Double mutant aox1a/ucp1 has compromised tolerance to low nitrogen stress
It has been reported that ALTERNATIVE OXIDASE 1a (AOX1a) and UNCOUPLING PROTEIN 1 (UCP1) respond to low nitrogen (N) stress at the transcriptional level in plants . To further investigate the function of AOX and UCP genes upon low-N condition, the physiological responses of AOX mutants [aox1a (Salk_084897), aox1c-1 (CS804611), aox1c-2 (CS877307), aox1d (CS390166) and aox2 (CS766955)] and UCP mutants [ucp1 (CS821384), ucp2 (Salk_080118C), ucp3 (Salk_006106), ucp4 (CS373159), ucp6 (Salk_111403C)] were analyzed under low-N condition. Fig. S1 and S2 showed that low N (0.1 N) resulted in the increased primary root length, accumulation of anthocyanin, but decreased fresh weight in Col-0 and the mutants. However, there were no significant differences among them. qRT-PCR results showed that low N significantly induced the expressions of AOX1a, AOX1c and AOX2, which were increased by 9.3-fold, 5.6-fold and 17.0-fold, respectively; while AOX1b and AOX1d expressions were decreased (Fig. S1d). Similarly, the transcripts of UCP1, UCP2 and UCP3 were increased by 1.8-fold, 2.0-fold and 1.3-fold, respectively; but UCP4, UCP5 and UCP6 transcripts were decreased (Fig. S2d). The highest expression of AOX1a in the AOX family and UCP1 in the UCP family suggests their essential function in Arabidopsis tolerance to low N.
To explore the function and relationship of AOX1a and UCP1 in Arabidopsis tolerance to N deficiency, the aox1a and ucp1 were crossed to generate the double mutant aox1a/ucp1. PCR analysis confirmed the screened homozygous lines (Fig. S3). Phenotypic analysis of 8-day-old single and cluster seedlings showed that there was no difference among Col-0, aox1a, ucp1 and aox1a/ucp1 on 1/2 MS and 1 N conditions (Fig. 1a). However, under 0.1 N and 0 N conditions, the primary root length of aox1a/ucp1 seedlings was shorter than that of Col-0 seedlings (Fig. 1a, d), and the difference reached the maximum at day 8 (Fig. S4). Under 0.1 N condition for 8 d, the primary root length of aox1a/ucp1 was reduced by 25.4% compared to Col-0 (Fig. 1d). Moreover, the anthocyanin content and the purple rate of aox1a/ucp1 cotyledons were higher than that of Col-0 and single mutants under 0.1 N condition (Fig. 1b, c, e). The cotyledon purple rate of Col-0, aox1a, ucp1 and aox1a/ucp1 seedlings was 60.2%, 58.4%, 61.1%, and 82.8%, respectively (Fig. 1e). Thus, AOX1a and UCP1 might be collaboratively involved in the adaptation of Arabidopsis seedlings to low-N stress.
UCP1 plays more important roles in regulating photosynthesis than AOX1a upon low-N condition
To further investigate the mechanism of AOX1a and UCP1 involvement in Arabidopsis adaption to low-N stress, we analyzed the rosette leaf area, shoot biomass and photosynthetic capacity of Col-0, aox1a, ucp1 and aox1a/ucp1 seedlings. In order to accurately control the N concentration in the medium, seedlings were grown hydroponically. Results showed that the rosette leaf area, fresh weight (FW) and dry weight (DW) of ucp1 and aox1a/ucp1 seedlings were markedly lower than that of Col-0 under moderate-N (5 N) and low-N (1 N) conditions, while aox1a had no significant difference compared to Col-0 (Fig. 2). Under low-N condition, the rosette leaf area, FW and DW in ucp1 were reduced by 24.4%, 29.0% and 27.7%, respectively, and by 44.6%, 26.3%, and 27.3% in aox1a/ucp1, respectively, compared to Col-0 (Fig. 2b-d). These results indicate that loss of UCP1 function results in more significant inhibition of growth than loss of AOX1a function under low-N condition.
