doi:10.1093/plphys/kiab206 PLANT PHYSIOLOGY 2021: 186: 2137–2151 Light signals generated by vegetation shade facilitate acclimation to low light in shade-avoider plants Downloaded from https://academic.oup.com/plphys/article/186/4/2137/6271415 by 81263600 user on 08 September 2021 Research Article Luca Morelli ,1,2 Sandi Paulisic,2 Wenting Qin,1,2 Ariadna Iglesias-Sanchez ,2 Irma Roig-Villanova ,2,† Igor Florez-Sarasa ,2 Manuel Rodriguez-Concepcion1,2,‡ and Jaime F. Martinez-Garcia 1,2,3,*,‡ 1 Institute for Plant Molecular and Cell Biology (IBMCP), CSIC-UPV, València 46022, Spain 2 Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Campus UAB Bellaterra, Barcelona 08193, Spain 3 Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluı́s Companys 23, Barcelona 08010, Spain *Author for communication: jaume.martinez@ibmcp.upv.es † Present address: Barcelona School of Agricultural Engineering (ESAB), Universitat Politècnica de Catalunya (UPC), Castelldefels, Barcelona 08860, Spain. ‡ Senior authors. M.R.-C. and J.F.M.-G. conceived the original research plan, directed, and coordinated the study. L.M., I.F.-S., A.I.-S., and M.R.-C. measured and analyzed photosynthetic parameters, respiration, and pigment levels; S.P., I.R.-V., and W.Q. performed all the other experiments. All authors analyzed their data and discussed the results. M.R.-C. and J.F.M.-G. wrote the paper with revisions and contributions or/and comments of all other authors. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is: Jaime F. Martinez-Garcia (jaume.martinez@ibmcp.upv.es) Abstract When growing in search for light, plants can experience continuous or occasional shading by other plants. Plant proximity causes a decrease in the ratio of R to far-red light (low R:FR) due to the preferential absorbance of R light and reflection of FR light by photosynthetic tissues of neighboring plants. This signal is often perceived before actual shading causes a reduc- tion in photosynthetically active radiation (low PAR). Here, we investigated how several Brassicaceae species from different habitats respond to low R:FR and low PAR in terms of elongation, photosynthesis, and photoacclimation. Shade-tolerant plants such as hairy bittercress (Cardamine hirsuta) displayed a good adaptation to low PAR but a poor or null response to low R:FR exposure. In contrast, shade-avoider species, such as Arabidopsis (Arabidopsis thaliana), showed a weak photo- synthetic performance under low PAR but they strongly elongated when exposed to low R:FR. These responses could be genetically uncoupled. Most interestingly, exposure to low R:FR of shade-avoider (but not shade-tolerant) plants improved their photoacclimation to low PAR by triggering changes in photosynthesis-related gene expression, pigment accumulation, and chloroplast ultrastructure. These results indicate that low R:FR signaling unleashes molecular, metabolic, and develop- mental responses that allow shade-avoider plants (including most crops) to adjust their photosynthetic capacity in antici- pation of eventual shading by nearby plants. Introduction outcomes of mutual shading, plants have developed re- Light is essential for plants as a source of energy and envi- sponse mechanisms based on the perception of light quality, ronmental information. Shading by nearby individuals can i.e. spectral information (Casal, 2013; Martinez-Garcia et al., reduce light quantity (i.e. photon supply) and hence com- 2010). The preferential absorbance of red (R) light and re- promise photosynthetic activity and growth, a problematic flection of far-red (FR) light by photosynthetic tissues results situation in intensive cropping systems. To deal with the in a decreased ratio of R to FR (R:FR) when light is reflected Received September 23, 2020. Accepted April 08, 2021. Advance access publication May 7, 2021 C American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com V 2138 | PLANT PHYSIOLOGY 2021: 186; 2137–2151 Morelli et al. from or filtered through green stems and leaves. The low photosynthetic capacity. The second one involves changes R:FR is a very reliable light signal that announces the close in the photosynthetic machinery, which appears to be more presence of nearby plants that may compete for resources. important in plant species from environments where tem- Plants growing in ecosystems where access to light is re- poral and spatial variations in light irradiance are common, stricted (e.g. in forest understories) show a shade-tolerant e.g. margins of woodlands. Combinations of these two main habit by adapting their light capture and utilization systems strategies give rise to the observed diversity in photoacclima- to low light intensity conditions. In contrast, plants growing tion. In the case of At and C. hirsuta (Ch), a differential re- in open habitats are shade-avoiders (also referred to as sponse to low R:FR in terms of photosynthetic pigment shade-intolerant or sun-loving). In shade-avoider plant spe- accumulation has been observed. Chlorophyll and caroten- cies, such as Arabidopsis (Arabidopsis thaliana) and most oid levels drop about 20% in At plants grown under low Downloaded from https://academic.oup.com/plphys/article/186/4/2137/6271415 by 81263600 user on 08 September 2021 sun-loving crops, perception of the low R:FR signal by the R:FR conditions, whereas the decrease is attenuated in Ch phytochrome photoreceptors activates a signaling pathway plants (Molina-Contreras et al., 2019). Whether photosyn- that eventually triggers a set of responses known as the thetic capacity and/or chloroplast ultrastructure is differen- shade-avoidance syndrome (SAS). The most prominent phe- tially impacted by low R:FR in these species remains notype following exposure to low R:FR is elongation (e.g. of unknown. In terms of light quantity, the shade-avoider At seedling hypocotyl, leaf petiole, and stem internode tissues), showed a lower capacity to acclimate to reduced photosyn- intended to overgrow neighboring competitors and outcom- thetically active radiation (low PAR) but a higher capacity to pete them in the access to light. If the neighboring individu- acclimate to intense light (high PAR) compared to the als overgrow and eventually shade the plant, the shade-tolerant Ch (Molina-Contreras et al., 2019). A similar consequent reduction in light quantity (i.e. in the amount physiological behavior has been described for shade-avoider of radiation available for photosynthesis) results