Coordination of carbon and nitrogen accumulation and translocation of winter wheat plant to improve grain yield and processing quality

The objective of this work was to characterize the accumulation of carbon (C) and nitrogen (N), and the translocation of wheat (Triticum aestivum L.) cultivars to achieve both high-quality and high-yield. Twenty-four wheat cultivars, including 12 cultivars containing high-quality gluten subunit 5 + 10 at Glu-D1, and 12 cultivars with no Glu-D1 5 + 10, were planted at Yuanyang and Xuchang in Henan Province, during 2016–2017, and 2017–2018 cropping seasons. Wheat cultivars containing Glu-D1 5 + 10 had an advantage in grain quality traits. Significant difference (P < 0.05) was observed for grain protein concentration (GPC) between 5 + 10 group and no 5 + 10 group. Grain yield (GY) was significantly correlated with kernel number (KN) (r = 0.778, P < 0.01), thousand-kernel weight (TKW) (r = 0.559, P < 0.01), dry matter accumulation at post-anthesis (r = 0.443, P < 0.05), and stem water-soluble carbohydrate (WSC) accumulation (r = 0.487, P < 0.05) and translocation amount (r = 0.490, P < 0.05). GPC, dough stability time (DST) and nitrogen agronomic efficiency (NAE) were significantly correlated with nitrogen accumulation (NAA) at maturity stage (r = 0.524, = 0.404, = 0.418, P < 0.01, < 0.05, < 0.05, respectively), and nitrogen translocation amount (r = 0.512, = 0.471, = 0.405, P < 0.05, < 0.05, < 0.05, respectively). These results suggest that good-quality, high-yield, and high-efficiency could achieve through the selection of high-quality wheat cultivars and coordination of C and N accumulation and translocation. High-quality gluten subunit gene Glu-D1 5 + 10 and stem WSC could be used as a selection index for breeding and production of high-quality and high-yield wheat.


Results
As listed in Table 1, analyses of variance were conducted for GY, kernel number (KN), thousand-kernel weight (TKW), plant height (PH), anthesis date (AD), aboveground biomass at jointing stage (AGBM-J), aboveground biomass at anthesis stage (AGBM-A), aboveground biomass at maturity stage (AGBM-M), WSC accumulation amount of stem peduncle (WSC-P), WSC accumulation amount of stem lower internodes (WSC-L), WSC accumulation amount of the total stem (WSC-T), WSC translocation amount of stem peduncle (WSCT-P), WSC translocation amount of stem lower internodes (WSCT-L), WSC translocation amount of the total stem (WSCT-T), leaf area index at jointing stage (LAI-J) leaf area index at anthesis (LAI-A), plant N accumulation at anthesis (NAA), plant N accumulation at maturity (NAM), N translocation amount to grain (NTA), N agricultural efficiency (NAE), grain protein concentration (GPC), WGC (wet gluten content), dough stability time (DST), dough water absorption rate (DWR). Cultivar, environment and cultivar × environment contributed significantly to variation in most of the traits. Significant environment effects were observed for GY, KN and LAI-J. No cultivar × environment effect was exerted on DST.

Source of variation
Correlations among grain quality, NAE and physiological traits. As shown in Figure  These results indicate that the N translocation amount from vegetative organs to grain play key roles in Table 2. Yield components, morphological and processing quality traits of 24 wheat cultivars averaged for four environments. The italicized data followed by different capital letters in the same column indicate a significant difference at P < 0.05. GY grain yield, KN kernels number, TKW thousand-kernel weight, PH plant height, AD anthesis date (from seedling emergence to anthesis), GPC grain protein concentration, WGC wet gluten content, DST dough stability time, DWR dough water absorption rate. www.nature.com/scientificreports/ influencing grain quality and NAE. Similar correlation relationship was also observed between grain quality and physiological traits of wheat cultivars within the same Glu-D1 allele (Table S1). NTA was positively correlated with GPC and DST in both Glu-D1 5 + 10 group and no Glu-D1 5 + 10 group. But, the significant correlation was only found within Glu-D1 5 + 10 group, with the value being r = 0.740 (P < 0.01), and = 0.720 (P < 0.01), respectively. Also, NAE was positively significantly correlated with WSC-T, WSCT-P, WSCT-L, and WSCT-T with r = 0.727 (P < 0.01), = 0.825 (P < 0.01), = 0.629 (P < 0.01), and r = 0.764 (P < 0.01) within no Glu-D1 5 + 10 wheat group, respectively.

