Responses of plant morphology and leaf development to low temperature stress
Observation on the morphological development of plants under low temperature stress
The Brassica napus L. was treated at low temperature for 24 h at 4 °C (Fig.1). The morphological changes of the plants showed that the leaves of 17NS and NF24 had wilting and sagging, of which the old leaves of sensitive NF24 were obviously wilted, the new were leaves slightly wilted, and the old leaves of cold tolerant 17NS were wilted while the new leaves barely changed. The two varieties after low temperature treatment were restored at room temperature for 24 hours. It was found that the old leaves of the sensitive variety NF24 all died, the new leaves returned to life, and the cold-tolerant variety 17NS recovered after 24 hours, the new leaves and the old leaves are green and revitalized. The results showed that the new leaves showed strong tolerance and adaptability in response to lo w temperature stress and the leaves of the cold-resistant variety 17NS have strong frost resistance under low temperature stress.
Observation chloroplast morphological under low temperature stress by transmission electron microscopy
Transmission electron microscopy observation of rapeseed leaves under low temperature stress showed that low temperature stress had a great influence on the structure and quantity of organelles in leaf cells, which were manifested in the morphological structure of chloroplasts, the number of hypersalotic particles and starch granules were changed. The varieties with different cold resistance have different degrees of changes in organelles. After 24 hours of cold tolerance of 17NS cold-resistant varieties, the granum were dispersed, the chloroplasts shrank, the volume became smaller, and the starch granules became smaller (Fig.2 -A1). Compared with the control (Fig.2-B1), the number of starch granules increased. There are up to 3 starch granules in the chloroplast, and the number of starvation granules increases (Fig.2-B2), the number and morphology of chloroplasts are not obvious (Fig.2-B3).In the sensitive variety NF24, the control (Fig. 2-C1) starch granules were faintly visible, disappeared after low temperature stress, and the thylakoid layer was significantly reduced (Fig.22-D1). The chloroplast changes from a spindle shape close to the cell wall to a paramecium shape far away from the cell wall, the number of mitochondria increased significantly (Fig.2-D2), and the number of chloroplasts in individual cells decreased (Fig.2-D3).It indicated that the varieties with different cold resistance had different responses to chloroplast photosynthesis and respiration after low temperature stress.
Response of Physiology and Biochemistry of Brassica napus L. to Low Temperature Stress
Response of cell membrane of Brassica napus L. to low temperature stress
When the plant tissue is damaged by the stress, the low temperature stress firstly damages the membrane system of the cell, the membrane function is impaired or the structure is destroyed, and the permeability is increased, the water-soluble substances in the cells, including the electrolyte, will have different degrees of extravasation. The relative conductivity measurement showed that the relative conductivity of the cold-tolerant variety 17NS and the sensitive variety NF24 was almost the same before the low temperature stress. After the low temperature stress, the relative conductivity increased significantly (Fig.3). The relative conductivity of the sensitive variety NF24 increased sharply. It is 0.869 ms/cm3, which is larger than the rising value of 17NS (0.760 ms/cm3), indicating that the electrolyte of the cell membrane of Brassica napus L. has a large amount of extravasation after low temperature damaging. Sensitive varieties have large electrolyte exosmosis, poor cold tolerance and adaptability while cold resistant variety have strong ability to maintain the relative stability and structural integrity of the cell membrane system, and better resistance to low temperature damage.
Effects of low temperature stress on photosynthetic parameter
The photosynthetic parameters of 0~24h under low temperature stress showed that the low temperature significantly reduced the net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr), and increased the intercellular CO2 concentration (Ci). ). Pn, Gs, Tr and Ci between different varieties were significantly different with the treatment time (Fig. 4). Before the low temperature stress, the Pn, Gs and Tr of the cold-tolerant variety 17NS were lower than the cold sensitive variety NF24, but the Ci was higher than the cold sensitive variety NF24. After 24 h of low temperature stress, the Tr, Gs and Pn of the cold-tolerant 17NS leaves were significantly lower than the control, with the reductions being 30%, 60% and 72%, respectively. During the low temperature stress of NF24, the Pn, Gs and Tr of the leaves increased first and then decreased, however, it was significantly higher than the control group after 24h of stress, Pn decreased by 66.13% compared with the control, and Tr and Gs increased by 205.01% and 280% compared with the control. The variation range of cold sensitive cultivars was more unstable than that of cold resistant cultivars at the low temperature stress. The Ci of the cold-tolerant variety 17NS and the cold-sensitive variety NF24 were first decreased and then increased. After 24 hours, the Ci increased by 16.29% and 20.01%, respectively. Therefore, low temperature significantly affects photosynthesis and stomatal gas exchange, and has a more greater impact on Tr, Gs and Pn of cold sensitive varieties.
