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plants

Review
Fruit Photosynthesis: More to Know about Where, How
and Why
Andreia Garrido 1 , Artur Conde 1 , João Serôdio 2 , Ric C. H. De Vos 3 and Ana Cunha 1, *

1 Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho,
Campus de Gualtar, 4710-057 Braga, Portugal; arturconde@bio.uminho.pt (A.C.)
2 Centre for Environmental and Marine Studies (CESAM), Department of Biology, University of Aveiro,
Campus de Santiago, 3810-193 Aveiro, Portugal; jserodio@ua.pt
3 Business Unit Bioscience, Wageningen Plant Research, Wageningen University and Research
Centre (Wageningen-UR), P.O. Box 16, 6700 AA Wageningen, The Netherlands; ric.devos@wur.nl
* Correspondence: accunha@bio.uminho.pt

Abstract: Not only leaves but also other plant organs and structures typically considered as carbon
sinks, including stems, roots, flowers, fruits and seeds, may exhibit photosynthetic activity. There
is still a lack of a coherent and systematized body of knowledge and consensus on the role(s) of
photosynthesis in these “sink” organs. With regard to fruits, their actual photosynthetic activity is
influenced by a range of properties, including fruit anatomy, histology, physiology, development
and the surrounding microclimate. At early stages of development fruits generally contain high
levels of chlorophylls, a high density of functional stomata and thin cuticles. While some plant
species retain functional chloroplasts in their fruits upon subsequent development or ripening, most
species undergo a disintegration of the fruit chloroplast grana and reduction in stomata functionality,
thus limiting gas exchange. In addition, the increase in fruit volume hinders light penetration and
access to CO2 , also reducing photosynthetic activity. This review aimed to compile information on
aspects related to fruit photosynthesis, from fruit characteristics to ecological drivers, and to address
the following challenging biological questions: why does a fruit show photosynthetic activity and
what could be its functions? Overall, there is a body of evidence to support the hypothesis that
photosynthesis in fruits is key to locally providing: ATP and NADPH, which are both fundamental
for several demanding biosynthetic pathways (e.g., synthesis of fatty acids); O2 , to prevent hypoxia in
Citation: Garrido, A.; Conde, A.; its inner tissues including seeds; and carbon skeletons, which can fuel the biosynthesis of primary and
Serôdio, J.; De Vos, R.C.H.; Cunha, A. secondary metabolites important for the growth of fruits and for spreading, survival and germination
Fruit Photosynthesis: More to Know of their seed (e.g., sugars, flavonoids, tannins, lipids). At the same time, both primary and secondary
about Where, How and Why. Plants metabolites present in fruits and seeds are key to human life, for instance as sources for nutrition,
2023, 12, 2393. https://doi.org/ bioactives, oils and other economically important compounds or components. Understanding
10.3390/plants12132393 the functions of photosynthesis in fruits is pivotal to crop management, providing a rationale for
Academic Editor: William L. Bauerle manipulating microenvironmental conditions and the expression of key photosynthetic genes, which
may help growers or breeders to optimize development, composition, yield or other economically
Received: 9 May 2023
important fruit quality aspects.
Revised: 13 June 2023
Accepted: 15 June 2023
Keywords: fruit characteristics; fruit tissues; photosynthetic activity; functions
Published: 21 June 2023

Copyright: © 2023 by the authors. 1. Introduction


Licensee MDPI, Basel, Switzerland. Fruits are important products derived from agriculture and their consumption is
This article is an open access article indispensable for the human diet. In addition to vitamins, minerals and fibers, several
distributed under the terms and phytochemicals present in fruits are generally regarded as beneficial to human health,
conditions of the Creative Commons
by reducing the risk of a wide range of cancers, cardiovascular diseases and other diet-
Attribution (CC BY) license (https://
related diseases, possibly through their antioxidant capacity [1]. Due to the high nutritional
creativecommons.org/licenses/by/
and economic value of fruits—a perishable staple—many biochemical, physiological and
4.0/).

Plants 2023, 12, 2393. https://doi.org/10.3390/plants12132393 https://www.mdpi.com/journal/plants


Plants 2023, 12, 2393 2 of 29

molecular studies have been performed, e.g., to improve their nutritional quality and shelf
life [2].
Evolutionary pressures have resulted in a wide diversity of fruits, ranging from small
dry seed capsules that burst to allow seed dispersal, to relatively large complex fleshy
fruits that have evolved bright colors and complex aromas to attract seed-dispersing birds
and other animals [3]. This diversity can be organized under the following dichotomies:
fleshy or dry (with/without a soft succulent pericarp) with the dry fruits being either
dehiscent or indehiscent (open/not open to discharge seeds). For instance, capsules,
siliques and legumes are dehiscent and dry; achenes, nuts and caryopsis of cereal grains
are indehiscent and dry; drupes, apples and berries are indehiscent and fleshy. With regard
to fruit ripening, the classification of climacteric or non-climacteric fruits is based on their
ethylene production and respiration rate [4]. The onset of ripening in climacteric fruits is
characterized by an increase in respiration with a simultaneous and well-characterized
peak in ethylene production (e.g., apple—[5]; tomato—[6]), while in non-climacteric fruits
the ripening process occurs more gradually without sudden changes (e.g., strawberry—[7];
grapes—[8]).
Photosynthesis essentially depends on irradiance, resistance to CO2 diffusion (from
the atmosphere to the sites of assimilation in the chloroplasts) and surface/depth contain-
ing photosynthetic pigments. In plants, photosynthesis predominantly occurs in green
leaves, which are the primary sources of photoassimilates to the whole plant. However,
it has been demonstrated that throughout the life cycle of the plant also other vegetative
and reproductive structures can be photosynthetically active, including green fruits, green
stems, green flower organs and even roots (as reviewed by Aschan and Pfanz [9], and
more recently by Brazel and Ó’Maoileídigh [10] and by Simkin et al. [11]) (Table 1). The
photosynthetic activity of these organs and structures may be seasonal, occurring at specific
developmental stages, and may vary between tissues within these structures. For instance,
in grape berries at the green phase, a clear tissue-specific distribution of photosynthetic com-
petence was observed by Breia et al. [12]: the exocarp revealed the highest photosynthetic
capacity, whereas the mesocarp exhibited very low fluorescence signals and photochemical
competence, and the seed outer integument revealed a photosynthetic ability similar to
that of the exocarp.

Table 1. Net photosynthetic (PN ) rates in different plant species and their structures. PN is expressed
in µmol CO2 m−2 s−1 with the exception of the case reported for Vitis vinifera L.

Species Structure PN
(µmol CO2 m−2 s−1 ) References
Fruit
Cucumis sativus L. Cucumber 2.1–2.4 [13]
Helleborus viridis L. agg. - 0.1 [14]
Olea europaea L. (cv. Leccino) Olive approx. 9 a [15]
Ficus carica L. Figs 12.9–18.2 [16]
Fragaria ananassa L. Strawberry 1–4 [17]
Floral Parts
Helleborus viridis L. agg. Sepals 2.3 [14]

Lilium hybrid L. (cv. Enchantment) Anther 2.3


[18,19]
Tepals 1.8
Flower 2.5
Spiranthes cernua L. Bud 3.7 [20]
Inflorescence 0.2
Caesalpinia virgata Torr. 7.8
[21]
Senna armafa L. 5.8
Prunus persica L. Stem 0.4–1.0 [22]
Spartium junceum L. approx. 8 [23]
Vitis vinifera L. approx. 0.8 (Fv /Fm ) [24]
Plants 2023, 12, 2393 3 of 29

Table 1. Cont.

Species Structure PN
(µmol CO2 m−2 s−1 ) References
Roots
Sonneratia alba Sm. 0.6
Pneumatophores [25]
Avicennia marina (Forssk.) 0.2
Tecticornia pergranulata (J.M.Black) Aquatic adventitious 0.5 [26]
K.A.Sheph. and Paul G.Wilson
a Measured as gross photosynthetic rate.

Early studies on fruit photosynthesis in climacteric apples and non-climacteric grapes


(both fleshy fruits), indehiscent fruits (e.g., cereal grain) and dehiscent fruits (e.g., pea pod)
were reviewed by Blanke and Lenz [27]. They reported that the photosynthetic profile
of each type of fruit was dependent on some morphological and anatomical character-
istics, as, for instance, pea pods have much less resistance to CO2 diffusion than fleshy
fruits [27]. Only a few studies have been performed on this research topic, often using
different experimental approaches. In addition, the relevant information is dispersed in the
literature and should be compiled for a better understanding of the drivers and constraints
for fruit photosynthesis to occur. Therefore, this review aims to compile and integrate
information focusing on the characteristics of fruits, including anatomical, physiological
and biochemical properties, as well as raising discussion on the possible functions of pho-
tosynthesis of fruits on their metabolism and development. Additionally, it also highlights
several physiological, biochemical and molecular aspects of the photosynthetic active grape
berry tissues, since recent studies brought new results and insights related to this research
topic [12,28–33].

2. Anatomical and Physiological Characteristics of Fruits


2.1. Cuticular Structure
Fleshy fruits are covered by an outer epidermis coated with a cuticle of variable
thickness, which is composed of cutin and impregnated with waxy or greasy layers [34].
During the development of fleshy fruits, the biosynthesis of cuticular wax is regulated by
environmental factors such as drought/humidity, light, temperature and pathogens (as
reviewed by Trivedi et al. [35]). Like in leaves, in fruits, the cuticle provides a waterproof
barrier between the epidermal cells and their relatively dry environment [36]. In addition,
during fruit growth and development, the cuticle maintains its integrity with the increasing
fruit volume and also plays a central role in protecting the fruit against biotic stresses (e.g.,
insects and fungi) and abiotic stresses (e.g., UV radiation) [34].
Several studies have been carried out with the aim of understanding cuticle formation
in fruits, including apple [37], grapes [38] and tomato [39]. In general, during fruit growth,
there is a rapid accumulation of wax in the cuticle, which makes it thicker and hinders
the diffusion of gases [27]. For instance, the cuticle of Riesling grape berries is present
since their earliest development, with an increasing rate of cutin and wax deposition at
the pre-veraison stage, while at later stages, along with the very rapid expansion of fruit
surface area, the cuticular material flattens out [40]. In oranges at later developmental
stages, several genes involved in the biosynthesis of wax, cutin and lignin are significantly
expressed, while genes involved in photosynthesis are repressed [41]. In pepper, the
removal of the cuticle increased the gas permeability of the fruit surface [42], suggesting
that the fruit cuticle is crucial in determining compositional gradients between the external
and internal atmosphere.
The pods of pea (Pisum sativum L.) have two distinct photosynthetic layers, i.e., the
outer (exocarp) and the inner (endocarp) epidermis, containing a thick and thin cuticle, re-
spectively. The exocarp controls the diffusion of CO2 from the outside atmosphere through
the stomata, while the endocarp is involved in the photoassimilation of CO2 diffusing from
the inner fruit cavity and mainly resulting from seeds respiration processes [43].
Plants 2023, 12, 2393 4 of 29

2.2. Stomata Density and Functionality


Although stomata are present in the outer epidermal layers of fruits, their density
is 10 to 100 times lower than in the abaxial epidermis of the respective plant leaves [9].
Despite this, in young fruits, the stomata are as sensitive to gases as in leaves and regulate
the rate of CO2 exchange. However, upon growing the fruit surface expands and the
density of stomata decreases, and in full-grown fruits, the diffusion resistance to CO2 is
mainly determined by the lenticels, which are small, round or elliptical, pore-like structures
derived from nonfunctional stomata [27]. Table 2 shows the stomatal density of some fruits
at a specific developmental stage.

