Research Progress on the Effect of Nitrogen on Rapeseed between Seed Yield and Oil Content and Its Regulation Mechanism

1. Introduction

Rapeseed is one of the major oil crops in China. Oil production depends not only on the seed yield but also on the oil content [1]. Nitrogen, as a living element of plants, is particularly vital for their growth and development. Applying nitrogen is an important way to ensure a strong and stable yield. However, most varieties of rapeseed are extremely sensitive to nitrogen and have a reduced seed oil content after nitrogen application. The oil content decreased by 1.6% for every increase of 100 kg·hm^-2 nitrogen application [2,3], and then equals a decrease of 4% in seed yield [3,4]. It is important to cultivate varieties of rapeseed that do not decrease in oil content after nitrogen application to ensure oil production. Moreover, the molecular mechanism of nitrogen-regulated oil accumulation in developing rapeseed is still poorly understood. Nitrogen levels affect the deposition of proteins and starch in maize and the accumulation of protein and oil in rapeseed [5,6,7]. Due to different target products, the mechanism of nitrogen on rapeseed is different from that of cereal crops such as maize and rice. It is important to study nitrogen’s effect on rapeseed grain yield and oil content.

2. Effects of Nitrogen on Plants

Sufficient nitrogen will insure large, vibrantly green leaves, additional branches, abundant flowers, and an increase in yield [8]. Excessive nitrogen can hinder the growth and development of plant roots. In Arabidopsis thaliana, lateral root elongation will be suppressed with increasing nitrogen concentration, while primary lateral root growth is insensitive to nitrogen [9]. Too much nitrogen can cause the stems and leaves to swell, reduce resistance to lodging and stress, and delay maturation time [7,10,11]. When nitrogen is insufficient, the organic matter in the plant will allocate to the root system priority and stimulate the growth of lateral roots [12]. Thus, the plant grows moderately, diminishes in size, gradually loses fresh leaves, becomes smaller, and has a lower chlorophyll content and redder leaves [13] and a reduction in the number of branches, flowers and grains [14]. The oil content of maize increases with the increase in nitrogen application rate [8]. In wheat, the effect of nitrogen on yield and protein yield is a quadratic curve [15]. In rapeseed, nitrogen can lead to a significant increase in yield, but can also cause a decrease in oil content. Protein, glucosinolate, and erucic acid content all tend to increase with higher nitrogen levels [1,7,10,16]. These results suggest different mechanisms by which nitrogen affects different plants.

3. Effects of Nitrogen on Rapeseed Yield

Nitrogen affects the growth and yields formation of rapeseed. It is reasonable to control the nitrogen application rate within 90~225kg·hm^-2 [17,18,19]. Appropriate nitrogen can boost photosynthetic capacity, metabolic levels, morphogenesis, and increase yield [13]. The shortage of nitrogen can limit the photosynthesis ability, deplete the nutrient supply required for pod development, and reduce the count of productive pods on the main stem and the number of seeds per pod, therefore decreasing the weight per 1,000 grains and yielding [7,20]. Excessive nitrogen can induce over-growth during the vegetative stage and reduce nitrogen use efficiencies and yield during the maturity stage. This is because pre-flowering rapeseed consists primarily of vegetative morphogenesis, such as stems and leaves; After flowering, nutrients are transported to the grain [7]. An overabundance of nitrogenous fertilizers can cause stems and branches to produce an excessive number of ineffective shoots and branches, and lead to fertilizer waste and ecological destruction [14]. The siliques number per plant and seeds number per pod does not increase significantly with the nitrogen application rate beyond 180 kg·hm^-2 [18].

