Isolation of viable protoplasts from the aleurone layers of commercial barley malting varieties

Abstract

The use of aleurone‐layer protoplasts as a model system for barley germination research offers several advantages over working with the whole tissue, including the ability to more precisely control the level of hormone exposure during experiments, and to more easily perform cell biology studies using microscopy. However, protoplasts from the cultivar Himalaya have dominated these studies, probably owing to the availability of a suitable isolation protocol tailored to these hull‐less grains. Here the development of a protocol to isolate aleurone‐layer protoplasts from commercial malting barley cultivars is described. By implementing the protocol, protoplasts were isolated from three commercial Australian varieties and their responses to incubation with gibberellic acid evaluated. Key properties of the protoplasts that make them a good model for the aleurone layer are demonstrated. The soluble proteome of the isolated protoplasts was shown to be highly similar to that of the intact tissue. The cell death response of the protoplasts to treatment with gibberellic acid, which involves increased vacuolization, was visualized as increasingly transparent protoplasts under microscopy. The use of isolated protoplasts offers several applications of great potential to further understand the behaviour of the aleurone layer during germination and thus during the malting process. Copyright © 2016 The Institute of Brewing & Distilling

The impact of multiple abiotic stresses on ns-LTP2.8 gene transcript and ns-LTP2.8 protein accumulation in germinating barley (Hordeum vulgare L.) embryos

“…Samples (mature root (separated from the soil by intensive rinsing with distilled water for 5 seconds), mature leaf (three best-developed leaves from each plant), crown tissue, mature shoot, aleurone layer and embryonal axes) from both types of experiments were collected during the day time and immediately frozen in liquid nitrogen after sampling. Aleurone layers and embryonal axes were collected according to the modified protocol of Daneri-Castro and Roberts [ 47 ].…”

“…Extracted proteins were used to screen presence of mature ns-LTP2.8 protein across all the tested barley tissues. Protein extracts (30 μg TSP/well) were denatured and separated by SDS-PAGE using a 6% stacking gel and 17% separating gel [ 47 ] in a Mini-PROTEAN Vertical Electrophoresis Cell (Bio-Rad). Proteins were transferred to a PVDF membrane (Immobilon ® PSQ, Merck) by semidry protein transfer (55 min, 76 mA–parameters determined experimentally).…”

Over-Expression of a Wheat Late Maturity Alpha-Amylase Type 1 Impact on Starch Properties During Grain Development and Germination

“…Sixty grains per line were used for tissue isolation according to the method described by Barrero et al (2013) and Daneri-Castro and Roberts (2016) . The husk and embryo from mature grains were isolated and collected after imbibition with 40 ml water at 4°C for 4 h. The crease tissue was removed from deembryonated grains and the remaining tissues were imbibed in 10 ml of Milli-Q water containing 150 μg/ml Cefotaxime and 50 μg/ml nystatin for 16 h in Petri dishes at 4°C.…”

Protoplast: A Valuable Toolbox to Investigate Plant Stress Perception and Response

“…Indeed, protoplasts are obtained from the digestion of tissues containing a mixture of differentiated cell types that can display different locations of specific proteins ( Faraco et al, 2011 ). Even though some protocols exist to isolate protoplasts of specific cell types such as guard cell ( Zhao et al, 2019 ), aleurone layer cell ( Daneri-Castro and Roberts, 2016 ), or from various root tissues ( Demidchik et al, 2003 ), the complementary use of transgenic plants is recommended if a tissue-specific behavior of the process studied is anticipated ( Sharma et al, 2018 ). Instead, if no tissue-specificity is expected, protoplasts offer a valuable model to study physiological processes as it is less time-consuming to obtain than transgenic plant and can be performed in a broad range of plant species, contrary to agroinfiltration in leaves that are mainly restricted to the plant host Nicotiana benthamiana ( Sharma et al, 2018 ).…”