Reducing the nitrate content of burley tobacco by grafting with ue-cured tobacco

Background: Nitrosition of tobacco pyridine alkaloids by nitrate-derived NOx is the origin of tobacco-specifc nitrosamines (TSNAs) formed in tobacco, which are among the most notable toxicants present in tobacco products and smoke. Burley tobacco is particularly susceptible to TSNA formation because the cultivars exhibit a chloroplast-decient and nitrogen-use-defciency phenotype which results in high accumulation of nitrate. Strategies to lower nitrate levels in tobacco could produce a corresponding decrease in TSNAs accumulation in leaves. Previous studies have showed that grafting with high nitrogen use eciency rootstock was able to improve the nitrogen utilization of ue-cured tobacco. In this study, a reciprocativel grafting experiment has been conducted with two varieties of different tobacco types (burley tobacco ‘Eyan No.1’ and ue-cured tobacco ‘K326’) to investigate whether replacing burley tobacco root with ue-cured tobacco by grafting can enhance pigment biosynthesis and photosynthesis parameters and reduce nitrate content of burley tobacco leaves, and to explore the corresponding mechanism. Results: The results showed that compared with the self-rooted burley tobacco seedlings, grafting signicantly increased the pigment content, net photosynthesis, biomass, total soluble sugar, reducing sugar, nitrate reductase activity, glutamine synthetase activities, NH 4 -N and soluble protein content of the leaves of Eyan No.1 while decreased the nitrate content and NO 3 -N/TN. Interestingly, transcription level analysis suggested that replacing burley tobacco root with ue-cured tobacco by grafting was able to up-regulate the genes involved in starch and sucrose metabolism, porphyrin and chlorophyll metabolism, carbon xation in photosynthetic organism metabolism, carotenoid biosynthesis and nitrogen metabolism of burley tobacco leaves. In addition, the PPI network revealed gene_68511, gene_35043 and gene_77508 had higher degrees via replacing burley tobacco root with ue-cured tobacco, which might be hub proteins to reduce nitrate accumulation. Conclusions: In conclusion, grafting with high nitrogen use eciency rootstock provided an exceptionally promising means of nitrate reduction in burley


