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Article
Chickpea, an important grain legume, suffers from considerable loss of yield due to Fusarium wilt disease. Inaccessibility of resistant gene pool among cultivars and lack of report of resistance, genes from alien sources have been the major constraints for resistance development in this valuable crop. However, along with some other transcription factors, MYB78 was significantly upregulated during chickpea—Fusarium interplay in resistant chickpea genotype. Being a highly recalcitrant species, the transformation of this important crop remained non-reproducible until recently. Following a tissue culture independent plumular meristem transformation protocol, introgression of CaMYB78 TF finally became feasible in chickpea. The overexpressed plants developed resistance against the pathogen but the anthocyanin production in transformed flowers was perturbed. In silico analyses of the anthocyanin biosynthetic key gene promoters reported the occurrence of multiple MYB-binding cis elements. Detailed molecular analyses establish the differential regulatory roles of CaMYB78, resistance response against Foc1 on one hand and suppression of pigmentation during flower development on the other, which is an innovative finding of its kind.
 
Article
Brachiaria, a genus from the Poaceae family, is largely cultivated as forage in Brazil. Among the most cultivated varieties of Brachiaria spp., B. brizantha cv. Marandu (syn. Urochloa brizantha) is of great agronomical importance due to the large areas cultivated with this species. This cultivar is apomictic and tetraploid. Sexual diploid genotype is available for this species. The difference in levels of ploidy among sexual and apomictic plants contributes to hindering Brachiaria breeding programs. The induction of haploids and double haploids is of great interest for the generation of new genotypes with potential use in intraspecific crosses. A key factor for the success of this technique is identifying adequate microspore developmental stages for efficient embryogenesis induction. Knowledge of the morphological changes during microsporogenesis and microgametogenesis and sporophytic tissues composing the anther is critical for identifying the stages in which microspores present a higher potential for embryogenic callus and somatic embryo through in vitro culture. In this work, morphological markers were associated with anther and pollen grain developmental stages, through histological analysis. Anther development was divided into 11 stages using morphological and cytological characteristics, from anther with archesporial cells to anther dehiscence. The morphological characteristics of each stage are presented. In addition, the response of stage 8 anthers to in vitro culture indicates microspores initiating somatic embryogenic pathway.
 
Article
Ascorbic acid (AsA) plays an important role in scavenging reactive oxygen species (ROS) and reducing photoinhibition in plants, especially under stress. The function of SlGGP which encodes the key enzyme GDP-L-galactose phosphorylase in AsA synthetic pathway is relatively clear. However, there is another gene SlGGP-LIKE that encodes this enzyme in tomato, and there are few studies on it, especially under salt stress. In this study, we explored the function of this gene in tomato salt stress response using transgenic lines overexpressing SlGGP-LIKE (OE). Under normal conditions, overexpressing SlGGP-LIKE can increase the content of reduced AsA and the ratio of AsA/ DHA (dehydroascorbic acid), as well as the level of xanthophyll cycle. Under salt stress, compared with the wild-type plants (WT), the OE lines can maintain higher levels of reduced AsA. In addition, OE lines also have higher levels of reduced GSH (glutathione) and total GSH, higher ratios of AsA/DHA and GSH/oxidative GSH (GSSR), and higher level of xanthophyll cycle. Therefore, the OE lines are more tolerant to salt stress, with higher photosynthetic activity, higher antioxidative enzyme activities, higher content of D1 protein, lower production rate of ROS, and lighter membrane damage. These results indicate that overexpressing SlGGP-LIKE can enhance tomato resistance to salt stress through promoting the synthesis of AsA.
 
Article
The medicinal plant Catharanthus roseus biosynthesizes many important drugs for human health, including the anticancer monoterpene indole alkaloids (MIAs) vinblastine and vincristine. Over the past decades, the continuous increase in pharmaceutical demand has prompted several research groups to characterize MIA biosynthetic pathways for considering future metabolic engineering processes of supply. In line with previous work suggesting that diversification can potentially occur at various steps along the vindoline branch, we were here interested in investigating the involvement of distinct isoforms of tabersonine-16-O-methyltransferase (16OMT) which plays a pivotal role in the MIA biosynthetic pathway. By combining homology searches based on the previously characterized 16OMT1, phylogenetic analyses, functional assays in yeast, and biochemical and in planta characterizations, we identified a second isoform of 16OMT, referred to as 16OMT2. 16OMT2 appears to be a multifunctional enzyme working on both MIA and flavonoid substrates, suggesting that a constrained evolution of the enzyme for accommodating the MIA substrate has probably occurred to favor the apparition of 16OMT2 from an ancestral specific flavonoid-O-methyltransferase. Since 16OMT1 and 16OMT2 displays a high sequence identity and similar kinetic parameters for 16-hydroxytabersonine, we postulate that 16OMT1 may result from a later 16OMT2 gene duplication accompanied by a continuous neofunctionalization leading to an almost complete loss of flavonoid O-methyltransferase activity. Overall, these results participate in increasing our knowledge on the evolutionary processes that have likely led to enzyme co-optation for MIA synthesis.
 
Pictorial presentation of Mentha species aMentha citrata, bMentha x piperita, cMentha pulegium, dMentha spicata, and eMentha suaveolens
Dose-responsive curve of each Mentha species aMentha citrata, bMentha x piperita, cMentha pulegium, dMentha spicata, and eMentha suaveolens and f positive controls, against eight human pathogens namely (E.c) E. coli, (P.a) P. aeruginosa, (S. t) S. typhi, (K.p) K. pneumoniae, (P.m) P. mirabilis, (B.s) B. subtilis, (S.a) S. aureus, and (C.a) C. albicans. Mean data with standard errors (± SE) exhibited significant variation (α < 0.05)
Article
The Mentha species of family Lamiaceae are famous for their flavor and are commercially used in many food products worldwide. They are widely used to cure digestive problems as well as a natural source of antioxidants and antimicrobials. In this report, the essential oils (EOs) of five Mentha species, namely Mentha citrata, Mentha x piperita, Mentha pulegium, Mentha spicata, and Mentha suaveolens were extracted and their chemical diversity was investigated through gas chromatography-mass spectroscopy (GC–MS). The differential doses (5, 10, and 15 µl) of EOs were tested for antimicrobial potential against two gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), five gram-negative bacteria (Escherichia coli, Pseudomonas aeroginosa, Salmonella typhi, Proteus mirabilis and Klebsiella pneumoniae) and a fungal strain (Candida albicans). The GC–MS results revealed the major components in the EOs were Carvone, Linalool, Hotrienol, Menthol, Isopulegone, Furanone, Piperitone, and Thymol. Moreover, the higher dose (15 µl) of EOs of M. citrata inhibited the growth of S. typhi and C. albicans (35.8 ± 2.4 and 35.2 ± 2.5 mm), M. x piperita inhibited E. coli, B. subtilis, and C. albicans (28.5 ± 3.5, 26.1 ± 2.1, and 25.4 ± 1.1 mm), M. pulegium inhibited K. pneumoniae, B. subtilis, and C. albicans (26.8 ± 1.8, 24.2 ± 2.2, and 25.3 ± 0.9 mm), M. spicata significantly inhibited S. typhi and B. subtilis (35.7 ± 2.7 and 36.3 ± 2.1 mm), and M. suaveolens inhibited K. pneumoniae, C. albicans, and S. typhi (30.8 ± 1.9, 26.9 ± 1.1, and 20.1 ± 0.8 mm) respectively. This study concluded that the EOs of M. citrata was effective against S. typhi and C. albicans. The M. x piperita exhibited strong activities against E. coli, B. subtilis, and C. albicans. Furthermore, the M. pulegium strongly inhibited the growth of K. pneumoniae and C. albicans. The EO of M. spicata was more potent against S. typhi and B. subtilis, while the M. suaveolens was comparatively more effective against S. typhi, K. pneumoniae, and C. albicans. These EOs offer a natural source of antimicrobial agents with high commercial values and social acceptance and could be scale up by food and pharmaceutical industries to control pathogenic diseases.
 
Embryos obtained from different genotypes, the same genotype with stationary culture and shake culture and embryo development. a Embryos from 18SY01. b Embryos from 18SY03. c Embryos from 18SY01 with stationary culture. d Embryos from 18SY01 with shake culture. e Embryos transferred to MS medium. f Regenerated plantlet
Identification of ploidy level of regenerated plants. a Flowers of different ploidy level plants (haploid, doubled haploid, polyploid, from left to right). b DNA content distribution of haploid plants. c DNA content distribution of doubled haploid plants. d DNA content distribution of polyploid plants
Morphological traits of pakchoi DH lines. a Pakchoi DH lines. b Donor plants 18SY01. c DH line BM002. d DH line BM012
Horticultural traits of pakchoi hybrid combinations. a Hybrid combination NB04. b Hybrid combination NB07
Article
Microspore embryogenesis is an effective method of obtaining double haploid (DH) lines in only 1 year. However, the microspore embryogenesis protocol was not efficient in pakchoi. This study aimed to establish an effective microspore culture protocol in pakchoi for hybrid breeding. The embryos were obtained from three genotypes (18SY01, 18SY02, 18SY03), but the frequency of microspore embryogenesis was significantly different. Globular embryos from three genotypes were placed into a rotary shaker (50 r/min, 25 ℃) for further culture to improve microspore embryogenesis and plantlet regeneration without callus development. Shake culture not only increased the frequency of cotyledonary embryos but also accelerated microspore embryogenesis in the NLN-13 liquid medium. Moreover, the doubled haploid rates of regenerated plants for the three genotypes were above 50%. The morphological characters and plot yield of DH lines were identified, and there were significant differences between them. According to the measurement of the self-compatibility index, all the DH lines were self-incompatible. Furthermore, the hybrid combination was prepared with the selected DH lines and the pakchoi genic male sterile line GMS010 to develop excellent hybrids. This work contributes to accelerating the application of microspore embryogenesis and supplying the DH lines in pakchoi hybrid breeding.
 
Article
Antisera were raised against the C-terminal amino acid sequences of the two rhodopsins ADY17806 and AEA49880 of Oxyrrhis marina . The antisera and affinity-purified antibodies thereof were used in western immunoblotting experiments of total cell protein fractions from cultures grown either in darkness or in white, red, green, or blue light. Furthermore, the rhodopsin abundances were profiled in cultures fed with yeast or the prasinophyte Pyramimonas grossii . The immunosignals of ADY17806 and AEA49880 were similar when O. marina was grown in white, green, or blue light. Signal intensities were lower under conditions of red light and lowest in darkness. Higher amounts were registered for both rhodopsins when O. marina was fed with yeast compared to P. grossii . Furthermore, total cell protein of cultures of O. marina grown under all cultivation conditions was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by tryptic in-gel digestion and mass spectrometric analysis of the 25-kDa protein bands. The rhodopsin ADY17809 was detected in all samples of the light quality experiments and in 14 of the 16 samples of the prey quality experiments. The rhodopsin ABV22427 was not detected in one sample of the light quality experiments. It was detected in 15 of the 16 samples of the prey quality experiments. Peptide fragments of the other rhodopsins were detected less often, and no clear distribution pattern was evident with respect to the applied light quality or offered prey, indicating that none of them was exclusively formed under a distinct light regime or when feeding on yeast or the prasinophyte. Fluorescence light microscopy using the affinity-purified antibodies revealed significant labeling of the cell periphery and cell internal structures, which resembled vacuoles, tiny vesicles, and rather compact structures. Immunolabeling electron microscopy strengthened these results and showed that the cytoplasmic membrane, putative lysosome membranes, membranes encircling the food vacuole, and birefringent bodies became labeled.
 
