physiological and molecular responses of a newly …...2020/02/20 · light with adequate (200...

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Short title: Adaptation of Chlamydomonas to limiting B12

Physiological and Molecular Responses of a Newly Evolved

Auxotroph of Chlamydomonas to B12 Deprivation

Freddy Bunbury1, Katherine E Helliwell2, Payam Mehrshahi1, Matthew P Davey1, Deborah

Salmon3, Andre Holzer1, Nicholas Smirnoff3 and Alison G Smith1*

1Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK

2Marine Biological Association of the UK, Citadel Hill, Plymouth, UK

3School of Biosciences, University of Exeter, Exeter, UK

*Corresponding author

Email: [email protected]

Address: Department of Plant Sciences, Downing Street, Cambridge, CB2 3EA, UK

Tel: +44-1223 333952

Fax: +44-1223-333953

One-sentence summary: Study of an artificially evolved strain of the model alga

Chlamydomonas provides insight into how algal B12 auxotrophy has arisen naturally but also

reveals the challenges to becoming B12 dependent.

Author contributions F.B., P.M. and A.G.S designed the research; F.B., D.L.S. and A.H. performed the research;

N.S., D.L.S. and M.P.D. contributed new reagents or analytic tools; F.B., D.L.S. and A.H.

analysed the data; F.B., A.G.S., K.E.H. and P.M. wrote the paper with input from all authors.

Plant Physiology Preview. Published on February 20, 2020, as DOI:10.1104/pp.19.01375

Copyright 2020 by the American Society of Plant Biologists

www.plantphysiol.orgon October 19, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

mailto:[email protected]

http://www.plantphysiol.org

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Abstract

The corrinoid B12 is synthesised only by prokaryotes yet is widely required by eukaryotes as an

enzyme cofactor. Microalgae have evolved B12 dependence on multiple occasions and we

previously demonstrated that experimental evolution of the non-B12-requiring alga

Chlamydomonas reinhardtii in media supplemented with B12 generated a B12-dependent mutant

(hereafter metE7). This clone provides a unique opportunity to study the physiology of a nascent

B12 auxotroph. Our analyses demonstrate that B12 deprivation of metE7 disrupts C1 metabolism,

causes an accumulation of starch and triacylglycerides, and leads to a decrease in

photosynthetic pigments, proteins, and free amino acids. B12 deprivation also caused a

substantial increase in reactive oxygen species (ROS), which preceded rapid cell death.

Surprisingly, survival could be improved without compromising growth by simultaneously

depriving the cells of nitrogen, suggesting a type of cross protection. Significantly, we found

further improvements in survival under B12 limitation and an increase in B12use efficiency after

metE7 underwent a further period of experimental evolution, this time in coculture with a B12-

producing bacterium. Therefore, although an early B12-dependent alga would likely be poorly

adapted to coping with B12 deprivation, association with B12-producers can ensure long-term

survival whilst also providing a suitable environment to evolve mechanisms to better tolerate B12

limitation.

Keywords

Chlamydomonas reinhardtii, symbiosis, experimental evolution, vitamin B12, auxotrophy, algae



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Introduction

Over 50% of algal species require an exogenous source of B12 for growth (Croft et al.,

2005), yet large areas of the ocean are depleted of this vitamin (Caterina Panzeca et al., 2009;

Sanudo-Wilhelmy et al., 2012). Eukaryotic algae cannot synthesise B12, and must instead obtain

it from certain B12-producing prokaryotes (Croft et al., 2005). Indeed, whilst dissolved B12

concentrations are positively correlated with bacterioplankton density (Gobler et al., 2007; C.

Panzeca et al., 2008), they have been found to negatively correlate with phytoplankton

abundance (Ohwada, 1973; Sañudo-Wilhelmy et al., 2006). Furthermore, nutrient amendment

experiments suggest that B12 limits phytoplankton growth in many aquatic ecosystems (Bertrand

et al., 2007; Browning et al., 2017; Cohen et al., 2017). Despite this, understanding of the

physiological and metabolic adaptations that B12-dependent algae employ to cope with B12

deprivation is rather limited.

In many algae, B12 is required as a cofactor for the B12-dependent methionine synthase

enzyme (METH) (Helliwell et al., 2011), although some algae encode a B12-independent isoform

of this enzyme (METE) and thus do not require B12 for growth. Bertrand et al. (2012), showed

that the B12-dependent marine diatom Thalassiosira pseudonana, which possesses only METH,

responds to B12 scarcity by increasing uptake capacity and altering the expression of enzymes

involved in C1 metabolism. Heal et al. (2019) found that despite these responses, B12

deprivation disrupted the central methionine cycle, transulfuration pathway, and polyamine

biosynthesis. Phaeodactylum tricornutum, a marine diatom which uses but does not depend on

B12 (encoding both METE and METH), responds similarly to T. pseudonana (Bertrand et al.,

2012), but can also rely on increasing expression of METE to maintain the production of

methionine. Phylogenetic analysis of the METE gene among diatoms shows no simple pattern

of gene loss or gain, as indeed is the case across the eukaryotes (Ellis et al., 2017; Helliwell et

al., 2013), but there is a clear link between the lack of a functional copy of the METE gene and

B12-dependence (Helliwell et al., 2011; Helliwell, 2017).

As with the diatoms, the phylogenetic distribution of METE within the Volvocales (a

family of green freshwater algae) points to gene loss on several independent occasions. The

genomes of two volvocalean algae, V. carteri and G. pectorale, contain METE pseudogenes,

indicating that B12 dependence has evolved relatively recently in these species (Helliwell et al.,

2015). Chlamydomonas reinhardtii is a related alga that possesses a functional copy of METE

and so is B12-independent. Helliwell et al. (2015) generated a METE mutant of C. reinhardtii by

experimental evolution in conditions of high vitamin B12 concentration, demonstrating that

sustained levels of B12 in the environment can drive METE gene loss. This mutant, which



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contained a Gulliver-related transposable element in the 9th exon of the METE gene, was

completely reliant on B12 for growth, but in the presence of the vitamin it was able to outcompete

its B12-independent progenitor. In the absence of B12, the METE mutant would sometimes revert

to B12 independence and resume growth. Reversion was found to be due to excision of the

transposon to leave behind a wild-type METE gene sequence, but there was a single case

where a 9-bp fragment of the transposon was left behind resulting in a stable B12-dependent

strain, hereafter called metE7.

