Investigations into factors controlling growth and lignin synthesis in spruce

The Norway spruce is a large coniferous tree native to northern, central and eastern Europe. Conifers make up a large portion of terrestrial biomass and serve as an important carbon sink, with the majority of carbon going into the cell walls of woody tissues. The economically important Norway spruce is no exception.

The Norway spruce is a typical gymnosperm species – woody plants that produce naked seeds, on cones, without forming flowers and fruits – its secondary cell wall (water-conducting vascular tissue, also called ‘xylem’) contains 27% of an important element. Phenolic polymer – lignin.

Lignin provides rigidity and structural support to the polysaccharides of the cell wall. They are also evaluated for the production of important biomaterials. Thus, Norway Spruce is of importance not only as an important timber crop but also as a source of rich organic chemicals.

As such, much research has been done over the years to unravel the complex metabolic pathways involved in the growth and production of metabolites in this species, led by researchers in Finland.

Now, Professor Kazuyuki Kochitsu at Tokyo University of Science (TUS), Japan, a leading researcher of reactive oxygen species (ROS) in plants has collaborated with Finnish scientists to study embryonic biosynthesis in spruce.

Previous research has shown that the final polymerization stages in lignin production involve the oxidation of monolignol to phenolic radicals, which are then coupled non-enzymatically, using either hydrogen peroxide (H₂a₂) – the use of peroxidase or oxygenation using lactase. With time, the role of vital ROS enzymes in lignin synthesis and spruce growth has also been determined.

ROS, such as the superoxide anion radicals, H₂a₂, and hydroxyl radicals, can be produced by a number of sources in the plasma membrane and cell walls of plants and enter the apoplast (the space outside the plasma membrane of a plant cell). These sources include various enzymes, for example, oxidase and peroxidase, as well as respiratory burst oxidase homologues (RBOHs, also known as NADPH oxidases).

Using cytoplasmic NADPH (nicotinamide adenine dinucleotide phosphate reductase) as an electron donor, the plant produces RBOH superoxide anion radicals, which then dissociate into H₂a₂. Professor Kochitsu’s research revealed that this ROS production mechanism is essential for many molecular processes in plants including pollen tube growth and fertilization.

In embryonic and developing xylem cell culture from Norway spruce, PaRBOH1 is the most highly expressed RBOH gene.

But how is PaRBOH1 regulated?

To answer this question, Professor Kochitsu’s team from TUS in collaboration with Finnish scientists studied the ROS production activity and the regulatory mechanism of PaRBOH1 in gymnosperms, including spruce. Their study revealed, for the first time, that PaRBOH1 is activated by calcium ions and phosphorylated to produce ROS.

In addition, protein kinase activity was observed in the cell extract from developing xylem, resulting in the phosphorylation of some serine and threonine residues of PaRBOH1.

These results have been published in Frontiers of plant science. The multinational team that participated in the study included Dr. Kenji Hashimoto of TUS; Kaloyan Nikolov from the University of Oulu, Finland; Dr.. Adrien Gauthier of the Aguile Unit, Institut Polytechnique Unila Salle, France; and Dr. Anna Karkkonen from the Natural Resources Institute of Finland (NIRF), Finland.

Results of RBOH regulation in the first gymnosperm species examined, the Norway spruce, demonstrate that all seed plants—gymnosperms (bare seeds) or angiosperms (enclosed seeds)—share the same mechanisms for controlling RBOH activity.

Professor Kochitsu explains why this is important: “Reactive oxygen species have traditionally been thought of as toxicants, but our study shows that many plant functions, including stress response, plant growth and plant reproduction, are regulated by ROS produced by ROS bioenzymes.”

Professor Kochitsu also sheds light on the practical applications of their findings. With their potential as new sources of energy and materials, research into the development of trees and the mechanisms that govern the valuable components of their cells is gaining momentum. “In the future, our research may promote tree growth and help develop technology to produce valuable materials,” notes Professor Kochitsu.