First investigation into the genetic control of meiosis in sugarcane.

The sugarcane (Saccharum spp.) genome is one of the most complex of all. Modern varieties are highly polyploid and aneuploid as a result of hybridization between Saccharum officinarum and S. spontaneum. Little research has been done on meiotic control in polyploid species, with the exception of the wheat Ph1 locus harboring the ZIP4 gene (TaZIP4-B2) which promotes pairing between homologous chromosomes while suppressing crossover between homeologs. In sugarcane, despite its interspecific origin, bivalent association is favored, and multivalents, if any, are resolved at the end of prophase I. Thus, our aim herein was to investigate the purported genetic control of meiosis in the parental species and in sugarcane itself. We investigated the ZIP4 gene and immunolocalized meiotic proteins, namely synaptonemal complex proteins Zyp1 and Asy1. The sugarcane ZIP4 gene is located on chromosome 2 and expressed more abundantly in flowers, a similar profile to that found for TaZIP4-B2. ZIP4 expression is higher in S. spontaneum a neoautopolyploid, with lower expression in S. officinarum, a stable octoploid species. The sugarcane Zip4 protein contains a TPR domain, essential for scaffolding. Its 3D structure was also predicted, and it was found to be very similar to that of TaZIP4-B2, reflecting their functional relatedness. Immunolocalization of the Asy1 and Zyp1 proteins revealed that S. officinarum completes synapsis. However, in S. spontaneum and SP80-3280 (a modern variety), no nuclei with complete synapsis were observed. Importantly, our results have implications for sugarcane cytogenetics, genetic mapping, and genomics.

In Planta Study Localizes an Effector Candidate from Austropuccinia psidii Strain MF-1 to the Nucleus and Demonstrates In Vitro Cuticular Wax-Dependent Differential Expression.

Austropuccinia psidii is a biotrophic fungus that causes myrtle rust. First described in Brazil, it has since spread to become a globally important pathogen that infects more than 480 myrtaceous species. One of the most important commercial crops affected by A. psidii is eucalypt, a widely grown forestry tree. The A. psidii-Eucalyptus spp. interaction is poorly understood, but pathogenesis is likely driven by pathogen-secreted effector molecules. Here, we identified and characterized a total of 255 virulence effector candidates using a genome assembly of A. psidii strain MF-1, which was recovered from Eucalyptus grandis in Brazil. We show that the expression of seven effector candidate genes is modulated by cell wax from leaves sourced from resistant and susceptible hosts. Two effector candidates with different subcellular localization predictions, and with specific gene expression profiles, were transiently expressed with GFP-fusions in Nicotiana benthamiana leaves. Interestingly, we observed the accumulation of an effector candidate, Ap28303, which was upregulated under cell wax from rust susceptible E. grandis and described as a peptidase inhibitor I9 domain-containing protein in the nucleus. This was in accordance with in silico analyses. Few studies have characterized nuclear effectors. Our findings open new perspectives on the study of A. psidii-Eucalyptus interactions by providing a potential entry point to understand how the pathogen manipulates its hosts in modulating physiology, structure, or function with effector proteins.

Foliar Calcium Absorption by Tomato Plants: Comparing the Effects of Calcium Sources and Adjuvant Usage.

The deficiency of calcium (Ca) reduces the quality and shelf life of fruits. In this scenario, although foliar spraying of Ca2+ has been used, altogether with soil fertilization, as an alternative to prevent deficiencies, little is known regarding its absorption dynamics by plant leaves. Herein, in vivo microprobe X-ray fluorescence was employed aiming to monitor the foliar absorption of CaCl2, Ca-citrate complex, and Ca3(PO4)2 nanoparticles with and without using adjuvant. We also investigated whether Sr2+ can be employed as Ca2+ proxy in foliar absorption studies. Moreover, the impact of treatments on the cuticle structure was evaluated by scanning electron microscopy. For this study, 45-day-old tomato (Solanum lycopersicum L., cv. Micro-Tom) plants were used as a model species. After 100 h, the leaves absorbed 90, 18, and 4% of aqueous CaCl2, Ca-citrate, and Ca3(PO4)2 nanoparticles, respectively. The addition of adjuvant increased the absorption of Ca-citrate to 28%, decreased that of CaCl2 to 77%, and did not affect Ca3(PO4)2. CaCl2 displayed an exponential decay absorption profile with half-lives of 15 h and 5 h without and with adjuvant, respectively. Ca-citrate and Ca3(PO4)2 exhibited absorption profiles that were closer to a linear behavior. Sr2+ was a suitable Ca2+ tracer because of its similar absorption profiles. Furthermore, the use of adjuvant affected the epicuticular crystal structure. Our findings reveal that CaCl2 was the most efficient Ca2+ source. The effects caused by adjuvant suggest that CaCl2 and Ca-citrate were absorbed mostly through hydrophilic and lipophilic pathways.