C. reinhardtii is a well-researched model organism that has been instrumental in

improving our understanding of algal photosynthesis, ciliogenesis, and responses to fluctuating

nutrient environments (Dubini et al., 2009; Grossman, 2000; Rochaix, 1995). We wanted to use

the metE7 mutant of C. reinhardtii to study how recently acquired B12 auxotrophy impacts an

organism’s fitness and physiology, and to provide insight into the metabolic challenges that

other B12-dependent algae might have faced when they first evolved. In this work, we

characterized the responses of metE7 to different vitamin B12 regimes and compared them to

the responses of its ancestral B12-independent strain as well as a closely related, naturally B12-

dependent alga Lobomonas rostrata. The responses of metE7 to B12 deprivation were quantified

by measuring changes in gene expression, cellular composition, photosynthetic activity, and

viability, and were contrasted against changes under nitrogen deprivation. To assess whether a

recently evolved algal B12 auxotroph could improve its survival during B12 deprivation relatively

quickly, we subjected metE7 to a further experimental evolution period of several months in

limited B12 or coculture with a B12-producing bacterium and characterised the resulting lines.

Results

B12 deprivation causes substantial changes to C1 metabolism in the metE7 mutant

Methionine synthase plays a central role in the C1 cycle (Fig. 1A), and thus facilitates

nucleotide synthesis and production of the universal methyl donor S-adenosylmethionine, which

is essential for many biosynthetic and epigenetic processes (Ducker & Rabinowitz, 2017; Lieber

& Packer, 2002). Wild-type (WT) C. reinhardtii can operate this cycle in the absence of B12 using

the methionine synthase variant METE, but metE7 relies solely on the B12-requiring METH

isoform. Although our main aim was to characterise the phenotype of a unique experimentally

evolved B12-dependent strain, metE7, we also wanted to confirm that the mutation in METE was

solely responsible for the B12 dependence of this strain. We therefore generated an independent

METE mutant line (metE4) using CRISPR/Cpf1 on a background strain suitable for genetic

manipulation (UVM4) (Ferenczi et al., 2017). This mutant has an in-frame stop codon resulting

in a truncated METE amino acid sequence and, as predicted, exhibits B12-dependence (Fig.



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S1). Suitably convinced of the role of METE in providing B12 independence, we decided to

return our focus to the experimentally evolved B12 dependent strain, metE7, which is perhaps

more reflective of B12-dependent algae that have arisen naturally.

Both the WT ancestral line and metE7 were precultured in TAP medium in continuous

light with adequate (200 ng·l-1) B12 to maintain a low cellular quota of the vitamin. The cells were

then pelleted, washed, and transferred to B12-replete (1000 ng·l-1) or B12-deprived (no B12) TAP

medium at 5×105 cells/ml and grown for 30 h. Steady-state transcript levels of six enzymes in

the C1 cycle were then investigated by RT-qPCR (Fig. 1B). In the WT, three transcripts (METE,

SAH1, and MTHFR) were significantly (p<0.05) upregulated by B12 deprivation, while in metE7

transcripts for all six enzymes (including METH, METM, and SHMT2) increased. Levels of the

methionine-cycle metabolites methionine, SAM, and SAH were quantified by HPLC-MS. In the

WT, there was no difference in methionine, SAM, or SAH levels between the two conditions

(Fig. 1C). However, in metE7 cells under B12 deprivation, methionine levels were raised 6-fold,

which was somewhat unexpected given that methionine synthase activity was impeded. SAH

levels were also significantly elevated, whereas there was no effect on SAM. Consequently, the

SAM:SAH ratio decreased by 10-fold to 3:1 under B12 deprivation. We then studied the

dynamics of these changes by measuring metabolites and RNA abundance at several points

during 3 days of B12 deprivation and then for 2 days following the addition of 1000 ng·l-1 B12. The

transcripts for all six tested C1-cycle genes increased rapidly in the first 6 h and then plateaued;

reintroduction of B12 led to an immediate reduction to near initial amounts (Fig. S2A). Similar

profiles were seen for the metabolites SAM and SAH, although the peak occurred later at 24 h

(Fig. S2B). Methionine levels were more variable, but nonetheless there was a similar trend of a

peak 24 h after removal of B12. More significantly, the SAM:SAH ratio fell sharply from 30 to less

than 1 within 24 h. A subsequent gradual increase occurred over the next 2 days, and resupply

of B12 increased this ratio further over the following 2 days. The likelihood therefore is that many

cellular processes would be impacted in B12-deprived metE7 cells.

B12 deprivation significantly impacts cell physiology and biochemical composition

Our data demonstrate a substantial impact of B12 limitation on the expression of C1

metabolic genes as well as the abundance of C1 metabolites. To elucidate downstream

consequences of perturbed C1 metabolism, we also characterised broader physiological

responses to B12 deprivation. As has been documented previously (Helliwell et al., 2015),

growth of metE7 cells was significantly impaired in B12-deprived conditions (Fig. S3A). However,

by day 2 the B12-deprived cells had a 36% larger diameter resulting in a 150% increase in

volume (Fig. 2A and Fig. S3B), indicating that cell division was more restricted than overall



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growth. Moreover, cell viability, which was assayed by the ability of cells to form colonies when

plated on B12-replete TAP agar, decreased to below 25% within 4 days of B12 limitation (Fig.

S3C). This was preceded by a reduction in photosystem II maximum efficiency (Fv/Fm) (Fig.

S3D), an often-used indicator of algal stress (Parkhill et al., 2001; White et al., 2011).

The biochemical composition of C. reinhardtii cells is altered considerably and similarly

under various nutrient deprivations and so we hypothesised that B12 limitation would also induce

broadly the same responses (Grossman, 2000; Juergens et al., 2016; S. Saroussi et al., 2017).

Therefore, metE7 cells were precultured as before in 200 ng·l-1 B12, then washed and

resuspended in TAP with or without B12 (1000 ng·l–1) and cultured mixotrophically for 4 days.

