Prunus Knotted-like Genes: Genome-Wide Analysis, Transcriptional Response to Cytokinin in Micropropagation, and Rootstock Transformation.

Knotted1-like homeobox (KNOX) transcription factors are involved in plant development, playing complex roles in aerial organs. As Prunus species include important fruit tree crops of Italy, an exhaustive investigation of KNOX genes was performed using genomic and RNA-seq meta-analyses. Micropropagation is an essential technology for rootstock multiplication; hence, we investigated KNOX transcriptional behavior upon increasing 6-benzylaminopurine (BA) doses and the effects on GF677 propagules. Moreover, gene function in Prunus spp. was assessed by Gisela 6 rootstock transformation using fluorescence and peach KNOX transgenes. Based on ten Prunus spp., KNOX proteins fit into I-II-M classes named after Arabidopsis. Gene number, class member distribution, and chromosome positions were maintained, and exceptions supported the diversification of Prunus from Cerasus subgenera, and that of Armeniaca from the other sections within Prunus. Cytokinin (CK) cis-elements occurred in peach and almond KNOX promoters, suggesting a BA regulatory role in GF677 shoot multiplication as confirmed by KNOX expression variation dependent on dose, time, and interaction. The tripled BA concentration exacerbated stress, altered CK perception genes, and modified KNOX transcriptions, which are proposed to concur in in vitro anomalies. Finally, Gisela 6 transformation efficiency varied (2.6-0.6%) with the genetic construct, with 35S:GFP being more stable than 35S:KNOPE1 lines, which showed leaf modification typical of KNOX overexpression.

Convergence between Development and Stress: Ectopic Xylem Formation in Arabidopsis Hypocotyl in Response to 24-Epibrassinolide and Cadmium.

Ectopic xylary element (EXE) formation in planta is a poorly investigated process, and it is unknown if it occurs as a response to the soil pollutant Cadmium (Cd). The pericycle cells of Arabidopsis thaliana hypocotyl give rise to EXEs under specific hormonal inputs. Cadmium triggers pericycle responses, but its role in EXE formation is unknown. Brassinosteroids (BRs) affect numerous developmental events, including xylogenesis in vitro, and their exogenous application by 24-epibrassinolide (eBL) helps to alleviate Cd-stress by increasing lateral/adventitious rooting. Epibrassinolide's effects on EXEs in planta are unknown, as well as its relationship with Cd in the control of the process. The research aims to establish an eBL role in pericycle EXE formation, a Cd role in the same process, and the possible interaction between the two. Results show that 1 nM eBL causes an identity reversal between the metaxylem and protoxylem within the stele, and its combination with Cd reduces the event. All eBL concentrations increase EXEs, also affecting xylary identity by changing from protoxylem to metaxylem in a concentration-dependent manner. Cadmium does not affect EXE identity but increases EXEs when combined with eBL. The results suggest that eBL produces EXEs to form a mechanical barrier against the pollutant.

Poly-(lactic-co-glycolic) Acid Nanoparticles Entrapping Pterostilbene for Targeting Aspergillus Section Nigri.

Poly-(lactic-co-glycolic) acid (PLGA) is a biodegradable, biosafe, and biocompatible copolymer. The Aspergillus section Nigri causes otomycosis localized in the external auditory canal. In this research, Aspergillus brasiliensis, a species belonging to the Nigri section, was tested. Coumarin 6 and pterostilbene loaded in poly-(lactic-co-glycolic) acid nanoparticles (PLGA-coumarin6-NPs and PLGA-PTB-NPs) were tested for fungal cell uptake and antifungal ability against A. brasiliensis biofilm, respectively. Moreover, the activity of PLGA-PTB-NPs in inhibiting the A. brasiliensis infection was tested using Galleria mellonella larvae. The results showed a fluorescence signal, after 50 nm PLGA-coumarin6-NPs treatment, inside A. brasiliensis hyphae and along the entire thickness of the biofilm matrix, which was indicative of an efficient NP uptake. Regarding antifungal activity, a reduction in A. brasiliensis biofilm formation and mature biofilm with PLGA-PTB-NPs has been demonstrated. Moreover, in vivo experiments showed a significant reduction in mortality of infected larvae after injection of PLGA-PTB-NPs compared to free PTB at the same concentration. In conclusion, the PLGA-NPs system can increase the bioavailability of PTB in Aspergillus section Nigri biofilm by overcoming the biofilm matrix barrier and delivering PTB to fungal cells.

A novel approach to control Botrytis cinerea fungal infections: uptake and biological activity of antifungals encapsulated in nanoparticle based vectors.

