Colorful flowers of rapeseed (Brassica napus L.) have been a hotspot for researchers, but the underlying mechanisms of pigment formation still need to be clarified. In this study, two stages of unopened rapeseed petals with red, white, and yellow colors were selected to identify the metabolites and genes involved in red pigment formation. Metabolomic analysis showed that flavonoids enriched the most co-differentially accumulated metabolites among all categories, and showed higher accumulation in red petal rapeseed than in white and yellow petal ones. RNA-seq analysis showed that among co-differentially expressed genes involved in red pigment formation, genes involved in anthocyanin (belonging to flavonoids) biosynthesis pathway were largely regulated by ANS, DFR, and UF3GT. The expression of those genes was higher in red petals of rapeseed than in white and yellow petals ones as well. Results of RNA interference of BnaA03.ANS in red rapeseed altered petal colors from raspberry red to beige red and zinc yellow under different interference levels, with the contents of pelargonidin, cyanidin, lutein, neoxanthin, β-carotene, and lycopene significantly decreased. However, overexpression of BnaA03.ANS in yellow rapeseed petals did not change the color of yellow petals. This study confirmed the important function of flavonoids, especially anthocyanins on red pigment formation, and for the first time, identified the irreplaceable role of BnaA03.ANS on red-flowered rapeseed.

Accelerating the differentiation of floral meristem (FM) from shoot apical meristems (SAM) which determines the conversion from vegetative to reproductive growth is of great significance for the production of rapeseed (Brassica napus L.). In this research, the mechanisms of different nitrogen (N) application rates (low N, N1; normal N, N2; and high N, N3) on different FM development stages triggering the regulation of FM differentiation genes through the auxin biosynthetic and signal transduction were investigated. We found that the stage of FM differentiation, which was identified through a stereomicroscope and scanning electron microscope, came 4 and 7 days earlier under high N rate than under normal and low N levels, with the seed yield increased by 11.1 and 22.6%, respectively. Analysis of the auxin and its derivatives contents showed that the main biosynthesis way of auxin was the indole acetaldehyde oxime (IAOx) pathway, with 3-Indole acetonitrile dramatically accumulated during FM differentiation. At the same time, an obvious decrease of IAA contents at each FM differentiation stage was detected, and then gradually rose. Results of the expression of genes involved in auxin biosynthesis, auxin signaling transduction, and FM identification under five FM differentiation stages and three nitrogen application rates showed that genes involved in auxin biosynthesis were regulated before the FM differentiation stage, while the regulation of FM identity genes appeared mainly at the middle and later periods of the five stages, and the regulation level of genes varied under different N rates. Taken together, a high nitrogen rate could accelerate the initiation of FM differentiation, and auxin involved a lot in this regulation.

Delayed planting date of rapeseed is an important factor affecting seed yield. However, regulation of the leaf carbohydrate metabolism in rapeseed by a late planting date at the reproductive stage is scarcely investigated. A two-year field experiment was conducted to assess the effect of planting dates, including early (15 September), optimal (1 October), late (15 October), and very late (30 October), on leaf growth and carbohydrate biosynthetic and catabolic metabolism at the reproductive stage. The results showed that leaf dry matter decreased linearly on average from 7.48 to 0.62 g plant−1 with an early planting date, whereas it increased at first and peaked at 14 days after anthesis (DAA) with other planting dates. Leaf dry matter was the lowest at the very late planting date during the reproductive stage. For leaf chlorophyll content, rapeseed planted at an optimal date maximized at 14 DAA with an average content of 1.51 mg g−1 fresh weight, whereas it kept high and stable at a very late planting date after 28 DAA. For the carbohydrate catabolic system, acid and neutral invertase (AI and NI, respectively) showed higher activity before 14 DAA, whereas both sucrose synthase (SS) and starch phosphorylase (SP) showed higher activity after 14 DAA. For the carbohydrate biosynthetic system, the activity of sucrose phosphate synthase (SPS) was the highest at the late planting date after 14 DAA, whereas it was at the lowest at the very late planting date. However, the activity of ADP-glucose pyrophosphorylase (AGPase) at the late and very late planting dates was significantly higher than that of the early and optimal plant dates after 21 DAA, which is in accordance with the leaf total soluble sugar content, suggesting that leaf carbohydrate metabolism is governed by a biosynthetic system. The current study provides new insights on leaf carbohydrate metabolism regulation by late planting in rapeseed at the reproductive stage.