Rapid seedling emergence of invasive Phytolacca americana is related to higher soluble sugars produced by starch metabolism and photosynthesis compared to native P. acinosa

Seedling emergence is an essential event in the life cycle of plants. Most invasive plants have an advantage in population colonization over native congeners. However, differential seedling emergence between invasive plants and native congeners, especially their mechanisms, have rarely been explored. In this study, we show that the seedlings of invasive Phytolacca americana emerge faster compared to native P. acinosa. Genome-wide transcriptomes of initially germinated seeds versus seedlings at 4 days after germination (DAG) suggested that differentially expressed genes (DEGs) in the photosynthesis-antenna proteins pathway were up-regulated in both P. americana and P. acinosa, while DEGs in starch and sucrose metabolism were significantly down-regulated in P. americana. Gene expression analysis indicated that photosynthesis-related DEGs reached their highest level at 3 DAG in P. americana, while they peaked at 4 DAG in P. acinosa. We also identified one β-amylase gene in P. americana (PameAMYB) that showed the highest expression at 1 DAG, and two β-amylase genes in P. acinosa that expressed lower than PameAMYB at 0 and 1 DAG. Enzymatic activity of β-amylases also suggested that P. americana had the highest activity at 1 DAG, which was earlier than P. acinosa (at 4 DAG). Soluble sugars, the main source of energy for seedling emergence, were showed higher in P. americana than in P. acinosa, and reached the highest at 4 DAG that positively affected by photosynthesis. These results indicate that the rapid seedling emergence of invasive P. americana benefited from the high soluble sugar content produced by starch metabolism and photosynthesis. Altogether, this work contributes to our fundamental knowledge on physiological and molecular mechanisms for plant invasion success.


Introduction
Seedling emergence occurs after seed germination, when the shoot and root of the seed are formed (Csontos, 2003), and is an essential prerequisite for vegetative growth, population colonization, and dispersal of plants (Verdúand Traveset, 2005;Leger et al., 2019;Larson et al., 2020).However, emerging seedlings are vulnerable to many extrinsic factors, such as water, pests, and maternal effects (Lamichhane et al., 2018;Chen et al., 2020).Seedto-seedling transition is also affected by intrinsic factors, including plant genotypes (Graebner et al., 2012;Souza and Fagundes, 2014;Yu et al., 2020).Therefore, short time intervals between seed germination and seedling emergence are usually helpful for successful establishment of seedlings.
Seedling emergence is highly dependent on plant physiological status.Typically, soluble sugars are the main source of energy for seedling emergence (Penfield et al., 2005).Hydrolytic enzymes, including a-amylase, b-amylase, and a-glucosidase, are the primary enzymes that break down starch into soluble sugars (Zeeman et al., 2010).In Triticum aestivum, post-germination seedling growth is positively regulated by starch degradation that is catalyzed by aamylase and a-glucosidase (Sun et al., 2020).The b-amylase activity in Cyclobalanopsis gilva was found to be higher in germinated seeds compared to non-germinated seeds (Zaynab et al., 2018).Therefore, high hydrolytic enzyme activity in germinated seeds is crucial for rapid seedling emergence.
Invasive plants cause serious ecological consequences and pose great threats to the economy and human health (Rands et al., 2010;Vila et al., 2011;Rai and Singh, 2020).Most invasive plants grow more rapidly than their native congeners, such as Copaifera langsdorffii versus C. oblongifolia, Spartina densiflora versus S. maritime, as well as Phytolacca americana versus P. acinosa (Fagundes et al., 2020;Infante-Izquierdo et al., 2020;Liu et al., 2022).Functional traits of plants, like specific leaf area and photosynthetic rate, have been an active point of research on the rapidity of plant growth (Feng et al., 2009;Liu et al., 2022).However, the mechanisms that underlie the physiological and molecular differences of plant growth between invasive and native congeners are still poorly understood, especially during the transition from seeds to seedlings.
P. americana is an herbaceous perennial plant native to North America that occupies a wide range of habitats in China, and is known to be poisonous to mammals (Xu et al., 2012).Meanwhile, P. acinosa is the congener of P. americana that is native to China.In this study, we compared the differences in seedling emergence of invasive P. americana and native P. acinosa.Firstly, the number of emerged seedlings for the two species was recorded in the 10 days after seed germination (DAG).Subsequently, genome-wide transcriptomes were sequenced for emerged seedlings at 0 and 4 DAG for both species.According to the differential metabolic pathways between P. americana and P. acinosa, photosynthesisrelated differentially expressed genes (DEGs) and differentially expressed b-amylase genes were further investigated.Additionally, gene expression and enzymatic activity of b-amylase, and total soluble sugar content were determined to investigate the effect of photosynthesis and starch metabolism on seedling emergence.
These results would help illustrate the physiological and molecular mechanisms of differential seedling emergence between the two congeners, and further reveal the invasion mechanisms of P. americana.

