Flávio Costa Miguens

Identification of compatible and incompatible interactions between Arabidopsis thaliana and Xanthomonas campestris pv. campestris and characterization of the hypersensitive response

Both compatible and incompatible interactions between Arabidopsis thaliana and Xanthomonas campestris have been identified and, for the first time, a strong hypersensitive response has been characterized. A highly reproducible mass spray inoculation protocol has been established and was used together with the more commonly used infiltration inoculation procedure to study the defense responses occurring in mature A. thaliana plants. A series of bacterial strains have been tested on A. thaliana ecotype Columbia (Col-O). X. c. pv. campestris was the most effective pathogen in these tests and was used for further detailed analysis. Several X. c. pv. campestris isolates were tested on A. thaliana Col-O, and one particular X. c. pv. campestris strain (147) was tested on 27 Arabidopsis ecotypes. Symptom development of compatible and incompatible interactions, including the hypersensitive response, was extensively characterized in A. thaliana Col-O. Lesion structure, bacterial distribution, accumulation of polyphenolic compounds, and the deposit of callose in inoculated leaves were documented by microscopic analysis. Activation of the defense-associated genes coding for phenylalanine ammonia lyase (PAL), beta-1, 3-glucanases, chitinases, and peroxidases was evaluated by Northern blot analysis.

Digestion of legume starch granules by larvae of Zabrotes subfasciatus (Coleoptera : Bruchidae) and the induction of α-amylases in response to different diets

Zabrotes subfasciatus larvae possess three alpha-amylase isoforms as determined by in gel assays following SDS-PAGE. The two minor isoforms present lower electrophoretic mobility than the major form, and seem to occur as a heterodimer. When developed inside Vigna unguiculata (cowpea) seeds, fourth instar larvae have minor quantities of the slow-migrating forms, but when reared on seeds of Phaseolus vulgaris (common bean) or Phaseolus lunatus, the two slow-migrating forms are expressed in higher amounts, while activity of the major form was independent of the host seed. Larvae developing inside cowpea seeds at the beginning of the fourth instar were fed on flour from cotyledons of cowpea or common bean. Larvae fed on the common bean flour started to express the dimer in higher amounts when compared with the control larvae fed on cowpea flour. In an attempt to correlate differences between starch granules and the induction of alpha-amylases, a detailed study on the digestive process of the granules was conducted. Incorporation of purified starch granules into artificial diets did not induce the two minor alpha-amylases. The in vitro hydrolysis rates of purified granules and the pattern of dextrins liberated by the different alpha-amylases were similar for the two legume species. The starch granules enter the midgut extensively damaged, which may facilitate the access to the more susceptible parts of the granules to enzymatic attack.

A scanning electron microscope study of normal and vitrified leaves from Datura insignis plantlets cultured in vitro

The surface anatomy of normal and vitreous leaves of plantlets obtained from Datura insignis Barb Rodr nodal segment cultures was compared using scanning electron microscopy. Normal and vitrified leaves are similar in several ways. They are both amphistomatic, and have similar distributions of glandular and non-glandular trichomes. Stomata have similar length, diameter and distribution in normal and vitreous plants. Immature stomata, which have closed pores, and plugged stomata, which contain an amorphous material between their guard cells, occur in both normal and vitrified leaves. Normal and vitreous leaves differ in the frequency of normal and abnormal stomata. Normal stomata have kidney-shaped guard cells and resemble closely those found in field-grown plants, whereas abnormal stomata have deformed guard cells. Normal stomata represent approximately 80% of the total number of stomata in normal leaves, but only 7% of the total number of stomata in vitreous leaves. Abnormal stomata represent 90% of the total number in vitreous leaves. The deformation of guard cells could possibly be a mechanical impediment to stomatal function.

Sugarcane cell wall structure and lignin distribution investigated by confocal and electron microscopy

Lignocellulosic plant cell wall is considered a potential source for second generation biofuels. The plant cell wall is a highly complex structure mainly composed of cellulose, hemicelluloses, and lignin that form a network of crosslinked fibers. The structural organization of the sugarcane cell wall has not been previously analyzed in detail, and this analysis is a prerequisite for further studies on the recalcitrance and deconstruction of its biomass. In this work, cellulose and lignin localization were investigated by confocal laser scanning microscopy. In addition, the internode sugarcane cell wall structural organization was analyzed by electron microscopy. Internode stem anatomy showed a typical monocot structure consisting of epidermis, hypoderm, and vascular bundles scattered throughout ground parenchyma tissue and surrounded by sclerenchyma fibers. Confocal images of safranin labeled sugarcane showed that lignin distribution was predominant in the vessel elements, cell wall corners (CC), and middle lamella (ML), while cellulose‐rich cell walls were randomly distributed in the ML and organized in the other cell wall layers. KMnO4 cytochemistry revealed that lignin was predominantly distributed in secondary cell walls, ML and CC. Cell wall sublayers (S1, S2, and S3) were identified and measured by transmission electron microscopy. Our results provide insights that may help further understanding of sugarcane cell wall organization, which is crucial for the research and technology of plant‐based biofuel production. Microsc. Res. Tech. 76:829–834, 2013.

