Characterization of CYCLOPHILLIN38 shows that a photosynthesis-derived systemic signal controls lateral root emergence

Characterization of CYCLOPHILLIN38 shows that a photosynthesis-derived systemic signal controls lateral root emergence Lina Duan , 1,2 Juan Manuel Pe´rez-Ruiz , 3 Francisco Javier Cejudo 3 and Jose´ R. Dinneny 1,2, * ,† 1 Biology Department, Stanford University, Stanford, CA 94305, USA 2 Department of Plant Biology, Carnegie Institution for Science, Stanford, CA 94305, USA 3 Instituto de Bioquı´mica Vegetal y Fotosı´ntesis, Universidad de Sevilla and Consejo Superior de Investigaciones Cientı´ficas, Avda Ame´rico Vespucio 49, 41092 Sevilla, Spain *Author for communication: [email protected] † Senior author. L.D., J.M.P., F.J.C., and J.R.D. designed this study. L.D. performed the mutant screen, phenotypic analysis, cloning, imaging, and performed the data analysis. J.M.P. performed the biochemistry studies and measurements of photosynthesis parameters. L.D., J.M.P, F.J.C., and J.R.D. wrote and revised themanuscript. 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) is: Jose´ R. Dinneny (di[email protected]). Abstract Photosynthesis in leaves generates fixed-carbon resources and essential metabolites that support sink tissues, such as roots. Two of these metabolites, sucrose and auxin, promote growth in root systems, but the explicit connection between photosynthetic activity and control of root architecture has not been explored. Through a mutant screen to identify pathways regulating root system architecture, we identified a mutation in the Arabidopsis thaliana CYCLOPHILIN 38 (CYP38) gene, which causes accumulation of pre-emergent stage lateral roots. CYP38 was previously reported to stabilize photosystem II (PSII) in chloroplasts. CYP38 expression is enriched in shoots, and grafting experiments show that the gene acts non-cell-autonomously to promote lateral root emergence. Growth of wild-type plants under low-light conditions phenocopies the cyp38 lateral root emergence defect, as does the inhibition of PSII-dependent electron transport or Nicotinamide adenine dinucleotide phosphate (NADPH) production. Importantly, these perturbations to photosynthetic activity rapidly suppress lateral root emergence, which is separate from their effects on shoot size. Supplementary exogenous sucrose largely rescued primary root (PR) growth in cyp38,butnotlateralroot growth. Auxin (indole-3-acetic acid (IAA)) biosynthesis from tryptophan is dependent on reductant generated during photosynthesis. Consistently, we found that wild-type seedlings grown under low light and cyp38 mutants have highly diminished levels of IAA in root tissues. IAA treatment rescued the cyp38 lateral root defect, revealing that photosynthesis promotes lateral root emergence partly through IAA biosynthesis. These data directly confirm the importance of CYP38-dependent photosynthetic activity in supporting root growth, and define the specific contributions of two metabolites in refining root architecture under light-limited conditions. Introduction The coordination of shoot and root system growth is highly dependent on environmental context (Bloom, 2005;Puig et al., 2012). Previous research has shown that the limitation of water or nutrients in soil can enhance the growth of the root system relative to the shoot (Rella´n-A ´lvarez et al., 2015). Conversely, shoot tissues can also have a positive effect on root system branching (Wightman and Thimann, Research Article Received August 18, 2020. Accepted October 29, 2020. Advance access publication November 27, 2020 V CAmerican Society of Plant Biologists 2020. All rights reserved. For permissions, please email: [email protected] doi:10.1093/plphys/kiaa032 PLANT PHYSIOLOGY 2021: 185: 503–518 Downloaded from https://academic.oup.com/plphys/article/185/2/503/6007478 by Universidad de Sevilla user on 06 April 2021 1980;Bhalerao et al., 2002). Initial studies investigating the role of shoot-derived signals in promoting root growth demonstrated that the excision of tissues from the seedling shoot inhibited the emergence of lateral root (LR) primordia. The critical signal transmitted from shoot to root was later identified as auxin (McDavid et al., 