Modulation of autophagy and protease activities by small bioactive compounds to reduce cell death and improve stress-induced microspore embryogenesis initiation in rapeseed and barley

Abstract

Microspore embryogenesis is a powerful biotechnological tool that is very useful in crop breeding for the rapid production of haploid and double-haploid embryos and plants. In this in vitro system, the haploid microspore is reprogrammed by the application of specific stress treatments. A high level of cell death after the stress is a major factor that greatly reduces embryogenesis yield at its initial stages. Autophagy is a degradation pathway that is present in all eukaryotes and plays key roles in a range of processes, including stress responses. Many proteases participate in autophagy and cell death; among them, cathepsins are the most abundant enzymes with a role in plant senescence and programmed cell death (PCD). Moreover, although plant genomes do not contain homologues of caspases, caspase 3-like activity (main executioner protease of animal cell death) has been detected in many plant PCD processes. Recent studies by our group in barley microspore cultures reported that the stress treatment required for inducing microspore embryogenesis (cold treatment), also produced reactive oxygen species (ROS) and cell death, concomitantly with the induction of autophagy, as well as cathepsin-like and caspase 3-like proteolytic activities. In the present study, we report new data on microspore embryogenesis of rapeseed that indicate, as in barley, activation of cell death and autophagy processes after the inductive stress. The results revealed that treatments modulating autophagy and proteases produced the same effect in the two plant systems, regardless of the stress applied, cold in barley or heat in rapeseed. Pharmacological treatments with small bioactive compounds that inhibit ROS, autophagy and specific cell death-proteases led to reduced cell death and an increased embryogenesis initiation rate in both, barley and rapeseed. Taken together, these findings open up new intervention pathways by modulating autophagy and proteases, which are very promising in terms of increasing the efficiency of in vitro microspore embryogenesis systems for biotechnological applications in crop breeding.

Impedance flow cytometry allows the early prediction of embryo yields in wheat (Triticum aestivum L.) microspore cultures

“…We easily monitored microspore viability evolution using IFC, from tiller harvesting to the first seven days of culture. The decrease in cell viability observed in our study is in agreement with other studies carried out on several isolated microspore culture systems [53][54][55]. The first viability decline (from D-21 to D0) may be associated with stress treatment effects.…”

“…Studies showed that in barley microspore cultures, autophagy is induced after pretreatment and could be responsible for stress-induced cell death within these microspores [54,55,57].…”

Advances in Plant Regeneration: Shake, Rattle and Roll

“…Recent reports have indicated that stress-induced cell death is a major factor that greatly reduces the yield of SE in various in vitro systems, particularly in microspore embryogenesis [77,78]. Markers of cellular death such as autophagy, the major catabolic process of eukaryotic cells, and cell death proteases (metacaspases, cathepsins and proteases with caspase 3-like activity) are activated during stress-induced microspore embryogenesis [71,[79][80][81][82]. Pharmacological treatments with inhibitors of autophagy and proteolytic activities lead to the reduction of cell death, consequently increasing the embryogenesis initiation rate [79][80][81].…”

Boosting autophagy in sexual reproduction: a plant perspective

“…Thus, a pollen grain generally contains one vegetative cell and two sperm cells (Berger & Twell, ). Although our current knowledge on the roles of autophagy in the differentiation and formation of plant sperm cells is limited, there are some studies showing that autophagic vacuole degradation participates in clearance of cytoplasm and organelles after the meiotic event to produce microspores and the subsequent mitotic event that leads to the formation of haploid gametophyte (McCue et al , ; Bárány et al , ; Pérez‐Pérez et al , ). The autophagic degradation of existing cellular components facilitates the rearrangement of the cytoplasm and formation of a new haploid nucleus in each microspore, which is essential for the transition from sporophyte to gametophyte (Zhao et al , ; Dündar et al , ).…”