Schematic representation of the evolution of WRKY transcription factor genes from unicellular early green lineage to multi-cellular plants
Multiple roles of WRKY transcription factors in plant defense under different environmental conditions (senescence, drought, salt, heat, cold, UV, carbon starvation, inorganic phosphate deficiency, and wounding stress) including the relationship with ABA hormonal signaling
Article
The recent advancements in sequencing technologies and informatic tools promoted a paradigm shift to decipher the hidden biological mysteries and transformed the biological issues into digital data to express both qualitative and quantitative forms. The transcriptomic approach, in particular, has added new dimensions to the versatile essence of plant genomics through the large and deep transcripts generated in the process. This has enabled the mining of super families from the sequenced plants, both model and non-model, understanding their ancestry, diversity, and evolution. The elucidation of the crystal structure of the WRKY proteins and recent advancement in computational prediction through homology modeling and molecular dynamic simulation has provided an insight into the DNA–protein complex formation, stability, and interaction, thereby giving a new dimension in understanding the WRKY regulation. The present review summarizes the functional aspects of the high volume of sequence data of WRKY transcription factors studied from different species, till date. The review focuses on the dynamics of structural classification and lineage in light of the recent information. Additionally, a comparative analysis approach was incorporated to understand the functions of the identified WRKY transcription factors subjected to abiotic (heat, cold, salinity, senescence, dark, wounding, UV, and carbon starvation) stresses as revealed through various sets of studies on different plant species. The review will be instrumental in understanding the events of evolution and the importance of WRKY TFs under the threat of climate change, considering the new scientific evidences to propose a fresh perspective.
 
Article
The present study reveals silica in sporoderms of micro- and megaspores of the modern quillwort Isoetes echinospora Durieu and homologizes layers of the sporoderm in spores of this plant. Here, the presence of silica in sporoderms of microspores has been documented for the first time, and observations of megaspore sporoderms were used to test various methods, such as energy dispersive (EDS) and electron energy loss (EELS) spectroscopies. The results elucidate the occurrence of silicon in the quillworts and will influence on the interpretation of their modern and fossil sporoderms.
 
Article
Watermelon and melon are members of the Cucurbitaceae family including economically significant crops in the world. The expansin protein family, which is one of the members of the cell wall, breaks down the non-covalent bonds between cell wall polysaccharides, causing pressure-dependent cell expansion. Comparative bioinformatics and molecular characterization analysis of the expansin protein family were carried out in the watermelon (Citrullus lanatus) and melon (Cucumis melo) plants in the study. Gene expression levels of expansin family members were analyzed in leaf and root tissues of watermelon and melon under ABA, drought, heat, cold, and salt stress conditions by quantitative real-time PCR analysis. After comprehensive searches, 40 expansin proteins (22 ClaEXPA, 14 ClaEXPLA, and 4 ClaEXPB) in watermelon and 43 expansin proteins (19 CmEXPA, 15 CmEXPLA, 3 CmEXPB, and 6 CmEXPLB) in melon were identified. The greatest orthologous genes were identified with soybean expansin genes for watermelon and melon. However, the latest divergence time between orthologous genes was determined with poplar expansin genes for watermelon and melon expansin genes. ClaEXPA-04, ClaEXPA-09, ClaEXPB-01, ClaEXPB-03, and ClaEXPLA-13 genes in watermelon and CmEXPA-12, CmEXPA-10, and CmEXPLA-01 genes in melon can be involved in tissue development and abiotic stress response of the plant. The current study combining bioinformatics and experimental analysis can provide a detailed characterization of the expansin superfamily which has roles in growth and reaction to the stress of the plant. The study ensures detailed data for future studies examining gene functions including the roles in plant growth and stress conditions.
 
Article
An in-depth understanding of the development and distribution of laticifer (latex secretory structure) will be important for the production of both rubber and medicines and will support studies on plant adaptations to their environments. We characterize here and describe the ontogenesis of the laticifer sytem in Calotropis procera (Apocynaceae), an invasive subshrub species in arid landscapes. Anatomical and histochemical evaluations of the primary and secondary structures of the stem were carried out on a monthly basis during a full year, with ultrastructural evaluations of laticifer on the stem apex during the rainy season. In the primary structure, laticifer differentiate early from procambium and ground meristem cells of the cortex and medulla and become concentrated adjacent to the external and internal phloem of the bicollateral bundles. In the secondary structure, laticifer differentiates from fusiform derivative cells of the phloem close to the sieve-tube elements. The laticifer is of the articulated, anastomosing, branched type, and it originates from precursor cells that loose the transversal and longitudinal walls by dissolution. Latex is a mixture of terpenes, alkaloids, flavonoids, mucilage, and proteins. The apical meristem and vascular cambium where the laticifer system begins its development are active throughout the year, including during the dry season. The vascular cambium produces phloem with laticifer precursor cells during the rainy season, with high temperatures and long days. The ability of C. procera to grow under water deficit conditions and produce laticifer throughout the year contribute to its wide distribution in arid environments.
 
Article
These YABBY genes are transcription factors (TFs) that play crucial roles in various developmental processes in plants. There is no comprehensive characterization of YABBY genes in a valuable Chinese orchid herb, Dendrobium officinale. In this study, a total of nine YABBY genes were identified in the D. officinale genome. These YABBY genes were divided into four subfamilies: CRC/DL, FIL, INO, and YAB2. Expression pattern analyses showed that eight of the YABBY genes were strongly expressed in reproductive organs (flower buds) but weakly expressed in vegetative organs (roots, leaves, and stems). DoYAB1, DoYAB5, DoDL1, and DoDL3 were abundant in the small flower bud stage, while DoDL2 showed no changes throughout flower development. In addition, DoDL1-3 genes were strongly expressed in the column, tenfold more than in sepals, petals, and the lip. DoYAB1 from the FIL subfamily, DoYAB2 from the YAB2 subfamily, DoYAB3 from the INO subfamily, and DoDL2 and DoDL3 from the CRC/DL subfamily were selected for further analyses. Subcellular localization analysis showed that DoYAB1-3, DoDL2, and DoDL3 were localized in the nucleus. DoYAB2 and DoYAB3 interacted strongly with DoWOX2 and DoWOX4, while DoYAB1 showed a weak interaction with DoWOX4. These results reveal a regulatory network involving YABBY and WOX proteins in D. officinale. Our data provide clues to understanding the role of YABBY genes in the regulation of flower development in this orchid and shed additional light on the function of YABBY genes in plants.
 
Article
Plants adjust their complex molecular, biochemical, and metabolic processes to overcome salt stress. Here, we investigated the proteomic and epigenetic alterations involved in the morphophysiological responses of Pfaffia glomerata, a medicinal plant, to salt stress and the demethylating agent 5-azacytidine (5-azaC). Moreover, we investigated how these changes affected the biosynthesis of 20-hydroxyecdysone (20-E), a pharmacologically important specialized metabolite. Plants were cultivated in vitro for 40 days in Murashige and Skoog medium supplemented with NaCl (50 mM), 5-azaC (25 μM), and NaCl + 5-azaC. Compared with the control (medium only), the treatments reduced growth, photosynthetic rates, and photosynthetic pigment content, with increase in sucrose, total amino acids, and proline contents, but a reduction in starch and protein. Comparative proteomic analysis revealed 282 common differentially accumulated proteins involved in 87 metabolic pathways, most of them related to amino acid and carbohydrate metabolism, and specialized metabolism. 5-azaC and NaCl + 5-azaC lowered global DNA methylation levels and 20-E content, suggesting that 20-E biosynthesis may be regulated by epigenetic mechanisms. Moreover, downregulation of a key protein in jasmonate biosynthesis indicates the fundamental role of this hormone in the 20-E biosynthesis. Taken together, our results highlight possible regulatory proteins and epigenetic changes related to salt stress tolerance and 20-E biosynthesis in P. glomerata, paving the way for future studies of the mechanisms involved in this regulation.
 
Mitotic metaphases (2n = 2x = 26), CMA banding, and 5S and 35S rDNA FISH signals (Columns from left to right). P. hispidinervum (a–c), P. aduncum (d–f), and P. affinis hispidinervum (g–i). Chromosomes were stained with DAPI (gray), arrowheads show tertiary constriction, CMA + bands are shown in yellow (b, e, h), and 5S and 35S rDNA probes in red and green, respectively (c, f, i). Bar: 10 µm
Karyotype ideograms of P. hispidinervum (a), P. aduncum (b), and P. affinis hispidinervum (c): primary constrictions (black), tertiary constriction (blue), 5S rDNA sites (red), and co-localization of CMA + and 35S rDNA (green) in the secondary constrictions. Bar: 10 µm
Mitotic metaphases (2n = 2x = 26) of Piper species hybridized with genomic probes. Species are identified in the lines: P. hispidinervum (a–c), P. aduncum (d–f), and P. affinis hispidinervum (g–i). Genomic probes of P. hispidinervum (green), P. aduncum, and P. affinis hispidinervum (red) are identified in the columns from left to right. Simultaneous hybridization with P. hispidinervum and P. aduncum genomic probes in P. affinis hispidinervum genome is shown in the last line (j–l). Chromosomes were stained with DAPI (gray). Bar: 10 µm
Article
The species Piper hispidinervum, Piper aduncum, and Piper affinis hispidinervum have essential oils with high levels of safrole, dillapiole, and sarisan, respectively. Safrole is important for pharmaceutical and chemical industries, while dillapiole and sarisan are promising compounds to control insects and fungi. These species are very similar morphologically and their taxonomy is controversial. Divergent hypotheses consider P. aduncum and P. hispidinervum either as a single species or as distinct taxa, while P. affinis hispidinervum is inferred to be a natural hybrid or a chemotype of P. hispidinervum. Delimiting the taxonomic boundaries would be helpful for germplasm conservation and breeding programs. This study aimed to undertake a detailed analysis of P. aduncum, P. hispidinervum, and P. affinis hispidinervum karyotype and rDNA sites. Genomic in situ hybridization (GISH) was used to establish genomic homology among species and to test the natural hybridization hypothesis for origin of P. affinis hispidinervum. Karyotype traits were similar for all three species: 2n = 26 small chromosomes, predominantly metacentric. All three species exhibited CMA+ bands on the secondary constriction of chromosome pair 4. A size-heteromorphic 35S rDNA site was co-localized with the CMA+ band. A 5S rDNA site was located in the proximal region of chromosome pair 7. The patterns of genomic hybridization revealed that the repetitive DNA fraction of the species is highly similar in terms of proportion of genome, sequence type, and distribution. Our findings did not allow us to differentiate the three species and point to the importance of deeper genomic studies to elucidate the taxonomic controversy.
 