Plant materials
Seeds of P. americana (24°49′ N, 102°52′ E) and P. acinosa (25°26′ N, 104°19′ E) were collected from the field in August 2020 in Yunnan, China.Mature racemes, that grew under adequate sunlight in the canopy, were randomly collected from each plant species.Seeds were obtained after the raceme flesh were removed (Liu D. et al., 2020).All seeds were stored in the laboratory at the Institute of Biodiversity, School of Ecology and Environmental Science, Yunnan University.The 1,000-seed weight of P. americana and P. acinosa was recorded as 7.55 ± 0.08 g and 7.56 ± 0.03 g, respectively.For germination, all seeds were soaked in 98% H 2 SO 4 for 15 min to break dormancy.Subsequently, seeds were separately placed in Petri dishes (d = 9 cm) with 20 mL of 1% agar medium (1g agar and 100 mL ddH 2 O) and cultured in a growth chamber (Yiheng, Shanghai, China) with a 14 h light: 10 h dark cycle at 27°C and 25°C, respectively.

Germination rate, seedling emergence, and seedling biomass measurement
To compare the difference in time intervals between seed germination and seedling emergence, we first calculated seed germination rate of P. americana and P. acinosa.Six seeds of one species were placed in a Petri dish with agar medium and cultured as described above.Ten replicates were performed.The seeds were considered to have germinated when approximately 1 mm of the radicle protruded from the seed coat.At day 3, the radicle of the seed emerged, and the number of germinated seeds was recorded.The germination rate was determined as the number of germinated seeds at day 3 versus the total number of tested seeds.
The seeds that germinated at day 3 were employed for seedling emergence tests and treated as 0 DAG.Twenty initially germinated seeds of P. americana and P. acinosa were separately transferred into another 1% agar medium and cultured as described above.Three replicates were conducted.Seedlings with spread cotyledons were used for seedling emergence analysis (Larson et al., 2020).The number of emerged seedlings was recorded daily for 10 days.At 10 DAG, the fresh weight of the seedlings was measured using an analytical balance (BSA223S, OLABO, Shandong, China).

RNA isolation, Illumina sequencing, and data processing
Seedlings at 4 DAG of each species were individually collected for transcriptome analysis, and 0 DAG seedlings were used as controls (Supplementary Figure S1).Eight germinated seeds were used for one replicate, and three biological replicates were conducted.Total RNA was extracted using the RNAprep Pure Plant Kit (Tiangen Biotech Co., Ltd, Beijing, China) following the manufacturer's instructions.RNA quality and purity were determined using a NanoPhotometer N60 (Implen, Germany) and an Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).A sample of 1 mg RNA was used, and cDNA libraries were constructed separately, as previously described (Liu D. et al., 2020).Transcripts at the genome-wide transcriptional level were separately determined using pair-end (150 bp) RNA sequencing (RNA-Seq) on an Illumina NovaSeq 6000 platform at Biomarker Technology Co. (Beijing, China).
Clean reads were obtained by removing adaptor, reads with Q30 (probability of incorrect base call = 1/1000) of less than 85%, and reads containing more than 10% unknown bases per read from raw reads.After trimming, transcripts were assembled by pooling clean reads for all individual plant treatments using Trinity (v2.5.1), with min_kmer_cov set to 2 (Grabherr et al., 2011).To quantify gene expression, clean reads were mapped back to the transcriptome dataset, and read numbers were obtained using RSEM (v1.2.19;Li and Dewey, 2011).The mapped reads were normalized according to the fragment per kilobase of exon model per million mapped reads (FPKM; Mortazavi et al., 2008).Transcripts with a P-adjusted (P-adj) value of < 0.05 between two groups (each group with three biological replicates) and absolute log 2 (0 DAG verse 4 DAG) > 1 were assigned as DEGs (Anders and Huber, 2010;Wu et al., 2021).Gene ontology (GO) enrichment analysis of the DEGs was performed using the topGO R package (v2.28.0) based on the Kolmogorov-Smirnov test (Alexa and Rahnehfuhrer, 2016).KOBAS2.0 software was used to test the statistical enrichment of DEGs in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (Xie et al., 2011).Raw data were deposited in the Sequence Read Archive of the National Center for Biotechnology Information (accession number: PRJNA992375).