in addi- and shade-tolerant species of the genus Tradescantia tional and stronger SAS responses such as reduced leaf size, (Benkov et al., 2019), a model to study the ecology of pho- attenuated defense mechanisms, and early flowering (Roig- tosynthesis and the mechanisms of photoacclimation in Villanova and Martinez-Garcia, 2016). plants (Ptushenko and Ptushenko, 2019). The possible con- The most extensively studied SAS response by far is hypo- nections between low R:FR signaling and photoacclimation cotyl elongation in A. thaliana (At). In this species, low R:FR responses in plants remain, however, virtually unknown. inactivates phytochrome B (phyB), releasing Here, we explored natural and engineered genetic diversity PHYTOCHROME INTERACTING FACTORs (PIFs) that can to investigate this connection using different Brassicaceae then regulate gene expression and promote elongation species. growth. This response is also repressed by negative SAS reg- ulators such as ELONGATED HYPOCOTYL 5 (HY5), Results amongst many others (Cifuentes-Esquivel et al., 2013; Ciolfi Different Brassicaceae species present divergent et al., 2013). Biological activity of these transcription factors photoacclimation responses can be modulated by additional components of the SAS reg- We previously showed that, compared to sun-loving At Col- ulatory network such as LONG HYPOCOTYL IN FAR-RED 1 0, shade-tolerant Ch Ox exhibits a better ability to maintain (HFR1, which binds PIFs to prevent their binding to target photosynthesis after transfer to low PAR but a stronger genes) and phytochrome A (phyA, which gets stabilized in chlorophyll loss when light intensity increases (Molina- shade and then promotes HY5 accumulation; Ciolfi et al., Contreras et al., 2019). To better characterize the photoaccli- 2013; Martinez-Garcia et al., 2014; Yang et al., 2018). Both mation responses of these two Brassicaceae species, both At HFR1 and phyA hence act as additional SAS repressors that and Ch were germinated and grown for 7 d under control were recently found to be instrumental for the adaptation conditions of a photosynthetic photon flux density (PPFD) to shade. Indeed, the shade-tolerant hairy bittercress in the PAR region of 20–24 mmol m2 s1 (W20). Then they (Cardamine hirsuta), a close relative of At, does not elongate were transferred to either lower PAR (W4, PPFD of 4 mmol when exposed to low R:FR unless the function of phyA or m2 s1) or higher PAR (W200, PPFD of 200 mmol m2 s1) HFR1 is genetically lost in mutant plants (Hay et al., 2014; for up to seven more days (Figure 1). Light curve analysis at Molina-Contreras et al., 2019; Paulisic et al., 2021). day 3 after the transfer already showed clearly opposite Differences between shade-avoider and shade-tolerant spe- responses of At and Ch, i.e. a better photosynthetic activity cies are not restricted to changes in elongation after expo- of Ch compared to At when transferred to W4 and a better sure to low R:FR. Photoacclimation (i.e. the ability of plants activity of At compared to Ch when transferred to W200 to adjust photosynthesis to changes in the incident light (Figure 1A). Derived parameters such as maximum electron with specific phenotypic changes) also diverges. Variation of transport rate (ETRm) and photosynthetic rate in light-lim- photoacclimation responses among species on day-to-week ited region of the light curve (alpha) also illustrated that At time scale has been associated to two main strategies performed better than Ch after transfer to higher light (Murchie and Horton, 1997; Ptushenko and Ptushenko, (W200) but worst after transfer to lower light (W4; 2019). The first one consists of an alteration of photosyn- Figure 1B). Other photosynthetic parameters such as maxi- thetic pigment content, which positively corresponds with mum quantum efficiency of PSII (Fv/Fm) and light use Plant Physiology, 2021, Vol. 186, No. 4 PLANT PHYSIOLOGY 2021: 186; 2137–2151 | 2139 Downloaded from https://academic.oup.com/plphys/article/186/4/2137/6271415 by 81263600 user on 08 September 2021 Figure 1 Arabidopsis thaliana and Cardamine hirsuta show antagonistic photoacclimation responses to higher and lower PAR. A, Light curves of At and Ch seedlings germinated and grown under white light of 20-mmolm2s1 PPFD (W20) for 7 d and then either kept under W20 or trans- ferred to either 200 (W200) or 4 (W4) mmolm2s1 PPFD for 3 more days. Values represent the mean and standard error of n ¼ 3 plants for treat- ment. B, ETRm and photosynthetic rate in the light-limited region of the light curve (alpha) calculated from the curves shown in (A). Asterisks mark statistically significant changes (t test *P < 0.05, **P < 0.01) in W4 or W200 relative to W20. C, Maximum photochemical efficiency of PSII in the dark-adapted state (Fv/Fm) and effective quantum yield calculated at growth light (UPSII) of seedlings germinated and grown for 7 d under W20 and then transferred to either W200 or W4 for seven more days. Data were taken at 0, 3, and 7 d after the transfer. Values are mean and stan- dard error of n ¼ 7 seedlings per treatment. Black asterisks mark statistically significant differences between At and Ch at each time point (t test *P < 0.05, **P < 0.01). Red asterisks indicate statistically significant differences between genotypes over time (two-way ANOVA, **P < 0.01). efficiency of PSII (UPSII) also showed differences between At and hypocotyl elongation in six different Brassicaceae species and Ch at day 3 after transfer, but these differences became or accessions, including At and Ch as controls. The selected clearer at longer times of exposure to either W200 or W4 wild mustards were alpine rock cress (Arabis alpina, Aa), (Figure 1C). Specifically, Fv/Fm values were lower in Ch than two accessions of shepherd’s purse (Capsella bursa-pastoris), in At after transfer to higher light, while the opposite was Freiburg-1 (Cb-F) and Strasbourg-1 (Cb-S), pink shepherd’s- observed when transferred to lower light. A similar trend purse (Capsella rubella, Cr), watercress (Nasturtium officinale, was observed in the case of UPSII (Figure 1C). These results No), and London rocket (Sisymbrium irio, Si). Initially, we together indicate that Ch tolerates better the transfer to aimed to classify them as shade-avoider or shade-tolerant lower PAR (consistent with Ch being more tolerant to based on photoacclimation responses. After germination shade), while an increase in light irradiance compromises