Modified mechanism of stem photosynthesis in M.
micrantha
Plants obtain carbohydrates from photosynthesis and consume them via
respiratory processes for maintaining metabolism (McDowell & Sevanto,
2010). The removal of leaves stops the primary carbohydrate source from
plants, causing them to experience carbon stress. Defoliation-induced
carbon stress can reduce nonstructural carbohydrate reserves and
increase both vulnerability to insect infestation and hydraulic
performance (Anderegg & Callaway, 2012). However, in some cases, a
reduction in leaf photosynthesis can be compensated for by the
mobilization of stored carbohydrates, reallocation of carbon or stem
photosynthesis (Eyles et al., 2009). Compared with leaf photosynthesis,
stem photosynthesis is less vulnerable to environmental stresses such as
seasonal changes and reduced water availability; therefore, stem
photosynthesis is able to compensate for the loss of leaf photosynthesis
under stress conditions (Nilsen & Bao, 1990; Nilsen et al., 1993). InM. micrantha , the response of soluble sugars to defoliation
treatment was slower than the response of stem photosynthesis (Figure
6), suggesting that stem photosynthesis rather than the use of stored
carbohydrates is preferentially used to compensate for the decrease in
leaf photosynthesis.
The carbon starvation-induced optimization of stem photosynthesis
ability in M. micrantha is an integration of a series of
interactive factors. In terms of the phenotype, removal of the leaves
decreased the internode length, diameter and anthocyanin pigmentation of
the stems (Figure 5). Although internode length is unrelated to stem
photosynthesis ability, a shorter internode length could provide more
opportunities for sprouting new leaves along the stems. By contrast,
thinner stems have a larger surface area-to-volume ratio that favors gas
exchange between the stems and external environment. From this
perspective, stems with a smaller diameter could promote stem
photosynthesis. However, as the low projected area of thin stems may
affect their light capture ability. Interestingly, this deleterious
effect could be offset by reducing the amount of anthocyanin pigments,
which act as light barriers, in the stem epidermis.
Anthocyanin pigments can act as light attenuators or as antioxidants to
protect the photosynthetic apparatus in plant vegetative tissues (Neill
& Gould, 2003). Since anthocyanins were distributed in the epidermis of
the stems of the plants in the present study, they were more likely to
play a light-barrier role rather than an antioxidant role. In fact,
anthocyanins accumulate in young stems but diminish with maturity. This
pigmentation pattern has a lot in common with that in leaves (Hughes et
al. , 2007; Zhu et al. , 2018), suggesting that different
kinds of tissues can utilize the same approach to protect immature
photosynthetic apparatuses. With regard to the native species, P.
nil and P. scandens had the same pigmentation pattern in their
stems as M. micrantha did (Figure 2 and S2). The stems ofP. lobata did not accumulate anthocyanins, but they were more
pubescent than the stems of the other species were. Pubescence can play
a similar role as anthocyanins with regard to photoprotection
(Liakopoulos et al. , 2006). Defoliation induced an improvement in
the stem photosynthesis ability, allowing the apparatuses to withstand
stronger irradiance. In this case, anthocyanins become an unnecessary
tool to protect the stem photosynthetic apparatus. After removal of the
leaves, the reduction in anthocyanins in the stems was observed to be
associated with decreased soluble sugar contents, indicating that sugars
are involved in the regulation of anthocyanins. This is consistent with
the findings of a recent study showing the regulatory mechanism of
anthocyanins in kiwifruit (Nardozza et al., 2020).
In addition to the removal of light-barrier pigments, carbon starvation
also induced stomatal opening and thereby removed the obstacle that
limited gas exchange between the stems and the atmosphere. Stomatal
opening was confirmed by microscopy-based observations and measurements
of stomatal conductance based on H2O exchange (Figure
5). However, because the stomatal densities were different, stomatal
conductance in the stems without leaves was observed still to be lower
than that in the leaves of the plants in the normal-growth group.
In addition to overcoming the barriers to light and CO2,M. micrantha was also able to optimize its cellular and
subcellular structure to increase photosynthesis efficiency. There were
3-4 layers of cells below the epidermis that became larger and
accumulated increased amounts of chlorophyll, indicating that
photosynthesis was mainly performed in this zone of the stems (Figure
2). Ultrastructural observations showed that chloroplasts in this zone
in the plants in normal-growth group were elliptica -shaped; however,
after the removal of their leaves, they became spindle shaped, and their
length dramatically increased (Figure 7). Such changes might increase
the light-receiving area of the chloroplasts. The modification of both
the Chl a /b ratio and plastoglobule size and the enhanced
turnover of D1 protein supported that chloroplasts were exposed to high
light in the stems of the defoliated plants.
