| Rice, rich in starch and high quality protein, is one of the major food crops in the world. But lysine is the first limiting essential amino acid in rice, which limited the nutritive value of rice as both food and feed. Efforts, including genetic engineering, thus far have not achieved adesirable level of lysine in rice. The synthesis and degradation of lysine is regulated by a complex metabolic network. In higher plants, the aspartate-family pathway, synthesizes, through several different metabolic branches, four essential amino acids including lysine. It has a strong metabolic connection with the other pathway such as the cellular energy metabolism, revealing the multifaceted role of aspartate-family pathway in plant metabolism.In previous studies, we engineered rice for increased lysine through expressing bacterial lysine feedback-insensitive aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS) to enhance lysine biosynthesis; through RNA interference or knockdown of rice lysine ketoglutaric acid reductase/saccharopinedehydropine dehydrogenase (LKR/SDH) to down-regulate its catabolism; and by combined expressing AK and DHPS and interference of LKR/SDH to achieve both metabolic effects. Results showed that the increase of lysine content was quite different among different transgenic events. Besides, it also needs more works to elucidate the nutritional value, field performance as well as the metabolic connection with other pathways.In this study, with the aim to better understand the characteristics of selected transgenic rice and further metabolic regulation of lysine accumulation in rice, five types of transgenic rice lines and their wild type (WT) were used, and the molecular characterization, quality and field performance, nutritional value, and metabolome and transcriptome were carefully analyzed and compared among different transgenic events. The five transgenic rice lines were all derived from the japonica cultivar Wuxiangjing 9 (WT), including:(1) 35S-15 transgenic line containing the bacterial AK and DHPS genes driven by the CaMV 35S promoter (indicated as 35S transgenic event); (2) GR-14 and GR-65 transgenic lines containing bacterial AK, DHPS and rice LKR-RNAi transgenes all driven by rice glutelin GluB-1 and Gtl promoters (GR transgenic event); (3) HFL1 and HFL2 are two pyramid transgenic lines from the cross between 35S-15 and GR-14 or GR-65, respectively, and identified to carry both 35S and GR events. All the transgenic rice was free of the selectable marker gene. The followings experiments were carried out and the main results were presented below. All the data and results will be made to understand biological processes associated with the lysine metabolism, as well as its regulation mechanisms, and its importance for optimal improvement of the nutritional quality of crops.1. Molecular characteristics, quality evaluation and field performance of high-lysine transgenic rice.The characteristics of different transgenic events, especially the two pyramid transgenic lines HFL1 and HFL2, were compared with their wild type (WT). The results from quantitative RT-PCR and Western blot analyses showed that the bacterial AK and DHPS genes had been successfully integrated into rice genome and highly expressed, and the endogenous LKR gene was suppressed in related transgenic rice. However, there was no or slight impact on the expression of rice endogenous AK and DHPS genes in transgenic rice. The free lysine content in mature seeds of 35S-15 transgenic line increased by 35% compared with WT. In mature seeds of GR-14 and GR-65 lines, the free lysine content significantly increased up to 10 and 8 folds compared with WT, respectively. For the case of pyramid transgenic events HFL1 and HFL2, the free lysine content highly accumulated, up to 25 and 20folds compared to WT, respectively. With the increase of free lysine level, the contents of other amino acids also increased in mature seeds of transgenic rice. In HFL1 and HFL2 mature seeds, the contents of total free amino acids, total lysine (including the protein-bound form), total amino acids and total proteins also significantly increased. The above data proved that it would be help to accumulate more lysine by pyramiding the 35S and GR transgenic events.The data of physical-chemical qualities were compared, and the results indicated that the apparent amylose content of HFL1 and HFL2 slightly decreased, and the gel consistency became soft. The RVA profiles of isolated starches were identical between transgenic rice and their wild type, implying no obvious impact of high lysine accumulation on starch quality in transgenic rice. The results from field trials showed that, expect of the plant height of 35S-15 transgenic rice, there was no or little impact on the agronomic traits as well as grain yield of transgenic rice. However, as the result of high accumulation of lysine in transgenic rice, some mature grain appears dark brown phenotype, especially in HFL transgenic events. Interestingly, it was shown that the germination vigor of high lysine transgenic rice was faster than WT, which is quite different from that in Arabidopsis and tobacco. A series of physiological and biochemical analyses during seed germination revealed that the starch hydrolysis in transgenic seeds was faster than WT during the early stage of germination, but it was comparable on the late stage of germination as well as the stage of seedling formation.2. Evaluation of nutritive value of high-lysine transgenic rice in rats.With the aim to evaluate the nutritive value of high-lysine transgenic rice, the milled rice from two pyramid transgenic lines HFL1 and HFL2 and their wild type (WT) were used to prepare for the diets for 60 days of animal (SD rats) feeding trials. Besides of the above three diets, other four diets containing the WT rice plus different level of synthetic lysine were also prepared. The supplement of lysine is 10%,20%,40% and 60% of the total lysine content of WT rice, and the rats fed with these diets were termed