Cultures were visually inspected by microscopy (Fig. 2A) and the amounts of various cellular

components were measured on day 2 and 4 (Fig. 2B and Fig. S4). Chlorophyll levels declined

considerably under B12 deprivation so that by day four the cells had a bleached appearance with

an 85% lower concentration than the B12-replete cells. Similarly, free fatty acids (FFA), polar

lipids, and proteins were at least 50% lower under B12-deprivation conditions on day 4. Starch

content on the other hand, showed the largest absolute increase from B12-replete to B12-

deprived cells (Fig. S4), and triacylglycerides (TAG) were 10-fold higher in B12-deprived cells

(Fig. 2B), which effectively balanced the loss of polar lipids and free fatty acids so that overall

lipid levels were roughly 8–10% of dry mass in both treatments. To look in more detail,

quantification of free amino acids and fatty acid composition of all lipid classes was carried out

(Fig. S5). By day 4, most of the amino acids decreased significantly under B12 deprivation.

Particularly noteworthy are the reduction in methionine, in contrast to its elevation at an earlier

timepoint, and the increase in glutamine, the only amino acid that is more abundant in B12-

deprived cells (Fig. S5A). Overall the degree of fatty acid saturation was higher under B12

deprivation, due mainly to an increase in the dominant saturated fatty acids palmitate (16:0) and

stearate (18:0) (Fig. S5B), although levels of several unsaturated fatty acids, in particular 16:2,

16:3(7,10,13), 18:1, and 18:2, were also elevated.

Responses to nitrogen deprivation improve survival under B12 deprivation

Our results demonstrate that B12 deprivation of metE7 causes several changes in

biochemical composition, including accumulation of TAG and starch and decreases in

chlorophyll and protein, akin to those exhibited following nitrogen deprivation of WT C.

reinhardtii (Cakmak et al., 2012; Park et al., 2015a; Yang et al., 2015). To further investigate this

comparison we measured growth, viability, and photosynthetic efficiency under both conditions

over a timecourse (Fig. S6). metE7 culture density increased more under B12 than nitrogen

deprivation (Fig. S6A), but started to decline after day 2, unlike under nitrogen deprivation



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where growth continued more slowly over 4 days. For cell viability, both conditions caused a

decline, but while loss of viability continued in B12-deprived cells, under nitrogen deprivation the

initial loss was followed by recovery (Fig. S6B). Maximum photosynthetic efficiency of

photosystem II, however, did not recover under either condition, and its decline was more rapid

in nitrogen-deprived cells (Fig. S6C).

The increased viability of metE7 under nitrogen deprivation compared to under B12

deprivation suggested that either the metabolic role of B12 would make it intrinsically more

difficult to cope without or that the evolutionary naivety of metE7 to B12 dependence would mean

it had little time to evolve protective responses to B12 limitation. We therefore tested whether

responses to nitrogen deprivation could afford some protection against B12 deprivation. Viability

measurements were monitored over several days, and cultures lacking either nitrogen or B12

behaved as previously (Fig. 3A). However, metE7 cells deprived of both nitrogen and B12

simultaneously were more like those starved of nitrogen, with an initial decrease in viability

followed by recovery to a level significantly higher than in B12 deprivation alone. As total growth

under B12- and nitrogen-deprivation conditions was not significantly different from that under B12

deprivation alone (Fig. S7), this apparent protective mechanism in response to nitrogen

deprivation is not simply a result of inhibiting growth and hence avoiding severe B12 starvation.

Instead, it seems likely that nitrogen deprivation would have elicited photoprotective responses,

such as increasing non-photochemical quenching in order to avoid the accumulation of

damaging reactive oxygen species (ROS) (Erickson et al., 2015; S. Saroussi et al., 2017, 2019;

S. I. Saroussi et al., 2016), initiated the quiescence cycle to mitigate the genomic damage

caused by ROS (Takeuchi & Benning, 2019), or activated the gametic survival program (Martin

& Goodenough, 1975).

To investigate whether the cell death observed under B12 deprivation of metE7 could be

due to ROS, the general ROS-sensitive dye dihydrodichlorofluorescein diacetate was incubated

with cells at different timepoints during nutrient deprivation. We found that ROS levels increased

under all nutrient-deprived conditions in the first two days but were highest in those cells

deprived of B12 alone (Fig. 3B). This peak coincided with the start of the substantial decline in

cell viability (Fig. 3A). The combination of B12 and nitrogen deprivation reduced ROS levels to

similar amounts to those seen in the nitrogen-deprived cells, and so may be a factor behind

reduced cell death.

Natural B12 auxotroph Lobomonas rostrata fares better under B12-limiting conditions than

metE7



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Considering that metE7 quickly lost viability in the absence of B12 while nitrogen

starvation invoked protective responses independent of B12 status, it is possible that as a novel

auxotroph, the response of metE7 to B12 deprivation is simply underdeveloped. To test this, we

compared the B12 physiology of metE7 with Lobomonas rostrata, a naturally B12-dependent

member of the same Volvocaceae family of chlorophyte algae (Provasoli, 1958; Sausen et al.,

2018). Cell viability was significantly greater in L. rostrata cells compared to the metE7 line after

2–4 days of B12 deprivation despite also growing to a greater density (Fig. S8). Moreover, a B12

dose-response experiment, in which the two species were each cultured mixotrophically in a

range of B12 concentrations, revealed that L. rostrata reached a higher optical density than

metE7 at all B12 concentrations below 90 ng·l-1, while the inverse was true above 90 ng·l-1 (Fig.

4A). This indicates that L. rostrata has a lower B12 requirement than metE7.

In the natural environment the ultimate source of B12 is from prokaryotes since they are

the only known B12 producers (Warren et al., 2002). In separate studies it was shown that B12-

dependent growth of L. rostrata and metE7 can be supported by the B12-synthesising bacterium

Mesorhizobium loti (Helliwell et al., 2015; Kazamia et al., 2012). We therefore directly compared

the growth of metE7 and L. rostrata in B12-supplemented (100 ng·l-1) axenic culture and in

coculture with M. loti in media lacking a carbon source (TP) (Fig. 4A). Even though metE7 grew

much more quickly and to a higher density than L. rostrata under axenic, B12-supplemented

conditions, it grew less well in coculture with M. loti (Fig. 4B), indicating that B12 provision from

the bacterium is less effective at supporting the growth of metE7 than of L. rostrata, perhaps

simply due to their different B12 requirements, but possibly due to more sophisticated symbiotic

interactions.