Background
Tobacco (Nicotiana tabacum L.) is a nonfood crop that is economically important worldwide and is grown in many countries [1 . Cured leaves of different tobacco market types ( ue-cured, burley and Oriental) are typically blended together in the production of cigarettes. Of these market types, burley tobacco is unique in that they display a chloroplast-de cient phenotype and is impaired in nitrogen use e ciency with reduced pigment content [2 . As a result, burley tobacco requires 3-5 times levels of N-fertilization than ue-cured tobacco to achieve comparable yields [3 . Lewis et al. [4 found reduced nitrogen use and utilization de ciencies in burley tobacco are attributed to two homozygous recessive mutant alleles at the Yellow Burley 1 (Yb1) and Yellow Burley 2 (Yb2) loci, which are probably the main explanations for the typically high fertilization level of burley tobacco with greater quantities of nitrogen, to achieve a yield that is commensurate with that of most other tobacco types. Carbohydrates are the material basis for plant metabolism. From the perspective of energy metabolism, an important reason for the increase in nitrate accumulation in burley tobacco was the reduction of carbon assimilation products induced by the decrease in photosynthesis [5 . The increased leaf nitrate nitrogen content is associated with the formation of tobacco-speci c nitrosamines (TSNAs), which is a class of toxicants that contribute to cancers formation, in burley tobacco [4 , since nitate could be reduced to nitrite by microbiols during leaf curing and could give rise to gaseous NOx during leaf storage [6][7] , which are important nitrosating agents. Therefore, technologies to decrease nitrate content in burley tobacco should be justi ed to decrease the risk exposed to the portion of the population that uses tobacco products.
Based on above information, parameters such as photosynthesis, pigment content, nitrate reductase acivity (NRA), glutamine synthetase acivity (GSA) and total nitrogen content are highly related to nitrate content, and all these traits should be taken into account when considering nitrate decrease. In recent years, many strategies including molecular breeding [8 , chemical regulation [9 and agronomic practises [10 have been conducted to reduce nitrate content in burley tobacco. Nevertheless, such approaches still have some limitations. For instance, the potential of chemical and agronomic regulation to reduce nitrate content in buley tobacco leaves is very limited. Overexpression or knockout of the key genes related to nitrate content can effectively alleviate nitrate accumulation in burley tobacco leaves, but it is costly and di cult to operate owing to the impact on quality and restrictions on genetically modi ed organisms [11][12] .
Grafting, as an ancient cultivation technique with a long history, has been reported as an effective strategy to enhance plant resistance [13 , increase yield [14 , improve photosynthesis [15 and reduce the alkaloid synthesis [16 . In particular, grafting has a signi cant impact on improving the absorption of nutrients by using cultivar rootstocks with more advantageous nitrogene cient absorption and enhanced nitrogen-use e ciency, thus alleviating the nitrate accumulation in tobacco [17 . Another described effect of grafting is the alteration of the hormonal balance between rootstock-scion [18][19] . Beyond that, grafting also plays a signi cant part in facilitating plant photosynthesis performance [20 . However, no data relating the impacts of grafting on nitrate content of burley tobacco leaves was publicly available. In our previous study, we observed that nitrogen use e ciency and carbon assimilation differed signi cantly between ue-cured and burley tobacco with ue-cured tobacco exhibiting much higher levels of nitrogen use e ciencu than burley tobacco [5 . The lower pigment content, week carbon xation and nitrogen assimilation were the main causes of abnormally high levels of nitrate accumulation in burley tobacco.
Taken above information into consideration, we hypothesized that grafting burley tobacco onto ue-cured tobacco can improve the photosynthesis and nitrogen utilization e ciency of burley tobacco, thus decreasing the nitrate content of burley tobacco leaves. Therefore, a pot experiment using burley tobacco as scion and ue-cured tobacco as rootstock was conducted. The physiological and transcriptome differences were determined aiming to clarify whether the replacement of burley roots by uecured tobacco roots is able to reduce the nitrate accumulation in burley tobacco leaves and to investigate the mechanism of this decrease.

Differences in pigment content and photosynthesis traits
The pigment content, net photosynthetic rate, biomass accumulation and carbohydrate content are displayed in Figure 1. There were signi cant differences in the pigment content and photosynthesis between different grafting groups. Contents of chlorophyll a, chlorophyll b, carotenoid and pigment were signi cantly lower in E/E than those in K/K (Figure 1.a-d). Also, the net photosynthesis rate, biomass accumulation and carbohydrate content were always lower in E/E than those in K/K (Figure 1.e-h).
Lower pigment content may have an in uence on carbon xation and lead to low biomass and carbohydrate accumulation in the leaves of E/E. The pigment content and photosynthesis were signi cantly improved in the grafting combination, chlorophyll a, chlorophyll b, carotenoid, pigment contents and net photosynthesis rate increased by 20.17%, 22.43%, 48.38%, 25.44% and 21.89% in the leaves of E/K in comparision to E/E (Figure 1.a-d). In addition, burley tobacco plants grafted onto 'K' rootstocks showed higher total soluble sugar and reducing sugar content of 21.05% and 79.44% of the leaves (Figure 1.g-h). Regardless of the leaves, stem or roots, the biomass accumulation in E/K plants was higher than that in E/E. More nitrogen could be absorbed by the roots of ue-cured tobacco, thus resulting in the increase of pigment content which contributed to the increase of photosynthesis and carbohydrate accumulation.

