Differential effects of synthetic media on long-term growth, starch accumulation and transcription of ADP-glucosepyrophosphorylase subunit genes in Landoltia punctata

Murashige & Skoog (MS) and Hoagland’s media were previously used for in vitro culture of Landoltia punctata. During subsequent ex vitro culture, the use of MS medium resulted in a higher growth rate, compared to Hoagland’s medium. Thus, a higher starch content of L. punctata in MS medium was previously hypothesized. Here, L. punctata strain 5632 was isolated and characterized using morphological characteristics and the atpF-atpH intergenic region. During early cultivation stage, fresh weight and relative growth rate in MS medium were lower than Hoagland’s medium. Conversely, starch content in MS medium was considerably higher than in Hoagland’s medium. Medium effects on expression of genes coding for starch-biosynthesis ADP-glucosepyrophosphorylase (AGPase) were determined. Genomic fragments of small (LeAPS) and large (LeAPL1) AGPase subunits were characterized. Differential expression between each AGPase subunit genes was observed in both media. Additionally, in MS medium, the highest correlation coefficients between starch content and gene expression was found with LeAPS (0.81) and followed by LeAPL3 (0.67), LeAPL2 (0.65) and LeAPL1 (0.28). In Hoagland’s medium, the coefficients of LeAPL3 (0.83) and LeAPL2 (0.62) were higher than LeAPS (0.18) and LeAPL1 (−0.62). This suggested different levels of contributions of these genes in starch biosynthesis in both media.


Results and Discussion
Morphological and molecular characterization. Duckweed samples were collected, and an axenic culture on Hoagland's medium was established from a single mother frond to ensure the genetic similarity among samples throughout the study. The strain was designated as strain 5632. It was important to note here that the nutrient compositions of Hoagland's medium were somewhat different from the original publication 29 . Originally, two different macronutrient solutions were described for the preparation of Hoagland's medium 29 . The main difference between them was the nitrogen source. While one solution contained only NO 3 − , the other one was supplemented with both NO 3 − and NH 4 + 29 . Hoagland's solution, used in this study, had nutrient compositions ( Table 1) that were more similar to the latter one. However, there was a distinction in the NO 3 − concentration in the original solution (1 mM) 29 and this study (6 mM). Additionally, iron was supplied as iron tartate in the original publication 29 , whereas it was given here as chelated iron. Basic morphological characteristics of strain 5632 were examined (Fig. 1). Fronds were inflated and oval-shaped. The upper surface was dark green. The lower surface was reddish, suggesting the accumulation of anthocyanins in the tissues. Several roots were present on the lower side of the fronds, indicating that strain 5632 was likely a member of the genus Landoltia. The presence and the number of roots were distinguishing morphological characteristics among the five genera of the family Lemnaceae 30 . For example, a single root was present on each frond of Lemna spp., whereas plants in genera Wolffia and Wolffiella did not produce roots. In contrast, several roots were present on each frond of members in genera Spirodela and Landoltia 31 . The number of roots of the genus Spirodela ranged from 7 to 21, while that of the genus Landoltia ranged from 2 to 7 32 . Additional characteristics, e.g., a medial series of papillae on the upper surface, frond prophyllum, frond nerves and external anther locules, could be used to differentiate Landoltia from Spirodela 32 . However, because of their small and highly reduced structures, identification of duckweeds at the species levels could be challenging without a special expertise in the Lemnaceae family 33 . Biochemical and DNA sequence data were previously combined with morphological and anatomical markers to study the phylogeny of the family Lemnaceae 34 . The phylogenetic tree confirmed the presence of the paraphyletic subfamily Lemnoideae, consisting of Spirodela, Landoltia, and Lemna, and the monophyletic subfamily Wolffioideae, comprising of Wolffia and Wolffiella. Additionally, a DNA barcode, based on the atpF-atpH intergenic region, was developed to aid the characterization of lemnaceous plants 33 . For identification of strain 5632, the atpF-atpH intergenic region was amplified from plastid DNA and sequenced. Using the blastn analysis, the highest identity level (100%) was found between the sequences of strain 5632 and L. punctata strains DW2701-1 (accession number: KJ630554) and DW2701-4 (KJ630555). To understand the phylogenetic relationship between strain 5632 and other L. punctata strains, the phylogenetic tree was reconstructed based on the atpF-atpH intergenic sequences. It showed that strain 5632 was closely related to L. punctata strains DW2701-1, DW2701-4 and 20d (Fig. 2). This was supported by the 100% confidence level. Consistent with its morphologies, the molecular characterization indicated that strain 5632 was a member of the species L. punctata.
