Effect of SP light intensity on RLC profiles and estimated parameters
The effect of SP light intensity on chl a fluorescence parameters under light conditions was studied by rapid light curves (RLC). Figure 3 shows typical effective quantum yield (ΦII) images recorded during the RLCs performed at each of the SP intensities tested. It is evident that ΦII could only be determined at a very narrow range of low actinic light (AL) intensities, as at an AL as low as 145 μmol m−2 s−1 the yield signal is almost lost. Also, there seems to be a SP-dependent decrease in yield for light intensities above SP3, particularly in inner tissues. At PFR = 0, which could be seen as an Fv/Fm of light-adapted samples, higher yield values were obtained at SP3, decreasing for higher SP intensities, in line with the observed dependency of Fm on SP (Fig. 2). At low AL intensities (e.g. PFR = 3 or 11 μmol m−2 s−1), the trend is similar but the yield values seemed to peak at SP4, suggesting that the SP effect may depend on the light-adaptation status of the sample. However, the decay in yield during the RLC with the increase in AL intensities was sharper for SP above SP3 also suggesting a synergistic inhibitory effect between SP and AL intensities.
Figure 3. Images of effective quantum efficiency of PSII (ΦII) of median transverse tissue slices of green berries (independent triplicates) recorded during RLCs at different SP intensities (see legend of Fig. 2). Intact grape berries were previously adapted to 50 μmol m−2 s−1 for 30 min. The color scale has the meaning of the one shown in Fig. 1. PFR has units of μmol m−2 s−1.
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In all tissues, F'm and ΦII were higher at SP3 and SP4 (Fig. 4). Both the initial values of these parameters and the rates of their decay with increasing PFR at each SP were tissue specific. Mesocarp tissue showed the lowest F'm and the smallest variation, ranging from ca 0.3 to 0.1. Highest F'm was measured in the seed outer integument, reaching near 0.8, but like the mesocarp, this tissue also presented a small amplitude decay (Fig. 4a). The exocarp also showed high F'm, but contrarily to the other tissues presented a larger variation, decreasing faster during the initial light steps and maintaining a small slope for higher AL intensities. The lowest values of ΦII were also measured in the mesocarp, especially at SP6 and SP8, which decreased to near zero soon after the initial light steps (Fig. 4b). Although seed outer integument displayed the highest maximum fluorescences— Fm (Fig. 2) and F'm (Fig. 4a)—, ΦII in this tissue was lower than in exocarp at all the SP intensities tested. Contrary to other tissues, ΦII in exocarp rarely dropped to zero even at high SP intensities.
Figure 4. F'm (a) and ΦII (b) mean values estimated for the exocarp, mesocarp and seed outer integument from the RLCs performed in median transverse tissue slices of light-adapted (50 μmol m−2 s−1) green berries. SP intensities are as reported in Fig. 2. F'm and ΦII means are plotted with respective ± SD (n = 13–18)
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The larger amplitude decrease in F'm observed in the exocarp when compared with the seed outer integument suggest that the photoprotective mechanisms may be better developed in this tissue. In accordance, PAM fluorometry studies in green Carica papaya fruit revealed that the exocarp presents both high photochemical yields and nonphotochemical quenching being its chloroplasts structurally similar to those of mesophyll cells .
Mean rETRm, which is a measure of tissue photosynthetic capacity, peaked at SP3 in all tissues (Table 1) and decreased with higher SP intensities, however, the exocarp was susceptible only to the highest light intensities (SP6 and SP8). At SP3, the highest values of rETRm were estimated in the exocarp, tissue with up to twice the values of the seed outer integument and mesocarp.