Discussion
Increasing the yield of wheat has always been one of the primary goals of wheat production, especially in a country like China with a large population. With the development of agricultural technology, the yield levels in the HHWWZ increased from 5,500 kg ha −1 in 1980s to over 7,500 kg ha −1 after 2010 32 . Since the 1980s, the quality of wheat has garnered attention, and some high-quality wheat cultivars (strong gluten wheat) have been bred. However, the quality improvement of these high-quality wheat cultivars is sometimes accompanied by the decrease of GY. The HHWWZ is an irrigated area, and the major wheat production objectives in this zone are to improve wheat yield and the processing quality for pan bread and noodles 24 . Thus, it is very important to achieve a high yield of high-quality wheat in HHWWZ. Here, five wheat cultivars, including 'Zhengmai 119' , 'Zhengmai 369' , 'Fengdencun 5' , 'Zhoumai 32' , and 'Sandemai 1' , not only have high GY (the average GY over 7,500 kg ha −1 ), but also have good quality traits (the average GPC, WGC, DST and DWR all meeting the standard of Qualify Classification of Wheat Varieties from the People's Republic of China 33 ). These results indicate that high-quality wheat cultivars could achieve high GY through coordinating C and N metabolism. It is generally considered that HMW-GS 5 + 10 play a great contribution to dough strength 7,30,31 . Here, wheat cultivars 'Zhengmai 119' , 'Zhengmai 369' and 'Fengdecun 5' containing the subunit pair 5 + 10 had better grain quality traits; the subunit 5 + 10 may be the basis for their high-quality. However, we also noticed that 'Zhoumai 32' and 'Saidemai 1' without HMW-GS 5 + 10 also processed high quality traits. These phenomena mainly contributed to other quality related genes, such as low molecular weight glutenin subunits (LMW-GS) 28 . But, we have to mention that Table 3. Physiological traits of 24 wheat cultivars averaged for four environments.  www.nature.com/scientificreports/ wheat cultivars with high-quality subunits (5 + 10) generally have more opportunities to have good grain quality. Especially, we found that cultivars genotyped with 7 + 8 (Glu-B1) and 5 + 10 (Glu-D1) got a significantly higher GPC, DST, DWR, and NTA than cultivars genotyped with no 5 + 10. As previous reported, selection for subunits/ alleles 1, 7 + 8, 5 + 10, and Glu-A3d would be more effective for improving gluten quality and pan bread quality 7 . It is known that the dry matter accumulation or biomass enrichment is the basis for high-yield of grain 34 . In our study, we found that the dry matter accumulation at different growth stages significantly correlated to GY, but the biomass at maturity showed the highest correlation coefficient. Additionally, post-anthesis dry matter accumulation also significantly positive correlated to GY. This suggests that the dry matter accumulation after anthesis than that before anthesis plays a greater role in increased grain-yield. Our results are consistent with the findings of Zhou et al. 35 and Jiang et al. 36 , who suggested that maintaining high dry matter accumulation after flowering is an effective way to increase wheat yield. WSC in stem and sheath is an important C source for wheat grain and positively correlates to grain yield 17,18 . Xue et al. 18 suggested that much of the WSC in stem during the late grain-filling period can be remobilized to grains. In our study, WSC accumulation and translocation positively correlated to GY, which indicate that increasing stem WSC accumulation during the early grain-filling period and promoting its translocation during the late grain-filling period can potentially increase wheat grain yield. It has been previously reported that different stem internodes respond differently to the environment 37 , and stem WSC in the lower internodes have a greater role in grain yield than the upper internodes under drought 38 . Here, we also found that GY was significantly correlated with WSC accumulation in lower internodes and WSC translocation amount. But, the correlation coefficient between peduncle and GY was slightly higher than that between lower internodes and GY. One possible reason could be that stem was only divided into peduncle and the rest parts of stem in this study. Another reason may be due to the environment conditions wheat cultivar planted. The underlying mechanism of high WSC accumulation and transport may lie in the expression profiles of fructan metabolism related genes; higher expression of genes related to fructan synthesis and degradation at WSC accumulation and translocation stage, respectively 37 . Thus, the close relationship between stem WSC and GY indicate that keeping high WSC content and accumulation could contribute to increasing GY. Slewinski 39 also pointed out that manipulating the stem WSC is an avenue to stabilize and increase wheat grain, especially Table 4. Nitrogen accumulation, translocation amount and nitrogen use efficiency of 24 wheat cultivars averaged for four environments. The italicized data followed by different capital letters indicate a significant difference at P < 0.05. NAA-A nitrogen accumulation amount at anthesis, NAA-M nitrogen accumulation amount at maturity, NTA nitrogen translocation amount, NAE nitrogen agricultural efficiency, NUtE N utilization efficiency. www.nature.com/scientificreports/ in the face of the flourishing population in future. Thus, WSC accumulation could be used as a morphological and physiological criterion for increasing GY in wheat production or high-yield wheat breeding. Moreover, the correlation relationship among GY, grain quality and physiological