Growth and starch accumulation of L. punctata 5632 in synthetic media. A previous study showed that the growth rate of L. punctata in MS medium was lower than that in Hoagland's medium 26 . However, after transferred to nutrient-limited water, L. punctata obtained from MS medium grew at a higher rate than those from Hoagland's medium 26 . This suggested a higher starch content of L. punctata in MS medium that could be used to sustain subsequent ex vitro growth. To test this hypothesis, strain 5632 was grown in MS and Hoagland's liquid media for 35 days, and fresh weight was determined every seven days (Fig. 3a). It was important to note that there were some differences, e.g. types of some micronutrient salts and some nutrient concentrations, between the compositions of Hoagland's solution used here (Table 1) and in the previous study 26 . However, during the first 14 days of cultivation, the fresh weight of strain 5632 in Hoagland's solution was higher than in MS medium. This was consistent with the previous study 26 . This continued until day 21 of cultivation. Conversely, on day 28, the fresh weight in MS medium (3.14 ± 0.04 g) became significantly higher (P = 0.038) than that in Hoagland's medium (3.04 ± 0.04 g). On day 35, it became 1.2 times of that in Hoagland's medium.
Relative growth rate of strain 5632 in both media were calculated and found relatively consistent with fresh weight analysis (Fig. 3b). During the first 14 days of cultivation, strain 5632 in MS medium exhibited lower relative growth rate, compared to Hoagland's medium. In contrast, on day 28 and 35 of cultivation, the relative growth rate in MS medium became substantially higher than in Hoagland's medium. This indicated a more rapid reduction in growth of strain 5632 in Hoagland's medium. A discrepancy between fresh weight and relative growth rate was observed on day 21. While the fresh weight in MS medium was lower than Hoagland's medium (Fig. 3a), the relative growth rate became considerably higher than Hoagland's medium (Fig. 3b). This was because of the differential increase between the fresh weight in both media. From day 14 to day 21, the fresh weight in MS medium elevated from 0.53 ± 0.04 g to 1.71 ± 0.08 g, respectively. This yielded the relative growth rate at 0.17 ± 0.004 day −1 . During the same period, the fresh weight in Hoagland's medium rose from 1.02 ± 0.07 g to 2.23 ± 0.12 g, and yielded relative growth rate at 0.11 ± 0.002 day −1 .

MS medium
Hoagland's medium  www.nature.com/scientificreports www.nature.com/scientificreports/ Starch content of strain 5632 in MS and Hoagland's media was also determined throughout the cultivation period (Fig. 3c). In MS medium, it continuously increased and reached the level of 3.31 ± 0.28% on day 28. This was followed by a decrease to 1.95 ± 0.25% on day 35. In contrast, starch content of strain 5632 in Hoagland's medium increased relatively slowly during the first three weeks of cultivation. Subsequently, it reached the highest level (4.01 ± 0.24%) on day 28 and slightly decreased to 3.91 ± 0.18% on day 35. Comparison between the fresh weight and starch content in both media revealed an inverse correlation. From day 7 to 21, the fresh weight of strain 5632 in MS medium was lower than Hoagland's medium, whereas starch content in MS medium was higher than the other medium. The opposite was found on day 28 and 35 of cultivation.