Article
This work aimed to evaluate the effects of lower water levels on leaf intercellular spaces and to assess their relations with the gas exchange, anatomy, and growth of Sorghum bicolor. Experiments were conducted in a greenhouse, in which plants were subjected to three water conditions (ten replicates, n = 30): well-irrigated, decreased irrigation, and limited irrigation. Lower water levels had no significant effect on the growth of S. bicolor but increased the biomass of the roots. Moreover, the number of leaves, leaf area, and leaf size as well as the chlorophyll content were not affected by lower water levels, and no significant changes were detected for whole plant photosynthesis, transpiration, or stomatal conductance. The water content of the plants and the water potential remained unchanged. However, compared with other treatments, the decreased irrigation decreased water loss and increased the water retention. Lower water levels increased the intercellular CO2 percentage, mesophyll area, and proportion of stomatal cavities and promoted minor changes in leaf tissue and stomatal traits. The increased stomatal cavities provided higher CO2 uptake and prevented excessive water loss. Thus, modifications to the intercellular spaces promoted conditions to avoid excessive water loss while concurrently improving CO2 uptake, which are important traits for drought-tolerant plants.
 
Article
The inflorescence is composed of spikes, and the spike is the carrier of grass seed formation and development, so the development status of inflorescence implies grass seed yield and quality. So far, the systematic analysis of inflorescence development of Kentucky bluegrass has not been reported. The development process of the female gametophyte of wild germplasm materials of Kentucky bluegrass in Gannan, Gansu Province of China (KB-GN), was observed. Based on this, the key developmental stages of inflorescence in KB-GN were divided into premeiosis (GPreM), meiosis (GM), postmeiosis (GPostM), and anthesis (GA), and four stages of inflorescence were selected to analyze the transcriptome expression profile. We found that its sexual reproduction formed a polygonum-type embryo sac. Transcriptome analysis showed that 4256, 1125, 1699, and 3127 genes were highly expressed in GPreM, GM, GPostM, and GA, respectively. And a large number of transcription factors (TFs) such as MADS-box, MYB and NAC, AP2, C2H2, FAR1, B3, bHLH, WRKY, and TCP were highly expressed throughout the inflorescence development stages. KEGG enrichment and MapMan analysis showed that genes involved in plant hormone metabolism were also highly expressed at the entire stages of inflorescence development. However, a few TFs belong to stage-specific genes, such as TRAF proteins with unknown function in plants was screened firstly, which was specifically and highly expressed in the GPreM, indicating that TRAF may regulate the preparatory events of meiosis or be essential for the development of megaspore mother cell (MMC). The expression patterns of 15 MADS-box genes were analyzed by qRT-PCR, and the expression results were consistent with that of the transcriptome. The study on the inflorescence development of KB-GN will be great significant works and contribution to illustrate the basic mechanism of grass seeds formation and development.
 
Article
The ultrastructure of the birefringent bodies of the dinoflagellate Oxyrrhis marina was investigated by transmission electron microscopy. Ultrathin sectioning revealed that the bodies consist of highly ordered and densely packed lamellae, which show a regular striation along their longitudinal axis. A lattice distance of 6.1 nm was measured for the densely packed lamellae by FFT (Fast Fourier Transformation) analysis. In addition, a rather faint and oblique running striation was registered. Lamellae sectioned rather oblique or almost close to the surface show a honeycombed structure with a periodicity of 7.2–7.8 nm. Freeze-fracture transmission electron microscopy revealed that the lamellae are composed of highly ordered, crystalline arrays of particles. Here, FFT analysis resulted in lattice distances of 7.0–7.6 nm. Freeze-fracture transmission electron microscopy further revealed that the bodies remained intact after cell rupture followed by ascending flotation of the membrane fractions on discontinuous sucrose gradients. The birefringent bodies most likely are formed by evaginations of membranes, which separate the cytoplasm from the food vacuoles. Distinct, slightly reddish-colored areas, which resembled the birefringent bodies with respect to size and morphology, were registered by bright field light microscopy within Oxyrrhis marina cells. An absorbance maximum at 540 nm was registered for these areas, indicating that they are composed of rhodopsins. This was finally proven by immuno-transmission electron microscopy, as antisera directed against the C-terminal amino acid sequences of the rhodopsins AEA49880 and ADY17806 intensely immunolabeled the birefringent bodies of Oxyrrhis marina .
 
Article
The present paper aims to shed light on the influence of N6-methyladenosine (m6A) long non-coding RNAs (lncRNAs) and immune cell infiltration on colorectal cancer (CRC). We downloaded workflow-type data and xml-format clinical data on CRC from The Cancer Genome Atlas project. The relationship between lncRNA and m6A was identified by using Perl and R software. Kyoto Encyclopedia of Genes and Genomes enrichment analysis was performed. Lasso regression was utilized to construct a prognostic model. Survival analysis was used to explore the relationship between clusters of m6A lncRNAs and clinical survival data. Differential analysis of the tumor microenvironment and an immune correlation analysis were used to determine immune cell infiltration levels in different clusters and their correlation with clinical prognosis. The expression of lncRNA was tightly associated with m6A. The univariate Cox regression analysis showed that lncRNA was a risk factor for the prognosis. Differential expression analysis demonstrated that m6A lncRNAs were partially highly expressed in tumor tissue. m6A lncRNA-related prognostic model could predict the prognosis of CRC independently. “ECM_RECEPTOR_INTERACTION” was the most significantly enriched gene set. PARP8 was overexpressed in tumor tissue and high-risk cluster. CD4 memory T cells, activated resting NK cells, and memory B cells were highly clustered in the high-risk cluster. All of the scores were higher in the low-risk group. m6A lncRNA is closely related to the occurrence and progression of CRC. The corresponding prognostic model can be utilized to evaluate the prognosis of CRC. m6A lncRNA and related immune cell infiltration in the tumor microenvironment can provide novel therapeutic targets for further research.
 
Article
Acid sulphate soil contains high amounts of iron (Fe) and aluminum (Al), and their contamination has been reported as major problems, especially in rainfed and irrigated lowland paddy fields. Rice is sensitive to Fe and Al grown in acid soil (pH < 5.5), leading to growth inhibition and grain yield loss. The objective of this study was to evaluate Fe and/or Al uptake, translocation, physiological adaptation, metal toxicity, and growth inhibition in rice genotypes grown in acid soil. Fe and Al in the root tissues of all rice genotypes were enriched depending on the exogenous application of either Fe or Al in the soil solution, leading to root growth inhibition, especially in KDML105 genotype. Expression level of OsYSL1 in KDML105 was increased in relation to metal uptake into root tissues, whereas OsVIT2 was downregulated, leading to Fe (50.3 mg g‒1 DW or 13.1 folds over the control) and Al (4.8 mg g‒1 DW or 2.2 folds over the control) translocation to leaf tissues. Consequently, leaf greenness (SPAD), net photosynthetic rate (Pn), stomatal conductance (gs), and transpiration rate (E) in the leaf tissues of genotype KDML105 under Fe + Al toxicity were significantly declined by 28.4%, 35.3%, 55.6%, and 51.6% over the control, respectively. In Azucena (AZU; Fe/Al tolerant), there was a rapid uptake of Fe and Al by OsYSL1 expression in the root tissues, but a limited secretion into vacuole organelles by OsVIT2, leading to a maintenance of low level of toxicity driven by an enhanced accumulation of glutathione together with downregulation of OsGR expression level. In addition, Fe and Al restriction in the root tissues of genotype RD35 were evident; therefore, crop stress index (CSI) of Fe + Al-treated plants was the maximum, leading to an inhibition of gs (53.6% over the control) and E (49.0% over the control). Consequently, free proline, total phenolic compounds, and ascorbic acid in the leaf tissues of rice under Fe + Al toxicity were significantly increased by 3.2, 1.2, and 1.5 folds over the control, respectively, indicating their functions in non-enzymatic antioxidant defense. Moreover, physiological parameters including leaf temperature (Tleaf) increment, high level of CSI (>0.6), SPAD reduction, photon yield of PSII (PSII) diminution, Pn, gs, and E inhibition in rice genotype IR64 (Fe/Al sensitive) under Fe + Al treatment were clearly demonstrated as good indicators of metal-induced toxicity. Our results on Fe- and/or Al-tolerant screening to find out the candidate genotypes will contribute to present screening and breeding efforts, which in turn help increase rice production in the Fe/Al contaminated acid soil under lowland conditions.
 
cGISH in diploid Cynodon species (2n = 2x = 18). Genomic DNA (gDNA, in magenta) from C. incompletus 2x hybridizing to C. dactylon 2x (a), C. nlemfuensis 2x (b), and C. incompletus 2x chromosomes (c). Genomic DNA from C. nlemfuensis 2x hybridizing to C. dactylon 2x (d), C. nlemfuensis 2x (e), and C. incompletus 2x chromosomes (f). Genomic DNA from C. dactylon 2x hybridizing to C. dactylon 2x (g), C. nlemfuensis 2x (h), and C. incompletus 2x chromosomes (i). Scale bar = 10 µm
Double-GISH in diploid Cynodon species (2n = 2x = 18). aCynodon dactylon chromosomes and nucleus: in green C. incompletus gDNA, in magenta C. nlemfuensis gDNA. bCynodon incompletus chromosomes and nucleus: in green C. nlemfuensis gDNA, in magenta C. dactylon gDNA. cCynodon nlemfuensis chromosomes and nucleus: in green C. incompletus gDNA, in magenta C. dactylon gDNA. Arrows indicate a c-metaphase chromosome from C. nlemfuensis and interphase nucleus with the possible cytomolecular marker. Scale bar = 10 µm
cGISH in polyploid Cynodon species. Genomic DNA (gDNA in magenta) from C. dactylon 2x hybridizing to C. dactylon 3x (a), 4x (b), and 5x chromosomes (c). Cynodon dactylon 4x chromosomes hybridized by gDNA of C. incompletus 2x (d), C. nlemfuensis 2x (e), C. dactylon 4x (f), and C. transvaalensis 4x (g). Cynodon transvaalensis 4x chromosomes hybridized by gDNA of C. dactylon 2x (h), C. incompletus 2x (i), C. nlemfuensis 2x (j), C. dactylon 4x (k), and C. transvaalensis 4x (l). Scale bar = 10 µm
A proposal for Cynodon evolution built from this study and published data: genome sizes and ploidy level (Wu 2011; Chiavegatto et al. 2016; Chaves et al. 2019; Zhang et al. 2020; Grossman et al. 2021), karyotype analysis (Chiavegatto et al. 2016, 2019; Chaves et al. 2019), meiosis behavior (Forbes and Burton 1963; Felder 1967; Harlan and De Wet 1969; Gupta and Srivastava 1970; Johnston 1975; Brilman et al. 1982; Dhaliwal and Gupta 2011; Silva et al. 2018), phylogenetic data (Jewell et al. 2012; Peterson et al. 2010; 2015; Muchut et al. 2017), and crossings (Felder 1967; Wu et al. 2004; Wu 2011). Arrows indicate the result of a crossing. Non-dashed lines indicate the best scenario for hybridization. Dashed lines indicate alternative hypotheses for hybridization. WGD, UG, and RG mean whole-genome duplication and unreduced and reduced gametes, respectively
Article
Polyploidy is the main mechanism for chromosome number variation in Cynodon. Taxonomic boundaries are difficult to define and, although phylogenetic studies indicate that some species are closely related, the degree of genomic similarity remains unknown. Furthermore, the Cynodon species classification as auto or allopolyploids is still controversial. Thus, this study aimed to investigate the genomic constitution in diploid and polyploid species using different approaches of genomic in situ hybridization (GISH). To better understand the hybridization events, we also investigated the occurrence of unreduced gametes in C. dactylon diploid pollen grains. We suggest a genomic nomenclature of diploid species as DD, D1D1, and D2D2 for C. dactylon, C. incompletus, and C. nlemfuensis, and DDD2D2 and DD2D1D1 for the segmental allotetraploids of Cynodon dactylon and C. transvaalensis, respectively. Furthermore, an evolutionary proposal was built based on our results and previous data from other studies, showing possible crosses that may have occurred between Cynodon species.
 
