Starch and sucrose dynamics
Starch metabolism and corresponding enzyme dynamics
Description of four stages of corm development was displayed in Additional file 1: Tab. S1. Dynamics of starch content and the enzyme activities are shown in Fig. 1. Over the new corm developmental stages, starch content saw an overall increasing trend with the minimum (30.94 mg/g.FW) and maximum value (65.04 mg/g.FW) appearing, respectively, at the beginning and the end of the sampling event. Starch content increased slowly within 110 days since mother corm embedding. Thereafter, the content rose rapidly reaching ~ 60 mg/g.FW from 110 to 130 d (transition from initial swelling stage to rapid swelling stage), followed by a slightly decrease downward (~ 130–150 d) and subsequent increase at the corm maturation stage (~ 150–190 d).
Beta-AMY activities were rather dynamic. Specifically, β-AMY activities increased from 273 to 408 µg/min/g.FW during the corm formation stage (60–90 d). After transient decrease, the enzyme activity increased sharply and peaked at 120 d (appearance of flower bud), reaching 498.33 µg/min/g.FW, which was followed by marked decrease in the next sampling event and ensuing increase with fluctuations during maturation stage. Throughout the corm lifecycle, an overall increasing trend of SBE activities was observed, especially at the rapid swelling stage. During the maturation stage (150–180 d), the increasing rate of enzyme activity was almost flat. The value of the highest enzymatic activity was ~ 4 fold that of the minimum at the beginning of sampling. Overall, APGase activities increased across the corm development stages, especially during the formation (60–90 d) and maturation stages (140–190 d), while fluctuations were observed during corm swelling (110–140 d). The minimum (~ 80 µg/min/g.FW) and maximum (~ 240 µg/min/g.FW) values appeared at the first and last sampling event, respectively.
Sucrose metabolism and corresponding enzyme dynamics
Figure 2 depicts changes of sugar content and corresponding enzyme activities across new corm development. On average, levels of sucrose and soluble sugars followed an increasing trend across the corm developmental process. By comparison, the increasing trend was more pronounced for sucrose than for soluble sugars for which more fluctuations were observed. At the formation stage (60–90 d), sucrose and soluble sugars both kept at a relatively constant level and then increased rapidly at times during corm swelling. Even at the maturation stage, sucrose content rose rapidly to reach a maximum (4.43 mg/g.FW), which was 3.69 times of the lowest value (1.2 mg/g.FW) at the first sampling event. For soluble sugars, the maximum and minimum values were 3.14 and 11.06 mg/g.FW, respectively.
SPS activities kept relatively constant (~ 350 µg/min/g.FW) during the formation stage (60–90 d), then increased sharply reaching maximum (~ 640 µg/min/g.FW) during corm swelling (100–140 d) and subsequently kept relatively stable except a marked drop at 150 d. Comparatively, the profile of SuSy activities exhibited a slightly different pattern. The enzymatic activity showed monotonous decline from 705 to 433 µg/min/g.FW during the formation stage, then rose rapidly arriving at maximum (~ 1006 µg/min/g.FW) during the swelling period and finally dropped back to the beginning level when corms began mature. NI activities increased continuously from 60 d (88.47 µg/min/g.FW) to 150 d (~ 180 µg/min/g.FW) followed by a sustaining drop arriving at 126.19 µg/min/g.FW at the end. Averagely, AI activities followed an initial increasing and subsequent decreasing trend as corms grew except the presence of a drop at 100 d. The maximum and minimum activities were 64.2 and 123.27 µg/min/g.FW, respectively.
Correlations between starch and sucrose content, enzyme activities and corm size
The correlation analysis shows that activities of SPS, AGPase and SBE were positively and significantly correlated with sucrose and corm diameter (P < 0.01), which all were significantly correlated to starch content (Table 1). Besides, NI exhibited a positive and significant relationship with starch content and corm dimeter (P < 0.01). Also, the correlation between SBE and NI and AGPase, and AGPase and β-AMY were significant (P < 0.01). By contrast, no significant association between NI, AI and β-AMY was observed. AI and SuSy showed no significant correlation with any other parameter at all.
Table 1
Pearson correlation between starch and sucrose content, enzyme activities and corm size
| NI | AI | β-AMY | SPS | SuSy | AGPase | SBE | Sucrose content | Starch content | Diameter |
NI | 1 | | | | | | | | | |
AI | 0.428 | 1 | | | | | | | | |
β-AMY | 0.327 | 0.148 | 1 | | | | | | | |
SPS | 0.591* | 0.372 | 0.478 | 1 | | | | | | |
SuSy | 0.622* | 0.601* | -0.047 | 0.439 | 1 | | | | | |
AGPase | 0.486 | 0.045 | 0.689** | 0.497 | 0.100 | 1 | | | | |
SBE | 0.800** | 0.154 | 0.548* | 0.651* | 0.392 | 0.878** | 1 | . | | |
Sucrose content | 0.573* | 0.000 | 0.559* | 0.682** | 0.256 | 0.856** | 0.879** | 1 | | |
Starch content | 0.760** | 0.255 | 0.585* | 0.662** | 0.458 | 0.857** | 0.957** | 0.921** | 1 | |
Diameter | 0.839** | 0.269 | 0.524 | 0.677** | 0.522 | 0.815** | 0.971** | 0.889** | 0.980** | 1 |
Note: “*” and “**” signify a significant level of 0.05 and 0.01, respectively. NI: neutral invertase; AI: acid invertase; AMY: amylase; SEB: starch branching enzyme; AGPase: adenosine diphosphoglucose pyrophosphorylase; SPS: sucrose phosphate synthase; SuSy: sucrose synthase |
Rna-seq And Transcriptome Assembly
The sequencing statistics based on a mixed cDNA library of freesia corms at different developmental stages are summarized in Additional file 2: Tab. S2 and Additional file 3: Tab. S3. After data filtering, 82.54 Gb of clean reads were generated with Q30 89.73 and GC ~ 48% averagely. Totally 100,999 unigenes were generated after assembly with a mean length of 772 nt. The N50 was 1,507 nt and 21,476 unigenes were longer than 1,000 nt. On average, 63% of the genes were uniquely mapped.
