Phytotoxic impact of bifunctionalized silver nanoparticles (AgNPs-Cit-L-Cys) and silver nitrate (AgNO3) on chronically exposed callus cultures of Populus nigra L.

Owing to the unique physicochemical properties and the low manufacturing costs, silver nanoparticles (AgNPs) have gained growing interest and their application has expanded considerably in industrial and agricultural sectors. The large-scale production of these nanoparticles inevitably entails their direct or indirect release into the environment, raising some concerns about their hazardous aspects. Callus culture represents an important tool in toxicological studies to evaluate the impact of nanomaterials on plants and their potential environmental risk. In this study, we investigated the chronic phytotoxic effects of different concentrations of novel bifunctionalized silver nanoparticles (AgNPs-Cit-L-Cys) and silver nitrate (AgNO3) on callus culture of Populus nigra L., a pioneer tree species in the riparian ecosystem. Our results showed that AgNPs-Cit-L-Cys were more toxic on poplar calli compared to AgNO3, especially at low concentration (2.5 mg/L), leading to a significant reduction in biomass production, accompanied by a decrease in protein content, a significant increase in both lipid peroxidation level, ascorbate peroxidase (APX), and catalase (CAT) enzymatic activities. In addition, these findings suggested that the harmful activity of AgNPs-Cit-L-Cys might be correlated with their physicochemical properties and not solely attributed to the released Ag+ ions and confirmed that AgNPs-Cit-L-Cys phytoxicity is associated to oxidative stress.

A Novel Role of Medicago truncatula KNAT3/4/5-like Class 2 KNOX Transcription Factors in Drought Stress Tolerance.

Class 2 KNOX homeobox transcription factors (KNOX2) play a role in promoting cell differentiation in several plant developmental processes. In Arabidopsis, they antagonize the meristematic KNOX1 function during leaf development through the modulation of phytohormones. In Medicago truncatula, three KNOX2 genes belonging to the KNAT3/4/5-like subclass (Mt KNAT3/4/5-like or MtKNOX3-like) redundantly works upstream of a cytokinin-signaling module to control the symbiotic root nodule formation. Their possible role in the response to abiotic stress is as-of-yet unknown. We produced transgenic M. truncatula lines, in which the expression of four MtKNOX3-like genes was knocked down by RNA interference. When tested for response to water withdrawal in the soil, RNAi lines displayed a lower tolerance to drought conditions compared to the control lines, measured as increased leaf water loss, accelerated leaf wilting time, and faster chlorophyll loss. Reanalysis of a transcriptomic M. truncatula drought stress experiment via cluster analysis and gene co-expression networks pointed to a possible role of MtKNOX3-like transcription factors in repressing a proline dehydrogenase gene (MtPDH), specifically at 4 days after water withdrawal. Proline measurement and gene expression analysis of transgenic RNAi plants compared to the controls confirmed the role of KNOX3-like genes in inhibiting proline degradation through the regulation of the MtPDH gene.

Effects of Microplastic Contamination on the Aquatic Plant Lemna minuta (Least Duckweed).

Microplastics are widely spread in aquatic environments. Although they are considered among the most alarming contaminants, toxic effects on organisms are unclear, particularly on freshwater plants. In this study, the duckweed Lemna minuta was grown on different concentrations (50, 100 mg/L) of poly(styrene-co-methyl methacrylate) microplastics (MP) and exposure times (T0, T7, T14, T28 days). The phytotoxic effects of MP were investigated by analyzing several plant morphological and biochemical parameters (frond and root size, plant growth, chlorophyll, and malondialdehyde content). Observations by scanning electron microscope revealed MP adsorption on plant surfaces. Exposition to MP adversely affected plant growth and chlorophyll content with respect to both MP concentrations and exposure times. Conversely, malondialdehyde measurements did not indicate an alteration of oxidative lipid damage in plant tissue. The presence of MP induced root elongation when compared to the control plants. The effects of MP on L. minuta plants were more evident at T28. These results contribute to a better understanding of MP's impact on aquatic plants and highlight that MP contamination manifests with chronic-type effects, which are thus detectable at longer exposure times of 7 days than those traditionally used in phytotoxicology tests on duckweeds.

