Molecular basis and evolutionary route of cell pluripotency in plant organ regeneration
De novo organ regeneration refers to the formation of adventitious shoots (i.e. de novo root regeneration) or roots (i.e. de novo shoot regeneration) after injury.
Under natural conditions, de novo organ regeneration allows plants regrow or replenish shoot or root branches, thereby contributing to shoot or root system architecture. Adventitious shoots can regenerate from the cambium region of the wounded stem after trimming of the tree crown. Adventitious roots can regenerate at the bottom wound of a stem or from detached leaves, such as root formation in cuttings.
In in vitro tissue culture, the ability to regenerate adventitious roots/shoots has long been exploited for vegetative propagation. In the 1950s, Skoog and Miller found that different concentrations of auxin and cytokinin in the medium were the key to induce the formation of callus, roots, and shoots. This discovery laid the foundation for the exploitation of organogenesis in tissue culture and was considered to be one of “the scientific roots of modern plant biotechnology” (Sussex, 2008, Plant Cell 20:1189-1198).
Theoretically, organ regeneration is based on plant cell pluripotency. How a single somatic cell can become a whole plant is a key question in the fields of plant cell biology and development (Vogel, 2005, Science 309:86).
2. Scientific questions
The overall goal of our research is to reveal the molecular basis and evolutionary route of cell pluripotency that allows plants to regenerate adventitious roots and shoots to replenish root/shoot system architecture.
With the progress of recent research on plant regeneration, it is now widely accepted that the root primordium (RP) or RP-like cell (e.g. callus) serves as the pluripotent stem cell for shoot and root regeneration under either natural conditions or in vitro tissue culture. Our specific scientific questions are: (1) how to establish RP or PR-like cells; (2) the molecular mechanism of RP pluripotency; (3) the evolutionary route of RP.
3. Establishment of methods to study de novo organ regeneration
The cellular and molecular mechanisms underlying adventitious rooting and shooting from wounded plants are barely understood because of the lack of efficient research methods. Therefore, we first tried to establish methods to study de novo organ regeneration mimicking natural conditions.
To study de novo shoot regeneration, we established a method in which Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) seedlings are decapitated at the hypocotyl, and then adventitious shoots regenerate from the decapitated hypocotyls. The adventitious shoot can normally develop to replace the decapitated shoot. No exogenous hormones are supplied in this system. Therefore, the shooting process is dependent on endogenous hormones in the plant and mimics adventitious shooting under natural conditions.
To study de novo root regeneration, we established a system in which Arabidopsis leaf explants are cultured on B5 medium without exogenous hormones. This simple method results in adventitious root formation from the wounded site on the leaf explant.
We have also used an in vitro tissue culture system for root and shoot regeneration. By comparing organ regeneration under natural conditions and in in vitro tissue culture, we have explored whether the regenerative abilities in in vitro tissue culture borrow from natural regenerative abilities.
4. Cell lineage and molecular framework of de novo organ regeneration
4.1 Establishment of RP
The RP establishment is a key step in de novo organ regeneration. Not all cells are able to initiate a RP, and only some regeneration-competent cells (e.g. pericycle, procambium, and some parenchyma cells) within the vasculature are able to undergo fate transition.
In de novo root regeneration, early signals (wound signals, environmental signals, and developmental status) may converge in mesophyll cells, leaf margin cells and some vascular cells (simply referred to as converter cells) to promote auxin production in the leaf explant. Auxin is then transported from converter cells into regeneration-competent cells (e.g. procambium cells) and triggers cell fate transition. The first step of cell fate transition is the priming of the regeneration-competent cell to become the root founder cell with specific expression of WOX11/12. The second step is the initiation of the RP from the root founder cell by cell division. In this step, WOX11/12 expression decreases while WOX5/7 and LBD16 are co-expressed in the RP cells.
In in vitro tissue culture, callus formation also requires two steps of cell fate transition. The first step is the priming of the regeneration-competent cell to become the callus founder cell through activation of WOX11/12 by exogenous auxin. The second step is initiation of the callus cells from the callus founder cell with cell division. The cellular nature of the newly formed callus is a group of RP-like cells that co-express WOX5/7 and LBD16.
Overall, establishment of the RP or RP-like callus requires two steps of cell fate transition guided by auxin. Expression of WOX11/12 marks founder cell formation, and co-expression of WOX5/7 and LBD16 marks the identity of the RP or RP-like callus.