Physiological responses of plants to in vivo X-ray damage from X-ray fluorescence measurements: insights from anatomical, elemental, histochemical, and ultrastructural analyses.

X-ray fluorescence spectroscopy (XRF) is a powerful technique for the in vivo assessment of plant tissues. However, the potential X-ray exposure damages might affect the structure and elemental composition of living plant tissues, leading to artefacts in the recorded data. Herein, we exposed in vivo soybean (Glycine max (L.) Merrill) leaves to several X-ray doses through a polychromatic benchtop microprobe X-ray fluorescence spectrometer, modulating the photon flux density by adjusting either the beam size, current, or exposure time. Changes in the irradiated plant tissues' structure, ultrastructure, and physiology were investigated through light and transmission electron microscopy (TEM). Depending on X-ray exposure dose, decreased K and X-ray scattering intensities and increased Ca, P, and Mn signals on soybean leaves were recorded. Anatomical analysis indicated the necrosis of epidermal and mesophyll cells on the irradiated spots, where TEM images revealed the collapse of cytoplasm and cell wall breaking. Furthermore, the histochemical analysis detected the production of reactive oxygen species and the inhibition of chlorophyll autofluorescence in these areas. Under certain X-ray exposure conditions, e.g. high photon flux density and long exposure time, XRF measurements may affect the soybean leaves structures, elemental composition, and cellular ultrastructure, inducing programmed cell death. Our characterization shed light on the plant's responses to the X-ray-induced radiation damage and might help to establish proper X-ray radiation limits and novel strategies for in vivo benchtop-XRF analysis of vegetal materials.

Histopathology of the Shoot Apex of Sugarcane Colonized by Leifsonia xyli subsp. xyli.

Colonization of the xylem of sugarcane by Leifsonia xyli subsp. xyli results in the occlusion of the vessels by a gum-like compound and compromises the elongation of the stalk leading to stunted plants. However, no study has been performed in the apical tissue where the elongation of the stalks initiates at the intercalary meristem (IM). Microscopic and histochemical analyses were performed in plants with lower and higher bacterial titers and revealed that in both cases L. xyli subsp. xyli is present in this tissue and colonizes the forming xylem vessels in a similar way as observed in developed internodes. In both cases, it was observed adhering to the secondary walls, but only in plants with higher titers were a mild degradation of the walls and a granular material filling the vessels observed. The mixed composition of lipids, proteins, and pectin indicates that the filling is not a bacterial extracellular polymeric substance. Plants with higher bacterial populations also presented lower starch content in the ground parenchyma at the node elements, possibly resulting from the reported downregulation of photosynthesis and increased accumulation of phenolics. Their second and third IMs presented fewer cells and reduced expression of genes related to the cell cycle and to the synthesis of ABA in the apical tissue. These results indicate that increased L. xyli subsp. xyli colonization affects the development of the IM, which ultimately would reduce the length of the internodes, resulting in the main symptom of the disease.

Double-Staining Method to Detect Pectin in Plant-Fungus Interaction.