1972;Rost, 1986). Using radiotracing to track transport, auxin applied to shoot tissues was later detected in roots (Rowntree and Morris, 1979;Kerk and Feldman, 1995;Reed et al., 1998). Furthermore, localized application of an auxin-transport inhibitor to the root/shoot junction prevented the transport of labeled auxin to roots and inhibited LR emergence. Later studies used transgenes to express orthogonal auxin-biosynthetic enzymes in shoot tissues and showed that auxin biosynthesis in the shoot is sufficient to promote LR emergence (Swarup et al., 2008). It has been suggested that the initiation of LRs is promoted by root tip-derived auxin, while emergence is promoted from the shoot (Laskowski et al., 1995;Bhalerao et al., 2002). The signaling pathways controlling the synthesis and transport of auxin from the shoot to root are currently being explored, but light intensity appears to be a critical component (Halliday et al., 2009). Hersch et al. showed that auxin levels increased as a consequence of increased irradiance of seedlings with light; however, light quality, manipulated by changing the ratio of red to far-red light, also influences auxin accumulation (Hersch et al., 2014). Bhalero et al. showed that LR emergence, and the associated shoot-derived auxin pulse that occurs after germination, is dependent on seedling exposure to light (Bhalerao et al., 2002). While light was shown to be necessary for shoot-mediated auxin accumulation in roots, whether this is dependent on photosynthetic activity or other light-signaling pathways has not been determined. Light perception by wavelength-specific photoreceptors regulates the production of auxin in the shoot to affect hypocotyl elongation (deWitetal.,2016). Auxinindependent mechanisms for light-regulated root growth also exist. ELONGATED HYPOCOTYL 5 (HY5) protein accumulates in a light-dependent manner, downstream of photoreceptor signaling, and translocates to the root to promote root branching (Chen et al., 2016;Van Gelderen et al., 2018; Zhang et al., 2019). Nevertheless, the relationship between these various light-sensitive pathways is still unclear, as is the potential role of photoreceptor-independent mechanisms in the systemic control of root architecture. From a resource-centric point of view, the plant body can be separated into two domains: source tissues, which generate resources, and sink tissues, which use these resources (Chang et al., 2017). Root system growth predominantly occurs below ground in most species, and requires the products of photosynthesis from shoots (Bloom, 2005). Sucrose is an important transport molecule that provides the energy for metabolism and the carbon-based building blocks for growth. Previous work has demonstrated that PR growth is limited in seedlings grown in the dark, while supplementing with exogenous sucrose can rescue this effect (Kircher and Schopfer, 2012). Photosynthesis also generates the reductant NADPH, which is used in the biosynthesis of a number of metabolites. In particular, the conversion of indole-3-pyru- vate acid (IPA) to indole-3-acetic acid (IAA), the natural form of auxin, utilizes the redox power of NADPH in the reaction (Mashiguchi et al., 2011;Zhao, 2012). What role these products of photosynthesis play in light-regulated LR growth has not been explored, which limits our ability to determine the relative contributions of photosynthesis-dependent metabolite production and photoreceptor-dependent signaling to root system architecture. LR emergence is a complex developmental process that integrates both new organ morphogenesis and structural change of the old root tissue. LRs form through oriented cell divisions within the pericycle tissue layer, which is hidden three layers deep inside the parent root (Van Norman et al., 2013). The positioning of LRs along the pericycle cell profile and the formation of LR founder cells are determined by the oscillation of auxin signaling (Moreno-Risueno et al., 2010). A dome-shaped lateral root primordia (LRP) is then formed after a series of cell divisions. The penetration of LRP through the outer tissues of the parent root requires auxin transport and signaling to change the mechanical properties of the three overlying