traits within the same Glu-D1 allele seem to indicate that there is a relatively high correlation coefficient between nitrogen accumulation and grain quality traits within Glu-D1 5 + 10 wheat cultivars. At the same time there is a relatively close correlation between carbon accumulation and transport and grain yield in wheat cultivars without Glu-D1 5 + 10 allele. These results further suggest that the coordination of carbon and nitrogen accumulation is very important for high yield and high quality of wheat. N assimilation and remobilization differ among different wheat cultivars 40 , and the increase in wheat GPC is largely dependent on the accumulation and remobilization of N before flowering 36 . In this study, N accumulation and translocation significantly positive correlated to GPC and ST, which indicate that the grain processing quality mainly depends on the accumulation and translocation of N, especially the remobilization of N accumulated before anthesis. The genetic factors affecting the relationship of GY-GPC have been explored. It was reported that AD regulated by photoperiod response gene (Ppd-D1) affect N uptake between pre-and post-anthsis; photoperiod sensitive alleles (Ppd-D1b) are prone to produce higher GY rather than GPC in comparison with insensitive allele (Ppd-D1a) 41 . Bogard et al. 42 suggested that AD may be the genetic factor for post-anthesis leaf senescence which contributes to the negative GPC-GY relationship. In this study, no significant correlation was observed between AD and GY, between AD and GPC. No significant positive correlation coefficients were also observed between GY and GPC, suggesting the possibility of breeding wheat cultivars with high protein contents and high grain yield. Additionally, even there were difference for AD among the test cultivars; all the wheat cultivars possess Ppd-D1a allele (Table S2). Further, the HMW-GS 5 + 10 wheat group in this study showed both higher quality traits (especially for GPC) and N accumulation, translocation than no HMW-GS 5 + 10 groups. The 5 + 10 at Glu-D1 may be one of the internal factors contributing to the GPC by affecting N metabolism. The N metabolism related enzymes activity or genes expression level and regulation factor also contribute to GPC 9,43,44 . Improved NUE would reduce environmental contamination caused by excessive application of N fertilizer, and in turn, increase economic benefit for farmers. Brasier et al. 41 found that Ppd-D1 affected NUE, but the difference between insensitive allele (Ppd-D1b) and sensitive allele (Ppd-D1a) allele varied with N fertilization manner. Here, no significant correlation appeared between NUtE, NAE and AD among these tested wheat cultivars. But, we found that NAE was significantly correlated with N accumulation and translocation amount, which are agreement with the relationship between N translocation and grain quality.

HMW-GS 5 + 10 Cultivar
The results indicate that increasing N accumulation and translocation would be better to improve grain quality and NAE. In fact, wheat cultivar 'Fengdecun 5' got the highest NAE (16.27 kg kg −1 ) and NUtE (37.16 kg kg −1 ), apart from high GY and quality traits. The results indicate that high-yield, good-quality and high-efficiency could be achieved through the selection of high-quality wheat varieties (genetic factor) and coordination of C and N accumulation and translocation.  www.nature.com/scientificreports/ conclusions Wheat cultivars containing HMW-GS 5 + 10 have more advantages to obtain high grain quality. High-quality gluten subunit gene Glu-D1 5 + 10 would be one of the genetic factors contributing to GPC, DST by affecting plant N accumulation and N translocation from vegetative to grain. Stem WSC accumulation and translocation are related to GY, and can be used as s a morphological and physiological selection criterion for increasing GY. The results suggest that good-quality, high-yield, and high-efficiency could be achieved through the selection of high-quality wheat cultivars and coordination of C and N accumulation and translocation.  Supplemental  Table S3.The mean temperature and precipitation at the planting sites during the wheat growing season are shown in Supplementary Figure S1. Twenty-four wheat cultivars were selected as representatives of high-quality and high-yield potential among the widely grown cultivars in the HHWWZ; the major wheat-producing region in China that accounts for about 2/3 of China's wheat production 6 . All the cultivars and their providers are listed in Supplemental Table S4. According to the HMW-GS type, the wheat cultivars were classified into two groups: with HMW-GS 5 + 10, and without HMW-GS 5 + 10. The composition of HMW-GS of these cultivars was listed in Table S2. The experiments were conducted according to a randomized block design with three replicates. The plot size was 3 m (width) × 5 m (length) and the planting density was 250 seed m -2 . Each plot had 12 rows with equal spacing between the rows. All the plots received 135 kg ha −1 of P 2 O 5 as triple superphosphate and 135 kg ha −1 of K 2 O as potassium chloride pre-sowing. 210 kg N ha −1 was applied as urea where 50% of the N was applied before sowing and another 50% was applied at the elongation stage. N0 (no N fertilization) treatment for each wheat cultivar with three replicates was used as control to calculate NAE. The growth and development period are listed in Supplementary Table S5. The plants in 9-m 2 area in each plot were harvested manually when the plants attained physiological maturity. The quality of wheat grains was evaluated after storing them at room temperature for 2 months.

