The role of proline in the adaptation of eukaryotic microalgae to environmental stress: An underestimated tool for the optimization of algal growth

Abstract

Microalgae are considered the most promising source of renewable fuels, high-value bio-products and nutraceuticals. Potentially, microalgae can satisfy many global demands, but in large-scale cultivation the average productivity of most industrial strains is lower than maximal theoretical estimations, mainly due to sub-optimal growth conditions. Although microalgae have developed complex strategies to cope with environmental stresses, cultivation in outdoor photobioreactors is limited to few species and it is not yet sufficiently remunerative. Indeed, most microalgal species are very sensitive to environmental conditions, and changes in solar irradiation, temperature, and medium composition can drastically decrease biomass yield. Developing new strategies for improving algal tolerance to stress conditions is thus greatly desirable. One of the first responses that occur in both higher plants and microorganisms following the exposure to abiotic stress conditions, is an increased synthesis and accumulation of the amino acid proline. While the role of proline accumulation in stress adaptation is well-recognized in higher plants, in microalgae the implication of proline in stress tolerance still awaits full elucidation. In this review we summarize available data on proline metabolism under environmental stress in eukaryotic microalgae. Possible implications toward optimization of algal growth for biotechnological purposes are also discussed.

Full text

Vol.:(0123456789)
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Journal of Applied Phycology (2023) 35:1635–1648
https://doi.org/10.1007/s10811-023-03017-9
REVIEW
The role ofproline intheadaptation ofeukaryotic microalgae
toenvironmental stress: Anunderestimated tool fortheoptimization
ofalgal growth
SimoneBarera1 · GiuseppeForlani1
Received: 2 March 2023 / Revised: 25 May 2023 / Accepted: 5 June 2023 / Published online: 24 June 2023
© The Author(s) 2023
Abstract
Microalgae are considered the most promising source of renewable fuels, high-value bio-products and nutraceuticals. Poten-
tially, microalgae can satisfy many global demands, but in large-scale cultivation the average productivity of most industrial
strains is lower than maximal theoretical estimations, mainly due to sub-optimal growth conditions. Although microalgae
have developed complex strategies to cope with environmental stresses, cultivation in outdoor photobioreactors is limited
to few species and it is not yet sufficiently remunerative. Indeed, most microalgal species are very sensitive to environmen-
tal conditions, and changes in solar irradiation, temperature, and medium composition can drastically decrease biomass
yield. Developing new strategies for improving algal tolerance to stress conditions is thus greatly desirable. One of the first
responses that occur in both higher plants and microorganisms following the exposure to abiotic stress conditions, is an
increased synthesis and accumulation of the amino acid proline. While the role of proline accumulation in stress adaptation
is well-recognized in higher plants, in microalgae the implication of proline in stress tolerance still awaits full elucidation.
In this review we summarize available data on proline metabolism under environmental stress in eukaryotic microalgae.
Possible implications toward optimization of algal growth for biotechnological purposes are also discussed.
Keywords Proline· Microalgae· Environmental stress· Biomass productivity· Heavy metals
Introduction
Microalgae are (mainly) unicellular photosynthetic organ-
isms living mainly in aquatic habitats, and show high adapt-
ability to a wide range of temperatures, salinities, pH values
and different light intensities, allowing the colonization of
oceans, lakes, rivers, ponds, and waste waters (Tirichine and
Bowler 2011). Their high biodiversity makes them a rich
source of interesting and useful metabolites. A large num-
ber of species are naturally lipid accumulator under stress,
and they can be exploited for next generation biofuels pro-
duction. Green microalgae include genera which are among
the most widely used for industrial applications, such as
Haematococcus, Nannochloropsis, Chlorella and Dunaliella
spp. Some species produce a wide range of bioproducts,
including polysaccharides, pigments, vitamins, antioxi-
dants and bioactive compounds. Moreover, microalgae can
be employed in wastewater treatment and atmospheric CO2
mitigation (Benedetti etal. 2018).
The interest in exploiting microalgae is increasing for sev-
eral reasons. In contrast to higher plants, algae do not require
arable lands and need far less freshwater for their growth.
Moreover, the possibility to cultivate algae in indoor photo-
bioreactors reduces a lot the effect of the seasonal cycle on
the biomass yield (Chu and Majumdar 2012). Despite these
potentialities, the biomass productivity in large-scale plants,
both indoor and outdoor, is still too low to make the whole
process profitable. Thus, an optimization of the cultivation
methodologies and genetic engineering/selection for micro-
algae growth enhancement are required.
Microalgal growth enhancement could also be obtained
through increased resistance to environmental stress con-
ditions. Several microalgal species are naturally resist-
ant to harsh environmental conditions, such as excess salt
* Simone Barera
brrsmn@unife.it
Giuseppe Forlani
flg@unife.it
1 Department ofLife Science andBiotechnology, University
ofFerrara, Via L. Borsari 46, 44121Ferrara, Italy
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1636 Journal of Applied Phycology (2023) 35:1635–1648
1 3
(Dunaliella salina (Bonnefond etal. 2017; Xu etal. 2018;
Ahmed etal. 2017)), cold and drought (Klebsormidium sp.
(Rippin etal. 2019)) and high irradiation (Chlorella ohadii
(Levin etal. 2021)). The cultivation of most of these species
is still limited to lab scale, yet the possibility to transfer some
of their abilities to microalgal species of commercial interest
could improve the productivity of the latter in large scale
cultivation. However, a proper knowledge of the metabolic
bases for increased stress tolerance is required for this aim.
