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.