Table 2. Stomatal density (number of stomata per mm2 ) of fruits of different species and at a specific
developmental stage.

Stomatal Density
Species Fruit Stage of Development References
(Number mm−2 )
1–2 Green and veraison
Vitis vinifera L. Grape berry [44]
<1 After veraison
200–300 Until 63 DAFB a
Citrus unshiu Marc. Satsuma mandarin [45]
~30 After 153 DAFB a
Cucumis sativa L. Cucumber 12.5 9 days after anthesis [13]
Ribes nigrum L. Black currant 0.18–0.20
Not referred [46]
Ribes rubrum L. Red currant 0.23–0.32
Malus domestica Borkh. Apple 10–20 At petal fall [27]
50–75 After anthesis
Persea americana Mill. Avocado [47]
<5 At maturation
6 After anthesis
Fragaria ananassa Duch. Strawberries 1–3 Before red color [17]
<1 Red fruit
Cicer arietinum L. Chickpea external pod wall 31 ± 3 Not referred [48]
30–35 At anthesis
Pisum sativum L. Pea pod [49]
24–26 Fully expanded pod
a DAFB—days after full bloom.

For instance, in grape berries, the stomata are functional until veraison, while after
that stage the stomata density decreases to less than one per mm−2 [44] and they become
nonfunctional (turning into lenticels covered by wax). These developmentally induced
changes result in a decrease in tissue transpiration and water loss, but also in higher CO2
and lower O2 concentrations inside the berry [50]. In cherry fruit the stomata are not
uniformly distributed over the surface of the epicarp: instead, they are found in high
numbers near the apex and only a few are present near the stem end [51,52]. In cucumber
fruit, the stomatal frequency is 12.5 mm−2 which is only 1.58% and 0.91% of the upper and
lower surfaces of leaves, respectively [13]. The stomata of a ripe apple are 30 times less
abundant than on the abaxial surface of the respective leaf [27]. In young peach fruits, the
stomata density is high, leading to a high conductance, while at maturity the stomata lose
their function and differentiate into lenticels [53]. Additionally, in chili peppers, stomata are
absent in the inner and outer epidermis of mature fruit, and gas exchange can only occur
through the cuticle [54]. Scanning electron micrography of mature olive fruits demonstrated
that the stomata are covered by a wax layer of complex architecture [15].
Stomata were also observed in the external surface of pods, but again at lower numbers
as compared to leaves of the same plant. For instance, in chickpeas, the external pod surface
has a mean stomatal density of 31 ± 3 mm−2 compared to 126 ± 6 mm−2 in leaves [48]. The
same study demonstrated a higher rate of transpiration and a lower water use efficiency
in old pods as compared to young pods, due to an increase in leakiness of the stomata
with pod aging. The inner epidermis of the endocarp of pea pods has thin cuticles lacking
Plants 2023, 12, 2393 5 of 29

stomata, while the outer epidermis, with thick cuticles, contains stomata at a density of
approximately 25% of that of the leaflet, suggesting its importance for the atmospheric
CO2 uptake [43] and regulation of water loss in the pods. In soybean pods, functional
stomata are present at the early stages of growth, thus enabling gas exchange [55]. Similarly,
the stomata of Brassica pods are more functional at early developmental stages compared
to later phases, with the stomatal conductance reaching a maximum at 30 days after
anthesis [56].
The grains of cereals have a green pericarp, in which stomata are occasionally present
on the external surface. In fact, there are contradictory data concerning this issue. Cochrane
and Duffus reported very few stomata in wheat pericarp, which are probably insufficient
for effective gas exchange with the outside environment [57]. On the other hand, Tambussi
et al. [58] did not find any stomata in the wheat pericarp, whereas high stomatal densities
were observed in the awns, glumes and lemmas. These latter authors suggested that the
photosynthetic activity of the green pericarp is dependent on CO2 internally generated by
the respiration of the endosperm cells.

2.3. Light Diffusion inside the Fruits


The morphology and anatomy of fleshy fruits (e.g., large volumetry) impose physical
constraints to light penetration into the inner tissues, eventually reducing the active pho-
tosynthetic zone to the outermost layers [12]. In addition to its intensity, also the quality
of light reaching the inner regions is influenced by the cells of the outer pericarp. The
presence of chlorophyll in green fruit strongly influences the spectral composition of the
light filtered through the fruit pericarp, as reviewed by Llorente et al. [59].
The photon flux density (PFD) transmission through the fleshy fruit skin ranges from
1 to 47% of the incident PFD and, generally, only 2% reaches the internal regions [9]. For
instance, the exocarp of grape berry transmits 47.1% of the incident PFD and that of apple
cv. Golden Delicious at about 31.4% [9]. In Citrus unshiu the photosynthesis was greater
in fruits than in leaves under considerably low PFD values (i.e., 13.5 to 68 µmol m−2 s−1 ),
suggesting that fruit photosynthesis resembles that of shade plants [45]. Green peel avocado
fruit transmitted only 1.5% of the incident light at 660 nm and 8.4% at 730 nm [47], consistent
with the spectral light absorbance characteristics of the chlorophylls present in the peel.
In inner tomato tissues, such as the locules, a high expression of genes associated with
photosynthesis can be observed [60]. Under a constant photosynthetically active radiation
(PAR) of 1750 µmol m−2 s−1 , the peel of young apple fruit transmitted 1–3% of incident
PAR at 400 nm, increasing to 10–12% at 850 nm [61], and with fruit development, this
light transmission into the fruit core decreased by up to 80%. Similarly, in olive fruits, the
penetration of PAR into the internal layers decreases upon development due to the increase
in fruit volume [15]. Chen and Cheng observed that the sun-exposed peel of apple fruit
had a higher photosynthetic O2 evolution capacity, as well as higher activities of enzymes
involved in the Calvin–Benson–Bassham (CBB) cycle, as compared to the shaded peel [62].
In the exocarp of cucumber fruit was verified the expression of key genes involved in the
photochemical phase, for example, associated with light-harvesting complexes (Lhca) of
photosystems I (PSI) and light-harvesting proteins (Lhcb) of photosystems II (PSII) [13]. In
the same fruit, the decrease in PAR from 200 to 50 µmol m−2 s−1 in the surface of cucumber,
led to a reduction of photosynthetic rate by 60–65% [63].
In pea pods up to 27 days after anthesis, the exocarp and mesocarp (pericarp) absorbed
about 67% of the incident PFD of 2200 µmol m−2 s−1 , while the endocarp received 10%
and the remaining 23% was transmitted to the seeds in the pod cavity [43]. At later
stages of pea pod development, the decline in chlorophyll content in the outer layers
resulted in an increase in PAR reaching both the endocarp and the seeds. In this manner,
there is a temporal separation between pods and seeds in their ripening-induced loss of
photosynthetic capacity. During day time, with an average of 1000 µmol m−2 s−1 sunlight,
developing soybean embryos receive moderate levels of light (5–30 µmol m−2 s−1 ) which
influences both the growth and the metabolism of the seed [64]. Similarly, in pods of
Plants 2023, 12, 2393 6 of 29

chickpeas, the light transmission through the seed coat increased with development, leading
to greater light utilization by the embryos [65].
In C3 cereals the complex structure of the ear makes it difficult for light to be trans-
mitted to the grain. In fact, the grain is surrounded by the lemma and palea, and it is
shaded by the glume, which results in low PFD levels reaching the green pericarp and
endosperm [58].

2.4. Chloroplasts and Photosynthetic Pigments


Microscopic observations revealed the presence of chloroplasts in different fruits, as
reviewed by Blanke and Lenz [27]. The density of chloroplasts in fruit tissues is generally
much lower than that in leaves, and thus the photosynthetic rate per unit of area (or per
gram of fresh or dry weight) is less as compared to that of leaves [9]. For instance, unripe
green strawberries contain 0.2–0.6 mg chlorophyll g−1 fresh weight, i.e., seven-fold less than
the respective leaves [17]. While in leaves chlorophyll is abundantly present in most cells, in
fruits its presence is mostly restricted to specific tissues or cell types (e.g., [12]). For instance,
in apple chloroplasts are only found in the hypodermal and inner perivascular tissue [66]. In
addition, chloroplasts from different fruit tissues may differ in their structure, composition
and function. Resorting to the apple example, the chloroplasts in the hypodermal layers
are smaller than those in the inner tissue, exhibit grana throughout fruit development and
contain starch granules, thus more resembling those found in leaves, and perform C3-type
photosynthesis [66]. On the other hand, chloroplasts from the perivascular tissue are larger
and present at smaller numbers per cell as compared to the respective leaf, apparently
lack starch grains and are characterized by a C4-type photosynthesis [66]. In the case
of cucumber, the chloroplasts appear in the inner walls of fleshy parenchyma cells and
contain grana stacks that are 1.7-fold larger than in leaves, while their quantity per unit
area is lower [13]. Avocado fruit contains sun-type chloroplasts, as found in leaves with C3
photosynthesis, that retain their structural integrity until harvest; these are comprised of
grana, with few thylakoids, of starch and of lower chlorophyll contents than the respective
leaves on the basis of surface area [47].
In grapes, the outer cell layer (i.e., the pericarp) of the flower ovary contains small
plastids with loosely arranged inter-granal lamellae and granal thylakoids and with abun-
dant starch grains; for this reason, they can be regarded as amyloplasts [67]. During the
post-anthesis period (i.e., between day 0 and approx. day 42 after anthesis) an increase in
chlorophyll content as well as in plastid density within the pericarp tissues is observed,
as a result of cell division and enlargement of the fruit [67]. Thereafter, the plastids ac-
quire a larger pleomorphic form, being largely devoid of starch granules until the last
stages of ripening while containing large lipid-like globules [67]. In the same study, it was
suggested that the plastids of the grape pericarp play a central role in the biosynthesis
of isoprenoids, including monoterpenes, and thus in the flavor and aroma of the mature
grapes and derived wine.
Chlorophyll fluorescence measurements show that several fruits species, at least dur-
ing their development and early ripening, possess a functional photochemical machinery—
e.g., mango [68], lemon [69], tomato [70], papaya [71], eggplant [72] and white grape
berries [12,28,29]. In fact, the light environment of fruits during their growth and de-
velopment is a determinant factor affecting fruit photosynthetic rate and the levels of
pigments and carbohydrates. For example, it has been demonstrated for mandarin cv.
Nules Clementine that fruits grown inside of the canopy had lower pigment concentrations
(both chlorophylls and carotenoids) and lower sugar levels as compared to those grown
outside of the canopy [73]. Similarly, the environmental light conditions influenced the
fluorescence ratio of each photosystem in eggplant fruits [72].
Throughout fruit development and maturation, the granal structure of the internal
chloroplasts disintegrates and the chlorophyll content decreases as a result of chlorophyl-
lase enzyme activity [74,75]. For instance, in ripening tomatoes, there is a fragmentation of
the thylakoid membrane of the green chloroplasts and the formation of colored chromo-
Plants 2023, 12, 2393 7 of 29