Plant cells use different structural mechanisms, either constitutive or inducible, to defend themselves from fungal infection. Encapsulation is an efficient inducible mechanism to isolate the fungal haustoria from the plant cell protoplast. Conversely, pectin, one of the polymeric components of the cell wall, is a target of several pectolytic enzymes in necrotrophic interactions. Here, a protocol to detect pectin and fungal hyphae through optical microscopy is presented. The pectin-rich encapsulation in the cells of coffee leaves infected by the rust fungus Hemileia vastatrix and the mesophyll cell wall modification induced by Cercospora coffeicola are investigated. Lesioned leaf samples were fixed with the Karnovsky solution, dehydrated, and embedded in glycol methacrylate for 2-4 days. All steps were followed by vacuum-pumping to remove air in the intercellular spaces and improve the embedding process. The embedded blocks were sectioned into 5-7 µm thick sections, which were deposited on a glass slide covered with water and subsequently heated at 40 °C for 30 min. Next, the slides were double-stained with 5% cotton blue in lactophenol to detect the fungus and 0.05% ruthenium red in water to detect pectin (acidic groups of polyuronic acids of pectin). Fungal haustoria of Hemileia vastatrix were found to be encapsulated by pectin. In coffee cercosporiosis, mesophyll cells exhibited dissolution of cell walls, and intercellular hyphae and conidiophores were observed. The method presented here is effective to detect a pectin-associated response in the plant-fungi interaction.

Genetic mapping reveals complex architecture and candidate genes involved in common bean response to Meloidogyne incognita infection.

Root-knot nematodes (RKNs), particularly Meloidogyne incognita, are among the most damaging and prevalent agricultural pathogens due to their ability to infect roots of almost all crops. The best strategy for their control is through the use of resistant cultivars. However, laborious phenotyping procedures make it difficult to assess nematode resistance in breeding programs. For common bean, this task is especially challenging because little has been done to discover resistance genes or markers to assist selection. We performed genome-wide association studies and quantitative trait loci mapping to explore the genetic architecture and genomic regions underlying the resistance to M. incognita and to identify candidate resistance genes. Phenotypic data were collected by a high-throughput assay, and the number of egg masses and the root-galling index were evaluated. Complex genetic architecture and independent genomic regions were associated with each trait. Single nucleotide polymorphisms on chromosomes Pv06, Pv07, Pv08, and Pv11 were associated with the number of egg masses, and SNPs on Pv01, Pv02, Pv05, and Pv10 were associated with root-galling. A total of 216 candidate genes were identified, including 14 resistance gene analogs and five differentially expressed in a previous RNA sequencing analysis. Histochemical analysis indicated that reactive oxygen species might play a role in the resistance response. Our findings open new perspectives to improve selection efficiency for RKN resistance, and the candidate genes are valuable targets for functional investigation and gene editing approaches.

Structural and Biochemical Aspects Related to Resistance and Susceptibility of Rubber Tree Clones to Anthracnose.

The aim of the present study was to evaluate structural and biochemical aspects related to the interaction of resistant (RRIM 937, IAC 502 and 507) and susceptible (RRIM 600) rubber tree clones with C. tamarillo. For such analysis, ultrathin sections of the leaf limb were embedded in historesin and differently stained to verify structural alterations and presence of starch grains, arginine, lipids, tannins and lignins. The total proteins and activity of the enzymes peroxidase and (PAL) were quantified. Stomatal density was also analyzed under a scanning electron microscope. Data indicated alterations in the cell content of resistant clones inoculated with the pathogen, as well as greater lignin and lipid accumulation in these samples. For tannins, there was no difference between inoculated and non-inoculated clones. Arginine was found at greater quantities in IAC 502 and 507. Starch grains were not detected in any of the analyzed samples. Protein level and stomatal density were lower in resistant clones. Peroxidase activity was more expressive in resistant clones. PAL activity, there was no significant difference between clones. The lignin and lipids, total protein, peroxidase activity and stomatal density may be related to the resistance of rubber tree clones to anthracnose.

The auxin-producing Bacillus thuringiensis RZ2MS9 promotes the growth and modifies the root architecture of tomato (Solanum lycopersicum cv. Micro-Tom).