Experimental evolution in coculture improves B12 use efficiency and resilience to B12

deprivation

Together, our data suggest that the newly evolved metE7 line is poorly adapted to coping

with B12 deprivation, but we wanted to determine whether the metE7 line could evolve improved

tolerance to B12 limiting conditions, so we employed an experimental evolution approach (Fig.

S9). We designed three distinct conditions, referred to as H, L, and C. Condition H (TAP

medium with high (1000 ng·l-1) B12) was a continuation of the conditions that had initially

generated metE7 (Helliwell et al., 2015). Condition L (TAP medium with low (25 ng·l-1) B12) was

chosen so that B12 would limit growth. Condition C (coculture with M. loti in TP medium) was a

simplification of an environmental microbial community. Eight independent cultures for each

condition were established from a single colony and then subcultured once per week over a

total period of 10 months. To account for the different growth rates in the three conditions, we



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applied the dilution rates of 10,000, 100, and 5 times per week in condition H, L, and C

respectively (Fig. S9). After 10 months under selective conditions, all 24 cultures had survived

and were then treated with antibiotics to remove the M. loti from condition C and to ensure that

there were no other contaminating bacteria. We then subcultured all 24 evolved lines alongside

eight replicates of the progenitor strain (which had been maintained on TP agar with 1000 ng·l-1

B12 without subculturing) in mixotrophic conditions with TAP + 200 ng·l-1 B12 three times over

nine days to ensure they were all acclimated to the same conditions. The behaviours of the

algal populations, hereafter referred to as metE7, metE7H, metE7L, and metE7C, were then

compared with one another for their responses to different B12 concentrations, B12 deprivation,

and growth in coculture with the B12-producer M. loti.

Under high levels of B12 (>320 ng·l-1), a similar carrying capacity was reached by the

progenitor metE7 strain and the metE7H and metE7C populations, whereas metE7L density

was significantly lower than the progenitor (Fig. S10A). When grown across a range of B12

concentrations to determine a dose response, the metE7C populations reached a significantly

higher optical density at the lower concentrations of 20 and 40 ng·l-1 B12 than the other lines

(Fig. 5A). The concentration of B12 required to produce half the maximum growth (EC50) of

metE7C was therefore much lower than the progenitor metE7 or metE7H (Fig. S10B) and this

was reflected in the higher B12 use efficiency i.e. the maximal increase in yield (OD730) that

results from an increase in B12 concentration (Fig. 5B). However, the maximal growth rate of

metE7C was significantly lower (Fig. S10C), and it is tempting to conclude that this is a

necessary trade-off. We also compared the viability of the experimentally evolved lines during

B12 deprivation (Fig. 5C). Fig. 5C shows that although all lines lost viability during B12

deprivation, metE7L and metE7C survived substantially better, with a median survival time more

than a day longer (Fig. 5D) than both the progenitor metE7 and metE7H. Finally, we compared

the growth of the evolved lines in coculture with M. loti, which showed, perhaps unsurprisingly,

that the metE7C lines grew better than the others (Fig. 5E), and at the end of the growth period

had a significantly higher number of algae supported per bacterium (Fig. 5F).

To elucidate which factors contributed to improved survival during B12 deprivation, we

performed a multi-variable physiological analysis (Fig. 6). Sixteen variables or parameters were

measured across the 32 metE7 populations and the dataset (Supplemental dataset S1) was

visualised in two ways. Fig. 6A displays the first two components of a principal component

analysis of the data, which confirmed that the experimental evolution populations tended

towards forming separate clusters, with the metE7C populations most diverged from the

progenitor metE7, due mainly to higher B12 use efficiency and median survival time, but lower

maximal growth rate, carrying capacity, and EC50 for B12. Fig. 6B is a correlation matrix of the



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parameters, which reveals those pairs that are most positively or negatively correlated with one

another, for example, median survival time was quite highly negatively correlated with Maximal

growth rate and positively correlated with B12 use efficiency.

A more definitive statistical approach was then used to determine the most important

measurements for predicting survival time during B12 deprivation: using stepwise minimisation of

the Bayesian information criterion of the full additive linear model, the 15 other measurements

(all 16 minus median survival time) were reduced to just three. These three measurements,

higher B12 use efficiency, lower ROS levels, and lower maximal growth rate, can therefore be

considered sufficient to explain longer survival time under B12 deprivation of the metE7

populations. Using the same method, we also investigated which values best predicted growth

in coculture with M. loti, using algae:bacteria ratio as a proxy for this. We found that

algae:bacteria ratio was also optimally predicted by just three measurements: higher algal B12

use efficiency and lower algal maximal growth rate, as for survival time, but also lower algal B12

uptake capacity. Together these results indicate that experimental evolution in coculture not

only improves growth in coculture but also increases B12 use efficiency and survival during B12

deprivation.

Discussion

In this study, we exploited a novel model system for the evolution of vitamin B12

dependence by analysing the physiological and metabolic responses to B12 deprivation of an

artificially evolved B12-dependent mutant of C. reinhardtii. Our analyses demonstrate that B12

deprivation has important consequences for C1 metabolism: we observed a significant increase

in the transcript abundance of C1-cycle enzymes in both the WT and metE7 strain, and a

decrease in the methylation index (SAM:SAH ratio) in metE7 only. Moreover, B12 deprivation of

metE7 causes a decrease in chlorophyll, protein, and amino acids, and an increase in starch,

lipids, and saturated fatty acids, characteristic of limitation responses to macronutrients such as

nitrogen (Cakmak et al., 2012; Juergens et al., 2016; Park et al., 2015b; Yang et al., 2015). The

rapid loss of viability seen under B12 deprivation could be averted if the metE7 cells were also

limited for nitrogen, suggesting that it is not the lack of B12 per se that causes cell death, but an

inability to respond appropriately. Together this suggests a newly evolved B12 auxotroph would

be poorly adapted to surviving in the natural environment where a B12 supply is not guaranteed.

However, we found that metE7 can be supported for several months by a B12-producing

bacterium, and experimental evolution under these conditions caused improved B12 use

efficiency and resilience to B12 deprivation.



