Physiological responses to low temperature stress in sugar beet
Compared with the controls, there was no significant change in the Chl content within 48 h of low temperature treatment. After 72 h of low temperature treatment, the Chl content increased significantly to 20% (Fig. 1a). No significant change occurred in the damage of plasma membranes with REL of all the samples compared with the controls (Fig. 1b). However, a significant increase of MDA content was observed after 12 h of low temperature treatment (Fig. 1c). The activity of SOD increased and then reached its highest level after 24 h (Fig. 1d). These findings suggest that sugar beet responded positively within 24 h of low temperature treatment and was not significantly damaged in chloroplast.
Analysis of the expression pattern of BvDREBs genes
To profile the transcriptional response, BvDREBs, orthologous genes in Arabidopsis thaliana that act essential regulators in cold response pathway, were identified and selected as marker genes (Bo et al. 2007). A total of 73 homologous genes were identified in sugar beet using bidirectional BLAST searches. A phylogenetic analysis of these 73 homologous genes showed that 15 clades that contained the DREB-A1, DREB-A4 and soloist subfamilies were resolved (Fig. 2). The DREB-A1 subfamily played an important role in low temperature stress. As physiological drought is often accompanied by low temperature, the adjacent DREB-A4 subfamily that responded to drought was also considered as candidate genes.
There were four sugar beet genes in the same branch as that of the AtDREB-A1 gene. However, only Bv3_066590_ignp (BvDREBA1) was significantly up-regulated in four candidate genes after low temperature treatment based on the transcriptome data. Among of DREB-A4 subfamily genes, the Bv2_032310_xjoh (BvDREBA4) gene also responds to low temperature (Fig. 3a). Subsequently, the dynamic changes of expression of BvDREBA1 and BvDREBA4 were measured by qRT-PCR. The results indicated that BvDREBA1 and BvDREBA4 changed significantly within 3 h of the low temperature treatment and reached their peak after 24 h of treatment with low temperature. The level of expression of BvDREBA1 and BvDREBA4 began to gradually decease, and the level of expression after 48 h of low temperature treatment was significantly lower than that after 24 h of low temperature treatment (Fig. 3b, c).
Chloroplast proteome identification
Based on physiological and transcriptional changes, sugar beets that had been treated by 24 h of low temperature, were selected as the samples to detect chloroplast proteomes in subsequent studies. After extraction of chloroplast protein (CK, 24 h), only protein samples with a highly abundant Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo), as characteristic protein in chloroplasts, observed near 60 kDa and without obvious protein degradation was used for subsequent MS analysis. A total of 103,562 peptides (CK, 24 h) were identified by LC-MS/MS, with 16,423 unique peptides that corresponded to 3,420 proteins. Moreover, it was notable that approximately 10,677 to 14,102 unique peptides that corresponded to 2,887 to 3,169 proteins were identified in each sample (Fig. 4a, b). The Pearson correlation coefficients between three biological repeats were > 0.97 in each group (Fig. S1a). The log2 values of peptide intensity in each sample ranged from 25 to 30, and the peptides with different signal intensities had a normal distribution (Fig. S1b). That finding demonstrated that not only are the biological samples in the group reproducible but also that the MS is highly stable.
A t-test was performed to investigate DEPs in response to low temperatures. The ratio fold changes log2(24 h/ck) > 1.5 and p-values < 0.05 were regarded as the thresholds for screening the DEPs between the CK and 24 h (Fig. 4c). A total of 416 DEPs were identified, including 91 up-regulated proteins and 111 down-regulated proteins, and 214 proteins were specifically expressed in the CK or 24 h (Fig. 4d, Table S2). An analysis of the specific expression proteins indicated that 164 up-regulated and 50 down-regulated DEPs were detected. Among the up-regulated and down-regulated proteins, the expression of 33 and 24 proteins changed by more than 2-fold, respectively.
Identification of proteins encoding from chloroplast DNA
According to the sugar beet chloroplast genome (SMRT sequencing only de novo assembly of the sugar beet (Beta vulgaris) chloroplast genome), a total of nine proteins (0.3%) encoded by the chloroplast genome were identified by BLASTP (Fig. S2a). They were primarily divided into three types, namely ribosomal proteins (5), RNA polymerases (3) and an ATP synthase (1) (Fig. S2b). Among them, there were three DEPs, including two ribosomal proteins and one ATP synthase, accounting for 0.7% of the DEPs (Fig. S2c, d). Additionally, the expression of these proteins has a high fold change in response to low temperature treatment. At 24 h of low temperature treatment, the ATP synthase protein was up-regulated 23-fold, and the protein of two ribosomes was up-regulated 6-fold and 2-fold, respectively. Large changes in protein abundance suggest that chloroplast DNA-encoded proteins also play an important role in response to low temperature or cold acclimation of sugar beet.
GO enrichment analysis of DEPs
To explore the biological functions of the low temperature responsive proteins, we used AgriGO to analyze the DEPs at low temperature responsive stages. A total of 143 GO terms were significantly enriched, of these, including 47 biological process, three molecular function and 93 cellular components. Based on the relationship between the directed acycline praph and the false discovery rate (FDR), we screened 16 significantly enriched GO terms that play a major role in response to the low temperature of chloroplasts in sugar beets (Fig. 5).