Several factors are involved in the algal response to
abiotic stress that could be exploited to increase algal bio-
mass production under stress conditions, such as amino
acid (Arora etal. 2022), osmolyte (Kaur etal. 2022) and
antioxidantmetabolism (Nowicka 2022). In higher plants,
the synthesis and accumulation of the amino acid proline
is believed to play a multifaceted role in stress tolerance,
being involved in the cellular response to excess salt, dehy-
dration, low temperature and oxidative stress (Forlani etal.
2019b). Much less is known about a possible protective role
of proline in microalgae, which have to cope with somehow
different environmental stress conditions. In this review, we
describe the state-of-the-art about the involvement of proline
metabolism in stress responses of eukaryotic microalgae.
Proline synthesis inhigher plants
andeukaryotic microalgae
Intracellular concentration of free proline is mainly deter-
mined by four metabolic processes: biosynthesis, degrada-
tion, use of proline for protein biosynthesis and release of
proline during protein degradation (Hildebrandt 2018). In
plants, free proline is detectable in the cytoplasm, where
its biosynthesis proceeds by the sequential action of two
enzymes: a δ1-pyrroline-5-carboxylate (P5C) synthetase
(P5CS) that reduces glutamic acid to γ-glutamic semial-
dehyde (GSA), which spontaneously cyclizes to P5C with
loss of one water molecule; and a P5C reductase (P5CR)
that reduces P5C to proline (Trovato etal. 2019). The for-
mer enzyme strictly requires NADPH as the electron donor
(Sabbioni etal. 2021) whereas the latter may use either
NADPH or NADH with various efficiency and affinities
(Forlani etal. 2015) (Fig.1A). In Arabidopsis thaliana
and other angiosperms, two isoforms of P5CS are present,
respectively encoded by P5CS1 and P5CS2 gene (Székely
etal. 2008). P5CS1 has been identified as the major con-
tributor to stress-induced proline accumulation, while
P5CS2 has been shown important for embryo development
and growth (Funck etal. 2020). Several papers reported
increased expression of P5CS genes in plants exposed to
environmental stress conditions such as drought, the pres-
ence of high salt or heavy metals concentrations, resulting
in increased free proline levels (Wang etal. 2015). Besides
this main pathway, in some species proline biosynthesis
from ornithine has also been described (da Rocha etal.
2012). Within the mitochondrion, GSA/P5C can be pro-
duced starting from ornithine, which is directlyproduced
by arginase or imported from the chloroplast and used
as substrate by an ornithine-δ-aminotransferase (OAT),
which transfers an amino group to α-ketoglutarate yield-
ing glutamate and GSA (Winter etal. 2015). OAT is not
essential for proline production (Funck etal. 2008), but its
overexpression seems to be correlated to the response to
some environmental stress, such as excess salt (Roosens
etal. 1998).
From the cytosol, proline also can be translocated to the
mitochondrion (Di Martino etal. 2006), where its catabolism
takes place by means of two enzymes. A proline dehydroge-
nase (ProDH) using FAD as the electron acceptor converts
proline to P5C, which is reduced back to glutamic acid by
a NAD-dependent P5C dehydrogenase (P5CDH) (Trovato
etal. 2019) (Fig.1A).
In microalgae, the information about proline metabolism
is still largely incomplete, and only a few studies investigated
in detail the metabolic routes involved. As in plants, also in
the model organism Chlamydomonas reinhardtii proline bio-
synthesis takes place in the cytosol, but two separate enzymes
are responsible for the initial conversion of glutamate to P5C
(Merchant etal. 2007). Two isoforms of γ-glutamyl kinase
(G5K and PROB, also known as GGK1 and GGK2) phos-
phorylate glutamic acid to γ-glutamylphosphate (G5P),
which is subsequently reduced to P5C by a GSA dehydro-
genase (GSD1) (Miyoshi etal. 2011; Zalutskaya etal. 2020)
(Fig.1B). The biosynthetic route is therefore more similar to
that found in prokaryotes than in higher plants (Vallon and
Spalding 2009). Recent studies carried out with Auxenochlo-
rella protothecoides have identified two genes encoding P5CS
(P5CS1 and P5CS2), two genes encoding P5CR (P5CR1 and
P5CR2) and one gene encoding PDH involved in proline deg-
radation (Xing etal. 2022).
In C. reinhardtii proline biosynthesis can also start from
ornithine in the mitochondrion through the enzyme OTA1,
which converts ornithine into GSA. P5C produced within the
mitochondrial matrix is oxidized by ALD1 into glutamate,
which is translocated to the cytosol and possibly re-utilized
for proline synthesis (Fig.1B).
As in most eukaryotes, proline catabolism proceeds
in the mitochondrion by means of two separate enzymes,
POX1 and ALD1 (Fig.1B). Recent studies demonstrated
the involvement of nitric oxide (NO) in adaption of C. rein-
hardtii to various environmental stresses. NO treatment led
to a significant accumulation of proline. In contrast, expres-
sion levels of the gene encoding OAT (OTA1) decreased
after treatment with NO, suggesting the predominance of the
glutamate pathway over the ornithine pathway under stress
(Zalutskaya etal. 2020).
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1637Journal of Applied Phycology (2023) 35:1635–1648
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Proline accumulation asanearly response
tovarious stress conditions
Microalgae and higher plants are constantly exposed to
abiotic stress conditions, leading to frequent adjustment
and remodelling of the cell defence machinery that involve
metabolic reconfiguration. When exposed to harsh envi-
ronment causing hyperosmotic stress, the cell often accu-
mulates an array of metabolites, known as compatible
osmolytes, such as sugars, polyols, quaternary ammonium
compounds and amino acids, among which proline is the
most common. An increased level of free proline repre-
sents also one of the earliest responses to a wider range of
stressful conditions that do not require osmotic adjustment
(Mattioli etal. 2009). Higher concentrations of free pro-
line were found for instance after the exposure to heating
and cooling treatments of A. thaliana plants (Hildebrandt
2018). Even if the pivotal role of proline accumulation in
stress adaptation is well-recognized, the molecular bases
of its beneficial effects are still unclear, and several mecha-
nisms have been hypothesized to explain them (Forlani
etal. 2019b).