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B12 deprivation of metE7 decreased the SAM:SAH ratio 10-fold, mainly due to an

accumulation of SAH, reflecting a recent B12 deprivation study of the diatom T. pseudonana

(Heal et al., 2019), numerous observational studies of B12 deficiency in humans (Guerra-

Shinohara et al., 2004; Stabler et al., 2006), and studies of disrupted C1 metabolism in A.

thaliana (Meï et al., 2016). As SAH is a competitive inhibitor of methyltransferases (Chiang et

al., 1996), this decrease would likely lead to general hypomethylation in metE7. The epigenetic

marks methyldeoxyadenosine and methylcytosine are similarly abundant in C. reinhardtii and

appear to mark active genes and repeat-rich regions respectively, so the consequences of

hypomethylation are unclear (Fu et al., 2015; Lopez et al., 2015). The reduced abundance of

B12-bound METH under B12 deprivation would hinder methionine synthesis and could cause the

observed reduction in protein abundance (Fig. 2B). However, methionine levels increased

between 12 and 24 h of B12 deprivation (Fig. S2B), suggesting a reduction in its use, proteolysis,

or increased synthesis due to higher METH expression or via alternative pathways such as the

S-methylmethionine cycle, which is known to play an important regulatory role in plants

(Ranocha et al., 2001).

METE transcript abundance showed a much higher dynamic range than METH during B12

deprivation and reintroduction (Fig. S2A), which is reflected by the higher diurnal range of METE

observed in global transcriptomics and proteomics datasets (Strenkert et al., 2019). However,

on average METE is approximately 60-fold more abundant than METH in C. reinhardtii

(Strenkert et al., 2019). This may be due to a lower maximal catalytic rate of METE, as has

been observed in E. coli (Gonzalez et al., 1992), or due to its role in the flagella, which contain

METE but not METH (Schneider et al., 2008). Under B12-deprivation conditions, the activity of

METH would be compromised, yet in both metE7 and the ancestral strains, it was upregulated.

This is more similar to the B12-dependent algae T. pseudonana and Tisochrysis lutea, which

also upregulate METH under B12 deprivation (Bertrand et al., 2012; Nef et al., 2019), than the

B12-independent P. tricornutum, which decreases METH expression (Bertrand et al., 2013).

However, in both T. pseudonana and P. tricornutum, B12 deprivation substantially upregulates

C1-cycle enzymes including homologs of METM, MTHFR, and SAH1 (Bertrand et al., 2012),

reflecting our findings and those of Helliwell et al. (2014). Under sulphur- and nitrogen-

deprivation conditions, these C1-cycle genes are downregulated (González-Ballester et al.,

2010; Schmollinger et al., 2014), suggesting that their upregulation during B12 deprivation is not

a general response to nutrient stress, but a nutrient-specific one, as indeed is the case for T.

lutea (Nef et al., 2019).

Chlorosis is a common symptom of nutrient deficiency in C. reinhardtii, evident in

nitrogen-, sulphur-, iron-, and zinc-limiting conditions, and so it is not surprising that B12



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deprivation of metE7 caused a substantial decline in total chlorophyll (Fig. 2B) (Juergens et al.,

2015; Kropat et al., 2011; Schmollinger et al., 2014). The decrease in total protein content

occurred more slowly and was less substantial (50% reduction over four days) than that

reported under nitrogen and sulphur deprivation (80% reduction within one day) (Cakmak et al.,

2012). During nitrogen and iron starvation in C. reinhardtii, membrane lipids decrease drastically

concomitant with the increase in TAGs (Siaut et al., 2011; Urzica et al., 2013). This is very much

like what we observed for metE7 under B12 deprivation, although here the level of free fatty

acids and polar lipids decreased by a roughly similar amount to the increase in TAGs indicating

there is little, if any, de novo fatty acid synthesis. In addition, B12 deprivation causes similar shifts

in fatty acid composition to nitrogen and iron deprivation, most notably a substantial increase in

palmitic acid (16:0) and decrease in polyunsaturated 16:4 fatty acid (Msanne et al., 2012; Urzica

et al., 2013). Despite these similarities, B12 deprivation may elicit an increase in TAGs by a

different pathway due to disrupted C1 metabolism, as has been observed in several organisms

(da Silva et al., 2014; Meï et al., 2016; Visram et al., 2018). This is thought to be due to a

reduction in the methylation potential limiting membrane lipid synthesis and hence diverting

more lipids towards TAGs (Malanovic et al., 2008; Visram et al., 2018). Therefore, B12

deprivation could provide a complementary approach to other nutrient deprivation experiments

in improving our understanding of lipid metabolism in C. reinhardtii and other algae.

From an evolutionary perspective, the prevalence of vitamin B12 dependence among

algae appears somewhat at odds with the severe fitness penalties that would be incurred were

they exposed to limiting dissolved B12 concentrations. This is made more surprising by the fact

that the fitness benefit of B12 dependence under laboratory conditions in replete B12, although

statistically significant, appears to be minimal (Helliwell et al., 2015). However, relative to

optimal axenic laboratory conditions in which the metE7 line evolved, in the environment

multiple nutrients may colimit growth, perhaps even eliciting responses that mitigate against B12

deprivation, as we observed here, and B12-producing bacteria may not simply co-occur with

algae but also actively engage in mutualistic interactions (Cooper et al., 2019; Croft et al., 2005;

Kazamia et al., 2012, 2016). Furthermore, our evidence suggests that selection under coculture

conditions led to the newly evolved B12 auxotroph developing increased B12 use efficiency and

becoming better adapted to tolerating B12 limitation, which could make this line more robust to

the unreliable B12 supply in the natural environment. However, these improvements appeared to

come at the expense of maximal growth rate in B12-replete conditions (Fig. S10C), which is not

unexpected in light of previous experimental evolution studies in C. reinhardtii (Collins & Bell,

2004). As one of the conserved responses of C. reinhardtii upon detecting depletion of various

nutrients is to decrease cell division, it is possible that slower growth might even be selected for

under B12 deprivation. Indeed, a low growth rate was found to be a significant predictor of



13

greater survival time under B12 deprivation, alongside low ROS levels and high B12 use

efficiency.