Phytoremediation potential of the duckweeds Lemna minuta and Lemna minor to remove nutrients from treated waters.

Phytoremediation potential of duckweeds (Lemna minuta, Lemna minor) to remove nutrients from simulated wastewater was analyzed. In two separate experiments, the two species were grown for 28 days in waters enriched with nitrate and phosphate to simulate nutrient concentrations of domestic wastewater. Water physical and chemical measurements (temperature, pH, conductivity, oxygen) and plant physiological and biochemical analysis (biomass, relative growth rate-RGR, nutrient and chlorophyll contents, peroxidative damage, bioconcentration factor-BCF) were made to test and compare the phytoremediation capacity of the two Lemna species. L. minuta biomass increased almost tenfold during the time-course of the treatment resulting in a doubling of the mat thickness and a RGR of 0.083 ± 0.001 g/g day. Maximum frond content of phosphate was reached by day 21 (increase over 165%) and nitrate by day 7 (10%). According to the BCF results (BCF > 1000), L. minuta was a hyperaccumulator for both nutrients. On the other hand, L. minor biomass and mat thickness decreased continuously during incubation (RGR = - 0.039 ± 0.004 g/g day). In L. minor fronds, phosphate content increased until day 14, after which there was a decrease until the end of the incubation. Frond nitrate content significantly decreased by day 7, but then remained relatively constant until the end of the experiment. L. minor proved to be hyperaccumulator for phosphates, but not for nitrates. Results indicated L. minuta has a greater potential than L. minor to remove both nutrients by bioaccumulation, especially phosphates, demonstrated also by better physiological and biochemical responses. However, during the incubation, the chlorophyll content of L. minuta mat did continuously decrease and peroxidative damage had increased until day 14, indicating that the system was under some kind of stress. Strategies to avoid this stress were discussed.

Transcriptome driven characterization of curly- and smooth-leafed endives reveals molecular differences in the sesquiterpenoid pathway.

Endives (Cichorium endivia L.) are popular vegetables, diversified into curly/frisée- and smooth/broad-leafed (escaroles) cultivar types (cultigroups), and consumed as fresh and bagged salads. They are rich in sesquiterpene lactones (STL) that exert proven function on bitter taste and human health. The assembly of a reference transcriptome of 77,022 unigenes and RNA-sequencing experiments were carried out to characterize the differences between endives and escaroles at the gene structural and expression levels. A set of 3177 SNPs distinguished smooth from curly cultivars, and an SNP-supported phylogenetic tree separated the cultigroups into two distinct clades, consistently with the botanical varieties of origin (crispum and latifolium, respectively). A pool of 699 genes maintained differential expression pattern (core-DEGs) in pairwise comparisons between curly vs smooth cultivars grown in the same environment. Accurate annotation allowed the identification of 26 genes in the sesquiterpenoid biosynthesis pathway, which included several g ermacrene A s ynthase, g ermacrene A o xidase and co stunolide s ynthase members (GAS/GAO/COS module), required for the synthesis of costunolide, a key precursor of lactucopicrin- and lactucin-like sesquiterpene lactones. The core-DEGs contained a GAS gene (contig83192) that was positively correlated with STL levels and recurrently more expressed in curly than smooth endives, suggesting a cultigroup-specific behavior. The significant positive correlation of GAS/GAO/COS transcription and STL abundance (2.4-fold higher in frisée endives) suggested that sesquiterpenoid pathway control occurs at the transcriptional level. Based on correlation analyses, five transcription factors (MYB, MYB-related and WRKY) were inferred to act on contig83192/GAS and specific STL, suggesting the occurrence of two distinct routes in STL biosynthesis.

Plant Cellular and Molecular Biotechnology: Following Mariotti's Steps.

This review is dedicated to the memory of Prof. Domenico Mariotti, who significantly contributed to establishing the Italian research community in Agricultural Genetics and carried out the first experiments of Agrobacterium-mediated plant genetic transformation and regeneration in Italy during the 1980s. Following his scientific interests as guiding principles, this review summarizes the recent advances obtained in plant biotechnology and fundamental research aiming to: (i) Exploit in vitro plant cell and tissue cultures to induce genetic variability and to produce useful metabolites; (ii) gain new insights into the biochemical function of Agrobacterium rhizogenes rol genes and their application to metabolite production, fruit tree transformation, and reverse genetics; (iii) improve genetic transformation in legume species, most of them recalcitrant to regeneration; (iv) untangle the potential of KNOTTED1-like homeobox (KNOX) transcription factors in plant morphogenesis as key regulators of hormonal homeostasis; and (v) elucidate the molecular mechanisms of the transition from juvenility to the adult phase in Prunus tree species.

