Understanding the molecular mechanisms of plant regeneration is important for various horticultural and biotechnological procedures. For instance, you can regenerate a new plant organ by manipulating key regulator genes.
Moreover, knowledge of the molecular basis of in vitro plant regeneration enables further plant propagation of target commercial crops.
To unravel the molecular mechanism of plant regeneration, researchers have answered several fundamental questions in developmental and cell biology. Several experimental systems have been developed in target plants to gain insights about how plant tissues respond to wounding or plant growth regulators in vitro and how these stimuli drive the downstream developmental process to end with organ formation.
In this article, I will explain the molecular mechanisms underlying callus formation, organ regeneration, somatic embryogenesis and epigenetic control of plant regeneration.
How plant tissues respond to the wound stress?
In vitro, researchers commonly induce plant regeneration upon loss or injury of plant body parts. Following the injury, plant cells dedifferentiate, proliferate, and acquire new fates to repair damaged tissues or develop new organs from wound sites.
Just to clarify, dedifferentiation is the process where cells become less specialized. Typically, in developmental biology, you hear of “differentiation,” where cells become specialized into cell types. Here, the case is the opposite. Cells will no longer be specialized and will go back to their original cellular state. Then, they will be given new cellular fates in order to repair plant tissue.
The whole process implies that there is a complex network of transcriptional cascades to reprogram development and promote new cell fates.
In general, the plant regeneration in response to wound stress is mainly mediated by:
- Modification of hormonal signaling and homeostasis (the state of steady internal, physical, and chemical conditions maintained by living systems).
- Transcriptional modulation of key meristem or embryonic regulators.
In the first case, the hormonal modification necessarily implies a regulation of the balance of homeostasis to enable the metabolism to adapt to new hormonal conditions.
In the case of the transcriptional regulation, the way a developmental response is exerted at molecular level is activating genes (it is named transcriptional regulation). So if the program is for developing an organ, then meristematic genes are turned on (or off) and same apply for embryo development.
You can cut or wound a plant to induce plant regeneration. In cutting, a root or shoot meristem (a tiny tissue consisting of undifferentiated cells or meristematic cells capable of cell division) can be detached from the plant. Sometimes cutting is enough to induce de novo organ formation (new organs are formed without a preexisting meristem). Instead wounding (e.g. punching a tissue) can induce the growth regulator-dependent organ regeneration.
In general, wounding triggers a quick influx of calcium ions into cells followed by an increase of reactive oxygen species (ROS) that finally activates downstream signaling cascades and transcriptional outputs.
For instance, when cucumber roots are cut, that triggers an increase in hydrogen peroxide (H2O2) thereby promoting root regeneration.
Biotechnologically speaking, H2O2 can be applied exogenously to induce plant regeneration, however its application is plant-specific and/or dosage dependent. For example, H2O2 external application in cucumber induce roots but in wheat induce somatic embryogenesis (Ikeuchi et al 2019).
Molecular basis of callus formation
A callus can be defined as a mass of undifferentiated cells. These cells retain the potential or acquire the potential, in vitro, to develop as an embryo, or organ if you provide the appropriate culture conditions.
In molecular biology, a callus works as an intermediate vehicle in which an explant (the portion of plant tissue used in culture techniques) is converted into a new organ or embryo.
You can induce the callus formation in tissue culture by applying:
- Plant growth regulators (auxins or cytokinins)
- Causing stress through wounding
- Causing stress through other types of severe stress (e.g. high light or drought).
Growth regulators application
When explants are placed in an auxin-rich, callus-inducing medium, the developed calli resembles the root meristem histologically.
Some of the key regulators playing a role in the spatial expression of root meristem are WOX5 (WUSCHEL-RELATED HOMEOBOX 5) and SHR (SHORT ROOT). In other words, calli formation in response to PGRs like auxin rely on root initiation pathways.
Wound stress induction
However, when a calli is formed in response to wound, it promotes transcriptional activation of genes encoding the cytokinin biosynthesis like IPT3 (ISOPENTENYL TRANSFERASE 3), LOG1, LOG4, and LOG5 (LONELY GUY 1/4/5).
Repressors of the callus formation
You will not always get callus formation, however, certain negative regulators can impact callus response. For instance, studies suggest that very-long-chain fatty acids (VLCFAs) and their derivatives negatively regulate callus formation on auxin-rich medium. The repression of callus growth by VLCFA-derived signals seems to be due to the repression of the expression of ALF4 (ABERRANT LATERAL ROOT FORMATION), a gene involved in auxin signaling (as auxin triggers changes in gene expression, plant cells are susceptible to auxin signals).
Molecular Basis of organ regeneration
In tissue culture you can have different responses upon the wounding. It can be root or shoot formation or embryo development. Although there are still gaps in knowledge about the molecular mechanisms underlying the plant regeneration, it is important to know the fundamental basis to identify a possible cause of the responses in vitro. What we already know is that for each of these in vitro development programs there is a different molecular cause which can share key regulators with other processes.
Molecular basis of root regeneration
Upon injury, a root can be regenerated in two ways. When the root meristem is completely cut, root formation can regenerate over a non-pre existing meristem (called de novo root formation) or when the root meristem is partially lost, inducing the root formation over a pre-existing root meristem (called root meristem restoration). In both cases auxins play an essential role.