tissue layers (Swarup et al., 2008; Lucas et al., 2013;Vermeer et al., 2014). In this study, we specifically explore the role that photosynthesis plays in the systemic regulation of LR emergence by shoots. We identified a mutant that strongly inhibits LR emergence and revealed that the causative mutation disrupts CYCLOPHILIN 38 gene function, which normally promotes the assembly of photosystem II (PSII; Fu et al., 2007). The effects of the cyp38 mutation on root architecture are systemic, shoot-specific, and mimicked by the growth of seedlings with reduced light irradiance or inhibitors of light-dependent electron transport. We reveal that the cyp38 mutation or low light intensity causes a dramatic reduction in auxin biosynthesis, and the effect of these perturbations on LR emergence can be rescued with exogenous auxin treatment. Finally, genetic analysis of hy5 mutants reveals that this photoreceptor-dependent pathway likely acts in parallel to the photosynthesis-dependent pathway defined here, thus revealing multiple levels of regulation that coordinate root growth with shoot physiology. Results CYP38 is necessary for LR emergence To identify factors that play a role in LR emergence, we conducted a mutant screen and characterized a recessive mutant (originally named prematurely stunted lateral organs, presto) that exhibits a severe reduction (85.4% reduction) in LR density under standard growth conditions, compared to wild-type (WT), while PR growth is also significantly reduced, but to a lesser extent (51% reduction; Figure 1, A– C). Next-generation ethyl methanesulfonate (EMS) mutation mapping using sequencing was used to identify the causative genetic locus, CYCLOPHILIN 38 (CYP38; Supplemental Figure S1). A T-DNA allele (cyp38-5) with an insertion in the sixth exon phenocopied the EMS allele (cyp38-4; Figure 1, 504 |PLANT PHYSIOLOGY 2021: 185; 503–518 L. Duan et al. Downloaded from https://academic.oup.com/plphys/article/185/2/503/6007478 by Universidad de Sevilla user on 06 April 2021 0 1 2 3 4 5 LR density (#/cm) WT cyp38-4 cyp38-5 WT cyp38-4 cyp38-5 AB H 0 1 2 3 4 WT cyp38-4 proCYP38:CYP38 /cyp38-4 LR density (#/cm) * cyp38-4 cyp38-5 C 5’ 3’ WT cyp38-5 WT WT WT WT D E cyp38-5 WT cyp38-5 cyp38-5 cyp38-5 cyp38-5 0.0 0.5 1.0 1.5 2.0 2.5 PR length (cm) WT cyp38-4 cyp38-5 F 0 3 6 9 Lateral root primordia # Ipre-E EMTotal * * * * * * * * * * * ** * * * * * * * * * (Salk_029448) (CAG->TAG) G K 0 5 10 15 123456 PR length (cm) LR number WT cyp38- 5 r2=0.902 r2=0.573                                                 1 2 3 4 LR density (#/cm)                                                  0.2 0.3 0.4 WT 10dpg cyp38-5 13dpg Leaf area (cm2) WT 10 dpg cyp38-5 13 dpg IJ * * * * * * WT 10dpg cyp38-5 13dpg Figure 1 CYP38 is required for LR emergence. A, Scanned root images of 11-dpg WT, cyp38-4 and cyp38-5. 6-dpg root tip positions are labeled with black marks. Scale bar = 5 mm. Band C, Quantifications of LR density (B) and PR length (C) in two alleles of cyp38. (***P50.001, pairwise t test with Hochberg correction, n410). D, Quantifications of LR density of the EMS allele, cyp38-4, and the complemented line. (***P50.001, pairwise ttest with Hochberg correction, six complemented T1 plants are used). E, Diagram describing the mutation position of cyp38-4 (red asterisk) and T-DNA insertion position of cyp38-5 (triangle). F, Quantifications of different stages of LRP in WT and cyp38-5. I represents the initiation stage, pre-E represents pre-emergence stage, E represents emerged LR stage, M represents matured LRs, and Total represents the sum of all four stages. (*P50.05, ***P50.01, ***P50.001, pairwise ttest with Hochberg correction, n48). G, Microscopic images of cleared WT and cyp38-5 roots. Arrows point to pre-emerged LRP. Scale bar = 50 mm. H, Scanned shoot and root images of 10-dpg WT and 13-dpg cyp38-5. Root tip positions of 6-dpg are labeled with black marks. Scale bars = 5 mm. Iand J, Quantifications of shoot area (I) and LR density (J) in 10-dpg WT and 13-dpg cyp38-5. (***P50.001, pairwise ttest, n420). K, Quantifications of LR number over PR length were made with 6- to 10-d-old WT and cyp38-5 roots. Data points obtained at each day are pooled to generate scatter plot. Linear