Leaf Tissue Analysis Research Articles

Physiological, transcriptomic, and metabolic analyses reveal that mild salinity improves the growth, nutrition, and flavor properties of hydroponic Chinese chive (Allium tuberosum Rottler ex Spr)

Environmental stressors such as salinity have pronounced impacts on the growth, productivity, nutrition, and flavor of horticultural crops, though yield loss sometimes is inevitable. In this study, the salinity influences were evaluated using hydroponic Chinese chive (Allium tuberosum) treated with different concentrations of sodium chloride. The results demonstrated that lower salinity could stimulate plant growth and yield. Accordingly, the contents of soluble sugar, ascorbic acid, and soluble protein in leaf tissues increased, following the decrease of the nitrate content, under mild salinity (6.25 or 12.5 mM NaCl). However, a higher level of salinity (25 or 50 mM NaCl) resulted in growth inhibition, yield reduction, and leaf quality deterioration of hydroponic chive plants. Intriguingly, the chive flavor was boosted by the salinity, as evidenced by pungency analysis of salinity-treated leaf tissues. UPLC-MS/MS analysis reveals that mild salinity promoted the accumulation of glutamic acid, serine, glycine, and proline in leaf tissues, and thereby enhanced the umami and sweet flavors of Chinese chive upon salinity stress. Considering the balance between yield and flavor, mild salinity could conduce to hydroponic Chinese chive cultivation. Transcriptome analysis revealed that enhanced pungency could be ascribed to a salt stress-inducible gene, AtuFMO1, associated with the biosynthesis of S-alk(en)yl cysteine sulphoxides (CSOs). Furthermore, correlation analysis suggested that two transcription factors, AtubHLH and AtuB3, were potential regulators of AtuFMO1 expressions under salinity. Thus, these results revealed the molecular mechanism underlying mild salinity-induced CSO biosynthesis, as well as a practical possibility for producing high-quality Chinese chive hydroponically.

The size of iron oxide nanoparticles determines their translocation and effects on iron and mineral nutrition of pumpkin (Cucurbita maxima L.)

The ability of nanoparticles (NPs) to migrate in the plant body is an important issue to ensure that the NPs reach the desired tissue and to be able to select the most efficient NPs for agricultural applications. In this study, the size impact of four different iron oxide NPs (8–10, 18–20, 20–40, and 30–50 nm referred as NP10, NP20, NP30, and NP40, respectively) on their translocation in pumpkin was elucidated. To assess the root-to-shoot translocation, phloem sap was examined under transmission electron microscope (TEM). In addition, vibrating sample magnetometer (VSM) and inductively coupled plasma optical emission spectrophotometry (ICP-OES) analyses of stem and leaf tissues were performed to confirm size-dependent translocation. TEM and VSM analyses verified root-to-stem translocation of all tested NPs. The NPs treatment significantly altered the abundances of Mn, Cu, K, P, Al, Mg, and Na in tissues. The iron (Fe) content was abundant in plants treated with NP30 and NP20, and the lowest in plants treated with NP10 and NP40. Together with, only NP30 was found to be significantly translocated to the leaves, where it was 393 mg/kg in DW, about 2.3 times that of control. These findings pointed out the size-dependent translocation of NPs. It seems that biological barriers in the vascular bundle appear to restrict the migration, especially for NPs with an average size of 40 nm and above in pumpkins. These findings are important for selecting the most suitable size of iron oxide NPs for use in agricultural practices.

EgNRT2.3 and EgNAR2 expression are controlled by nitrogen deprivation and encode proteins that function as a two-component nitrate uptake system in oil palm

Oil palm (Elaeis guineensis Jacq.) is an important crop for oil and biodiesel production. Oil palm plantations require extensive fertilizer additions to achieve a high yield. Fertilizer application decisions and management for oil palm farming rely on leaf tissue and soil nutrient analyses with little information available to describe the key players for nutrient uptake. A molecular understanding of how nutrients, especially nitrogen (N), are taken up in oil palm is very important to improve fertilizer use and formulation practice in oil palm plantations. In this work, two nitrate uptake genes in oil palm, EgNRT2.3 and EgNAR2, were cloned and characterized. Spatial expression analysis showed high expression of these two genes was mainly found in un-lignified young roots. Interestingly, EgNRT2.3 and EgNAR2 were up-regulated by N deprivation, but their expression pattern depended on the form of N source. Promoter analysis of these two genes confirmed the presence of regulatory elements that support these expression patterns. The Xenopus oocyte assay showed that EgNRT2.3 and EgNAR2 had to act together to take up nitrate. The results suggest that EgNRT2.3 and EgNAR2 act as a two-component nitrate uptake system in oil palm.

