Stepwise evolution of the developmental and symbiotic functions of DELLA in land plants

This study reveals that while the developmental functions of DELLA proteins are evolutionarily conserved in the GA-lacking liverwort *Marchantia paleacea*, their role in arbuscular mycorrhizal symbiosis evolved later in vascular plants, likely driven by the integration of gibberellic acid-mediated systemic control.

The Gist

The Big Picture: The "Brake Pedal" of Plant Growth

Imagine a plant as a car. In flowering plants (like roses or corn), there is a specific gas pedal called Gibberellic Acid (GA) that tells the car to speed up. There is also a "brake pedal" called DELLA.

Normally, when the gas pedal (GA) is pressed, it signals the brake (DELLA) to be removed from the car so the plant can grow tall and fast. If the brake is stuck on (a mutation in DELLA), the plant stays short and dwarfed. This is how the "Green Revolution" created high-yield crops: scientists tweaked the brakes so they were easier to release.

But here is the mystery: DELLA proteins exist in ancient plants (like liverworts) that don't even have a gas pedal (GA). They have the brake, but no way to press the gas. So, what is this brake doing in a car that has no engine?

The Experiment: Testing the Ancient Brake

The researchers decided to investigate this using a liverwort called Marchantia paleacea. Think of this plant as a "living fossil"—it's a simple, non-vascular plant that hasn't changed much in hundreds of millions of years. It lacks the ability to make or sense the GA hormone.

1. Cutting the Brake (The Mutants)
The scientists used a genetic "scissors" (CRISPR/Cas9) to cut out the DELLA gene in these liverworts. They wanted to see what happens if you remove the brake pedal entirely.

• The Result: The plants became smaller, grew slower, and produced fewer "baby plants" (gemma cups). They also turned a lighter shade of green (less chlorophyll).
• The Analogy: It's like taking the brake pedal out of a car and realizing the car actually drives better with it, or perhaps the brake was actually helping the engine run smoothly in a different way. In this case, the "brake" (DELLA) was actually acting as a positive regulator for growth in these ancient plants. It wasn't stopping growth; it was helping coordinate it with light and energy.

2. The Symbiosis Test: The "Roommate" Agreement
In modern flowering plants, DELLA plays a huge role in symbiosis. This is when plants invite fungi (like Rhizophagus irregularis) to live inside their roots. The fungi trade nutrients for sugar. In modern plants, DELLA acts like a manager that helps negotiate this deal. If you remove DELLA in modern plants, the fungi can't get in, and the partnership fails.

The researchers asked: Did this ancient liverwort need DELLA to make friends with fungi?

• The Result: Surprisingly, no. Even without the DELLA gene, the liverworts successfully invited the fungi in. The fungi grew inside the plant, formed their complex structures (arbuscules), and the partnership worked perfectly.
• The Analogy: Imagine a modern office where a specific manager (DELLA) is required to sign off on new hires. If you fire that manager, the office stops hiring. But in this ancient liverwort "office," the manager wasn't needed at all. The hiring process (symbiosis) happened just fine without them.

The Evolutionary Story: How the Job Changed

So, why does DELLA help with symbiosis in modern plants but not in ancient ones? The authors propose a fascinating evolutionary story:

1. The Ancient Era: In early land plants, DELLA existed but had a job related to growth and light. It helped the plant grow in the right shape and color, independent of the GA hormone.
2. The Upgrade: Later, when vascular plants (plants with "veins" or tubes) evolved, they invented the GA hormone and its receptor (GID1). This created a new system: Gas (GA) removes the Brake (DELLA) to allow growth.
3. The Co-option: As plants evolved to live in complex ecosystems, they needed a way to control their "roommate" relationships (symbiosis) more strictly. They took the existing DELLA protein and hijacked it.
   • Now, DELLA became a systemic controller. Because GA travels through the plant's "veins" (vascular system), it can tell DELLA to step in or step out from far away.
   • This allowed the plant to say, "We have enough nutrients, so stop the symbiosis," or "We need help, so let the fungi in," all controlled by the hormone system.

The Conclusion

The paper concludes that the developmental role of DELLA (helping the plant grow) is ancient and conserved. However, the symbiotic role (helping the plant talk to fungi) is a newer invention.

The Metaphor:
Think of DELLA as a universal remote control.

• In ancient times, it only controlled the TV volume (growth/light).
• When the "Smart Home" (vascular plants) was built, someone figured out how to use that same remote to also control the security system (symbiosis).
• The ancient liverworts still only use the remote for the TV. They don't have a security system to control, so they don't need the remote for that job.

In short: DELLA didn't evolve to help with fungi; it evolved to help with growth. Later, flowering plants got clever and added "fungi control" to the same remote, using the hormone GA as the signal to switch modes.

Technical Summary

1. Problem Statement

DELLA proteins are master growth repressors in flowering plants, regulated by the hormone gibberellic acid (GA). In angiosperms, DELLA proteins play dual roles:

1. Development: They repress growth and are degraded upon GA binding to the receptor GID1.
2. Symbiosis: They are essential positive regulators for establishing arbuscular mycorrhizal (AM) symbiosis with fungi.