regression lines are added to show their trend. Plant Physiology, 2021, Vol. 185, No. 2 PLANT PHYSIOLOGY 2021: 185; 503–518 |505 Downloaded from https://academic.oup.com/plphys/article/185/2/503/6007478 by Universidad de Sevilla user on 06 April 2021 A–C), which has an early stop codon in the second exon (Figure 1E;Supplemental Figure S1). Expression of CYP38 using its native promoter fully complemented cyp38-4 (Figure 1D), indicating that CYP38 is indeed the causative gene. To determine the developmental basis for the cyp38 phenotype, we classified LR growth into four distinct stages (Malamy and Benfey, 1997;Duan et al., 2013;Orman-Ligeza et al., 2016): initiation (I, including stages I–III), pre-emergence (Pre-E, including stages IV–VII), emergence (E), and mature LR (M; Supplemental Figure S1B). Compared to WT, where the majority of LRs have typically progressed to the maturation stage in 8-d post germination (dpg) seedlings, we found that cyp38- 5mutants had a significant enrichment of pre-emergence stage LRs (Figure 1F). LRP exhibited a flattened shape in cyp38-5 instead of the typical dome shape observed in WT (Figure 1G). This defect has also been observed in other mutants that specifically affect the emergence stage (Pe´ret et al., 2012;Ferna´ndez-Marcos et al., 2017). It is likely that initiation and/or founder cell specification stages may also be disrupted to account for the full reduction in LR development. Previous work studying the effects of mutations in CYP38 demonstrated that shoot growth is reduced, particularly under high-light intensity growing conditions (Fu et al., 2007). Since the amount of shoot tissues corresponds with root growth, this raises the possibility that the observed changes in LR development in cyp38 are merely an indirect effect of the overall slower growth of the seedling rather than a direct effect of changes in the photosynthetic activity. Indeed, the total number of LRs (pre- and post-emergence) per seedling was reduced in cyp38 mutants, compared to WT, consistent with an overall slower rate of seedling development (Figure 1F). However, a closer examination of the LR growth defect revealed that development was not uniformly affected. For example, the number of pre-emergence stage LRs showed a trend opposite of what we expected, with an increase relative to WT. Further evidence that shoot size differences do not fully explain the changes in LR development come from comparisons between seedlings of different ages. In 13-dpg cyp38-5 seedlings, shoot size was measured to be 23.9% larger than WT, while LR emergence was still 32% reduced relative to WT at 10 dpg (Figure 1, H–J). We also analyzed the allometric relationship between PR length andLRnumberina5-dtimecourse(rootswereimagedevery24hfrom6to10dpg).Plottingofthesedatashow that WT PR length shows a linear relationship with LR number and that this correlation is less positive for cyp38 (r 2 = 0.902, WT; 0.573, cyp38-5;Figure 1K). These differences are inconsistent with the hypothesis that the cyp38 growth is simply delayed as one would expect allometric relationships to be maintained. Together these data strongly suggest that the developmental program of cyp38 is altered substantially and specifically, leading to a change in root architecture as a consequence of defects in LR emergence, and that these defects are not fully explained by the reduction in shoot size. CYP38 is primarily localized to the chloroplast As previously reported, CYP38 encodes a cyclophilin-related protein important in the assembly and stability of PSII under high light (Fu et al., 2007;Sirpio¨ et al., 2008;Vasudevan et al., 2012). However, limited characterization of the tissuespecific and subcellular localization of the protein has been performed. A transcriptional fusion, proCYP38::erGFP,and quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) indicated that the gene is expressed predominantly in shoot tissues (mesophyll and hypocotyl; Figure 2, A and B). While we observed low levels of reporter expression in root regions that are close to the hypocotyl, very little fluorescence could be detected in LRP and root tips (Figure 2A). Transient expression of a Ypet-tagged translational fusion to CYP38 in