Plastoglobuli are lipoprotein particles present in both
nonphotosynthetic and photosynthetic plastids in plants (van Wijk &
Kessler, 2017). In chloroplasts, they play functional roles in
chloroplast biogenesis, redox and photosynthetic regulation and
senescence by exchanging metabolites with the thylakoid membrane (Vom
Dorp et al. , 2015). They can serve as extrathylakoid storage
sites for excess isoprenoid lipids, such as α-tocopherol (vitamin E),
plastoquinone-9, and traces of xanthophylls (Lichtenthaler, 2007). Under
high-light conditions, as the essential components of thylakoids,
α-tocopherol and plastoquinone-9 often accumulate in excess amounts in
chloroplasts and are stored in plastoglobuli. The degradation of
epidermal anthocyanins and changes in chloroplast shape caused the
chloroplasts in the stem tissues to be exposed to high light. This could
explain why, compared with those of intact plants, the chloroplasts in
the stems of the defoliated plants had a higher number and a larger size
of plastoglobuli (Figure 7).
During defoliation, the Chl a content increased rapidly in the
stems of M. micrantha , whereas the Chl b content was
maintained at a similar level (Figure 2). As a consequence, the Chla /b ratio dramatically increased, which is in agreement
with the findings in leaves exposed to high irradiance (Kitajima &
Hogan, 2003; Sarijeva et al. , 2007). The differences in Chla /b ratios between sun and shade leaves are due to the
high-irradiance-adaptation response of the photosynthetic machinery of
sun leaves, which have a much lower quantity of light-harvesting Chla /b proteins (LHCII) and a greater number of PSII cores
than shade leaves do (Lichtenthaler et al., 1982). Since the Chla /b ratio was found to be positively correlated with the
ratio of PSII cores to LHCII (Terashima & Hikosaka, 1995), this ratio
is used as an indicator of N partitioning within a leaf (Kitajima &
Hogan, 2003). It is likely that the stems experienced N limitation
during defoliation, as the removal of leaves caused energy starvation in
the roots, which could in turn reduce the uptake of N. If so, the
adjustment of the stem Chl a /b ratio during the removal of
leaves was a result of irradiance and N availability acting together. In
fact, differences in N partitioning in stem chloroplasts between
defoliated plants and intact plants could be confirmed by the fact that
the stems of the defoliated plants had more D1 protein and a lower
amount of soluble protein than the stems of the control plants did
(Figure 6) Moreover, the Rubisco content in the defoliated stems also
seemed to be slightly reduced compared with that in the stems of intact
plants.
The photosynthesis of plants depends on the function of photosystem II
(PSII), which is a large multisubunit protein complex integrated within
the thylakoid membrane (Andersson and Barber, 1994). The PSII reaction
center contains the homologous D1 and D2 proteins, PsbI, PsbW and
cytochrome b559. This study showed that the amount of D1 protein
dramatically increased in the stems of M. micrantha after removal
of the leaves (Figure 6). The increase in D1 protein was positively
associated with the Chl a /b ratio, the ETR andΦ PSII (Figure S3), indicating that more PSII
reaction centers were assembled in the stem chloroplasts during
defoliation. However, the increase in D1 protein relative to that of the
control was evidently greater than the increase in total Chl and the Chla /b ratio. In fact, the D1 protein in PS II is prone to
irreversible damage caused by reactive oxygen species that are formed in
the light, and there is an intricate repair mechanism involving
degradation of the damaged D1 reaction center protein and insertion of
the newly synthesized copy into the photosystem for maintaining
photosynthesis (Lindahl et al., 2000). Generally, the rate of D1
impairment does not exceed the rate of its repair under optimal growth
conditions; therefore, no adverse effects on photosynthesis efficiency
are manifested. Stress conditions such as high light can disrupt the
balance between D1 protein impairment and its repair, resulting in
photoinhibition and lowering the quantum yield of photosynthesis and the
ETR of PSII (Andersson & Aro, 2006). Thus, D1 protein turnover is
crucial for plasticity of the photosynthetic apparatus. In the stems ofM. micrantha , the high rate of D1 synthesis guaranteed the
maintenance of high photosynthesis efficiency in the absence of
anthocyanin-mediated photoprotection.
We demonstrate here that chloroplast morphology, anthocyanins, stomata,
photosynthetic pigments and photosynthesis-related proteins are involved
in improving the photosynthesis efficiency of the stems of M.
micrantha during defoliation. However, the details through which such
processes occur are far from clear. The regulatory mechanisms underlying
anthocyanin degradation and stomatal behavior in the stems need to be
investigated. Moreover, the mechanism through which N partitioning
between thylakoid membrane proteins and soluble proteins contributes to
improved photosynthesis efficiency should be clarified.