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% significantly 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 significant except for in A2 strains (P < 0.05). Meanwhile, the PN 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 significant lower in NB plants of A1 and A4. Except for A2, the PN of all BT plants were significantly higher than that of NB plants (Fig. 2b). In May, the leaves of BT plants turned yellow and even withered, the PN as well as the Chl content decreased rapidly. Therefore, Chl content and PN were significantly higher in all NB plants compared with BT plants(Fig. 2c). Meanwhile, the NB plants showed higher photosynthetic parameters involved in gs, Tr and Ci in March and especially in May (Table 1). But there was no consistent difference between NB and BT plants from different strains in April.
Table1 Stomatal conductance, transpiration rate and intercellular carbon dioxide concentrations of the leaves of different cultivars (strains) of normal and early bolting Angelica dahurica.
Mean values ± standard error; statistically significant differences between genotypes are indicated with asterisks (* – P<0.05; ** – P<0.01).
|
Cultivars(Strains)
|
gs
|
E
|
Ci
|
March
|
NBA1
|
0.234±0.013 **
|
3.596±0.272 **
|
402.852±17.733**
|
|
BTA1
|
0.216±0.008
|
2.713±0.139
|
279.155±1.902
|
|
NBA2
|
0.225±0.014 **
|
2.477±0.077 **
|
287.385±21.049 *
|
|
BTA2
|
0.154±0.011
|
2.290±0.067
|
269.551±12.636
|
|
NBA3
|
0.176±0.005
|
2.123±0.043 **
|
340.583±28.138 **
|
|
BTA3
|
0.167±0.011
|
2.104±0.038
|
278.706±6.492
|
|
NBA4
|
0.273±0.023 **
|
3.324±0.806
|
271.624±1.185
|
|
BTA4
|
0.257±0.018
|
3.435±0.399
|
284.916±4.68 *
|
|
NBB2
|
0.287±0.021
|
3.614±0.094 **
|
248.221±5.425
|
|
BTB2
|
0.255±0.024
|
2.730±0.187
|
285.755±14.141*
|
|
NBB3
|
0.299±0.018 **
|
2.369±0.15 **
|
305.056±7.897
|
|
BTB3
|
0.250±0.020
|
2.199±0.071
|
294.157±8.278
|
April
|
NBA1
|
0.234±0.013 **
|
3.596±0.272 **
|
402.852±17.733**
|
|
BTA1
|
0.216±0.008
|
2.713±0.139
|
279.155±1.902
|
|
NBA2
|
0.225±0.014 **
|
2.477±0.077 **
|
287.385±21.049 *
|
|
BTA2
|
0.154±0.011
|
2.290±0.067
|
269.551±12.636
|
|
NBA3
|
0.176±0.005
|
2.123±0.043 **
|
340.583±28.138 **
|
|
BTA3
|
0.167±0.011
|
2.104±0.038
|
278.706±6.492
|
|
NBA4
|
0.273±0.023 **
|
3.324±0.806
|
271.624±1.185
|
|
BTA4
|
0.257±0.018
|
3.435±0.399
|
284.916±4.68 *
|
|
NBB2
|
0.287±0.021
|
3.614±0.094 **
|
248.221±5.425
|
|
BTB2
|
0.255±0.024
|
2.730±0.187
|
285.755±14.141*
|
|
NBB3
|
0.299±0.018 **
|
2.369±0.15 **
|
305.056±7.897
|
|
BTB3
|
0.250±0.020
|
2.199±0.071
|
294.157±8.278
|
May
|
NBA1
|
0.249±0.011 **
|
4.322±0.196 **
|
310.27±6.337 **
|
|
BTA1
|
0.224±0.028
|
2.955±0.335
|
266.129±1.198
|
|
NBA2
|
0.27±0.016
|
3.466±0.65
|
230.503±17.904 *
|
|
BTA2
|
0.293±0.005*
|
3.489±0.239
|
216.324±6.963
|
|
NBA3
|
0.298±0.012
|
4.257±0.243
|
234.522±16.475
|
|
BTA3
|
0.290±0.029
|
4.606±0.333
|
254.033±15.142 *
|
|
NBA4
|
0.333±0.009 *
|
4.691±0.27 *
|
239.224±4.704
|
|
BTA4
|
0.280±0.02
|
3.488±0.183
|
261.58±11.477 *
|
|
NB
|
0.286±0.014 **
|
3.652±0.382 **
|
248.162±6.651
|
|
BTB2
|
0.210±0.006
|
2.092±0.104
|
260.699±19.23 *
|
|
NBB3
|
0.331±0.008 **
|
4.127±0.448
|
281.056±10.594
|
|
BTB3
|
0.206±0.009
|
4.045±0.442
|
330.818±5.384 **
|
|
NBA1
|
0.196±0.013 **
|
2.636±0.127 **
|
332.852±17.733 **
|
|
BTA1
|
0.066±0.007
|
0.693±0.063
|
130.129±5.896
|
|
NBA2
|
0.152±0.027 **
|
2.133±0.083 *
|
217.82±13.105 **
|
|
BTA2
|
0.059±0.003
|
0.382±0.034
|
124.91±11.519
|
|
NBA3
|
0.197±0.018 **
|
1.617±0.061 **
|
238.813±12.826 **
|
|
BTA3
|
0.079±0.005
|
0.56±0.028
|
112.699±3.519 I
|
|
NBA4
|
0.17±0.016 **
|
2.191±0.279 **
|
239.624±4.464 **
|
|
BTA4
|
0.065±0.004
|
0.818±0.051
|
123.914±6.539
|
|
NBB2
|
0.167±0.019 **
|
1.419±0.021 **
|
236.221±20.353**
|
|
BTB2
|
0.049±0.008
|
0.467±0.05 F**
|
124.699±7.901
|
|
NBB3
|
0.198±0.029 **
|
2.339±0.174 **
|
302.621±7.421 **
|
|
BTB3
|
0.068±0.006
|
0.662±0.115
|
138.485±1.848
|
Table 2 Correlation analysis on the chlorophyll content and photosynthetic parameters of the leaves of different cultivars(strains) of Angelica dahurica. Mean values ± standard error; statistically significant differences between genotypes are indicated with asterisks (** – P<0.01).
|
Chl
|
gs
|
E
|
Ci
|
PN
|
Chl
|
1.000
|
0.212
|
0.171
|
0.408**
|
0.693**
|
gs
|
\
|
1.000
|
0.895**
|
0.580**
|
0.065
|
E
|
\
|
\
|
1.000
|
0.217
|
0.100
|
Ci
|
\
|
\
|
\
|
1.000
|
0.305
|
PN
|
\
|
\
|
\
|
\
|
1.000
|
In order to confirm whether the enzyme activity was influenced 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 significantly higher than that of normal plants in May(Fig. 3). It suggested a higher carbon assimilation efficiency 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 significant difference between the NB and BT plants (Fig. 4).
Furthermore, it was found that uroporphyrinogen, coproporphyrin and protochlorophyllide contents in the Chl synthesis pathway significantly 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, significantly 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 significantly down-regulated compared with the NB plants.
Only AdhemH was found to be significantly 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 significantly 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 significantly 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 significantly down-regulated in the BT plants leaves, indicated that the photosynthetic electron transfer efficiency of BT plants was inhibited, resulting in the decline of photosynthesis (Fig. 6f).The transcriptional levels of AdACACA, AdKCS and AdFATB were significantly up-regulated, while the transcriptional levels of AdFAdA and AdACSL were significantly 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).