Optogenetic control of transgene expression in Marchantia polymorpha

Abstract

Premise: The model liverwort Marchantia polymorpha is an emerging testbed species for plant metabolic engineering but lacks well-characterized inducible promoters, which are necessary to minimize biochemical and physiological disruption when over-accumulating target products. Here, we demonstrate the functionality of the light-inducible plant-usable light-switch elements (PULSE) optogenetic system in Marchantia and exemplify its use through the light-inducible overproduction of the bioplastic poly-3-hydroxybutyrate (PHB).

Methods: The PULSE system was used to drive expression of luciferase as a reporter and characterize its induction in transgenic M. polymorpha. Additionally, PULSE was used to drive expression of the PHB biosynthetic pathway; the accumulation of PHB under light-inducible control was compared to constitutive overexpression.

Results: PULSE was fully functional and minimally leaky in M. polymorpha. The presence of the PULSE construct, even in the absence of induction, resulted in a developmental phenotype. Constitutive and inducible expression resulted in similar PHB accumulation levels.

Discussion: PHB biosynthesis in plants is known to adversely affect plant health, but placing its production under optogenetic control alleviated negative effects on biomass accumulation in some instances. The work presented here represents a significant expansion of the toolbox for the metabolic engineering of M. polymorpha.

Keywords: acetyl‐CoA; liverwort; metabolic engineering; poly‐3‐hydroxybutyrate (PHB); toolbox.

Figure 1

Characterization of the PULSE optogenetic system in Marchantia polymorpha. (A) Dynamics and distribution of expression through PULSE in M. polymorpha. Counts per second (cps) normalized by the final mass of each plant are shown in response to monochromatic red‐light treatment; the inlay represents raw data obtained from a photon‐counting camera, showing signal heterogeneity where red represents highest counts and blue lowest counts (n = 4). Shaded regions on the graph represent standard error of the mean (SEM). Bar above the figure represents the light conditions used, where red is monochromatic red light and white is white light with no supplemental far‐red light. (B) Response of the PULSE system to different intensities of red light; intensities are shown in units of μmol photons·m⁻²·s⁻¹ (n = 3). Error bars represent SEM. (C) Response of the PULSE system to different ratios of blue to red light; ratios of blue to red light intensities are shown in units of μmol photons·m⁻²·s⁻¹ (n = 3). Error bars represent SEM. (D) Response of the PULSE system to different white light conditions (n = 3). W+FR, white light with supplemental far‐red; W, white light with no supplemental far‐red; FR, far‐red. Error bars represent SEM. (E) Long‐term induction behavior of PULSE over a 6‐d period. Horizontal lines represent expression levels at t = 0 (n = 3). Shaded regions represent SEM. (F) Induction of PULSE in response to multiple three‐day pulses of red light (n = 3). Shaded regions represent SEM. For E and F, a bar above the figure represents the light condition used, where red is monochromatic red light and light purple is W+FR. (G) Phenotype of three PULSE lines and wild‐type M. polymorpha Tak1 and Tak2 plants after 25 d of growth from gemmae in W+FR conditions. Scale bar is 5 mm. gFW, gram fresh weight; OE, overexpressor/transgenic lines; WT, wild‐type.

Figure 2

Optogenetic control over PHB production in Marchantia polymorpha. (A) PHB yield in three selected high‐PHB‐producing constitutive lines grown for 39 d from gemmae (n = 6). Error bars represent SEM. (B) Representative image of Nile Blue A–stained PHB granules in a gemma of line #25. Granules are indicated in the inset with white arrows. Scale bar is 10 μm. (C) Images of three selected high‐PHB‐producing constitutive lines and wild‐type M. polymorpha after 29 d of growth. Scale bar is 5 mm. Black arrows indicate enlarged pore‐like structures. (D) Induction scheme used for characterization of inducible PHB lines. The sampling point is indicated with a black arrow. (E) PHB levels expressed as percent PHB per gram fresh weight (%PHB/gFW) in both constitutive (#18, 25, 3) and inducible (#112, 88) lines grown according to the scheme shown in D (n = 6). Error bars represent SEM. *** represents a P value < 0.001, ** a P value < 0.01, * a P value < 0.05, and n.s. a P value > 0.05 (Mann–Whitney U). (F) Images of three inducible PHB lines with severe branching phenotypes that were not taken forward for further analysis. (G) Images of three selected high‐inducible PHB‐producing lines and wild‐type M. polymorpha after 31 d of growth and induction. Scale bar is 5 mm. Black arrows indicate enlarged pore‐like structures. (H) Induction scheme used for characterizing PHB accumulation dynamics. Sampling points are indicated with black arrows. (I) The change in PHB contents expressed in terms of %PHB/gFW of thallus cuttings from both constitutive and inducible PHB‐producing plants over time. PHB levels in inducible lines grown for the same amount of time but not induced are also shown (0bis) (n = 3). (J) The change in PHB contents expressed as total milligrams per plant of thallus cuttings from both constitutive and inducible PHB‐producing plants over time (n = 3). Legend applies to both (I) and (J), and error bars represent SEM. OE, overexpressor/transgenic lines; WT, wild‐type.