Proline may protect proteins from high ion concentra-
tions by a direct interaction with water molecules present
on their surface. High salt concentration affects the stability
of protein hydration shell, destabilizing protein conforma-
tion. Proline acts as a kosmotropic molecule by lowering
the entropy under hyperosmolarity and maintaining the
water layer surrounding proteins intact. The pyrrolidine ring
allows the interaction with hydrophobic surface residues of
proteins, thereby increasing their hydrophilic area (Arakawa
and Timasheff 1983, 1985) and counteracting the negative
chaotropic effect of high salt concentration. On the other
hand, due to its amphipathic nature, proline stabilizes mem-
branes by intercalation between phospholipid head groups
(Rudolph etal. 1986).
Fig. 1 Pathways for proline biosynthesis and degradation in Arabi-
dopsis thaliana (A) and Chlamydomonas reinhardtii (B). (A) Pro-
line biosynthesis proceeds in plants by the sequential action of two
enzymes: an ATP- and NADPH-dependent P5CS that reduces glu-
tamic acid to GSA, which spontaneously cyclizes to P5C with loss
of one water molecule; a P5CR that reduces P5C to proline. P5CR
may use either NADPH or NADH as cofactor with various efficiency
and affinities. Proline degradation takes place in mitochondria, where
a FAD-dependent ProDH converts proline to P5C, and a P5CDH oxi-
dizes P5C back to glutamic acid. In mitochondria GSA is also pro-
duced starting from ornithine by the action of OAT. (B) Within the
cytosol, P5C biosynthesis in C. reinhardtii is accomplished by two
separate enzymes: G5K/PROB, which phosphorylates glutamic acid
into G5P, and GSD1, which reduces G5P to P5C. In the mitochon-
drion, POX1 oxidizes proline to P5C, which is in turn oxidized to
glutamate by ALD1
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1638 Journal of Applied Phycology (2023) 35:1635–1648
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Interestingly, several studies reported that proline is
involved also in stress defence mechanisms induced by
presence of high concentration of heavy metals. It has been
formerly proposed that proline is capable of reactive oxy-
gen species (ROS) detoxification. However, it has been
later demonstrated that its chemical properties do not allow
scavenging of singlet oxygen, superoxide, NO and nitrogen
dioxide (Signorelli etal. 2016). According to recent studies,
it seems that proline can act exclusively as a hydroxyl radical
scavenger (Signorelli etal. 2015). Notwithstanding this, the
exogenous supply of proline to A. thaliana roots resulted in
reduced ROS levels (Cuin and Shabala 2007). Moreover, the
activity of antioxidative enzymes, such as peroxidase(POD)
and superoxide dismutase (SOD), was enhanced in presence
of high concentration of exogenous proline in tobacco cell
suspensions exposed to high salts concentrations (Hoque
etal. 2007). It seems therefore that proline may contribute
indirectly to ROS scavenging, but this topic is still subject
of debate. Notably, proline synthesis and degradation may
play also a key role in redox balancing between cytosol and
mitochondria, since these processes cause a significant fluc-
tuations of NAD(P)+/NAD(P)H pools (Giberti etal. 2014).
Microalgae occupy mostly aquatic habitats, and their
life cycle is strictly relater to water. Aquatic ecosystems are
constantly subjected to changing in temperature, due to sea
currents or the presence of geothermal sources. Also light,
which reachesthe inner layers of rivers and seas, is strongly
influenced in quality and quantity by rapid and constant
movements of water masses. In open pond large-scale cul-
tivation, in which water masses are constantly moved, the
continuous light-shade alternation generates light stress and
affects algal growth. In addition, also fluctuations in salt
concentration and the presence of pollutants and heavy met-
als can act as stressors (Fig.2). As a consequence, microal-
gae have developed complex defense systems against rapid
changing of these environmental factors.
Proline involvement instress defence
towardsharsh environmental conditions
inmicroalgae
Salt stress
During recent years, the ongoing climate change and human
interventions caused salinization of a significant part of
freshwater resources on a global level. Large-scale microal-
gal cultivation needs massive amount of freshwater, there-
fore the availability of halotolerant strains is very attrac-
tive. High salinity is a challenging environmental stress for
organisms to overcome, and most of green microalgae are
extremely vulnerable to excess salt, not only due to ionic
imbalance, but also to the generated ROS interfering with
the photosynthetic machinery (Fal etal. 2022).
Fig. 2 Schematic representa-
tion of major abiotic stresses
that affect plant and microalgal
growth. The thickness of the
arrows represents the relative
importance of each stressor in
the two cases
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1639Journal of Applied Phycology (2023) 35:1635–1648
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To face hyperosmotic stress, microalgae developed a wide
range of physiological, metabolic and molecular responses
(Wang etal. 2018), including the accumulation of carbohy-
drates and lipids as storage molecules to maintain microalgae
survival (Anand etal. 2019). Microalgae exposed to high
salinity accumulate ROS-detoxifying enzymes such as SOD,
ascorbate peroxidase (APX), catalase (CAT), glutathione
reductase (GR), andPOD, as well as osmoprotectant mol-
ecules, such as glycine betaine, glycerol and proline (Pancha
etal. 2015) (Ismaiel etal. 2018). Such osmolytes prevent
water loss and contribute to maintain the osmotic balance
(Anand etal. 2019). Contrary to higher plants, in microalgae
mannitol and sorbitol are also used in salinity adaptation,
especially in brown algae (Wegmann 1986). In addition to
organic solutes, inorganic ions also play an important role in
osmoregulation (Hellebust 1985).