3.1. Effects of Nitrogen on Photosynthetic Assimilation of Rapeseed

Nitrogen affects the efficiency of photosynthetic energy transport in leaves and then regulates the photosynthetic product. After nitrogen is assimilated, it is mainly transported to the mesophyll cells in the top of the plant through the xylem in the form of glutamine, aspartic acid, glutamic acid, and asparagine for carbon assimilation. Leaves are the primary organ of photosynthesis [14]. Chloroplasts are the sole site of photosynthetic carbon assimilation and the primary site of nitrogen assimilation in cells [21]. In chloroplasts, chlorophyll a and chlorophyll b have the function of collecting and transmitting sunlight energy [22]. The energy previously converted into chemical energy by sunlight is temporarily stored in ATP and NADPH for CO₂ assimilation in dark reactions [23]. The way of CO₂ assimilation is through the Calvin cycle pathway (C3) [24]. Triose phosphate (TP) is the net product of carbon assimilation that is assimilated through the pentose phosphate reduction pathway (RPPP) [14]. Further, TP is a major precursor to other biosynthetic reactions, including starch and sucrose biosynthesis, nitrogen and sulfur metabolism, fatty acid biosynthesis, cell wall biosynthesis, secondary metabolism, and other metabolic pathways [25]. The CO₂ assimilation capacity is mainly affected by the nitrogen content of the leaves and is generally positively correlated with nitrogen content [26]. This is because rapeseed leaves capture sunlight energy processes, electron transport processes, and enzymatic reactions in carbon metabolism require large amounts of nitrogen to synthesize ATP, NADPH, and various proteases [26].

Under nitrogen deficiency conditions, the chlorophyll content of leaves decreased, the original chlorophyll in the cytoplasm of mesophyll cells disappeared, fresh chlorophyll could not be formed, and the photosynthetic rate decreased [13,27]. Increasing nitrogen can promote chlorophyll synthesis and increase Rubisco content, while the high activity of Rubisco can promote photosynthetic rate [7,15,28,29,30]. In addition, nitrogen can also regulate CO₂ concentration in chloroplasts by increasing the surface area of mesophyll cell walls and chloroplast to regulate photosynthetic rates [31].

3.2. Nitrogen Affects the Substance Distribution of Rapeseed

The amino acids produced in the process of nitrogen assimilation are transported between different organs through the phloem and xylem [33]. Phloem mainly transfers amino acids from photosynthetic tissue (source organs) to non-photosynthetic tissues (sink organs) [34]. The phloem sap of rapeseed contains up to 650 millimolar of free amino acids [35,36]. In the source leaves, sucrose and amino acids are excreted from mature and senescent leaves, loaded into the phloem through cytoplasmic pathways or extracellular phloem loading, and transported over long distances to developing sink organs [37]. The transport and distribution of carbon and nitrogen assimilation products in different parts and at different times are regulated by the physiological state, source strength, and expression of the transporters. In the vegetative growth phase, the assimilates in the rapeseed leaves are mainly transported to the immature leaves and developing roots [38,39]. During the reproductive growth phase, assimilates in the leaves are transported to the flowers, siliques, and grains via phloem loaders and transporters, including sugar and amino acid transporters [40].

Nitrogen deficiency will accelerate leaf senescence and result in a decrease in protein content in leaves. This may change the amino acid transport activity but increase nitrogen remobilization [36,41,42]. Leaf proteins are degraded when carbon and nitrogen are needed to support the growth of the carbon sink organ. Appropriate application of nitrogen can significantly promote the accumulation of carbon and nitrogen products in leaves, pods, and stems and promote their transfer from source to sink in the late growth stage [7,17,19,43]. The seed is the primary sink tissue for photosynthetic products. Carbohydrates and amino acids are metabolized and then stored in the seed as proteins and oils [44]. To enable the transport and accumulation of carbon and nitrogen products in the seed, the seed coat and seed sink tissue need to coordinate through a membrane that transports sucrose and amino acids [21]. This membrane plays a crucial role in the movement of nutrients between the two tissues, and its effectiveness is closely related to the quality and quantity of seed development. After the nutrients in the seed coat are transmitted through the cytoplasm of the seed coat parenchyma, the assimilates are exported to the extracellular cavity and subsequently absorbed by the developing seeds [21].