Article
The development of scales and the sequence of epidermal layers during snake embryogenesis has been studied by immunofluorescence for the localization of cell adhesion, adherens, and communicating cell junctional proteins. At about 2nd/3rd of embryonic development in snakes the epidermis forms symmetric bumps at the beginning of scale formation, and they rapidly become asymmetric and elongate forming outer and inner surfaces of the very overlapped scales seen at hatching. The dermis separates a superficial loose from a deeper dense part; the latter is joined to segmental muscles and nerves, likely acting on scale orientation during snake movements. N-cam is present in the differentiating epidermis and mesenchyme of forming scales while L-cam is only/mainly detected in the periderm and epidermis. Mesenchymal N-cam is associated with the epidermis of the elongating dorsal scale surface and with the beta-differentiation that occurs in the overlapping outer surface of scales. Beta-catenin and Connexin-43 show a similar distribution, and they are mainly present in the periderm and differentiating suprabasal keratinocytes likely forming an intense connectivity during epidermal differentiation. Beta-catenin also shows nuclear localization in differentiating cells of the shedding and beta-layers at late stages of scale morphogenesis, before hatching. The study suggests that intensification of adhesion and gap junctions allows synchronization of the differentiation of suprabasal cells to produce the ordered sequence of epidermal layers of snake scales, starting from the shedding complex and the beta-layer.
 
Article
Excessive heavy metal (HM) levels in soil have become a source of concern due to their adverse effects on human health and the agriculture industry. Soil contamination by HMs leads to an accumulation of reactive oxygen species (ROSs) within the plant cell and disruption of photosynthesis-related proteins. The response of tobacco lines overexpressing flavodoxin (Fld) and betaine aldehyde dehydrogenase (BADH) to cadmium (Cd) toxicity was investigated in this study. PCR results demonstrated the expected amplicon length of each gene in the transgenic lines. Absolute qRT-PCR demonstrates a single copy of T-DNA integration into each transgenic line. Relative qRT-PCR confirmed overexpression of Fld and BADH in transgenic lines. The maximum quantum yield of photosystem II (Fv/Fm) was measured under Cd toxicity stress and revealed that transgenic lines had a higher Fv/Fm than wild-type (WT) plants. Accumulation of proline, glycine betaine (GB), and higher activity of antioxidant enzymes alongside lower levels of malondialdehyde (MDA) and hydrogen peroxide (H2O2) was indicative of a robust antioxidant system in transgenic plants. Therefore, performing a loop in reducing the ROS produced in the photosynthesis electron transport chain and stimulating the ROS scavenger enzyme activity improved the plant tolerance to Cd stress.
 
Article
Nitric oxide (NO) is a signaling molecule controlling several steps of plant development and defense process under stress conditions. NO-induced alleviation of manganese (Mn) toxicity was investigated on bean plants submitted for 28 days to 500 µM MnCl2. Manganese excess decreased plant dry weight and elongation and increased levels of reactive oxygen species and lipid peroxidation leading to up-regulation of superoxide dismutase, catalase, and ascorbate peroxidase activities. The inhibitory effects of Mn on plant growth were associated to reduction of light-saturated carbon assimilation (Amax), stomatal conductance (gs), and transpiration (E). By contrast, Mn induced significant increase in the apparent quantum yield (ɸ) and light compensation point (LCP). Interestingly, intracellular CO2 (Ci) remains stable under Mn stress. Concomitantly, leaf membrane lipids have drastically reduced under high Mn concentration. After Mn exposition, leaf fatty acids exhibited a significant loss of linolenic acid, accompanied by an accumulation of palmitoleic, stearic, and linoleic acids leading to alteration of lipid desaturation. NO supply reversed Mn toxicity as evidenced by enhancement of growth biomass and recovery of Amax, E, ɸ, and LCP. Similarly, NO addition has positive effects on leaf lipid content and composition leading to restoration of lipid unsaturation. The modulation of fatty acid composition can be a way to reduce leaf membrane damages and maintain optimal photosynthesis and plant growth. Despite the absence of enough evidences in how NO is involved in lipid and photosynthesis recovery under Mn stress conditions, it is assumed that NO beneficial effects are attributable to NO/Mn cross-talk.
 
Article
Tetradesmus is a microalgal genus with biotechnological potential due to its rapid production of biomass, which is plenty in proteins, carbohydrates, lipids, and bioactives. However, its morphology and physiology need to be determined to guide better research to optimize the species cultivation and biocompounds processing. Thus, this study describes the biochemistry and morphology of the strain Tetradesmus obliquus BR003, isolated from a sample of freshwater reservoirs in a Brazilian municipality. In the T. obliquus BR003 dry biomass, we identified 61.6% unsaturated fatty acids, and 3.4% saturated fatty acids. Regarding other compounds, 28.50 ± 1.47 g soluble proteins/100 g, 0.14 ± 0.009 g carotenoids/100 g, 0.76 ± 0.013 g chlorophyll a/100 g, and 0.42 ± 0.015 g chlorophyll b/100 g with a chlorophyll a/b ratio of 1.8 were detected. The main chemical elements found were S, Mg, and P. The cells of BR003 were elliptically curved at the ends and without appendages. Histochemical tests showed carbohydrates distributed in the cytoplasm and pyrenoids, some lipid droplets, and proteins. The cytoplasm is rich in vacuoles, rough endoplasmic reticulum, mitochondria, and chloroplasts. The nucleus has a predominance of decondensed chromatin, and the cell wall has three layers. Chloroplasts have many starch granules and may be associated with a spherical central pyrenoid. To the best of our knowledge, this was the first biochemical description combined with ultrastructural morphological characterization of the strain T. obliquus BR003, grown under standard conditions, to demonstrate specific characteristics of the species.
 
Article
The individual impact of silicon (Si) and nitric oxide (NO) on secondary metabolism in several plant species has been reported, but their combined effect has never been evaluated yet. Therefore, in this study, single and combined impacts of NO and Si on the biosynthesis of rosmarinic acid (RA) and essential oil (EO) content in leaves of Salvia officinalis were investigated under both non-stress and Cu stress conditions. The results indicated that high Cu concentration decreased biomass and the content of polyphenols, but elevated electrolyte leakage, while lower Cu concentrations, especially 200 μM Cu, increased the content of polyphenols, EO, and antioxidant capacity in leaves of S. officinalis. The foliar application of sodium silicate (1 mM Si) and sodium nitroprusside (200 μM SNP as a NO donor) alone and particularly in combination improved shoot dry biomass, restored chlorophyll and carotenoids, increased EO content, the amounts of flavonoids, and phenolic compounds especially RA, and enhanced antioxidant capacity in the leaves of S. officinalis under both non-stress and Cu stress conditions. Copper treatment increased NO content, upregulated expression of PAL, TAT, and RAS genes, and enhanced phenylalanine ammonia-lyase activity in the leaves, which were responsible for improving the production of phenolic compounds, particularly rosmarinic acid. Foliar spraying with Si and SNP intensified these attributes. All responses were more pronounced when NO and Si were simultaneously applied under Cu stress. These findings suggest that NO and Si synergistically modulate secondary metabolism through upregulation of related gene expression and enzyme activities under both non-stress and Cu stress conditions.
 
Article
The growing zone (GZ) of the unicellular coenocytic sporangiophore of Phycomyces blakesleeanus represents the site of stimulus reception (light, gravity, gas) and stimulus response, i.e., local modulations of the elongation growth, which may result, in dependence of the stimulus direction, in tropic bending. Until now, evidence for a possible participation of the columella in sensory reception is absent. We confirm with light microscopy earlier studies that show that the GZ and the columella are not separated by a membrane or cell wall, but rather form a spatial continuum that allows free exchange of cytoplasm and organelle transport. Evidence is presented that the columella is responsive to external stimuli. Columellae, from which spores and sporangial cell wall had been removed, respond to exogenous auxin with a local depolarization of the membrane potential and an increased growth rate of the GZ. In contrast, auxin applied to the GZ causes a decrease of the growth rate irrespective of the presence or absence of sporangia. The response pattern is specific and relevant for the sensory reception of Phycomyces, because the light-insensitive mutant C148carAmadC, which lacks the RAS-GAP protein MADC, displays abnormal IAA sensitivity and membrane depolarization. We argue that the traditional concept of the GZ as the only stimulus-sensitive zone should be abandoned in favor of a model in which GZ and columella operate as a single entity capable to orchestrate a multitude of stimulus inputs, including auxin, to modulate the membrane potential and elongation growth of the GZ.
 
Article
Maxillaria crassifolia (Lindl.) Rchb.f. belongs to the polyphyletic genus Maxillaria Ruiz & Pav., which currently is the subject of several taxonomic research. There are conflicting descriptions of megasporogenesis, megagametogenesis, and embryogenesis in orchids from the tribe Cymbidieae, in general, and in the genus Maxillaria, in particular. In the present report, all stages of embryonic development of M. crassifolia were examined using confocal fluorescence microscopy. Some features of the development of the ovule and embryo, which distinguish M. crassifolia from other species of the tribe Cymbidieae were identified. The T-shaped arrangement of megaspores is formed by dividing the micropylar megaspore of the dyad. The megagametophyte develops according to the modified Polygonum-type with an unstable number of nuclei in the embryo sacs. The nucleus of the central cell varies in composition and may include unfused micropylar and chalazal nuclei and daughter nuclei formed during their division. The sequence of embryonal divisions is strictly structured. A special variant of embryogenesis, the Cymbidium-type Maxillaria-variant, has been described. Its characteristic features are the strictly apical nature of embryonic divisions, the absence of basal cell (cb) division, the formation of one to three pairs of tubular suspensor cells, and the localization of all suspensor cells within the inner integument.
 