4.2 Organ formation from pluripotent RP
The RP cells are pluripotent and can give rise to either roots or shoots.
In de novo root regeneration from leaf explants, the RP could be patterned into a root apical meristem with continuous cell division. In this step, endogenous cytokinin is upregulated and the auxin maximum is gradually restricted to the stem cell niche surrounded by cytokinin within the root apical meristem. At the molecular level, LBD16 expression decreases and WOX5/7 expression is gradually restricted to the stem cell niche. After that, the mature root tip can emerge from the leaf explant.
In de novo shoot regeneration from wounded hypocotyls, endogenous auxin first promotes the formation of a root founder-like cell mediated by WOX11/12, and then an RP-like cell mediated by WOX5/7. Meanwhile, cytokinin is transported from the root to the hypocotyl to activate the shoot progenitor marker gene WUS in the RP-like cell to fulfill its transition from an RP-like cell to a shoot progenitor cell. The shoot progenitor cell eventually differentiates into the shoot apical meristem, in which WUS is restricted to the stem cell niche.
In in vitro tissue culture, the root apical meristem can be patterned from PR-like callus on root-inducing medium with a low level of auxin to form an adventitious root. The RP-like callus cell can also change fate to become a shoot progenitor cell with WUS expression on shoot-inducing medium with a high level of cytokinin to form an adventitious shoot. Therefore, the ability of callus to regenerate roots and shoots in in vitro tissue culture might ‘borrow’ from the natural mechanism of adventitious rooting and shooting in wounded plants.
Overall, adventitious roots and shoots form from the RP or PR-like callus. A high auxin/low cytokinin ratio promotes the RP to differentiate into a root apical meristem. A high cytokinin level transdifferentiates the RP to become a shoot progenitor cell, which then differentiates into a shoot apical meristem guided by a high cytokinin/low auxin ratio.
4.3 Molecular mechanism of RP pluripotency
If auxin is the dominant hormone in the RP, then the RP will pattern into a root apical meristem with functional domains and finally differentiate to become an adventitious root. In contrast, if cytokinin becomes the dominant hormone in the RP, then the RP will change its fate to a shoot progenitor cell with WUS expression and then pattern into a shoot apical meristem to become an adventitious shoot. Therefore, auxin and cytokinin determine the fate of the pluripotent RP to undergo the rooting or shooting pathway, respectively.
Our preliminary data have shown that a group of RP-related genes including WOX5/7 and LBD16 are involved in pluripotency acquisition. Mutations in these genes can cause a loss of shoot and root formation from the RP or RP-like callus. Our hypothesis is that WOX5/7 might be involved in creating a cytokinin-hypersensitive environment in the RP, thereby allowing the RP to switch fate to become a shoot progenitor cell by cytokinin induction. We are now trying to understand: 1) the molecular mechanism of WOX5/7 and LBD16 in pluripotency acquisition; and 2) the molecular pathway to switch the fate of a differentiated somatic cell to a pluripotent cell.
5. Molecular evolution of RP
The appearance of vascular plants was a great step in the colonization of land by plants during evolution (Pires and Dolan, 2012, Phil. Trans. R. Soc. B 367:508-518). The vascular plants evolved into several lineages, two of which survive today: lycophytes and euphyllophytes. The euphyllophytes include psilotophytes, ferns, seed plants, etc. We have collected typical model plants to study the initiation and evolution of different types of roots.
Early vascular plants on earth formed only the shoot without roots and leaves. Based on the fossil evidence and the root anatomy of extant vascular plants, there were separate root-evolution events that resulted in different types of roots in extant vascular plants. In the lycophyte lineage, Selaginella kraussiana has only one type of root, i.e. the bifurcating root, in which the division of the root apical meristem gives rise to independent autonomous twin root meristems. In the euphyllophyte lineage, the psilotophyte Psilotum nudum has no roots; the fern Ceratopteris richardii has adventitious roots and endodermis-derived lateral roots; and the seed plant Arabidopsis thaliana has a primary root, adventitious roots, and pericycle-derived lateral roots. Using these model plants, we have attempted to analyze the evolutionary route of RP initiation and reveal how pluripotent RP stem cells evolved in plants.