3.3. Molecular Mechanism of Nitrogen Affecting Rapeseed Yield

With the rapid development of molecular biology techniques and intensive research on crop nitrogen use efficiency, genes that regulate plant nitrogen uptake, transport and utilization efficiency have been discovered. External nitrate and ammonium are absorbed by roots and then are assimilated by the nitrate transporter family (NRTs) and the ammonium transporter family (AMTs) in the roots [45,46,47]. Typically, the uptake of NO₃^- by roots is limited. Most of NO₃^- is reduced to NH₄⁺ by nitrate reductase (NR) and nitrite reductase (NIR) in shoots [48]. NH₄⁺ is then further assimilated into organic forms through the glutamine synthetase/glutamate synthetase (GS/GOGAT) cycle and participates in carbon metabolism and biosynthesis of other compounds in plants [50]. Once NH₄⁺ is accumulated, plants will produce toxic symptoms [44,46,50,51]. A high concentration of NH₄⁺ as the only nitrogen source will cause stress, and the nutrition of NO₃^- or NO₃^- plus a slight amount of NH₄⁺ is suitable for rapeseed growth [52]. In addition, nitrogen levels had a significant effect on nitrate reductase (NR) activity in rapeseed, maize, and wheat [7,8,53,54]. Sucrose and amino acids, the products of plant carbon and nitrogen assimilation, are released from mature and aged leaves, packed into the phloem, and transported to the sink tissues by the sucrose transporters family (SUTs/SUCs/SWEETs) and amino acid transporters [44].

Nitrogen levels affect the expression levels of ZmSWEETs and ZmSUTs in different parts of the maize during the vegetative stage [55]. The expression levels of ZmSWEETs and ZmSUTs in the leaves are higher in the low nitrogen condition than in the sufficient nitrogen condition, while the expression levels of ZmSWEETs and ZmSUTs in the internodes are higher in the sufficient nitrogen condition than in the low nitrogen condition [55]. These results show that sucrose transport from source to sink is more efficient at sufficient nitrogen levels than that at low nitrogen levels [55]. Sucrose synthase (SS) is a regulatory enzyme for sucrose degradation. It facilitates the decomposition of sucrose transported from the source organ and provides the basic substances for the synthesis and accumulation of fats and oils, and proteins in grains [56]. The activity of sucrose synthetase (SS) in rapeseed seeds is related to nitrogen level, and the SS activity is low under low nitrogen treatment [57]. Phytohormones are a class of naturally occurring small organic molecules that play an important role in regulating plant growth and development [58]. Nitrogen signaling and metabolic networks were regulated by hormone signals in plants [60,61,62]. The gene Dehydration-ResponsiveElement-Binding Protein 1C (OsDREB1C) belongs to the APETALA2/ethylene-responsive element binding factor (AP2/ERF) family. Its overexpression can significantly increase nitrogen uptake, transport, and utilization efficiency in rice, as well as the number of grains per panicle, grain weight, yield, and harvest index [62].

NO₃^- and NH₄⁺ can act as signaling molecules that interact with various plant hormone signals to regulate plant nitrogen uptake, transport, and assimilation. The regulatory factor DNR1 modulates nitrogen metabolism in rice by affecting auxin biosynthesis and signal transduction pathways [63]. The transcription factor GROWTH-REGULATING FACTOR 4 (OsGRF4) and the growth inhibitor DELLA confer co-regulation of the absorption, assimilation, and transport of NH₄⁺, regulate the photosynthetic carbon fixation capacity of crops, and enable high nitrogen utilization efficiency, increasing the yield of rice, maintaining homeostasis [64]. In general, there have been numerous studies of the molecular mechanisms of the nitrogen effect on production, but most of them have focused on Arabidopsis, maize, rice, and other plants, and extremely little has been done in the field of rapeseed.

Table 1. Research progress of carbon and nitrogen metabolism genes related to yield.