The total chlorophyll content (chlt) in ucp1 and aox1a/ucp1 plants were decreased by 7.4% and 9.2%, respectively, in comparison with that in Col-0 upon low-N condition. However, in aox1a, it did not change (Fig. 3a). Under the control condition, the net photosynthetic rate (A) and electron transfer rate (ETR) in ucp1 were significantly lower than that in Col-0. However in aox1a and aox1a/ucp1 plants, A and ETR were not different from that in Col-0 (Fig. 3b, c). Under low-N condition, A and ETR in aox1a was similar to that in Col-0. In contrast, they were significantly lower in ucp1 and aox1a/ucp1 than that in Col-0. A and ETR were reduced by 27.4% and 31.0%, respectively, in ucp1, and by 29.8% and 26.5%, respectively, in aox1a/ucp1 (Fig. 3b, c). The analysis result of light response curve showed that the light saturation point in ucp1 was markedly lower than that in Col-0 when the light intensity exceeded 200 µmol photons m− 2 s− 1 (PAR) under the control condition (Fig. 3d). Under low-N condition, the light saturation point in both ucp1 and aox1a/ucp1 plants were significantly lower than that in Col-0 and aox1a, but there was no difference between ucp1 and aox1a/ucp1 (Fig. 3e). These results indicate that aox1a/ucp1 and ucp1 plants have low photosynthetic capacity under low-N stress. UCP1 plays more important roles in regulating photosynthesis than AOX1a does in Arabidopsis tolerance to low-N stress.
Mutation of AOX1a and UCP1 decreases C and N contents in the shoots of aox1a/ucp1
Above results confirmed that mutation of AOX1a and UCP1 affects the photosynthetic capacity of aox1a/ucp1. We subsequently analyzed the changes of C and N contents under low-N stress. Upon low-N and moderate-N conditions, the percentage of C in Col-0, aox1a, ucp1, and aox1a/ucp1 shoots was decreased. Interestingly, there was a marked difference only between ucp1 and Col-0 under 1 N condition (Fig. 4a). Low N similarly decreased the percentage of N in all plants (Fig. 4b). The total C and N contents in ucp1 and aox1a/ucp1 shoots were significantly lower than that in Col-0 upon low-N condition (Fig. 4c, d), which were reduced by 21.6% and 26.1%, respectively, in ucp1 and by 24.2% and 23.11%, respectively, in aox1a/ucp1 under 5 N condition. Under 1 N condition, the total C and N contents were reduced by 29.6% and 28.6%, respectively, in ucp1 and by 27.0% and 27.9%, respectively, in aox1a/ucp1. However, in aox1a, the total C and N contents were similar to that in Col-0 under all N conditions. Thus, the photosynthetic C fixation and N assimilation capacity in ucp1 and aox1a/ucp1 were lower than that in Col-0 and axo1a plants under low-N condition, and this explained the decreased biomass in ucp1 and aox1a/ucp1 plants.
Expression patterns of AOX1a and UCP1 and changes of respiratory rates upon low-N condition
To investigate the expression patterns of AOX1a and UCP1 upon low-N condition, the proAOX1a:GUS and proUCP1:GUS transgenic lines were generated. GUS staining results showed that 0.1 N treatment induced marked increase of AOX1a and UCP1 expression in 6-day-old seedlings. AOX1a was expressed in the whole seedling, whereas UCP1 was mainly expressed in cotyledons and the maturation zone of primary roots (Fig. 5a-d). Under low-N condition, the AOX1a expression was higher than UCP1 in rosette leaves (Fig. 5e-h). At the reproductive stage, low N also increased the expression of AOX1a and UCP1 in flowers and stamens (Fig. 5i-p). Specifically, the expression of AOX1a was mainly increased in the anthers of stamens (Fig. 5n), while UCP1 was mainly expressed in the filaments of stamens (Fig. 5p).