Growth and starch content of strain 5632 were likely affected by the differences between the concentrations of both NH 4 + and NO 3 − in MS and Hoagland's media (Table 1). During the early phase of cultivation, the slower growth in MS medium was likely caused by the high concentration of NH 4 + in MS medium (20.6 mM). A previous study showed that NH 4 + was preferable over NO 3 − for the uptake by L. punctata 35 . Thus, cultivation of strain 5632 in the medium likely caused excessive accumulation of NH 4 + in strain 5632. This resulted in lower biomass production, compared to the use of Hoagland's medium, that was supplemented with 1 mM NH 4 + . When present at a high concentration, NH 4 + was known to negatively affect plant growth 36 . This was also demonstrated in other duckweed species. Relative growth rate of S. polyrhiza decreased upon the increase of NH 4 + concentration 37 . Growth inhibition and frond chlorosis were also found in Lemna minor, grown in Hoagland's medium supplemented with 280 and 840 mg l −1 NH 4 + -N, which equaled to 20 and 60 mM NH 4 + , respectively 38 . During the later cultivation period, growth of strain 5632 in Hoagland's medium declined more rapidly, compared to that in MS medium. This may be attributed to the NO 3 − concentration in Hoagland's medium (10 mM) that was only 25.4% of that in MS medium (39.4 mM). The result suggested that this level of NO 3 − , in Hoagland's medium, was insufficient to sustain growth of strain 5632 for the entire cultivation period. Additionally, another nutrient that may cause the distinction in growth of strain 5632 in the two media was iron. It is an important microelement in various plant metabolic processes 39 . Generally, FeSO 4 was added together with Na 2 EDTA to generate chelated iron that ensured its transport into plant cells 40 . Here, the concentrations of FeSO 4 and Na 2 EDTA, in Hoagland's medium, were approximately 9% of those in MS medium and may be insufficient to sustain growth during the extended cultivation period. On the other hand, starch biosynthesis was previously known to be upregulated under nutrient starvation conditions, which also resulted in growth retardation 4,5,22,41,42 . This was somewhat consistent with the increase in starch content of strain 5632 in MS medium, where growth was relatively lower than in Hoagland's medium. However, as mentioned above, both NH 4 + and NO 3 − in MS medium were supplied at much higher levels than Hoagland's medium. This suggested that differential starch accumulation of strain 5632 in the two media was likely a secondary effect that resulted from growth modulation by the nitrogen sources.

Nucleotide variations in full-length cDNA sequences of LeAPL1 and LeAPS. Full-length LeAPS
and LeAPL1 cDNA were previously sequenced in L. punctata strain 0202 that was collected in China 4 . However, strain 5632 was obtained from a local pond in Thailand. Thus, cDNA sequences of both genes were determined to investigate nucleotide variations that may exist between the two genes of these strains. Full-length LeAPS  Table 2). Two of these resulted in changes of the deduced amino acid residues. For LeAPL1, there were three nucleotide variations at positions 563, 680 and 754, all of which resulted in changes of corresponding amino acid residues (Table 2). To investigate whether these amino acid variations were inherent to strain 5632, the deduced LeAPL1 and LeAPS protein sequences of strain 5632 were used for multiple alignment analysis with those of strain 0202 and their orthologs from other plant species. The analysis indicated that amino acid positions 9, 192 and 257 of LeAPS (Fig. 4a) and 188 and 227 of LeAPL1 (Fig. 4b) of strain 5632 were more similar to the orthologs than strain 0202. These differences between the deduced amino acid sequences of strains 5632 and 0202 were possibly derived from DNA amplification errors during the cloning step. LeAPS and LeAPL1 cDNA of strain 0202 were amplified, using rTaq DNA polymerase 4 . In contrast, the cloned sequences of strain  www.nature.com/scientificreports www.nature.com/scientificreports/ 5632 were obtained with Q5 high-fidelity DNA polymerase, of which the error rate was described as 280 times lower than regular Taq DNA polymerase (www.neb.com). Thus, these amino acids of strain 5632 likely represented the actual residues of LeAPS and LeAPL1. The only exception was the amino acid position 252 of LeAPL1 that was derived from its corresponding codon containing the nucleotide variant position 754. The amino acid sequences of strain 5632 and other orthologs carried methionine at this position, as opposed to valine found in L. punctata 0202 and S. tuberosum sequences (Fig. 4b). Thus, this nucleotide position may represent a genotypic variation between LeAPL1 of strains 5632 and 0202.