Candidate gene selection
We selected DEGs from KEGG pathways with P < 0.05 between the two developmental stages, and DEGs with FPKM value > 10 and log 2 foldchange (0 DAG versus 4 DAG) > 2 or < -2 in each pathway were filtered and then analyzed in R (4.1.3,R Core Development Team, 2020) using the GOplot package.
We found that DEGs enriched in the photosynthesis-antenna proteins pathway were up-regulated in both P. americana and P. acinosa.To better analyze the phylogenetic relationship among these DEGs, we constructed a phylogenetic tree according to their deduced amino acid sequences with MEGA 11.0 using the Neighbor-Joining method with 1000 bootstrap replicates (Hall and Notes, 2013).FPKM values for the DEGs were also presented using iTOL (https://itol.embl.de/).To further analyze gene expression, some homologous DEGs between the two species were randomly selected.These genes were amplified with the primers listed in Table 1 and cloned into a sequencing vector BluntSimple (TransGen, China) for full sequencing.Quantitative polymerase chain reaction (qPCR) was then performed to determine the gene expression of the seedlings at 0, 1, 2, 3, and 4 DAG.
We also found that b-amylase genes in starch and sucrose metabolism were significantly down-regulated compared with other DEGs in the selected KEGG pathways in P. americana.An additional phylogenetic tree was constructed based on the deduced amino acid sequences for the b-amylase DEGs in P. americana and P. acinosa together with b-amylases with reviewed functions in UniProt (Hall and Notes, 2013).According to the reference genomes of P. americana and P. acinosa, candidate bamylase genes were amplified with the primers listed in Table 2, and

Name
Sequences 5'-3' For sequence amplification  then sequenced.Substrate-binding and protein active sites of the deduced amino acid sequences from the amplified sequences were verified according to the homologous b-amylase in Ipomoea batatas (Yoshida and Nakamura, 1991) using SnapGene (4.2.4,GSL, Biotech, USA).qPCR was also conducted to analyze the bamylase gene expression of the seedlings at 0, 1, 2, 3, and 4 DAG.

cDNA preparation and qPCR analysis
Emerged seedlings at 0, 1, 2, 3, and 4 DAG of the two species were separately collected and stored at -80°C after flash freezing in liquid nitrogen.Total RNAs were extracted as described above, and cDNA was subsequently prepared using 1.0 mg RNA using the FastQuant RT kit (Tiangen Biotech Co., Ltd, Beijing, China) according to the manufacturer's instructions.
SnapGene was used to design the primers to quantify target gene expression (Tables 1, 2).qRCR analysis was conducted using SuperReal PreMix Plus kits (SYBR Green; Tiangen Biotech Co., Ltd., Beijing, China) and an ABI QuantStudio 7 Flex PCR system (Applied Biosystems, Carlsbad, CA, USA).Samples were detected separately in 20 mL reaction mixture volumes with an initial thermal profile of 15 min at 95°C, followed by 40 cycles of 10 s at 95°C and 32 s at 60°C.Three technical replicates were performed for each sample, and three biological samples were used.
For qPCR analysis, we used the absolute quantitative method where the copy number of each gene was determined according to a standard curve.The standard curve was generated using a 10-fold serial dilution of standard plasmid ranging from 1 × 10 6 to 1 × 10 2 copies/mL target fragments (Liu et al., 2016).
b-amylase activity and soluble sugar content in P. americana and P. acinosa Emerged seedlings collected at 0, 1, 2, 3, and 4 DAG of the two species were also used to determine b-amylase activity and soluble sugar content.The b-amylase activity and soluble sugar content of each sample were separately determined following the instructions of b-amylase kits (Grace Biotechnology Co., Ltd.Suzhou, China) and soluble sugar content kits (Grace Biotechnology Co., Ltd.Suzhou, China) using a microplate reader (SuperMax 3100, Flash, China).
To validate the role photosynthesis played in the production of soluble sugars during seedling emergence, we cultured initial germinating seeds of the two species in the dark, and then collected emerged seedlings at 0, 1, 2, 3, and 4 DAG of the two species.Soluble sugar content of each sample was determined as described above, and three replicates were performed for each treatment.