plasts with new carotenoid-bearing structures [76], as detected by confocal laser scanning
microscopy [77]. The decrease in chlorophyll content during ripening is observed in several
fruits, including papaya [78], olive [15], tomato [79], mandarin [73], apple [80], coffee [81]
and white grape berry [28,29].
In berry tissues of the white grape cv. Alvarinho, the chlorophyll content in the exocarp
decreased six-fold during ripening, from 120 to 20 µg g−1 fresh weight, while in the seed
integument, it decreased two-fold, from 80 to 40 µg g−1 fresh weight [28]. However,
transcriptional analysis of exocarp and seed tissue showed that the Chlorophyll Synthase
gene (VvChlSyn) keeps its relative expression of transcripts in both tissues until later stages
of development [31]. Interestingly, a recent work using plastids purified from berry skins
of the red grape variety Vinhão, showed an increase in the total chlorophyll content from
green to mature berries, suggesting that in this variety the ripening-associated transition
from green to red is not associated with a loss of chlorophylls [33]. The authors proposed
that the skin of these ripening berries maintains its chloroplasts (with chlorophylls), but
the green berry color is masked by the accumulation of anthocyanins [33]. Whether these
effects of ripening on chlorophylls in berries also hold true for other white or red grape
varieties still needs to be investigated.
During fruit ripening carotenoids and other pigments in fruits can undergo dramatic
changes in their content and composition, which are for instance dependent on the specific
fruit tissue and maturation stage (for more details see the recent reviews by Kappor
et al. [82] and by Simkin et al. [83]). Lado et al. [84] provided a comprehensive overview
concerning the main carotenoid profiles in fleshy fruits and the pattern of changes during
ripening and the different regulatory levels responsible for the diversity of carotenoid
accumulation in fruit tissues. In grape berries, β-carotene and lutein are the predominant
carotenoids and there is a steady decline of these compounds after veraison, which appears
to be related to chloroplast disappearance and to the formation of carotenoid-derived
norisoprenoid volatiles (e.g., β-ionone and β-damascenone) [85–87]. These volatiles are
important for wine aroma, because of their low olfactory perception threshold [88]. In
agreement with this, the total carotenoids content (i.e., the sum values of α-carotene, β-
carotene and lutein) in grape exocarp and the seed of the white cv Alvarinho decreased
during berry development [29]. Similarly, the expression of genes coding for key enzymes
of the carotenoid pathway in exocarp (i.e., phytoene synthase 1, VvPSY1; and lycopene
epsilon cyclase 1, VvLECY1) also decreased throughout berry development [31]. Overall,
these results suggest that the carotenoid pathway in both grape berry tissues favors the
direction towards α-carotene (i.e., the lutein-epoxide cycle) at the green stage, whereas at
veraison and mature stages, it relies on the violaxanthin cycle; in seeds both xanthophyll
cycles appear to operate at these later stages of development.
In addition, the light microclimate in the canopy also influences the pigments content of
both the exocarp and seed of grape berries: at the green stage of development, tissues from
berries growing at a high light microclimate (HL, receiving approx. 150 µmol photons m−2 s−1
on average) had higher contents of chlorophylls and carotenoids than those from low light
microclimate (LL, receiving approx. 50 µmol photons m−2 s−1 on average) [29]. Similar
HL-LL contrasting effects of light microclimate were observed for the photosynthetic
activity of both tissues, as determined by in vivo chlorophyll fluorescence [29]. Likewise,
the relative expression levels of genes coding for the photosynthesis-related enzymes, such
as chlorophyll synthase (VvChlSyn) and the expression of some key genes in the carotenoid
pathway (e.g., VvPSY1; lycopene betacyclase 2, VvLBCY2; and VvLECY1), were increased
by HL microclimate in both tissues, as compared to the LL condition [31]. In particular,
HL significantly increased VvChlSyn expression in seeds from berries at veraison and
mature stages [31], which result may be partly explained by the high dependency of this
specific fruit tissue on incoming light, which is consistent with HL seeds being better able
to acclimate their photosynthetic capacity to higher light intensities than LL seeds [28].
Pea pod walls also have chloroplasts in different cell layers. The outer epidermis of
the exocarp contains few chloroplasts, but they are abundant in the inner epidermis of the
Plants 2023, 12, 2393 8 of 29

endocarp (with smaller starch grains), as well as in the parenchyma layers of mesocarp [43].
In the case of chickpea pod walls, similar low levels of chlorophyll were detected in both the
embryo and the cotyledons, while the seed coat contained a layer of chloroplast-rich cells
directly below the epidermis [65]. The changes in chlorophyll levels occurring in soybean
pods are similar to that in fleshy fruits, with a decrease throughout development [55]. These
soybean pods contained 9.2–12.4 times less chlorophyll per gram fresh weight than their
respective leaves. However, the gross photosynthetic rates on a per mg chlorophyll basis
were greater in pods than in leaves, which may be important for the rapid seed-filling
period of pods [55].
In cereals, the total chlorophyll content on a dry weight basis is lower in ear parts than
in the flag leaf [89]. This difference may be explained by the distinct light microenvironment
of these two structures, with the ear localized at the top of the cereal plant and the flag leaf
more shaded below. In fact, these authors relate the lower chlorophyll/carotenoid ratio in
glumes, lemmas and awns of the wheat ears, as compared to the flag leaf, to their higher
need for photoprotection. During the development of the wheat grains, the maximum
chlorophyll content in the pericarp green layer was observed 20 days after anthesis, when
there was also a peak of photosynthetic activity [90].

2.5. Assimilation and Refixation of Internal CO2


Fruit gas exchange with the external atmosphere takes place mainly through stomata
and depends on a diversity of morphological and physiological aspects, namely: fruit type
and size, fruit ontogeny stage, fruit temperature, shading and incident PFD, and chlorophyll
content [91]. For instance, Aschan and Pfanz [9] mentioned that fleshy fruits perform
basically internal CO2 refixation, whereas dry fruits, especially during their younger stages,
are also able to assimilate atmospheric CO2 . The incident light also influences the rate of
CO2 fixation: e.g., in mandarin fruit, the light-saturated net CO2 assimilation rate (Amax )
(measured with a portable photosynthesis system using a chamber) was significantly higher
in fruits located in the outer canopy than in those in the inner canopy [73]. Overall, fruits
assimilate less atmospheric CO2 via ribulose-1,5-bisphosphate carboxylase/oxygenase
(RuBisCO) when compared to the respective leaves (Blanke and Lenz, 1989). In oranges, as
an example, fruits assimilate 50% to 75% less atmospheric CO2 than leaves [73].
As outlined above, at early development the cuticular and stomata characteristics
enable gas exchange in both fleshy and dry fruits. Then, during fruit ontogeny, the great
accumulation of wax on its surface leads to a reduction of CO2 atmospheric exchange
rate (e.g., 10-fold in the case of fleshy fruits) [27] and a simultaneous reduction of water
loss via transpiration. Moreover, the increase in mitochondrial respiratory processes,
predominantly fueled by imported photoassimilates from leaves at these later stages,
results in a rise in the internal CO2 concentration [27]. Thus, in the cytosol, the “excess”
of internal CO2 can be converted by carbonic anhydrase into bicarbonate ions (HCO3 − ),
which can be subsequently re-fixed by phosphoenolpyruvate carboxylase (PEPC).
In green and red-turning tomato fruit, PEPC enzyme activity is two times higher
than RuBisCO activity [70]. Similar results have been obtained for coffee green pericarp,
in which the ratio PEPC/RuBisCO activity was higher than in the respective leaves [92].
In the mesocarp of pre-climacteric avocado fruits, the CO2 recycling occurs by PEPC,
with an enzyme activity around 2.5 µmol CO2 s−1 g−1 fresh weight [47]. In mandarin
fruit uncovered with paper bags, the high CO2 refixation by PEPC was suggested to
contribute to a higher final sugar content at harvest, when compared with fruits covered
with bags [93]. More recently, the same authors suggested the presence of a C4/CAM
(crassulacean acid metabolism) photosynthetic mechanism in mandarin fruits, based on
14 CO labeling experiments [45]. Additionally, by using 14 CO incorporation, it was
2 2
calculated that PEPC enzyme activity in the skin and mesocarp of grape berries was about
17 times higher before veraison than after, leading to a higher concentration of malate and
therefore to acidic berries [94]. Further, in apple fruit, PEPC activity was accompanied
by malic acid synthesis and a simultaneous regulation of cytoplasmic pH [95], as it is
Plants 2023, 12, 2393 9 of 29

in tomato fruit [96]. More recently, a first quantitative study on in vivo respiratory CO2
recapture in non-climacteric fleshy fruits showed that cucumbers, which can assimilate
atmospheric CO2 via RuBisCO, capture approximately 88% of respiratory CO2 via PEPC;
this fixation route was quantitatively more important than the direct CO2 fixation from the
atmosphere [13].
Additionally, in the ears of cereals, such as barley and bread/durum wheat, CO2
refixation is considered a quantitatively relevant process (as reviewed by Hu et al. [97]
and Tambussi et al. [58]). In fact, awns and the external surface of glumes (where stomata
are abundant) are the main structures responsible for external CO2 assimilation, while the
green pericarp and internal surfaces of lemmas (facing the grain), the recycling of respired
CO2 is the principal process (reviewed by Tambussi et al. [58]). Indeed, it was observed that
PEPC plays a role in CO2 respiratory refixation, both in barley ears [98] and in chloroplasts
of pericarp cells and glumes of durum wheat [99]. Thus, the recycling of respired CO2 (i.e.,
refixation) could have a considerable impact on the final yield of cereal crops, by preventing
C loss [97].
In legumes, respiratory CO2 released from the embryo is re-fixed by a layer of cells
on the inner pod wall. In planta, experiments with isotopically labeled CO2 (i.e., 13 CO2
injected into the pod space) showed that more than 80% of the label was fixed by the pod
walls of chickpea, rather than the seed tissues [65]. This internal recycling of CO2 inside
the pod may assist in maintaining seed filling in water-stressed chickpea plants [48].

