic tomato and tobacco scions after grafting onto the Sd1 rootstocks and that virus resistance was induced in the scions.     

Genetic mapping reveals a candidate gene (<Emphasis Type="Italic">ClFS1</Emphasis>) for fruit shape in watermelon (<Emphasis Type="Italic">Citrullus lanatus</Emphasis> L.)

Key message

A 159 bp deletion in ClFS1 gene encoding IQD protein is responsible for fruit shape in watermelon.

Abstract

Watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] is known for its rich diversity in fruit size and shape. Fruit shape has been one of the major objectives of watermelon breeding. However, the candidate genes and the underlying genetic mechanism for such an important trait in watermelon are unknown. In this study, we identified a locus on chromosome 3 of watermelon genome controlling fruit shape. Segregation analysis in F₂ and BC₁ populations derived from a cross between two inbred lines “Duan125” (elongate fruit) and “Zhengzhouzigua” (spherical fruit) suggests that fruit shape of watermelon is controlled by a single locus and elongate fruit (OO) is incompletely dominant to spherical fruit (oo) with the heterozygote (Oo) being oval fruit. GWAS profiles among 315 accessions identified a major locus designated on watermelon chromosome 3, which was confirmed by BSA-seq mapping in the F₂ population. The candidate gene was mapped to a region 46 kb on chromosome 3. There were only four genes present in the corresponding region in the reference genome. Four candidate genes were sequenced in this region, revealing that the CDS of Cla011257 had a 159 bp deletion which resulted in the omission of 53 amino acids in elongate watermelon. An indel marker was derived from the 159 bp deletion to test the F₂ population and 105 watermelon accessions. The results showed that Cla011257 cosegregated with watermelon fruit shape. In addition, the Cla011257 expression was the highest at ovary formation stage. The predicted protein of the Cla011257 gene fitted in IQD protein family which was reported to have association with cell arrays and Ca²⁺-CaM signaling modules. Clear understanding of the genes facilitating the fruit shape along with marker association selection will be an effective way to develop new cultivars.

     

The crucial role of roots in increased cadmium-tolerance and Cd-accumulation in the pea mutant <Emphasis Type="Italic">SGECd</Emphasis><Superscript><Emphasis Type="Italic">t</Emphasis></Superscript>

Elucidation of mechanisms underlying plant tolerance to cadmium, a widespread toxic soil pollutant, and accumulation of Cd in plants are urgent tasks. For this purposes, the pea (Pisum sativum L.) mutant SGECd^t (obtained by treatment of the laboratory pea line SGE with ethylmethane sulfonate) was reciprocally grafted with the parental line SGE, and four scion/rootstock combinations were obtained: SGE/SGE, SGECd^t/SGECd^t, SGE/SGECd^t, and SGECd^t/SGE. They were grown in hydroponics in the presence of 1 μM CdCl₂ for 30 d. The SGE and SGECd^t scions on the SGECd^t rootstock had a higher root and shoot biomass and an elevated root and shoot Cd content compared with the grafts having SGE rootstock. Only the grafts with the SGE rootstock showed chlorosis and roots demonstrating symptoms of Cd toxicity. The content of nutrient elements in roots (Fe, K, Mg, Mn, Na, P, and Zn) was higher in the grafts having the SGECd^t rootstock, and three elements, namely Ca, Fe, and Mn, were efficiently transported by the SGECd^t root to the shoot of these grafts. The content of other measured elements (K, Mg, Na, P, and Zn) was similar in the root and shoot in all the grafts. Then, the non-grafted plants were grown in the presence of Cd and subjected to deficit or excess concentrations of Ca, Fe, or Mn. Exclusion of these elements from the nutrient solution retained or increased differences between SGE and SGECd^t in growth response to Cd toxicity, whereas excess of Ca, Fe, or Mn decreased or eliminated such differences. The obtained results assign a principal role of roots to realizing the increased Cd-tolerance and Cdaccumulation in the SGECd^t mutant. Efficient translocation of Ca, Fe, and Mn from roots to shoots appeared to counteract Cd toxicity, although Cd was actively taken up by roots and accumulated in shoots.     

Improved watermelon quality using bottle gourd rootstock expressing a Ca2+/H+ antiporter

Bottle gourd (Lagenaria siceraria Standl.) has been commonly used as a source of rootstock for watermelon. To improve its performance as a rootstock without adverse effects on the scion, the bottle gourd was genetically engineered using a modified Arabidopsis Ca²⁺/H⁺ exchanger sCAX2B. This transporter provides enhanced Ca²⁺ substrate specificity and decreased Mn²⁺ transport capability. Our previous work demonstrated that sCAX2B bottle gourds were more robust and nutrient dense than controls. Here, several cucurbit crops were test-grafted onto the transgenic bottle gourd to determine its effect on the scions. The grafted watermelons and melons onto the transgenic rootstocks appeared to show more robust growth than the controls 35 days after greenhouse transplanting. Watermelon fruits with the watermelon/transgenic bottle gourd (scion/rootstock) combination demonstrated higher osmotic pressure and more soluble solids than controls. These results suggest that sCAX2B expression in the bottle gourd rootstock facilitates improved watermelon quality through the translocation of nutrients and/or water toward enhancing the biomass of scion.     