Botrytis cinerea, responsible for grey mold diseases, is a pathogen with a broad host range, affecting many important agricultural crops, in pre and post harvesting of fruits and vegetables. Commercial fungicides used to control this pathogen are often subjected to photolysis, volatilization, degradation, leaching, and runoff during application. In this context, the use of a delivery system, based on poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) represents an innovative approach to develop new pesticide formulations to successfully fight B. cinerea infections. In order to study NPs uptake, B. cinerea conidia and mycelium were treated with PLGA NPs loaded with the high fluorescent probe coumarin 6 (Cu6-PLGA NPs) and analyzed under ApoTome fluorescence microscopy. The observations revealed that 50 nm Cu6-PLGA NPs penetrated into B. cinerea conidia and hyphae, as early as 10 min after administration. Pterostilbene, a natural compound, and fluopyram, a synthetic antifungal, were entrapped in PLGA NPs, added to B. cinerea conidia and mycelium, and their antifungal activity was tested. The results revealed that the compounds loaded in NPs exhibited a higher activity against B. cinerea. These results lay the foundations for the use of PLGA NPs as a new strategy in plant pest management.

The Effect of Fusarium verticillioides Fumonisins on Fatty Acids, Sphingolipids, and Oxylipins in Maize Germlings.

Fusarium verticillioides causes multiple diseases of Zea mays (maize) including ear and seedling rots, contaminates seeds and seed products worldwide with toxic chemicals called fumonisins. The role of fumonisins in disease is unclear because, although they are not required for ear rot, they are required for seedling diseases. Disease symptoms may be due to the ability of fumonisins to inhibit ceramide synthase activity, the expected cause of lipids (fatty acids, oxylipins, and sphingolipids) alteration in infected plants. In this study, we explored the impact of fumonisins on fatty acid, oxylipin, and sphingolipid levels in planta and how these changes affect F. verticillioides growth in maize. The identity and levels of principal fatty acids, oxylipins, and over 50 sphingolipids were evaluated by chromatography followed by mass spectrometry in maize infected with an F. verticillioides fumonisin-producing wild-type strain and a fumonisin-deficient mutant, after different periods of growth. Plant hormones associated with defense responses, i.e., salicylic and jasmonic acid, were also evaluated. We suggest that fumonisins produced by F. verticillioides alter maize lipid metabolism, which help switch fungal growth from a relatively harmless endophyte to a destructive necrotroph.

Aspergillus flavus Exploits Maize Kernels Using an "Orphan" Secondary Metabolite Cluster.

Aspergillus flavus is a saprophytic cosmopolitan fungus, capable of infecting crops both pre- and post-harvest and exploiting different secondary metabolites, including aflatoxins. Aflatoxins are known carcinogens to animals and humans, but display no clear effect in host plants such as maize. In a previous study, we mined the genome of A. flavus to identify secondary metabolite clusters putatively involving the pathogenesis process in maize. We now focus on cluster 32, encoding for fungal effectors such as salicylate hydroxylase (SalOH), and necrosis- and ethylene-inducing proteins (npp1 domain protein) whose expression is triggered upon kernel contact. In order to understand the role of this genetic cluster in maize kernel infection, mutants of A. flavus, impaired or enhanced in specific functions (e.g., cluster 32 overexpression), were studied for their ability to cause disease. Within this frame, we conducted histological and histochemical experiments to verify the expression of specific genes within the cluster (e.g., SalOH, npp1), the production of salicylate, and the presence of its dehydroxylated form. Results suggest that the initial phase of fungal infection (2 days) of the living tissues of maize kernels (e.g., aleuron) coincides with a significant increase of fungal effectors such as SalOH and Npp1 that appear to be instrumental in eluding host defences and colonising the starch-enriched tissues, and therefore suggest a role of cluster 32 to the onset of infection.

Intimate functional interactions between TGS1 and the Smn complex revealed by an analysis of the Drosophila eye development.