Several halotolerant microalgal species have been isolated
that can grow in saline environments. Among them, either
freshwater or marine water species of the same genus, such
as C. reinhardtii and Ch. vulgaris (freshwater species) and
Chlamydomonas pulsatilla and Chlorella salina (marine
species). Salt-resistant green microalgae are characterized
by the capability to over-produce compatible solutes, and
often by the presence of a cellulosic cell wall.
In Chlamydomonas spp., high salinity adaptation involves
changes in cell morphology and aggregation state, termed
as palmelloid. In palmelloid of C. reinhardtii, 4–16 cells are
clustered, lose flagella and increase secretion of exopolysac-
charides (Shetty etal. 2019). These strategies are necessary
to face both temporary and prolonged osmotic shock. When
C. reinhardtii cells were exposed to salt stress and subse-
quently treated with exogenous proline, the treatment ame-
liorated the negative effects of high salinity (Reynoso and
de Gamboa 1982). Chlamydomonas pulsatilla can withstand
hypersaline environments by increasing glycerol levels, and
can survive at lethal salt concentrations by forming resting
spores (Hellebust 1985). In C. reinharditii the endogenous
content of free proline increased during 24h of treatment
at NaCl concentration above 100mM (Mastrobuoni etal.
2012). Further evidences have shown increased expressions
of proline synthesis metabolic pathway genes under osmotic
stress in Chlamydomonas sp. ICE-L, which is periodi-
cally exposed to extreme salinity concentrations inside the
brine channels in the Antarctic Sea ice (Zhang etal. 2020).
Stressed cells of C. reinhardtii accumulated carotenoids and
proline as osmoprotectant (Fal etal. 2022).
Chlorella and Dunaliella spp. do not usually form
palmelloids after the exposure to high salinity stress, and
show different defence mechanisms. Chlorella spp. are
characterized by a rigid cellulosic cell wall that limits
their ability to change cell volume. Therefore, osmoregu-
lation is maintained through the production of organic sol-
utes and accumulation of inorganic ions. On the contrary,
Dunaliella lacks a rigid cell wall, allowing rapidly changes
in cells volume during high salinity stress by adjusting ion
and glycerol concentration within the cell (Chen etal.
2009). Although Chlorella and Dunaliella spp. show great
differences in cell morphology, a number of studies found
a similar increase of free proline content immediately after
high salinity treatments.Chlorellavulgaris is considered
a freshwater species, but it can tolerate up to 0.8M NaCl,
while Ch. salina, a marine species, can survive up to 2M
NaCl. Chlorella autotrophica, another marine species, is
able to grow in environments characterized by fourfold
seawater salt concentration, about 2.4M (Ahmad and Hel-
lebust 1984). It has been found that in Ch. vulgaris and Ch.
autotrophica free proline content increases under salinity
stress, whereas in Ch. salina it remains constant (Farghl
etal. 2015). When Ch. vulgaris and Chlorococcum humi-
cola (a halotolerant microalga) were compared, proline
content in cells treated with different NaCl concentrations
was found in both cases to increase with increased salin-
ity. Maximal level (threefold that in untreated controls)
was found at 500mM NaCl for Ch. vulgaris, but only
at above 1000mM NaCl for Chl. humicola (Singh etal.
2019). In another study on Ch. vulgaris, cells were treated
for 30days with NaCl concentrations ranging between 0.1
and 0.4M. In this case also, proline increased with the
treatment. Remarkably, the content of glycine betaine was
also found to increase, showing that the adaptive response
of Ch. vulgaris can involve other compatible solutes (Hire-
math and Mathad 2010). More recently, lipid accumulation
was induced in Ch. vulgaris strain YH703 by means of
NaCl treatments, which caused high H2O2 production and
increased APX activity and proline contents. Since Ch.
vulgaris YH703 strain can mitigate ROS generated under
salinity stress, a possible involvement of proline in ROS
scavenging has been hypothesized (Yun etal. 2019).
Several studies have revealed the implication of proline
metabolism in high salinity stress response also in Scened-
esmus, a genus that has been actively studied for bio-diesel
production, and has one of the highest biomass productivity
among green algae, mainly under heterotrophic growth (da
Silva etal. 2009; Mandal and Mallick 2009; Yoo etal. 2010;
El-Sheekh etal. 2013). Some Scenedesmus strains contain
also a considerable amount of carbohydrates (about half of
their dry weight), which makes them attractive candidates
for bio-ethanol production (John etal. 2011). In Scenedes-
mus sp. CCNM 1077, a dose-dependent increase in proline
content (up to 1.9-fold) was found under salinity stress in
the midpoint of the treatment, but it decreased thereafter
(Pancha etal. 2015). Scenedesmus sp. IITRIND2 exposed to
high salt concentration exhibited an increase of proline and
glycine betaine content that was paralleled by an increase of
the activity levels of some antioxidant enzymes, in particular
CAT and APX (Arora etal. 2017, 2019).
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1640 Journal of Applied Phycology (2023) 35:1635–1648
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Proline is the major osmolyte detected in halotolerant
Picochlorum sp. (Henley etal. 2004). It has been reported
that Picochlorum soloecismus could be a new platform for
the production of renewable fuels (Gonzalez-Esquer etal.