KNAT3/4/5-like class 2 KNOX transcription factors are involved in Medicago truncatula symbiotic nodule organ development.

We investigated the role of KNOX genes in legume root nodule organogenesis. Class 1 KNOX homeodomain transcription factors (TFs) are involved in plant shoot development and leaf shape diversity. Class 2 KNOX genes are less characterized, even though an antagonistic function relative to class 1 KNOXs was recently proposed. In silico expression data and further experimental validation identified in the Medicago truncatula model legume three class 2 KNOX genes, belonging to the KNAT3/4/5-like subclass (Mt KNAT3/4/5-like), as expressed during nodulation from early stages. RNA interference (RNAi)-mediated silencing and overexpression studies were used to unravel a function for KNOX TFs in nodule development. Mt KNAT3/4/5-like genes encoded four highly homologous proteins showing overlapping expression patterns during nodule organogenesis, suggesting functional redundancy. Simultaneous reduction of Mt KNAT3/4/5-like genes indeed led to an increased formation of fused nodule organs, and decreased the expression of the MtEFD (Ethylene response Factor required for nodule Differentiation) TF and its direct target MtRR4, a cytokinin response gene. Class 2 KNOX TFs therefore regulate legume nodule development, potentially through the MtEFD/MtRR4 cytokinin-related regulatory module, and may control nodule organ boundaries and shape like class 2 KNOX function in leaf development.

Insights into the Sesquiterpenoid Pathway by Metabolic Profiling and De novo Transcriptome Assembly of Stem-Chicory (Cichorium intybus Cultigroup "Catalogna").

Stem-chicory of the "Catalogna" group is a vegetable consumed for bitter-flavored stems. Type and levels of bitter sesquiterpene lactones (STLs) participate in conferring bitterness in vegetables. The content of lactucin-and lactucopocrin-like STLs was higher in "Molfettese" than "Galatina" landrace stalks, regardless of the cultivation sites, consistently with bitterness scores and gustative differences. The "Galatina" transcriptome assembly resulted in 58,872 unigenes, 77% of which were annotated, paving the way to molecular investigation of the STL pathway. Comparative transcriptome analysis allowed the identification of 69,352 SNPs and of 1640 differentially expressed genes that maintained the pattern independently of the site. Enrichment analyses revealed that 4 out of 29 unigenes were up-regulated in "Molfettese" vs "Galatina" within the sesquiterpenoid pathway. The expression of two germacrene A -synthase (GAS) and one -oxidase (GAO) genes of the costunolide branch correlated positively with the contents of lactucin-like molecules, supporting that STL biosynthesis regulation occurs at the transcriptional level. Finally, 46 genes encoding transcription factors (TFs) maintained a differential expression pattern between the two varieties regardless of the growth site; correlation analyses among TFs, GAS, GAO gene expressions and STLs contents suggest that one MYB and one bHLH may act in the pathway.

TALE and Shape: How to Make a Leaf Different.

The Three Amino acid Loop Extension (TALE) proteins constitute an ancestral superclass of homeodomain transcription factors conserved in animals, plants and fungi. In plants they comprise two classes, KNOTTED1-LIKE homeobox (KNOX) and BEL1-like homeobox (BLH or BELL, hereafter referred to as BLH), which are involved in shoot apical meristem (SAM) function, as well as in the determination and morphological development of leaves, stems and inflorescences. Selective protein-protein interactions between KNOXs and BLHs affect heterodimer subcellular localization and target affinity. KNOXs exert their roles by maintaining a proper balance between undifferentiated and differentiated cell state through the modulation of multiple hormonal pathways. A pivotal function of KNOX in evolutionary diversification of leaf morphology has been assessed. In the SAM of both simple- and compound-leafed seed species, downregulation of most class 1 KNOX (KNOX1) genes marks the sites of leaf primordia initiation. However, KNOX1 expression is re-established during leaf primordia development of compound-leafed species to maintain transient indeterminacy and morphogenetic activity at the leaf margins. Despite the increasing knowledge available about KNOX1 protein function in plant development, a comprehensive view on their downstream effectors remains elusive. This review highlights the role of TALE proteins in leaf initiation and morphological plasticity with a focus on recent advances in the identification of downstream target genes and pathways.

