An international research team investigated the genetic regulation of stem cell division in plant stems. During their investigations, they revealed that the key gene coordinating stem cells, WOX4, is controlled by the vital plant hormone auxin via auxin response proteins. The results are valuable for both fundamental biology and ecology. Since the studied mechanism allows controlling wood formation it could help to solve economic and environmental problems. The research has been published in the journal Nature Communications.
Scientists from the Centre for Organismal Studies (Germany), Gregor Mendel Institute (Austria), Institute of Cytology and Genetics (Russia), Novosibirsk State University (Russia), Wageningen University (Netherlands) and Institute for Plant Molecular and Cellular Biology, Polytechnic University of Valencia (Spain) showed a key role of auxin response regulatory proteins in the cambium (the stem cell niche responsible for wood formation, and also for specified spatial coordination features of gene activity.
Although an influence of auxin on the activity of WOX4 was known before, the exact mechanism was unclear. In this work, the authors demonstrated a direct regulation of WOX4 by auxin signaling factors.
"Due to the strong focus on individual cell types, our analysis represents an important milestone for our research on cambium regulation. We more and more understand the exciting complexity of the regulatory network important for a very dynamic growth process which produce a large part of the biomass present on this planet," said Thomas Greb professor of the Heidelberg University Centre for Organismal Studies and principal investigator of this study.
The amazing cambium and the great auxin
The cambium mediates wood formation which represents a large proportion of terrestrial biomass. The cambium is unique due to producing two different tissues at the same time -- xylem also known as wood and phloem -- in a bidirectional manner.
The xylem transports water together with dissolved nutrients and the phloem transports sugars essential for growth and development of cells. «Adult» vascular tissues are formed by dead cells and grow constantly in volume due to divisions of stem cells sitting in the cambium. It is important that xylem and phloem are not only produced during the embryonic period but throughout the plant life cycle. When the cambium «dies», so does the stem.
Scientists managed to study wood formation mechanisms in model plant Arabidopsis thaliana, the weed that doesn't form thick wood itself. Intriguing, but Arabidopsis has all the genetic mechanisms required for wood formation. Thus, all the advantages of the model plant -- a small size, a short life cycle and well-annotated genome - are used to investigate the complicated mechanism.
It was shown on Arabidopsis that a small molecule auxin is a King among plant hormones. It's responsible for a huge amount of different jobs -- following the sunlight by plants, responding to gravity, as well as controlling development of all plant organs, including embryos, roots, leaves and stems.
Genetic manipulations with plants depicted relations between cambium regulatory proteins and auxin
Cambium activity has been tightly associated with the plant hormone auxin for a long time, but at the level of gene regulation (the «reading» of the genetic information), the process was studied with such a high accuracy for the first time. To do that the collaborators from Germany, Austria and Spain created transgenic plants with visualized regulatory proteins, responsible for cambium formation and further differentiation (WOX4, ARFs and others). With these plants, it was possible to observe the spatial distribution of auxin signaling components and auxin responses in the cambium area.
**Vocabulary: Transgenic plants are the ones, whose DNA is modified using genetic engineering techniques. The aim is to introduce a new trait to the plant which does not occur naturally in the species. For scientific purposes the trait might be an ability to visualize a certain protein under a fluorescent microscope. **
Firstly, using transgenic plants the authors detected auxin response signal in the xylem-associated cambium. Secondly, study of transgenic lines visualizing regulatory proteins in the cambium discovered that WOX4 meets the auxin response factor ARF5 exactly in the xylem-associated cambium.
Other auxin response factors ARF3, ARF4 were also active in cambium, but their expression domains were broader.
The authors showed that it was no coincidence that the cambium cells express both WOX4 and ARF5 regulatory proteins together with the auxin response sensor. To prove that they all are pieces of one puzzle, the scientists performed a rigorous study of the regulatory protein expression in the mutant plants. Mutant plants had a changed activity of the key regulatory proteins: the regulator was either not synthesized at all, or was «broken» with a reduced or enhanced activity. Mutants for key regulators of cambium growth had significant differences in the thickness of the cambium layer and arf5 and wox4 mutant plants had opposite effects on cambium: it was wider than normal in arf5 and smaller in wox4. In both mutants the corresponding genes were damaged, thus, ARF5 and WOX4 proteins were not synthesized leading to the disruption of cambium proliferation functioning.
*Note: don't be confused: genes and proteins synthesized from them are differently marked in text. The gene itself is marked with italic script and small letters, and the protein -- with straight text and capital letters.*
Moreover, higher level of WOX4 protein was detected in arf5 mutant. Simply this means that when ARF5 protein is broken it doesn't affect WOX4 gene expression, so that the gene activity increases. Together these observations proposes that ARF5 fulfils its function partly by directly attenuating WOX4 activity.
ARF5 protein action on WOX4 gene activity might be direct
The next question arises if ARF5 affects WOX4 directly, or there were other mediators. The scientists from Novosibirsk, Russia predicted the presence of two binding sites for auxin response factor ARF5 in WOX4 regulatory region. One of the sites have been shown bound by ARF5 in vitro.
**Vocabulary: Binding site is a short segment of DNA sequence that are specifically bound by transcriptional factor (regulatory protein) and initiates RNA synthesis from the located nearby gene. There could be several binding sites in a gene proximity. **
"In the genetic engineering experiments our colleagues investigated that auxin response factor ARF5 influences the expression of transcription factor - WOX4. There could be many mechanisms of such regulation, one of them is following: the regulatory protein binds DNA in the particular location - binding site - upstream of the gene. To identify potential ARF5 binding sites in WOX4 regulatory region was a challenge. To solve this task, we analyzed previously validated ARF5 binding sites in the upstream regions of other genes. As a result of bioinformatic analysis, we predicted two 8 nucleotides in length binding sites near WOX4 gene," said Daria Novikova, Ph.D student at the Institute of Cytology and Genetics SB RAS and Wageningen University, engineer at the Computational Transcriptomic and Evolutionary Bioinformatics Lab, Novosibirsk State University
The international group of scientists enlighten a long-observed role of auxin signalling in radial plant growth. Three auxin response factors -- ARF3, ARF4 and ARF5 have been identified in regulating of cambium activity. The distribution of the response factors together with other cambium regulators was observed in plant tissues in detail in comprehensive gene-engineering experiments.
The paradox is that although ARF5 is crucial for promoting plant growth, however it acts as a repressor for WOX4 and thus restricts the cambium formation. At the same time, WOX4 depends positively on auxin signalling overall. So the particular role of other auxin response factors (except ARF5) in control of cambium activity still remains puzzling and has to be discovered further.
The study was supported by the German Research Foundation, Heisenberg professorship to Thomas Greb, Russian Science foundation.