The inhibition of photosynthesis of Angelica dahurica caused by early bolting

Background Ealy bolting affects the photosynthesis of plants. However, there are positive or negative impacts in different plants. The mechanism of photosynthesis pathway respond to early bolting is still a mystery. Angelica dahurica is a traditional Chinese medical plant that has been used as the raw material of medicine and food. Early bolting of Angelica dahurica occurring in the eld production, which decreases quality and yield of A. dahurica. Results In this research, we rstly revealed the damage of early bolting on the photosynthesis of A. dahurica. The photosynthetic capacity signicantly decreased after bolting. The metabonomic analysis showed that the chlorophyll synthesis metabolism was repressed and the accumulation of saturated fatty acid increased after early bolting. Meanwhile, transcriptomic analysis indicated a down-regulation of photosynthetic electron transport and up-regulation of saturated fatty acid synthesis. Interestedly, an unexpected raise in Rubisco and PEPC activities of the leaves of early bolting plants was found in our research. Conclusion The is Meanwhile, carboxylase; Ad: Acetyl coenzyme KCS: protein

Rubisco enzyme is the key rate limiting enzyme of carbon assimilation. It is a kind of bifunctional enzyme in the mesophyll. It can not only x the CO 2 absorbed by the leaves through carboxylation, but also catalyze the oxidation of ribulose-1,5-diphosphate to participate in photorespiration, which plays an important role in photosynthesis. The rapid Rubisco accumulation that depends on su cient PEP activity is important for normal seedling growth [9]. Phosphoenolpyruvate carboxylase (PEPC) widely exists in organisms, and its carbon assimilation e ciency and speci city are dozens of times higher than that of Rubisco. Previous studies showed that PEPCs proteinsare were up-regulated under low CO 2 to carry on a rearrangement of carbon metabolism to support C4 photosynthetic carbon assimilation in Thalassiosira pseudonana [10]. As an important photoreceptor, chlorophyll is essential for photosynthesis of plants like affecting the photosynthetic electron transfer e ciency of plants and thus affects their photosynthesis [11]. However, the Chl content cannot fully re ect leaf age, because even completely green leaves began to senescence at the molecular level. Compared with single leaf senescence parameters, comprehensive senescence parameters at the whole plant level, such as the proportion of aged leaves, can better re ect the molecular state of plants [12]. Transcription factors regulating chlorophyll metabolism have been gradually resolved. For example, OsMYB102 gene delays chloroplast apoptosis by down-regulating ABA accumulation signal induction pathway, thus delaying leaf senescence and prolonging photosynthesis [13].
There is no doubt that photosynthesis changed signi cantly before and after bolting. However, the effect of early bolting on plants is quiet a controversial topic. The analysis of 48 lineages of Arabidopsis thaliana distributed under different environmental gradients suggested that the higher tness was associated with earlier bolting, greater early allocation to increased numbers of in orescences, reduction in rosette leaf photosynthesis and earlier fruit ripening [14]. A research on early owering high-quality rice showed that its photosynthesis was higher, and the contents of carbohydrate, protein and amino acid were also higher.
In conclusion, the effect of early bolting on photosynthesis of different plants are different or even opposite. Although the early bolting is identi ed a harmful trait on A. dahurica by preliminary studies, the mechanism of occurrence and in uence are still a mysterious. In this study, we suggested a novel suppressed mechanism on the photosynthesis of A. dahurica by early bolting.

Plant material and culture condition
The 6 varieties (strains)of A. dahurica named A1, A2, A3, A4, B2 and B3 were all bred and cultivated by our laboratory. A1 was a certi ed variety bred by professor Wu Wei (Agronomy College, Sichuan Agricultural University) and approved by Sichuan crop variety Approval Committee. A2-B3 strains were stable breeding line for more than 3 successive generations by our laboratory. The experimental strains were all planted in the same led of Modern R&D Base of Sichuan Agricultural University (103.67, 30.63).

Determination of Agronomic Characters and photosynthetic parameters
After the early bolting of Angelica dahurica in March 2019, the photosynthetic parameters of normal and early bolting plants were measured by Li-6400 portable photosynthesis analyzer from the 25th to 28th of each month. The light intensity was set at 1600 µmol·m − 2 ·s − 1 in 6 times repeatedly. Meanwhile, the plant height and basal culm thickness were measured by ruler and electronic vernier caliper of 10 normal and early bolting plants, respectively.

Chlorophyll Content
The 0.1g fresh plants leaves samples were stored in ice box in the eld. The chlorophyll was extracted with mortar and pestle in 80% chilled acetone plus, after centrifugation at 4°C the resultant solution were determined spectro-photometrically at 663 nm and 646 nm, repeated 4 times.