2019). In addition, Picochlorum strain SENEW3 accu-
mulated proline under high salt stress conditions through
upregulation of one gene involved in proline synthesis
(Foflonker etal. 2016). An increase of free proline content
was also detected in Acutodesmus dimorphus, a halotoler-
ant microalga able to accumulate a large amount of lipids
under heat stress (Chokshi etal. 2015, 2017), and in Neo-
chloris oleoabundans, an oleaginous microalgal species that
can be cultivated both in freshwater and salt water (Jaeger
etal. 2018). Another microalgal species, Micractinium sp.,
following exposure to high NaCl concentrations showed
increased free proline level, together with higher lipid accu-
mulation (Yang and Hu 2020).
Dessication is often related to hypersaline environments,
and it is doubtless one of major abiotic stressors that affect
microalgal growth. Recently two microalgal clades, Pseu-
dostichoccus and Deuterosticoccus, were found able to sur-
vive in total absence of water in both saline and non-saline
conditions. Pseudostichococcus, which was able to recover
fully after desiccation with or without salinity stress, showed
a higher proline:sorbitol ratio compared to untreated con-
trols. These results suggest the possible implication of pro-
line in desiccation resistance, and open the way for further
investigations on the possible applications of this genus in
salinity bioremediation (Van and Glaser 2022).
Although much evidence of increased free proline con-
tent under osmotic stress has been obtained in a wide range
of microalgal species, it is still unclear whether this amino
acid acts mainly as osmoprotectant, or it is involved in the
activation of ROS detoxification response. Hence, proline
could act also as signal molecule involved in activation of
multiple responses under osmotic stress. Unfortunately, lack
of complete overview of how many and which genes of pro-
line metabolism are upregulated under saline stress, and their
interconnection with ROS detoxification pathways, hampered
to date full understanding the role of proline in salt resistance.
Heavy metal stress
The presence in soil and water of an excess of heavy metals
caused by anthropogenic pollution represents one of the harsh-
est conditions to cope with. Heavy metals comprise both essen-
tial micronutrients (Cu, Zn, Co, Fe, Mn, Mo) and cations that
do not play any function in living organisms (Ag, As, Cd, Cr,
Hg, Ni, Pb, Sb, U, V, W). Accumulation of heavy metals in the
cell induces oxidative damages by generating ROS, which lead
to enzyme inactivation, protein degradation, pigment bleaching
and lipid peroxidation (Nowicka 2022) One of the first defence
mechanisms adopted by microalgae is a fast antioxidant response.
One of the first studies to correlate increased proline con-
tent with an excess of heavy metals ions in microalgae was
published in the 90’s, when high levels of free proline were
detected in Ch. vulgaris cells exposed to high concentra-
tions of Cu ions. If proline was exogenously supplied before
the treatment, Cu uptake was drastically reduced (Wu etal.
1998). Further studies revealed that free proline content
increases drastically within a few hours after the exposure of
Ch. vulgaris cells to increasing concentrations of Cu, Cr, Ni
and Zn cations. The authors suggested that rapid accumula-
tion of free proline may confer resistance by inhibiting heavy
metal-induced lipid peroxidation (Mehta and Gaur 1999).
Excess Cu affected also the growth of C. reinhardtii caus-
ing lipid peroxidation and generation of NO and induced
a consistent accumulation of free proline. Higher expres-
sion levels of enzymes involved in proline synthesis were
detected in presence of NO analogues, suggesting a regu-
latory function of NO in proline metabolism under heavy
metal stress (Zhang etal. 2008). Exposure of C. reinhardtii
cells to high concentrations of Hg ions induced accumula-
tion of free proline and significantly increased the expression
of ROS scavenging enzymes (Elbaz etal. 2010) (Wei etal.
2011). The importance of proline in heavy metal detoxifica-
tion was strengthened by the results obtained overexpress-
ing mothbean P5CS in C. reinhardtii. Recombinant cells
showed 80% more free proline than the wild type, and were
able to grow better in Cd-supplemented medium, most likely
because of higher ROS scavenging, and higher GSH levels
that facilitate phytochelatin synthesis and Cd sequestration
(Siripornadulsil etal. 2002).
Free proline content alterations in response to heavy
metal treatment were assessed in many other microalgal spe-
cies. Studies conducted on Scenedesmus obliquus revealed
significant increasing in both proline and polyphenol con-
tents after Pb2+ exposure (Danouche etal. 2020). Treat-
ment of Scenedesmus sp. with Cu or Zn ion concentrations
below 10μM induced hyperaccumulation of heavy metals
and increased proline biosynthesis. Notably, concentrations
above 10μM of Cu ions had, on the contrary, an inhibitory
effect on proline synthesis. Cu and Zn ions induced strong
oxidative stress in Scenedesmus sp. by increasing mem-
brane permeability and lipid peroxidation. A pre-treatment
with 1mM proline for 30min increased GR activity levels
and totally prevented lipid peroxidation, suggesting that
it acts by scavenging ROS rather than by chelating metal
ions (Tripathi and Gaur 2004). Further evidences of proline
involvement in Cu ions detoxification in Scenedesmus sp.
were reported in (Tripathi and Gaur 2004) and (Kováčik
etal. 2010). The exposure of Scenedesmus quadricauda
cells to high concentration of Cd2+ ions inhibited growth
and pigmentation, and induced accumulation of proline and
malondialdehyde, reflecting high level of lipid peroxidation
(Çelekli etal. 2013). The uptake of other heavy metals such
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1641Journal of Applied Phycology (2023) 35:1635–1648
1 3
as As enhanced proline accumulation in Scenedesmus sp.
IITRIND2A (Arora etal. 2018).
Several microalgal species showed a high tolerance
against a variety of heavy metals concomitant with the
accumulation of proline, as in Euglena gracilis (Cervantes-
García etal. 2011) and in Desmodesmus sp. (Buayam etal.