Genes	Plants	Functions
LBD gene family (LBD37, LBD38, LBD39)	Arabidopsis	LBD transcription factors inhibition of anthocyanin biosynthesis and nitrogen availability signal [65]
NRT1.1; NRT2.1; NRT2	Arabidopsis	Nitrate transport [66,67,68,69]
MYB	Soybean; Sugarcane ; Foxtail millet	Regulate nitrate transporters [70,71,72]
NLP7	Arabidopsis	Regulation of nitrate assimilation [73]
		Nitrate sensors and transcriptional activators initiate nitrate-mediated transcriptome signaling [74]
NLP5	Maize	Regulation of nitrate assimilation [75]
CLC gene family	Arabidopsis	Regulation of nitrate transport [76,77]
CBL, CIPK8	Arabidopsis	Regulation of nitrate transport and assimilation [78]
AMT1.3; AMT2.1; AMT2.2; AMT2.3; AMT3.1; AMT3.2;	Rice	Regulation of ammonium salt absorption and transport [79]
SWEETs	Maize; Arabidopsis	Sucrose transport; the expression affected by nitrogen [55,80,81]
SUTs	Arabidopsis; Maize	Sucrose transport; expression is affected by nitrogen [55,82]
NAS1 /NAP1	Soybean	Regulating the flow distribution of PEP to maintain carbon and nitrogen balance [83]
PBF1	Maize	Regulate carbon and nitrogen metabolism in a nitrogen-dependent manner [5]
ANR1	Arabidopsis； Chrysanthemum	ANR1-mediated auxin response [9,84]
DNR1	Rice	DNR1-mediated auxin response [63]
GRF4	Rice	Regulation of nitrogen absorption, assimilation, and transport [64]
DREB1C	Rice	Regulation of nitrogen utilization; Ethylene-mediated [62]

Table 1. Research progress of carbon and nitrogen metabolism genes related to yield.

Genes	Plants	Functions
LBD gene family (LBD37, LBD38, LBD39)	Arabidopsis	LBD transcription factors inhibition of anthocyanin biosynthesis and nitrogen availability signal [65]
NRT1.1; NRT2.1; NRT2	Arabidopsis	Nitrate transport [66,67,68,69]
MYB	Soybean; Sugarcane ; Foxtail millet	Regulate nitrate transporters [70,71,72]
NLP7	Arabidopsis	Regulation of nitrate assimilation [73]
		Nitrate sensors and transcriptional activators initiate nitrate-mediated transcriptome signaling [74]
NLP5	Maize	Regulation of nitrate assimilation [75]
CLC gene family	Arabidopsis	Regulation of nitrate transport [76,77]
CBL, CIPK8	Arabidopsis	Regulation of nitrate transport and assimilation [78]
AMT1.3; AMT2.1; AMT2.2; AMT2.3; AMT3.1; AMT3.2;	Rice	Regulation of ammonium salt absorption and transport [79]
SWEETs	Maize; Arabidopsis	Sucrose transport; the expression affected by nitrogen [55,80,81]
SUTs	Arabidopsis; Maize	Sucrose transport; expression is affected by nitrogen [55,82]
NAS1 /NAP1	Soybean	Regulating the flow distribution of PEP to maintain carbon and nitrogen balance [83]
PBF1	Maize	Regulate carbon and nitrogen metabolism in a nitrogen-dependent manner [5]
ANR1	Arabidopsis； Chrysanthemum	ANR1-mediated auxin response [9,84]
DNR1	Rice	DNR1-mediated auxin response [63]
GRF4	Rice	Regulation of nitrogen absorption, assimilation, and transport [64]
DREB1C	Rice	Regulation of nitrogen utilization; Ethylene-mediated [62]

4. Effects of Nitrogen on the Oil Content of Rapeseed

Oil is the ultimate target product of oil crops. Protein is the basic material of the protoplasm. Nitrogen is a major component of protein in plants, and many intermediates of sugar metabolism are major sources of protein synthesis. At different levels of nitrogen, the oil content of rapeseed without nitrogen application is the highest, and the oil content gradually decreases and the protein content gradually increases with the increase of nitrogen application [1,57,85]. In the case of low nitrogen is adverse to the biosynthesis of protein and glucosinolate but is beneficial to the formation and accumulation of lipid in seed [61].