Table 1. Values for rETRm, α, β and Ek parameters estimated after Platt et al.  fitting of RLCs replicates. All the values are the mean of 13–18 replicates, except those in gray (3 ≤ n ≤ 6). For each tissue and parameter, different lowercase letters indicate statistically different mean values. For each SP intensity and parameter, tissues with different uppercase letters have statistically different mean values. The values of SP intensities are indicated in Fig. 2. Ek has units of μmol photons m−2 s−1 and rETRm of μmol electrons m−2 s−1
|SP levels||Exocarp||Mesocarp||Seed outer integument|
|rETRm||α||β|| E k ||rETRm||α||β|| E k ||rETRm||α||β||Ek|
|SP2||5.37a, A||0.241a, AB||−0.015a, A||23.33a, A||2.28a, B||0.183a, A||−0.015a, A||12.69a, B||3.72a, C||0.255 a, B||−0.015 a, A||14.04 a, A|
|SP3||6.49a, A||0.264ab, A||−0.016a, A||25.30a, A||3.07b, B||0.197a, B||−0.011a, A||17.15a, B||4.37a, C||0.253a, AB||−0.014a, A||20.12b, A|
|SP4||6.35a, A||0.316b, A||−0.013a, A||20.71ac, A||2.13a, B||0.420b, B||−0.010a, A||5.06bc, B||3.32b, C||0.318a, A||−0.008a, A||10.89a, C|
|SP6||3.27b, A||0.266ab, A||0.022b, A||11.88bc, A||2.25ab, A||0.236a, A||0.085b, B||9.57ac, A||2.01c,,A||0.223a, A||0.068b, B||9.41a, A|
|SP8||3.25b, A||0.394c, A||0.023b, A||8.45b, A||0.56c, B||0.275a, B||0.131b, B||2.01c, A||2.18cb, AB||0.252a, B||0.109b, A||8.46a, A|
Some cautious has to be taken on the analysis of the α parameter because there is a substantial photoinhibition at high SP intensities, and therefore, less empirical data are available for fitting what may lead to biased estimations. Some estimated values (see Table 1; exocarp at SP8 and mesocarp with SP4) might be an expression of this problem. Having this in consideration, it is plausible that α tend to peak at SP3/SP4. At these SP settings, the exocarp and the seed outer integument presented similar high values, and at SP3 they were higher than those of the mesocarp.
The near zero values of β—a parameter related to photoinhibition— at low SP intensities (SP2–SP4) increased significantly at SP6 and SP8 in all tissues, suggesting susceptibility of PSII to photoinhibition at these SP intensities. At SP6 and SP8, β estimates were significantly higher for both the mesocarp and seed outer integument, with similar values, and the exocarp is only marginally affected, revealing that this tissue is the most resistant to the SP-induced photoinhibition.
The Ek parameter—an index associated with an adaptation to low light intensities—was also affected by SP intensity peaking at SP3. At this SP intensity, the exocarp presented a significantly higher Ek than the mesocarp and seed outer integument, both with similar values (Table 1), what may translate the fact of these tissues being naturally adapted to low light regimes.
These results revealed remarkable differences between tissues' photosynthetic phenotypes. The exocarp is the tissue with the highest photochemical competence, showing the higher photosynthetic efficiency (α) and capacity (rETRm) and the lower susceptibility to photoinhibition (β). Also, it seems to be adapted to higher light intensities (Ek). On the other hand, the mesocarp showed the lowest photochemical competence, having the lowest photochemical efficiency and the highest susceptibility to photoinhibition. The seed outer integument, although located in the central region of the fruit, justifying the low-light-adaptation phenotype and susceptibility to high light, presented photochemical efficiencies similar to the exocarp and also relatively high photochemical capacity.
Regarding the effects of the saturating pulse intensities, the SP2 level induced generally lower fluorescence and also lower photochemical parameters' mean values in all tissues, when compared with those obtained at SP3, in agreement with the experiments performed under dark conditions, revealing an incomplete reduction in the plastoquinone pool . At the highest SP light intensities a photoinhibitory effect becomes evident because all the estimated parameters (except α) were significantly affected in all tissues, at SP6 and SP8. However, experiments performed under light-adapted conditions brought up other interesting points, namely that the exocarp was the only tissue where none of the photochemical parameters analyzed were affected when SP4 intensity was used (Table 1) and that the SP-induced photoinhibitions registered were much stronger than that observed in experiments under dark conditions. For instance, for rETRm and comparing the values obtained at SP3 with those obtained at the SP where significant inhibition of this parameter firstly occurred (at SP6 for the exocarp and at SP4 for the other tissues), reductions of 25–50% were registered (Table 1). In dark conditions, maximum SP-induced reductions in Fm reached only less than 10% when comparing means between SP3 and SP8 (Fig. 2). This suggests not only that there might be a cumulative effect due to exposure to repetitive saturating pulses—that are nonphotoinhibitory or only moderately photoinhibitory when given as single pulses—, but also that the magnitude of this effect is dependent of tissue type.