Article
Drought has been identified as a major factor restricting maize productivity worldwide, especially in the rainfed areas. The objective of the present study was to investigate the physiological adaptation strategies and sugar-related gene expression levels in three maize (Zea mays L.) genotypes with different drought tolerance abilities (Suwan4452, drought tolerant as a positive check; S7328, drought susceptible as a negative check; Pac339, drought susceptible) at the seedling stage. Ten-day old seedlings of maize genotypes were subjected to (i) well-watered (WW) or control and (ii) water-deficit (WD) conditions. Leaf osmotic potential of cv. S7328 under WD was significantly decreased by 1.35–1.45 folds compared with cv. Pac339 under WW, whereas it was retained in cv. Suwan4452, which utilized total soluble sugars as the major osmolytes for maintaining leaf greenness, Fv/Fm, ΦPSII, and stomatal function (Pn, net photosynthetic rate; gs, stomatal conductance; and E, transpiration rate). Interestingly, sucrose degradation (65% over the control) in cv. Pac339 under WD was evident in relation to the downregulation of the ZmSPS1 level, whereas glucose enrichment (1.65 folds over the control) was observed in relation to the upregulation of ZmSPS1 and ZmSUS1. Moreover, CWSI (crop water stress index), calculated from leaf temperature of stressed plants, was negatively correlated with E, gs, and Pn. Overall, growth characteristics, aboveground and belowground parts, in the drought-susceptible cv. Pac339 and cv. S7328, were significantly decreased (> 25% over the control), whereas these parameters in the drought-tolerant cv. Suwan4452 were unaffected. The study validates the use of leaf temperature, CWSI, Pn, gs, and E as sensitive parameters and overall growth characters as effective indices for drought tolerance screening in maize genotypes at the seedling stage. However, further experiments are required to validate the results observed in this study under field conditions.
 
Article
In unicellular algae with a single chloroplast, two mechanisms coordinate cell and chloroplast division: the S phase–specific expression of chloroplast division genes and the permission of cell cycle progression from prophase to metaphase by the onset of chloroplast division. This study investigated whether a similar mechanism exists in a unicellular alga with multiple chloroplasts using the glaucophyte alga Cyanophora sudae, which contains four chloroplasts (cyanelles). Cells with eight cyanelles appeared after the S phase arrest with a topoisomerase inhibitor camptothecin, suggesting that the mechanism of S phase–specific expression of cyanelle division genes was conserved in this alga. Inhibition of peptidoglycan synthesis by β-lactam antibiotic ampicillin arrested cells in the S–G2 phase, and inhibition of septum invagination with cephalexin resulted in cells with two nuclei and one cyanelle, despite inhibition of cyanelle division. This indicates that even in the unicellular alga with four chloroplasts, the cell cycle progresses to the M phase following the progression of chloroplast division to a certain division stage. These results suggested that C. sudae has two mechanisms for coordinating cell and cyanelle division, similar to the unicellular algae with a single chloroplast.
 
Article
Microbial volatile organic compounds (mVOCs) play important roles in inter- and intra-kingdom interactions, and they are also important as signal molecules in physiological processes acting either as plant growth-promoting or negatively modulating plant development. We investigated the effects of mVOCs emitted by PGPR vs non-PGPR from avocado trees (Persea americana) on growth of Arabidopsis thaliana seedlings. Chemical diversity of mVOCs was determined by SPME–GC–MS; selected compounds were screened in dose–response experiments in A. thaliana transgenic lines. We found that plant growth parameters were affected depending on inoculum concentration. Twenty-six compounds were identified in PGPR and non-PGPR with eight of them not previously reported. The VOCs signatures were differential between those groups. 4-methyl-2-pentanone, 1-nonanol, 2-phenyl-2-propanol and ethyl isovalerate modified primary root architecture influencing the expression of auxin- and JA-responsive genes, and cell division. Lateral root formation was regulated by 4-methyl-2-pentanone, 3-methyl-1-butanol, 1-nonanol and ethyl isovalerate suggesting a participation via JA signalling. Our study revealed the differential emission of volatiles by PGPR vs non-PGPR from avocado trees and provides a general view about the mechanisms by which those volatiles influence plant growth and development. Rhizobacteria strains and mVOCs here reported are promising for improvement the growth and productivity of avocado crop.
 
Article
Picrorhiza kurroa Royle ex Benth is a valuable medicinal herb of North-Western Himalayas due to presence of two major bioactive compounds, picroside-I and picroside-II used in the preparation of several hepatoprotective herbal drugs. These compounds accumulate in stolons/rhizomes; however, biosynthesized in different organs, viz., picroside-I in shoots and picroside-II in roots. As of today, no information exists on what transporters are transporting these metabolites from shoots and roots to the final storage organ, stolon, which ultimately transforms into rhizome. The ATP-binding cassette (ABC) transporters are reported to transport majority of secondary metabolites, including terpenoids in plants, therefore, we mined P. kurroa transcriptomes to identify and shortlist potential candidates. A total of 99 ABC transporter-encoding transcripts were identified in 3 differential transcriptomes, PKSS (shoots), PKSTS (stolons), and PKSR (roots) of P. kurroa, based on in silico comparative analysis and transcript abundance. 15 of these transcripts were further validated for their association using qRT-PCR in shoots, roots and stolon tissues in P. kurroa accessions varying for picroside-I and picroside-II contents. Organ-specific expression analysis revealed that PkABCA1, PkABCG1, and PkABCB5 had comparatively elevated expression in shoots; PkABCB2 and PkABCC2 in roots; PkABCB3 and PkABCC1 in stolon tissues of P. kurroa. Co-expression network analysis using ABC genes as hubs further unravelled important interactions with additional components of biosynthetic machinery. Our study has provided leads, first to our knowledge as of today, on putative ABC transporters possibly involved in long distance and local transport of picrosides in P. kurroa organs, thus opening avenues for designing a suitable genetic intervention strategy.
 
Article
Elaborate petals are highly diverse in morphology, structure, and epidermal differentiation and play a key role in attracting pollinators. There have been few studies on the elaborate structure of petals in the tribe Isopyreae (Ranunculaceae). Seven genera in Isopyreae (Aquilegia, Semiaquilegia, Urophysa, Isopyrum, Paraquilegia, Dichocarpum, and Leptopyrum) have petals that vary in morphology, and two genera (Enemion and Thalictrum) have no petals. The petals of nine species belonged to 7 genera in the tribe were studied to reveal their nectary structure, epidermal micromorphology and ancestral traits. The petal nectaries of Isopyreae examined in this study were located at the tip of spurs (Aquilegia yabeana and A. rockii), or the bottom of shallow sacs (Semiaquilegia adoxoides, Urophysa henryi, Isopyrum manshuricum, and Paraquilegia microphylla), a cup-shaped structure (Dichocarpum fargesii) and a bilabiate structure (Leptopyrum fumarioides). The petal nectary of eight species in Isopyreae (except A. ecalcarata) was composed of secretory epidermis, nectary parenchyma, and vascular tissues, and some sieve tubes reached the secretory parenchyma cells. Among the eight species with nectaries examined in the present study, A. yabeana had the most developed nectaries, with 10–15 layers of secretory parenchyma cells. The epidermal cells of mature petals of the nine species were divided into 11 types. Among these 11 types, there were two types of secretory cells and two types of trichomes. Aquilegia yabeana and A. rockii had the highest number of cell types (eight types), and I. manshuricum and L. fumarioides had the lowest number of cell types (three types). Aquilegia ecalcarata had no secretory cells, and the papillose conical polygonal secretory cells of D. fargesii were different from those of the other seven species with nectaries. Trichomes were found only in Aquilegia, Semiaquilegia, Urophysa, and Paraquilegia. The ancestral mode of nectar presentation in Isopyreae was petals with hidden nectar (70.58%). The different modes of nectar presentation in petals may reflect adaptations to different pollinators in Isopyreae.
 
Article
One of the classes of the plant developmental programmed cell death (PCD) is vacuolar cell death or autolysis. The results of the transmission electron microscope (TEM) studies indicated that this type of PCD occurs during the petal senescence of Antirrhinum majus “Legend White” flowers. The major hallmarks of the process related to the ultrastructure of the cells involved chloroplast degradation, vacuolation, chromatin condensation, cell wall swelling, degradation of Golgi apparatus, protoplasmic shrinkage, degradation of the endoplasmic reticulum, nuclear fragmentation, rupture of tonoplast, and plasma membrane. Macroautophagy and microautophagy processes were also clearly observed during vacuole formation. As in yeasts, in the present study, Golgi apparatus became autophagosome-like structures during degradation that had autophagy activity and then disappeared. Our results revealed a type of selective microautophagy, piecemeal microautophagy of the nucleus (PMN), in nuclear degradation during PCD of petals that has not previously been reported in plants. Moreover, vesicular structures, such as paramural and multilamellar bodies, were observed in some stages.
 