and growth for 7 d under W, seedlings were either kept un- photosynthetic efficiency in Ch more than in shade-avoider der control W20 or transferred to lower light (W4). Light At. Based on these results, we used light curve analysis at curve analyses at day 1 after the transfer already showed dif- day 3 or earlier to estimate photoacclimation to lower PAR ferential responses that served to classify the accessions in and Fv/Fm measurements at day 7 to estimate photoaccli- two groups (Figure 2). Similar to the shade-avoider At, seed- mation to higher PAR. lings of Cb-F, Cb-S, and Cr showed a lowering of the curve Besides At and Ch, the Brassicaceae family (mustards) under W4 conditions, whereas those of Aa, No, and Si be- includes many food crops (e.g. cauliflower, broccoli, radish, haved as the shade-tolerant Ch and showed virtually identi- cabbage, kale, and similar green leafy vegetables) and a di- cal light curves under W20 and W4 (Figure 2A). ETRm and versity of wild species from forested and open habitats. As a alpha values also illustrated that the W4 treatment led to first step to explore the possible connection between low decreased photosynthetic performance in At, Cb-F, Cb-S, PAR and low R:FR responses, we analyzed photoacclimation and Cr but not in Ch, Aa, No, and Si (Figure 2B; 2140 | PLANT PHYSIOLOGY 2021: 186; 2137–2151 Morelli et al. Downloaded from https://academic.oup.com/plphys/article/186/4/2137/6271415 by 81263600 user on 08 September 2021 Figure 2 Brassicaceae plants can be grouped with either A. thaliana or C. hirsuta based on their photoacclimation responses. A, Light curves of Arabidopsis thaliana (At), Capsella bursa-pastoris (Cb-F and Cb-S), Capsella rubella (Cr), Cardamine hirsuta (Ch), Arabis alpina (Aa), Nasturtium officinale (No), and Sysimbrium irio (Si) seedlings germinated and grown under white light (W20) for 7 d and then either kept under W20 or trans- ferred to lower PAR (W4) for one more day. Values represent the mean and standard error of n ¼ 3 plants for treatment. B, ETRm values calcu- lated from the curves shown in (A). C, Fv/Fm values of seedlings grown for 7 d under W20 and then transferred to higher PAR (W200) for seven more days. Mean and standard error of n ¼ 9 seedlings per treatment are represented. Asterisks in (B) and (C) mark statistically significant changes (t test, **P < 0.01) relative to W20. Supplemental Figure S1). We next analyzed photoacclima- based on their photoacclimation features corresponded with tion to increased irradiation quantifying Fv/Fm before or af- their elongation response to low R:FR. After germination ter transferring 7-d-old W20-grown seedlings to W200 for and growth for 3 d under W20 (R:FR ¼ 1.5–3.3), seedlings seven additional days. Again, At grouped together with the were either kept under W20 or transferred to FR-supple- two accessions of Cb and with Cr as they acclimated much mented W20 (W20þFR, R:FR ¼ 0.02) for four additional days, better to high PAR compared to the group formed by Ch, and then hypocotyl length was measured (Figure 3). Similar Aa, No, and Si (Figure 2C). Together, these photoacclimation to At, the Cb-F accession showed a strong hypocotyl elonga- results led to classify the former group as shade-avoiders, tion response, whereas Cb-S, Cr, and No elongated moder- and the latter as shade-tolerant species. ately in response to low R:FR. In contrast, Ch, Aa, and Si did not elongate in response to low R:FR (Figure 3A). These Photoacclimation responses can be uncoupled from results confirm that the elongation response to low R:FR shade-driven hypocotyl elongation cannot be fully predicted based on the photoacclimation Next, we investigated whether the classification of the se- phenotype of a particular accession. Nonetheless, accessions lected mustard species as shade-avoider or shade-tolerant classified as shade-avoider based on their photoacclimation Plant Physiology, 2021, Vol. 186, No. 4 PLANT PHYSIOLOGY 2021: 186; 2137–2151 | 2141 Downloaded from https://academic.oup.com/plphys/article/186/4/2137/6271415 by 81263600 user on 08 September 2021 Figure 3 The hypocotyl elongation response to low R:FR is plastic in Brassicaceae plants. A, The indicated genotypes were germinated and grown under W20 for 3 d and then either kept under W20 or transferred to low R:FR (W20þFR) for four more days. Then, pictures were taken and hypo- cotyl length was measured. B, Hypocotyl length of the indicated mutants grown as indicated in (A). In both (A) and (B), mean and standard error of measurements from at least 20 seedlings in n ¼ 3 independent experiments per treatment are represented. Asterisks mark statistically signifi- cant changes in W20þFR relative to W20 (t test, *P < 0.05, **P < 0.01). behavior (i.e. poor photoacclimation to decreased PAR but the hypersensitive Ch mutant lines appeared to gain a partial good photoacclimation to increased PAR) exhibit a range of shade-avoider phenotype in terms of photoacclimation to low elongation responses to low R:FR (i.e. from moderate to PAR, as lower values of light curves (Figure 4A) and ETRm strong elongation), whereas plant species with a shade-toler- (Figure 4B) were observed under W4 compared to W20. ant photoacclimation responses display either no elongation However, photoacclimation to increased PAR (W200) estimated or a mild shade-avoider phenotype in terms of hypocotyl from Fv/Fm values and also from chlorophyll levels (Molina- elongation when exposed to low R:FR (e.g. No). Contreras et al., 2019) was similar for Ch-WT, Ch-sis1, and Ch- The shade-avoider or shade-tolerant elongation phenotype in hfr1 plants (Figure 4C). We therefore concluded that manipula- response to low R:FR can be reversed by manipulating the levels tion of the plant ability to elongate in response to proximity of specific SAS regulators. Previous results have shown that At shade hardly impacts their photoacclimation capacity, at least lines overexpressing HY5 (At-HY5ox) display an attenuated hypo- when plants are growing in the absence of the low R:FR signal. cotyl response to low R:FR (Ortiz-Alcaide et al., 2019), whereas a similar but weaker response was observed in a quadruple mu- Activation of low R:FR signaling causes a decrease in tant defective in all members of the photolabile PIF quartet pigment levels and photosynthetic activity (PIFQ), PIF1, PIF3, PIF4 and PIF5 (At-pifq; Figure 3B). Despite the Low R:FR signals not only influence hypocotyl elongation