Effect of SP light intensity on Fv/Fm recovery after RLC experiments
To investigate possible PSII photoinhibition caused by repetitive exposure to SP even at SP operational conditions (SP3 and SP4), the short-term recovery dynamics of Fv/Fm after RLC experiments were studied in all tissues. For this, Fv/Fm values were determined in discs of previously dark-adapted berries for 30 min (control), and after a dark adaptation period of 10 and 20 min following a RLC experiment.
As can be observed in Fig. 5, at SP3, no differences in mean Fv/Fm were found when comparing the three experimental conditions in any tissue, meaning that Fv/Fm fully recovered even for the lowest recovery time tested (RLC + 10'dark) or that PSII reaction centers were not affected at all by the previous light challenge (AL + SP). However, at SP4, none of the tissues was able to fully recover Fv/Fm after 20 min in the dark, but tissue-dependent responses were also detected.
Figure 5. Fv/Fm mean values (n = 18, + SD) of exocarp, mesocarp and seed outer integument, determined in median transverse tissue slices of grape berries dark adapted for 30 min (dark adapted), and for 10 (RLC+10' dark) and 20 min (RLC+20' dark) after a RLC experiment using SP3 and SP4 (see legend of Fig. 2). For each tissue, same letters or absence of notation indicates that mean values were not statistically different.
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After 10 min of dark recovery, Fv/Fm in the exocarp was 92% of the control and increased significantly to 96% after 20 min. The seed outer integument recovered only to 82% after 10 min, but like the exocarp, increased significantly after 20 min (to 89%). The mesocarp was the only tissue that did not present any Fv/Fm recovery after 20 min. These results suggest that tissue exposure to SP4 intensities during RLC experiments may cause photodamage of PSII, at least in the mesocarp. To recover from photodamage leaves must spend energy, which is believed to preferentially come from photophosphorylation coupled to electron transport rate . In this sense it is reasonable to think that the relatively high photosynthetic capacities of the exocarp and seed outer integument (Table 1) might be contributing to support eventual repair costs allowing an Fv/Fm recovery that was not observed in the mesocarp (Fig. 5).
From the results above it stands out that the repetitive exposure to SP, even as low as 3200 μmol m−2 s−1, may potentiate photodamage. For this, and although in dark conditions a SP light intensity of 3200 μmol m−2 s−1 (SP4) is adequate to assess Fv/Fm with this grape berry disc model, when running RLC, a SP light intensity of 2500 μmol m−2 s−1 (SP3) would be necessary to avoid SP-induced photoinhibitory effects. PSII inactivation by the cumulative exposure to short high light pulses during the induction period of photosynthesis was also observed in leaves of various species . Also, lower ΦII values where found when pea leaves were treated with light pulses of 10 000 μmol photons m−2 s−1 . Thus, a careful adjustment of the operational SP intensity to each model and to each experimental light condition is crucial to avoid erroneous estimations of photosynthetic responses.
The seed outer integument photosynthetic performance is also of particular interest. Although seed photosynthesis had been reported for several species, the majority of these are legume species, where seeds develop in pods, or other dry fruits typically with a thin pericarp, in which the light intensity at the seed level reach up to 30% of the incident light [33, 34]. However, high photosynthetic performances in the innermost tissues of unripe grape berries, a fruit with a large pericarp, were somewhat puzzling. A structural feature that may help to explain the high photochemical activity observed in the grape seed is the transparency of the light path inside the fruit, mainly composed by large, thin-walled and highly vacuolized mesocarp cells.