De novo root formation
Polar auxin transport (auxin transport from cell-to-cell in a directional way, from the root to the shoot and vice versa) is critical for auxin-mediated promotion of de novo root formation.
In the case of de novo root formation, the mechanism for regeneration will follow this process:
First auxin becomes detectable between 12-18 h after cutting near wound sites, especially in the vasculature (conducting tissue in plants which allows for the transport of water, minerals and nutrients). After, de novo roots emerge from this vasculature.
For instance, key auxin transporters, such as PIN1 (PIN-FORMED 1), PIN2, PIN3 and AUX1 (AUXIN RESISTANT 1), have been identified by playing roles in root regeneration from leaf explants.
Root meristem restoration
In the case of root meristem restoration, the mechanism for regeneration will follow this process:
When the farthest part of the meristem is lost by injury, the quiescent center (a special group of cells in the root tip) transfers this signal (the injury) to the surrounding cells promoting the root meristem restoration. The way in which this quiescent center transfers the information is altering auxin content in the surrounded cells.
In order to regulate the process, there are some key regulators that are induced locally in the area where the original root meristem was wounded or amputated. These regulators include:
ERF115 (ETHYLENE RESPONSE FACTOR 115)
PAT1 (PHYTOCHROME A SIGNAL TRANSDUCTION 1).
These two regulators promote reconstruction of the meristem.
For plant researchers, these regulators are critical to know because using molecular biology approaches, these gene regulators can be overexpressed to speed or increase the response for root formation.
Furthermore, the way these auxins are distributed within a plant is potentially what provides global special information for root regeneration. Auxin distribution within a plant also provides the plant with information about when and where to activate mitosis (part of the cell cycle in which replicated chromosomes are separated into two new nuclei). Auxins do this by reprogramming regulators which enables spatially controlled cell proliferation and patterning during root meristem reconstruction.
Molecular basis of shoot regeneration
The mechanism for shoot regeneration relies more on cytokinins. Cytokinins commonly induce shoot regeneration from competent cells (those with capability to acquire a new cell fate). Here, different molecular components associated with cytokinin sensing and signaling mediate the shoot regeneration.
For instance, the cytokinin receptor WOL (WOODEN LEG) plays a major role in shoot regeneration. When ARR1 (ARABIDOPSIS RESPONSE REGULATOR 1), a downstream gene of WOL is overexpressed in plants, they regenerate shoots in the absence of exogenous cytokinin application.
Also, when WUSCHEL (WUS), a gene underlying cytokinin-induced shoot regeneration is overexpressed, it is enough to trigger shoot formation ectopically (the abnormal gene expression within a cell or tissue type) in a cytokine-free medium.
Interestingly, only certain cells express WUS in callus cultured on a cytokinin-rich medium. Why? Well, it seems that other transcriptional factors like PHB (PHABULOSA), PHV (PHAVOLUTA), and REV (REVOLUTA) are also required for cytokine-independent WUS induction and subsequent shoot regeneration.
Molecular basis of somatic embryogenesis
Zygotic embryos from Arabidopsis plant species have been used to understand the molecular basis for somatic embryogenesis. Generally, these zygotic embryos cultured in auxin-rich medium produce calli. Subsequently, after the transfer to auxin-free medium, auxin response maxima (point within cells where auxin content is the highest) is established at external regions within the callus via the gene PIN1, an auxin transporter.
Furthermore, within 24 hours after media transfer, WUS and WOX5 start to be expressed in subsets of cells near the auxin response maxima and lead to specify the shoot and root poles within the embryo, respectively.
Although the cause of how somatic cells gain an embryonic fate under this culture condition remains unanswered, several genome-wide transcriptome analyses have revealed important transcriptional regulators highly interconnected that participate in embryogenesis like LEC1 and LEC2 (LEAFY COTYLEDON 1/2), AGL15 (AGAMOUS-LIKE 15) and BBM (BABY BOOM).
Many of these embryonic regulators appear to promote embryogenesis by modulating auxin biosynthesis and signaling.
Epigenetic control over plant regeneration
Accumulating evidence suggests the transcription of many reprogramming genes (which cause dynamic changes in gene expression and subsequently lead to changes in cell fate) are epigenetically regulated.
Epigenetics means that heritable phenotype (what you see in a plant like color and shape) is produced by changes that do not involve alterations in the DNA sequence. In other words, changes occur in proteins associated with DNA like histones but not in the DNA sequence. With epigenetics, genes can be induced or repressed in the right cells at the correct developmental window of time.
How does it happen? Well, maybe you have heard that epigenetic changes influence the chromatin environment through the modifications of histones by acetylation and methylation. The alterations in the histones are responsible for the activation or repression of gene expression.
For instance, PRC2 (POLYCOMB REPRESSIVE COMPLEX 2) is an evolutionarily conserved protein complex mediating the methylation of Histone 3. In vitro, PRC2 represses the expression of genes encoding embryonic regulators, including LEC2 and BBM in embryogenesis and WIND3 (WOUND-INDUCED DEDIFFERENTIATION 1) in callus formation. Thus, when above genes are ectopically expressed in PCR2 mutants, they lead to spontaneous somatic cellular dedifferentiation, callus formation, and embryoid development.
In summary, plant growth regulators and stress signaling (by cutting or wounding) are essential for defining cell fate in vitro. Thus, the uncovering of the gene regulatory network that underlies this crosstalk between PGRs and wounding will be fundamental to improve further horticultural and biotech processes.
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