The fact that metE7 survived a 10-month period either with limited artificial

supplementation of B12 or by relying completely on bacterial B12 provision does suggest that

even a newly evolved and poorly adapted B12 auxotroph would have ample opportunity to adapt

further. What adaptations are likely to improve growth and survival under B12 deprivation are not

altogether clear, but it is not unreasonable to assume that exaptation of existing nutrient

limitation responses would play a major role. B12 dependence is certainly a risky evolutionary

strategy, and one which may have ended in extinction countless times, but our work suggests

that even the simplest of symbioses with B12-producing bacteria may be sufficient to ensure the

survival and drive the continued evolution of B12-dependent algae.

Materials and Methods

Strains

Mesorhizobium loti (MAFF 303099) was a gift from Prof. Allan Downie at the John Innes

Centre, Norwich, UK. Algal strains used in this study are shown in Table S1 and include

Lobomonas rostrata (SAG 45/2), as well as several Chlamydomonas reinhardtii strains derived

from strain 12 of wild type 137c or the cell wall-deficient strain cw15. The stable B12-dependent

metE7, the unstable B12-dependent (S-type) as well as the B12-independent revertant line (R-

type) all evolved from the strain 12 of wild type 137c (Ancestral) as described by Helliwell et al.

(2015). Another B12-dependent mutant (metE4) was generated by targeted (CRISPR/Cpf1)

knockout of the METE gene in the UVM4 strain using the protocol described in Ferenczi et al.

(2017 ; (21). The guide RNA and single- stranded donor oligonucleotide used for homology-

directed repair are given in Table S2, alongside sequencing primers used to confirm that the

modification resulted in a premature stop codon producing an 89 amino- acid protein rather than

the full length sequence of 815 amino acids sequence.

Culture conditions and growth measurements

Algal colonies were maintained quarterly on Tris-acetate phosphate (TAP) (Table S5) +

1000 ng·l-1 cyanocobalamin (B12) agar (1.5% w/v) in sealed transparent plastic tubes at room

temperature and ambient light. Cultures were grown in TAP or Tris min medium under

continuous light or a light-dark period of 16- hr light: - 8-hr dark cycle, at 100 µE·m-2·s-1, at a

temperature of 25⁰°C, with rotational shaking at 120 rpm in an incubator (InforsHTMultitron;



14

Switzerland). For nutrient starvation experiments the pre-culture TAP medium contained 200

ng·l-1 of B12, and when cell densities surpassed 106 cells·ml-1 or an OD730 nm of 0.2, cultures

were centrifuged at 2,000 g for 2 minutes, followed by supernatant removal and resuspension of

the cell pellet in media. For nitrogen deprivation, ammonium chloride was omitted from the

media with no replacement.

Algal cell density and optical density at 730 nm were measured using a Z2 particle count

analyser (Beckman Coulter Ltd.) with limits of 2.974–-9.001 µm, and a FluoStar Optima (BMG

labtech) or Thermo Spectronic UV1 spectrophotometer (ThermoFisher), respectively. Mean cell

diameter was also quantified on a Z2 particle analyser (Beckman Coulter Ltd.). Dry mass was

measured by filtering 20 ml of culture through pre-dried and weighed grade- 5 WhatmannTM filter

paper (Sigma-Aldrich WHA1005090), drying at 70°⁰C for 24 hoursh, followed by further

weighing on a Secura mass balance (Sartorius). Algal and bacterial viable cell density were

determined by plating serial dilutions on solid media and counting colonies to calculate colony-

forming units per ml (CFU·ml-1).

Measurement of photosynthetic parameters

200 µl of cultures with an OD730 nm>0.1 were transferred to a 96- well plate which was

then incubated at 25°°C in the dark for 20 minutes. F0 was measured prior to, and Fm during, a

saturating pulse at 6172 µE·m-2·s-1. The light intensity was increased to 100 µE·m-2·s-1 and the

cells allowed to acclimate for 30 seconds prior to another set of fluorescence measurements

before and during a saturating pulse. From these fluorescence measurements, the CF imager

software calculated non-photochemical quenching (Fm/Fm’-1), PSII maximum efficiency

(Fv’/Fm’), and the coefficient of photochemical quenching (Fq’/Fv’).

Measurement of cellular biochemical composition

Lipids were extracted from the cell pellet from 10 ml of culture using the

chloroform/methanol/water method, and triacylglycerides (TAGs), polar lipids and free fatty

acids in the total lipid extract and total fatty acid methyl esters (FAMEs) were analysed by GC-

FID and GC-MS, as described in Supplemental Materials and Methods supplemental file 1 and

Davey et al. (2014) (22). A 1- ml aliquot of algal culture was used for pigment and starch

quantification as described in Davey et al. (2014), and a 10 -ml aliquot for protein quantification

using a Bradford assay and amino acids by HPLC as described in Supplemental Materials and

Methods supplemental file 1 and Helliwell et al. (2018) (23).



15

Reactive oxygen species quantification

2 µl of 1 mM 2’,7’ Dichlorofluorescein diacetate (Sigma-Aldrich) dissolved in DMSO was

added to 198 µl of cell culture in a black f-bottom 96 -well plate (Greiner bio-one) and incubated

at room temperature in the dark for 60 minutes before recording fluorescence at 520 nm after

excitation at 485 nm in a FluoStar Optima Spectrophotometer (BMG labtech). Fresh cell culture

media devoid of any cells was used as a blank.

Vitamin B12 quantification

A 1- ml aliquot of the culture to be tested was boiled for 5 minutes to release B12 into

solution and then the growth response of a B12-dependent strain of Salmonella typhimurium

(AR3612) incubated for 16 hours h at 37°C in 50% (v/v) 2*M9 media + 50% (v/v) boiled extract

was quantified by measuring optical density at 600 nm. B12 concentration was calculated by

comparing OD600 nm to a standard curve of known B12 concentrations using a fitted logistic

model. To calculate B12 uptake by algal cells, B12 was added to an aliquot of algal culture to a

concentration of 1000 ng·l-1, followed by incubation for 1 hour h under previously described

growth conditions, then measuring the B12 remaining in the media (supernatant of the

centrifuged aliquot). B12 uptake (Initial B12 – remaining B12) was divided by OD730 nm of the

aliquot to give B12 uptake capacity in ng·l-1·OD730 nm-1.