Trimethylguanosine synthase 1 (TGS1) is a conserved enzyme that mediates formation of the trimethylguanosine cap on several RNAs, including snRNAs and telomerase RNA. Previous studies have shown that TGS1 binds the Survival Motor Neuron (SMN) protein, whose deficiency causes spinal muscular atrophy (SMA). Here, we analyzed the roles of the Drosophila orthologs of the human TGS1 and SMN genes. We show that the Drosophila TGS1 protein (dTgs1) physically interacts with all subunits of the Drosophila Smn complex (Smn, Gem2, Gem3, Gem4 and Gem5), and that a human TGS1 transgene rescues the mutant phenotype caused by dTgs1 loss. We demonstrate that both dTgs1 and Smn are required for viability of retinal progenitor cells and that downregulation of these genes leads to a reduced eye size. Importantly, overexpression of dTgs1 partially rescues the eye defects caused by Smn depletion, and vice versa. These results suggest that the Drosophila eye model can be exploited for screens aimed at the identification of genes and drugs that modify the phenotypes elicited by Tgs1 and Smn deficiency. These modifiers could help to understand the molecular mechanisms underlying SMA pathogenesis and devise new therapies for this genetic disease.

Unsaturated Lipids Change in Olive Tree Drupe and Seed during Fruit Development and in Response to Cold-Stress and Acclimation.

The olive tree is a plant of economic value for the oil of its drupe. It is a cultigen complex composed of genotypes with differences in cold-hardiness. About 90% of the oil is stored in oil bodies (OBs) in the drupe during the oleogenic phase. Phenols and lipids contribute to oil quality, but the unsaturated fatty acid (FA) fraction is emerging as the most important for quality, because of the very high content in oleic acid, the presence of ω6-linoleic acid and ω3-linolenic acid, and the very low saturated FA content. Another 10% of oil is produced by the seed. Differences in unsaturated FA-enriched lipids exist among seed coat, endosperm, and embryo. Olive oil quality is also affected by the environmental conditions during fruit growth and genotype peculiarities. Production of linoleic and α-linolenic acids, fruit growth, fruit and leaf responses to low temperatures, including cuticle formation, and cold-acclimation are related processes. The levels of unsaturated FAs are changed by FA-desaturase (FAD) activities, involving the functioning of chloroplasts and endoplasmic reticulum. Cold induces lipid changes during drupe and seed development, affecting FADs, but its effect is related to the genotype capability to acclimate to the cold.

OeFAD8, OeLIP and OeOSM expression and activity in cold-acclimation of Olea europaea, a perennial dicot without winter-dormancy.

MAIN CONCLUSION: Cold-acclimation genes in woody dicots without winter-dormancy, e.g., olive-tree, need investigation. Positive relationships between OeFAD8, OeOSM , and OeLIP19 and olive-tree cold-acclimation exist, and couple with increased lipid unsaturation and cutinisation. Olive-tree is a woody species with no winter-dormancy and low frost-tolerance. However, cold-tolerant genotypes were empirically selected, highlighting that cold-acclimation might be acquired. Proteins needed for olive-tree cold-acclimation are unknown, even if roles for osmotin (OeOSM) as leaf cryoprotectant, and seed lipid-transfer protein for endosperm cutinisation under cold, were demonstrated. In other species, FAD8, coding a desaturase producing α-linolenic acid, is activated by temperature-lowering, concomitantly with bZIP-LIP19 genes. The research was focussed on finding OeLIP19 gene(s) in olive-tree genome, and analyze it/their expression, and that of OeFAD8 and OeOSM, in drupes and leaves under different cold-conditions/developmental stages/genotypes, in comparison with changes in unsaturated lipids and cell wall cutinisation. Cold-induced cytosolic calcium transients always occurred in leaves/drupes of some genotypes, e.g., Moraiolo, but ceased in others, e.g., Canino, at specific drupe stages/cold-treatments, suggesting cold-acclimation acquisition only in the latter genotypes. Canino and Moraiolo were selected for further analyses. Cold-acclimation in Canino was confirmed by an electrolyte leakage from leaf/drupe membranes highly reduced in comparison with Moraiolo. Strong increases in fruit-epicarp/leaf-epidermis cutinisation characterized cold-acclimated Canino, and positively coupled with OeOSM expression, and immunolocalization of the coded protein. OeFAD8 expression increased with cold-acclimation, as the production of α-linolenic acid, and related compounds. An OeLIP19 gene was isolated. Its levels changed with a trend similar to OeFAD8. All together, results sustain a positive relationship between OeFAD8, OeOSM and OeLIP19 expression in olive-tree cold-acclimation. The parallel changes in unsaturated lipids and cutinisation concur to suggest orchestrated roles of the coded proteins in the process.

Overexpression of AtPCS1 in tobacco increases arsenic and arsenic plus cadmium accumulation and detoxification.