There were eight significantly enriched GO terms in the biological process, among them, the most significantly enriched GO term was translation (GO:0006412), and its FDR value was 2.1E-5. Response to cold (GO:0009409), response to cadmium ion (GO:0046686) and small molecule metabolic process (GO:0044281) followed with FDR values of 1.4E-4, 2.2E-4 and 6.8E-4, respectively. Significantly enriched GO terms reflect that sugar beet chloroplasts initiate a response to low temperature through Ca2+ signaling and translate more novel proteins that are involved in the resistance to cold. RNA binding (GO:0003723), translation factor activity (GO:0008135) and structural molecule activity (GO:0005198) were significantly enriched in the molecular function, with FDR values of 0.022, 0.022 and 0.033, respectively. Five GO terms in the cell component were significantly enriched. The FDR values of chloroplast stroma (GO:0009570), chloroplast envelope (GO:0009941) and chloroplast thylakoid membrane (GO:0009535) were 3.3E-24, 3.1E-16 and 2.3E-13, respectively, which were the most significant in all of the GO terms. This finding indicates that the chloroplast stroma, chloroplast envelope and chloroplast thylakoid membrane are the primary components of low temperature response.
The functions of the DEPs located on chloroplast components were classified in more detail. A total of 112 DEPs were involved in the low temperature response in chloroplast stroma (GO:0009570), chloroplast envelope (GO:0009941) and chloroplast thylakoid membrane (GO:0009535) (Fig. 6a).
The number of proteins related to substance metabolism and protein synthesis was the largest in the chloroplast stroma (GO:0009570), with 18 and 17, accounting for 26.9% and 25.4%, respectively (Fig. 6b). Five proteins were up-regulated, and 13 proteins were down-regulated in substance metabolism. These types of substance metabolism involved the pentose phosphate pathway, starch metabolism and synthesis. In addition, the DEPs related to protein synthesis primarily included the 50S large subunit and 30S small subunit that form ribosomes, and protein translation and folding related proteins, such as tRNA aminotransferase, translation elongation factor and peptidyl prolyl cis-trans isomerase. Four ribosomal proteins significantly increased after 24 h of low temperature treatment. Moreover, there were also a large number of proteins related to stress defense (9) and light reactions (8), accounting for 13.4% and 11.9%, respectively. In stress and defense, Cu/Zn-SOD, CAT, peroxiredoxin (Prx) and thioredoxin (Trx) were involved to activate the H2O2 signaling-related proteins and ROS scavenging.
The number of transport-related proteins was the largest chloroplast envelope in (GO:0009941). They totaled 12, accounting for 22.2%, primarily including Toc-Tic complexes, glucose transporters, aquaporins, and ADP/ATP transport among others (Fig. 6c). Protein synthesis followed with 10 DEPs, accounting for 18.5%, including the 50S large subunit and 30S small subunit. There were seven unknown proteins accounting for 13%, which could be related to the low temperature response. The role of these proteins in low temperature response merit additional study in the future.
The main low temperature responsive proteins in chloroplast thylakoid membrane (GO:0009535) were concentrated in the light reaction and included 13 proteins, accounting for 38.2% (Fig. 6d). These proteins are involved photosystem I (PS I), photosystem II (PS II), quinone oxidoreductase, and ATP synthase. Additionally, nine unknown proteins still responded to low temperature stress, accounting for 26.5%. The function and relationship with the light reaction still merit further study.
Molecular mechanisms of the chloroplast that underlie low temperature in sugar beet
This analysis revealed the following processes that sugar beets use to manage low temperature stress in chloroplast of sugar beet (Fig. 7). After low temperature treatment for 24 h, a large number of proteins significantly increased in the photosynthetic system. Notably, most photosynthetic proteins were expressed in the nuclear genomes and then entered the chloroplasts through the Toc-Tic complex, including BvTOC100, under the control of BvLTD. In additional, chloroplast genes were also induced by low temperature to initiate transcription and translation. ATP synthase was highly expressed under the induction of chloroplast ribosomal protein. The α subunit in particular was up-regulated 23-fold. An increased abundance of these proteins may lead to an increase in glucose content in chloroplasts. These glucose molecules can be used as osmotic regulators by transportation outside of the chloroplasts via BvGlcT. Moreover, it can also be synthesized as amylose. Amylose is further metabolized to amylopectin under the action of enzymes, such as BvPU1. The abundance of starch degradation-related proteins (BvISA3 and BvGWD3) decreased significantly, indicating that low temperature could induce the formation of starch granules in sugar beet chloroplasts. Finally, to protect the ROS generated through photosynthetic electron transport, the ROS scavenging system composed of BvCu/Zn-SOD and BvCAT was also induced by low temperature. The up-regulation of BvPrx and BvTrx protein not only enhanced the removal of H2O2 but were also involved in the oxidation of signaling proteins, such as transcription factors and phosphatase, enabling signaling via the transmission of H2O2 to the nucleus and up-regulating the expression of stress and defense-related proteins.