SummaryHistochemical analyses in plants are commonly performed on hand-made sections of fresh materials. The disadvantages of embedding in historesin, paraffin or paraplast® are the alterations to cellular contents, the high costs and few evident results, depending on the test. Polyethylene-glycol (PEG), as a low cost, hydrophilic medium that maintains most of the cellular features similar to fresh conditions, may be useful for obtaining good histochemical results in thinner and homogeneous sections. The current study aimed to compare the efficiency of PEG as an embedding medium for histochemical analyses of primary and secondary metabolites accumulation. Using hand-made sections of fresh samples (T1) as a comparison, we tested the influence of the use of Karnovsky's solution as a fixative (T2) versus embedding in PEG (T3). The samples herein analyzed comprise leaves, stems, seeds and insect galls of different plant species. Neither the Karnovsky's fixative nor the embedding in PEG altered the histochemical results for starch, lipids, terpenoids, proteins and reducing sugars in T1, T2, and T3. However, PEG binds to phenols, such as tannins, flavonoids and lignins, thereby presenting false negatives in T3. (J Histochem Cytochem 62:577-583, 2014)

Insect galls may be study models to test the distribution of pectins and arabinogalactan-proteins (AGPs) and their related functions during plant cell cycles. These molecules are herein histochemically and immunocitochemically investigated in the kidney-shaped gall induced by Baccharopelma dracunculifoliae (Psyllidae) on leaves of Baccharis dracunculifolia DC. (Asteraceae) on developmental basis. The homogalacturonans (HGAs) (labeled by JIM5) and the arabinans (labeled by LM6) were detected either in non-galled leaves or in young galls, and indicated stiffening of epidermal cell walls, which is an important step for cell redifferentiation. The labeling of HGAs by JIM7 changed from young to senescent stage, with an increase in the rigidity of cell walls, which is important for the acquaintance of the final gall shape and for the mechanical opening of the gall. The variation on the degree of HGAs during gall development indicated differential PMEs activity during gall development. The epitopes recognized by LM2 (AGP glycan) and LM5 (1–4-β-D-galactans) had poor alterations from non-galled leaves towards gall maturation and senescence. Moreover, the dynamics of pectin and AGPs on two comparable mature kidney-shaped galls on B. dracunculifolia and on B. reticularia revealed specific peculiarities. Our results indicate that similar gall morphotypes in cogeneric host species may present distinct cell responses in the subcelular level, and also corroborate the functions proposed in literature for HGAs.

The dynamics of cell wall components during gall development are related to structural specialization to meet the defensive or nutritional requirements of gall tissues. Cell wall features have been studied mostly in galls induced by hemipterans (Psylloidea), while galls induced by Cecidomyiidae have been little explored. We applied monoclonal antibodies to epitopes of proteins and pectins in the cell walls of non-galled leaves and galls induced by Clinodiplosis sp. (Diptera; Cecidomyiidae) on Croton floribundus Spreng. (Euphorbiaceae). The complexity of tissue zonation in Clinodiplosis galls reflected the impairment of the activity of the pectin-methylesterases during development. The labeling of the epitopes of extensins in young galls denoted cell enlargement with resistant cell walls, while the labeling of epitopes of the arabinogalactan proteins in senescent galls indicated the involvement of these proteins with programmed cell death, at the end of cell cycles at the gall development site. We conclude that the cell wall profile in Clinodiplosis galls implies an imbalance between tissue porosity, cell-to-cell signaling, and resistance linked to tissue structural and functional compartments. Current data confirm the presence of the epitopes of extensins in young galls, and the compartmentalization of homogalacturonans and rhamnogalacturonan I in galls as an independent taxon feature.

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