Arabidopsis protoplasts showed a chloroplast-specific localization pattern (Figure 2C). In stably transformed plants, only chloroplast localization was observed (Figure 2D). These data are consistent with a chloroplast-specific function for CYP38 in Arabidopsis. CYP38 regulates LR emergence through systemic signals We next used grafting to resolve whether CYP38 function in the shoot or root is responsible for the LR growth defects observed. Both WT and cyp38-5 mutant scions were grafted to WT or cyp38-5 root stocks. Root branching was quantified 12-d post-grafting. Because we observed senescence of the PR tip after formation of the graft junction, we quantified the total number of branches formed instead of LR density. Interestingly, grafting of a WT scion onto a cyp38-5 root stock significantly rescued the LR emergence defect while a cyp38-5 scion caused the WT root stock to exhibit a cyp38- like phenotype (Figure 3, A and B). These data suggest that the LR emergence phenotype is determined primarily by the genotype of the shoot. As previously reported in tomato, the CYCLOPHILIN protein DIAGETROPICA promotes root branching by moving from the shoot to root (Ivanchenko et al., 2015;Spiegelman et al., 2015). To investigate whether CYP38 mRNA or protein is able to translocate across the shoot–root junction, we compared expression in shoot and root tissues of the four grafted combinations described above. Interestingly, the CYP38 transcript remained low in the WT/cyp38-5 rootstock despite the LR development being rescued. The transcript level in cyp38-5/WT scion is increased relative to the cyp38- 5mutant root system; however, the levels are very low compared with the WT shoot (Figure 3C). Thus, we conclude that there is little to no movement of CYP38 mRNA across the shoot–root junction. Similarly, we also examined protein movement by grafting the shoot of proUBQ10:CYP38-Ypet/ cyp38-5 transgenic lines with a cyp38-5 mutant root or shoot. Confocal microscopy was used to detect yellow fluorescence at the shoot–root junction after the graft junction formed successfully (about 5- to 7-d post-grafting). No substantial fluorescence could be detected across the grafted junction (Figure 3, D and E). Grafting of proUBQ10:CYP38- Ypet/cyp38-5 onto cyp38 mutant root stocks resulted in a 506 |PLANT PHYSIOLOGY 2021: 185; 503–518 L. Duan et al. Downloaded from https://academic.oup.com/plphys/article/185/2/503/6007478 by Universidad de Sevilla user on 06 April 2021 rescue of LR emergence, indicating the fusion protein was functional (Supplemental Figure S2). In summary, the chloroplast localization pattern of CYP38 is consistent with previous findings on the importance of CYP38 in photosystem stability. The limited mobility of both mRNA and protein indicates that CYP38 promotes LR emergence nonautonomously from photosynthetic shoot tissues. Reduced quantity of light suppresses LR emergence To determine whether the defect in LR emergence in cyp38 mutants is a result of changes in overall photosynthetic activity, we first examined what effect light intensity had on root architecture. Importantly, while previous studies have demonstrated that treatment of seedlings with darkness inhibits LR emergence, this extreme change in light intensity may have many indirect effects on physiology. We manipulated light intensities by layering sheets of window screen to block varying amounts of light, which we found reduces the intensity of the light without affecting the spectrum, particularly the red/far red light ratio (Supplemental Figure S3, A and B). Arabidopsis seedlings were grown under standard light conditions (140 mEm –2 s –1 ) for 5 d and then transferred to several reduced light intensities and grown for an additional 4 d. A severe reduction in emerged LRs (up to 92% reduction) and total LR length (up to 96.6% reduction) was observed when light intensity declined (Figure 4, A and Band Supplemental Figure S3C), while PR growth was moderately affected (up to 32% reduction; Figure 4, A and C). Intriguingly, light intensity affected LR emergence rapidly in seedlings transferred from standard to low light conditions; after 1 d of low-light treatment we observed a significant reduction in the number of