An Integrative Transcriptional Network Revealed Spatial Molecular Interplay Underlying Alantolactone and Inulin Biosynthesis in Inula racemosa Hook f.

Inula racemosa Hook. f. (Pushkarmula), a perennial Himalayan herb known for its aromatic and phytopharmaceutical attributes, is not yet explored at genomic/transcriptomic scale. In this study, efforts were made to unveil the global transcriptional atlas underlying organ-specific specialized metabolite biosynthesis by integrating RNA-Seq analysis of 433 million sequenced reads with the phytochemical analysis of leaf, stem, and root tissues. Overall, 7242 of 83,772 assembled nonredundant unigenes were identified exhibiting spatial expression in leaf (3761), root (2748), and stem (733). Subsequently, integration of the predicted transcriptional interactome network of 2541 unigenes (71,841 edges) with gene ontology and KEGG pathway enrichment analysis revealed isoprenoid, terpenoid, diterpenoid, and gibberellin biosynthesis with antimicrobial activities in root tissue. Interestingly, the root-specific expression of germacrene-mediated alantolactone biosynthesis (GAS, GAO, G8H, IPP, DMAP, and KAO) and antimicrobial activities (BZR1, DEFL, LTP) well-supported with both quantitative expression profiling and phytochemical accumulation of alantolactones (726.08 μg/10 mg) and isoalantolactones (988.59 μg/10 mg), which suggests “roots” as the site of alantolactone biosynthesis. A significant interaction of leaf-specific carbohydrate metabolism with root-specific inulin biosynthesis indicates source (leaf) to sink (root) regulation of inulin. Our findings comprehensively demonstrate the source-sink transcriptional regulation of alantolactone and inulin biosynthesis, which can be further extended for upscaling the targeted specialized metabolites. Nevertheless, the genomic resource created in this study can also be utilized for development of genome-wide functionally relevant molecular markers to expedite the breeding strategies for genetic improvement of I. racemosa.

INFLUÊNCIA DA DENSIDADE E DISTÂNCIA DE Digitaria horizontalis Willd NOS PARÂMETROS BIOMÉTRICOS E NUTRICIONAIS DE MUDAS PRÉ-BROTADAS DE CANA-DE-AÇÚCAR

ABSTRACT Sugarcane is one of the main agricultural crops in Brazil and the presence of weeds in production areas can affect growth, development and accumulation of nutrients of the crop, which may relate to the density and area of influence of the weeds in the field. Therefore, this study aimed to evaluate the effects of the density and distance of crabgrass (Digitaria horizontalis Willd) on the development of pre-sprouted seedlings (PSS) of sugarcane of the variety IAC SP 95-5000. We conducted the experiment in a greenhouse in a randomized design with four repetitions. We evaluated height, leaf area, aerial dry biomass, and nutritional analysis of leaf tissue of the PSS after the weeds flowered at 84 days. The crabgrass did not interfere in the biometric and nutritional parameters when they were 18 and 24 cm away from the crop. Regardless of the planting density and the density of 80 plants m-2 of crabgrass for all distances, we considered the distance of six cm between the crop and the weeds critical for the crop, since they significantly reduced height, leaf area, and aerial dry biomass of the PSS. The crabgrass was a potential competitor for N, P, K, Mn, Fe, and Zn with the PSS in the conditions of 2, 6, and 12 cm when they were in 40 and 80 plants m-2.

RNAi silencing of wheat gliadins alters the network of transcription factors that regulate the synthesis of seed storage proteins toward maintaining grain protein levels.