Data analysis
Data were analyzed using R. Student's t-tests were used to compare germination rates, the number of emerged seedlings on the same day, and the fresh weight of the 10 days old seedlings between P. americana and P. acinosa.Likewise, expression levels of target genes, b-amylase activity, and soluble sugar content of the samples collected at the same time were also compared using t-tests.The number of emerged seedlings, expression level of target genes, bamylase activity, and soluble sugar content of the same species at different times were analyzed using one-way analysis of variance (one-way ANOVA) and pair-wise post hoc analyses by least significant difference (LSD) in the agricolae package.Statistical significance was set at P < 0.05.

Results
Germination rate, seedling emergence, and seedling fresh weight for P. americana and P. acinosa After being treated with H 2 SO 4 to break seed dormancy, we found P. americana and P. acinosa seeds began to germinate at day 3, and their germination rates were not significantly different (t-test, P = 0.1152; Figure 1A).These germinated seeds were then used for the following experiments.
After culturing the initially germinated seeds for 10 days, we found that P. americana seedlings began to emerge at 4 DAG (one- way ANOVA, P Pame < 0.001), while P. acinosa began to emerge at 5 DAG (one-way ANOVA, P Paci < 0.001).At 10 DAG, approximately 80% of the tested P. americana seeds developed into seedlings, while less than 10% of the P. acinosa seedlings developed.Additionally, the number of emerged P. americana seedlings was significantly higher than that of P. acinosa (t-test; P 5 DAG = 0.004; P 6 DAG = 0.001; P 7 DAG < 0.001; P 8 DAG < 0.001; P 9 DAG < 0.001; P 10 DAG < 0.001; Figure 1B).At 10 DAG, the fresh weight of P. americana seedlings was also significantly higher than that of P. acinosa seedlings (t-test, P = 0.004; Figure 1C).Together, these results indicate that the time intervals between seed germination and seedling emergence of invasive P. americana were shorter than that of native P. acinosa.

Genome-wide transcriptome data analysis
RNA-Seq data of the tested samples were evaluated using Pearson's correlation coefficient (r) and showed high consistency (r > 0.94) within groups (Supplementary Figure S2).The average FPKM of each group was then used for DEG analysis.In total, 7,806 and 11,340 DEGs were identified in the 0 vs 4 DAG seedlings of P. americana and P. acinosa, respectively.Of these, 4,341 and 3,465 genes in P. americana, and 6,779 and 4,561 genes in P. acinosa were up-and down-regulated, respectively (Figure 2A).In both P. americana and P. acinosa, 5,705 DEGs showed more than 50% similarity and were treated as the intersection of the Venn diagram (Figure 2B; Supplementary Material).We also found 2,101 DEGs only in the 0 vs 4 DAG seedlings for P. americana, and 6,511 DEGs only in the 0 vs 4 DAG seedlings for P. acinosa.
According to the annotations on the GO database, P. americana seedling DEGs were highly enriched in the metabolic and cellular processes in the biological process category, cell and cell part in the cellular component category, as well as binding and catalytic activity in the molecular function category.The same trend was also observed in P. acinosa seedlings.However, the number of DEGs in P. acinosa was higher than that in P. americana (Supplementary Figure S3).KEGG pathway enrichment analysis suggested that 26 pathways were differentially enriched by the DEGs in P. americana, while 19 pathways were enriched in P. acinosa (Supplementary Figure S4).Among these KEGG pathways, 12 pathways were only found in P. americana seedlings, while only five were in P. acinosa seedlings.Furthermore, energy metabolism pathways, such as butanonate metabolism, pentose phosphate pathway, and starch and sucrose metabolism, were only found in P. americana.According to the FPKM values, DEGs for the photosynthesis-antenna proteins pathway in both species were highly up-regulated.However, some DEGs for starch and sucrose metabolism were significantly downregulated in P. americana (Supplementary Figures S5, S6).

DEGs in photosynthesis-antenna proteins pathway
In total, we filtered 23 photosynthesis-antenna proteins pathway DEGs in the 0 vs 4 DAG seedlings for P. americana and 37 DEGs in P. acinosa seedlings.The phylogenetic tree suggested that photosynthesis-antenna proteins pathway DEGs between P. americana and P. acinosa were highly similar.Even though these DEGs were significantly up-regulated, many DEGs in P. acinosa seedlings were more highly expressed compared to their homogeneous genes in P. americana (Figure 3).
Additionally, we compared the gene expression among seedlings collected at 0, 1, 2, 3, and 4 DAG, and found that the tested DEGs in P. americana had the highest expression at 3 DAG, while in P. acinosa they showed the highest expression at 4 DAG (Figure 4).