4.1. Nitrogen Affects the Synthesis of Lipids and Proteins in Rapeseed

The reason for the negative correlation between oil content and protein content in plants is that the lipid and protein in plants come from the product of glycolysis-pyruvate (PRY), so there is substrate competition in the synthesis of the two products [86,87]. After entering reproductive growth, sucrose in the vegetative body is transported to the developing seed for protein and lipid synthesis [88]. Oil is a higher fatty acid glyceride synthesized by fatty acids and glycerol and is stored in seeds in the form of triacylglycerol [89]. The synthesis of lipids in plants is complex and is divided into three main stages: 1. Formation of fatty acids and fatty acid precursor acetyl-CoA: Sucrose is converted to hexose by glycolysis and hexose is oxidized to acetyl-CoA and one molecule of CO₂ [11]. In this process, every two carbon atoms are converted to acetyl-CoA, and one carbon atom (one-third of carbon) is lost as CO₂ [21]. Acetyl-CoA carboxylates to malonyl-CoA, which further synthesizes saturated fatty acids in the plastids, or desaturates to form unsaturated fatty acids [90]; 2. Production of 3-phosphoglycerol: In the cytoplasm, hydroxyacetone phosphate produced by the glycolytic pathway is reduced to 3-phosphoglycerol by the catalysis of 3-phosphoglycerol dehydrogenase [11]; 3. Assembly of fatty acyl-CoA and 3-phosphoglycerol: Fatty acyl-CoA and 3-phosphoglycerol were dehydrated and condensed in the endoplasmic reticulum to produce triacylglycerol [14,91]. In rapeseed, there are numerous enzymes and proteins involved in the synthesis of lipids and nitrogen is an important element in the formation of proteins. An adequate supply of nitrogen increases protein, and protein synthesis precedes fat synthesis, but in protein synthesis large amounts of photosynthetic products are consumed, resulting in a weakening of the library source allocated for lipid, and thus affecting oil content.

4.2. Molecular Mechanism of Nitrogen Level Affecting Lipid Synthesis in Rapeseed

The oil content is a complex quantitative trait influenced by multiple genes and subject to complex genetic interactions with the environment. The balance between lipid and protein synthesis in plants depends on the activity of key enzymes in their metabolism [88]. Pyruvate carboxylase (PEPC) is a key enzyme controlling the ratio of protein/oil content and is involved in the regulation of seed storage protein and fatty acid metabolism in plant seeds [56,86]. The activity of PEPC may be coordinated through post-translational regulation, with the overexpression of PEPC genes accelerated after nitrogen application [6,92]. Acetyl-CoA carboxylase (ACCase) is the rate-limiting enzyme in fatty acid biosynthesis [93]. The expression of the ACCase gene in rapeseed treated with different nitrogen changes significantly after 31 days of pollination [6]. After nitrogen application, the expression of the ACCase gene decreases in the high oil-content material, while it increases in the high protein-content material [6]. Phospholipid phosphatase (PPase) is the rate-limiting enzyme in the biosynthesis of triacylglycerol [56]. The oil content of seeds is related to the activity of the PPase gene. PPase activity in oilseeds is highest at 35 and 55 days after flowering under high nitrogen treatment, followed by medium and low nitrogen treatment [57]. Diacylglycerol acyltransferase (DGAT2) catalyzes the synthesis of triacylglycerol (TAG) from diacylglycerol (DAG) in plants and is the last step and the rate-limiting step of TAG synthesis [6]. Overexpression of the DGAT2 gene in tobacco can increase the oil content and the content of oleic acid and linoleic acid in leaves [6,94]. Similarly, the expression of the DGAT2 gene in the accession with high oil content was decreased by 12-fold after 31 days of pollination, compared to the case without nitrogen application [6]. Pyruvate kinase (PK) catalyzes phosphoenolpyruvate (PEP) to pyruvate (PYR) and is the last irreversible enzyme in the glycolysis pathway [95]. Glycerin-3-phosphate dehydrogenase (G3PDH) is involved in energy metabolic pathways such as glycolysis, gluconeogenesis, and the Calvin cycle, and plays an important role in promoting lipid synthesis [96]. The expression of the G3PDH and PK genes is found to be enhanced with increasing nitrogen application levels [97]. While many genes regulate lipid synthesis after nitrogen application, the regulatory network behind it is complex and huge, as oil content in rapeseed is a complex trait. Therefore, the molecular mechanism of nitrogen levels affecting the oil content of rapeseed still needs to be further investigated.

