Balin and Bluemoon were obviously different in morphological and chromosomal characteristics. The natural vegetative plant height in Balin was significantly higher than that in Bluemoon, and its growth rate was 8.7 times of Bluemoon (Fig. 1a and 1b). The green intensity of the leaves reflects the quality of the turf. The darker the green leaf color, the higher the satisfaction with the turf quality (Fig. 1e). Data measured by SPAD 502 showed Bluemoon had higher green intensity than Balin (Fig. 1c), which indicated that the turf appearance of Bluemoon was better than Balin. ‘FISH’ results at the metaphase of mitosis showed that Balin was a hendecaploid with 83 chromosomes, while Bluemoon was a decaploid with 75 chromosomes (Fig. 1f and 1 g).
2.2 N Content, C/N Ratio And NUE
Two cultivars showed significant differences in N accumulation and saturated N content in leaves when N supply was higher than 8.0 mM (Fig. 1d). The N accumulation continued to increase to the maximum until the N supply concentration was at 15 mM, and then the N content in the leaves remained steady for both cultivars. At this point, N accumulation in Balin was 81.8% higher than Bluemoon (Fig. 1d). Therefore, the concentration of 15 mM could be regarded as the optimal N supply level for two cultivars. When plants were exposed to low N condition, the N content decreased in both cultivars, but the reduction rate of N content in Balin was greater than that of in Bluemoon. Normally, the N content in Bluemoon leaves was much lower than that in Balin under the condition of adequate N supply (Fig. 2a), however, during lower N process, the losses of N in Balin was remarkably greater and with the N content 11.4% lower comparing with Bluemoon. The ratio of carbon to nitrogen (C/N) is crucial to reflect the state of plant growth when plant enduring environmental stress. Under optimal N condition, the ratio of C/N in Bluemoon was slightly higher by 3.7% comparing with Balin, however, after treated with lower N, the ratio of C/N increased rapidly and varied significantly in two cultivars (Fig. 3e). Comparing with the optimal N supply, the average increase of C/N ratio in Balin was 36% higher than that of in Bluemoon. In addition, high NUE after low N stress indicated that Bluemoon possessed the trait of low N requirement (Fig. 2b).
2.3 Assay Of Enzymes Activities
During low N stress, the Nr activity in Bluemoon increased fast and evidently differences from other treatments (Fig. 2c), suggesting that N assimilation in Bluemoon at the N short stage was more strongly regulated by Nr than that of in Balin. The activities of NiR and GOGAT were showed in a similar trend with Nr in different N treatments. Low levels of nitrogen reduced NiR and GOGAT activities for both cultivars, while the activities of NiR and GOGAT in Bluemoon were 17.3% and 16.2% higher comparing with Balin (Fig. 2d and 2f). Under optimal N condition, GS activity was much higher in Balin than that in Bluemoon, however, under low N situation, GS activity increased in Bluemoon by 48.3% relative to Balin (Fig. 2e).
2.4 Chlorophyll And Photosynthetic Rate
Under optimal N supply, there was no significant difference in Chl content between the two cultivars, the Chl content in Bluemoon was only 1.2% higher than that of in Balin, but under low N condition, the decrease of Chl in Balin was remarkably greater and was 15.6% lower than Bluemoon (Fig. 3f). Chls are important components in photosynthetic center. Low N reduced Pn and Ci, while the rate of Pn and the concentration of Ci in Bluemoon were still 29.7% and 9.8% greater respectively than that of in Balin (Fig. 3a and 3b). Under optimal N condition, the maximum quantum yield in photosystemⅡ(Fv/Fm) was notably higher (P˂0.05) in Balin than in Bluemoon; however, under low N condition, Fv/Fm was 12.0% higher and NPQ was 20.1% lower in Bluemoon than that of Balin (Fig. 3c and 3d).
2.5 RNA-seq Results
2.5.1 Overview the RNA-seq
To identify the regulatory genes involved in N assimilation and photosynthesis carbon fixation, we conducted transcriptome sequencing of Balin and Bluemoon leaves. A total of 522,590 unigenes with an average length of 800 bp were achieved (Fig. S1a). The species distribution analysis revealed that KB had a number of homologous sequences in many plant species. The majority of transcripts had a significant level of sequence identity to Brachypodium distachyon, Aegilops tauschii, Hordeum vulgare, which accounted for 19.2%, 19.1%, 13.9%, of the total transcripts, respectively (Fig. S1b). This result suggested that the genome of Brachypodium distachyon may serve as a reference for the transcriptome analysis of KB. There were 189,919 (36.3% of all unigenes), 165,273 (31.6%), 53,260 (10.2%), 112,180 (21.5%), 133,162 (25.5%), 136,033 (26.0%), and 35,085 (6.7%) unigenes were found in the NR, NT, KO, SwissProt, PFAM, GO, and KOG databases, respectively. When the sequences were compared with all databases, only 14,672 (2.8%) were annotated in total, while all 269,020 unigenes (51.47%) were annotated in at least one database according to Blastx search (Table. 1).
As for GO clustering (Fig. 4a), the dominant subcategories were, ‘cellular process’ and ‘metabolic process’ in Biological Process (BP), ‘cell’ and ‘cell part’ in Cellular Component (CC), and ‘binding’ in Molecular Function (MF). Regarding KEGG categorization, 41,031 annotated genes were assigned to 137 pathways belonging to 19 metabolic groups (Fig. 4c); ‘carbohydrate metabolism’ and ‘translation’ represented the most abundant classes in ‘Metabolism’(D) and ‘Genetic information processing’ (C), respectively. In KOG analysis, 39,408 annotated genes were assigned to 25 groups. Among these annotated unigenes, several related to N reduction and assimilation were the most enriched in ‘Nitrogen metabolism’, and ‘Alanine, Aspartate and Glutamate Metabolism’ pathways abundant within the 2 macro-groups were ‘translation, ribosomal structure and biogenesis’ (J) and ‘posttranslational modification, protein turnover, chaperones’ (O) (Fig. S1c).