3. Biochemical Pathways Proposed for Fruit Photosynthesis


More than 30 years ago, Blanke and Lenz [27] proposed a biochemical process for
the respiratory CO2 refixation, called the malate-CO2 shuttle (Figure 1). However, this
process could not be generalized for all fruits, as it may vary with the type of fruit, type of
tissue within a fruit, the stage of development, etc. Indeed, and based on the available data,
Blanke and Lenz suggested that fruits have their own type of photosynthesis, which is an
Plants 2023, 12, x FOR PEER REVIEWintermediate status between C3 and C4/CAM photosynthesis, 10 of 32 rather than categorizing
them strictly in C4-type photosynthesis [27].

Figure 1. Malate-CO2 shuttle proposed by Blanke and Lenz (1989). Abbreviations: HCO3−,
Figure 1.ion;
bicarbonate Malate-CO shuttle proposed
NAD-MDH, 2NAD-linked by Blanke
malate dehydrogenase; and Lenz
NADP-ME, (1989).malic
NADP-linked Abbreviations: HCO3 − , bicarbon-
enzyme;
ate ion;PEPC, phosphoenolpyruvate
NAD-MDH, NAD-linkedcarboxylase;
malatePPDK, pyruvate orthophosphate
dehydrogenase; NADP-ME,dikinase; NADP-linked malic enzyme;
RuBisCO, ribulose-1,5-bisphosphate carboxylase/oxygenase; TCA, tricarboxylic acid cycle. Adapted
PEPC,
from phosphoenolpyruvate
Blanke carboxylase;
and Lenz (1989) [27]. Reproduced PPDK,
with permission frompyruvate orthophosphate
Michael Blanke, Fruit dikinase; RuBisCO,
photosynthesis; published by John Wiley & Sons Ltd., 1989.
ribulose-1,5-bisphosphate carboxylase/oxygenase; TCA, tricarboxylic acid cycle. Adapted from
Blanke
Here,and
the Lenz
excess (1989) [27]. Reproduced
of respiratory with permission
CO2 is firstly transformed from
into HCO3− by Michael
carbonic Blanke, Fruit photosynthesis;
anhydrase in the cytosol. Subsequently, a β-carboxylation step of phosphoenolpyruvate
published by John Wiley− & Sons Ltd., 1989.
(PEP) with the produced HCO3 takes place, irreversibly catalyzed by PEPC, to synthesize
oxaloacetate (OAA), which in turn is reduced to malate by a cytosolic NAD-dependent

malate Here, the excess
dehydrogenase of respiratory
(NAD-MDH). Malate can beCO 2 is firstly
translocated to the transformed
chloroplast and into HCO3 by carbonic
then decarboxylated by NADP-malic enzyme, resulting in CO 2 and pyruvate; the latter
anhydrase in the cytosol. Subsequently, a β-carboxylation step of phosphoenolpyruvate
can be regenerated into phosphoenolpyruvate by pyruvate phosphate dikinase (PPDK).
(PEP) with
The released COthe produced HCO3 −in takes
2 can be fixated by RuBisCO the CBBplace, irreversibly
cycle, while pyruvate andcatalyzed
malate by PEPC, to synthesize
oxaloacetate
can (OAA),
be directed to other which
metabolic in turn
pathways, such is
as reduced to malate
the tricarboxylic acid (TCA)by a cytosolic NAD-dependent
cycle
and gluconeogenesis or, in the case of malate, accumulated in the vacuole.
Despite the similarities between this mechanism and C4-type photosynthesis, it is
argued that the fruits cannot be cataloged as C4-types, since they do not present Kranz
anatomy [27]. However, it is also mentioned that C4 photosynthesis may be also achieved
by compartmentalization within the cell [100] without the presence of Kranz anatomy
[101]. Additionally, a proxy to CAM mechanism does not imply a spatial separation or
Kranz architecture, but a temporal separation of the two carboxylation reactions,
Plants 2023, 12, 2393 10 of 29

malate dehydrogenase (NAD-MDH). Malate can be translocated to the chloroplast and


then decarboxylated by NADP-malic enzyme, resulting in CO2 and pyruvate; the latter can
be regenerated into phosphoenolpyruvate by pyruvate phosphate dikinase (PPDK). The
released CO2 can be fixated by RuBisCO in the CBB cycle, while pyruvate and malate can
be directed to other metabolic pathways, such as the tricarboxylic acid (TCA) cycle and
gluconeogenesis or, in the case of malate, accumulated in the vacuole.
Despite the similarities between this mechanism and C4-type photosynthesis, it is
argued that the fruits cannot be cataloged as C4-types, since they do not present Kranz
anatomy [27]. However, it is also mentioned that C4 photosynthesis may be also achieved
by compartmentalization within the cell [100] without the presence of Kranz anatomy [101].
Additionally, a proxy to CAM mechanism does not imply a spatial separation or Kranz
architecture, but a temporal separation of the two carboxylation reactions, eventually
associated with the fruit developmental stage.
Recently, Henry et al. [102] reviewed the pathways of photosynthesis in non-leaf
tissues, including stems, petioles, seeds and fruits. They verified that, in general, the C4
pathway has been reported in these non-leafy tissues in plants that employ C3 photosyn-
thesis in the leaf. Moreover, the authors proposed a process of C4 photosynthesis for those
structures, partitioned between the inner shaded tissues (e.g., cells of the endosperm
Plants 2023, 12, x FOR PEER REVIEW 11 of 32 in
seeds of wheat), which are characterized by high levels of respiratory CO2 , and the outer
green tissues (e.g., pericarp of wheat) which are more exposed to the light (Figure 2).

Figure 2. Photosynthesis in non-leaf tissues in outer and inner parts of the organ, proposed by Henry
Figure 2. Photosynthesis in non-leaf tissues in outer and inner parts of the organ, proposed by
et al. [102]. Key reactions in inner tissues (without light) to capture respired carbon: PEPC,
Henry et al. [102]. Keycarboxylase;
phosphoenolpyruvate reactions inMDH,
innermalate
tissuesdehydrogenase.
(without light) to capture
Reactions respired
in outer carbon:
tissues (with PEPC,
phosphoenolpyruvate
light): ME, malic enzyme; carboxylase;
RuBisCO,MDH, malate dehydrogenase.
Ribulose-1,5-bisphosphate Reactions in outer PPDK,
carboxylase/oxygenase; tissues (with
Pyruvate
light): ME,orthophosphate
malic enzyme; dikinase. Adapted
RuBisCO, from Henry et al. [102]. Reproduced
Ribulose-1,5-bisphosphate with permission PPDK,
carboxylase/oxygenase;
from Robert
Pyruvate Henry, Pathways
orthophosphate of Photosynthesis
dikinase. Adapted from in Non-Leaf
Henry Tissues; published
et al. [102]. by MDPI,
Reproduced 2020.
with permission
from Robert Henry, Pathways of Photosynthesis in Non-Leaf Tissues; published by MDPI, 2020.
This internal recycling of respired CO2 can be an evolutive adaptation of fruits and
other non-foliar
This internal photosynthetic tissues toCO
recycling of respired avoid carbon losses and thus locally improve
2 can be an evolutive adaptation of fruits and
their carbon balance [9]. In fact, Henry
other non-foliar photosynthetic tissues to avoid et al. [102] stated that losses
carbon the main andcontribution
thus locally of the
improve
photosynthesis in non-leaf tissues seemed to be associated with the
their carbon balance [9]. In fact, Henry et al. [102] stated that the main contribution of need to re-capture
carbon, especially in storage organs that have high respiration rates.
the photosynthesis in non-leaf tissues seemed to be associated with the need to re-capture
For grape berries, Beriashvili and Beriashvili suggested the operation of two main
carbon, especially in storage organs that have high respiration rates.
photosynthetic routes, whereby CO2 was assimilated primarily as malate in the early
For grape berries, Beriashvili and Beriashvili suggested the operation of two main
stages of development (C4-type photosynthesis, involving PEPC) and subsequently
photosynthetic routes, whereby CO2 was
primarily as sugars in ripening berries
assimilated primarily as malate in the early stages
(C3-type photosynthesis) [103]. This hypothesis is
ofconsistent
development (C4-type photosynthesis,
with the strong malic acid vs. sugar involving PEPC)
accumulation and subsequently
patterns during developmentprimarily as
sugars in ripening
and ripening, berries (C3-type
respectively. The declinephotosynthesis)
in expression [103].
of twoThis hypothesis
putative is consistent
PEPC isogenes and with
the strongenzyme
of PEPC malic acid vs. sugar
activity accumulation
from green to veraison patterns during
stages [104], development
supports andofripening,
this theory a
ripening-induced photosynthetic switch. In fact, Sweetman et al. [105] suggested that the
accumulation of malic acid is in large part due to de novo synthesis in grape berry fruits,
through the metabolism of photoassimilates translocated from leaf tissues, as well as those
resulting from the photosynthetic activity within the fruit itself. In agreement with this
Plants 2023, 12, 2393 11 of 29