Gluten proteins are responsible for the unique viscoelastic properties of wheat dough, but they also trigger the immune response in celiac disease patients. RNA interference (RNAi) wheat lines with strongly silenced gliadins were obtained to reduce the immunogenic response of wheat. The E82 line presents the highest reduction of gluten, but other grain proteins increased, maintaining a total nitrogen content comparable to that of the wild type. To better understand the regulatory mechanisms in response to gliadin silencing, we carried out a transcriptomic analysis of grain and leaf tissues of the E82 line during grain filling. A network of candidate transcription factors (TFs) that regulates the synthesis of the seed storage proteins (SSPs), α-amylase/trypsin inhibitors, lipid transfer proteins, serpins, and starch in the grain was obtained. Moreover, there were a high number of differentially expressed genes in the leaf of E82, where processes such as nutrient availability and transport were enriched. The source-sink communication between leaf and grain showed that many down-regulated genes were related to protease activity, amino acid and sugar metabolism, and their transport. In the leaf, specific proline transporters and lysine-histidine transporters were down- and up-regulated, respectively. Overall, the silencing of gliadins in the RNAi line is compensated mainly with lysine-rich globulins, which are not related to the proposed candidate network of TFs, suggesting that these proteins are regulated independently of the other SSPs. Results reported here can explain the protein compensation mechanisms and contribute to decipher the complex TF network operating during grain filling.

Comparative transcriptome analysis of leaf, stem, and root tissues of Semiliquidambar cathayensis reveals candidate genes involved in terpenoid biosynthesis.

Semiliquidambar cathayensis is a traditional medicinal plant and endemic species in China. Its roots, branches, leaves, bark, and nectar are known to have therapeutic effects against rheumatoid arthritis, lumbar muscle strain, and several other diseases. However, limited knowledge regarding the molecular properties of S. cathayensis highlights the need for further research in order to elucidate the underlying pathways governing the synthesis of its active ingredients and regulation of its accumulation processes. We conducted transcriptome sequencing of the leaf, stem and root epidermises, and stem and root xylems of S. cathayensis with three biological replicates. Moreover, candidate genes involved in terpenoid biosynthesis, such as IDI, FPPS, DXR, SQS, GPPS, and HMGR were selected for quantitative real-time PCR analysis. We identified 88,582 unigenes. Among which, 36,144 unigenes were annotated to the nr protein database, 21,981 to the Gene Ontology database, 11,565 to the Clusters of Orthologous Groups database, 24,209 to the Pfam database, 21,685 to the SWISS-PROT database, and 12,753 to the Kyoto Encyclopedia of Genes and Genomes (KEGG), with 5072 unigenes common to all six databases. Of those annotated using the KEGG database, 187 unigenes were related to the terpenoid metabolism pathway, and expression analysis of the related genes indicated that the mevalonate and methylerythritol 4-phosphate pathways play different roles in terpenoid biosynthesis in different tissues of S. cathayensis. These findings greatly expand gene resources of S. cathayensis and provide basic data for the study of the biosynthetic pathways and molecular mechanisms of terpenoids.

Unraveling the Host-Selective Toxic Interaction of Cassiicolin with Lipid Membranes and Its Cytotoxicity.

Cassiicolin (Cas), a toxin produced by Corynespora cassiicola, is responsible for Corynespora leaf fall disease in susceptible rubber trees. Currently, the molecular mechanisms of the cytotoxicity of Cas and its host selectivity have not been fully elucidated. Here, we analyzed the binding of Cas1 and Cas2 to membranes consisting of different plant lipids and their membrane disruption activities. Using high-speed atomic force microscopy and confocal microscopy, we reveal that the binding and disruption activities of Cas1 and Cas2 on lipid membranes are strongly dependent on the specific plant lipids. The negative phospholipids, glycerolipids, and sterols are more sensitive to membrane damage caused by Cas1 and Cas2 than neutral phospholipids and betaine lipids. Mature Cas1 and Cas2 play an essential role in causing membrane disruption. Cytotoxicity tests on rubber leaves of Rubber Research Institute of Vietnam (RRIV) 1, RRIV 4, and Prang Besar (PB) 255 clones suggest that the toxins cause necrosis of rubber leaves, except for the strong resistance of PB 255 against Cas2. Cryogenic scanning electron microscopy analyses of necrotic leaf tissues treated with Cas1 confirm that cytoplasmic membranes are vulnerable to the toxin. Thus, the host selectivity of Cas toxin is attained by the lipid-dependent binding activity of Cas to the membrane, and the cytotoxicity of Cas arises from its ability to form biofilm-like structures and to disrupt specific membranes.