Difference in enzyme activities related to nitrogen metabolism and nitrogen compounds
Two key enzyme activities, namely, NR and GS, and nitrogen compounds were measured in tobacco seedling tissues, as shown in Data processing Nine cDNA libraries were established for RNA-seq analysis. A total of 131.07, 128.42, and 128.62 million raw reads were identi ed in the K/K, E/E and E/K samples, respectively. After data processing, 129.38 (98.71%), 126.30 (98.35%) and 126.79 (98.58%) million clean reads were obtained for further analysis (Table 1). More than 94% of the clean reads across all the samples were mapped to the tobacco reference genome using Hisat2 [21 , most of which had unique location in that genome. And more than 92.05% of cDNA sequencing quality was ≥ 30 (Q30), implying successful library construction and reliable for subsequent bioinformatics analysis.
Gene expression and correlation analysis between samples As shown in Figure 3.a, the FPKM expression levels for each sample were calculated. In addition, Pearson's correlation coe cient and principle component analysis (PCA) of the data pro les from all 9 samples revealed high correlation among all the samples (Figure 3.b-c). These analysis demonstrated that the sequencing data in this study was adequately representative and valid.

GO enrichment and KEGG pathway analysis of DEGs Response to Grafting
To explore the DEGs in E/E plants improved by using the root of ue-cured, venn digram analysis between E/K_E/E_up and E/E_K/K_down was conducted ( Figure 5.a). GO and KEGG were then conducted to examine the potential gene function and metabolism pathway of DEGs. Venn analyses showed that 1590 DEGs were enriched between E/K_E/E_up and E/E_K/K_down. The GO and all KEGG pathways are presented in Figure 5.b-c. The genes were signi cantly enriched in the pathway of starch and sucrose metabolism, porphyrin and chlorophyll metabolism, carbon xation in photosynthetic organism metabolism and carotenoid biosynthesis.

DEGs Involved in Carbon and Nitrogen Metabolism
Transcriptome sequencing technology provides a large amount of information regarding the DEGs that are involved in speci c biological responses. To further understand how the nitrogen and carbon metabolism-related genes in E/K plants changed compare to E/E tobacco seedlings, we extracted the DEGs involved in starch and sucrose metabolism, porphyrin and chlorophyll metabolism, carbon xation in photosynthetic organism metabolism, carotenoid biosynthesis and nitrogen metabolism ( Figure   6.a-e).The results showed that DEGs were down-regulated in E/E tobacco than in K/K tobacco while up-regulated in E/K tobacco seedlings, which might also be the cause for lower nitrate content in E/K-treated tobacco.
PPI network construction and network degree analysis The PPI networks were constructed (Figure 7.a-c).There were 283 protein nodes and 300 protein pairs in the PPI network. After network degree analysis, gene_68511 had highest degrees in networks, followed by gene_35043 and gene_77508, which might be hub proteins.