Strains of Bacillus thuringiensis (Bt) are commonly commercialized as bioinoculants for insect pest control, but their benefits go beyond their insecticidal property: they can act as plant growth-promoters. Auxins play a major role in the plant growth promotion. However, the mechanism of auxin production by the Bacilli group, and more specifically by Bt strains, is unclear. In previous work, the plant growth-promoting rhizobacterium (PGPR) B. thuringiensis strain RZ2MS9 increased the corn roots. This drew our attention to the strain's auxin production trait, earlier detected in vitro. Here, we demonstrate that in its genome, RZ2MS9 harbours the complete set of genes required in two pathways that are used for Indole acetic acid (IAA) production. We also detected that the strain produces almost five times more IAA during the stationary phase. The bacterial application increased the shoot dry weight of the Micro-Tom (MT) tomato by 24%. The application also modified MT root architecture, with an increase of 26% in the average lateral root length and inhibition of the axial root. At the cellular level, RZ2MS9-treated MT plants presented elongated root cortical cells with intensified mitotic activity. Altogether, these are the best characterized auxin-associated phenotypes. Besides that, no growth alteration was detected in the auxin-insensitive diageotropic (dgt) plants either with or without the RZ2MS9 inoculation. Our results suggest that auxins play an important role in the ability of B. thuringiensis RZ2MS9 to promote MT growth and provide a better understanding of the auxin production mechanism by a Bt strain.

X-ray fluorescence spectroscopy (XRF) applied to plant science: challenges towards in vivo analysis of plants.

X-ray fluorescence spectroscopy (XRF) is an analytical tool used to determine the elemental composition in a myriad of sample matrices. Due to the XRF non-destructive feature, this technique may allow time-resolved plant tissue analyses under in vivo conditions, and additionally, the combination with other non-destructive techniques. In this study, we employed handheld and benchtop XRF to evaluate the elemental distribution changes in living plant tissues exposed to X-rays, as well as real-time uptake kinetics of Zn(aq) and Mn(aq) in soybean (Glycine max (L.) Merrill) stem and leaves, for 48 hours, combined with transpiration rate assessment on leaves by an infrared gas analyzer (IRGA). We found higher Zn content than Mn in stems. The latter micronutrient, in turn, presented higher concentration in leaf veins. Besides, both micronutrients were more concentrated in the first trifolium (i.e., youngest leaf) of soybean plants. Moreover, the transpiration rate was more influenced by circadian cycles than Zn and Mn uptake. Thus, XRF represents a convenient tool for in vivo nutritional studies in plants, and it can be coupled successfully to other analytical techniques.

Novel Insights Into the Early Stages of Ratoon Stunting Disease of Sugarcane Inferred from Transcript and Protein Analysis.

Despite of the importance of ratoon stunting disease, little is known on the responses of sugarcane to its causal agent, the vascular bacterial endophyte Leifsonia xyli subsp. xyli. The transcriptome and proteome of young plants of a susceptible cultivar with no symptoms of stunting but with relative low and high bacterial titers were compared at 30 and 60 days after inoculation. Increased bacterial titers were associated with alterations in the expression of 267 cDNAs and in the abundance of 150 proteins involved in plant growth, hormone metabolism, signal transduction and defense responses. Some alterations are predicted to benefit the pathogen, such as the up-regulation of genes involved in the synthesis of methionine. Also, genes and proteins of the cell division cycle were all down-regulated in plants with higher titers at both times. It is hypothesized that the negative effects on cell division related to increased bacterial titers is cumulative over time and its modulation by other host and environmental factors results in the stunting symptom.

The Eucalyptus Cuticular Waxes Contribute in Preformed Defense Against Austropuccinia psidii.