Organization of genomic LeAPS and LeAPL1. Although their cDNA sequences were previously characterized 4 , the genomic sequences of LeAPS and LeAPL1 were still unavailable. Here, amplification of genomic LeAPS with the CLeAPS-F and LeAPS-R primers was successful. The length of the amplified fragment was 2,621 bp. It was important to note that the fragment was only partial and did not cover the remaining 128 nucleotides on the 3′ region of its cDNA. Eight exons and seven introns were identified in the sequence (Fig. 5a). The numbers of exons and introns found in genes coding for AGPase small subunits were variable among different plant species. A study on the genomic structure of maize Bt2 showed that it consisted of ten exons and nine  www.nature.com/scientificreports www.nature.com/scientificreports/ introns 11 . In contrast, sAGP of potato 43 and ibAGP1 and ibAGP2 of sweet potato 44 were similarly organized into nine exons and eight introns. On the other hand, the genomic fragment of LeAPL1 (3,161 bp) was obtained using the CLeAPL1-F and CLeAPL1-R primers. The genomic sequence consisted of 14 exons and 13 introns (Fig. 5b). The average length of the exons was 111 bp. Exon 1 was the longest (210 bp), while exon 13 was the shortest (61 bp). The organization of exons and introns in LeAPL1 was different from its ortholog, SpAPL1 of S. polyrhiza, where 15 exons and 14 introns were characterized 28 .

Expression of AGPase subunit genes and correlation with starch accumulation. Expression of
AGPase subunit genes, including LeAPS, LeAPL1, LeAPL2 and LeAPL3, was analysed, in order to understand the molecular responses towards nutrient compositions in MS and Hoagland's media. Based on their genomic sequences, new primers were designed and used to quantify expression of LeAPS and LeAPL1. Unlike the previously described primers 4 , they spanned over intron regions to ensure the amplified products were derived from cDNA of processed mRNA molecules. For LeAPL2 and LeAPL3, the primers used in the analysis were from the previous study 4 , because their genomic sequences were unavailable. Additionally, the expression analysis here covered the cultivation period of five weeks. This was much longer than other previous studies where it ranged from seven to ten days 4,25,42 and enabled the monitoring of the long-term gene regulation in both media. Expression levels of each gene during the cultivation in both media are shown in Fig. 6. In MS medium, the highest expression levels of LeAPS (0.65 ± 0.07), LeAPL2 (1.92 ± 0.88) and LeAPL3 (1.48 ± 0.77) were observed on day 21 and approximately 8.2 times of the level on day 0. In contrast, LeAPL1 expression increased and reached the maximum level (0.66 ± 0.14) on day 14, which was approximately 2.6 times of that on day 0. Similar to LeAPS, the highest levels of LeAPL2 and LeAPL3 expression occurred on day 21 and were 4.0 and 8.1 folds of day 0, respectively. In Hoagland's medium, expression of LeAPS (0.72 ± 0.10) and LeAPL1 (1.02 ± 0.15) similarly peaked on day 14 and were 10.3 and 4.2 folds of day 0, respectively. LeAPL2 expression became the highest on day 28 (2.38 ± 0.80) and was 9.5 times of day 0. In contrast, expression of LeAPL3 continuously increased throughout the cultivation period and was the highest on day 35 (1.48 ± 0.61) and 3.6 folds of day 0. Significant differences were also observed between expression levels of some AGPase subunit genes in both media. On day 21, LeAPS in MS medium was expressed at a level significantly higher (P = 0.049) than in Hoagland's medium. On the other hand, LeAPL1 in MS medium was expressed at levels significantly lower than in Hoagland's medium on day 14 (P = 0.039) and 21 (P = 0.029). However, its expression in MS medium became significantly higher than in Hoagland's medium on day 28 (P = 0.03) and 35 (P = 0.014). In contrast, differences in LeAPL2 and LeAPL3 expression levels observed in MS and Hoagland's media were not substantial throughout the cultivation period. The result observed here indicated differential regulation of each AGPase subunit gene. This was also previously found with L. punctata strain 0202, that was grown under nitrogen and phosphorus starvation conditions 4 . In response to nitrogen starvation, upregulation of LeAPL1 and LeAPL3 was observed earlier than LeAPL2 4 . Under www.nature.com/scientificreports www.nature.com/scientificreports/ phosphorus deprivation, a dramatic increase in LeAPL1 and LeAPL2 expression occurred more rapidly than LeAPL2 4 . In contrast, LeAPS was highly expressed throughout the nitrogen starvation period and during the early stage of phosphorus starvation 4 . ABA may play a role in regulating expression of AGPase genes in this study, since its application on L. punctata was previously shown to increase starch content and AGPase activity 20,45 . Consistently, a transcriptomic study demonstrated that both ABA content and expression levels of AGPase large subunit genes were elevated, when L. punctata was treated with uniconazole 24,25 . Reports in S. polyrhiza 28 and rice cell culture 46 suggested that each AGPase subunit gene may respond to ABA differently. Expression of SpAPL2 and SpAPL3 was upregulated by the ABA treatment 28 . In contrast, the expression level of SpAPL1 remained relatively unchanged and was much lower than SpAPL2 and SpAPL3. On the other hand, application of ABA to rice cell culture resulted in elevated expression of OsAPL3 and OsAPS1, while its effects on other rice AGPase subunit genes were much less pronounced 46 . These previous observations suggested that ABA effects on expression of AGPase genes may also vary among different plant species.