Heightened miR6024-NLR interactions facilitate necrotrophic pathogenesis in tomato.

miR6024 acts as a negative regulator of R genes, hence of Tomato plant immunity, and facilitates disease by the necrotrophic pathogen A. solani. Plant resistance genes or Nucleotide-binding leucine-rich repeat (NLR)genes, integral components of plant disease stress-signaling are targeted by variable groups of miRNAs. However, the significance of miRNA-mediated regulation of NLRs during a pathogen stress response, specifically for necrotrophic fungus, is poorly understood. A thorough examination of Tomato NLRs and miRNAs could map substantial interactions of which half the annotated NLRs were targets of Solanaceae-specific and conserved miRNAs, at the NB subdomain. The Solanaceae-specific miR6024 and its NLR targets analysed in different phytopathogenic stresses revealed differential and mutually antagonistic regulation. Interestingly, miR6024-targeted cleavage of a target NLR also triggered the generation of secondary phased siRNAs which could potentially amplify the defense signal. RNA-seq analysis of leaf tissues from miR6024 overexpressing Tomato plants evidenced a perturbation in the defense transcriptome with the transgenics showing unwarranted immune response-related genes' expression with or without infection with necrotrophic Alternaria solani, though no adverse effect could be observed in the growth and development of the transgenic plants. Transgenic plants exhibited constitutive downregulation of the target NLRs, aggravated disease phenotype with an enhanced lesion, greater ROS generation and hypersusceptibility to A. solani infection, thus establishingthat miR6024 negatively impacts plant immune response during necrotrophic pathogenesis. Limited knowledge about the outcome of NLR-miRNA interaction during necrotrophic pathogenesis is a hindrance to the deployment of miRNAs in crop improvement programs. With the elucidation of the necrotrophic disease-synergistic role played by miR6024, it becomes a potent candidate for biotechnological manipulation for the rapid development of pathogen-tolerant solanaceous plants.

Transcriptome Analysis of Green and White Leaf Ornamental Kale Reveals Coloration-Related Genes and Pathways.

Leaf color is a crucial agronomic trait in ornamental kale. However, the molecular mechanism regulating leaf pigmentation patterns in green and white ornamental kale is not completely understood. To address this, we performed transcriptome and pigment content analyses of green and white kale leaf tissues. A total of 5,404 and 3,605 different expressed genes (DEGs) were identified in the green vs. white leaf and the green margin vs. white center samples. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analysis showed that 24 and 15 common DEGs in two pairwise comparisons were involved in chlorophyll metabolism and carotenoid biosynthesis, respectively. Seventeen genes related to chlorophyll biosynthesis were significantly upregulated in green leaf tissue, especially chlH and por. Of the 15 carotenoid biosynthesis genes, all except CYP707A and BG1 were lower expressed in white leaf tissue. Green leaf tissue exhibited higher levels of chlorophyll and carotenoids than white leaf tissue. In addition, the DEGs involved in photosystem and chlorophyll-binding proteins had higher expression in green leaf tissue. The PSBQ, LHCB1.3, LHCB2.4, and HSP70 may be key genes of photosynthesis and chloroplast formation. These results demonstrated that green and white coloration in ornamental kale leaves was caused by the combined effects of chlorophyll and carotenoid biosynthesis, chloroplast development, as well as photosynthesis. These findings enhance our understanding of the molecular mechanisms underlying leaf color development in ornamental kale.

Multidimensional Gene Regulatory Landscape of Motor Organ Pulvinus in the Model Legume Medicago truncatula.

