Cold acclimation can specifically inhibit chlorophyll biosynthesis in young leaves of Pakchoi

Abstract

Background: Leaf color is an important trait in breeding of leafy vegetables. Y-05, a pakchoi (Brassica rapa ssp. chinensis) cultivar, displays yellow inner (YIN) and green outer leaves (GOU) after cold acclimation. However, the mechanism of this special phenotype remains elusive.

Results: We assumed that the yellow leaf phenotype of Y-05 maybe caused by low chlorophyll content. Pigments measurements and transmission electron microscopy (TEM) analysis showed that the yellow phenotype is closely related with decreased chlorophyll content and undeveloped thylakoids in chloroplast. Transcriptomes and metabolomes sequencing were next performed on YIN and GOU. The transcriptomes data showed that 4887 differentially expressed genes (DEGs) between the YIN and GOU leaves were mostly enriched in the chloroplast- and chlorophyll-related categories, indicating that the chlorophyll biosynthesis is mainly affected during cold acclimation. Together with metabolomes data, the inhibition of chlorophyll biosynthesis is contributed by blocked 5-aminolevulinic acid (ALA) synthesis in yellow inner leaves, which is further verified by complementary and inhibitory experiments of ALA. Furthermore, we found that the blocked ALA is closely associated with increased BrFLU expression, which is indirectly altered by cold acclimation. In BrFLU-silenced pakchoi Y-05, cold-acclimated leaves still showed green phenotype and higher chlorophyll content compared with control, meaning silencing of BrFLU can rescue the leaf yellowing induced by cold acclimation.

Conclusions: Our findings suggested that cold acclimation can indirectly promote the expression of BrFLU in inner leaves of Y-05 to block ALA synthesis, resulting in decreased chlorophyll content and leaf yellowing. This study revealed the underlying mechanisms of leaves color change in cold-acclimated Y-05.

Keywords: 5-aminolevulinic acid (ALA); BrFLU; Chlorophyll biosynthesis; Cold acclimation; Leaf color conversion; Pakchoi.

Fig. 1

The inner leaves of cold-acclimated Y-05 exhibit decreased chlorophyll content and undeveloped thylakoids. a The phenotype of G-04 and Y-05 before and after cold acclimation. For cold acclimation, two-month old Y-05 plants were grown 3 weeks at 4 °C, and then return to 23 °C for continues grown. Before, before cold acclimation. After, after cold acclimation. Bar = 5 cm. b The total chlorophyll content of outer and inner leaves from Y-05 and G-04 before and after cold acclimation. c The net photosynthetic rate (P_n) of outer and inner leaves from Y-05 and G-04 after cold acclimation. PAR, photosynthetic active radiation. Three individual plants of each cultivar were quantified, and the total chlorophyll content and P_n were measured three times. Error bars represent SE (±SE, n = 3). Different letters indicated statistically significant differences at the level of p < 0.05. d-k Chloroplast ultrastructure of outer and inner leaves from G-04 and Y-05 after cold acclimation. v = vacuole, s = starch grains, gt = granum thylakoids, st = stroma thylakoids. In the Fig. 1d, f, h, j, Bar = 4 μm. In the Fig. 1e, g, i, k, Bar = 1 μm

Fig. 2

The enriched GO pathway of the differentially expressed genes (DEGs) between TOU and TIN of pak choi Y-05. -log10 (KS) represents the statistical significance of GO term. The bigger of -log10 (KS), the more significant enriched

Fig. 3

The top 20 enriched KEGG pathway of the differentially expressed genes (DEGs) between TOU and TIN of pak choi Y-05. Each point represents a KEGG pathway, ordinate represents pathway name, and abscissa represents the enrichment factor. The larger the enrichment factors, the more significant the enrichment level of DEGs are showed. The color of the circle represents q-value, lower q-value means the more reliable results. And the size of the circle represents the number of genes enriched in the pathway