Because of their differential expression, each AGPase subunit genes may contribute to starch accumulation of strain 5632 in MS and Hoagland's media differently. Correlation coefficients between the expression levels of AGPase subunit genes and the starch content were determined. In MS medium, LeAPS had the highest correlation coefficient (0.81) and was followed by LeAPL3 (0.67) and LeAPL2 (0.65). A small correlation (0.28) was observed with LeAPL1. In Hoagland's medium, expression of LeAPL3 was the most correlated (0.83) with starch content. An intermediate level of correlation was found with LeAPL2 (0.62). In contrast, the correlation coefficient of LeAPS was only 0.18, while LeAPL1 was negatively correlated (−0.62). The intermediate and high correlation coefficients of LeAPL2 and LeAPL3 were consistent with their expression levels that were found comparable in both media (Fig. 6c,d). This suggested the roles of LeAPL2 and LeAPL3 in starch biosynthesis may be non-specific to the media. In contrast, LeAPS contribution may be more pronounced in MS medium. On the other hand, the correlation coefficients of LeAPL1 in the two media were highly distinctive and ranged from low to negatively intermediate levels. This indicated its input in starch content was likely dependent on medium compositions. This was also reflected on the differences between its expression levels in both media (Fig. 6b). The correlation coefficients observed here were consistent with a previous study, where L. punctata strain 0202 was grown in Hoagland's medium under nitrogen-and phosphorus-deficient conditions for seven days 4 . The correlations between LeAPL2 and LeAPL3 expression and starch content were either intermediate or high under both conditions. LeAPS was found correlated to starch accumulation specifically under phosphorus starvation. In contrast, low or negative correlations were also found with LeAPL1 under nitrogen and phosphorus deprivation.

Conclusions
Long-term (35 days) physiological and molecular responses were demonstrated in L. punctata strain 5632 cultured in MS and Hoagland's media. During the early stage, the use of MS medium resulted in lower growth and higher starch content, compared to Hoagland's medium. The situation was reverse during the last 14 days of the experiment. Expression of AGPase subunit genes was differentially regulated in both media. Based on the correlation coefficients between starch content and gene expression, LeAPL2 and LeAPL3 were likely important for starch biosynthesis in both media, while LeAPS was more specific to MS medium. In contrast, low or negative correlation coefficients were associated with LeAPL1.

Methods
Plant materials and cultivation. L. punctata (G. Mey.) Les & D. J. Crawford was collected from a local pond in Bangkok, Thailand. To establish in vitro culture, fronds were surface-sterilized, using 10% (v/v) NaClO solution and a few drops of Tween-20 for two minutes with vigorous shaking. They were washed in sterilized distilled water three times. Surface-sterilized fronds were grown on Hoagland's solid medium (PhytoTechnology Laboratories, USA), containing 2% (w/v) sucrose and 0.7% (w/v) agar, pH 5.7. Growth conditions were 25 ± 2 °C and 16-hour-light/8-hour-dark photoperiods. Photosynthetically active radiation (PAR) flux was 65 µmol m −2 s −1 . Daughter fronds derived from a single mother frond were obtained. Morphological characteristics were examined. This duckweed strain was registered to the Rutgers Duckweed Stock Cooperative as strain 5632. where W t = fresh weight on day 7, 14, 21, 28 and 35 of cultivation; W t-1 = fresh weight of prior sample collection and time = the time interval (7 days). One-hundred milligrams of fresh samples collected on each day were used for the analysis of starch content with Total Starch Assay Kit (Megazyme, Ireland), according to the manufacturer's protocol. Water was used as the blank control, and 1 mg ml −1 D-glucose was used as the standard. The experiment was done in triplicate. Two-tailed student's t-test was used to determine statistically significant differences (P < 0.05) between fresh weight, relative growth rate, and starch content, in MS and Hoagland's media on each day.