as groups of WT+10%Lys, WT+20%Lys, WT+40%Lys and WT+60%Lys, respectively.During 60 days of feeding trials, the rats of each group grew normally, and no abnormal death phenomenon was observed. The results showed that the biological properties of body weight, body weight gain and food utilization were much higher in rats fed with HFL1 or HFL2 diets than those with WT diet. All the above indexes were similar between two HFL groups and WT+20%Lys one, which was supplied with additional 20% lysine. The data from N-balance analysis revealed that the apparent digestibility, protein efficiency ratio and lysine availability were also similar between the rats with HFL diets and WT+20%Lys one, but quite higher than those with WT diet. It indicated that biofortification of lysine in transgenic rice obviously enhanced their nutritional value. Besides, it was found that the supplement of synthetic lysine should be optimal to about 20-40%. Moreover, there was no obvious difference between theorgan/body weight ratio in rats fed with high-lysine transgenic rice and WT rice, implying no obvious undesirable effect of high-lysine transgenic rice in rats.3. Comparison of metabolome and transcriptome between high-lysine transgenic rice and wild type.In order to improve the lysine content in plant without adverse effects, it is necessary to explore the metabolic connection of lysine metabolism with other related pathways. In present study, the metabolomics platform with integration of LC-MS/MS and GC-MS techniques was used to analyze the metabolome of leaf, developing and mature seeds. Meanwhile, the transcriptome of either leaf or developing seeds was detected by RNA-Seq and compared between transgenic rice and their wild type.The results from metabolomics analyses indicated that regulation of lysine metabolism pathway in GR-14 and GR-65 lines, in which the foreign genes were driven by the endosperm-specific Gtl promoter, had limited effects on the metabolites’level in leaves and developing seeds at 10 days after flowering (DAF). But, the number of different compounds was larger in leaves and developing seeds of 35S-15 line than other transgenic events. In the case of mature seeds, there were much more differential metabolites in transgenic rice when compared with WT, especially in pyramid transgenic lines HFL1 and HFL2. These differential compounds were mainly involved in the pathways of amino acids, and then peptides, lipids, nucleotides, and carbohydrates.After comparison of the transcriptome of developing seeds at 10 DAF or 15 DAF with wild type rice, it was found that there were much more differential expressed genes (DEGs) in GR-65 and its derived line HFL2, but relatively few DEGs in 35S-15, GR-14 and their derived pyramid line HFL1. This suggested that regulation of lysine metabolism pathway altered more genes expression level in GR-65 and HFL2 than that in GR-14 and HFL1, which might be due to the difference of individual transgenic events. GO analysis showed that the DEGs were enriched in the pathways related to cellular metabolic processes, primary metabolic processes and small molecular metabolic processes. Based on the KEGG database, these DEGs were classified into the pathways of amino acid metabolism, plant stress response, lipid metabolism, and carbohydrate metabolism. Interestingly, it was revealed that the number of DEGs in developing seeds at 15 DAF was less than that at 10 DAF (P<0.05), and much more DEGs in developing seeds at late filling stage were identified to be involved in response to plant stress. This suggested that lysine metabolic pathway, deriving from aspartate family pathway, might be associated with many other metabolic pathways and played multiple roles in higher plant.4. Dissection of the metabolic connection for the dark-brown kernels in high-lysine transgenic rice.An unexpected change in high-lysine transgenic rice is the dark-brown phenotype in some mature seeds. The analyses from multiple comparison of metabolome and transcriptome revealed that it was altered of the lysine metabolism, TCA cycle and glycolysis, aromatic amino acids and secondary metabolism, carbohydrate metabolism, purine and pyrimidine metabolism and lipid metabolism pathway in transgenic rice, especially the pyramid lines with very high level of free lysine. Further analysis revealed that the specific significant accumulation in dark-brown seeds might be trytamine and serotonin from the pathway of tryptophan metabolism. The data from LC-MS analysis confirmed that there was high level of trytamine and serotonin in purified dark-brown substrates. Meanwhile, the up-regulated expression levels of TDC and T5H genes, two key members involving in trytophan metabolism, were also confirmed by using quantitative RT-PCR. In addition, over-expression of TDC1 or TDC3 gene in rice further confirmed that much trytamine and serotonin accumulated in rice seed, resulted in dark-brown phenotype. Thus, it was obvious that the dark-brown phenotype in mature seeds of high-lysine transgenic rice might be due to the high level of trytamine and serotonin.Using differential metabolites analysis among different phenotype accompanied with transcription analysis, we found that the accumulation of lysine in seeds induced TDC expression by jasmine acid pathway, and then promoted the accumulation of trytamine and serotonin. Furthermore, the dark-brown substance in seeds could be induced by low temperature, and it accumulated significantly in low temperature and enhanced dark-brown phenotype, while no or little dark-brown phenotype was found under high temperature. Besides, there were massive metabolites related to plant stress response highly accumulated in HFL1 and HFL2 seeds. Taken together, it could be concluded that regulation of lysine metabolic pathway in rice might cause the change of plant stress related pathway, and then induce the tryptophan metabolism and accumulate more trytamine and serotonin, and, finally, produce the dark-brown seeds. |
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