respectively. The decline in expression of two putative PEPC isogenes and of PEPC enzyme
activity from green to veraison stages [104], supports this theory of a ripening-induced pho-
tosynthetic switch. In fact, Sweetman et al. [105] suggested that the accumulation of malic
acid is in large part due to de novo synthesis in grape berry fruits, through the metabolism
of photoassimilates translocated from leaf tissues, as well as those resulting from the pho-
tosynthetic activity within the fruit itself. In agreement with this suggestion, proteomic
studies revealed that the skin of ripe berries still contains detectable amounts of proteins
with functions related to photosynthesis and carbon assimilation [106]. Indeed, both the
large subunit and the subunit binding protein alpha of RuBisCO were more abundant in
the skin than in the pulp (mesocarp), in accordance with previous studies [107,108]. Thus,
the expression pattern of the carbon assimilation proteins indicates that the skin might
retain a functional photosynthetic apparatus [106]. In accordance with this, the results of
our group using chlorophyll fluorescence analysis demonstrated that grape berry exocarp
exhibits a much higher photochemical efficiency than the mesocarp [12], and it keeps
this photosynthetic activity until later stages of development [28,29]. In addition, light
microclimate also influenced the photosynthesis-related transcripts: at the green stage, HL
exocarps showed a significantly higher expression of VvRuBisCO (>2-fold) in comparison to
LL exocarps [31], in line with the results of their photosynthetic activity [29]. Moreover, the
relative expression of VvRuBisCO in exocarp increases during berry development, similar
to the expression patterns of the VvSPS1 gene [31], which encodes the sucrose-phosphate
synthase enzyme that is involved in sucrose biosynthesis. These results suggest an active
photosynthetic/CBB cycle function until late in berry development in both grape berry
tissues, and particularly in the exocarp. Thus, from the veraison stage onwards the C-
intermediates (e.g., triose-phosphates, erythrose-4-phosphate and 3-phosphoglyceric acid)
inside the berry chloroplasts or from the chloroplasts can fuel the biosynthesis of other
metabolites.
In apple, a similar malate metabolism and simultaneous recycling of respiratory CO2
was verified by Blanke [95]. In fact, the PEPC of apple fruit is a very efficient enzyme with
high affinity to its two substrates, HCO3 − and phosphoenolpyruvate, similar as in CAM or
C4 photosynthesis. Here, PEPC gene expression was high only until fruit set, decreasing
thereafter during maturation; thus, the further fruit metabolism mostly depends on its
initial PEPC protein resource. Malate dehydrogenase was 80-fold in excess in comparison
with PEPC, but both were concentrated in the vascular tissue linking the calyx, core and
peduncle.
In addition to the reported differences between fruits, it is possible that these mecha-
nisms can occur in the same fruit (spatially or temporally separated) and may or may not be
interconnected. In this way, photosynthesis can occur in a typical C3 way (photochemical
phase and CBB cycle with CO2 fixation by RuBisCO); in a type C4/CAM way, with internal
CO2 refixation with production of malate, which after being decarboxylated, is fixed by
RuBisCO in the CBB cycle; or the CO2 fixation being just/mainly internal HCO3 − refixation
with malate production, which can be used for other purposes. All these mechanisms may
depend on the tissue, developmental stage, light microclimate, among other factors.

4. Possible Roles of Photosynthesis in Fruits


Considering all the anatomical, histological, physiological and biochemical aspects,
as well as temporal/developmental effects discussed above, in the present section the
possible roles of photosynthesis in fruits will be addressed, as well as the particular case of
photosynthesis in seeds.

4.1. The Relationship between Photosynthesis and Metabolism in Fruits


The role of photosynthesis in fruit metabolism, and consequently on its quality, has
been extensively discussed in tomato [109–113]. The main objective of those works was to
obtain answers for the questions: What is the contribution of fruit photosynthesis to fruit
metabolism before ripening? Does it have any effect on the final quality of the fruit?
Plants 2023, 12, 2393 12 of 29

In tomato fruit, genetic and molecular approaches have been applied to understand
the relationship between photosynthesis and fruit metabolism, composition and nutritional
value. For instance, experiences with transgenic tomato plants, where the expression of
chloroplastidial isoform of fructose 1,6-bisphosphatase (cp-FBPase) was inhibited using
antisense technology, were performed [109]. The FBPase catalyzes the conversion of
fructose 1,6-bisphosphate to fructose 6-phosphate. In green plant tissues there are two
isoforms, being the plastidial isoform an important enzyme for the control of CBB cycle. The
results showed that the transgenic lines had few changes in their carbohydrate metabolite
levels, probably due to its ability to import sugars from leaves. The authors explained
this by considering that if there is indeed repression of photosynthesis in the fruits of
the transgenic lines, an increased sucrose import becomes necessary as compensation. In
addition, the same authors observed that the repression of cp-FBPase enzyme led to a
reduction in the average weights of fully ripe fruits. Interestingly, this decrease in weight
was quantitatively similar to the estimated contribution of the fruit to the photoassimilates
production, that is, around 15–20%, although it represents a minor component when
compared to that imported from the leaves [113].
In another study in tomato, a downregulation of an auxin response factor (i.e., ARF4,
which is from a class of transcription factors that regulate auxin-mediated gene expression)
resulted in a higher transient starch accumulation at the early stages, together with an
enhancement of chlorophyll content and photochemical efficiency, which is consistent with
the idea that the photosynthetic activity of fruit may be responsible, at least partially, for the
production of photoassimilates and, consequently, for the high levels of starch [112]. In ad-
dition, it was verified that the overexpression of the transcription factor SlGLK2 (important
for chlorophyll accumulation and distribution in developing fruit), led to an enhancement
in the expression of genes associated with fruit photosynthesis and chloroplast develop-
ment, and also to an increase in carbohydrates and carotenoids in ripe tomato fruit [111].
These studies in tomato point to a contribution of photosynthesis in supplying assimilates,
such as sugars/starch and other metabolites needed for the growth of the fruit (e.g., lipids)
at the early stages of fruit development, whereas at later stages this photosynthesis may
supply carbon sources to other biosynthetic pathways, such as carotenoids or compounds
important for the final quality of the fruit.
Moreover, these studies also pointed to the importance of fruit photosynthesis to
carbon use efficiency, and its implications on fruit yield and quality, as recently reviewed by
Simkin et al. [11]. Ultimately, manipulation of key processes to increase fruit photosynthesis
could provide a novel target for breeding programs or genetic engineering, with the prime
objective to improve fruit and cereal crop yield, quality and nutritional value, as reviewed
by Simkin et al. [11,114,115]. For instance, transgenic wheat plants with increased activity
of the CBB cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase), revealed increased
gross photosynthesis in their ears relative to the wild-type control [116]. Similarly, in
tomatoes, an increase in SBPase activity results in a significant (>30%) increase in plant
biomass [117].
The contribution of fruit photosynthesis to the overall carbon budget (as shown in
Table 3), was assessed by: (1) calculations using CO2 gas exchange measurements, dry
weight and carbon content (e.g., [118]); (2) 14 CO2 feeding and gas exchange [13]; or (3) by
calculations using electron transport rates, determined by pulse amplitude modulated
(PAM) fluorometry, and surface area [119]. However, it is important to be aware of the
challenges and methodological limitations associated with measuring photosynthesis in
non-foliar tissues, as reviewed by Lawson and Milliken [120] and by Simkin et al. [11]. For
instance, the gas exchange technique can underestimate photosynthetic rate, due to the
two possible sources of CO2 , the atmosphere and the mitochondrial respiration (or other
internal sources). Alternatively, combining thermography and other approaches, such as
chlorophyll fluorescence, can provide insights into spatial and temporal stomatal behavior,
which may be valuable to elucidate and quantify atmospheric CO2 fixation from other CO2
sources [120].
Plants 2023, 12, 2393 13 of 29

Table 3. Fruit photosynthesis contribution to total carbon (%).

Fruit Photosynthesis
Fruit Stage of Development Tissue Contribution to Total References
Carbon (%)
Grape berry Several Whole/attached fruit 10 [121]
Tomato Green (near mature) Skin 15 [119]
Blueberry From anthesis through fruit ripening Whole/attached fruit 15 [118]
Mango Mature (around 1–2 weeks before ripening) Skin 1 [68]
Several: from 24 days after flowering until
Peach Whole/attached fruit 5–9 [122]
harvest (in biweekly intervals)
Pea pod -- -- 16–20 [123]
Olive Several: from full bloom to harvest Whole fruit 40 [15]
Coffee -- -- 20–30 [92]
Cucumber Mature (i.e., 9–10 days after anthesis) Exocarp (i.e., skin) 9.4 [13]
-- Not referred.

However, this is only about carbon, and photosynthesis can provide more than car-
bohydrates or even C-skeletons. Therefore, understanding the various functions of fruit
photosynthesis is crucial, since it can provide a potential route for manipulating key pho-
tosynthetic genes to enhance fruit development, composition, yield or nutritional quality,
particularly under conditions of stress when leaf photosynthesis may be compromised.
In that regard, reviews concerning the photosynthetic characteristics of non-foliar organs
in C3 cereals concluded that overall, the non-foliar organs (e.g., the ears), had higher tol-
erance to environmental stresses (e.g., water stress), as compared with the leaves [58,97].
A possible explanation for this difference is that water relations in these organs are con-
siderably different to foliar tissue and stomata could play a key role in this regulation (as
reviewed by Tambussi et al. [58] and Lawson and Milliken [120]). In fact, stomata have
been found on both the adaxial and abaxial sides of glumes and lemma of the ears [11],
and therefore, the stomata can support both atmospheric CO2 uptake from one side of the
tissues and refixation of respiratory CO2 uptake on the other side [120]. Thus, the capacity
of photosynthetic components under drought (delayed senescence), might help the ear
to continue to fix CO2 late in the grain-filling period [58,97]. Still concerning the cereal
crops, recently, Araus et al. [124] and Sanchez-Bragado et al. [125] reviewed the different
strategies to increase the photosynthetic rates and the impact of these strategies in terms of
increasing biomass and/or yield of non-laminar organs. The authors presented a summary
of the net photosynthesis rate in different green photosynthetic organs of C3 cereals. For
instance, they observed that the ear photosynthesis per unit area is, in general, lower than
flag leaf photosynthesis, but, when expressed per whole organ, ear photosynthetic values
may be even higher than in the flag leaf and could significantly contribute to total canopy
photosynthesis [124].
This theme is complex and still controversial. In fact, Lytovchenko et al. [126] men-
tioned that tomato fruit photosynthesis is not important for photoassimilate accumulation,
including those metabolites impacting taste, and consequently it is not required for fruit
metabolism and development. However, the same authors verified that tomato photosyn-
thesis had a considerable role in seed development, impacting on seed set, composition,
and morphology during the early developmental stage. In the same way, a recent review
about the primary metabolism of apples, suggested that apple photosynthesis may be
mainly used to support seed development and accumulate malate via the malate-CO2
shuttle [127]. This hypothesis was based on the low rates of CO2 assimilation in immature
apples, as compared to those of the leaves, meaning that the CO2 fixed in the peel also has
little effect on growth and development in this crop [128,129].
Regarding grape berry, studies applying transcriptome [104,130–132], proteome [106,133,134]
and metabolome [130,134,135] analyses, and integration of these data into omics networks
to identify putative stage-specific biomarkers [136,137], as well as gene co-expression
networks [138], identified components directly related to photosynthetic activity before
Plants 2023, 12, 2393 14 of 29