different degrees of elongation response to low R:FR, these two but they are also known to reduce the contents of photo- lines showed photoacclimation responses to lower PAR very synthetic pigments (chlorophylls and carotenoids) in many similar to those of wild-type (At-WT) controls (Figure 4). Both plant species (Roig-Villanova et al., 2007; Cagnola et al., light curves (Figure 4A) and ETRm values (Figure 4B) were al- 2012; Patel et al., 2013; Bou-Torrent et al., 2015; Molina- most identical in At-WT plants and mutants hyposensitive to Contreras et al., 2019). The reduction is observed in both low R:FR. In the case of Ch, lines deficient in phyA (Ch-sis1) or elongating (At-WT) and nonelongating (Ch-WT) seedlings, HFR1 (Ch-hfr1) gain the ability to elongate when exposed to but it is stronger in the former (Figure 5). Cardamine hirsuta low R:FR (Molina-Contreras et al., 2019; Paulisic et al., 2021; mutants that gained the ability to elongate in response to Figure 3B). In contrast to the shade-hyposensitive At mutants, shade, such as Ch-sis1 and Ch-hfr1, also displayed stronger 2142 | PLANT PHYSIOLOGY 2021: 186; 2137–2151 Morelli et al. Downloaded from https://academic.oup.com/plphys/article/186/4/2137/6271415 by 81263600 user on 08 September 2021 Figure 4 Mutations that alter sensitivity to low R:FR do not impact photoacclimation responses. A, Light curves of At and Ch wild-type and mu- tant seedlings germinated and grown under W20 for 7 d and then either kept under W20 or transferred to lower PAR (W4) for one more day. Values represent the mean and standard error of n ¼ 3 plants for treatment. B, ETRm values calculated from the curves shown in (A). C, Fv/Fm values and HPLC-determined relative chlorophyll levels of seedlings grown for 7 d under W20 and then transferred to higher PAR (W200) for seven more days. Mean and standard error of n ¼ 9 seedlings (Fv/Fm) or n ¼ 3 independent pools (HPLC) per treatment are represented. Asterisks in (B) and (C) mark statistically significant changes (t test, *P < 0.05, **P < 0.01) relative to W20. reductions in photosynthetic pigment contents relative to Proximity shade signals have also been found to impact Ch-WT after low R:FR exposure (Figure 5A; Molina- photosynthesis at the level of gene expression. Analyses of Contreras et al., 2019). Conversely, At mutants with a re- low R:FR-triggered transcriptomic changes showed reduced duced ability to elongate in response to shade, such as At- levels of transcripts encoding photosynthesis-related pro- pifq and At-HY5ox (Figure 3B), showed attenuated reduction teins (e.g. enzymes involved in chlorophyll and carotenoid of pigment contents relative to At-WT when exposed to biosynthesis, components of the photosynthetic apparatus, low R:FR (Figure 5A). and/or members of the carbon fixation process) in several To test whether decreases in photosynthetic pigment lev- species, including alfalfa (Lorenzo et al., 2019), maize (Shi els driven by simulated shade exposure might affect photo- et al., 2019), tomato (Cagnola et al., 2012), and At (Leivar synthetic activity, we next measured Fv/Fm and UPSII in et al., 2012). Interestingly, the changes in the expression of seedlings grown either under W20 or under W20þFR photosynthesis-related genes triggered by low R:FR are at- (Figure 5B; Supplemental Figure S2A). Indeed, low R:FR was tenuated in the At-pifq mutant compared to At-WT seed- found to result in decreased photosynthetic activity in the lings (Figure 6). This is particularly evident in the case of low lines with strong pigment loss responses independently on R:FR-repressed photosynthetic genes (Figure 6), suggesting the species (At-WT, Ch-sis1, and Ch-hfr1). ETRm and alpha that the PIF-mediated regulation of gene expression in re- parameters also tended to be lower in WþFR-exposed At- sponse to low R:FR is instrumental for the observed changes WT, Ch-sis1, and Ch-hfr1 seedlings compared to W controls in photosynthesis (Figure 5). (Figure 5C; Supplemental Figure S2B). The effect of low R:FR on photosynthesis was much less dramatic in the rest of the Exposure of shade-avoider plants to low R:FR lines (At-pifq, At-HY5ox, and Ch-WT), which consistently improves their photoacclimation to low PAR displayed a reduced impact of W20þFR exposure on their The observation that exposure of low R:FR caused a de- photosynthetic pigment levels (Figure 5). creased in photosynthetic activity of At-WT seedlings and Plant Physiology, 2021, Vol. 186, No. 4 PLANT PHYSIOLOGY 2021: 186; 2137–2151 | 2143 shade-hypersensitive Ch mutants prompted us to analyze whether this light signal may also cause changes in chloro- plast ultrastructure. Cotyledons from At-WT seedlings ger- minated and grown for 2 d under W20 and then either kept in W20 or transferred to W20þFR for five additional days were collected and used for transmission electron micros- copy (TEM). Chloroplasts from low R:FR-exposed samples were found to exhibit larger grana stacks and contain less and smaller plastoglobules compared to W-grown controls (Figure 7). Interestingly, similar changes are associated to Downloaded from https://academic.oup.com/plphys/article/186/4/2137/6271415 by 81263600 user on 08 September 2021 low PAR photoacclimation (Rozak et al., 2002; Lichtenthaler, 2007; Wood et al., 2018). We therefore reasoned that expo- sure to low R:FR in the absence of any light intensity change might trigger responses to anticipate a foreseeable shading involving a decrease in PAR. To test this hypothesis, we ana- lyzed light curves of WT and mutant seedlings grown in ei- ther W20 or W20þFR and then transferred to lower PAR (W4) for 3 d (Figure 8). Pre-exposure of At-WT seedlings to low R:FR (W20þFR) resulted in a strongly attenuated reduc- tion in ETRm after their transfer to lower PAR (Figure 8A). In contrast, At mutants with reduced SAS elongation responses also lost the response to low R:FR in terms of im- proved photoacclimation to lower PAR (W4; Figure 8A). Pre- treatment with W20þFR had virtually no effect on the pho- toacclimation of Ch-WT seedlings to lower PAR (W4) but caused a slight but significant improvement of ETRm in shade-hypersensitive Ch mutants at day 1 after transfer to W4 (Figure 8A). When analyzing photoacclimation to higher PAR, pre-exposure of At-WT or Ch-WT seedlings to W20þFR resulted in no improvement compared to W20- grown controls (Figure 8B). If anything, Ch-WT seedlings grown under W20þFR photoacclimated worse than W20- grown