To find N related genes, DEGs between optimal and low N treatments in two cultivars were compared. There were 36,432 and 45,671 DEGs between Balin and Bluemoon (BCK vs LCK, BL vs LL), and 10,156 and 838 DEGs between the optimal and low N (BCK vs BL, LCK vs LL) as shown in Venn diagram, respectively (Fig. 4b).
2.5.2 Genes Involved In N And C-N Metabolism
To confirm the functions of the notable transcripts in response to low N in two cultivars, specific DEGs with eliminating genetic background differences (Fig. 4b) were identified in Bluemoon (336) and Balin (968). KEGG analysis showed that these DEGs enriched in the pathways of ‘nitrogen metabolism’ (ko00910; FDR = 1.06e-14), ‘pyruvate metabolism’ (ko00620; FDR = 6.04e-06) and ‘carbon fixation in photosynthetic organisms’ (ko00710; FDR = 6.10e-04) in Bluemoon (Fig. 4d). Nevertheless, the most significantly enriched pathways were ‘fatty acid elongation’ (ko00062; FDR = 3.38e-05) and ‘phenylpropanoid biosynthesis’ (ko00940; FDR = 4.75e-03). The pathway ‘nitrogen metabolism’ (FDR = 0.21) was not significantly enriched, and no pathway related to C metabolism was significantly enriched in Balin (Fig. 4e). GO enrichment was basically consistent with KEGG analysis, pathways related to C/N metabolism ‘carbon fixation’ (GO: ko00062; FDR = 2.90e-08), ‘carbohydrate metabolic process’ (GO: 0005975; FDR = 2.03e-07), ‘citrate metabolic process’ (GO: 0006101; FDR = 9.08e-08), ‘nitrate metabolic process’ (GO: 0042126; FDR = 2.09e-05) and ‘nitrate assimilation’ (GO: 0042128; FDR = 2.09e-05), which were notable enriched in Bluemoon (Fig. S2), but not enriched in Balin (Fig. S3).
Genes related to N transportation and assimilation induced by N starvation were differentially expressed in two cultivars. NRT2 (TRINITY_DN140116_c2_g3) encoding nitrate transporter proteins and NR (TRINITY_DN118471_c0_g1) encoding nitrate reductase were highly expressed in ‘Balin’ at low N stress. NiR (TRINITY_DN72083_c1_g1) encoding nitrite reductase was expressed preferentially in Balin than that in Bluemoon at optimal N treatment, but there was no significant differences in gene expression between two cultivars at low N stress. By contrast, GDH (TRINITY_DN129678_c2_g2) encoding glutamate dehydrogenase, GS (TRINITY_DN111472_c0_g1 and 122245_c2_g1) encoding glutamine synthetase, GOGAT (TRINITY_DN94356_c1_g2, 123883_c1_g3 and 106544_c0_g2), AspAT (TRINITY_DN115099_c0_g2 and 79352_c0_g1) encoding glutamate synthetase, aspartate aminotransferase were highly expressed in Bluemoon at low N status, respectively (Fig. 5a).
Since nitrate conversion to ammonium consumes one NADH and six reduced ferredoxins, and the energy needed are directly or indirectly derived from the processes of Pn, glycolysis and respiration, we examined several key genes involving in these C and N metabolic processes. Under low N, gene expressions of CA (TRINITY_DN137763_c1_g1 and 148777_c16_g1) encoding carbonic anhydrase, PEPc (TRINITY_DN118112_c0_g2) encoding phosphoenolpyruvate carboxylase, ENO (TRINITY_DN81908_c0_g1) encoding enolase, MDH (TRINITY_DN103121_c1_g1 and 76243_c1_g2) encoding malate dehydrogenase, and OGDH (TRINITY_DN117626_c0_g1 and 107170_c1_g1) encoding 2-oxoglutarate (2-OG) dehydrogenase were higher in Bluemoon than that in Balin (Fig. 5a).
To better understand the roles of DETFs involved in N metabolism in KB, all DEGs were analyzed with the PlantTFDB. Finally, 1606 sequences had hits in the plant transcription factor database and were assigned into 34 families. The most frequently represented DETF families were C2H2 (13.9%), FAR1 (7.9%), bHLH (5.6%), MYB (4.2%) (Fig. S1d). 22 MYB DETFs unigenes in relation to N metabolism were selected from four comparisons, and were blasted against the MYB family of Brachypodium distachyum to determine their sub-family. Two of them were MYB-related types, and 13 were MYB-R2R3 types. The expression levels of DETFs analysis was confirmed by qPCR.
It was sure that the MYB transcription factor binds to the GS promoter. So, we have attained a promoter sequence (unpublished data) of GS (TRINITY_DN111472_c0_g1) via genome-walking technique. In addition to the basic components such as CAAT-box and TATA-box, a CCAAT-box along with the function of MYBHv1 (R2R3-MYB in Hordeum vulgare) binding site was also observed, its binding TFs of KB were found by blast in transcriptome (TRINITY_DN138437_c5_g1, 138437_c5_g3). The DETFs’ expression levels were up-regulated in two cultivars, and the highest level expression was observed in Bluemoon. The GS promoter also contains three cis-elements including TAACCA-motif, CAACCA-motif and CCGTTG-motif that were corresponded to MYB (Fig. 5b). Therefore, we hypothesized that the trancripts (TRINITY_DN138437_c5_g1, 138437_c5_g3) in KB could bind to the promoter of GS, thereby promoting N assimilation in GS/GOGAT cycling.