A new method for determination of the photosynthetic pathway in grasses

Abstract

An easy and inexpensive method of determining the photosynthetic pathway in grasses using a dye widely used in microscopy. To evaluate the efficiency of a new histochemical test for determination of the photosynthetic pathway in grasses (Poacea). Leaves of 58 grass species were sectioned transversally, and the sections treated with a 2% sodium hypochlorite solution to clarify the tissue. After discoloration, sections were washed with distilled water and double-stained with astra blue and safranin (1% each in 50% ethanol) for 1 min. Sections were then mounted between microscopy glass slides and coverslips using water. Grass species showing red staining of the bundle sheath cells were considered C4, and species with translucent bundle sheath were considered C3. The results of the histochemical test were then compared with results from carbon isotope composition analysis and the relevant scientific literature. Observations from the histochemical test were congruent with results from δ13C isotope composition analysis, and with data previously presented in the scientific literature. The proposed histochemical test proved efficient for characterization of the photosynthetic pathway in the tested grasses; however, the method should be further tested in a greater number of grass species, encompassing, preferably, all Poacea subfamilies. Future studies may elucidate if the proposed method can effectively be used in other botanical families. Furthermore, additional investigations may determine whether the phenolic compounds indicated by the histochemical test are exclusive to the bundle sheath of C4 grasses and if possible relations exist between these phenolic compounds and the C4 photosynthetic pathway in grasses.

Red Panda feces from Eastern Himalaya as a modern analogue for palaeodietary and palaeoecological analyses

“…C 3 plants in the region are mainly represented by arboreal angiosperms and ferns. However, members of the Bambusoideae are also C 3 plants and display a range of carbon isotope values between − 26.4‰ and − 30.8‰ 54 , which closely overlaps with our data. Other angiosperms and ferns may also have contributed to these values.…”

Ferulic acid in C<sub>4</sub> grasses.

“…The authors also observed that only bundle sheath cells of C4 grasses stained red, indicating that safranin reacts with some unknown phenolic substances present exclusively in C4 grasses bundle sheath cells. Neto and Guerra [3] performed the histochemical test in 58 grass species with different photosynthetic pathways (C3 and C4 of all three types), comparing the results with isotope composition analysis and relevant literature. The pattern observed by Neto, Mardegan and Barbosa [1] was confirmed by Neto and Guerra [3], proving the efficacy of the test and suggesting the possible involvement of the reactive phenolic substance(s) in the photosynthesis of C4 grasses.…”

“…We scrutinized the identity of the phenolic compounds responsible for the positive response in the C4-safranin histochemical test comparing two grass species: Arundo donax (C3) and Urochloa brizantha (C4). Both species were previously identified and investigated in the study by Neto and Guerra [3].…”

“…The results found by Neto, Mardegan and Barbosa [1] and Neto and Guerra [3] suggest the presence of one or more phenolic substances exclusively stored in the protoplasm of C4 grasses' bundle sheath cells. This apparently compartmentalized location suggests a possible physiological function, which could differ from previously known functions of ferulic acid in grasses (Carpita [17]; Buanafina [9]; Buanafina et al [18]; Buanafina et al [19]).…”

“…Neto and Guerra [3] used the C4-safranin histochemical test to screen 58 grass species (C3 and C4 of different types: NAD dependent malic enzyme (ME-NAD), NADP dependent malic enzyme (ME-NADP) and phosphoenolpyruvate carboxykinase (PCK)), confirming the efficacy of the method in grasses, although apparently it does not apply in Cyperaceae. Furthermore, these authors observed that the phenolic substances are located within the protoplast of bundle sheath cells, not on the cell walls.…”