seedlings when exposed to higher light intensity (Figure 8B). The battery of mustards that grouped together with At in terms of photoacclimation responses (Cb-F, Cb-S, and Cr; Figure 2; Supplemental Figure S1) also showed improved photoacclimation to reduced PAR when pre-exposed to low R:FR, whereas the simulated shade signal did not have an ef- fect on those clustered with Ch (Aa, No, and Si; Figure 8A). This low R:FR-dependent phenotype was independent of the growing light intensity and photoperiod, as it was also observed in At-WT seedlings growing under W200 or W200þFR for 8 h or 16 h a day (i.e. under long day or short day conditions, respectively) and then transferred to W15 (Supplemental Figure S3). Because both the response of shade-avoider plants to low R:FR and the acclimation to low light involve a reduced respiration rate to cope with the lim- ited generation of photoassimilates and hence contribute to quantified spectrophotometrically. B, Fv/Fm values of seedlings germi- Figure 5 Activation of low R:FR signaling reduces photosynthetic pig- nated and grown as indicated in (A). Lower pictures show false-color ment levels and activity. A, The indicated genotypes were germinated images in wild-type seedlings. (C) ETRm values of seedlings germi- and grown under W20 for 3 d and then either kept under W20 or nated and grown as indicated in (A). Mean and standard error of transferred to low R:FR (W20þFR) for four more days. Then, the levels n ¼ 3 independent pools of seedlings (A) or n ¼ 9 seedlings (B and C) of photosynthetic pigments (carotenoids and chlorophylls) were per treatment are represented. Asterisks mark statistically significant changes in W20þFR relative to W20 (t test, *P < 0.05). 2144 | PLANT PHYSIOLOGY 2021: 186; 2137–2151 Morelli et al. Downloaded from https://academic.oup.com/plphys/article/186/4/2137/6271415 by 81263600 user on 08 September 2021 Figure 6 Exposure to low R/FR triggers changes in photosynthetic gene expression that are attenuated in the hyposensitive At-pifq mutant. Data were extracted from a publicly available experiment (Leivar et al., 2012). At-WT and At-pifq lines were germinated and grown under 19 mmolm2s1 PAR white light (W20, R:FR of 6.48) for 2 d and exposed to low R:FR (W20þFR, R:FR of 0.006) for 0, 1, 3, or 24 h. Plots represent the number of DEGs either up- or downregulated in W20þFR versus W20 that are involved in photosynthetic pigment biosynthesis (Kyoto Encyclopedia of Genes and Genomes pathways ath00906 and ath00860), photosynthesis (ath00195 and ath00196), and carbon fixation (ath00710). carbon balance (Cagnola et al., 2012; Casal, 2013), we next tolerance refers to the capacity of a given plant to tolerate measured changes in respiration in whole wild-type At and low light levels, but it is also associated with a wide range of Ch seedlings exposed or not to low R:FR and then trans- traits, including phenotypic plasticity to optimize light cap- ferred to reduced PAR (Supplemental Figure S4). In W20 ture (Valladares and Niinemets, 2008). Analyzing a range of controls, respiration (estimated as total oxygen consumption caulescent herbs, it was suggested that the elongation re- in darkness) was reduced in At seedlings when they were sponse upon exposure to low R:FR was dependent on the moved to W4. When exposed to W20þFR, however, respira- shade habit, the shade-avoiders elongating the most and the tion was already lower and did not significantly change after shade-tolerant showing a mild or no elongation response transferring to lower PAR. In contrast, Ch seedlings showed (Smith, 1982). Indeed, elongation might not be the best so- similar respiration values in all conditions (Supplemental lution for plants that spend all their lives under a canopy or Figure S4). Based on these data we conclude that detection permanently shaded by other plants. Another important pa- and transduction of low R:FR signals not only allows shade- rameter to ascertain the degree of shade tolerance of a plant avoider plants to overgrow their neighbors but also to pre- is photoacclimation capacity, which is essential for plant fit- adapt their photosynthetic and respiratory machinery to ness in environments with changing light input conditions foreseeable conditions of actual shading involving reduced (e.g. those where the growth of nearby plants may suddenly PAR. In contrast, shade-tolerant plants have a better compromise access to light). By taking into account both adapted capacity to grow under reduce PAR and do not parameters (the hypocotyl elongation response and the ca- seem to use the low R:FR signal. pacity to acclimate to low or high PAR), here we analyzed the shade tolerance of several Brassicaceae species, including Discussion the closely related mustard model systems At and Ch. As a Plants have been traditionally classified as shade avoider and rule of thumb, we observed that Ch and other species show- tolerant based mostly on their natural habitat, although vir- ing a good photoacclimation response to lower PAR (and tually all plants are exposed to at least some degree of shade badly performing after transfer to higher PAR) showed a during their lifetime. As an ecological concept, shade poor or null elongation response to low R:FR (Figures 2 and Plant Physiology, 2021, Vol. 186, No. 4 PLANT PHYSIOLOGY 2021: 186; 2137–2151 | 2145 Downloaded from https://academic.oup.com/plphys/article/186/4/2137/6271415 by 81263600 user on 08 September 2021 Figure 7 Low R:FR triggers ultrastructural changes in At chloroplasts. At-WT seeds were germinated and grown under W20 for 2 d and then either kept under W20 or transferred to low R:FR (W20þFR) for 5 more days. Cotyledons were then used for TEM analysis of chloroplast ultrastructure. Representative pictures at different scales (numbers indicate micrometer) are shown. Boxplots show quantification of the indicated parameters from the images. Boxes show the values between the upper and the lower quartile, the cross represents the mean and the horizontal line the me- dian. Whiskers (the upper and lower extremes) and circles represent single data and the ones located outside of the whiskers limit are the outliers (data with the same numerical value are visualized as a single point). For quantifying grana thickness, all the distinguishable structures were used (W20 n ¼ 30, W20þFR n ¼ 20). For quantifying grana layers, four major grana complexes from higher magnifications were measured. For quantify- ing the number of plastoglobules, at least six individual chloroplasts for each treatment were used. Plastoglobule area was measured for all the plastoglobules (W20 n ¼ 87, W20þFR n ¼ 22). PG, plastoglobules; G, grana. 