2019) exposed to excess Cu2+ (Liu etal. 2020). Increased
proline content goes hand in hand with higher GSH level,
yet it is not clear how proline acts in redox balancing. In
the model proposed by Siripornadulsil and co-workers, Cd-
induced hydroxyl radicals react directly with free proline
(Siripornadulsil etal. 2002). This hypothesis is consistent
with results obtained in Brassica juncea showing that proline
was able to quench free radicals in thylakoids isolated from
plants exposed to high light intensities (Alia and Mohanty
1997). Proline could react with hydroxyl radical with the
formation of hydroxyproline, but no evidence supporting
this scavenging mechanism has been reported (Smirnoff and
Cumbes 1989; Matysik etal. 2002). Further studies demon-
strated in plants that proline reacts with hydroxyl radicals to
form P5C, which is converted back to proline by P5CR in
the so called Pro-Pro cycle (Signorelli etal. 2014).
Stress generated bythepresence ofpollutants
Besides heavy metals, wastewater contains a wide range
of compounds such as herbicides, insecticides, cleaning
detergents, plastic processing wastes etc., which can be
toxic for aquatic organisms. To cope with water contami-
nation, during the last decades many works have exploited
the capability of microalgae of sequestering and detoxifying
chemical pollutants, the so-called microalgae wastewater
bioremediation. In several cases, proline metabolism has
been found involved in providing tolerance to such toxic
chemical compounds. Studies carried out on a freshwater
Chlamydomonas mexicana strain exposed to high doses of
the insecticides acephate and imidacloprid showed increased
SOD and CAT activity levels and high accumulation of pro-
line (Kumar etal. 2016). Significant build up of proline and
glycine betaine was found in D. salina following the treat-
ment with polyethylene glycol, whose presence in wastewa-
ter limits the use of large water resources. Activity levels
of some antioxidant enzymes, namely CAT and APX, were
simultaneously enhanced (Tafvizi etal. 2020). Perfluorooc-
tanoic acid (PFOA) has been found in various ecosystems
and is receiving growing attention due to its biomagnifi-
cation properties that increase its toxicity in aquatic envi-
ronments. PFOA inhibited the growth of C. reinhardtii but
only partially affected growth of S. obliquus. In both cases,
the exposure to this pollutant enhanced proline content (Hu
etal. 2014). All these studies clearly showed a correlation
between the treatment with chemical pollutants and proline
accumulation, yet the results do not allow to understand
whether the increase of proline content is simply due to the
need of scavenging ROS generated within the cell by toxic
chemicals, or proline metabolism may play a more specific
role in their detoxification.
Stress induced bytemperature andlight
fluctuations
The major constraints in outdoor microalgae mass cultiva-
tion is the susceptibility of many strains of commercial inter-
est to climate variations, as temperature and light intensity
fluctuations drastically affect microalgal productivity. Under
open ponds conditions, microalgae are subjected to night-
day and season cycles in which temperature and sunlight
irradiation may vary sharply. Heat stress affects pigments
metabolism, damages mitochondrial function, and causes
lipid peroxidation. Cold stress causes membrane rigidifica-
tion, affects the stability of proteins or protein complexes
and induces intracellular H2O2 accumulation (Choudhury
etal. 2017; Ding et al. 2019; Chokshi etal. 2020). The
intracellular responses induced by temperature fluctuations
reflect an imbalance in ROS homeostasis (Muhlemann etal.
2018; Wang etal. 2019; Chokshi etal. 2020). As a conse-
quence, microalgal yield is strongly reduced. In Ch. vulgaris
a variation of 5 and 10oC above the temperature optimum
decreased productivity by 50% and 100%, respectively (Con-
verti etal. 2009). In S. obliquus grown under a range of
temperatures lower than the optimum, an up to 80% yield
reduction was found (Chalifour and Juneau 2011).
Studies on A. protothecoides pointed out different effects
of heat and cold stress. High temperature treatments caused
a drastic increase of ROS levels. Consequently, the expres-
sion of key enzymes involved in ROS detoxification was
enhanced, but no significative changing in proline levels
was found. On the contrary, under cold stress free proline
content raised up to 3.8 fold respect to untreated controls
(Xing etal. 2022). When the expression levels of the two
isoforms of P5CS and P5CR genes and PDH gene were
determined under cold stress, P5CS and P5CR were found
up-regulated, while PDH was down-regulated, showing that
proline accumulation relies upon both increased biosyn-
thesis and reduced catabolism. Under heat stress, only an
increase of P5CR expression was found (Xing etal. 2022).
Conversely, C. reinhardtii did not show any fluctuation in
free proline content under cold stress conditions (Valledor
etal. 2013; Cvetkovska etal. 2022). The relationship between
proline metabolism and heat stress was investigated also in
Acutodesmus dimorphus, a potential species for biofuel pro-
duction. In cells exposed to high temperature (45 and 50°C),
free proline content initially showed a rapid increase up to
12h, but decreased drastically thereafter. Once again, SOD
and CAT were also found to increase (Chokshi etal. 2020).
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1642 Journal of Applied Phycology (2023) 35:1635–1648
1 3
Concerning the effect of light fluctuations, the maximal
theoretical efficiency of photosynthetically active radiation
(400–700nm) solar energy conversion into biomass under low
light conditions at the lab scale is about 27%, but the efficiency
drops to about 6% in outdoor cultivations under high light
(Weyer etal. 2010; Zhu etal. 2010). Under low-irradiance,
the photosynthetic rate increases linearly with light intensity,
while at high irradiance the increase is not linear and reaches
a plateau at light saturation (Pmax) (Li etal. 2009). Above Pmax,
photoinhibition and ROS generation occur, and excess energy
is dissipated into heat, with a consequent drop in productivity.