Determination of growth and starch content.
Amplification and phylogenetic analysis of the atpF-atpH intergenic region. Fronds from axenic culture were used for DNA extraction with a FavorPrep Plant Genomic DNA Extraction Mini Kit (Favorgen, Taiwan), according to the manufacturer's protocol. Amplification and sequencing of the atpF-atpH intergenic region were done with the atpF-atpH forward (5′-ACTCGCACACACTCCCTTTCC-3′) and reverse (5′-GCTTTTATGGAAGCTTTAACAAT-3′) primers, according to the previous study 33 . The sequence was analysed with blastn. Multiple alignment of the sequence with those of other duckweed strains in the GenBank database used the CLUSTAL W program version 1.81 47 . Gaps were manually removed and adjusted. A phylogenetic tree was reconstructed, using unweighted pair group method with arithmetic mean (UPGMA), in MEGA 7.0 48 . Bootstrap analysis 49 with 1,000 re-samplings determined the confidence level of each clade.
Cloning and sequencing of LeAPS and LeAPL1 cDNA. Total RNA was extracted from 100 mg of fronds, using a FavorPrep Plant Total RNA Mini Kit (Favorgen, Taiwan), according to the manufacturer's protocol. Genomic DNA was removed with DNaseI (Promega, USA) by the on-column method, described in the protocol. cDNA of all samples was synthesized from 500 ng of total RNA, using iScript cDNA Synthesis Kit (Bio-Rad, USA). Oligo-dT supplied with the kit was used as the primer for the first-strand synthesis. CLeAPS-F (5′-ATGGCGGCGACGAGCTTC-3′) and CLeAPS-R (5′-TCATATGATGGTTCCGCTAGGG-3′) primers were used for amplification of LeAPS cDNA, while CLeAPL1-F (5′-ATGGCGCTGCGGATTGAG-3′) and CLeAPL1-R (5′-TCAGATGACAAGGCCATCCTT-3′) primers were used for LeAPL1 cDNA. The primer sequences and amplification cycles were according to the previous study 4 . Q5 High-Fidelity DNA polymerase (New England Biolabs, USA) was used for the amplification of full-length cDNA. The nucleotide sequences were compared with those of L. punctata strain 0202, using blastn. Multiple alignment analysis was performed between the deduced amino acid sequences of LeAPS and LeAPS and their homologs, using Clustal Omega (https://www.ebi.ac.uk/ Tools/msa/clustalo/). Gene expression analysis. Total RNA was extracted from 100 mg of strain 5632 fronds, collected at each time point. The extraction and cDNA synthesis followed the methods mentioned above. Quantitative reverse transcription PCR was done using SensiFast Real-Time PCR kit (Bioline, USA), on CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). New primers for quantification of LeAPS and LeAPL1 expression were designed. APSQ-F1 (5′-TCCCAGATTTCAGCTTCTATGATCGG-3′) and APSQ-R1 (5′ TGAATCTTGCAGTTCTTAATCACGC-3′) primers were for LeAPS, while APL1Q-F1 (5′-AGAACTCGAAGATCAGGAACTGC-3′) and APL1Q-R1 (5′-TCTTTCGGCTTCTTGGATTCCCTC-3′) primers were for LeAPL1. Previously described primers were used to quantify expression of LeAPL2 (LeAPL2-F/ LeAPL2-R) and LeAPL3 (LeAPL3-F/LeAPL3-R) 4 . Gene expression levels were determined according to a previously described method 28 . Briefly, five μl of cDNA of all samples were combined. A standard dilution series of 1x, 10 −1 X, 10 −2 X and 10 −3 X concentrations were made. The quantitation cycle (C q ) values of the standards and all samples were determined. Standard curves of standard cDNA concentrations and C q values were generated. Gene expression levels of all samples were determined by normalizing the sample C q values against the standard curves. The experiment was performed in triplicate. Two-tailed student's t-test was used to determine statistically significant differences (P < 0.05) between expression levels of each AGPase subunit genes in MS and Hoagland's media on each day. Correlation coefficients between the averages of starch content and expression levels were determined for each gene.