Effect of municipal solid waste compost and sewage sludge use on wheat (Triticum durum): growth, heavy metal accumulation, and antioxidant activity.

BACKGROUND: Inappropriate utilisation of biosolids may adversely impact agrosystem productivity. Here, we address the response of wheat (Triticum durum) to different doses (0, 40, 100, 200 and 300 t ha(-1)) of either municipal solid waste (MSW) compost or sewage sludge in a greenhouse pot experiment. Plant growth, heavy metal uptake, and antioxidant activity were considered. RESULTS: Biomass production of treated plants was significantly enhanced at 40 t ha(-1) and 100 t ha(-1) of MSW compost (+48% and +78% relative to the control, respectively). At the same doses of sewage sludge, the increase was only 18%. Higher doses of both biosolids restricted significantly the plant growth, in concomitance with the significant accumulation of heavy metals (Ni2+, Pb2+, Cu2+ and Zn2+), especially in leaves. Leaf activities of antioxidant enzymes (ascorbate peroxidase, glutathione reductase, catalase and superoxide dismutase) were unchanged at 40 t ha(-1) MSW compost or sewage sludge, but were significantly stimulated at higher doses (200-300 t ha(-1)), together with higher leaf concentration of reduced glutathione. CONCLUSION: This preliminary study suggests that a MSW supply at moderate doses (100 t ha(-1)) could be highly beneficial for wheat productivity.

The peach (Prunus persica [L.] Batsch) homeobox gene KNOPE3, which encodes a class 2 knotted-like transcription factor, is regulated during leaf development and triggered by sugars.

Class 1 KNOTTED1-like transcription factors (KNOX) are known to regulate plant development, whereas information on class 2 KNOX has been limited. The peach KNOPE3 gene was cloned, belonged to a family of few class 2 members and was located at 66 cM in the Prunus spp. G1 linkage-group. The mRNA localization was diversified in leaf, stem, flower and drupe, but recurred in all organ sieves, suggesting a role in sap nutrient transport. During leaf development, the mRNA earliest localized to primordia sieves and subsequently to mesophyll cells of growing leaves. Consistently, its abundance augmented with leaf expansion. The transcription was monitored in leaves responding to darkening, supply and transport block of sugars. It peaked at 4 h after darkness and dropped under prolonged obscurity, showing a similar kinetic to that of sucrose content variation. Feeding leaflets via the transpiration stream caused KNOPE3 up-regulation at 3 h after fructose, glucose and sucrose absorption and at 12 h after sorbitol. In girdling experiments, leaf KNOPE3 was triggered from 6 h onwards along with sucrose and sorbitol raise. Both the phloem-associated expression and sugar-specific gene modulation suggest that KNOPE3 may play a role in sugar translocation during the development of agro-relevant organs such as drupe.

Characterization of KNOX genes in Medicago truncatula.

We isolated three class I and three class II KNOX genes in Medicago truncatula. The predicted amino acid sequences suggested a possible orthology to the Arabidopsis homeodomain proteins STM, KNAT1/BP, KNAT3 and KNAT7 that was confirmed by phylogenetic and conserved structural domain analyses. Moreover, the STM-like MtKNOX1 and MtKNOX6 proteins were shown to retain the capability to interact with the Arabidopsis BELL protein partners of STM and KNAT1/BP. Amino acid residues that characterize the different classes of KNOX proteins were identified. Gene expression studies revealed organ-specificity, possible cytokinin-dependent transcriptional activation of two MtKNOXs and expression of one STM-like and a BP/KNAT1-like MtKNOX in roots. Interestingly, mRNA localization studies carried out on class I MtKNOX genes revealed important differences with previously characterised legume KNOXs. M. truncatula transcripts were not down-regulated in leaf primordia and early stages of leaf development, features shared with the more distant compound-leaved species Solanum lycopersicum.