Metabolomic and transcriptomic analysis
The results came from our previous metabolomics and transcriptomics data of normal and early bolting A1 cultivar leaves.

Statistical analysis
All statistical analysis were conducted using SAS software (Pro 9.4 SAS Institute Inc., Cary, NC, USA). Student's t-test was used for comparison of two means, and linear regression analysis was used for comparison of two variables.

Analysis of Agronomic Characters
In March, after the early bolting occurred, the different strains of early bolting A. dahurica exhibited 6.04% 13.06% higher height than the normal one, while their stem base diameter were 17.41 ~ 55.94% larger than that of NB plants (Fig. 1a).
In April, the higher height and wider stem were also showed in NB plants (Fig. 1b). However, in May, the plant heights of BT plants were 8.82% ~ 30.05% signi cantly higher than that of NB plants, while the stem base diameter were 9.55% ~ 45.43% smaller in BT plants compared with NB plants (Fig. 1c).

Determination of Chl content and Photosynthetic Characteristics
In March, Chl contents of different BT plants were 3.44-31.83% lower compared with NB ones, the difference was not signi cant except for in A2 strains (P < 0.05). Meanwhile, the P N also differed much higher in NB plants (7.11% ~ 23.84%) (Fig. 2a).
In April, the plunge of Chl contents occurred in the NB plants, while Chl contents of BT plants decreased slowly. Among them, the Chl contents were higher in A3, B2 and B3 BT plants, but signi cant lower in NB plants of A1 and A4. Except for A2, the P N of all BT plants were signi cantly higher than that of NB plants ( Fig. 2b). In May, the leaves of BT plants turned yellow and even withered, the P N as well as the Chl content decreased rapidly. Therefore, Chl content and P N were signi cantly higher in all NB plants compared with BT plants Fig. 2c . Meanwhile, the NB plants showed higher photosynthetic parameters involved in g s , Tr and C i in March and especially in May (Table 1). But there was no consistent difference between NB and BT plants from different strains in April. Mean values ± standard error; statistically significant differences between genotypes are indicated with asterisks (* -P<0.05; ** -P<0.01). In order to con rm whether the enzyme activity was in uenced under early bolting, we examined the PEPC and Rubisco enzyme activities of all strains. Interestingly, PEPC/Rubisco enzyme activity of all strains of BT plants leaves was signi cantly higher than that of normal plants in May Fig. 3 . It suggested a higher carbon assimilation e ciency in the early bolting plants.

The Analysis of Metabolome
We selected the A1 variety with stable genetic characters which was bred by our laboratory for metabonomic and transcriptomic analysis. Enrichment map indicated the photosynthesis pathway was signi cant difference between the NB and BT plants (Fig. 4).
Furthermore, it was found that uroporphyrinogen, coproporphyrin and protochlorophyllide contents in the Chl synthesis pathway signi cantly decreased in the leaves of BT plants compared with NB plants in March. (Fig.5a, 5b, 5c). However, both of the contents of octadecanamide and palmitic acid, representing the accumulation of saturated fatty acid, signi cantly increased in leaves of BT plants (Fig. 5d, 5e) The transcripmic analysis of carbon assimilation, electron transport and chlorophyll metabolism In the photosynthetic carbon assimilation pathway, phosphoglycerate kinase (PGK) in chloroplast mainly regulates plant metabolism and optimal growth, and the down-regulation of AdPGK would reduce the photosynthetic capacity of plant leaves. The function of RPI isomerase is to catalyze the conversion of xylose-5-phosphate to ribose-5-phosphate.
The results indicated that the transcription levels of AdRPI (Fig. 6a) and phosphoglycerate kinase gene 1 AdPGK (Fig. 6b) in the calvin cycle pathway of BT plants were signi cantly down-regulated compared with the NB plants.
Only AdhemH was found to be signi cantly up-regulated in the leaves of BT plants in Chl transcription pathways (Fig. 6c). The PSI and PSII are the main pathways of photosynthesis electron transport in the higher plants. It was found the transcription level of AdPsaB in the leaves of BT plannts was signi cantly up-regulated (Fig. 6d). The main function of PSbW is to maintain the stability of supramolecular structure of PSII photoreaction center. The expression of the AdPsbW encoding the PSII reaction center was signi cantly down-regulated as shown in (Fig. 6e) revealed that the stability of PSII protein complex was damaged, which affected its normal function. In addition, the AdFdrA, which regulated the important electron carrier in the photosynthetic electron transport chain, was also signi cantly down-regulated in the BT plants leaves, indicated that the photosynthetic electron transfer e ciency of BT plants was inhibited, resulting in the decline of photosynthesis (Fig. 6f).The transcriptional levels of AdACACA, AdKCS and AdFATB were signi cantly up-regulated, while the transcriptional levels of AdFAdA and AdACSL were signi cantly down-regulated. The results showed that the synthesis pathway of fatty acid in early mossy plants was up-regulated and fatty acid degradation pathway was down-regulated which was basically consistent with the results of metabolome (Fig. 7).