emerged LRs (Figure 4, D and E). We found that as the light intensity declined, LRs were increasingly enriched at the pre-emergence stage (Figure 4F), indicating that this response reflected a specific inhibitory effect on the process of LR emergence rather than a general reduction in growth. LR development is not necessarily always most restricted when seedling growth is inhibited, as our previous study showed that treatment with 1-aminocy- clopropane-1-carboxylic acid inhibited PR growth more effectively than LRs (Duan et al., 2013). To investigate whether the specific developmental response to light intensity is due to the direct perception of light by the root system in our tissue culture conditions, we utilized the Growth and Luminescence Observatory for Roots system to recapitulate a more physiologically relevant condition where roots are shielded from light and grown in soil (Rella´n-A ´lvarez et al., 2015). Using this system, we C D proUBQ10:CYP38-Ypet proCYP38:erGFP A B 18 20 22 Shoot Root CYP38 expression level ( Ct) * * * Mesophyll Hypocotyl Hypocotyl-root junction Lateral root primordia 1 Lateral root primordia 2 Early maturation Meristem proUBQ10:CYP38-Ypet Figure 2 CYP38 is highly expressed in shoots and localized in plastids. A, Confocal images of proCYP38:erGFP transgenic line. Magenta indicates autofluorescence of chloroplasts in mesophyll cells together with PI staining in the hypocotyl and various regions of the root, while green indicates GFP fluorescence. Scale bars = 50 mm. B, Quantitative RT-PCR of CYP38 transcript level in WT shoot and root tissue. The numbers are presented as log 2 transformed values (DDC t ). (***P50.001, pairwise ttest, n=3). C, Confocal images of WT protoplast that was transiently transformed with proUBQ10:CYP38-Ypet. Magenta indicates chloroplast autofluorescence, while yellow indicates Ypet fluorescence. Scale bars = 10 mm. D, Confocal images of proUBQ10:CYP38-Ypet transgene fluorescence in various tissues including (left to right) mesophyll, stomata, mature root and root tip. Magenta indicates PI staining and autofluorescence from chloroplasts while yellow indicates Ypet fluorescence. Scale bars =20 mm. Plant Physiology, 2021, Vol. 185, No. 2 PLANT PHYSIOLOGY 2021: 185; 503–518 |507 Downloaded from https://academic.oup.com/plphys/article/185/2/503/6007478 by Universidad de Sevilla user on 06 April 2021 observed a significant reduction in the number of LRs in cyp38-5 mutant plants compared to WT; a similar effect was observed in plants grown under low-light conditions (Supplemental Figure S3, D and E). These data indicate that in both tissue culture conditions and soil conditions, the amount of light perceived by the shoot has a specific promoting effect on LR development that is distinguishable from the overall effect on growth. Photosystem stability and chloroplast redox state regulate LR emergence To directly test whether the activity of the photosynthetic apparatus is required for LR emergence, we treated seedlings with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which is a well-characterized inhibitor of electron transport between PSII and PSI. While we observed a nearly complete block of LR emergence with 2 mM DCMU, 1 mMDCMU caused a less severe reduction (41.5% reduction; Figure 5, A and B). Consistent with other experiments affecting the photosynthetic activity, PR growth was affected to a lesser extent than LRs (18.2% reduction; Figure 5A; Supplemental Figure S4A). We hypothesized that the products of the electron transport chain, such as NADPH or other downstream reducing agents may play important roles in modulating LR development. Consistent with this hypothesis, a reduction in LR WT WT WT cyp38-5 WT cyp38-5 cyp38-5 cyp38-5 B C D A WT/WT cyp38-5/cyp38-5 WT/cyp38-5 cyp38-5/WT E a 20 40 60 Total branching number WT/WT cyp38-5/cyp38-5 WT/cyp38-5 cyp38-5/WT b a b shoot root CYP38 transcript level ( Ct) WT/WT cyp38-5/cyp38-5 WT/cyp38-5 cyp38-5/WT 0 10 20 30 aa b c a a b b cyp38-5 proUBQ10:CYP38-Ypet/cyp38-5 proUBQ10:CYP38-Ypet/cyp38-5 cyp38-5 Figure 3 The activity of CYP38 in shoots is essential for LR emergence. A, Scanned root images of 17-d-old grafted plants. Seedlings were grafted 5 dpg, graft junction was