SAM and SAH quantification

10 ml of samples were centrifuged at 2,000 g for 2 minutes, supernatant removed, and

cell pellet lyophilised at <-40⁰C and <10 pascals for 12–-24 h. 300 µl of 10% methanol (v/v) (LC-

MS grade) spiked with stable isotope-labelled amino acids (L-amino acid mix, Sigma-Aldrich,

Co., St. Louis, MO, USA) was added to each sample. They were vortexed 3 times, every 10

min, before sonicating for 15 min in an iced water bath then centrifuging (16,100 x g) for 15 min

at 4°oC. Quantitative analysis was performed on 150 µl of supernatant using an Agilent 6420B

triple quadrupole (QQQ) mass spectrometer (Agilent Technologies, Palo Alto, USA) coupled to

a 1200 series Rapid Resolution HPLC system. Details of the HPLC-MS are given in

supplemental file 1 Supplemental Materials and Methods and Table S3.

Transcript quantification



16

Total RNA extraction was performed on the cell pellet from 10 ml of algal culture using

the RNeasy® Plant Mini Kit (QIAGEN). DNase treatment was carried out using TURBO DNA-

free™ kit (Ambion), and cDNA synthesis using SuperScript®III First-Strand synthesis system for

RT-PCR (Invitrogen) according to the manufacturer’s instructions. RT-qPCR was performed as

described in supplemental file 1Supplemental Materials and Methods and Helliwell et al. (2018;

(24), using primers listed in Table S4.

Artificial Evolution setup

A culture of metE7 cells was plated on TAP + 1000 ng·l-1 B12 agar, then 8 colonies

picked and resuspended in TAP + 200 ng·l-1 B12 in a 96- well plate. Each well was split into 3

wells, each in a different 96- well plate containing 200 µl of a different media: TAP +1000 ng·l-1

B12, TAP +25 ng·l-1 B12, and TP medium. M. loti was prepared in a similar manner to metE7,

except preculturing was performed in TP + 0.01% glycerol (w/v). M. loti was added to the TP

culture containing metE7 at a density roughly 20 times greater than the alga. The 96 -well plates

were incubated at 25°C, under continuous light at 100 µE·m-2·s-1, on a shaking platform at 120

rpm. Each week the cultures were diluted: tThose in TAP +1000 ng·l-1 B12 were diluted 10,000-

fold, TAP +25 ng·l-1 B12 = 100-fold, and TP = 5-fold. Every three weeks, 10 µl of serial dilutions

of each culture was also spotted onto TAP agar + Ampicillin (50 µg·ml-1) and Kasugamycin (75

µg·ml-1) and TAP agar + 1000 ng·l-1 B12 to check for B12-independent C. reinhardtii, or bacterial

contaminants and to act as a reserve in the case of contamination. If cultures were found to be

contaminated, then at the next transfer they were replaced by colonies from the same well that

had grown on the TAP agar plates. At four points during the 12-month evolution period, all

cultures were transferred to TAP agar plates where they were stored for 2 weeks during an

absence from the lab, meaning that the total time in liquid culture was 10 months. See Fig. S9

for an illustration of the experimental evolution setup and the tests of B12 dose- response viability

during B12 deprivation and growth in coculture with the B12- producer M. loti that were performed

on all the evolved lines.

Accession Numbers

Names and gene IDs of genes referred to in the text are given in Supplemental Table S4.

Supplemental Data

Supplemental Figure S1. Testing the B12 requirement of several C. reinhardtii strains.



17

Supplemental Figure S2. C1 metabolism enzyme transcript and metabolite abundances in

metE7 during B12 deprivation and resupply.

Supplemental Figure S3. Characteristics of metE7 cells cultured in B12-replete and B12-

deprived conditions.

Supplemental Figure S4. Composition of metE7 cells cultured in B12-replete and B12-deprived

conditions.

Supplemental Figure S5. Amino acid and fatty acid composition of metE7 cells in B12-replete

and -deprived conditions.

Supplemental Figure S6. Growth and survival of metE7 under nitrogen- and B12-deprivation

conditions.

Supplemental Figure S7. Growth and survival of metE7 under a combination of nitrogen- and

B12-deprivation conditions.

Supplemental Figure S8. Growth and survival of L. rostrata and metE7 under B12-deprivation

conditions.

Supplemental Figure S9. Diagram for the experimental evolution setup and the experiments

used to analyse the evolved lines.

Supplemental Figure S10. Growth parameters of the metE7 progenitor strain, and its

experimental evolution descendants.

Supplemental Table S1. Information about C. reinhardtii strains used in this study.

Supplemental Table S2. CRISPR/Cpf1 guide RNAs and ssDNA repair templates

Supplemental Table S3. Optimised values for Mass-spectroscopic analysis of methionine cycle

metabolites MRM - Positive Polarity.

Supplemental Table S4. RT-qPCR primer sequences.

.

Supplemental Table S5. TAP medium composition.

Supplemental Dataset S1.

Supplemental Materials and Methods.

Acknowledgements

This work was supported by the BBSRC Doctoral Training Partnership BB/M011194/1 (FB,

AGS), BBSRC BB/M018180/1 (PM, AGS), BB/I013164/1 (KEH & AGS), Leverhulme Trust RPG

2017-077 (MPD, AGS), Natural Environment Research Council NE/R015449/1 (KEH), and

Gates Cambridge Trust PhD scholarship (AH).

Competing interests

The authors declare no competing interests.



18

Figure legends

Figure 1. C1 cycle metabolites and transcripts increase during B12 deprivation of metE7.

(A) Metabolic map of a portion of the C1 cycle centred around METE and METH, with enzyme

abbreviations in black, metabolite abbreviations in grey, and arrows depicting enzyme-catalysed

reactions. (B) Abundances of six transcripts for enzymes of the C1 cycle measured by RT-

qPCR on RNA extracted from the ancestral line and metE7 after 30 h of incubation in

mixotrophic conditions with (1000 ng·l-1) or without B12. (C) Abundances of Met, SAM, and SAH

metabolites measured by HPLC-MS on the same samples as above. Metabolite and transcript

abundances are expressed as levels in B12-deprived conditions relative to B12-replete conditions

and presented on a log2() scale. Error bars = SD, n=3–4, ‘ns’=not significant, *=p<0.05,

**=p<0.01, ***=p<0.001, Welch’s t-test. WT = ancestral B12-independent strain, metE7 =

experimentally evolved B12-dependent line. See also Fig. S2.