qRT-PCR results reported that low N induced the expression of AOX1a and UCP1 at almost every developmental stage. When Arabidopsis seeds were germinated and grown for 6 d in 0.1 N medium, the expression of AOX1a and UCP1 were enhanced by 9.3-fold and 1.8-fold, respectively (Fig. 5q). In rosette leaves, the response of AOX1a expression to low-N condition was higher than that of UCP1 (Fig. 5r). However, in flowers and siliques, the AOX1a expression increased about 1.5-fold and 2.1-fold upon low-N condition, respectively, while the UCP1 expression was increased by 1.7-fold and 2.3-fold (Fig. 5s, t).
To clarify the relationship between AOX1a and UCP1 as well as the effect of low N on the alternative pathway capacity (Valt) and UCP pathway activity (Vucp), the respiratory rates were analyzed. Fig. S5 showed that low N significantly increased Valt and Vucp in Col-0. Under high-N (control) condition, Valt in ucp1 was decreased by 84.3% and Vucp in aox1a was decreased by 42.1% compared to Col-0. Interestingly, the mutation of UCP1 in ucp1 stimulated the Valt by 63.5% upon low-N condition (Fig. S5a). Similarly, Vucp was also increased in aox1a and Col-0 upon low-N condition. Thus, there should be the close relationship between AOX1a and UCP1 under normal growth condition.
Mutation of AOX1a and UCP1 affects N metabolism upon low-N condition
To investigate the effects of AOX1a and UCP1 mutations on N metabolism in Arabidopsis in response to low N, the differences of the uptake, assimilation and transport of N were analyzed in Col-0, aox1a, ucp1, aox1a/ucp1 plants. The results showed that low N decreased the NO3− content and NR activity, but increased NiR activity in Col-0, aox1a, ucp1 and aox1a/ucp1 shoots (Fig. 6a-c). Compared to Col-0 under 1 N condition, the NO3− content, NR and NiR activities were reduced by 11.5%, 9.5%, 15.0%, respectively, in ucp1 and by 34.0%, 21.8%, 13.3%, respectively, in aox1a/ucp1; however, there was no significant difference between aox1a and Col-0.
To analyze the uptake rate of NO3− and the transport from root to shoot, Arabidopsis seedlings were starved under 0 N condition for 24 h, and then transferred to the growth solution containing 10 mM 15N-KNO3 (control) or 1 mM 15N-KNO3 (1 N) for 6 h. Results showed that low N decreased the uptake rate of NO3− in Col-0, aox1a, ucp1 and aox1a/ucp1 roots (Fig. 6d). However, compared to Col-0, the uptake rate of NO3− in aox1a, ucp1 and aox1a/ucp1 were elevated by 15.4%, 16.7% and 21.6%, respectively, under the control condition, and by 6.9%, 12.0% and 21.2%, respectively, under 1 N treatment (Fig. 6d). In contrast, the transport activity of NO3− from root to shoot in aox1a, ucp1 and aox1a/ucp1 was significantly decreased compared to Col-0 under both control and low-N conditions (Fig. 6e). Based on these results, although mutations of AOX1a and UCP1 resulted in acceleration of NO3− uptake under low-N stress, the low assimilation rate and transport activity of N in aox1a/ucp1 seedlings impaired the N metabolism.
Mutation of AOX1a and UCP1 decreases the seed yield and the C/N content in aox1a/ucp1 under low-N stress
N deficiency inevitably limits the reproductive growth of plants. Our results showed that low N significantly reduced the silique number and seed yield of Col-0, aox1a, ucp1 and aox1a/ucp1 plants (Fig. 7a, b). Loss of function of AOX1a and UCP1 resulted in more decrease of silique number and seed yield in aox1a/ucp1 (by 22.3% and 23.6%, respectively) than that in Col-0. A similar trend was also found in ucp1 mutant. However, there was no difference between aox1a and Col-0 (Fig. 7a, b). Figure 7c, d showed that the percentage of N was decreased in seeds of four genotypes upon low-N condition. However, the percentage of C in seeds had no significant changes compared to the control. Further result confirmed that the AOX1a mutation alone in aox1a did not affect the total C and N contents in seeds, but in ucp1 and aox1a/ucp1 seeds, the total C content was reduced by 21.1% and 21.8%, respectively, and the total N content was reduced by 27.4% and 34.1%, respectively (Fig. 7e, f). Thus, the decrease of C and N distribution in seeds accounted for the low seed yield in both ucp1 and aox1a/ucp1 plants.