Austropuccinia psidii, the causal agent of myrtle rust, is a biotrophic pathogen whose growth and development depends on the host tissues. The uredospores of A. psidii infect Eucalyptus by engaging in close contact with the host surface and interacting with the leaf cuticle that provides important chemical and physical signals to trigger the infection process. In this study, the cuticular waxes of Eucalyptus spp. were analyzed to determine their composition or structure and correlation with susceptibility/resistance to A. psidii. Twenty-one Eucalyptus spp. in the field were classified as resistant or susceptible. The resistance/susceptibility level of six Eucalyptus spp. were validated in controlled conditions using qPCR, revealing that the pathogen can germinate on the eucalyptus surface of some species without multiplying in the host. CG-TOF-MS analysis detected 26 compounds in the Eucalyptus spp. cuticle and led to the discovery of the role of hexadecanoic acid in the susceptibility of Eucalyptus grandis and Eucalyptus phaeotricha to A. psidii. We characterized the epicuticular wax morphology of the six previously selected Eucalyptus spp. using scanning electron microscopy and observed different behavior in A. psidii germination during host infection. It was found a correlation of epicuticular morphology on the resistance to A. psidii. However, in this study, we provide the first report of considerable interspecific variation in Eucalyptus spp. on the susceptibility to A. psidii and its correlation with cuticular waxes chemical compounds that seem to play a synergistic role as a preformed defense mechanism.

Novel insights into the early stages of ratoon stunting disease of sugarcane inferred from transcript and protein analysis

Despite of the importance of ratoon stunting disease, little is known on the responses of sugarcane to its causal agent, the vascular bacterial endophyte Leifsonia xyli subsp. xyli. The transcriptome and proteome of young plants of a susceptible cultivar with no symptoms of stunting but with relative low and high bacterial titers were compared at 30 and 60 days after inoculation. Increased bacterial titers were associated with alterations in the expression of 267 cDNAs and in the abundance of 150 proteins involved in plant growth, hormone metabolism, signal transduction and defense responses. Some alterations are predicted to benefit the pathogen, such as the up-regulation of genes involved in the synthesis of methionine. Also, genes and proteins of the cell division cycle were all down-regulated in plants with higher titers at both times. It is hypothesized that the negative effects on cell division related to increased bacterial titers is cumulative over time and its modulation by other host and environmental factors results in the stunting symptom.

Novel insights into the early stages of ratoon stunting disease of sugarcane inferred from transcript and protein analysis

Despite of the importance of ratoon stunting disease, little is known on the responses of sugarcane to its causal agent, the vascular bacterial endophyte Leifsonia xyli subsp. xyli. The transcriptome and proteome of young plants of a susceptible cultivar with no symptoms of stunting but with relative low and high bacterial titers were compared at 30 and 60 days after inoculation. Increased bacterial titers were associated with alterations in the expression of 267 cDNAs and in the abundance of 150 proteins involved in plant growth, hormone metabolism, signal transduction and defense responses. Some alterations are predicted to benefit the pathogen, such as the up-regulation of genes involved in the synthesis of methionine. Also, genes and proteins of the cell division cycle were all down-regulated in plants with higher titers at both times. It is hypothesized that the negative effects on cell division related to increased bacterial titers is cumulative over time and its modulation by other host and environmental factors results in the stunting symptom.

Structural and biochemical characteristics of citrus flowers associated with defence against a fungal pathogen.

The constitutive characters of plants can be structural or biochemical and play an important role in their defence against pathogens. Citrus postbloom fruit drop (PFD) caused by Colletotrichum spp. is one of the most important fungal diseases of citrus. The pathogen infects the flowers, leading to premature fruit drop and reducing citrus production. However, flower buds smaller than 8 mm long are usually not infected by Colletotrichum spp. Thus, this study investigated whether there are constitutive mechanisms in flower buds related to Colletotrichum spp. infection. We studied flower buds that were 2, 3, 4, 8, 12 and 15 mm long and petals, after anthesis, of sweet orange 'Valência' using light and scanning electron microscopy and histochemistry. We evaluated the effect of volatile organic compounds (VOCs) in flowers (R-limonene and linalool) on the in vitro growth of Colletotrichum acutatum. We found that the arrangement of the epidermal papillae in the petal primordia, the occurrence of prismatic crystals and the distribution of oil glands are the main differences between buds smaller than 8 mm and buds 8-15 mm long. Osmophores at the tips of petals produced and accumulated phenols, terpenes and lipophilic compounds. Flower buds smaller than 8 mm long have constitutive structural and biochemical barriers to Colletotrichum spp. infection. In addition, this is the first time that osmophores have been reported in citrus. Our study shows that natural terpenes of Citrus flowers inhibit the fungal growth in vitro, highlighting the potential use of terpenes for the chemical control of PFD in citrus.