Discussion
It has long been reported that the growth and yield of nitrogen-e cient genotypes was higher than those of nitrogen-ine cient genotypes under normal or low nitrogen conditions [22][23] . Studies by lewis et al. [4 and Shi et al. [5 indicated that burley tobacco had a weak nitrogen utilization e ciency and nitrogen assimilation capacity, thus leading to lower biomass and higher nitrate accumulation. In this study, we found that grafted burley tobacco with a ue-cured tobacco rootstock had reduced levels of nitrate accumulation in leaves (Figure 2.e). Our study also showed that the root, stem and leave biomass were lower in selfgrafted burley tobacco (E/E) compared with self-grafted ue-cured tobacco (K/K) (Figure 1.f ). However, grafting burley tobacco onto ue-cured tobacco (E/K) improved the growth of burley tobacco, indicating rootstock indeed had signi cant in uence on scion growth to some extent. The results are in line with that of Huang Yuan [24 , who reported that grafting with wild watermelon rootstocks with high nitrogen use e ciency substantially improved whole plant growth.
Chlorophyll content is used as an indicator of photosynthesis which functions to provide both energy and carbon skeletons for plant growth and N assimilation [25,8 . Burley tobacco is a chloroplast-de cient mutant with reduced pigment content and lower photosynthetic capacity [2 . In this study, it was concluded that grafting burley tobacco onto ue-cured tobacco enhanced pigment content, net photosynthesis rate and photosynthesis products of burley tobacco (Figure 1.a-h), and also promoted the total nitrogen and nitrogen accumulation in root (Figure 2.g-h). This nding suggests that the ue-cured tobacco rootstocks has an increased nutrient acquisition capacity, which was translated into higher leaf chlorophyll content and photosynthesis products [26][27] . These results are similar to those found in other eld crops and tomato, in which an increase of absorption and utilization of nitrogen and photosynthesis was observed in grafted plants [28,20 . In addition, studies have also shown that grafting signi cantly affected the content of endogenous hormones in plant leaves, thereby having regulatory effect on photosynthetic capacity of plant leaves [29][30] , which need to be veri ed in our study. Furthermore, transcriptome analysis revealed 5777 DEGs between burley tobacco/ ue-cured tobacco (E/K) and burley tobacco self-grafted combination (E/E), which were signi cantly enriched in the pathways of starch and sucrose metabolism, porphyrin and chlorophyll metabolism, carotenoid biosynthesis, carbon xation in photosynthetic organism, and nitrogen metabolism (Figure 4.a, k-i). Further investigation of down-regulated DEGs between burley tobacco self-grafted and ue-cured tobacco selfed-grafted combination and up-regulated DEGs between burley tobacco/ uecured tobacco and burley tobacco self-grafted combination suggested ue-cured tobacco rootstock promoted pigment biosynthesis and photosynthesis of burley tobacco leaves ( Figure 5.a-c). Expression of genes of POR (gene_13949), PBG (gene_76264) and GSA (gene_69664) were greatly up-regulated by grafting burley tobacco onto ue-cured tobacco (Figure 6.b). Beyond that, the genes related to carbon xation in photosynthetic organism and starch and sucrose metabolism were all upregulated by grafting burley tobacco onto ue-cured tobacco, including SBP (gene_47109), which encodes a key enzyme in ribulose-1,5-bisphosphate (RuBP) regeneration and Calvin cycle process [31 , FBA (gene_57322) , which has a major in uence on the growth and photosynthesis of tobacco [32][33] .
Nitrogen metabolism needs energy and carbon skeleton provided by photosynthesis and carbon xation [ 34 ,8 . In this study, nitrate content and NO 3 -N/TN of burley tobacco was signifcantly decreased by using an ue-cured tobacco as rootstock, indicating that the replacement of root system reversed the disadvantage of scion to some extent. Nitrogen metabolism is one of the most important and basic metabolisms in plants and nitrate content is highly correlated with the capacity of nitrogen assimilation [35,5 . Nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS) are considered to play key roles in nitrogen assimilation [36][37] . Previous work published elsewhere has indicated that grafting improved tomato yield by enhancing the activities of GS [28 . Our results demonstrated that grafting burley tobacco onto ue-cured tobacco increased the activities of NR and GS in burley tobacco. The greater e ciency in NO 3 − reduction and assimilation in grafted plants was con rmed by the results for NH 4 -N, proteins and total N, which were all higher in grafted burley tobacco with an ue-cured tobacco rootstock compared with self-grafted burley tobacco. Moreover, expression of genes NiR (gene_84305) and GS (gene_61180) were signi cantly up-regulated, indicating that nitrogen metabolism was markedly stimulated in burley tobacco with a ue-cured tobacco rootstock.

Conclusion
In conclusion, in this experiment, we found that reduction in nitrate was accompanied by signi cant increases in pigment content, photosynthesis, carbohydrate and nitrogen assimilation capacity, indicating rootstocks from plants with better NUE traits can be used in grafting to improve this characteristic in less e cient shoots. In a word, grafting in tobacco plants can be de ned as a rapid and effective alternative for enhancing pigment biosynthesis and photosynthesis, thereby increasing yield and decreasing nitrate and subsequent TSNA accumulation in tobacco.