veraison, namely light harvesting complexes, the photosystem II oxygen evolving complex
and CBB cycle enzymes. In particular, the downregulation of photosynthetic genes after
veraison was first analyzed by expressed sequence tag (EST) profiling [139,140] and con-
firmed by microarray analysis in grape berries [104,137,141,142], and specifically in berry
skins [143], wherein photosynthesis-related transcripts are more abundant [131]. Overall,
these results are in line with our data obtained by chlorophyll fluorescence in the grape
tissue exocarp and seed [12,28,29]. Interestingly, a recent proteomic work using the red
grape berry skins (cv. Vinhão), showed that several proteins of the light reactions (e.g.,
antenna complex, PSII, PSI, cytochrome b6/f, and ATP synthase) were significantly accumu-
lated in the chloroplasts at the mature stage as compared to green stage [33]. Moreover, the
transition from the green to mature stage was accompanied by a strong decrease in proteins
involved in the biosynthetic reactions of the CBB cycle [33]. The authors suggest that at
the green stage the CBB cycle can provide carbon compounds to the biosynthesis of amino
acids, whereas at the mature stage the chloroplasts may provide adenosine triphosphate
(ATP) for cell maintenance and metabolism (energy-demanding processes) or even O2 to
feed the respiratory demand of inner tissues [33]. Clearly more studies, preferably with
different grape varieties, are needed to conform these proteomic results and draw general
conclusions.
Indeed, the CBB cycle can supply precursors for pathways of primary and secondary
metabolism (as reviewed for tomato by Cocaliadis et al. [113]; and for apple by Tijero
et al. [127], who highlighted the importance of apple photosynthesis for its primary
metabolism). Primary metabolites, such as sugars, organic acids, and amino acids, pro-
duced at the early stages of development, determine most of the final fruit quality, as they
are the main substrate for many reactions of secondary metabolism. Secondary metabo-
lites, such as phenolic compounds, play important roles in plant defense against biotic
and abiotic factors [144]. Additionally, the diversity of compounds contributes for the
nutritional quality of fruits and for its organoleptic properties, which are important to make
them attractive and palatable. For instance, in grape berries, phenolics contribute to the
color, taste, texture and astringency of the wine, as well as to its antioxidant properties and
beneficial effects on health [145,146]. These compounds are mainly present in the exocarp
and seeds of the grape berries [30,147], both photosynthetically active [12,28,29].
In particular, large-scale untargeted metabolomics analysis showed that the exocarp
from the green stage had relative high levels of flavan-3-ols and of stilbenes, including
resveratrol, when compared to the mature stage [30]. Similarly, the expression of genes
from the phenylpropanoid (i.e., phenylalanine ammonia lyase 1, VvPAL1) and stilbenoid
(i.e., stilbene synthase 1, VvSTS1) pathways, as well as of those associated with flavan-3-ols
biosynthesis (i.e., dihydroflavonol reductase, VvDFR; leucoanthocyanidin reductase 2,
VvLAR2; and anthocyanidin reductase, VvANR), was also higher at the green stage than
in subsequent stages [31]. Moreover, the exocarp showed an increase in the abundance
of several flavonol glycosides along berry ripening [30], which was consistent with the
expression of flavanol synthase 1 gene (VvFLS1) [31]. Thus, the CBB cycle in the exocarp,
which is still active until later stages of berry development (c.f. VvRuBisCO expression)
can contribute with carbon skeletons needed in the flavonoid pathway. However, further
studies are required to better understand this possible crosstalk/relationship between
photosynthesis/photochemistry/CBB cycle and secondary metabolism in grape berry.

4.2. The Particular Case of Photosynthesis in Seeds


In addition to fruit tissues, some seeds are green (chlorophyll-containing) during part
of their development (e.g., grape berry seeds—[28]; immature coffee grains—[148]), or
even until germination [149]. During maturation, the seeds lose their green color due to
a decrease in chlorophylls and carotenoids, as seen for grape berry seeds [29] and coffee
grains [148]. Although the machinery and the mechanism of the photosynthesis of leaves
and green seeds seem to be quite similar, there are differences in characteristic features
between the leaf and the green seed photosynthesis in higher plants, as recently revised
Plants 2023, 12, 2393 15 of 29

by [149]. For instance, as compared to the leaves, the photosynthetic green seeds have: low
chlorophyll a and b content; high chlorophyll a to b ratio; chloroplast with broad granal
stacks; abundant PSII associated to proteins; higher PSI activity than PSII activity; and high
potential to harvest at low PFD [149].
Research has been conducted to understand the specific physiological functions of
photosynthesis in these organs [64,149–153]. In general, seed photosynthesis may con-
tribute in three distinct ways: (1) supply of oxygen to prevent and/or reduce hypoxia and
hence to aid the high respiration rates during seed germination; (2) production of nicoti-
namide adenine dinucleotide phosphate (NADPH) and adenosine triphosphate (ATP),
both fundamental for energetically demanding biosynthetic pathways in the chloroplast
such as fatty acid synthesis; (3) provision of C-intermediates for primary and secondary
metabolism by the CBB cycle; and (4) re-fixation of respiratory CO2 by RuBisCO, which
process can improve the energy efficiency of seeds.

4.2.1. Possible Functions of O2 , ATP and NADPH from the Photochemical Phase
Most seeds have some peculiarities that hinder the absorption of oxygen, such as
a thick coat and the accumulation of proteins and oils that become a glassy matrix dur-
ing desiccation [154], or being located deep in dense mesocarps as in some fleshy fruits
as the grape berry [12]. Thus, during the maturation and desiccation process, there is a
decrease in oxygen diffusion into the dense inner seed tissues. This situation of hypoxia
causes restrictions to the production of ATP by mitochondria (oxidative phosphorylation),
which is pivotal for various metabolic pathways during seed development and embryo
maturation [155–157]. Hypoxia can directly or indirectly affect several other processes
in seeds, such as nutrient uptake (e.g., wheat—[155]), storage activity and metabolite
distribution (e.g., soybean—[151]), assimilate partitioning between endosperm and em-
bryo (e.g., maize—[158]), and enzymatic activities associated with lipid metabolism (e.g.,
rapeseed—[157]). It is appealing to assume seed photosynthesis to the production of O2 in
the photochemical phase thus avoiding hypoxia conditions inside of seeds.
Monocotyledonous barley caryopsis (Hordeum vulgare L.) has a green pericarp with
chlorophyll (called chlorenchyma), where photosynthesis occurs in the mid-storage stage [159].
Chlorophyll fluorescence images of the effective quantum yield of PSII allowed to verify that
the photosynthetic activity was restricted to the chlorenchymatous regions of the pericarp,
and the photosynthetic activity in these regions was responsible for the production of
3.5 µmol NADPH h−1 and 2.3 µmol ATP h−1 , both important for storage [152]. The oxygen
distribution (“oxygen maps”) in barley allows to better understand the photosynthetic
activity in its tissues [160]. In general, the pericarp exhibits high levels of oxygen, while in
the central regions, there is an oxygen deficiency. In the dark, the oxygen levels decrease
dramatically in the inner endosperm region and in the transfer cells (transport pathway of
assimilates to endosperm) [161]. On the other hand, in the light, this reduction was not so
great, suggesting that the oxygen released by photosynthesis may play an important role in
nutrient transport to the endosperm. In addition, the expression of photosynthesis-related
genes peaks before the assimilate storage phase [162].
Legume seeds, such as soybean (Glycine max (L.) Merril), present an embryo that
turns green at the early stages of development. Approximately only 10% of incident light
is available to the embryo surface [151]. However, soybean embryos exhibit specialized
chloroplasts with high grana stacking. The oxygen map of soybean showed that the seed
coat presents high concentrations of oxygen (at the early storage stage), but these values
decrease to minimum levels within the liquid endosperm. Furthermore, when measured in
the dark, the oxygen concentration within the embryo is much lower (2 µM) comparatively
to that under light conditions (220 µM) [151]. At the late-storage stage, soybean embryos
have less oxygen concentration and a lower variation (in the same conditions), suggesting
a decline in the capacity to balance oxygen consumption with its supply [151]. Pulse
amplitude modulated (PAM) fluorescence analysis allowed confirming this situation. In
fact, the effective quantum yield of PSII showed a homogeneous pattern for small embryos
Plants 2023, 12, 2393 16 of 29

at the early storage stage, but at the mid-/late storage stage, there was a gradient declining
towards the interior of the embryo, suggesting a gradual loss of photosynthetic ability [163].
Studies in grape berries using chlorophyll fluorescence imaging have demonstrated
that, in addition to the exocarp, the outer integument of the green seed had a very high
fluorescence signal corresponding to high effective quantum efficiencies of PSII [12]. Later
on, it was shown that the seed integument had higher values for photochemical efficiency
and capacity at green stages of development, similar to those of the exocarp, decreasing at
later stages [28,29]. Despite the increase in the volume of the grape berry throughout the
development stages, seeds can receive diffuse transmitted light (2.3%), as already reported
by Aschan and Pfanz [9], allowing photosynthetic activity at later stages even if at lower
levels. Therefore, and based on the light intensity that we previously measured at the grape
berry surface level [29], the grape berry seeds received an estimated mean light intensity
as low as 1.15 and 3.35 µmol photons m−2 s−1 , at LL and HL microclimate, respectively,
which nevertheless support distinct photosynthetic activities in the seeds [29].
This photosynthetic activity of grape seeds can provide the O2 necessary to avoid the
hypoxia that exists in grape berries [164]. Moreover, oxygen can fuel energy-generating
biochemical pathways, including mitochondrial respiration, or others that require the
consumption of O2 such as lignification [165]. In accordance with this, results from our
own studies showed that from the green to veraison stage, seeds had an increase in a
lignan-type [30], which is associated with lignin synthesis [165], in agreement with the
degree of lignification of grape seeds along development [166].
Additional research in developing seeds (soybean, rapeseed and oilseed rape) sug-
gested that the photosynthetic activity can supply the energy (ATP) and reduction power
(NADPH) necessary for lipid biosynthesis, storage metabolism and redox modulation of
biosynthetic enzymes [150,151,163,167]. Furthermore, the biosynthesis of fatty acids is
a light-dependent reaction using the reducing power and adenosine triphosphate (ATP)
generated during the photochemical phase of photosynthesis [168,169]. In this line, studies
with legume seeds point to an effect of light on lipid metabolism via seed photosynthe-
sis [64,167] and Ruuska et al. [150] showed that Brassica napus seeds in siliques exposed to
light in planta produce more fatty acids than seeds from shaded siliques.
Previously, we showed that in grapes at the green stage, the photosynthetic capacity of
HL seed was higher compared to that of LL seed, whereas in the mature stage, the values
were similar for both light microclimates [29]. In addition, our metabolomic results showed
that the light microclimate influences seed lipid profiles in grapes at all developmental
stages: at the green stage, HL seeds had higher levels of ceramides, while at the mature
stage, they contained higher relative levels of triacylglycerols and glycerophospholipids, as
compared to LL seeds [32]. Thus, at the green stage, seed photosynthesis can supply energy,
reducing power and oxygen to fuel the de novo biosynthesis of fatty acids and ceramides.
In particular, the HL-induced increase in the relative abundance of ceramides may be
associated with seed morphogenesis, cell growth and differentiation that occurs at early
developmental stages [170,171]. On the other hand, at the mature stage, the photosynthesis
of HL seeds can contribute to the biosynthesis of storage lipids/oils (which are associated
with the reserve accumulation), and thus contribute to the final quality of the grape seeds.