formed after 5 d on filter paper and then the whole grafted plants were transferred back to regular medium for 7 d to recover. Scale bar = 5 mm. B, Quantification of total branch number in four different grafted combinations. Significantly different groups are indicated with letters (P50.05, pairwise ttest with Hochberg correction, n48). C, Quantitative RT-PCR for CYP38 transcript levels in both shoot and root tissues of the grafted plants. The numbers are presented as log 2 transformed values (DDC t ). Significantly different groups are denoted with letters (P50.05, pairwise ttest with Hochberg correction, n=3) and analyzed separately for shoot and root samples. Dand E, Confocal images of the hypocotyl junction in grafted plants. proUBQ10:CYP38-Ypet is used as the scion in (D), or used as the rootstock in (E). Arrows mark the grafting junction. Scale bars = 50 mm. 508 |PLANT PHYSIOLOGY 2021: 185; 503–518 L. Duan et al. Downloaded from https://academic.oup.com/plphys/article/185/2/503/6007478 by Universidad de Sevilla user on 06 April 2021 growth was reported in mutant plants devoid of NADPH- dependent thioredoxin (Trx) reductase C (NTRC; Kirchsteiger et al., 2012), a chloroplast enzyme (Serrato et al., 2004) that plays a central role in redox regulation of chloroplast proteins. NTRC is the most efficient electron do- nor to 2-Cys peroxiredoxins (Prxs), thereby the NTRC-2-Cys Prx redox system maintains the reductive capacity of chloroplast Trxs, allowing the proper regulation of their targets (Pe´rez-Ruiz et al., 2017). To further characterize the contribution to LR development of these redox systems, NTRC-2-Cys Prx and Trxs, we evaluated the various mutants reported, which experience impaired redox homeostasis to different extents (Pe´rez-Ruiz et al., 2017). Interestingly, although only mild reduction in LR emergence was observed AB 0 1 2 3 4 5 LR density (#/cm) C 0 1 2 3 4 PR length (cm) a a b c c aab cc 140 110 70 45 25 µE m-2 s-1 140 110 70 45 25 µE m-2 s-1 140 110 70 45 25 Initiation Pre-emergence Emergence Mature LR Total 140 110 70 45 25 140 110 70 45 25 140 110 70 45 25 140 110 70 45 25 140 110 70 45 25 0 3 6 9 LRP number n.s. aa b bc c n.s. a b c d d aabc ab bc c F 1.6% 7.3% 26.4% 32% 11.3% 36.9% 82.9% 92% µE m-2 s-1 µE m-2 s-1 140 25 µE m-2 s-1                                                                                                                                                                                                                    RL LL 0 1 2 3 4 LR density (#/cm)   1234 1234 Days after transfer * * * E D * * Figure 4 Reducing light quantity inhibits LR emergence. A, Scanned root image of 9-dpg WT seedlings grown under 140-lE light for 5 d and then transferred to different light intensities for another 4 d. Root tip positions of 5-dpg are labeled with white marks. Band C, Quantification of LR density (B) and PR length (C) in WT under different light intensities. Percentage reduction compared with control light condition (140 mEm –2 s –1 )is labeled on each box. Significantly different groups are indicated with letters (P50.05, pairwise ttest with Hochberg correction, n416). D,Scanned root images. WT was grown under standard light for 6 d and then transferred to low light for 1 d. Red asterisks mark the emerged LRP. E, Quantification of LR density in WT after 1–4 d treatment of regular (RL) or low light (LL). (***P50.001, pairwise ttest, n425). F,Quantificationof different stages of LRP in WT under different light intensities. Total represents the sum of all previous four stages. Significantly different groups are indicated with letters (P50.05, pairwise ttest with Hochberg correction, n412). Plant Physiology, 2021, Vol. 185, No. 2 PLANT PHYSIOLOGY 2021: 185; 503–518 |509 Downloaded from https://academic.oup.com/plphys/article/185/2/503/6007478 by Universidad de Sevilla user on 06 April 2021 in ntrc, a strong inhibition was observed in ntrc-trxf1f2 triple mutants (Figure 5C and Supplemental Figure S4B). This strong defect was completely rescued by the ntrc-D2cp and ntrc-trxf1f2-D2cp mutants, which lack 2-Cys Prx B and contains a small amount of 2-Cys Prx A in ntrc or ntrc-trxf1f2 background, in line with previous results showing that decreased