2146 | PLANT PHYSIOLOGY 2021: 186; 2137–2151 Morelli et al. Downloaded from https://academic.oup.com/plphys/article/186/4/2137/6271415 by 81263600 user on 08 September 2021 Figure 8 Pre-exposure to low R:FR improves the photoacclimation to low PAR in shade-avoider plants. A, The indicated genotypes were germi- nated and grown under W20 for 3 d, transferred to either W20 or W20þFR for 4 d, and then exposed to W4. Mean and standard error of ETRm val- ues at 0, 1, 2, and 3 d after transfer to W4 are shown (n ¼ 3 seedlings per treatment). Asterisks indicate statistically significant differences between treatments (W20 or W20þFR) over time (two-way ANOVA, *P < 0.05, **P < 0.01). B, Wild-type At and Ch lines were germinated and grown under W20 for 2 d, transferred to either W20 or W20þFR for 5 d, and then exposed to W200 for 7 more days. Fv/Fm values and HPLC-quantified chloro- phyll levels were determined. Mean and standard error of n ¼ 7 seedlings (Fv/Fm) or n ¼ 3 independent pools (HPLC) per treatment are repre- sented. Asterisks mark statistically significant differences between values before and after exposure to W200 (t test, *P < 0.05; **P < 0.01). 3). Mustards such as At that photoacclimated poorly to R:FR, but there were exceptions of poorly elongating species lower PAR but better to higher PAR tended to more con- such as No (Figures 2 and 3). Furthermore, mutation of spicuously elongate their hypocotyls in response to low genes encoding SAS regulators can dramatically change the Plant Physiology, 2021, Vol. 186, No. 4 PLANT PHYSIOLOGY 2021: 186; 2137–2151 | 2147 elongation response to low R:FR without improving the thylakoid layers per granum but more small grana spread photoacclimation phenotype (Figure 4). Together, these throughout the chloroplast compared to end-of-day R con- results confirm that the capacity for photosynthetic acclima- trols (Kasperbauer and Hamilton, 1984). While these differ- tion to changing irradiance is a species-specific trend (Bailey ences in chloroplast ultrastructure might derive from et al., 2001) and a reliable indicator of shade tolerance. The distinct treatments being applied to diverse species, both shade-induced hypocotyl elongation response should only solutions likely contribute to optimize photosynthesis in the be used as a complementary phenotype to classify a plant shade, when relatively less photons would strike a leaf. as shade-tolerant (badly adapted to higher PAR exposure, Indeed, leaves that develop under low PAR have chloroplasts well adapted to live under lower PAR and poorly responsive with less plastoglobules (which are derived from thylakoid to low R:FR) or shade-avoider (well adapted to higher PAR, membranes) and more thylakoids per granum (Rozak et al., Downloaded from https://academic.oup.com/plphys/article/186/4/2137/6271415 by 81263600 user on 08 September 2021 poor performers under lower PAR that elongate when ex- 2002; Lichtenthaler, 2007; Wood et al., 2018). Based on these posed to low R:FR). results, we suggest that the chloroplast ultrastructural Our results also unveiled that an activation of low R:FR changes observed in At-WT plants grown under low R:FR signaling in shade-avoider species such as AtAt-WT and are most likely aimed to acclimate their photosynthetic ma- shade-tolerant Ch plants with mutations causing low R:FR chinery to perform better under low PAR by, for instance, hypersensitivity (Ch-sis1 and Ch-hfr1) regulated photosyn- allowing a more efficient energy transfer. In agreement, pre- thesis at multiple levels. We confirmed that exposure to treatment with low R:FR improved photoacclimation to low WþFR caused a substantial decrease in the levels of photo- PAR of At-WT seedlings but had no effect in At mutants synthetic pigments (chlorophylls and carotenoids) in these defective in low R:FR signaling (Figure 8). Further experi- lines (Roig-Villanova et al., 2007; Bou-Torrent et al., 2015; ments showed that the observed positive effect of low R:FR Molina-Contreras et al., 2019; Paulisic et al., 2021) and exposure for acclimation to low PAR can be observed in At- proved that the changes had a direct impact on decreasing WT plants growing under different light conditions phytosynthetic activity (Figure 5). Low R:FR treatments are (Supplemental Figure S3) and in other shade-avoider known to trigger changes in gene expression within minutes Brassicaceae (Cb-F, Cb-S, and Cr), but not in shade-tolerant (Kohnen et al., 2016). These changes, which are often instru- species such as Ch, Aa, No, and Si (Figure 8A). mental for altering rapid growth responses, such as hypo- At low irradiances, a proper balance between carbon allo- cotyl or petiole elongation, are usually mediated by PIFs cation to growth and to respiration is important to meet (Hornitschek et al., 2009; Galstyan et al., 2011; Cifuentes- the challenges associated with a shade environment. Wild- Esquivel et al., 2013; de Wit et al., 2015; Gallemi et al, 2017). type At (shade-avoider) but not Ch (shade-tolerant) seed- PIFs were also found to regulate longer-term changes in lings showed a drop in dark respiration when irradiation gene expression such as those affecting photosynthetic was reduced (Supplemental Figure S4), likely to reduce car- genes (Figure 6). Because loss of PIFQ function in the At- bon loss for a better carbon balance. This adaptive mecha- pifq mutant resulted in a much attenuated response to nism might contribute to explain why shade-avoider and WþFR compared to At-WT in terms of photosynthetic shade-tolerant species appear to show little or no differences gene expression (Figure 6) but it also prevented photosyn- in carbon balance under low light conditions (Sterck et al., thetic pigment and activity loss (Figure 5), we propose that 2013; Pons and Poorter, 2014). Similar to that observed for stabilization of PIFQ proteins following low R:FR exposure photosynthetic activity (Figure 8), the respiration drop ob- triggers a reprogramming of photosynthesis-related gene ex- served in At-WT seedlings was attenuated by pre-exposure pression that eventually results in lower pigment levels and to low R:FR (Supplemental Figure S4). Interestingly, there is reduced photosynthetic activity. Based on the results