4.2.2. Intermediates from Photosynthesis Used in Seed Metabolism and RuBisCO as a CO2
Rescue Mechanism
Seeds have high concentrations of internal CO2 , mainly produced by respiration, but
the CO2 released during oil synthesis also impacts the overall carbon economy of seed [172].
In addition, the seed coat can be quite thick and hard, making it difficult for CO2 to escape.
In this sense, the refixation of the respiratory CO2 could be an adaptive “strategy” of the
green seeds to cope with the cellular acidification caused by this high partial pressure of
CO2 [150].
Furthermore, during embryogenesis, seeds receive photoassimilates from the phloem
that are used for growth and for the synthesis of reserves, being this metabolic pathway
Plants 2023, 12, x FOR PEER REVIEW 18 of 32
Plants 2023, 12, 2393 17 of 29

of the green seeds to cope with the cellular acidification caused by this high partial
pressure of CO2 [150].
characterized
Furthermore,byduring
the conversion
embryogenesis,of seeds
sucrose to pyruvate,
receive through
photoassimilates from glycolysis,
the which is then
phloem that areby
transformed used for growth
pyruvate and for the synthesis
dehydrogenase of reserves,
(PDH) being this metabolic
in acetyl-CoA. This is the main precursor of
pathway characterized by the conversion of sucrose to pyruvate, through glycolysis,
biosynthesis of fatty acids,
which is then transformed whichdehydrogenase
by pyruvate are then used towards
(PDH) triacylglycerides
in acetyl-CoA. This is the or triacylglycerols
synthesis (TAG,oforbiosynthesis
main precursor storage lipids or oils)
of fatty acids,[173].
whichThisare conversion of sugars results in the loss
then used towards
oftriacylglycerides
carbon, in the or triacylglycerols
form of COsynthesis (TAG, or storage lipids or oils) [173]. This
2 , for each acetyl-CoA unit produced. According to Ruuska
conversion of sugars results in the loss of carbon, in the form of CO2, for each acetyl-CoA
etunit
al.produced.
[150], RuBisCO provides another route for the fixation of CO2 released by the PDH.
According to Ruuska et al. [150], RuBisCO provides another route for the
Corroborating thisby
fixation of CO2 released view, from
the PDH. a study with
Corroborating embryos
this view, of Brassica
from a study napus L. (oilseed rape),
with embryos
Schwender et al.
of Brassica napus L. [173]
(oilseeddescribed a newetmetabolic
rape), Schwender pathway,
al. [173] described in metabolic
a new which RuBisCO acts without
pathway, in which RuBisCO acts without the CBB cycle, in a mechanism previously
the CBB cycle, in a mechanism previously unknown (Figure 3).
unknown (Figure 3).

Figure 3. Metabolic pathway of transformation of sugars into fatty acids and highlighting the
Figure 3. Metabolic pathway of transformation of sugars into fatty acids and highlighting the possible
possible contributes of photosynthesis. Abbreviations: Glc-6-P, glucose-6-phosphate; GAP,
contributes of photosynthesis.
glyceraldehydes-3-phosphate; Abbreviations: Fru-6P,
PEP, phosphoenolpyruvate; Glc-6-P, glucose-6-phosphate;
fructose-6-phosphate; PGA, 3- GAP, glyceraldehydes-
phosphoglyceric acid; PDH, pyruvate dehydrogenase; AcCoA, acetyl-CoA; FAS, fatty acid
3-phosphate; PEP, phosphoenolpyruvate; Fru-6P, fructose-6-phosphate; PGA, 3-phosphoglyceric
synthesis; OPPP, oxidative pentose phosphate pathway; PRK, phosphoribulokinase; E4P, erythrose-
acid; PDH, RuBP,
4-phosphate; pyruvate dehydrogenase;TAGs,
ribulose-1,5-bisphosphate. AcCoA, acetyl-CoA;
triacylglycerides. FAS,from
Adapted fatty acid synthesis; OPPP, oxidative
[64,150,173].
Reproduced with permission from John Ohlrogge, The capacity of green oilseeds to utilize
pentose phosphate pathway; PRK, phosphoribulokinase; E4P, erythrose-4-phosphate;
photosynthesis to drive biosynthetic processes, 2004, 136, page 2706, and by permission of Oxford
RuBP, ribulose-
1,5-bisphosphate.
University Press. TAGs, triacylglycerides. Adapted from [64,150,173]. Reproduced with permission
from John Ohlrogge, The capacity of green oilseeds to utilize photosynthesis to drive biosynthetic
processes, 2004, 136, page 2706, and by permission of Oxford University Press.

This metabolic pathway involves three main steps. The first is the conversion of
hexose- and triose-phosphates to ribulose-1,5-bisphosphate by the non-oxidative reactions
of the oxidative pentose phosphate pathway (OPPP) together with phosphoribulokinase
(PRK); the second, the conversion of RuBP and CO2 (most of which is produced by pyru-
vate dehydrogenase—PDH) to 3-phosphoglyceric acid (PGA) by RuBisCO; and third, the
metabolism of PGA to pyruvate and acetyl-CoA, and then to fatty acids. Alternatively, the
carboxylation of RuBP by CO2 released by other internal pathways could also keep the
CBB cycle active in non-foliar tissues, mostly in case of a short supply of sugars from leaves
with reduced photosynthetic activity (e.g., under abiotic stresses, such as water stress).
Therefore, both mechanisms would avoid loss of carbon by recycling internal CO2 , as
well as provide several intermediates of the CBB cycle for distinct pathways of metabolism.
In fact, intermediates of OPPP pathway or RuBP regeneration phase of CBB cycle, as
erythrose-4-phosphate, together with PEP can be used for amino acid synthesis, which in
Plants 2023, 12, 2393 18 of 29

turn can contribute as precursors for the shikimate pathway. This biosynthetic pathway
is responsible for the production of phenylalanine, as well as other aromatic amino acids,
such as tyrosine and tryptophan. Phenylalanine in the first substrate of a key secondary
metabolic pathway, the phenylpropanoid pathway [174]. In this way, seed photosynthesis
is essential to provide the nutrients for the growing embryo, such as storage compounds
such as starch and oil bodies, rather than producing transportable carbohydrates as is in
leaf photosynthesis [149]. In line with this, Schwender et al. [173] showed that this new
pathway provides 20% more acetyl-CoA for fatty acid synthesis and resulted in 40% less
loss of carbon as CO2 , comparatively to glycolysis. Similarly, Allen et al. [64] verified that
in soybean embryos, RuBisCO re-fixed 11% of the CO2 released by lipid synthesis and the
TCA cycle, and consequently, the CBB cycle contributed to the carbon economy.
In the grape berry seeds, despite the decrease in the photosynthetic activity at the
mature stage [29], our recent transcriptional data showed that the relative expression of
VvRuBisCO was maintained in high levels throughout the three developmental stages of
seeds, in values similar to those found in the exocarps, and it was also up-regulated by
HL microclimate [31]. Therefore, this may suggest that the CBB cycle can be active, until
later stages of seed development, especially in HL seeds, as a CO2 rescue mechanism,
as proposed by Ruuska et al. [150] and Schwender et al. [173]. Thus, the CBB cycle may
indirectly provide acetyl-CoA needed for de novo synthesis of fatty acids in the chloroplast
and hence fueling the synthesis of triacylglycerols, sterols and glycerophospholipids in
these mature seeds [32]. Additionally, the CO2 rescue mechanism can provide other
intermediates for primary and secondary metabolism. For instance, it can be important to
support the high relative abundance of procyanidins at the green stage and of viniferins at
the veraison and mature stages [30].

4.3. Photosynthesis and the Vascular System of Fruits


Until the later stages of development, sugars are transported from source organs to
the fruits, through the phloem. The unloading of these sugars can occur by symplastic or
apoplastic pathways, the latter a mechanism dependent on energy [175]. The photochem-
ical phase of photosynthesis can provide the energy (ATP) necessary for the apoplastic
unloading [176], an energetically demanding process.
Hibberd and Quick [177] verified that the cells surrounding the peripheral vascular
system of stems and petioles of tobacco have chlorophyll and photosynthetic activity
(C4-type). Similar results were observed in young shoots and chlorenchyma of lignified
shoots of grapevine [24]. Although it was refereed that in grape berries the unloading is
predominantly symplastic in the early stages of development, becoming the apoplastic
pathway dominant with the onset of ripening [178], our previous work in white grape berry
tissues using the chlorophyll fluorescence imaging technique, showed that there was a high
concentration of chlorophyll/photochemical activity in perivascular cells of the peripheral
dorsal system [12], what is consistent with an association of the unloading process with
photochemistry. Moreover, our transcriptional results pointed to a possible crosstalk
between photosynthesis and sucrose unloading and breakdown/carbon usage in the berries.
In fact, the relatively high levels of transcripts of genes involved in sucrose catabolism
(i.e., VvSuSy1, sucrose synthase gene), as suggested by Wang et al. [179], combined with
the low levels of sucrose-phosphate synthase gene (VvSPS1), in the exocarp of the green
stage, may support our hypothesis that photosynthesis contributes with energy to that
unloading process [31]. Thus, at the initial stage of development, the main part of the
sucrose imported through the dorsal vascular system from the leaves, rather than sucrose
produced locally in the berry, is crucial to meet the relatively high demand for carbon and
energy that sustain the grape high growth rate [180].
Another interesting putative role for photosynthesis in the vascular bundles was
observed in cucumber fruit, where the PEPC is present [13]. The refixation of respiratory
CO2 by PEPC, followed by the synthesis of organic acids that can accumulate in the vacuole,
can provide the turgor pressure necessary for cell expansion and fruit growth [13]. The
Plants 2023, 12, 2393 19 of 29

same was suggested for tomato [70,96]. In grape berry, immunohistochemical studies
verified that PEPC is present in the vasculature, in the parenchyma cells of the pericarp
and within the developing seeds, leading to the hypothesis that PEPC may play a role in
the metabolism of the assimilates after their delivery to the fruit [107], and also into the
seed at the appropriate time during its development [181].