Nyctinastic leaf movement of Fabaceae is driven by the tiny motor organ pulvinus located at the base of the leaf or leaflet. Despite the increased understanding of the essential role of ELONGATED PETIOLULE1 (ELP1)/PETIOLE LIKE PULVINUS (PLP) orthologs in determining pulvinus identity in legumes, key regulatory components and molecular mechanisms underlying this movement remain largely unclear. Here, we used WT pulvinus and the equivalent tissue in the elp1 mutant to carry out transcriptome and proteome experiments. The omics data indicated that there are multiple cell biological processes altered at the gene expression and protein abundance level during the pulvinus development. In addition, comparative analysis of different leaf tissues provided clues to illuminate the possible common primordium between pulvinus and petiole, as well as the function of ELP1. Furthermore, the auxin pathway, cell wall composition and chloroplast distribution were altered in elp1 mutants, verifying their important roles in pulvinus development. This study provides a comprehensive insight into the motor organ of the model legume Medicago truncatula and further supplies a rich dataset to facilitate the identification of novel players involved in nyctinastic movement.

Reduction of leaf tip burns of Ornithogalum dubium by controlling the temperature during bulb storage and greenhouse forcing to produce quality plants

Production of quality potted Ornithogalum dubium Houtt. plants were investigated under multiple conditions: pre-planting treatment at 10, 16, and 22°C for 40 days from Sept. 21 (stage A; ST-A) during bulb storage and then bulbs were potted. After potting, post-planting treatment at 15/12, 18/15, and 21/18°C (day/night) during stage B for 35 days from Nov. 2 (stage B; ST-B), and at 15/12 and 21/18°C during stage C for 30 days from Dec. 7 (stage C; ST-C) during greenhouse forcing was applied. Leaf tissue analyses for macro- and micro-nutrients were performed to investigate the cause of leaf tip burn symptom (LTB). Three criteria for quality of the plants at flowering were established: (1) LTB occurs on less than 1.5 leaves per plant. (2) the number of days to flower is less than 115 days, the length of the third leaf counted from the crown (the junction of the shoot and roots) is shorter than 11.5 cm, and the width is narrower than 2.5 cm; the scape length is shorter than 15 cm, and there are more than 45 flowers. (3) the leaf spread and morphology (leaf spread) and the pattern of the scape curvature (scape growth) have a score of less than 1.5. The following conditions are optimal to produce quality plants based on these three criteria: (1) Pre-planting bulbs treatment was applied at 10 or 16°C during ST-A, and forcing was performed at 15/12°C during ST-B and 21/18°C during ST-C. These conditions accelerated flowering, produced straight scape growth and upward (erect) growing leaves, and yielded acceptable leaf length and width. (2) The incidence of LTB was minimal at 10°C or possibly 16°C during ST-A, and at 15/12°C in ST-B and at 21/18°C in ST-C during greenhouse forcing. Leaf tip burn symptom was observed in both young and old leaves and was caused by a high boron (B) concentration (218-230 ppm) and possibly a high zinc (Zn) concentration (155-159 ppm) in O. dubium. A low calcium (Ca) concentration was not the cause of LTB. Although LTB cannot be avoided, it can be minimized by temperature manipulation during pre- and post-planting phase to produce high quality potted plants.

Metal Accumulation Properties of Eight Traditionally Utilized Nutritional Plants and Their Potential as Suitable Crops for Cultivation on Acidic Soils of the Northern Province Uíge, Angola

High metal contents of acidic soils from sub-Saharan Africa often prevent the cultivation of crops and lead to a low livestock yield. The carbohydrate rich diet of the Angolan population is low in minerals and vitamins, resulting in various deficiency syndromes and a high child mortality rate. Eight traditionally utilized plants (Anisophyllea quangensis, Annona stenophylla subsp. cuneata, Canarium schweinfurthii, Eugenia malangensis, Landolphia lanceolata, Raphionacme madiensis, Tristemma mauritianum, Vitex madiensis subsp. madiensis) with nutritional value for the Angolan population were analyzed for their soil and growing conditions. The species are adapted to the local conditions and can serve as crops for the unfavorable soils of the province Uíge. Chemical and physical characteristics of the uppermost soil (0–5 cm) and in 30 cm depth were analyzed. The plant-available macro-and micronutrients were determined using Mehlich 3 extraction. Data are completed with leaf tissue analyses, examining the uptake of minerals. As aluminum (Al) and manganese (Mn) are plant-available in high amounts, local plants evolved mechanisms dealing with those metals. These Al accumulators with foliar contents above 1000 mg/kg are Anisophyllea quangensis (7884 mg/kg), Landolphia lanceolata (6809 mg/kg), Tristemma mauritianum (4674 mg/kg), and Eugenia malangensis (13,989 mg/kg). All four species bear edible fruits with nutritional potential. The domestication and commercialization of those plants seem to be promising, utilizing local soils without expensive amelioration techniques.