However, the evolutionary history of these functions remains unclear. Non-vascular plants (bryophytes) like the liverwort Marchantia polymorpha and moss Physcomitrium patens possess DELLA proteins but lack the genes for bioactive GA biosynthesis and the GID1 receptor. While developmental defects have been observed in bryophyte DELLA mutants (often via overexpression), the symbiotic function of DELLA in non-vascular plants has never been tested, as these lineages generally lack AM symbiosis. The liverwort Marchantia paleacea is a unique model because it retains the ancestral ability to form AM symbiosis. The study aims to determine if the symbiotic role of DELLA is an ancient, conserved trait or a later evolutionary innovation in vascular plants, and to dissect the GA-independent functions of DELLA in a non-vascular context.

2. Methodology

The researchers employed a combination of genetic engineering, phenotypic analysis, and evolutionary biology approaches using Marchantia paleacea:

• Mutant Generation: Using CRISPR/Cas9, the team generated:
  • Single mutants for MpaDELLA (five independent alleles).
  • Single mutants for MpaGRAS13 (a closely related paralog specific to liverworts).
  • Double mutants (Mpadella/gras13) to test for functional redundancy.
• Reporter Lines: Generated MpaDELLApro:GUS lines to monitor tissue-specific expression patterns under various conditions.
• Phenotypic Characterization:
  • Developmental: Plants were grown under low, medium, and high light regimes. Metrics included thallus size, gemma cup number, chlorophyll content (a and b), and growth rates over 57 days.
  • Symbiotic: Plants were inoculated with the AM fungus Rhizophagus irregularis. Symbiosis was assessed via:
    • Presence of AMS-induced purple pigment.
    • Quantification of fungal colonization progression (exclusion zone measurement).
    • Microscopic imaging (WGA-Alexafluor 488 staining) to visualize arbuscule formation and morphology.
• Evolutionary Analysis: Phylogenetic reconstruction of DELLA homologs across 26 land plant species (14 vascular, 12 bryophytes) to trace gene duplication and loss events.

3. Key Contributions

• First Functional Analysis of DELLA in AM-Symbiotic Bryophytes: This is the first study to generate viable DELLA knockout mutants in a liverwort capable of AM symbiosis, moving beyond the overexpression studies previously limited to M. polymorpha.
• Decoupling of Functions: The study provides evidence that the developmental and symbiotic roles of DELLA evolved independently.
• Identification of Redundancy: The discovery and functional characterization of the liverwort-specific paralog GRAS13, which shares developmental redundancy with DELLA but not symbiotic function.
• Evolutionary Model: Proposes a stepwise evolutionary model where the symbiotic function of DELLA was co-opted only after the emergence of the GA-GID1 signaling module in vascular plants.

4. Results

A. Developmental Role (GA-Independent)

• Phenotype: Mpadella single mutants exhibited significant growth retardation, reduced thallus size, fewer gemma cups, and lower chlorophyll content compared to wild-type controls.
• Light Interaction: Under high light, della mutants showed asymmetric growth patterns, suggesting a link between DELLA, light signaling (potentially via PIFs), and chlorophyll biosynthesis.
• Redundancy: The gras13 single mutant showed no strong developmental defects. However, the della/gras13 double mutant displayed severe developmental defects (complete absence of gemma cups, severe asymmetry), indicating that GRAS13 acts redundantly with DELLA in development.
• Conclusion: DELLA acts as a positive regulator of growth and reproduction in M. paleacea in a GA-independent manner.

B. Symbiotic Role

• Colonization: Unlike in angiosperms (where della mutants fail to form arbuscules), Mpadella single mutants and della/gras13 double mutants successfully formed arbuscules.
• Quantitative vs. Qualitative: While the della mutants showed a slight quantitative reduction in colonization progression (likely secondary to their smaller size and reduced photosynthetic capacity), the formation and morphology of arbuscules were not impaired.
• Conclusion: DELLA is not essential for the establishment of AM symbiosis in non-vascular plants. The symbiotic defects seen in flowering plants are not conserved in M. paleacea.

C. Evolutionary Context

• Phylogenetic analysis confirmed that DELLA and GRAS13 arose from a duplication event in bryophytes.
• The GA receptor GID1 and bioactive GA biosynthesis are absent in bryophytes but present in vascular plants.
• The study posits that the ancestral function of DELLA was developmental (GA-independent). The symbiotic function evolved later in vascular plants, likely because the emergence of the GA-GID1 module allowed DELLA to serve as a systemic, hormone-regulated "switch" for symbiosis.

5. Significance

This paper fundamentally shifts the understanding of DELLA protein evolution:

1. Uncoupling of Traits: It demonstrates that the developmental and symbiotic functions of DELLA are distinct evolutionary modules. The developmental role is ancient and conserved in non-vascular plants, while the symbiotic role is a derived trait specific to vascular plants.
2. Mechanism of Co-option: It suggests that the evolution of the GA signaling pathway (GID1 receptor) provided a new regulatory layer that was co-opted to control symbiosis. This allowed vascular plants to integrate symbiotic establishment with systemic growth regulation via GA.
3. Model for Symbiosis Evolution: By showing that the "common symbiosis pathway" components (like CYCLOPS and CCaMK) are conserved and functional in M. paleacea without DELLA, the study clarifies that DELLA is a vascular-specific addition to the symbiotic toolkit, rather than a core ancestral component.

In summary, the authors propose that DELLA's role in symbiosis is a "stepwise" evolutionary innovation in vascular plants, leveraging the pre-existing GA signaling network to add a systemic control layer to the ancient, conserved machinery of plant-fungal mutualism.