Ultrastructure and immunolabelling of fungi cells treated with Vigna unguiculata vicilins (7S storage proteins)

Abstract Scanning electron microscopy showed that vicilins from cowpea seeds affect the growth and inhibit spore germination of Fusarium oxysporum and leads to abnormal development (sporulation) of Saccharomyces cerevisiae cells. Control treatments induced normal hyphal and cell development. Fluorescence microscopy and immunolabelling analysis showed that anti-vicilin serum strongly labelled the cell surfaces of yeast and filamentous fungi, previously incubated with these seed proteins. These results suggest that the inhibitory effects of cowpea vicilins on fungi growth may be exerted through their ability to bind to the cell surfaces of the micro-organisms, cell wall and/or plasma membrane.

Fine structure of phloematic trypanosomatid–coconut tree interaction

Phytomonas wilt or Hartrot is a fatal disease of palm (Arecaceae) species including Cocos nucifera (coconut) and is caused by a phloematic trypanosomatid, a promastigote parasite that inhabits phloem sieve elements of disease palms. In the present work, we described the morphology of the interaction between a phloematic trypanosomatid (Phytomonas staheli) and C. nucifera. Two varieties and one ecotype of the adult coconut palm from northeast and southeast Brazil were analyzed, totaling 34,000 plants. Coconut palm losses due to Hartrot varied according to the variety or ecotype and geographic area. Occurrence of Hartrot was insignificant in Rio de Janeiro state (southeast), but in Bahia state (northeast) losses were substantial when appropriate cultural practices were not applied. Symptomatic and healthy palm tissues were analyzed by light and electron microscopy. Laboratory diagnoses revealed the twisted promastigote form of phloematic trypanosomatids in the extracts of shoot apex, leaves, stems and inflorescence in diseased plants, but not in the healthy ones. No parasites were found in the roots. Although the general anatomy of healthy and diseased palms was similar, callose deposition in the sieve plates was revealed by histochemistry and immunocytochemistry in the diseased tissue. Plugging by the P-protein and plastid alterations was also observed. Our observations strongly suggest that parasite traffic between sieve elements took place, although their cell bodies were larger than the sieve pores. Phloematic trypanosomatid proliferation in the sieve tube elements might interrupt the transport of phloem or/and consume plant nutrients. In addition, an association between the percentage of sieve elements colonized by pathogen in palm tissues and disease severity was established.

Report of Balanorchis anastrophus in Pará state with surface topography by scanning electron microscopy

Balanorchis anastrophus Fischoeder, 1901, from the reticulum of Bos taurus is reported for the first time in the State of Pará, Brazil. The surface topography as revealed by scanning electron microscopy is presented.

Tillandsia stricta Sol (Bromeliaceae) leaves as monitors of airborne particulate matter—A comparative SEM methods evaluation: Unveiling an accurate and odd HP‐SEM method

Airborne particulate matter (PM) has been included among the most important air pollutants by governmental environment agencies and academy researchers. The use of terrestrial plants for monitoring PM has been widely accepted, particularly when it is coupled with SEM/EDS. Herein, Tillandsia stricta leaves were used as monitors of PM, focusing on a comparative evaluation of Environmental SEM (ESEM) and High‐Pressure SEM (HPSEM). In addition, specimens air‐dried at formaldehyde atmosphere (AD/FA) were introduced as an SEM procedure. Hydrated specimen observation by ESEM was the best way to get information from T. stricta leaves. If any artifacts were introduced by AD/FA, they were indiscernible from those caused by CPD. Leaf anatomy was always well preserved. PM density was determined on adaxial and abaxial leaf epidermis for each of the SEM proceedings. When compared with ESEM, particle extraction varied from 0 to 20% in air‐dried leaves while 23–78% of particles deposited on leaves surfaces were extracted by CPD procedures. ESEM was obviously the best choice over other methods but morphological artifacts increased in function of operation time while HPSEM operation time was without limit. AD/FA avoided the shrinkage observed in the air‐dried leaves and particle extraction was low when compared with CPD. Structural and particle density results suggest AD/FA as an important methodological approach to air pollution biomonitoring that can be widely used in all electron microscopy labs. Otherwise, previous PM assessments using terrestrial plants as biomonitors and performed by conventional SEM could have underestimated airborne particulate matter concentration.

Analytical Microscopy as a Tool in Sediments Study

Historically, microscopes were used for descriptions of animals, plants and non-living specimens. In 19 th century, the shape-function relationship marked microscopic studies. The later 20 th century witnessed the nanometric special resolution provided by electron microscopes. As we enter the 21 st century, the analytical electron microscopy promises to change our comprehension about the nano-world. Chemical analyses of the shape will redesign our understanding of the structure and functioning of animals, plants and non-living specimens, including sediments.