Transcriptional Analysis of Maize Leaf Tissue Treated With Seaweed Extract Under Drought Stress

Kappaphycus alvarezii seaweed extract (KSWE) has been known for its plant biostimulant and stress alleviation activities on various crops. However, very few reports are available depicting its impact at the molecular level, which is crucial in identifying the mechanism of action of KSWE on plants. Here, maize leaf tissue of control and KSWE-treated plants were analyzed for their transcriptional changes under drought stress. KSWE was applied foliarly at the V5 stage of maize crop under drought, and leaf transcriptome analysis was performed. It was found that a total of 380 and 631 genes were up- and downregulated, respectively, due to the application of KSWE. Genes involved in nitrate transportation, signal transmission, photosynthesis, transmembrane transport of various ions, glycogen, and starch biosynthetic processes were found upregulated in KSWE-treated plants, while genes involved in the catabolism of polysaccharide molecules such as starch as well as cell wall macromolecules like chitin and protein degradation were found downregulated. An overview of differentially expressed genes involved in metabolic as well as regulatory processes in KSWE-treated plants was also analyzed via Mapman tool. Phytohormone signaling genes such as cytokinin-independent 1 (involved in cytokine signal transduction), Ent-kaurene synthase and GA20 oxidase (involved in gibberellin synthesis), and gene of 2-oxoglutarate-dependent dioxygenase enzyme activity (involved in ethylene synthesis) were found upregulated while 9-cis-epoxycarotenoid dioxygenase (a gene involved in abscisic acid synthesis) was found downregulated due to the application of KSWE. Modulation of gene expression in maize leaf tissue in response to KSWE treatment elucidates mechanisms to ward off drought stress, which can be extended to understand similar phenomenon in other crops as well. This molecular knowledge can be utilized to make the use of KSWE more efficient and sustainable.

Evaluation of different artificial light technologies for indoor aquaponic production of Bibb lettuce, Lactuca sativa var. capitata, and compact basil, Ocimum basilicum var. Genovese

AbstractAquaponic production in temperate climates is often conducted in insulated buildings to reduce heating costs and therefore must rely on artificial lighting to replace natural sunlight. However, there are several different light technologies available to producers. We conducted two aquaponic growth trials to compare four lighting technologies for the indoor production of Bibb lettuce, Lactuca sativa var. capitata (Trial 1), and basil, Ocimum basilicum var. Genovese (Trial 2). Light types evaluated included metal halide (MH), fluorescent (FLO), light‐emitting diode (LED), and induction (IND). Using a complete block design, each of the four identical aquaponic systems included all four light types. Juvenile Nile tilapia, Oreochromis niloticus, (Trial 1:145 g; Trial 2:169 g) were stocked into each replicate system and fed at a rate of 60 g feed m−2 of plant grow‐space per day. In Trial 1, Bibb lettuce plants grown under LED lights had significantly higher (p ≤ 0.05) average individual weights (164 g), higher production per unit area (3118 g m−2), and higher production per unit energy (84 g m−2 kWh−1) compared to those grown under the other light types. Bibb lettuce grown under IND and FLO lights had significantly higher (p ≤ 0.05) average individual weights (82.0 and 78.0 g, respectively) and production per unit of area (1762 and 1700 g m−2, respectively) than those grown under MH lights (1382 g m−2). In Trial 2, Basil grown under LED lights had significantly higher (p ≤ 0.05) average individual weights (188 g), production per unit area (4970 m−2), and production per unit of energy (1483 g m−2 kWh−1) than basil grown under the other three light types. There was no significant difference (p > 0.05) in average individual weight or production per unit area among basil plants grown under MH, IND or FLO. Analysis of leaf tissues indicated several statistically significant differences among the light treatments. However, the actual magnitudes of these differences were relatively small. Evaluation of emitted wavelengths indicate that production differences may be related to the amount of red light spectra (600–699 nm), and the ratio of red light to blue light (400–499 nm) (R:B ratio) produced by the different light types. The LED had a greater production of red spectra and higher R:B ratios than other lights which appears to be advantageous for growth of Bibb lettuce and basil.