MAIN CONCLUSION: The heterologous expression of AtPCS1 in tobacco plants exposed to arsenic plus cadmium enhances phytochelatin levels, root As/Cd accumulation and pollutants detoxification, but does not prevent root cyto-histological damages. High phytochelatin (PC) levels may be involved in accumulation and detoxification of both cadmium (Cd) and arsenic (As) in numerous plants. Although polluted environments are frequently characterized by As and Cd coexistence, how increased PC levels affect the adaptation of the entire plant and the response of its cells/tissues to a combined contamination by As and Cd needs investigation. Consequently, we analyzed tobacco seedlings overexpressing Arabidopsis phytochelatin synthase1 gene (AtPCS1) exposed to As and/or Cd, to evaluate the levels of PCs and As/Cd, the cyto-histological modifications of the roots and the Cd/As leaf extrusion ability. When exposed to As and/or Cd the plants overexpressing AtPCS1 showed higher PC levels, As plus Cd root accumulation, and detoxification ability than the non-overexpressing plants, but a blocked Cd-extrusion from the leaf trichomes. In all genotypes, As, and Cd in particular, damaged lateral root apices, enhancing cell-vacuolization, causing thinning and stretching of endodermis initial cells. Alterations also occurred in the primary structure region of the lateral roots, i.e., cell wall lignification in the external cortex, cell hypertrophy in the inner cortex, crushing of endodermis and stele, and nuclear hypertrophy. Altogether, As and/or Cd caused damage to the lateral roots (and not to the primary one), with such damage not counteracted by AtPCS1 overexpression. The latter, however, positively affected accumulation and detoxification to both pollutants, highlighting that Cd/As accumulation and detoxification due to PCS1 activity do not reduce the cyto-histological damage.

Recent Advances on Genetic and Physiological Bases of In Vitro Somatic Embryo Formation.

Somatic embryogenesis involves a broad repertoire of genes, and complex expression patterns controlled by a concerted gene regulatory network. The present work describes this regulatory network focusing on the main aspects involved, with the aim of providing a deeper insight into understanding the total reprogramming of cells into a new organism through a somatic way. To the aim, the chromatin remodeling necessary to totipotent stem cell establishment is described, as the activity of numerous transcription factors necessary to cellular totipotency reprogramming. The eliciting effects of various plant growth regulators on the induction of somatic embryogenesis is also described and put in relation with the activity of specific transcription factors. The role of programmed cell death in the process, and the related function of specific hemoglobins as anti-stress and anti-death compounds is also described. The tools for biotechnology coming from this information is highlighted in the concluding remarks.

Tapetum and middle layer control male fertility in Actinidia deliciosa.

BACKGROUND AND AIMS: Dioecism characterizes many crop species of economic value, including kiwifruit (Actinidia deliciosa). Kiwifruit male sterility occurs at the microspore stage. The cell walls of the microspores and the pollen of the male-sterile and male-fertile flowers, respectively, differ in glucose and galactose levels. In numerous plants, pollen formation involves normal functioning and degeneration timing of the tapetum, with calcium and carbohydrates provided by the tapetum essential for male fertility. The aim of this study was to determine whether the anther wall controls male fertility in kiwifruit, providing calcium and carbohydrates to the microspores. METHODS: The events occurring in the anther wall and microspores of male-fertile and male-sterile anthers were investigated by analyses of light microscopy, epifluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL assay) and transmission electron microscopy coupled with electron spectroscopy. The possibility that male sterility was related to anther tissue malfunctioning with regard to calcium/glucose/galactose provision to the microspores was also investigated by in vitro anther culture. KEY RESULTS: Both tapetum and the middle layer showed secretory activity and both degenerated by programmed cell death (PCD), but PCD was later in male-sterile than in male-fertile anthers. Calcium accumulated in cell walls of the middle layer and tapetum and in the exine of microspores and pollen, reaching higher levels in anther wall tissues and dead microspores of male-sterile anthers. A specific supply of glucose and calcium induced normal pollen formation in in vitro-cultured anthers of the male-sterile genotype. CONCLUSIONS: The results show that male sterility in kiwifruit is induced by anther wall tissues through prolonged secretory activity caused by a delay in PCD, in the middle layer in particular. In vitro culture results support the sporophytic control of male fertility in kiwifruit and open the way to applications to overcome dioecism and optimize kiwifruit production.

Potato virus X movement in Nicotiana benthamiana: new details revealed by chimeric coat protein variants.