Effect of low temperature on chlorophyll content of Brassica napus L
It can be seen from Fig.5 that the chlorophyll a and chlorophyll b contents showed a significant downward trend after 24 h of low temperature stress in the functional leaves of different Brassica napus L., and the chlorophyll a and chlorophyll b contents of sensitive NF24 sharp fell compared with the cold resistant variety 17NS, which was 0.184 mg/ g FW and 0.387 mg/g FW. The carotenoid content increased with the decrease of temperature, and the cold resistant variety 17NS increased faster, reaching 0.337 mg/g FW. It can be seen that the content of chlorophyll a and chlorophyll b in Brassica napus L. decreased with low temperature stress, and the content of carotenoids increased, and the decrease of chlorophyll content of cold resistant varieties was smaller than that of sensitive varieties, indicating that the cold resistant varieties had stronger resistance after low temperature stress. The strong photosynthetic capacity of the cold resistant variety is conducive to maintaining the normal growth of seedlings for adversity injury.
Screening of differentially expressed genes in Brassica napus L. under low temperature stress
According to the transcriptome data of Brassica napus L. under low temperature stress, the difference gene expression was analyzed by using edgeR software. The differential gene was screened by FDR and log2FC. The screening criteria were FDR<0.05 and |log2FC|>1. Among the different cold cultivars, NF24t0-17NSt0 was paired to obtain 18978 genetic difference genes (Table 1.). NF24t-17NSt paired to obtain 22019 differential genes, and the genetic background difference was removed to obtain significant differences in low temperature induction. A total of 3041 genes were expressed (739 differential genes up-regulated expression and 2302 differential genes down-regulated expression).
KEGG pathway analysis
By KEGG Pathway analysis of differential genes, the pathway with Qvalue ≤ 0.05 was chosen as the pathway for differential gene enrichment. The results showed that in all differentially expressed genes, after 24 h of low temperature stress, 4229 differentially expressed genes of NF24t0-17NSt0 and 4675 differentially expressed genes of NF24t-17NSt were enriched in 131 and 132 pathways, respectively. Twenty of the most significant pathways were screened by P<0.05 (Fig.6), and Ribosome and Metabolic pathway were the most significant pathways before and after low temperature stress, respectively.
Light and regulation related differential expression gene analysis
The analysis of pathways related to photosynthetic characteristics after low temperature stress revealed that Photosynthesis and Photosynthesis-antenna proteins are significant difference in energy metabolism, of which Photosynthesis-antennas were ranked in the top 20 in the NF24 t-vs-17NS t comparison group (Fig.6). Using Venny online software (http://bioinfogp.cnb.csic.es/tools/venny/) to analyze the DEGs(Differentially Expressed Genes) of different varieties under low temperature stress (Fig. 7), it was found that the significantly DEGs of Photosynthesis have 50 and the significantly DEGs of Photosynthesis-antennas have 12 in the NF24t0-17NSt0 comparison group, and the significantly DEGs Photosynthesis have 69 and the significantly DEGs of Photosynthesis-antennas have 30 in the NF24t-17NSt comparison group.
Analysis of photosynthesis regulation pathway induced by low temperature stress
After low temperature stress, in the photosynthesis and photosynthesis-antenna proteins related to photosynthetic regulation, DEGs repeated with NF24t0-vs-17NSt0 were removed from the DEGs of NF24t-vs-17NSt. 64 significant DEGs were obtained (As shown in the blue part of Fig.8). There were 38 DEGs in the Photosynthesis pathway, of which 7 DEGs up-regulated expression and 31 DEGs down-regulated expression, 26 DEGs in the Photosynthesis-antenna proteins pathway, of which one DEGs up-regulated expression and 25 DEGs down-regulated expression (Fig.9 and Fig.10).