Although proline metabolism, as emphasized above, is
often involved in ROS detoxification, it is still unclear whether
proline may play a role in microalgae resistance to the oxi-
dative stress generated by excess light. In plants, a possible
correlation has been hypothesized based on the high rate of
NADPH and ATP consumption required for proline synthesis
from glutamate (Hare and Cress 1997). Proline production
dissipating excess reducing power was proposed as a com-
pensatory strategy to sustain photosynthesis and prevent pho-
toinhibition under excess light in A. thaliana mutants lacking
a chloroplast NADP-dependent malate dehydrogenase (Heb-
belmann etal. 2012). Using reducing equivalents for enhanced
proline biosynthesis could limit the generation of ROS through
pseudocyclic photophosphorylation, and avoid the conse-
quent cell damage (Ben Rejeb etal. 2014). Tissue-specific
differences in proline metabolism, where proline synthesis in
photosynthetic tissues regenerates NADP+, while its catabo-
lism in meristematic and expanding cells sustains growth by
increased energy availability, further strengthened this hypoth-
esis (Sharma etal. 2011).
A similar correlation has been suggested in C. rein-
hardtiithe use of excess reducing equivalents for proline
accumulation ameliorates the redox imbalance caused by high
light irradiance in the chloroplast. Moreover, proline can be
subsequently translocated into the mitochondrion and used
to sustain the increased growth rate observed in high light
acclimation (Davis etal. 2013). Consistently, a four–fivefold
increase of free proline content was found in Asteracys sp. cells
grown mixotrophically under high irradiances respect to the
cells grown under low light (Agarwal etal. 2019). Increased
proline concentrations were recently found also in Ch. humi-
cola exposed of UV-B light, but in this case a direct role in
scavenging the ROS produced under these conditions seems
more likely (Singh etal. 2019).
Proline metabolic engineering, state
oftheart andperspectives
During the last decades the interest in microalgae for bio-
technological applications has increased, yet the large-
scale cultivation of most microalgal species is still not
sustainable. Currently, only the green algae Ch. vulgaris,
D. salina and Haematococcus pluvialis are cultivated on
a large scale (Borowitzka 2018), but for the production
of single products and with several limitations. Most of
the industrial scale microalgal growth for food/feed pro-
duction is performed in closed systems in mixotrophy
or heterotrophy under axenic conditions. This implies a
consequent high energy consumption for the maintenance
of optimal temperature, light irradiation, gas fluxes, stir-
ring and sterilization cycles. The possibility to find new
approaches for lowering costs of large-scale cultivations
would open new perspectives and widen microalgal appli-
cations. Several solutions have been already proposed,
including recycling media, using nutrients derived from
wastewater, phototrophic growth in sunlight, and amelio-
rating of photobioreactor architecture. Another possible
approach to maximise productivity is represented by the
identification of microalgal strains resistant to abiotic
stress conditions. This result could be obtained by con-
ventional selection of spontaneous or chemically-induced
mutants. However, the newest genetic engineering tech-
niques are opening the way to engineer strains with highly
desirable traits, such as hyper salinity tolerance, resistance
to excess light, heat and cold stress, capability to growth
under non-axenic conditions and in presence of pollutants.
Even more interestingly, these techniques could allow the
transfer of these traits from donor strains to commercial
strains with high productivity.
Genetic manipulation through CRISPR/Cas9 genome
editing has demonstrated the high potential and plastic-
ity of microalgae. For instance, several C. reinhardtii
strains with characteristics of commercial interest have
been obtained by transforming nucleus or chloroplast
(Benedetti etal. 2018), and the introduction of PtxD gene
allowed increased control of pest contamination (Loera-
Quezada etal. 2016; Changko etal. 2020; Cutolo etal.
2020). Engineering of C. reinhardtii opened the possibility
to utilize microalgae also as cell bio-factories to produce
high-value compounds at low cost, with application in
pharma, cosmetics, human nutrition (García etal. 2017)
and treatment of lignocellulosic biomass waste (Benedetti
etal. 2021). Although results demonstrated the robustness
of these techniques, it will be necessary to apply them to
microalgal species with higher productivity.
Genetic transformation of microalgal strains of com-
mercial interest as Chlorella spp. is extremely challenging,
one of the major constraints being represented by the pres-
ence of a cellulosic cell wall (absent in Chlamydomonas),
which hinders the insertion of exogenous DNA. The first
successes in chloroplast biolistic transformation of non-
model microalgae have been described only recently. Effi-
cient transformation of Ch. vulgaris chloroplast for the
production of two foreign peptides (Wang etal. 2021),
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1643Journal of Applied Phycology (2023) 35:1635–1648
1 3
and transformation of Nannochloropsis gaditana and Tet-
raselmis sp. with reporter genes (Cui etal. 2014, 2021)
have been reported. Another group succeeded in trans-
forming Picochlorum renovo, a green microalga charac-
terized by high-productivity, halo- and thermotolerance
(Dahlin etal. 2019). These studies represent an important
breakthrough in microalgae genetic manipulation, but they
are still far from the production of strains with charac-
teristics exploitable on industrial scale. The attainment
of improved growth rates can be pursuit by alternative
strategies, such as random mutagenesis and subsequent
selection of strains characterized by desirable phenotypic
traits. With this approach, Chlorella sp. and N. gaditana
strains characterized by high productivity and low sensi-
tivity to oxidative stress under high light irradiance have
been obtained (Cazzaniga etal. 2014; Perin etal. 2015;
Guardini etal. 2021).
Under this perspective, proline metabolism represents
an attractive target. As discussed above, proline metabo-
lism seems strictly involved in the cell response to a wide
range of environmental stresses, and in many instances a
higher proline content, or a more active proline synthesis,
was found to correlate with a higher tolerance to abiotic
stress(Forlani etal. 2019a). In plants, successful strategies
in proline metabolic engineering were developed. Over-
expression of a Vigna aconitifolia P5CS in tobacco led to
a higher proline accumulation under stress (Kishor etal.