Phylogenetic relationship of MusaF3′5′H-1. Diverse protein sequences used to generate a neighbour joining tree with MusaF3′5′H-1 (GSMUA_Achr2T00210_001) were Vitis vinifera VvF3′5′H (BAE47007.1), Petunia x hybrida PhF3′5′H (AUI38391.1), Antirrhinum majus AmF3′H (ABB53383.1), Oryza sativa OsF3′5′H (XP_015633235.1), Fragaria x ananassa FaF3′H (BAL63027.1), Arabidopsis thaliana AtF3′H (NP_196416.1), Populus trichocarpa PtF3′5′H (XP_002314004.2), Elaeis guineensis EgF3′5′H (XP_010922086.1), Phoenix dactylifera PdF3′5′H (XP_038980681.1), Ananas comosus AcF3′5′H (CAD1833806.1), Solanum lycopersicum SlF3′5′H (NP_001234840.2) and Triticum dicoccoides TdF3′5′H (XP_037455343.1). MusaF3′5′H-1 shows close association with F3′5′H of EgF3′5′H and PdF3′5′H
Amino acid sequence analysis of MusaF3′5′H-1. Conserved motifs in MusaF3′5′H-1 sequence were detected after alignment of MusaF3′5′H-1 sequence with F3′5′H sequences of Vitis vinifera VvF3′5′H (BAE47007.1), Oryza sativa OsF3′5′H (XP_015633235.1) and Petunia x hybrida PhF3′5′H (AUI38391.1). Conserved amino acid residues among different sequences are shaded and different motifs detected in MusaF3′5′H-1 sequence such as proline-rich region, oxygen-binding motif, EXXR motif, heme-binding domain, domain for hydroxylation activity (CR1) and substrate recognition sites marked as SRS1-SRS6 are boxed
Quantitative RT-PCR analysis and identification of cis-elements. Transcript abundance of MusaF3'5'H-1 in banana plants was detected after exposure of plants to signaling molecules such as (A) salicylic acid (2 mM), (B) methyl jasmonate (200 μM), (C) ABA (100 μM), and under influence of (D) high salinity and (E) drought. Fold value change over control expression in leaves of banana plants exposed to stress stimuli was calculated at different time points and expressed as mean ± SD from three replications. The different bar colors denote expression of MusaF3'5'H-1 in banana plants at various time points as indicated in the graphs. (F) PLACE software was used to predict presence of various cis-elements in a 936 bp upstream regulatory region of MusaF3'5'H-1 (PMusaF3'5'H-1). Important cis-elements related to stress responses, guard cell preferred expression and responses to phytohormone applications are boxed in color. The color coding of various boxes is indicated near the promoter sequence
Generation of transgenic tobacco lines and tissue-specific activity of PMusaF3'5'H-1. (A) T-DNA map of binary vector used to transform tobacco leaf discs. This T-DNA harbours PMusaF3'5'H-1 cloned upstream of GUS in pCAMBIA1301 and expression cassette of hygromycin resistance gene wherein hpt-II is placed under control of CaMV35S promoter. (B, C) Leaf discs of tobacco transformed with T-DNA harbouring PMusaF3'5'H-1-GUS showed emergence of putatively transformed shoots on shoot development medium supplemented with hygromycin. (D) Individual transgenic lines were cultured in vitro and rooted for further analysis. (E) T-DNA integration in the genome of transgenic lines was confirmed by PCR amplification of hpt-II from genomic DNA of transgenic lines, L1–L3. M: 1 kb DNA ladder; WT: PCR product from genomic DNA of wild-type tobacco plants. (F) GUS histochemical staining observed in leaf of L1. (G–I) Close-up of (F) showing GUS expression in guard cells of leaf of transgenic line L1. (J) GUS staining in stem of transgenic line L1. (K) Root of L1 after overnight incubation in GUS staining solution. (L) GUS expression under control of PMusaF3'5'H-1 in shoot of transgenic line L2. (M–O) Close-up of (L) showing the expression of GUS due to activity of PMusaF3'5'H-1 in guard cells of leaves. (P) GUS staining in stem of transgenic line L2. (Q) Root of transgenic line L2 after overnight incubation in GUS staining solution. (R) GUS staining in shoots of transgenic line L3. (S–U) Close-up of leaf in (R) showing expression in guard cells and vascular elements. (V) Activity of PMusaF3'5'H-1-GUS in stem and root of transgenic line L3 as demonstrated by GUS histochemical staining
Response of transgenic lines to stress applications. GUS histochemical staining in shoots of transgenic tobacco harbouring PMusaF3′5′H-1-GUS under the influence of salicylic acid, methyl jasmonate and abscisic acid and after exposure to high salinity and drought. (A–F) Representative pictures of GUS staining profile in transgenic shoot under (A) control conditions and after (B) 1 h, (C) 3 h, (D) 6 h, (E) 12 h and (F) 24 h of application of salicylic acid. (G–K) Representative pictures of GUS histochemical staining in transgenic shoots after (G) 1 h, (H) 3 h, (I) 6 h, (J) 12 h and (K) 24 h of application of abscisic acid. (L–P) Demonstrative pictures of GUS expression in shoots of transgenic lines under application of methyl jasmonate after (L) 1 h, (M) 3 h, (N) 6 h, (O) 12 h and (P) 24 h of hormonal application. (Q–U) Representative pictures of GUS expression in transgenic shoots harbouring PMusaF3′5′H-1 -GUS after (Q) 1 h, (R) 3 h, (S) 6 h, (T) 12 h and (U) 24 h of high-salinity treatment of transgenic lines. (V–Z) Demonstrative pictures of GUS staining in transgenic lines after (V) 1 h, (W) 3 h, (X) 6 h, (Y) 12 h and (Z) 24 h of drought commencement. (α–γ) GUS activities in leaves of transgenic lines at different time points after application of phytohormones (α) salicylic acid (2 mM), (β) abscisic acid (100 μM) and (γ) methyl jasmonate (200 μM MeJA). Total protein was isolated from leaves at different time points after application of phytohormone and MUG assay was conducted to determine the GUS activity [(pmol 4-MU/min)/μg protein] and data was visualized as fold change in GUS activity over the activity in control conditions
Article
Generation of crops with broad-spectrum tolerance to biotic and abiotic stress conditions depends upon availability of genetic elements suitable for varied situations and diverse genotypes. Here, we characterize the 5′-upstream regulatory region of flavonoid 3′5′-hydroxylase-1 (F3′5′H-1) gene from banana and analyzed its tissue-specific and stress-mediated activation in genetic background of tobacco plants. MusaF3′5′H-1 is a stress-responsive gene as its expression is induced in banana after application of salicylic acid and methyl jasmonate while its transcript levels were drastically reduced in response to drought, high salinity and abscisic acid. PMusaF3′5′H-1 harbours cis-elements associated with stress conditions and those responsible for tissue-specific expression. Transgenic lines harbouring PMusaF3′5′H-1-GUS displays strong GUS expression in guard cells of stomata indicating guard cell preferred activity of PMusaF3′5′H-1 while its activity was undetectable in roots. Drought and high salinity induce strong expression of GUS in transgenic tobacco lines and exposure to abscisic acid, salicylic acid and methyl jasmonate revealed distinct profiles of GUS expression in transgenic lines confirming involvement of F3′5′H-1 in plant stress responses. Fluorescent β-galactosidase assay revealed induction profiles of PMusaF3′5′H-1 at different time points in transgenic lines exposed to salicylic acid and abscisic acid while strong suppression in GUS expression was observed after application of methyl jasmonate. The guard cell preferred activity of PMusaF3′5′H-1 and stress-mediated expression profiles of MusaF3′5′H-1 indicated the suitability of PMusaF3′5′H-1 for generating stress-enduring crops and analyzing guard cell functions.
 
Article
Heavy waterlogging and high temperatures occur frequently in North China, yet the effects of changing environments on photochemical reactions and carbon metabolism have not been described in ginger. To determine the impact of waterlogging and high temperature on ginger, in this study, treatment groups were established as follows: (a) well-watered at ambient temperature (28 °C/22 °C) (CK), (b) well-watered at moderate temperature (33 °C/27 °C) (MT), (c) well-watered at high temperature (38 °C/32 °C) (HT), (d) waterlogging at ambient temperature (CK-WL), (e) waterlogging at moderate temperature (MT-WL), and (f) waterlogging at high temperature (HT-WL) during the rhizome growth period. We analyzed the effect of different treatments on the photosynthetic performance of ginger. Here, our results showed that waterlogging and high temperature irreversibly decreased the photosynthetic pigment content, increased the ROS content of leaves, inhibited leaf carbon assimilation and limited PSII electron transport efficiency. In addition, waterlogging in isolation and high temperature in isolation affected photosynthesis to varying degrees. Taken together, photosynthesis was more sensitive to the combined stress than to the single stresses. The results of this research provide deep insights into the response mechanisms of crop photosynthesis to different water and temperature conditions and aid the development of scientific methods for mitigating plant damage over time.
 
Article
Scilla siberica subsp. armena is known as Siberian Squill which is naturally distributed in Lebanon-Syria, Transcaucasus, and Turkey. It is a perennial bulbous plant with a short vegetation period from March to May. In this study, the antimicrobial, antioxidant, α-amylase, and α-glucosidase inhibitory activities of corm, leaf, and flower methanolic extracts were examined. The anatomy and morphology of the plant organs were investigated by light and electron microscopes. The anatomy of S. siberica subsp. armena was investigated for the first time by electron microscopy in this study. The corm, leaf, and flower methanolic extracts were assessed against E. coli ATCC 8739, S. aureus ATCC 6538, B. subtilis ATCC 19,659, C. albicans ATCC 10,231, C. krusei ATCC 14,243, and C. tropicalis ATCC 750. Among the extracts, that obtained from the blue pollen showed the best antimicrobial activity against C. tropicalis ATCC 750 strain with a MIC value of 312.5 µg/mL. The highest phenolic content was determined in leaf extract with 53.59211 µg GAE/mg extract value. The extract showed the best anti-lipid peroxidation activity with 376.69 µg/mL value. Using DPPH· and ABTS·+ tests, it was determined that the flower and leaf extracts have the best activity (IC50 = 756.13 μg/mL and IC50 = 94.07 μg/mL, respectively). The flower extract exhibited α-glucosidase inhibitory activity with the IC50 value of 5239 µg/mL. Based on the presented results of the in vitro antimicrobial, antioxidant, and antidiabetic activities of the S. siberica subsp. armena, we suggest that natural compounds from S. siberica subsp. armena are of potential use for the improvement of an antidiabetic, antioxidant, and antimicrobial agent. Graphical abstract
 
Article
Since the discovery of the anticancer drugs vinblastine and vincristine, Catharanthus roseus has been intensively studied for biosynthesis of several terpene indole alkaloids (TIAs). Due to their low abundance in plant tissues at a simultaneously high demand, modes of production alternative to conventional extraction are mandatory. Plant cell fermentation might become one of these alternatives, yet decades of research have shown limited success to certain product classes, leading to the question: how to preserve the intrinsic ability to produce TIAs (metabolic competence) in cell culture? We used the strategy to use the developmental potency of mature embryos to generate such strains. Two cell strains (C1and C4) from seed embryos of Catharanthus roseus were found to differ not only morphologically, but also in their metabolic competence. This differential competence became manifest not only under phytohormone elicitation, but also upon feeding with alkaloid pathway precursors. The more active strain C4 formed larger cell aggregates and was endowed with longer mitochondria. These cellular features were accompanied by higher alkaloid accumulation in response to methyl jasmonate (MeJA) elicitation. The levels of catharanthine could be increased significantly, while the concurrent vindoline branch of the pathway was blocked, such that no bisindole alkaloids were detectable. By feeding vindoline to MeJA-elicited C4 cells, vincristine became detectable; however, only to marginal amounts. In conclusion, these results show that cultured cells are not “de-differentiated”, but can differ in metabolic competence. In addition to elicitation and precursor feeding, the cellular properties of the “biomatter” are highly relevant for the success of plant cell fermentation.
 
Article
Aerenchyma formation plays an important role in the survival of Potamogeton perfoliatus in submerged environment. To understand the regulatory role of reactive oxygen species (ROS) and caspase 3-like protein signaling molecules in aerenchyma formation, we investigated the effects of exogenous NADPH oxidase inhibitor (diphenyleneiodonium chloride, DPI), catalase inhibitor (3-amino-1,2,4-triazole, AT), and caspase-3-like protein inhibitor (AC-DEVD-CHO, DEVD) on morphological and physiological characteristics and aerenchyma formation in P. perfoliatus. The results showed that after DPI treatment, caspase-3-like protein activity decreased, ROS-related enzyme activities increased, and H2O2 content decreased, thereby inhibiting aerenchyma formation. When the concentration of DPI was approximately 1 μmol/L, the inhibitory effect was the most obvious. On the contrary, after the AT treatment, caspase-3-like protein activity increased, ROS-related enzyme activities decreased, and the H2O2 content increased, ultimately promoting aerenchyma formation, and the promotion was the most obvious under treatment with approximately 500 μmol/L AT. After DEVD treatment, the inhibition of vegetative growth caused by DPI or AT treatment was alleviated, significantly reducing caspase-3-like activity and inhibiting aerenchyma development. The results of this study show that ROS has a positive regulatory effect on aerenchyma formation, and caspase-3-like protein is activated to promote ROS-mediated aerenchyma formation. This experiment provides a new theoretical basis for further exploration of the signal transduction effects of ROS and caspase-3-like protein in plant cells and their roles in plant development.
 