Drought-induced proline synthesis depends on root-to-shoot communication mediated by light perception

Proline accumulation in roots and shoots is one of the most evident responses to environmental stresses such as drought, which is currently one of the main threats for agriculture. Based on this response, in this work, we hypothesize that proline accumulation is dependent on root-to-shoot communication through light perception. Thus, we used exaggerated light response (hp1) and phytochrome-deficient (au) mutants of tomato, which were combined through self-grafting and reciprocal grafting and subjected to drought stress, for posterior determination of shoot and root growth and proline content. Light-affected proline metabolism, as hp1, had the highest accumulation, while au presented the lowest proline values. Reciprocal grafting showed that hp1 and MT as scion or rootstock improved MT and au proline content, respectively, indicating shoot-to-root and root-to-shoot communication modulate the metabolism of this compatible osmolyte. Dry weight, leaf area, and root area presented similar patterns to proline content, indicating the importance of this compound for plant growth under stress conditions. These results provide a new perspective on light mediation of long-distance proline translocation in stressed plants.     

<Emphasis Type="Italic">UGP</Emphasis> gene expression and UDP-glucose pyrophosphorylase enzymatic activity in grafting annonaceous plants

Grafting is commonly used to propagate commercial fruit species to ensure that the genetic characteristics of selected clones are maintained. However, the biochemical and molecular mechanisms involved in the graft incompatibility of woody trees are not well understood. We investigated the effect of grafting in vegetative growth, UDP-glucose pyrophosphorylase expression and activity of Annonaceous grafted plants: atemoya (Annona cherimola Mill. x Annona squamosa L.) ‘Thompson’ grafted onto different rootstocks, araticum-de-terra-fria (Annona emarginata Schltdl. H. Rainer “var. terra-fria”), araticum-mirim (Annona emarginata Schltdl. H. Rainer “var. mirim”) and biribá (Annona mucosa Schltdl. H. Rainer) at different post-grafting times. The growth of atemoya grafted onto araticum-mirim was lower than that of the rootstocks araticum-de terra-fria and biribá. The results also indicated that grafting alters UGPase gene expression; showing the combination atemoya grafted onto araticum-de-terra-fria (a compatible union) the higher levels of gene expression during the early stages of grafting development. However, no significant differences were detected in UGPase enzyme activity between the graft combinations. In addition, SDS-PAGE and MALDI-TOF analyses detected similar UGPase amino acid sequences in ungrafted atemoya samples to cherimoya (Annona cherimola Mill.), a female parent of the atemoya hybrid. These findings suggest that expression of the UGPase protein is related to graft compatibility in grafted Annona plants.     

Overexpression of <Emphasis Type="Italic">AtGRDP2</Emphasis> gene in common bean hairy roots generates vigorous plants with enhanced salt tolerance

Proteins with glycine-rich repeats have been identified in plants, mammalians, fungi, and bacteria. Plant glycine-rich proteins have been associated to stress response. Previously, we reported that the Arabidopsis thaliana AtGRDP2 gene, which encodes a protein with a glycine-rich domain, plays a role in growth and development of A. thaliana and Lactuca sativa. In this study, we generated composite Phaseolus vulgaris plants that overexpress the AtGRDP2 gene in hairy roots generated by Agrobacterium rhizogenes. We observed that hairy roots harboring the AtGRDP2 gene developed more abundant and faster-growing roots than control hairy roots generated with the wild type A. rhizogenes. In addition, composite common bean plants overexpressing the AtGRDP2 gene in roots were more tolerant to salt stress showing increments in their fresh and dry weight. Our data further support the role of plant GRDP genes in development and stress response.     

The first archaeobotanical evidence of <Emphasis Type="Italic">Lagenaria siceraria</Emphasis> from the territory of Hungary: histology,phytoliths and (a)DNA

Bottle gourd (Lagenaria siceraria (Molina) Standl.) is accepted as typical ‘New World’ cultivated economic plant. Currently, two subspecies are known: L. siceraria ((Molina) Standl.) ssp. siceraria has an African origin, whilst ssp. asiatica is recognized to be originating from Asian territories. It only seldom appears in European archaeological contexts, however finds from the Roman period appear sporadically. Part of a Late Middle Age (14th–15th century) settlement was excavated near the town of Pócspetri (SE Hungary) and one of the refuse pits contained waterlogged remains of several dozen dicot leaves and small sized branches (e.g. Populus sp.). In addition an entire dog skeleton and an apparently 10 cm by 7 cm bottle gourd pericarp fragment were also found in a well-preserved pot. This fortunate recovery accounts for the first evidence of bottle gourd in the archaeobotanical record of Hungary and Eastern Europe. Due to its favourable preservation not only precise morphological analysis could be undertaken, but ancient DNA (aDNA) extraction, PCR amplification and sequencing were used to identify more closely its possible origin and taxonomic relations. Different diagnostic phytolith morphotypes of the pericarp were matched to reference material phytolith assemblages. The macro-archaeobotanical record of the feature reflects the botanical elements of the natural environment of this medieval site and places the unique bottle gourd find in context.     