4.4. Ecological Advantages of Green Fruits and Seeds


The advantages or “services” of fruit photosynthesis can also be examined from an
ecological point of view, and we can start this topic by saying that a green-colored fruit,
located among green leaves, does not facilitate zoochoric seed dispersal. In fact, fruit color
influences their ability to be dispersed by animals, namely birds [182]. In this manner,
the ripening process composes the mutualistic relationship between fleshy-fruit plants
and seed-disperser animals [183]. In addition to the visibility conditions and the visual
aptitude of the receiver, the visual signal detectability is determined by its contrast against
the background, that is, the conspicuousness of the signal [182]. Young fruits are usually
green, but upon ripening their colors can range from red, blue, yellow and orange to brown
or even black. Indeed, chlorophyll degradation is generally accompanied by a conversion
of chloroplasts into chromoplasts that progressively accumulate high levels of carotenoids.
However, some fruits are green even when they are ripe (“green-ripe” or chlorophyllous),
for example, cucumber, kiwi, pea, green pepper and green apple varieties [184].
In nature, the dispersion of seeds of fleshy fruits is commonly performed by animals
(zoochory) that follow their visual and olfactory senses [185]. For diurnal seed dispersers
such as birds, the visual stimuli are particularly important, whereas many of the nocturnal
seed dispersers, such as bats and other mammals, rely to a large extent on olfactory
stimuli. “Green-ripe” fruits are less conspicuous than fruits of other colors, and are mostly
dispersed by mammals such as bats [184]. In this way, the fruits that maintain green until
full maturation tend to be dispersed by a limited variety of frugivores. Cipollini and
Levey [184] suggested that the ecological advantage of the “green-ripe” fruits consists in
their ability to actively perform photosynthesis, which reduces the plants energy costs
for fruit production and may enrich the fruit pulp in nutrient rewards for the frugivores.
The same authors showed that at high light levels these green-ripe fruits have a positive
carbon balance, while at low light levels their high rates of respiration often result in net
CO2 losses. The “green-ripe” fruits also produce higher seed and pulp masses, resulting in
a quantitatively larger offer for their seed dispersers. In addition, the presence of strong
odors in these fruits is also indicative that some secondary metabolic pathways may be still
active, eventually fueled by photosynthesis [184].
In grape berries the photosynthetic tissues are mainly the exocarp and the outer seed
integument [12], which both contain a relative high level of tannins, especially accumulated
at green stage, decreasing afterwards [30]. Moreover, the two distinct light microclimates
in the canopy (LL and HL) led to differences in the total flavan-3-ols in the exocarp at both
green and mature stages [30]. For instance, at the green stage the exocarps of high light (HL)
grown berries contained more hydroxy-procyanidin trimers, as compared to low light (LL)
ones, whereas at the mature stage the flavan-3-ols monomers (e.g., catechin), dimers (e.g.,
procyanidin B1) and trimers were lower in HL exocarps. From a sensory standpoint, these
compounds are correlated with astringency and bitterness of the wine [186]. In terms of eco-
physiological functions, they confer protection against fungal and bacterial pathogens, pest
insects and larger herbivores (as reviewed by Barbehenn and Constabel [187]). Together
these observations suggest roles for fruit photosynthesis with relevance at an ecological
level.
Table 4 summaries the principal and putative functions of photosynthesis in non-foliar
organs/tissues, which were described in Section 4.
Plants 2023, 12, 2393 20 of 29

Table 4. Overview of the principal and putative functions of photosynthesis in non-foliar organs/tissues.

Putative Functions Organ/Tissue Species References


Mandarin fruit Citrus unshiu Marc. [93]
Avocado fruit (mesocarp) Persea americana Mill. [47]
Grape berry (skin and mesocarp) Vitis vinifera L. [94]
Apple Malus domestica Borkh. [95]
Tomato Lycopersicon esculentum Mill. [96]
Re-fixation (recycling) of CO2 Cucumber fruit Cucumis sativa L. [13]
contributing to carbon economy
Ears cereals: the green pericarp and internal Several species Reviewed by [58,97]
surfaces of lemmas (facing the grain)
Ears of barley Hordeum vulgare L.
Triticum durum Desf. [98]
and durum wheat
Durum wheat (pericarp cells and glumes) Triticum durum Desf. [99]
Pods of chickpea (pod wall) Cicer arietinum L. [48,65]
Supply of oxygen Legume seeds (Several species) Reviewed by [188]
Broad bean Vicia faba L.
[189,190]
Pea seed Pisum sativum L.
Soybean seeds Glycine max L. [151]
- Supporting needs associated with storage activity and metabolite distribution
Barley grains Hordeum vulgare L. [160]
- Supporting needs associated with resource partitioning between endosperm and embryo Maize grains Zea mays L. [158]

- Supporting the increased respiratory rate/energy demand associated with lipid biosynthesis Soybean seeds Glycine max L. [163]
Rapeseed Brassica napus L. [167]
Soybean seeds Glycine max L. [150,151]
Supply of energy (ATP) and reduction power (NADPH)
Broad bean Vicia faba L.
[190]
Pea seed Pisum sativum L.
- ATP demand of unloading at the vascular system Grape berry exocarp Vitis vinifera L. [12,31,107]
Tomato seeds Solanum lycopersicum L. [126]
Seed development (e.g., impact on seed set, composition and morphology)
Apple seeds Malus domestica Borkh. [127]
- Seed filling Soybean pods Glycine max L. [55,64,150]
Plants 2023, 12, 2393 21 of 29

Table 4. Cont.

Putative Functions Organ/Tissue Species References


Supply carbon intermediates to the biosynthesis of/metabolic pathways:
- Isoprenoids Grape berry pericarp [67]

- Malic acid Grape berry Reviewed by [105]

- Amino acids Red grape berry (at green stage) Vitis vinifera L. [33]

- Flavan-3-ols and of stilbenes Grape berry exocarp (at green stage)


[30,31]
- Flavonol glycosides Grape berry exocarp (at later stages)

- Carbohydrates Tomato Lycopersicon esculentum L. [109]

- Starch Tomato [112]


Solanum lycopersicum L.
- Carbohydrates and carotenoids Tomate (ripe) [111]

- Primary metabolism Apple Malus domestica Borkh. [127]

- Primary and secondary metabolism Tomato -- Reviewed by [113]

- Fatty acid synthesis and TCA cycle Oil rape seed Brassica napus L. [173]

-- Not referred.
Plants 2023, 12, 2393 22 of 29

5. Concluding Remarks and Future Perspectives


Fruits are vital organs in plant sexual reproduction and indispensable foods in our
diet. Their quality depends on various physiological and biochemical mechanisms that in
the end contribute to the accumulation of a plethora of compounds. The main purpose of
this review was to compile and integrate information on anatomical, physiological and
biochemical features and constraints of different types of fruits and of their seeds, in order
to unveil potential biological functions of the photosynthesis performed by some of their
green tissues. A diversity of photosynthetic mechanisms or, in some cases, the utilization
of part of “old” photosynthetic routes in new solutions to meet tissue-specific demands or
alleviate biochemical pressures is discussed. The biological relevance of photosynthesis
in fruits is clearly supported by many findings. Overall, the evidences point to some
specific roles and functions, such as: (i) the supply of energy (ATP) and reducing power
(NADPH), both produced during the photochemical phase, and which can be important
for energy-dependent biochemical processes, such as the unloading of sugars from the
vascular system or even the synthesis of fatty acids; (ii) the production of oxygen that can
prevent and/or reduce the hypoxia inside of seeds; (iii) the re-fixation of respiratory CO2 by
RuBisCO in the CBB cycle and/or by PEPC (C4-type photosynthesis); and (iv) supply of the
carbon skeletons, derived from the CBB cycle, or RuBisCO action outside the cycle coupled
to OPPP, that can fuel pathways of primary and secondary metabolism, and thus contribute
for the nutritional and organoleptic properties of fruits, as well as to their adaptive value
on an ecological scale.
Photosynthesis of non-foliar tissues, in particular in fruits, is a fascinating topic studied
for over 30 years and from very different perspectives. However, due to the complexity of
fruit photosynthesis in space (different tissues, compartments, fruit geometries), in time
(variation associated with development) and its dependency on environmental factors, key
information is still lacking or conflicting. Well-controlled studies and model systems are
needed to better identify cause and effect relationships and to integrate global approaches
such as systems biology and omics networks. As an example, in vitro culture of cells
from different grape berry tissues with distinct photosynthetic competences may enable
studying the link between photosynthetic activity and specific changes at molecular and
biochemical levels in a more direct and controlled manner. Moreover, it is also important
to understand the complexities of coordination between environmental stresses, and fruit
photosynthesis and stress responses, since this may provide key information not only for
crop improvement in the context of the ongoing climatic changes, but also to meet the
growing demand for high quality food produced in a sustainable and safe manner.

Author Contributions: Conceptualization, A.G. and A.C. (Ana Cunha); writing—original draft
preparation, A.G.; writing—review and editing, A.G., A.C. (Artur Conde), J.S., R.C.H.D.V. and
A.C. (Ana Cunha); supervision, A.C. (Artur Conde), R.C.H.D.V. and A.C. (Ana Cunha); project
administration, A.C. (Ana Cunha). All authors have read and agreed to the published version of the
manuscript.
Funding: This research was funded by FCT—Portuguese Foundation for Science and Technology,
via a grant provided to AG (PD/BD/128275/2017), under the Doctoral Programme “Agricultural
Production Chains—from fork to farm” (PD/00122/2012), and by the European Social Funds and the
Regional Operational Programme Norte 2020. FCT also supported the work via CITAB and CBMA
research units under the projects UIDB/04033/2020 and UIDB/04050/2020, respectively. The work
was also support by project I&D&I “AgriFood XXI”, ref. NORTE-01-0145-FEDER-000041, co-financed
by the European Regional Development Fund (FEDER), through NORTE 2020 (Northern Regional
Operational Program 2014/2020). This work was also supported by CESAM (UIDP/50017/2020 +
UIDB/50017/2020 + LA/P/0094/2020). Ric C. H. De Vos was financed by the business unit Bioscience
of Wageningen University and Research Centre.
Data Availability Statement: Not applicable.
Plants 2023, 12, 2393 23 of 29

Acknowledgments: The authors acknowledge the consent of Michael Blanke (and John Wiley and
Sons permission), Robert Henry and of John Ohlrogge (and Oxford University Press permission) to
reproduce an adapted version of the figures from their previous published articles.
Conflicts of Interest: The authors declare no conflict of interest.

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