a The procedure of measuring the effect of K⁺, EGTA, and/or Ca²⁺ on the hydraulic resistance (Lp⁻¹) of a Chara cell. First, Lp⁻¹ of the cell was measured by means of the transcellular osmosis induced by 0.1 M sorbitol (I). Then the cell was treated with KCl for 1200 s, or with EGTA for 600 s and subsequently with CaCl2 for 600 s (II). After each treatment, the cell was washed with water twice and the Lp⁻¹ after treatment was measured (III). b Preparation of twin cells from an internodal cell by ligation and cutting. The cell was bathed in 0.2 M sorbitol to lower the cell turgor and ligated with strips of polyester thread at two loci of the cell (arrows). The sorbitol solution was discarded and water was added to the cell to recover the cell turgor. The cell was ligated again to prevent a possible leak from the knots. After the ligation, the cell was cut between the ligated knots. The resulted cell halves are, for convenience, called as a-cell and b-cell, respectively
Hydraulic resistances of Chara cells (Lp⁻¹) before (H2O) and after (K⁺) treatment with 10 mM KCl. Each bar shows the mean ± SD (n = 10)
Hydraulic resistances of the cell (Lp⁻¹), the cell wall (Lpw⁻¹), and the membrane (Lpm⁻¹) of Chara cells before (open column) and after (closed column) treatment with K⁺ or EGTA. a Cells were treated with 100 mM KCl. b Cells were treated with 5 mM EGTA. Twin cells, a-cell and b-cell, were used. Lp⁻¹ was measured in the a-cell before and after treatment with 100 mM KCl for 1200 s or with 5 mM EGTA for 600 s. Using the cell wall tube prepared from the b-cell, Lpw⁻¹ was measured before and after treatment of the cell wall tube with 100 mM KCl for 1200 s or with 5 mM EGTA for 600 s. Lpm⁻¹ was calculated from Lp⁻¹ and Lpw⁻¹ using Eq. (1). Each bar shows the mean ± SD (n = 14 (a) and 18 (b)). Significant differences (p < 0.05) between “before” and “after” treatments are indicated by asterisks
Relative hydraulic resistance of Chara cells (Lp⁻¹) before (H2O) and after treatment of cells first with 100 mM KCl (K⁺) and then with CaCl2 (Ca²⁺) or MgCl2 (Mg²⁺). a 70 mM CaCl2 was used in the last treatment. b 1 mM CaCl2 was used in the last treatment. c 70 mM MgCl2 was used in the last treatment. Each bar shows the mean ± SD (n = 7 (a), 11 (b), and 5 (c)). Significant differences (p < 0.05) vs “before” treatment (H2O) are indicated by asterisks
Relative hydraulic resistance of Chara cells (Lp⁻¹) or the cell wall (Lpw⁻¹). aLpw⁻¹ before (H2O) and after treatment of cells with 100 mM KCl and subsequently with 70 mM CaCl2 (K⁺-Ca²⁺). Twin cells were used. With a-cells the control Lpw⁻¹ was measured and with b-cells Lpw⁻¹ after successive treatments of cells with K⁺ and Ca²⁺ was measured. bLpw⁻¹ before (H2O) and after treatment of isolated cell wall tubes first with 100 mM KCl (K⁺) and subsequently with 70 mM CaCl2 (Ca²⁺). c Cells were sequentially plasmolyzed and deplasmolyzed, and then Lp⁻¹ was measured in H2O (H2O) and then after successive treatment of cells with 100 mM KCl (K⁺) and 70 mM CaCl2 (Ca²⁺). Each bar shows the mean ± SD (n = 4 (a), 15 (b), and 6 (c)). Significant differences (p < 0.05) vs “before” treatment (H2O) are indicated by asterisks
Article
The hydraulic resistance (the reciprocal of the hydraulic conductivity Lp) Lp⁻¹ was measured in cells of Chara corallina by the method of transcellular osmosis. Treatment of cells with 100 mM KCl decreased Lp⁻¹ significantly. Subsequent treatment of the cells with 70 mM CaCl2 recovered the decreased Lp⁻¹ to the original value. To know whether K⁺ or Ca²⁺/Mg²⁺ acts on the cell wall and/or the membrane, the hydraulic resistances of the cell wall (Lpw⁻¹) and that of the membrane (Lpm⁻¹) were determined in one and the same cell. For this, a pair of cells (twin cells) were made from an internodal cell, one used for measurement of Lp⁻¹ and the other used for the measurement of Lpw⁻¹. From Lp⁻¹ and Lpw⁻¹, Lpm⁻¹ was calculated. Both Lp⁻¹ and Lpw⁻¹ were decreased by K⁺, while Lpm⁻¹ was not affected by K⁺. The same result was obtained with 5 mM EGTA. Lpw⁻¹ was decreased more than it was by KCl but Lpm⁻¹ remained constant after EGTA treatment. The recovery of the K⁺-decreased Lp⁻¹ with Ca²⁺ can be explained exclusively by the recovery of Lpw⁻¹ with Ca²⁺. The Ca²⁺ recovery of Lpw⁻¹ was observed in the intact cell wall but not in the cell wall tube isolated from an internodal cell. The different response to Ca²⁺ between the intact cell wall and the isolated cell wall was discussed in relation to the tension in the cell wall which may be an important factor for the ionic regulation of hydraulic conductivity.
 
Clustered diagram representing (A) the schemes of sustainable development and its relationship in developing (B) the biofortified crops using various approaches. Developing miRNA-based biofortified crops with enhanced nutrient efficiency is a novel paradigm in circumventing Hidden Hunger Challenge
Diagram depicting networking of plant-nutrient-MiRNA interaction during normal and stressed situations. A Plant and nutrient homeostasis: (1) Soil is the main source of nutrients available to the plants. (2) Unidirectional flow of nutrients (red) uptake via xylem vessels. (3) Bi-directional transport of nutrients via phloem sap (red/blue). (4) Nutrients are further stored (red) and remobilized (blue) via vacuoles in plants. B miRNA and nutrient homeostasis: (1) During nutrient deficiency, plant signaling cascade gets active and passes the information utilizing signaling molecules like transcription factors (TF), microRNAs (miRNAs), and ubiquitin-mediated protein (UQ). (2a) Stress signaling can either activate or (2b) repress miRNA target genes. Differential expression of miRNA can result in either activation (3a) of stress-responsive proteins or inactivation (3b) of stress-responsive proteins. Thus, miRNA can act as either a positive regulator or negative regulator during nutrient stress in plants
miRNA regulation in nutrient homeostasis. The miRNAs responsive during nutrient stress have been illustrated above. Single miRNAs have been reported to be involved in multiple nutrient homeostasis and uptake. The multiple targets of miRNAs can be identified and utilized to create biofortified crops. Kong and Yang (2010), Liang et al. (2012), Li et al. (2013), Paul et al. (2015; 2016), Li et al. (2017c), Liang et al. (2017), Dos santos et al. (2019), Bao et al. (2019), Zeng et al. (2019), Liu et al. (2020a, b), Shi et al. (2020), Li et al. (2020), Wang et al. (2021), and Ding et al. (2021)
miRNA-based engineering for nutrient biofortification in bioengineered plants
Article
During their lifespan, sessile plants have to cope with bioavailability of the suboptimal nutrient concentration and have to constantly sense/evolve the connecting web of signal cascades for efficient nutrient uptake, storage, and translocation for proper growth and metabolism. However, environmental fluctuations and escalating anthropogenic activities are making it a formidable challenge for plants. This is adding to (micro)nutrient-deficient crops and nutritional insecurity. Biofortification is emerging as a sustainable and efficacious approach which can be utilized to combat the micronutrient malnutrition. A biofortified crop has an enriched level of desired nutrients developed using conventional breeding, agronomic practices, or advanced biotechnological tools. Nutrient homeostasis gets hampered under nutrient stress, which involves disturbance in short-distance and long-distance cell–cell/cell-organ communications involving multiple cellular and molecular components. Advanced sequencing platforms coupled with bioinformatics pipelines and databases have suggested the potential roles of tiny signaling molecules and post-transcriptional regulators, the microRNAs (miRNAs) in key plant phenomena including nutrient homeostasis. miRNAs are seen as emerging targets for biotechnology-based biofortification programs. Thus, understanding the mechanistic insights and regulatory role of miRNAs could open new windows for exploring them in developing nutrient-efficient biofortified crops. This review discusses significance and roles of miRNAs in plant nutrition and nutrient homeostasis and how they play key roles in plant responses to nutrient imbalances/deficiencies/toxicities covering major nutrients—nitrogen (N), phosphorus (P), sulfur (S), magnesium (Mg), iron (Fe), and zinc (Zn). A perspective view has been given on developing miRNA-engineered biofortified crops with recent success stories. Current challenges and future strategies have also been discussed.
 
Article
Our aim was to unravel the underlying mechanisms of pollen wall development in Cymbalaria muralis. By determining the sequence of developing substructures with TEM, we intended to compare it with that of other taxa and clarify whether physical processes of self-assembly and phase separation were involved. In parallel, we tried to simulate in vitro the substructures observed in Cymbalaria muralis exine development, using colloidal mixtures, to determine whether purely physical self-assembly processes could replicate them. Exine ontogeny followed the main stages observed in many other species and was initiated by phase separation, resulting in heterogeneity of the homogeneous contents of the periplasmic space around the microspore which is filled with genome-determined substances. At every stage, phase separation and self-assembly come into force, gradually driving the substances through the sequence of mesophases: spherical micelles, columns of spherical micelles, cylindrical micelles arranged in a layer, laminate micelles. The final two of these mesophases define the structure of the columellate ectexine and lamellate endexine respectively. Structures obtained in vitro from colloidal mixtures simulated the developing exine structures. Striking columella-like surface of some abnormal tapetal cells and lamella-like structures in the anther medium confirm the conclusion that pattern generation is a feature of colloidal materials, after genomic control on material contents. Simulation experiments show the high pattern-generating capacity of colloidal interactions.
 
Article
The protocol optimized for Petunia hybrida cv. Mirage Rose produced high protoplast yields in 3 out of other 11 cultivars (Damask White, Dreams White, and Opera Supreme White). Factors optimized in the protoplast transfection process showed that the best transfection efficiency (80%) was obtained using 2.5 × 10⁵ protoplast density, 40% polyethylene glycol (PEG) concentration, 10 µg plasmid DNA, and 15 min of transfection time. Assessing the usability of the protocol for other cultivars (Damask White, Dreams White, and Opera Supreme White), a reasonable protoplast transfection efficiency (⁓50%) was observed in the cultivars Dreams White and Opera Supreme White, with lower efficiency (⁓50%) observed in the cv. Damask White. The transient expression of enhanced green fluorescent protein (eGFP) in the nucleus of the transfected protoplasts of all cultivars was confirmed using PCR. This system could be valuable for genome editing of unwanted genes in petunias using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology. Furthermore, it could contribute to other studies on protein subcellular localization, protein–protein interactions, and functional gene expression in the petunias.
 
Article
Wheat (Triticum aestivum L.) is one of the most important food crops in the world. Somatic embryogenesis is an event that is triggered by the presence of auxin hormone for the induction of somatic cells to get converted to embryonic cells. Somatic embryogenesis represents the most important process of totipotency of plants. The role of auxins is widely understood during various stages of embryogenesis including polarity establishment, de-differentiation, re-differentiations, and morphogenesis. Many of the Aux/IAAs and ARFs which are part of auxin signaling components have been identified to play various roles during embryogenesis. In this analysis, the Aux/IAAs and ARFs of T. aestivum have been analyzed at the genome-scale; their structure, function, and evolutionary relatedness were determined. Several Aux/IAAs and ARFs components of T. aestivum have been found to exclusively regulate axis formation, meristem commitment, and other re-differentiation processes by differential expression studies.
 