The Role of virulence gene inducing factors in the improvement of in planta transformation of wheat (Triticum aestivum).

Agrobacterium tumefaciens-based plant transformation is a natural and ideal way of introducing genes into plant genomes. The system is based primarily on tissue culture techniques, including cell differentiation and plant regeneration, which might produce somatic cell mutations. In the present research, wheat plants were transformed by a non-tissue culture approach. Accordingly, mature embryos were inoculated with A. tumefaciens at an early stage of germination. A variety of different treatments, such as Agrobacterium strains (EHA105 and LBA4404 harboring pCAMBIAl105.1R), levels of glucose, concentrations of acetosyringone (0, 50, 100 and 200 µM), and types of wheat cultivars (Azar2, Alvand, and Sardari) were studied. The transformation efficiency was determined by using the number of resistant leaves to hygromycin, histochemical GUS analysis of leaf tissues, as well as PCR analysis of three transgenes located within the T-DNA region. The results show maximum efficiency obtained when 200 µM acetosyringone in combination with 15% glucose are used in the induction medium. In addition, the relative expression analysis of virulence genes by qRT-PCR revealed that VirB2 and VirD2 are significantly up-regulated when 200 µM acetosyringone and 15% glucose are used as inducers and carbon sources. Therefore, Vir genes inducing factors as well as three-step culture of Agrobacterium should be considered as major components of any wheat transformation protocol. The Putative transgenic T1 seeds were soaked and germinated in hygromycin solution, which the seedlings further analyzed with aforementioned methods. Based on the results, a modified Agrobacterium-mediated transformation protocol is presented. The current results suggest an inexpensive, quick, and efficient approach to transform wheat genome.

Using Fractionation Profile of Potentially Toxic Elements in Soils to Investigate Their Accumulation in Tilia sp. Leaves in Urban Areas with Different Pollution Levels

Optimal uptake of micronutrients (B, Cu, Fe, Mn, and Zn) and managing the potentially toxic elements (PTEs) (Co, Cr, Ni, Pb, and Sr) in the ranges not detrimental to plant function may be linked to improving plants’ healthy growth and the ability to provide ecosystem services. We investigated concentrations, mobility, and potential availability of potentially toxic elements (PTEs) in soil samples from polluted and non-polluted municipal parks in Reading (UK) and Belgrade (Serbia) and their impact on elemental concentrations in Tilia leaves. We aimed to identify common limiting factors potentially affecting the growth/healthy function of this widely-used tree species. Levels of all elements in soil were below limits established by the directive of European Communities, except for Ni at Belgrade sites. Content of Co, Cr, Cu, Fe, Ni, Pb, and Zn in soluble fraction at all locations was <10%, indicating low mobility; B showed moderate mobility (11.1–20.7%), Mn (6.5–55.6%), and Sr—high (44–76.3%). Principal Component Analysis of Tilia leaf tissues showed a different capacity for uptake/accumulation of PTEs in different locations. Findings indicate the complexity of local edaphic influences on plants’ elemental uptake and the risk of those leading to deficiency of important micronutrients, which may impede trees’ function and thus the ability to optimally provide ecosystem services.