Distinct transcript profiles in Col-0, aox1a, ucp1 and aox1a/ucp1 plants under low-N stress
The different physiological response of Col-0, aox1a, ucp1, and aox1a/ucp1 seedlings to low-N stress might be caused by differential expressions of downstream genes involved in different metabolic pathways. To test the hypothesis and elucidate the molecular metabolism of UCP1 and AOX1a in Arabidopsis tolerance to low N, we compared gene expression profiles in Col-0, aox1a, ucp1, and aox1a/ucp1 shoots by using RNA sequencing. Compared to Col-0, transcriptomes of aox1a, ucp1, and aox1a/ucp1 had marked changes (Fig. S6). With thresholds of log2(FC) ≥ 2 and FDR < 0.01, there were 1605 up-regulated genes and 1559 down-regulated genes in Col-0 upon low-N condition. The up- and down-regulated genes were 1656 and 1153, respectively, in aox1a, 687 and 1378, respectively, in ucp1, and 2321 and 1796, respectively, in aox1a/ucp1 (Fig. S6a-d). KEGG functional enrichment analysis indicated that the DEGs in Col-0 were mainly enriched in ribosome, plant hormone signal transduction, biosynthesis of amino acids, and plant-pathogen interaction. DEGs were mainly enriched in ribosome, biosynthesis of amino acids and carbon metabolism in aox1a; in plant hormone signal transduction, plant-pathogen interaction and photosynthesis in ucp1; and in carbon metabolism, biosynthesis of amino acids, and photosynthesis in aox1a/ucp1 (Fig. S6e-f). Thus, mutation of UCP1 and AOX1a in aox1a/ucp1 markedly disturbed the photosynthesis and carbon metabolism pathways upon low-N condition.
To further investigate the mechanism of decreased resistance to low N in aox1a/ucp1, DEGs in Col-0 and aox1a/ucp1 were analyzed. Venn diagram showed that 1233 and 923 genes were co up- and down-regulated, respectively, in Col-0 and aox1a/ucp1 (Fig. 8a). KEGG results showed that DEGs in both Col-0 and aox1a/ucp1 were mainly enriched in ribosome, biosynthesis of amino acids, photosynthesis and N metabolism. DEGs in Col-0 only (1961 genes) were mainly enriched in ribosome, plant hormone signal transduction, plant-pathogen interaction. DEGs only in aox1a/ucp1 (1008 genes) were mainly enriched in C metabolism, amino acid synthesis, C fixation in photosynthesis organisms, and glycolysis (Fig. 8b-d). The numbers of DEGs involved in the N metabolism, C metabolism, photosynthesis, por and chl metabolism and ribosome were 4, 44, 11, 6, 12, respectively, in aox1a/ucp1, but were 2, 3, 1, 2, 57, respectively, in Col-0 (Fig. 8e, f). Figure 8g further showed that most DEGs in both Col-0 and aox1a/ucp1 in the aforementioned metabolism pathways were generally down-regulated. However, the decrease was more significant in aox1a/ucp1 than in Col-0 under low-N stress (Fig. 8g). qRT-PCR was performed to validate some of the DEGs in Col-0 and aox1a/ucp1. Among the six genes examined, they all showed similar expression patterns as the RNA-seq results (Fig. 8h). Thus, the decreased expressions of genes related to C metabolism, photosynthesis, N metabolism in aox1a/ucp1 could be one of the reasons for the low photosynthetic capacity and C/N level in seeds.