Potato virus X coat protein is necessary for both cell-to-cell and phloem transfer, but it has not been clarified definitively whether it is needed in both movement phases solely as a component of the assembled particles or also of differently structured ribonucleoprotein complexes. To clarify this issue, we studied the infection progression of a mutant carrying an N-terminal deletion of the coat protein, which was used to construct chimeric virus particles displaying peptides selectively affecting phloem transfer or cell-to-cell movement. Nicotiana benthamiana plants inoculated with expression vectors encoding the wild-type, mutant and chimeric viral genomes were examined by microscopy techniques. These experiments showed that coat protein-peptide fusions promoting cell-to-cell transfer only were not competent for virion assembly, whereas long-distance movement was possible only for coat proteins compatible with virus particle formation. Moreover, the ability of the assembled PVX to enter and persist into developing xylem elements was revealed here for the first time.

The rolD oncogene promotes axillary bud and adventitious root meristems in Arabidopsis.

The expression of the Agrobacterium rhizogenes rolD oncogene induces precocious floral transition and strong flowering potential in tobacco and tomato. Here, we describe specific developmental effects induced by expression of rolD in Arabidopsis. We show that floral transition, as histologically monitored, occurred in rolD- plants earlier than in wild type, and this was coupled with a premature and enhanced formation of vegetative and reproductive axillary bud meristems. Furthermore, CYP79F1/SUPERSHOOT/BUSHY (SPS), a gene that negatively controls shoot branching in Arabidopsis and involved in glucosinolate metabolism and in cytokinin and auxin homeostasis, was down-regulated in rolD plants. The multiplication of post-embryonic meristems was also observed in the root system, with enhanced adventitious root formation. This result was confirmed by thin cell layer response in vitro, both under hormone-free and standard rooting conditions. However, the formation of lateral root meristems was not affected by rolD expression. Our results show that rolD accelerates and enhances specific post-embryonic meristems in Arabidopsis.

Modulation of intracellular proline levels affects flowering time and inflorescence architecture in Arabidopsis.

We reported previously that the plant oncogene rolD anticipates and stimulates flowering in Nicotiana tabacum, and encodes ornithine cyclodeaminase, an enzyme catalysing the conversion of ornithine to proline. To investigate on the possible role of proline in flowering, we altered the expression of AtP5CS1, encoding the rate-limiting enzyme of proline biosynthesis in plants. Accordingly we characterized a mutant line containing a T-DNA insertion into AtP5CS1 and introduced in Arabidopsis thaliana AtP5CS1 under the control of the CaMV35S promoter. As expected homozygous p5cs1 mutants behaved as late flowering. In addition p5cs1 mutants exhibited a shorter size and contained lower levels of proline, compared to wild type. 35S-P5CS1 plants, manifested, early in development, overexpression of P5CS1 and accumulation of proline, leading to early flowering, both under long- and short-day conditions. Later in development, down-regulation of P5CS1 occurred in 35S-P5CS1 leaves, leading to proline reduction, and, in turn, impaired bolting and stunted growth. Salt-stress restored expression of P5CS1 and proline accumulation in P5CS1-transformed plants, as well as rescuing growth. Our data suggest that proline plays a key role in flower transition, bolting and coflorescence formation.

Expression of rolB in tobacco flowers affects the coordinated processes of anther dehiscence and style elongation.

The effect of auxin on stamen and pistil development in tobacco flowers was investigated by means of the localized expression of rolB (root loci B), an Agrobacterium oncogene that increases auxin sensitivity in a cell-autonomous fashion. When rolB is driven by the promoter of the meiosis-specific Arabidopsis gene DMC1 (disrupted meiotic cDNA 1), expression occurs earlier in male than in female developing organs, resulting in a delay in anther dehiscence with respect to normal timing of pistil development. As a consequence of this developmental uncoupling, self-pollination is prevented in pDMC1:rolB plants. Histological analysis of pDMC1:GFP plants indicates that in tobacco, this promoter is active not only in meiocytes but also in somatic tissues of the anther. In contrast, simultaneous expression of rolB in anther and pistil somatic tissues, achieved by expressing a construct containing rolB under the control of the promoter of the petunia gene FBP7 (floral binding protein 7), results in a concomitant delay of both anther dehiscence and pistil development without affecting self-pollination of the plants. Analysis of plants harboring the pFBP7:GUS construct shows that in tobacco, this promoter is active not only in the ovules, as described for petunia, but also in pistil and anther somatic tissues involved in the dehiscence program. The delay in anther dehiscence and pistil development could be phenocopied by exogenous application of auxin. Jasmonic acid (JA) could not rescue the delay in anther dehiscence. These results suggest that auxin plays a key role in the timing of anther dehiscence, the dehiscence program is controlled by the somatic tissues of the anther, and auxin also regulates pistil development.