Figure 1. Distribution of carbon and nitrogen source-sink nutrients in rapeseed. The sympathetic transporter (NRT) transports nitrate (NO₃^-) to mesophyll cells, and the nitrate reductase (NR) converts nitrate (NO₃^-) to nitrite (NO₂^-). Nitrite (NO₂^-) binding H⁺ is transferred to the stroma of chloroplasts and converted to ammonium (NH₄⁺) by nitrite reductase (NiR). Ammonium (NH₄⁺) is converted to glutamate by glutamine synthetase (GS) and glutamate synthetase (GOGAT). Glutamic acid is converted to other amino acids in the cytoplasm [29]. The root absorbs N and transports it to the leaves for photoassimilation, and the assimilated product is transported through the phloem to the developing leaves and growing roots. Nutrients from old leaves are remobilized into developing seeds for lipid and protein synthesis. The green line shows the path of nitrogen transfer and the red line shows the path of redistribution of photosynthetic products. PEP: phosphoenolpyruvate; PYR: pyruvate.

Figure 1. Distribution of carbon and nitrogen source-sink nutrients in rapeseed. The sympathetic transporter (NRT) transports nitrate (NO₃^-) to mesophyll cells, and the nitrate reductase (NR) converts nitrate (NO₃^-) to nitrite (NO₂^-). Nitrite (NO₂^-) binding H⁺ is transferred to the stroma of chloroplasts and converted to ammonium (NH₄⁺) by nitrite reductase (NiR). Ammonium (NH₄⁺) is converted to glutamate by glutamine synthetase (GS) and glutamate synthetase (GOGAT). Glutamic acid is converted to other amino acids in the cytoplasm [29]. The root absorbs N and transports it to the leaves for photoassimilation, and the assimilated product is transported through the phloem to the developing leaves and growing roots. Nutrients from old leaves are remobilized into developing seeds for lipid and protein synthesis. The green line shows the path of nitrogen transfer and the red line shows the path of redistribution of photosynthetic products. PEP: phosphoenolpyruvate; PYR: pyruvate.

5. Discussion and Expectation

Rapeseed oil production is highest when nitrogen is applied at a rate of 180 kg·hm^-2 [4,7,19]. Most of the current research has focused on the effect of nitrogen application on the yield of rapeseed or the effect of nitrogen application on the oil content, but little work has been done on the mechanisms by which nitrogen application affects the yield and oil content. It is necessary to investigate the mechanisms by which nitrogen affects yield and oil content, to balance the relationship between yield and oil content, and to achieve the goal of reducing nitrogen application without reducing oil production from rapeseed. In the future, to increase oil production by nitrogen application, the following suggestions have been made:

(1) Improving the cultivation level and the proper application of nitrogen. The relationship between protein and oil content should be balanced. Suitable varieties of rapeseed should be selected first, and then the amount of nitrogen should be applied to improve the yield and quality. The amount of nitrogen can be adjusted according to the needs in the range of 0 ~ 337.5 kg·hm^-2 [7]. Studies have also shown that a nitrogen application rate maintained in the range of 90 ~ 180 kg·hm^-2 can get higher oil content in the case of increased yield, but nitrogen agronomy efficiency, nitrogen partial productivity, nitrogen apparent efficiency, and nitrogen physiological efficiency of rapeseed are all at high values when the rate is 180 kg·hm^-2 [19].

(2) Excavation of rapeseed for carbon and nitrogen-efficient germplasm resources. Most of the previous studies used a small number of accessions to analyze the relationship between nitrogen application rate and oil content but lacked representation. There are abundant germplasm resources of rapeseed with genetic diversity in China. It is necessary to ascertain whether any varieties produced an almost non-decreasing effect in the oil upon application of nitrogen. If such variety resources exist, high yield and oil content varieties with significantly increased oil yield after nitrogen application can be cultivated in the future.

(3) Studying the mechanism of carbon and nitrogen balance and identifying the important regulatory genes. Carbon and nitrogen partitioning in rapeseed growth and development is controlled by multiple genes and is involved in multiple metabolic pathways. A large number of previous studies have found that genes such as ACCase, PEPC, and DGAT2 control or affect carbon source partitioning [6,21,57]. 448 candidate genes related to acyl lipids and 11 genes related to storage proteins in rapeseed were identified [91]. However, the complex regulatory effects of nitrogen on oil and protein synthesis remain unclear. Functional genomics methods can be combined with transcriptome data to identify genes that control carbon and nitrogen partitioning, which is also an essential direction for future research.