Materials and methods
Measurements. Twenty stems of each plot were sampled at heading, at anthesis, 10 days after anthesis (DAS), and at the maturity stages. At maturity, the plants were separated into leaves, stems with leaf sheaths (these parts were cut into three segments: peduncle and lower internodes, according to Hou et al. 37 ), chaff, and grain. The fresh samples were put into an oven at 105 °C for 30 min and then dried at 80 °C until they reached a constant weight for dry weight determination.
Dry matter translocation. Different parameters related to the dry matter were calculated according to Liu et al. 45 .
Nitrogen accumulation and translocation, nitrogen efficiency. N content of the samples were determined using the Kjeldahl (K1100) procedure, and the accumulation and translocation of N in wheat plants were calculated as follows 22,46 : N accumulation amount (kg ha −1 ) = N concentration (%) × dry matter accumulation amount (kg ha −1 ). Pre-anthesis N translocation (Pre-NT, kg ha −1 ) = Total aboveground N accumulation amount at anthesis − N of vegetative parts at maturity. N agronomic efficiency (NAE) = (Grain yield in N fertilizer treatments − Grain yield in control treatment)/ the amount of nitrogen fertilizer applied. N utilization efficiency (NUtE) = Grain yield/Above-ground N accumulation at maturity.
Water-soluble carbohydrates (WSc). WSC content was determined according to the method of Hou et al. 37 . Briefly, the stem sample (0.10 g) was extracted with 80 °C water for 40 min, centrifuged (4,500 r min −1 , 20 min), and the supernatant was collected. The extraction process was then repeated twice with 4 ml of 80% ethanol. The WSC content was quantified based on the total sugar content obtained by absorption at 620 nm. WSC translocation amounts (kg ha −1 ) = WSC accumulation amounts at 10 DAA − WSC accumulation amounts at maturity. flour quality traits. Wheat grains were milled using a laboratory test mill (Brabender Junior) based on an approved method 26-21A (AACC, 1995). The flour protein concentration was determined by a near-infrared transmittance analyzer (Foss Tecator 1241), and the wet gluten content was tested by a gluten testing system (Perten Glutenmatic 2200). The stability time and dough water absorption rates were determined by a Farinograph (Brabender Farinograph-E, Duisburg, Germany) according to an approved method AACC 54-21 (AACC, 1995). www.nature.com/scientificreports/ Identification of HMW-GS and photoperiod genes allele. Glutenin protein extracts were prepared according to He et al. 7 , and the fraction was separated by SDS-PAGE analysis according to Singh et al. 47 . Nomenclature of Payne and Lawrence 48 was used to classify HMW-GS, and wheat cultivar 'CS' with N, 7 + 8, 5 + 10 was used as a reference. The photoperiod genes were determined using gene-specific markers reported by Beales et al. 49 . The HMW-GS composition and photoperiod genes allele were listed in Table S2.
Statistical analysis. Analysis of variance (ANOVA) was performed using GLM in SPSS 19.0 software (Statistical Program for Social Science) for all traits, with cultivars and environments as fix factors. Differences among cultivars were tested using Tukey's test. Pearson's linear correlation analysis was conducted by correlatebivariate. All the figures were drawn using Origin 9.0 (Origin Lab Corporation, USA).