1995), and the removal of feed-back inhibition allowed to
obtain plants showing water stress tolerance (Zhang etal.
1995). Similar results were obtained in chickpea and rice
(Karthikeyan etal. 2011), protecting plants from drought
(Surekha etal. 2014). Proline metabolism tuning, obtained
by expressing P5CR under the control of a heat-inducible
promoter, conferred drought tolerance in soybean (De Ronde
etal. 2004).Arabidopsisthaliana antisense mutants defec-
tive in ProDH expression showed increased free proline
content and enhanced tolerance to excess salt and low tem-
perature (Nanjo etal. 1999), without any detrimental effect
on plant development (Mani etal. 2002). More recently, the
same approach has been pursued with the CRISPR/Cas9
genome editing system, and prodh knock-out rice plants
were obtained that showed increased proline content and
displayed heat stress resistance (Guo etal. 2020).
In microalgae, however, only very few studies have
investigated the feasibility of modifying proline pathways
for biotechnological purposes. Two reasons may explain
this delay. The first one is that most microalgal species of
commercial interest are still recalcitrant to genetic trans-
formation, mainly due to the cellulosic cell-wall surround-
ing the microalgal cell, and the low frequency of transgene
integration into the algal genome (Cutolo etal. 2022). In
fact, to the best of our knowledge, the only study in which
proline synthesis has been engineered to date was carried
out with the model organism C. reinhardtii, a cellulose
cell-wall lacking species. In that case the insertion of a
P5CS gene from mothbean allowed to obtain increased
free proline content, which was paralleled by an increased
tolerance to Cd ions and to salt-induced stress (Siripor-
nadulsil etal. 2002). However, as emphasized above,
some efficient transformation methods have been recently
described, and this limitation could be overcome soon.
The other reason is that, contrary to plants, the meta-
bolic pathways for proline metabolism and their regulatory
switches under stress have been poorly investigated to date
in microalgae. Although several efforts have been made to
understand the correlation between environmental stress and
fluctuations in proline metabolism, there are still insufficient
information to obtain a detailed picture on how proline
metabolism could protect microalgae against abiotic stress
conditions, and –mainly– on the molecular mechanisms that
underlie its modulation.
The selection of proline-overproducing strains, or a tar-
geted modification of proline metabolism so as to obtain
increased proline production/accumulation under stress,
could increase algal capability to withstand environmental
stress conditions. This would not affect production costs,
and should be cost effective also for the algal cell, because
the metabolic charge for increased proline synthesis would
be paid off by the reduced negative effects of stress condi-
tions. Yet, it would remain to verify whether an increased
carbon flux toward proline synthesis, which requires ATP
and reducing power, could be detrimental under non-stress
conditions. It is quite likely that a constitutive production
of proline, mainly if deriving from the overexpression of
the biosynthetic genes under the control of strong pro-
moters, can reduce the growth of overproducing strains
with respect to wild-type controls in the absence of stress.
But this limitation could be easily overcome by the use of
stress-inducible promoters, thereby avoiding the occur-
rence of pleiotropic effects. On the other hand, increased
proline synthesis would require increased availability of
both carbon skeletons and nitrogen moieties. The first
one should not be a problem for photosynthetic organ-
isms that have been demonstrated to be able to modulate
photosynthesis as a function of energy demand (Li etal.
2009). The second requirement may be more challenging,
because higher nitrate/ammonia concentrations should be
added to the culture medium. However, this apparent limi-
tation could on the contrary turn into an advantage, as it
would allow the (at least partial) use for algal cultivation
of wastewater containing high levels of inorganic nitrogen.
The presence of increased proline content in the result-
ing biomass would cause no negative effects for down-
stream processing and product quality. If the biomass is
to be used as food or feed, the taste would be substan-
tially unchanged, since proline belongs to sweet amino
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1644 Journal of Applied Phycology (2023) 35:1635–1648
1 3
acids (Yuxiao etal. 2023) and does not contribute to bitter
or umami taste. Although being not essential, increased
proline level may on the contrary increase the nutritional
value of the algal biomass, taking into account that emerg-
ing evidence indicates dietary essentiality of non-essential
amino acids for animals and humans (Hou etal. 2015).
Even in the case that the algal biomass is to be processed
for the isolation of bioproducts, the presence of high lev-
els of free proline could be beneficial because, as a kos-
motropic substance, proline has been reported to increase
macromolecule solubility and stability.
Investments in both basic and applied research with the aim
to elucidate the metabolic pathways for proline synthesis in
eukaryotic microalgae and their regulation under stress would
be greatly desirable and would provide the basis for the devel-
opment of future strategies for proline metabolic engineering.
Hopefully, this will open new perspectives to achieve micro-
algae stress tolerance, with a consequent lowering of cultiva-
tion costs and enhancing of biomass yield, ensuring suitable
productivity also in outdoor large-scale cultivations.
Author’s contribution GF and SB identified patterns and trends in the
literature and designed the structure of the review. SB prepared figures.
GF and SB contributed to searching for relevant literature, and carried
out a critical analysis of the literature, discussed together and wrote
the manuscript. All authors read and approved the final manuscript.
Funding Open access funding provided by Università degli Studi di
Ferrara within the CRUI-CARE Agreement. Project funded under the
National Recovery and Resilience Plan (NRRP), Mission 04 Compo-
nent 2 Investment 1.5 – NextGenerationEU, Call for tender n. 3277
dated 30/12/2021. Award Number: 0001052 dated 23/06/2022.
Declarations
Competing interests The authors declare that they have no competing
interests.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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