Self-GISH on metaphase chromosomes and nuclei of Humulus lupulus, Humulus japonicus, and Cannabis sativa. Self-GISH with: male DNA as a probe (a–c), female DNA as a probe (d–f). Sex chromosomes (g–i). Self-GISH on nuclei (j–l)
Self-GISH and cGISH on metaphase chromosomes H. lupulus, H. japonicus, and C. sativa. Self-GISH with different ratio of male DNA as a probe and male DNA as a block (1:50, 1:100, 1:200) (a–c). Results of cGISH on metaphase chromosomes of H. lupulus with H. japonicus DNA as probe (d), on metaphase chromosomes of H. japonicus with H. lupulus DNA as probe (e), on metaphase chromosomes of C. sativa with H. japonicus DNA as probe (f)
Article
In plants, dioecy is relatively rare, and it involves sex chromosome systems that often developed independently over time. These characteristics make dioecious plants an attractive model to study sex chromosome evolution. To clarify the patterns of plant sex chromosome evolution, studies should be performed on a wide range of dioecious species. It is interesting to study the sex chromosomes in related species that evolved during a long period of independent sex chromosome evolution. The Cannabaceae family includes three dioecious species with heteromorphic sex chromosomes. Cannabis sativa and Humulus lupulus use the XX/XY chromosome system, whereas Humulus japonicus contains multiple sex chromosomes (XX/XY1Y2). To better understand sex chromosome evolution and the level of genomic divergence of these three related species, we undertook self-GISH and comparative GISH analyses. The self-GISH allowed visualization of the Y chromosomes of C. sativa, H. lupulus, and H. japonicus. The self-GISH signal was distributed along the entire Y chromosome, excluding the pseudo-autosomal region (PAR). Our results indicate that the male-specific region of the Y chromosome (MSY) spans the overwhelming majority of the Y chromosomes of all three species studied. The self-GISH results reveal the accumulation of repetitive DNA sequences in the Y chromosomes of all three species studied. This sequences presented in autosomes and/or chromosome X at a lower copy number than in Y. In comparative GISH experiments where the probe DNA of one species was applied to another species, a weak signal was exclusively detected on 45S rDNA sites, indicating a high level of genomic differentiation of the species used in this study. We demonstrate small PAR size and opposing large MSY and its positions on Y chromosomes. We also found that these genomes are highly differentiated. Furthermore, the data obtained in this study indicate a long period of independent and advanced sex chromosome evolution. Our study provides a valuable basis for future genomic studies of sex and suggests that the Cannabaceae family offers a promising model to study sex chromosome evolution.
 
Plant hormone concentration in germinating spruce pollen. CKs (a) and ABA (b) concentration in Picea pollen cultivated in vitro for 2, 9, or 14 h. ZR, zeatin riboside; ZN, zeatin nucleotide. Values are presented as the means: SEM ± standard error. Asterisks indicate a significant difference between the means (*p < 0.05, **p < 0.01). Data on hormone concentration were expressed per unit of the initial dry weight of pollen subjected to cultivation
Immunolocalization of zeatin and ABA in spruce pollen grains. Confocal images of zeatin (blue, a–c, g, h) and ABA (magenta, d–f, i) immunolocalization in Picea pollen grains germinating in vitro. Pollen suspension was incubated for 2 h (a–f) and 9 h (g–i). AC, antheridial cell; acn, antheridial cell nucleus; PC, prothallial cell; TC, tube cell; tcn, tube cell nucleus; some plastids with high hormone level are marked by arrowheads. Scale bar, 20 µm (a–f), 30 µm (g–i)
Immunolocalization of zeatin and ABA in spruce pollen tubes. Confocal images of zeatin (blue, a, b) and ABA (magenta, c-e) immunolocalization in Picea pollen tubes growing in vitro. Pollen suspension was incubated for 14 h. AC, antheridial cell; acn, antheridial cell nucleus; PC, prothallial cell; TC, tube cell; tcn, tube cell nucleus; some annular plastids are marked by arrowheads. Scale bar, 20 µm
Averaged sums of pixel fluorescence intensities per each pollen grain. Fluorescence intensity of Picea pollen grains treated with antibodies to CKs (a) and ABA (b) registered with confocal microscope and estimated with the ImageJ software. Asterisks indicate a significant difference between the means (*p < 0.05, **p < 0.01)
Immunolocalization of zeatin and ABA in tobacco pollen. Confocal images of zeatin (blue, a–c) and ABA (magenta, d–f) immunolocalization in Nicotiana tabacum pollen grains germinating in vitro. Pollen suspension was incubated for 10 (a, d), 30 (b, e), and 50 (c, f) min. GC, generative cell; gcn, generative cell nucleus; vcn, vegetative cell nucleus; pollen tube tips or aperture areas with high hormone levels are marked by arrowheads. Scale bar, 30 µm
Article
We used the enzyme-linked immunosorbent assay (ELISA) to assess the level of endogenous hormones in spruce pollen, and immunolocalization and confocal microscopy to study hormone localization in spruce and tobacco pollen. During pollen activation, the levels of ABA, zeatin, and its riboside significantly decreased. After the initiation of polar growth, the levels of all cytokinins increased sharply; ABA level also increased. In dormant spruce pollen grains, zeatin and ABA were localized uniformly throughout the cytoplasm. Zeatin was not detected in the nuclei, and the antheridial cell showed higher levels than the vegetative cell; ABA signal was detected in the cytoplasm and the nuclei. In germinating pollen, both hormones were detected mainly in plastids. The similar pattern was found in growing pollen tubes; signal from ABA also had a noticeable level in the cytosol of the tube cell, and was weaker in the antheridial cell. Zeatin fluorescence, on the other hand, was more pronounced in the antheridial cell. In non-germinated grains of tobacco, zeatin was localized mainly in organelles. ABA in dormant pollen grains demonstrated uniform localization, including the nuclei and cytoplasm of both cells. After germination, zeatin was accumulated in the plasmalemma or cell wall. ABA signal in the cytoplasm decreased; in the nuclei, it remained high. In growing tubes, the strongest zeatin and ABA signals were observed at the plasma membrane. The differences in ABA and cytokinin localization between species and dynamic changes in their level in spruce pollen highlight the key spatial and temporal parameters of hormonal regulation of gymnosperm pollen germination.
 
Suitable inflorescence and flower buds for microspore culture. a Donor florescence. b Suitable buds
Embryos yield of flowering Chinese cabbage genotypes cultivated in NLN-13 medium supplemented with different concentrations of CSN and embryo development. a Embryos from 19AY05, 0.1 mg· L⁻¹ sodium nitrophenol. b Embryos from 19AY15, 0.01 mg· L⁻¹ 5-nitrophenol. c Embryos transferred to MS medium. d Embryos developed on MS medium after 5 days. e Regenerated plantlet. f Plantlet transplanted to nutrient soil
Identification of ploidy level of regenerated plants, morphological characters of flowers among different levels of ploidy and self-compatibility of doubled haploid (DH) lines from 19AY05. a Flowers of different ploidy level plants (haploid, doubled haploid, polyploid, from let to right). b DNA content distribution of haploid plants. c DNA content distribution of doubled haploid plants. d DNA content distribution of polyploid plants. e DH02. f DH05. g DH11. h Self-pollination of buds. i Self-pollination of flowers
Traits of flowering Chinese cabbage hybrid combinations (YB01, GMS019 × DH05). a Single plant of YB01 showing multiple lateral branches. b Lateral branches harvested from YB01 at one time
Article
Isolated microspore culture has been implemented in breeding programs to produce doubled haploid (DH) lines and thus accelerates the breeding process. However, low microspore embryogenesis frequency in flowering Chinese cabbage remains a key obstacle to the practical application of this technique. This study aimed to establish an efficient microspore culture protocol for flowering Chinese cabbage that would be applied for heterosis breeding. Microspores of five genotypes, 19AY05, 19AY06, 19AY10, 19AY12, and 19AY15, were successfully induced to produce embryos in NLN-13 medium. Microspores of two genotypes, 19AY05 and 19AY15, were cultivated in NLN-13 medium supplemented with different concentrations (0, 0.01, 0.05, 0.1, or 0.2 mg·L−1) of compound sodium nitrophenol (sodium nitrophenol, 5-nitrophenol) to enhance microspore embryogenesis and plant regeneration without an intervening callus phase. The results showed that 0.05 ~ 0.1 mg· L−1 sodium nitrophenol and 0.01 ~ 0.2 mg· L−1 of 5-nitrophenol significantly promoted the induction of microspore embryogenesis of two genotypes, and the best concentrations required for different genotypes are different. Moreover, 0.1 mg· L−1 sodium nitrophenol can significantly increase the plant regeneration rate of the two genetypes. The 5-nitrophenol at 0.01 mg·L−1 significantly increased rate of embryos directly convert to plant in 19AY15. In addition, the average doubled haploid rates in the five genotypes were close to 63%. Horticultural traits of DH lines from 19AY05 were identified and all of them were self-incompatible lines. They showed a high uniformity and consistency that can be directly used for hybrid breeding. Furthermore, the hybrid combination was prepared with the selected DH lines and the Guangdong nucleus genic sterile line GMS019 to screen the excellent hybrid combination for the flowering Chinese cabbage breeding program. This method accelerates the application of microspore culture in hybrid breeding of flowering Chinese cabbage.
 
Article
Unprecedented growth in the communication sector and expanded usage of the number of wireless devices in the past few decades have resulted in a tremendous increase in emissions of non-ionizing electromagnetic radiations (EMRs) in the environment. The widespread EMRs have induced many significant changes in biological systems leading to oxidative stress as well as DNA damage. Considering this, the present study was planned to study the effects of EMRs at 900 MHz frequency with the power density of 10.0 dBm (0.01 W) at variable exposure periods (0.5 h, 1 h, 2 h, 4 h, and 8 h per day for 7 days) on percentage germination, morphological characteristics, protein content, lipid peroxidation in terms of malondialdehyde content (MDA), and antioxidant defense system of Trigonella foenum-graecum test system. The genotoxicity was also evaluated using similar conditions. It was observed that EMRs significantly decreased the germination percentage at an exposure time of 4 h and 8 h. Fresh weight and dry weight of root and shoot did not show significant variations, while the root and shoot length have shown significant variations for 4 h and 8 h exposure period. Further, EMRs enhanced MDA indicating lipid peroxidation. In response to exposure of EMRs, there was a significant up-regulation in the activities of enzymes such as ascorbate peroxidase (APX), superoxide dismutase (SOD), glutathione-S-transferase (GST), guaiacol peroxidase (POD), and glutathione reductase (GR) in the roots and shoots of Trigonella-foenum graecum. The genotoxicity study showed the induction of chromosomal aberrations in root tip cells of the Trigonella foenum-graecum test system. The present study revealed the induction of oxidative stress and genotoxicity of EMRs exposure in the test system.
 
Top-cited authors
Mohamed Al-whaibi
  • King Saud University
Shamsul Hayat
  • Aligarh Muslim University
Narendra Tuteja
  • International Centre for Genetic Engineering and Biotechnology
Parvaiz Ahmad
  • GDC Pulwama J&K INDIA/ King Saud University Riyadh Saudi Arabia
Mohammad Abass