Figure 2. B12 deprivation of metE7 causes cell enlargement and significant changes in

biochemical composition. (A) Micrographs taken at 1000x magnification (with scale bars

showing 10 µm) of metE7 cells grown in TAP medium with (1000 ng·l-1) or without B12 over a

period of 4 days (B) Biochemical composition of B12-deprived cells on day 2 and day 4 of the

growth period expressed as mass of those compounds normalised to total cell dry mass and

then expressed relative to the amounts in B12 replete conditions. Error bars = SD, n = 5.

**=p<0.01, ***=p<0.001, Welch’s t-test. See also Fig. S3, Fig. S4, and Fig. S5.

Figure 3. metE7 survives better and produces lower levels of reactive oxygen species

(ROS) when limited for both N and B12 than just B12 alone. (A) Percentage of cells that could

form colonies (a measure of viability) on nutrient replete agar when removed at different

timepoints from various nutrient deprivation conditions (Indicated in panels above the graphs as

follows: +B12-N: 1000 ng·l-1 B12 and 0 mM NH4Cl, -B12+N: 0 ng·l-1 B12 and 7.5 mM NH4Cl,

or -B12-N: 0 ng·l-1 B12 and 0 mM NH4Cl). (B) ROS measured by dichlorofluorescein diacetate

(DCFDA) fluorescence and normalised both on a per cell basis and to the nutrient replete

treatment (+B12+N). Error bars = SD, n = 3–6. See also Fig. S6 and Fig. S7.



19

Figure 4. L. rostrata grows better than metE7 in coculture with a B12-producing

bacterium, in part due to its lower demand for B12. (A) Precultures of the algae, grown with

200 ng·l-1 B12, were washed and inoculated at roughly 100 cells·ml-1 then grown mixotrophically

(TAP medium in continuous light), with B12 concentrations from 0 to 200 ng·l-1. Optical density at

730 nm was measured after 5 and 9 days of growth for metE7 and L. rostrata, respectively. (B)

Cultures were grown photoautotrophically (TP media in 16-h light (100 µE·m-2·s-1):8-h dark

cycles) in axenic culture (with 100 ng·l-1 B12) or coculture (with the B12-producing bacterium M.

loti) over a period of 16 days with cell density measurements performed every 1–2 days. For

both panel A and B, black = L. rostrata, grey = metE7, error bars = SD, n=4. See also Fig. S8

Figure 5. Experimental coevolution of metE7 with the bacterium M. loti selects for

improved algal growth in coculture, increased B12 use efficiency, and better resilience to

B12 deprivation. (A) Maximum optical density achieved by mixotrophically-grown cultures of

experimentally evolved lines of metE7 grown over a period of 12 days in six different

concentrations of B12. (B) B12 use efficiency of evolved lines calculated using a fitted Monod

equation and expressed as the maximum rate of increase in OD730 that would result from an

increase in B12 concentration. (C) Viability (measured as the percentage of cells capable of

forming colonies on B12-replete agar) of mixotrophically-grown cultures of experimentally

evolved lines of metE7 over a 5-day period cultured in 40 ng· l-1 B12. (D) Median survival time of

evolved lines after dilution of culture to 40 ng· l-1 B12 calculated using a fitted Verhulst equation.

(E) Algal cell density of photoautotrophically grown cocultures of experimentally evolved lines of

metE7 with M. loti over a 9-day period. (F) Ratio of algae to bacteria on the final day (day 9) of

growth in coculture. metE7H = metE7 evolved in TAP + 1,000 ng· l-1 B12 for 10 months; metE7L

= metE7 evolved in TAP media + 25 ng· l-1 B12; metE7C = metE7 evolved in Tris minimal

medium in coculture with the B12-producing bacterium M. loti. Mean of 7–8 independently

evolved lines are shown. Error bars = 95% confidence interval, letters above error bars indicate

statistical groupings provided by Tukey’s range test, which was performed following a significant

ANOVA result. See also Fig. S9 and Fig. S10.

Figure 6. Analysis of a range of measurements made or parameters calculated from the

metE7 progenitor strain and its experimental evolution descendants. (A) Plot of the first

two principal components derived from PCA applied to these measured variables and calculated

parameters. Each point represents an evolved line or replicate of the progenitor strain and is

given a colour and shape according to experimental evolution condition, which is specified in the



20

key in the top left corner. Ellipses are created to show the standard deviation of the eight lines

belonging to these experimental evolution groups. (B) Correlation matrix of all the measured

parameters in which white circles represent positive correlation, black circles represent negative

correlation, and the size of the circle represents the magnitude of the R2 value. B12 use

efficiency, carrying capacity, EC50 for B12, and max growth rate were calculated using all

timepoints from the B12 dose response experiment (Figure 5A). Median survival time was

calculated using all measurements of viability during B12 deprivation (Fig. 5C). Algae:Bacteria

ratio was taken from the final day (day 9) of coculturing the strains with the bacterium M. loti

(Fig. 5E). All other variables [B12 uptake (B12 absorbed in one h per unit OD), max PSII

efficiency (Fv/Fm), CFU density (Colony forming units/mL), OD 730nm (Optical density of

cultures at 730nm), cell density (Total cells/ml), NPQ (Non-photochemical quenching), ROS

(Reactive oxygen species per unit OD), Fq/Fv (coefficient of photochemical quenching), protein

(Protein concentration per unit OD), and lipid (Lipid concentration per unit OD)] were the

averages of all available measurements taken during B12 deprivation.

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https://www.ncbi.nlm.nih.gov/pubmed?term=Malanovic N%2C Streith I%2C Wolinski H%2C Rechberger G%2C Kohlwein SD %26 Tehlivets O %282008%29 S%2Dadenosyl%2DL%2Dhomocysteine hydrolase%2C key enzyme of methylation metabolism%2C regulates phosphatidylcholine synthesis and triacylglycerol homeostasis in yeast%3A Implications for homocysteine as a risk factor of atherosclerosis%2E J Biol Chem 283%3A 23989%26%23x&dopt=abstract

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physiological and molecular responses of a newly …...2020/02/20 · light with adequate (200...

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