evidence for the specific activation/deactivation of respira- obtained with other mutants (Figure 5), we speculate that tory pathways by the phytochrome system at different levels this signaling network is further influenced by factors such (Ribas-Carbo et al., 2008; Igamberdiev et al., 2014). as HFR1 and HY5, which prevent PIF binding to target genes Regardless of the signaling pathway connecting low R:FR by heterodimerization (Hornitschek et al., 2009) or competi- perception to reduced photosynthesis and respiration, this is tion for promoter binding sites (Toledo-Ortiz et al., 2014), likely part of an anticipation mechanism for shade-avoider respectively. plants to prepare for the foreseeable reduction in PAR asso- Concomitant with the described molecular and physiolog- ciated with shading. Indeed, low R:FR signals are perceived ical changes, we discovered that low R:FR treatment of At- before actual shading takes place and light becomes limiting, WT seedlings triggered ultrastructural changes in the chloro- and hence they are considered to act as a warning signal plast endomembrane systems resembling those occurring af- that shading might occur (Martinez-Garcia et al., 2010; ter transfer to low PAR (Figure 7). Grana with more Casal, 2013). When shade-avoider plants such as At and thylakoid layers and increased thickness were observed in most crops (including tomato, cereals, or legumes) grow the chloroplasts of At seedlings exposed to simulated shade. among taller plants or in a forest understory, they will use In contrast, chloroplasts from tobacco (Nicotiana tabacum) the low R:FR signals coming from a closing canopy to elon- leaves that received end-of-day-FR treatments (considered gate (to overgrow its neighbors) but also to readapt its pho- to induce similar shade responses as low R:FR) showed fewer tosynthetic and respiratory machinery to low PAR before 2148 | PLANT PHYSIOLOGY 2021: 186; 2137–2151 Morelli et al. actual shading takes place. In contrast, shade-tolerant plants previously described (Koenig et al., 2019). Seeds of S. irio are adapted to grow under dim light and hence photoaccli- were collected from wild populations in Bellaterra mation to low PAR is hardly improved even when hypersen- (Barcelona, Spain, coordinates: 41.497731, 2.109558). Seeds of sitive mutants that show shade-avoider responses in terms watercress (Nasturtium officinale) were provided by a seed of elongation (Figure 3) and photosynthesis (Figure 6) are company (www.semillasfito.es). Arabidopsis thaliana and Ch pre-exposed to low R:FR (Figure 8). mutant and transgenic lines were previously available in our While the observed decrease in respiration and photosyn- laboratories (Molina-Contreras et al., 2019; Ortiz-Alcaide thetic pigment and activity levels in shade-avoider plants et al., 2019; Paulisic et al., 2021). appears to be part of the anticipation mechanism to an For the light acclimation experiments seedlings were ger- eventual reduction in PAR, a too committed response might minated and grown in Petri dishes containing solid medium Downloaded from https://academic.oup.com/plphys/article/186/4/2137/6271415 by 81263600 user on 08 September 2021 be detrimental if light conditions change (e.g. if shading without sucrose (0.5 MS): 2.2 gL1 MS basal salt mixture does not occur or shade plants become exposed again to di- (Duchefa), 1% (w/v) agar, 0.25 gL1 2-(N-morpholino)etha- rect sunlight). We have previously shown that a compensa- nesulfonic acid (MES; Sigma Aldrich), pH 5.7). Normal light tion mechanism exist that represses the response to low conditions refer to white light (W) produced by cool-white R:FR when the photosynthetic capacity of chloroplasts is vertical fluorescent tubes of a photosynthetic photon flux compromised (Ortiz-Alcaide et al., 2019). The retrograde (i.e. density in the PAR region (PPFD) of 20-24 mmol m2 s1 chloroplast-to-nucleus) pathway that adapts low R:FR per- (W20) with a R:FR of 1.5–3.3. Low light and high light condi- ception and signaling to the photosynthetic status of the tions corresponded to W of PPFD of 4 (W4) and 200 (W200) plant involves the antagonistic factors PIFs and HY5, which mmol m2 s1, respectively, produced by horizontal fluores- also participate in retrograde signaling when underground cent tubes. Low R:FR treatment was produced by supple- seedlings are illuminated and start their photomorphogenic menting W20 with FR (W20þFR). FR was emitted from a (i.e. photosynthetic) development (Ruckle et al., 2007; GreenPower LED module HF FR (Philips), providing a R:FR Martin et al., 2016; Xu et al., 2016; Ortiz-Alcaide et al., 2019). of 0.02 (Martinez-Garcia et al., 2014). For the light acclima- The balance of positive and negative regulators together tion experiments shown in Supplemental Figure S3, seedlings with the chloroplast-mediated control of SAS likely contrib- were germinated and grown in Petri dishes, as previously de- ute to prevent an excessive response to shade, hence pre- scribed, but exposed to long-day (16-h light/8-h darkness) venting photooxidative damage (resulting from light or short-day (8-h light/16-h darkness) photoperiods. The intensity exceeding the photosynthetic capacity of the plant) light part of the photoperiod was produced by cool-white and facilitating the return to high R:FR conditions if the low horizontal fluorescent tubes of 200–210 mmol m2 s1 of R:FR signal disappears (e.g. if a commitment to the shade- PPFD (W200) with R:FR of 2–3.5). In that case, low light con- avoidance lifestyle is unnecessary). Together, our work dem- ditions corresponded to values of 15 mmol m2 s1 PPFD onstrates that regulation of photosynthetic (chloroplast) (W15). In this set-up, low R:FR treatment was produced by performance is both an output and an input of the re- supplementing W200 with the same FR lamps described sponse of plants to shade. Our results therefore contribute above (W200þFR), obtaining a R:FR of 0.2–0.25. Light fluence to a better understanding of how plants respond to shade, a rates were measured with a Spectrosense2 meter (Skye Instruments Ltd), which provides PPFD (400–700 nm), and knowledge that will contribute to optimally grow crop photon flux density in 10 nm windows of R (664–674 nm) plants closer together or/and under canopies (e.g. in inter- and FR (725–735 nm) regions to calculate the R:FR cropping settings). (Martinez-Garcia et al., 2014). Full spectra photon distribu- Materials and methods tion of W and