Discussion
Previous studies have shown that the quality and yield of A. dahurica in the production eld are affected by the early bolting [16]. In this research we further describe the inhibition of photosyhthesis pathway caused by early bolting, this is probably a straight correction with production loss. At the initial stage of the early bolting, the P N and Chl contents of NB plants were higher than the early bolting plants. Although the difference became less obvious in April, the photosynthetic characteristics of BT plants nally plunged in May. The result of correlation analysis suggested the decrease of P N be positively correlated with the Chl content of A. dahurica. The Chl content of early bolting A. thaliana lines decreased faster than the normal ones, indicating that the senescence of early bolting plants was accelerated during the process ower bud differentiation to bolting [17]. Interestingly, we found the activities of PEPC and Rubisco enzyme in BT plants of all varieties were higher than that in NB. A. duhurica owned both higher PEPC activity and lower intercellular CO 2 concentration in the late stage of early bolting. This feature is the reminiscent of the phenotype of cauli ower under low concentration CO 2 treatment. It was found that PEPC enzyme activity was nearly 2 times higher compared with the control [18]. The same phenomenon was also found in Hydrilla verticillata treated with low concentration CO 2 [19]. The improvement of Rubisco enzyme activity could rescue the delayed greening of FLN2 mutant [9]. We assumed that the increase of Rubisco activity in BT plants of A. dahurica be a response to the sharp decrease of Chl content, which could alleviate the chlorosis of leaves to a certain extent, so as to maintain the basic photosynthesis. The higher activity of PEPC could provide more organic acids for the development of carbon assimilation products such as α-keto acid for reproductive growth under the condition of leaf senescence and signi cant decrease of P N of A. dahurica. Previous studies have shown that inhibition of photosynthesis was consistent with senescence and the decrease of carbon assimilation enzyme activity [20]. However, the results of our study showed that the repressed photosynthsis did not induce the decrease of carbon assimilation enzyme activity, but increased it. It referred this mechanism might relate to the lower concentration intercellular CO 2 and Chl contents.
Our studies showed that Chl synthesis was one of the important factors affecting photosynthetic capacity of A. dahurica under early bolting. The metabolic analysis revealed that prochlorophyllate, copolyporogen and urea porphyrinogen III contents were down-regulated in BT plants leaves. The Chl synthesis pathway was inhibited when early bolting began. Therefore, it is assumeed that Chl synthesis metabolic be signi cantly repressed by the early bolting and nally cause serious damage to the process of photosynthesis.
The uroporphyrinogen III is the common precursors of siroheme (synthesized in bacteria, yeast and plants) and coproporphyrinogen III. It should be noted that the down-regulation of uroporphyrinogen III did not cause the down-regulation of sirohemon synthesis, but downstream coproporphyrinogen III and protochlorophyllide showed similar trend in BT plants.
Despites signi cantly different Chl precursors content, no genes were to be found down-regulated in Chl synthesis pathway in BT plants. However, the transcription level of AdhemH was up-regulated in BT plants. The hemH catalyzes the chelation of protoporphyrin IX with Fe to form heme, which essentially competes with chlorophyll synthesis pathway for protoporphyrin IX [21]. In cucumber seedlings under salt stress, the transcription level of protoporphyrin iron chelatase increased, while the transcription level of several genes in chlorophyll synthesis pathway was down-regulated. Exogenous 5-ALA treatment could signi cantly reduce the transcription level of feme and increase the transcription level of chlorophyll synthesis pathway [22].
In A. thaliana, inhibition of feme transcription level could improve the Chl content and photosynthetic capacity in PS II damaged mutants. The transcription level of protoporphyrin iron chelatase gene signi cantly increased in the leaves of early bolting A. dahurica. Therefore, it was speculated that the upregulated of Feme may affected the photosynthesis of early bolting A. dahurica plants. In addition, the expression levels of AdPGK and AdRPI were signi cantly down-regulated in the Calvin cycle pathway of early bolting A. dahurica leaves. Phosphoglycerate kinase gene in chloroplast mainly regulates plant metabolism and optimizes plant growth. The decrease of AtPGK1 expression level could reduce the photosynthetic capacity of plant leaves. It was found that the photosynthetic capacity and starch content decreased in the pgk A. thaliana, which was due to the impaired glycolysis caused by mutation of PGK enzyme [23].
In A. thaliana, AtRPI contains a chloroplast transport sequence, so it plays a regulatory role in plant chloroplast function. The activity of RPI enzyme did not differ signi cantly, chloroplast structure was to be found destroyed in rpi2 resulting in decreased photosynthetic capacity and promoted premature cell senescence [24]. The down-regulation of AdRPI might lead to the inhibition of ribose-5-phosphate regeneration and chloroplast function, which was consistent with the inhibition of Chl synthesis shown by metabolome.