Candidate genes of b-amylase
We then selected and compared DEGs involved in the synthesis of 21 enzymes for starch and sucrose metabolism.Genes for bglucosidase and b-amylase in P. americana were differentially expressed during the first four days after germination (Figure 5).The phylogenetic tree of b-amylase in P. americana and P. acinosa together with other plants reported in UniProt were constructed and showed that five DEGs for b-amylases in P. americana and three DEGs in P. acinosa were clustered with b-amylase in I. batatas (IbatAMYB; Figure 6A).
Further PCR amplification and sequence alignment revealed that four of the five b-amylase genes in P. americana were amplified and showed the consensus sequence (named PameAMYB).However, the remaining transcript could not be amplified.Additionally, two of the three transcripts for b-amylase in P. acinosa were confirmed to be the same and were named PaciAMYB1 and PaciAMYB2.Further, sequence alignment of Differentially expressed genes (DEGs) enriched in the photosynthesis-antenna proteins pathway between Phytolacca americana and Phytolacca acinosa.DAG: day(s) after germination.FPKM represent the fragment per kilobase of exon model per million mapped reads value of each DEG, and log 2 FC is the log2foldchange (0 DAG versus 4 DAG) of each DEG.EVM is the DEG in P. americana, and Pac is the DEG in P. acinosa.Liu et al. 10.3389/fpls.2024.1255698Frontiers in Plant Science frontiersin.org IbatAMYB, PameAMYB, PaciAMYB1 and PaciAMYB2 suggested that all the binding and active sites were present among the deduced amino acid sequences (Figure 6B).Together, these results suggest the presence of functional b-amylase in both P. americana and P. acinosa.
The enzymatic activity measurements suggested that b-amylase activity was the highest at 1 DAG in P. americana and decreased at 4 DAG to a level similar to 0 DAG.However, b-amylase activity was not significantly different during those four days (one-way ANOVA, P Pame = 0.265; Figure 7B).In P. acinosa, the b-amylase activity gradually increased (one-way ANOVA, P Paci < 0.001) and began to be higher than P. americana at 2 DAG (t-test, P 0 DAG < 0.001, P 1 DAG < 0.001, P 2 DAG < 0.001, P 3 DAG < 0.001, P 4 DAG < 0.001), and this trend could be related to the expression of PaciAMBY2, since it was up-regulated at 0 vs 4 DAG in P. acinosa (log 2 FC of PaciAMBY2 = 4.75, Supplementary Material).

Total soluble sugars determination
Total soluble sugar content in P. americana increased in the first two days after germination, and reached its highest level at 4 DAG (one-way ANOVA, P Pame < 0.001; Figure 8A).However, total soluble sugars in P. acinosa increased from 0 to 3 DAG, but then decreased (one-way ANOVA, P Paci < 0.001).At 0 DAG, the soluble sugar content was not significantly different between the two species (t-test, P = 0.748), and was higher at 1 (t-test, P = 0.045) and 2 (ttest, P = 0.009) DAG in P. americana compared to P. acinosa, but then decreased at 3 DAG (t-test, P = 0.149).However, it was markedly higher in P. americana than in P. acinosa at 4 DAG (t-test, P = 0.001).
When the initially germinated seeds were cultured in the dark, the two species showed a similar variation in total soluble sugar content (one-way ANOVA; P Pame < 0.001; P Paci = 0.035), even though the sugar content in P. americana was higher than that in P. acinosa (t-test, P 0 DAG = 0.443, P 1 DAG = 0.034, P 2 DAG = 0.011, P 3 DAG = 0.259, P 4 DAG = 0.006).Moreover, soluble sugar content in P. americana did not significantly increase at 4 DAG (Figure 8B).