The seed outer integument revealed to be more susceptible to photoinhibition than the exocarp, but it also presented similar Fv/Fm recovery dynamics, photosynthetic efficiencies under low light conditions and the highest Fm of all tissues. This is in accordance with the specialized nature of the chloroplasts present in seeds, whose thylakoids contain chlorophyll–protein complexes similar to leaf chloroplasts but a greater proportion of granal stacking [35, 36]. This seed outer integument photosynthetic competence, but also that of the exocarp, probably enable the maintenance of relatively high photosynthetic rates under variable but generally very low light regimes, as grape berries are often covered by leaves. Grape berries are typically moderately shadowed and subjected to mean light intensities of 30–50 μmol photons m−2 s−1, but heavily shadowed grape berries could develop in light intensities as low as 1–5 μmol photons m−2 s−1 . Altogether, the photosynthetic competence exhibited by the seed outer integument suggests it might have a key role in grape berry seed physiology.
Although various fruit species have green seeds, the role(s) of photosynthesis in seed tissues is not fully disclosed. It is believed that it does not contribute to net sugar production and accumulation, as most of the seed carbon is imported from the phloem, mainly as sucrose and amino acids . It is rather consensual that chloroembryophyta species, namely green oilseeds, utilize embryo photosynthesis to drive biosynthetic processes, such as the energetically expensive fatty acid synthesis . In simple dry fruits with seed involved by green pericarps, such as barley and wheat caryopsis, the photosynthetic production of oxygen is fundamental for the translocation of assimilates to the starchy endosperm . In this sense, seed outer integument photosynthesis in the unripe grape berry may be also providing ATP and NADPH to drive fatty acid synthesis (13% of seed dry mass is accounted by oils of which more than 95% are neutral lipids ) and other seed storage products, like proteins. It may also supply the O2 needed to sustain mitochondrial respiration inside the metabolically active developing seed, as the low permeability to gases of the seed coat may otherwise induce seed hypoxia and limit metabolic fluxes, as it was reported for other species . It was stated that if O2 availability is insufficient to meet the demand this might lead to abnormal seed development and even seed abortion . In fleshy fruits with large watery pericarps, such as grape and tomato berries, the oxygen diffusion is particularly limited as gas diffusion is several thousand-fold slower through liquid medium than through air, and hence, an O2 source closer to the seed tissues seems pivotal to meet seed metabolic demands and its normal development. In fact, when chlorophyll synthesis was reduced in tomato by knockout experiments, a reduction in seed development was observed .
Besides these already discussed seed photosynthesis functions, mainly anchored on light reactions products, grape berry seed photosynthesis could be also contributing with Calvin cycle-derived precursors for the synthesis of secondary metabolites, such as tannins, which are important defense molecules mainly produced in the green phase and particularly abundant in grape berry seeds and skins. The high photosynthetic competence found in the grape berry exocarp supports this view. To sustain a functional Calvin cycle in seed tissues, light but also CO2 concentration minimums should be met. It was reported that light-regulated Calvin cycle enzymes could be active even under the very low light intensities at the seed level, and that the high concentrations of CO2 there registered, maintained by respiratory mechanisms and by the low diffusivity of the seed coat, allow fully carbamylation and activation of RuBisCO . Globally, these observations and our results support the idea of an operational Calvin cycle together with an efficient photochemistry in the grape berry seed that may be fundamental for several physiological and developmental seed processes.
The inhibitory effects of high SP intensities over most of the photochemical parameters tested in grape berry cross sections were clearly demonstrated in this study. It was also shown that the photoinhibitory threshold is dependent on the tissue type and on the overall light conditions experienced by the berry, suggesting a synergistic interaction between AL, SP intensities and the repetitive incidence of SP flashes on the sample. The results emphasize as well the importance of the optimization of SP intensity, particularly in studies of photochemical heterogeneous surfaces with different susceptibilities to light.