Ferritin is an important electron transport carrier of PSI. The down-regulation of AdFdrA transcription level indicated the decrease of electron transfer e ciency of PSI, indicating that plants are vulnerable to photoinhibition and affect photosynthesis. P700 protein is the main electron donor of PSI [25]. In studies on A. thaliana, AtPsaB regulated P700 protein. The expression of AtPsaB was related to the damage of photosynthetic organs by reactive oxygen species [26]. In this study, the signi cantly up-regulated AdPsaB was in the leaves of early bolting A. dahurica might reduce the destructive effect of photoinhibition to the BT plants.
PsbW is a low molecular weight protein unique to photosynthetic eukaryotes, which is related to the composition of PSII 680 protein complexes [27] .The results showed that the loss of PsbW in A. thaliana could destroyed the supramolecular structure of PSII. In the absence of PsbW protein, PSII-LHCII hypercomplex could not be detected or isolated, and the phosphorylation of PSII core protein decreased signi cantly, and the redox state of plastid quinone (PQ) in dark adapted leaves changed. Compared with wild type, PsbW protein deletion leads to a rapid transition state 1 to state 2 of PQ, that is, the redox change is faster. The down regulation of PsbW transcription level in early bolting A. dahurica leaves might also distructe the stability of PSII protein complex. In this research, the photosynthetic electron transport pathway appeard to be down-regulated at the transcriptional level in BT plants. It is widely believed that unsaturated degree of membrane lipid could signi cantly affect the photosynthesis of the plants [28].
The contents of octadecanamide and palmitic acid in the leaves of early bolting A. dahurica leaves were signi cantly higher than those in the normal one. The results showed that the accumulation of saturated fatty acids in leaves increased after early bolting. Previous study indicated that the increasing accumulation of saturated fatty acids in the early senescence stage could improve the sensitivity to ethylene and promote the owering of Petunia hybrida [29]. And long-chain fatty acids were involved in the transport of polar auxin in A. thaliana [21]. In addition, studies on membrane lipids of photosynthetic organs in leaves showed that the increase of unsaturated fatty acid ratio helps to protect plant PSII damage under stress, maintain the stability of thylakoid membrane, and improve the photosynthetic heat resistance of plants. The saturated fatty acid content of old leaves was also higher than that of new leaves [30,31]. IAA can induce the accumulation of palmitic and stearic while decreasing the synthesis of linoleic and α-linolenic in Chlorella vulgaris [32]. In our previous research, we found that the accumulation of auxin in early bolting A. dahurica also increased, which might be related to the accumulation of longchain saturated fatty acids in leaves. The transcriptional levels of several genes involved in the biosynthesis and metabolic of long-chain saturated fatty acids signi cantly increased, and the downregulation of long-chain acyl CoA synthase and acetylcoa acyltransferase was also found in the fatty acid degradation pathway, indicating that early bolting could increase the accumulation of long-chain saturated fatty acids in leaves of A. dahurica at the molecular level. At present, we do not know the mechanism of this phenomenon, but many studies have shown that the accumulation of saturated fatty acids is not conducive to maintaining the stability of various organelle membranes, and plants are vulnerable to various stress environment damage, which may lead to early bolting of A. dahurica leaf photosynthetic organs vulnerable to strong light damage and accelerate aging.
Early bolting could decreases both the quality and yield of lettuce. However, a comparative proteomics analysis suggested a high temperature enhances the function of photosynthesis to promote the process of bolting of lettuce [33]. This conclusion contradicted our ndings. Therefore, we speculate that the effect of early bolting on plant photosynthesis be related to to species differences.

Conclution
These present ndings com rmed a negative effect of early bolting on photosynthetic pathway of A. dahurica. The repression of Chl synthesis at the early stage of bolting seems not directly related to the change on transcript level of genes in Chl synthesis pathway. Futhermore, a special response mechanism on inhibited photosynthesis under early bolting by increasing activities of PEPC and Rubisco increased at the late stage of bolting of A. dahurica. It provides a novel way to expolre the different effect on photosyhthesis of early bolting among different plants.

Availability of data and materials
All data generated or analyzed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.

Funding
The work is supported by Supported by Sichuan Science and Technology Program,

Figure 4
Page 19/21 Enrichment map of differential metabolism.  Transcriptomic analysis on fatty acid metabolism of the leaves of early bolting and normal Angelica dahurica.