Biolistic transformation of Carrizo citrange (<Emphasis Type="Italic">Citrus sinensis</Emphasis> Osb. × <Emphasis Type="Italic">Poncirus trifoliata</Emphasis> L. Raf.)

Key message

The development of transgenic citrus plants by the biolistic method.

Abstract

A protocol for the biolistic transformation of epicotyl explants and transgenic shoot regeneration of immature citrange rootstock, cv. Carrizo (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.) and plant regeneration is described. Immature epicotyl explants were bombarded with a vector containing the nptII selectable marker and the gfp reporter. The number of independent, stably transformed tissues/total number of explants, recorded by monitoring GFP fluorescence 4 weeks after bombardment was substantial at 18.4 %, and some fluorescing tissues regenerated into shoots. Fluorescing GFP, putative transgenic shoots were micro-grafted onto immature Carrizo rootstocks in vitro, confirmed by PCR amplification of nptII and gfp coding regions, followed by secondary grafting onto older rootstocks grown in soil. Southern blot analysis indicated that all the fluorescing shoots were transgenic. Multiple and single copies of nptII integrations were confirmed in five regenerated transgenic lines. There is potential to develop a higher throughput biolistics transformation system by optimizing the tissue culture medium to improve shoot regeneration and narrowing the window for plant sampling. This system will be appropriate for transformation with minimal cassettes.

     

Phosphorus deprivation effects on water relations of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> plant via reducing plasma membrane permeability

Plants grown in phosphorus-deprived solutions often exhibit disruption of water transport due to reduction in root hydraulic conductivity (Lp_r). To uncover the relationship between root Lp_r and water permeability coefficient (P_f) of plasma membrane and the role of aquaporins, we evaluated P_f of plasma membrane and also PIP-type aquaporin gene expression in tobacco (Nicotiana tabacum L.) plant roots after seven days P-deprivation. The results showed significant reduction in sap flow rate (J_v) and osmotic root hydraulic conductivity (L_pr-o) in P-deprived roots. These effects were reversed 24 h after P-resupplying. Interestingly, the P_f of root protoplasts was 57% lower in P-deprived plants compared with P-sufficient ones. The expression of NtPIP1;1 and NtPIP2;1 aquaporins did not change significantly in P-deprived plants compared with P-sufficient ones, but the copy number of NtAQP1 increased significantly in P-deprived plants. P-deprivation did not change Lpr-o significantly in antisense NtAQP1 plants. Taken together, these findings suggest that P-deprivation may play an important role in modulation of root hydraulic conductivity by affecting P_f in transcellular pathway of water flow across roots and aquaporins. Finally, we concluded that dominant water transport pathway under P-deprivation was transcellular one.     

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.     

Vetch aphid, <Emphasis Type="Italic">Megoura crassicauda</Emphasis> (Hemiptera: Aphididae), parasitism does not reduce the bean production of narrow-leaved vetch, <Emphasis Type="Italic">Vicia sativa</Emphasis> subsp. <Emphasis Type="Italic">nigra</Emphasis> (Fabaceae)

Megoura crassicauda Mordvilko (Hemiptera: Aphididae) is a dominant aphid species found on Vicia sativa subsp. nigra (L.) Ehrh. (Fabaceae) in the spring. Worker ants of Formica japonica, the dominant ant species attracted to the extrafloral nectaries of V. s. nigra, often attack ladybirds (Coccinella septempunctata), which are aphid enemies. However, the workers of F. japonica do not attack or exclude M. crassicauda, the non-myrmecophilous aphid. It appears that the “bodyguard” retained by the plant guards the plant’s herbivore by attacking the herbivores’ enemies, rather than guarding the plant itself. The relationship between V. s. nigra and M. crassicauda was observed in the field to examine and evaluate the cost of parasitism. Parasitism by M. crassicauda delayed flower bud formation markedly in V. s. nigra but did not kill the plants. V. s. nigra plants that were parasitized showed a net bean production similar to that of the non-parasitized controls. The parasitism rate of M. crassicauda increased when extrafloral nectaries were used by F. japonica. These results may indicate that M. crassicauda provides V. s. nigra with benefits by preventing other serious disadvantages.