Go function classification of specific expression genes
Through the function annotation (Fig.11) of Go function, it is found that 64 specific DEGs related to photosynthetic regulation in the differentially expressed genes under low temperature stress are mostly annotated into cell component processes and biological processes, and the number of differentially expressed genes are least in molecular function. It indicated that low temperature stress affected the growth and development of Brassica napus L. Among them are mainly cellular processes, metabolic processes, cells, cellular components, organelles, binding, catalytic activity and transport activity.
qRT-PCR validation of specific expression genes
The 64 low-temperature-induced specific DEGs (Table 2), mainly with photosynthetic system I, photosynthetic system II, oxygen-releasing protein, iron Oxygen-reducing protein-NADP reductase, chlorophyll protein, and ATP synthase are related,most of the genes of sensitive NF24 and cold-tolerant cultivar 17NS were down-regulated after low temperature stress. From Photosynthesis pathway screening one up-regulated DEGs and 7 up-regulated DEGs, from Photosynthesis - antenna proteins pathway screening one gene up-regulated DEGs and 8 down-regulated performed with qRT-PCR. The results (Fig. 12) of qRT-PCR indicate that were consistent with the results of RNA-Seq analysis,the expression of 17 DEGs in qRT-PCR and RNA-Seq showed the same trend, confirming the reliable results of RNA-Seq analysis.
Low temperature stress inhibited the expression of ferredoxin-coenzyme ii reductase, photosystem I reaction, oxygen-releasing protein, ATP synthase, photosystem II repair protein PSB27-H1, photosystem I chlorophyll a/b binding protein, chlorophyll a-b binding protein and so on, and induces expression of ferredoxin and chlorophyll a-b binding protein CP26. It is indicated that the inhibition of photosynthesis by low temperature mainly occurs in chloroplasts, and photosynthesis is inhibited by inhibiting photosynthetic pigments and photoreaction systems.
Analysis of cold resistance of Brassica napus L
To promote the breeding process of Brassica napus L. were used, the existing with the aid of Brassica genome information, analysis identified 1173 flowering related genes of Brassica napus L.[34], Two QTLS related to the content of fatty glycosides in seeds [35] and Twenty abiotic drought-related genes[36], There were 1651 up-regulated genes and 405 down-regulated genes in tea trees after low temperature stress and are closely related to photosynthesis regulation[37, 38]. At present, there are many researches on the analytical mechanism of plant cold tolerance, for example, RbohB gene[26], COLD1[39] and chitinase gene (BnCHB4)[28], COR[40], ICE[41], HSF[42], CBF[43] and other antifreeze proteins. This study found that low temperature stress could cause to the chloroplast number of Brassica napus L. reduce were used, the volume contraction of the chloroplast, grana lamella fuzzy, addicted to hungry particle number increases, chlorophyll a, chlorophyll b content decreased significantly, the photosynthetic rate (Pn), transpiration rate (Tr) and stomatal conductance (Gs) and intercellular CO2 concentration (Ci) go down and then it goes up. It indicates that plants can alleviate the damage of low temperature by adding osmotic regulatory substances, and the varieties with stronger cold resistance are hindered in CO2 supply and CO2 assimilation capacity [44-46], which eventually leads to decreased photosynthetic capacity and affects photosynthetic response [47-49], which is consistent with previous research results[22, 24, 25, 50].
Transcriptome analysis under low temperature stress
Through the transcriptome analysis, get two significantly photosynthesis regulation pathways with Photosynthesis and Photosynthesis-antenna proteins. NF24t-v-17NSt0 for comparison, NF24t-v-17NSt for processing, 7 DEGs has up-regulated and 31 DEGs has down-regulated in Photosynthesis pathway,1 DEGs has up-regulated and 25 DEGs has down-regulated in Photosynthesis-antenna proteins pathways, a total of 64 specific gene expression. Analyzed found that the low temperature stress lead to PS Ⅰ and PS Ⅱ be suppressed[51], was mainly related to PS Ⅰ, PS Ⅱ, increasing oxygen protein, Ferrix -NADP reductase put oxygen reductase, phyllochlorin, ATP synthesis enzyme and so on, partially restore ferredoxin is used for by PETH reduction NADP +, which is used for producing NADPH carbon assimilation and other biosynthetic pathway, and can also be mediated electron flow to protect leaf light system [38]. Genetic variations of top up or down expression by qRT - PCR analysis, test and verify the reliability of the RNA - Seq results, and access to 17 stable expression of photosynthetic regulation candidate genes, expression of 15 genes, 2 raised expressed genes, confirmed that the low temperature stress on the stress of Brassica napus L. were used mainly by inhibition of photosynthesis and light reaction system. Further research is needed on the molecular mechanism of candidate genes.