Discussion
Invasive P. americana shows faster seedling emergence compared to native P. acinosa It has been reported that invasive plants show faster seedling emergences compared to native ones, such as Bromus tectorum (Griffith et al., 2014), Sorghum halepense (Reichmann et al., 2016), and Spartina densiflora (Infante-Izquierdo et al., 2020).In our study, even though P. americana and P. acinosa seeds began to germinate at the same time and showed similar germination rates, the invasive P. americana seedlings emerged more rapidly than native P. acinosa (Figures 1A, B).Typically, emerged seedlings use resources for growth and, therefore, gain competitive ability.Under resource competition, early seedling emergence could lead to disproportional resource utilization and create fecundity advantages (Dyer et al., 2000).We found that P. americana showed seedling emergence one day earlier than P. acinosa, which suggest that invasive P. americana could utilize resources earlier than native P. acinosa.Ross and Harper (1972) reported that 95% of plant biomass variation could be accounted for by time since emergence.Here, the biomass of P. americana seedlings at 10 DAG was significantly higher than P. acinosa seedlings (Figure 1C).This is also similar to the previous data that P. americana had a higher total biomass and exhibited a higher reproductive capacity compared to P. acinosa (Liu et al., 2022).Additionally, to avoid the effect of seed batch on seed germination and seedling emergence, we compared the data among seeds collected in 2019, 2021 to 2023, and found similar data among one particular species during the years (Supplementary Figures S7, S8).
Our transcriptome data also showed that many genes were differentially expressed at 0 vs 4 DAG in both P. americana and P. acinosa seedlings.Total DEGs in P. acinosa were higher than in P. americana (Figure 2), which may be related to the ploidy level of these plants, since P. americana is diploid and P. acinosa is autotetraploid.Similar results were found in autotetraploid rice and its diploid donor (Wang et al., 2022).GO analysis of the 0 vs 4 DAG seedlings of P. americana and P. acinosa also suggest that the two species undergo similar development (Figure 3).However, the KEGG pathway enrichment analysis suggests that the differential KEGG pathways between the two congeners are not entirely consistent (Supplementary Figure S3).More pathways are activated in invasive P. americana than in native P. acinosa, especially pathways related to energy metabolism.Studies found that genes in slowly and rapidly developing Panicum virgatum are differentially expressed in pathways related to diterpenoid biosynthesis, thiamine metabolism, and circadian rhythm (Zhang et al., 2019).The inconsistent results might be caused by the varying developmental stages of tested samples.
FIGURE 1 Germination rate at day 3 after seed dormancy breaking treatment (A), number of emerged seedlings with spread cotyledons (B) and weight of the 10 DAG seedlings (C) of Phytolacca americana (Pame) and Phytolacca acinosa (Paci).DAG: day(s) after germination.Values represent the mean ± standard error.Different letters in the same shade as the line indicate that the means differ significantly among the tested time points under one-way ANOVA followed by least significant difference (LSD) test.Asterisks indicate a significant difference between the two species at the same time point under Student's t-test.*P < 0.05, **P < 0.01, ns P > 0.05.

FIGURE 4
FIGURE 4Expression level of homologous fragments of genes involved in photosynthesis-antenna proteins pathway between Phytolacca americana and Phytolacca acinosa.DAG: day(s) after germination.Values represent the mean ± standard error.Different letters in the same color as the column indicate that the means differ significantly among the tested time points under one-way ANOVA followed by least significant difference (LSD) test.Asterisks indicate a significant difference between the two species at the same time point under Student's t-test.*P < 0.05, **P < 0.01, ***P < 0.001, ns P > 0.05.
FIGURE 5 Starch and sucrose metabolism pathway and the related differentially expressed genes (DEGs) in Phytolacca americana and Phytolacca acinosa.(A) Starch and sucrose metabolism pathway that DEGs were detected in P. americana and P. acinosa.(B) Heatmap of the DEGs based on the fragment per kilobase of exon model per million mapped reads values.DAG: day(s) after germination.## indicates the sequence is related to more than one enzyme.DAG01, DAG02, and DAG03 are the three replicates of 0 DAG seedlings, while DAG41, DAG42, and DAG43 are the three replicates of 4 DAG seedlings.
FIGURE 6 b-amylase in Phytolacca americana and Phytolacca acinosa.(A) Phylogenetic tree of b-amylase in P. americana and P. acinosa together with other reported b-amylases in Uniprot.(B) Sequence alignment of b-amylase in P. americana (PameAMYB), P. acinosa (PaciAMYB1 and PaciAMYB2), and Ipomoea batatas (IbatAMYB).EVM is the DEG in P. americana, and Pac is the DEG in P. acinosa.Red font represents the target sequences.Red triangles indicate the substrate binding sites of the proteins, and red pentangles show the active sites of the proteins.

TABLE 1
Primers for genes involved in photosynthesis-antenna proteins pathway.

TABLE 2
Primers for b-amylase genes.