WþFR treatments have been described else- where (Molina-Contreras et al., 2019). Plant material and growth conditions Alpine rock cress (Arabis alpina, pep1-1 mutant; Wang et al., Measurement of hypocotyl length 2009), Arabidopsis (Arabidopsis thaliana, Col-0 accession), For hypocotyl measurement, about 30 seeds of each geno- hairy bittercress (Cardamine hirsuta, Oxford, Ox accession; type were germinated and grown on plates containing 0.5 Molina-Contreras et al., 2019), shepherd’s purse (Capsella MS solid media. For quantification of hypocotyl length, at bursa-pastoris, accessions Strasbourg-1, Str-1 and Freiburg-1, least 20 seedlings were analyzed with the FIJI-ImageJ soft- Fre-1), pink shepherd’s-purse (Capsella rubella), and London ware (Schindelin et al., 2012), as described (Roig-Villanova rocket (Sysimbrium irio) plants were grown in the green- et al., 2019). All experiments were repeated at least three house under long-day photoperiods (16-h light and 8-h times with consistent results. Hypocotyl measurements from dark) to produce seeds, as described (Gallemi et al., 2017). all the different experiments were averaged. Seeds of C. bursa-pastoris were collected by Ruben Alcazar (University of Barcelona, Spain) from wild populations in Photosynthetic measurements and pigment Strasbourg (France, coordinates: 48.612436, 7.767881; Str-1) quantification and Freiburg (Germany, coordinates: 47.994945, 7.861979; Whole seedlings were harvested, ground in liquid nitrogen, Fre-1). Seeds of C. rubella, collected from wild populations in and the resulting powder was used for quantification of Crete (Greece, coordinates 35.29, 24.42; accession 879) were chlorophylls and carotenoids either spectrophotometrically Plant Physiology, 2021, Vol. 186, No. 4 PLANT PHYSIOLOGY 2021: 186; 2137–2151 | 2149 or by high performance liquid chromatography (HPLC) as Transmission electron microscopy described (Bou-Torrent et al., 2015). Chlorophyll fluores- TEM was carried out as described (Flores-Perez et al., 2008). cence measurements were carried out on seedlings using a Chloroplast features in the pictures were quantified by using MAXI-PAM fluorometer (Heinz Walz GmbH) as described the FIJI-ImageJ software (Schindelin et al., 2012). (Molina-Contreras et al., 2019). Briefly, for every measure- ment the whole cotyledons of seven seedlings were consid- Accession numbers ered. Effective quantum yield of photosystem II (PSII) under Sequence data from this article can be found in the EMBL/ growth light, UPSII, was measured as DF/Fm0 , where DF cor- Genbank and C. hirsuta genetic and genomic resource responds to Fm0 F (the maximum minus the minimum (http://chi.mpipz.mpg.de) data libraries under the following fluorescence of light-exposed plants). Maximum quantum accession numbers: AT1G02340 (AtHFR1), AT5G11260 Downloaded from https://academic.oup.com/plphys/article/186/4/2137/6271415 by 81263600 user on 08 September 2021 yield of PSII, Fv/Fm, was calculated as (FmFo)/Fm, where (AtHY5), AT2G20180 (AtPIF1), AT1G09530 (AtPIF3), Fm and Fo are, respectively, the maximum and the mini- AT2G43010 (AtPIF4), AT3G59060 (AtPIF5), CARHR001660 mum fluorescence of dark-adapted samples. For dark accli- (ChHFR1), and CARHR009540 (SIS1/ChPHYA). mation, plates were incubated for at least 30 min in darkness to allow the full relaxation of photosystems. Light curves were constructed with 10 incremental steps of actinic Supplemental data irradiance (E; 0, 20, 55, 110, 185, 280, 395, 530, 610, 700 The following materials are available in the online version of lmol photonsm2s1of PPFD). For each step, UPSII was this article. monitored every minute and electron transport rate (ETR) Supplemental Figure S1. Alpha values calculated from was calculated as EUPSII0.84  0.5 (where 0.84 is the the light curves shown in Figure 2A. light absorptance by an average green leaf and 0.5 is the Supplemental Figure S2. Activation of low R:FR signaling fraction of absorbed quanta available for PSII). The light re- reduces photosynthetic activity. sponse and associated parameters ETRm (maximum elec- Supplemental Figure S3. Pre-exposure to low R:FR tron transport rate) and alpha (photosynthetic rate in light- improves photoacclimation to lower PAR in A. thaliana limited region of the light curve) were characterized by fit- plants grown under photoperiods. ting iteratively the model of the rETR versus E curves using Supplemental Figure S4. Exposure to low R:FR differen- MS Excel Solver (Platt et al., 1980). The fit was very good in tially impacts respiration rate of shade-avoider and shade- all the cases (r > 0.98). tolerant plants. Acknowledgments Respiration measurements Seedlings were germinated and grown on 0.5 MS plates, as We thank Ma Rosa Rodrı́guez (CRAG) for technical support, described (Supplemental Figure S4). Before the measure- and George Coupland (MPI for Plant Breeding Research, ments, seedlings were placed in the dark for about 30 min Cologne, Germany), Rubén Alcazar (Universitat de Barcelona, to avoid light-enhanced dark respiration. Five to 10 seedlings Spain) and Ignacio Rubio (CRAG) for providing mustard were then collected, immediately weighed, and placed into seeds. the respiration cuvette containing the respiration buffer (30- mM MES, pH 6.2, 0.2-mM CaCl2). Oxygen uptake rates were Funding measured in darkness using a liquid-phase Clark-type oxygen L.M. received a predoctoral fellowships from La Caixa electrode (Rank Brothers Ltd.) as previously described Foundation (INPhINIT fellowship LCF/BQ/IN18/11660004). (Florez-Sarasa et al., 2009) at a constant temperature of W.Q. is a recipient of a predoctoral Chinese Scholarship 23 C. Council (CSC) fellowship. A.I.-S. is supported by a predoc- toral fellowship from MICINN (PRE2018-083610). I.F.-S. has Microarray data analyses received funding from the European Union’s Horizon 2020 Microarray data corresponding to Col-0 At-WT and At-pifq research and innovation programme under the Marie seedlings exposed to low-R:FR for 0, 1, 3, and 24 h (Leivar Skłodowska-Curie grant agreement no. 753301. Our research et al., 2012) were analyzed to select for differentially is supported by grants from MICINN-FEDER (BIO2017- expressed genes (DEGs) specifically related to photosynthe- 85316-R, and BIO2017-84041-P) and AGAUR (2017-SGR1211, sis. The reported list of DEGs was further filtered using cut- 2017-SGR710 and Xarba) to J.F.M.-G. and M.R.-C. 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