Fig. 4

The enriched KEGG pathway of the differentially expressed metabolites (DEMs) between inner-yellow (MIN) and outer-green (MOU) leaves of pakchoi Y-05. Short time-series expression miner (STEM) was used to analyze the metabolites expression pattern. The number of DEMs in each profile was labeled above the frame. The bar represents the proportion metabolites in each profile of the total annotated metabolites

Fig. 5

Exogenous application of ALA can rescue the yellow leaves phenotype in cold-acclimated Y-05. a The leaf phenotype of ALA-treated plants compared with CK plants. ALA, cold-acclimated Y-05 sprayed by 1 mM ALA. CK, cold-acclimated Y-05 sprayed by water as control. Bar = 5 cm. b The chlorophyll content of ALA-treated plants was higher than CK plants. Chlorophyll a, Chlorophyll b and total chlorophyll abbreviated as Chl a, Chl b, Total Chl, respectively. c The ALA content of ALA-treatment plants was higher than CK plants. Three individual plants of each cultivar were quantified, and the chlorophyll and ALA content were measured three times. Error bars represent SE (±SE, n = 3). Different letters indicated statistically significant differences at the level of p < 0.05. d-k Observation of chloroplast ultrastructure showed that ALA-treated plants possessed mature chloroplasts and granum thylakoids. v = vacuole, gt = granum thylakoids, st = stroma thylakoids. In the Fig. 4d, f, h, j, bar = 2 μm; in the Fig. 4e, g, bar = 500 nm; in the Fig. 4i, k, bar = 800 nm

Fig. 6

Gabaculine-treated Y-05 seedlings show yellow leaves phenotype without cold acclimation. a The gabaculine-treated plants showed yellow phenotype without cold-acclimation. Before, plants before treated with gabaculine. After, plants after treated with gabaculine. 0 μM and 50 μM represented CK plants and gabaculine-treated plants, respectively. Bar = 5 cm. b The inner leaves of gabaculine-treated plants showed low chlorophyll content compared with CK plants. c The inner leaves of gabaculine-treated plants showed low ALA content compared with CK plants. Three individual plants of each cultivar were quantified, and the chlorophyll and ALA content were measured three times. Error bars represent SE (±SE, n = 3). * represents p < 0.05, ** represents p < 0.01

Fig. 7

Silencing of BrFLU in Y-05 is critical for ALA biosynthesis. a The phenotype of pTY plants (CK) and pTY-FLU (BrFLU-silenced) plants. Bar = 5 cm. b The relative expression level of BrFLU significantly decreased in pTY-FLU plants. The inner leaves were selected to confirm the BrFLU expression. c The pTY-FLU plants showed higher ALA content than pTY plants. d The pTY-FLU plants showed higher chlorophyll content than pTY plants. e The phenotype of pTY and pTY-FLU plants after cold acclimation. Bar = 1 cm or 2 mm. f The pTY-FLU plants showed higher chlorophyll content than pTY plants after cold acclimation. Chlorophyll a, Chlorophyll b and total chlorophyll abbreviated as Chl a, Chl b, Total Chl, respectively. Three individual plants of each cultivar were quantified, and the chlorophyll content was measured three times. Error bars represent SE (±SE, n = 3). * represents p < 0.05, ** represents p < 0.01

Fig. 8

The proposed model of chlorophyll biosynthesis in Y-05 under normal or low temperature. At normal temperature, the expression of BrFLU remains stable, and GLU converts to ALA to maintain normal biosynthesis of chlorophyll. Under low temperature, cold acclimation can trigger an unknown regulator, inducing BrFLU upregulation and the interaction between BrFLU and GLU-TR to block ALA synthesis, resulting in decreased chlorophyll content and leaf yellowing in Y-05. L-Glutamic acid, glutamyl-tRNA reductase, 5-Amino-levulinate and total chlorophyll were abbreviated as Glu, GluTR, ALA and Chl, respectively. Red and green arrow represents up-regulated and down-regulated of compounds, respectively