Insight into the regulatory networks underlying the high lipid perennial ryegrass growth under different irradiances

Abstract

Under favourable conditions, perennial ryegrass (Lolium perenne) engineered to accumulated high lipid (HL) carbon sink in their leaves was previously shown to also enhance photosynthesis and growth. The greater aboveground biomass was found to be diminished in a dense canopy compared to spaced pots. Besides, the underlying genetic regulatory network linking between leaf lipid sinks and these physiological changes remains unknown. In this study, we demonstrated that the growth advantage was not displayed in HL Lolium grown in spaced pots under low lights. Under standard lights, analysis of differentiating transcripts in HL Lolium reveals that the plants had elevated transcripts involved in lipid metabolism, light capturing, photosynthesis, and sugar signalling while reduced expression of genes participating in sugar biosynthesis and transportation. The plants also had altered several transcripts involved in mitochondrial oxidative respiration and redox potential. Many of the above upregulated or downregulated transcript levels were found to be complemented by growing the plants under low light. Overall, this study emphasizes the importance of carbon and energy homeostatic regulatory mechanisms to overall productivity of the HL Lolium through photosynthesis, most of which are significantly impacted by low irradiances.

Fig 1. Box and whisker plots of relative growth rate (RGR, g g^-1d^-1).

A RGR of total mass, B leaf RGR, C sheath RGR and D root RGR of high lipid (HL) Lolium and non-transformant (NT) control grown under standard and low lights. ^A-DAlphabets indicated significant differences (p < 0.05). df = 71 (n = 9 or 10). ¹Data were analyzed using log-transformation. RGR calculated as (ln W₂ –ln W₁)/ (t₂-t₁), where W₁ and W₂ were plant dry weights at times t₁ and t₂, respectively.

Fig 2. The relative gene expression level of differentially expressed genes (DEGs).

Data represent the means of normalized transcript per million (TPM) with the error bar of the SE. DEGs were identified in high lipid (HL) ryegrass compared with non-transformant (NT) plants grown under standard (white graph area) and low (shading graph area) irradiance levels. ^A-DAlphabets indicated significant differences (p < 0.05, n = 3). DEGs grouped using Gene Ontology analysis as following: A lipid metabolism: Glycerol kinase (GK, XP_015636240.1), glycerol-3-phosphate-2-O-acyltransferase (GPAT, XP_015626270.1), peroxisomal enoyl-CoA hydratase (ECH1, XP_015632322.1), phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT, XP_015644243.1), pyruvate dehydrogenase E1 component (PDC, XP_015636508.1), and sugar-dependent triacylglycerol lipase (SDP1, XP_015651228.1), B carbohydrate metabolism and glycolysis: Sucrose transporter protein 2 (SUT2, XP_015619709.1), fructan:fructan 1-fructosyltransferase (6G-FFT, XP_015625788.1), sucrose synthase (SUSY, XP_025882333.1), glyceraldehyde-3-phosphate dehydrogenase (G3PDH, XP_015625382.1), cytosolic pyruvate kinase (PK, XP_015616831.1) and enolase 1 (XP_015643741.1), C photosynthesis pathway: Ferredoxin NADP reductase (FNR, XP_015640980.1), and cytochrome b6-f complex (Cytb6-f, XP_015647138.1).

Fig 3. Expression of genes involved in sugar signalling pathway in high lipid (HL) lines.

A Data represent the means of normalized transcript per million (TPM) with the error bar of the SE, identified in HL Lolium grown under standard (white graph area) and low (shading graph area) irradiance levels, compared to non-transformant (NT) control. ^A-EAlphabets indicated significant differences (p < 0.05, n = 3). B Validation of selected genes for relative expression. * indicates p < 0.05, ** for p < 0.01 and *** for p < 0.001 (Student’s t-test). Bars represent the means and SE of five biological replicates. Gene ontology grouped in sugar signalling pathway: Hexokinase 7 (HK7, XP_015637554.1); sucrose-nonfermented related protein kinase 1 subunit gamma (SnRK1-γ, XP_015635849.1); trehalose-6-phosphate synthase 1 (TPS1, XP_015640390.1); trehalose 6-phosphate synthase 6 (TPS6, XP_015611910.1); trehalose-phosphate phosphatase 7 (TPP7, XP_15651449.1).

Fig 4. Immunoblot analysis of specific protein abundance in high lipid (HL) Lolium compared with non-transformant (NT) control.

Abbreviations: Cytochrome c oxidase (COX, XP_015648280.1); malate dehydrogenase 2 (MDH2, XP_015621604.1); L-ascorbate oxidase (AO, XP_015611052.1); L-ascorbate peroxidase (APX, XP_015630498.1); sucrose-nonfermented related protein kinase 1 subunit gamma (SnRK1-γ, XP_015635849.1); and trehalose-6-phosphate synthase 1 (TPS1, XP_015640390.1).

Fig 5. Validation of selected genes in high lipid (HL) lines and non-transformant (NT) plants for relative expression.

* indicates p < 0.05, ** for p < 0.01 and *** for p < 0.001 (Student’s t-test). Bars represent the means and SE of five biological replicates. Selected DEGs grouped using Gene Ontology analysis as following: A Mitochondrial respiration: Ubiquinol oxidase (UbiQ-Ox, XP_015635413.1); alternative NAD(P)H: Ubiquinone oxidoreductase (UbiQ-OxRd, XP_015637913.1); fumarate hydratase (FH, XP_015633139.1); malate dehydrogenase 2 (MDH2, XP_015621604.1); and 2-oxoglutarate dehydrogenase (2-OGDH, XP_015646480.1). B Redox potential: L-ascorbate oxidase (AO, XP_015611052.1); L-ascorbate peroxidase (APX, XP_015630498.1).