A NAC transcription factor BrNAC087 is involved in gibberellin-delayed leaf senescence in Chinese flowering cabbage

Phytohormone gibberellins (GAs) regulate leaf senescence. A global view of the regulatory pathways associated with GA-mediated inhibition of leaf senescence, especially in economically important leafy vegetables such as Chinese flowering cabbage, remains unclear. In this study, exogenous supply of gibberellin (GA3) delayed postharvest Chinese flowering cabbage leaf senescence. By comparative transcriptome analysis of leaf tissue, a total of 4682, 5059, and 2362 differentially expressed genes (DEGs) were identified between the comparison of 0d (cabbages at the harvest day) and CK3d (control cabbages at day 3 post-harvest), 0d and GA3d (GA3-treated cabbages at day 3 post-harvest), and CK3d and GA3d, respectively. Further analysis of identified DEGs involved in chlorophyll and GA degradation during leaf senescence, and the activity of chlorophyll catabolic genes (CCGs) BrPPH and BrRCCR, and bioactive GA degradation gene BrGA2ox1, was greatly reversed with GA3 application during leaf senescence. More importantly, a NAC transcription factor (TF), BrNAC087, was found to be associated with leaf senescence. BrNAC087 is a nuclear localized gene with transcriptional activity, highly expressed during senescence but downregulated by GA3. Significantly, BrNAC087 positively regulated BrPPH, BrRCCR and BrGA2ox1 expression by binding to NAC-binding sequences in their promoters in vitro and in vivo. These findings collectively provide evidence for a new molecular regulatory pathway explaining, at least in part, a mechanistic basis for gibberellin-mediated leaf senescence inhibition in postharvest Chinese flowering cabbage.

Transcriptome sequencing and functional characterization of new sesquiterpene synthases from Curcuma wenyujin

Tubers of Curcuma wenyujin are rich in essential oils, mainly various sesquiterpenes, showing antibacterial, anti-viral and anti-tumor effects. However, the molecular mechanism of C. wenyujin is deficient and related sesquiterpene synthases are still unclear. In this study, the transcriptome data of tubers and leaves from C. wenyujin were obtained and assembled into 78 092 unigenes. Of them, 244 unigenes were predicted to be involved in terpenoid biosynthesis while 131 unigenes were categorized as the “Terpenoid backbone biosynthesis” (TBB) term. Twenty-two unigenes possessed terpene synthase domain; five were predicted to be sesquiterpene synthases. Of the 208 unigenes annotated as cytochromes P450, 8 unigenes with full-length coding sequences were part of the CYP71 clade that primarily may perform hydroxylations of specialized metabolites. Furthermore, Ten DEGs related to the C5 precursor supply and sesquiterpene synthesis were validated by Real-time PCR; that showed a close correspondence with transcriptome sequence. A novel germacrene B synthase (CwGBS) and α-santalene synthase (CwSS) were identified in metabolically engineering E. coli. This study provided the first de novo transcriptome comparative analysis of leaf and tuber tissues from C. wenyujin, aiming to understand genetic mechanisms. Key genes involved in the biosynthesis of sesquiterpene will help for revealing the underlying mechanisms of C. wenyujin.

Site-specific, silicon-induced structural and molecular defence responses against powdery mildew infection in roses.

Silicon (Si) application to miniature potted roses can decrease severity of powdery mildew (Podosphaera pannosa) and this is associated with increased accumulation of callose and hydrogen peroxide (H2 O2 ) as well as hypersensitive (HR) cells. We used microscopy, gene expression and specific inhibitors of callose and H2 O2 to determine how effective these plant responses are in stopping infection. Pathogen arrest in Si-treated (Si+) plants was accompanied by increased accumulation of callose and H2 O2 in papillae and HR cells, respectively. These responses were reduced by application of specific inhibitors (2-deoxy-d-glucose for callose and catalase for H2 O2 ), which increased disease severity in Si+, but not in Si- plants. As markers for HR and callose, expression of the HR-specific gene hsr203J and the wound-related callose synthase GSL5, respectively, was studied. An up-regulation of expression was only seen after isolation of HR cells with laser capture microdissection. The up-regulation was higher in Si+ than in Si- plants and occurred concomitantly with more efficient photosynthesis in Si+ plants at high disease severity as compared to Si- plants. Silicon-mediated activation of callose and H2 O2 are decisive factors in the defence of rose against P. pannosa and these responses were accompanied with more efficient photosynthesis to strengthen the plant. Only by isolation of HR cells using laser capture microdissection as compared to analysis of whole leaf tissues allowed detection of elevated transcript levels of hsr203J and GSL5 at infection sites as markers for HR. © 2021 Society of Chemical Industry.