Unigene Annotation And Functional Classification
In the current study, totally 44,405 unigenes were annotated with 41,175 annotated in Nr, 13,753 in COG, 25,142 in GO, 15,743 in KEGG, 23,015 in KOG, 24,441 in Swissport and 28,454 in Pfam (see Additional file 4: Tab. S4). Figure 3 shows that 13,753 sequences were classified into 24 COG categories, covering majority of life activities. Apart from “general function prediction only” (1,561 unigenes, 10.18% of annotated) and “translation, ribosomal structure and biogenesis” (1,460 unigenes, 9.52%), “carbohydrate transport and metabolism” term took the central position with 1432 unigenes annotated (9.34%).
There are 25,142 unigenes annotated in GO database, which were classifies into three big functional categories, i.e., cellular component, molecular function and biological process (Fig. 4). The subcategories in the principle category of cellular component was most represented by “cell”, “membrane”, “cell part” and “organelles”. Within the principle category of molecular function, most genes participated in activities related to catalysis and binding. In the principle category of biological process, genes were mostly involved in metabolic process, cellular process and single-organism process, indicative of occurrence of important metabolic and cellular activities in freesia corms. Notably, a considerable portion of genes were assigned to reproductive processes and development processes. All of these annotation information provides insights into investigation of potential genes involved in developmental processes of freesia corms.
Distribution And Function Assignment Of Degs
The expression levels of unigenes at different developmental stages were compared. As shown in Fig. 5, 3,427 DEGs in total from three pairwise comparisons were detected with the largest number distributed in the third pair (120 vs.190 d), where 2037 DEGs were identified with 1,551 down regulated and 486 upregulated. In comparison, 723 (290 up-regulated and 433 down-regulated) and 667 DEGs (520 up-regulated and 147-down regulated) were found in other two pairs (60 vs. 90 d and 90 vs. 120 d, respectively). The contrasting results suggested that more complex biochemical activities may occur during the late developmental stages especially during the rapid swelling period.
GO and COG functional and KEGG enrichment pathway analysis was performed to gain further insights into DEGs. The major categories of GO terms in three pairwise comparisons (see Additional file 5: Fig. S1) were similar to that for the combined unigenes at four developmental stages as described in Fig. 4. Notably, DEGs between 120 and 190 d were enriched in “nutrient reservoir activity” pathway, which was void in other two comparisons. Comparatively, differences in COG enrichment profiles of DEGs in three comparisons were more pronounced (see Additional file 6: Fig. S2). Within the 60 vs. 90 d comparison, the term “posttranslational modification, protein turnover, chaperones” was at the top, followed by “carbohydrate transport and metabolism” and “general function prediction only”. In the 90 vs. 120 d comparison, the top representative terms were “signal transduction mechanisms”, “carbohydrate transport and metabolism” and “posttranslational modification, protein turnover, chaperones” sequentially. While within the 120 vs. 190 d comparison, “carbohydrate transport and metabolism” ranked the first, further suggesting that carbohydrate metabolic pathway might be most active during late stages of corm development. Pathway enrichment analysis revealed top 20 enriched pathways of DEGs in Additional file 7: Fig. S3. Generally, DEGs were, though in small quantity (< 5 or 10), significantly enriched in terms like riboflavin metabolism and diterpenoid biosynthesis in 60 vs. 90 d comparison, linoleic acid metabolism and monoterpenoid biosynthesis in 90 vs. 120 d comparison, and anthocyanin biosynthesis in 120 vs. 190 d comparison. Notably, DEGs enriched in starch and sucrose metabolism in the 120 vs. 190 d comparison was quantitatively superior to other pathways, attenuating the greater activity of starch and sucrose metabolism in late developmental stages, in line with the results from GO and COG analysis.
Verification Of Gene Expression By Qrt-pcr
A keyword search in RNA-seq annotation generated a total of 39 putative genes encoding six carbohydrate-metabolizing enzymes investigated in this study, i.e., SPS, AGPase, SBE, β-AMY, SuSy and INV. The expression of these genes were displayed in Additional file 8: Tab. S5. To confirm the gene expression profiles of the enzymes from RNA-seq results, 6 out of the 39 genes (shown in bold and italic in Additional file 8: Tab. S5), including β-AMY1 (c62599.graph_c1), INV2 (c87855.graph_c0), SPS1 (c91035.graph_c1), SuSy (c93394.graph_c2), SBE4 (c78179.graph_c0) and AGPGase5 (c93923.graph_c0), were selected for qRT-PCR. Primer sequences of the genes for PCR were specified in Additional file 9: Tab. S6. The results of agarose gel electrophoresis evidenced that all 6 primer pairs amplified a single band (see Additional file 10: Fig. S4). Overall, the expression profiles of selected genes revealed by qRT-PCR matched that by RNA-seq across four developmental stages (see Additional file 11: Fig. S5). Correlation analysis shows that the gene expression levels from qRT-PCR and RNA-seq are positively and significantly related (r2 = 0.7712, p < 0.001)(Fig. 6). Among the 39 putative genes, averagely, most of them were differentially expressed across the corm developmental stages with homologues of AGPase, INV and SPS the most actively expressed (see Additional file 8: Tab. S5).