levels of 2-Cys Prxs restore chloroplast redox homeostasis of NTRC-deficient mutants (Pe´rez-Ruiz et al., 2017). Chloroplast cyclophilins have been identified as binding partners of Trx (Motohashi et al., 2003). As reported by Vasudevan et al. (Vasudevan et al., 2012), the mature CYP38 protein contains a single cysteine at position 256 in the cyclophilin domain. We checked whether this cysteine could mediate protein dimerization, through a disulfide bridge, by visualizing the protein on a nonreducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel. However, no apparent dimerization band was found (Supplemental Figure S4C). We also mutated this cysteine to serine and transformed the mutated coding sequence back into cyp38-4. Full rescue of the mutant phenotype was observed (Figure 5D), indicating that the function of CYP38 is not dependent on the cysteine and the protein is unlikely to be redox regulated. Because CYP38 has been identified as a potential partner of 2-Cys Prxs (Cerveau et al., 2016), we determined whether this cyclophilin acts as an upstream regulator of these peroxidases. Thus, we evaluated the redox status of 2-Cys Prxs and fructose bisphosphatase (FBPase), included here as a well-established target of the Trx pathway, in mutant plants lacking CYP38. While no difference was observed for 2-Cys Prxs (Figure 5, E and G), lightdependent FBPase reduction was similarly impaired in cyp38 D E 0 1 2 3 4 5 WT cyp38-4 UBQ10:CYP38C256S /cyp38-4 LR density (#/cm) C F Control 200 nM DPI 200 nM DPI 200 nM DPI 0 2 4 LR density (#/cm) Control Control WT WT-LL cyp38-5 * * * * * WT ntrc cyp38-5 D LL RL D LL RL D LL RL 2SH 1SH 0SH anti-2-Cys Prx WT ntrc ntrc- 2cp cyp38-5 D LL RL D LL RL D LL RL D LL RL red ox anti-FBPase aa b AB 0 2 4 6 LR density (#/cm) WT ntrc ntrc-trxf1f2 ntrc-trxf1f2- 2cp ntrc- 2cp a ab ab b c 01 2 µM DCMU G                                                                                         0 1 2 3 4 LR density (#/cm) 012 µM DCMU * * * D LL RL WT ntrc cyp38−5 WT ntrc cyp38−5 WT ntrc cyp38−5 40 60 80 100 Reduced 2−Cys−Prx (%)  D LL RL 0 25 50 75 100 Reduced FBPase (%) WT ntrc ntrc- 2cp cyp38−5 WT ntrc ntrc- 2cp cyp38−5 WT ntrc ntrc- 2cp cyp38−5 H I aa aa a b a b aa aa a a aa aa bb ab Figure 5 Chloroplast redox status regulates LR emergence. A, Scanned images of 10-dpg WT seedlings on 0, 1, and 2 lM DCMU treatment. Root tip positions of 6-dpg are labeled with black marks. The same control panel image is used as in Figure 1H.B, Quantification of LR density in WT with and without DCMU treatment (***P50.001, pairwise ttest with Hochberg correction, n430). C, Quantifications of LR density in WT, ntrc, ntrc-D2cp, ntrc-trxf1f2, and ntrc-trxf1f1-D2cp mutants under standard growth conditions. Significantly different groups are indicated with letters (P50.05, pairwise ttest with Hochberg correction, n416). D, Quantification of LR density in WT, cyp38-4, and complemented lines with cysteine to serine mutation in CYP38 coding sequence. Significantly different groups are indicated with letters (P50.05, pairwise ttest with Hochberg correction, n413 individual T1 complemented lines). Eand F, WT and mutant plants, as indicated, were grown under long-day conditions for 4 weeks at a light intensity of 125 mEm –2 s –1 . In vivo redox status of 2-Cys Prx (E) and FBPase (F) proteins was determined at the end of the dark period (D) and after 30 min of illumination at 50 mEm –2 s –1 (LL) or 175 mEm –2 s –1 (RL). 0SH, 1SH, and 2SH indicate the reduction of zero, one, or two cysteine residues, respectively, of 2-Cys Prx (E). Red indicates reduced state and ox marks the oxidized state of FBPase (F). G,H, Quantifications of the proportion of reduced protein from (E) and (F). Significantly different groups are indicated with letters (P50.05, pairwise t test with Hochberg correction, n53). I, Quantifications of LR density in WT, cyp38-5 under standard light conditions (140 mEm –2 s –1 ) and WT under low light (25 mEm –2 s –1 ), with or without 200 nM DPI treatment (**P50.01, ***P50.001, two-way Analysis of variance (ANOVA) comparing WT under control and DPI treatment, n421). 510 |PLANT PHYSIOLOGY 2021: 185; 503–518 L. Duan et al. Downloaded from https://academic.oup.com/plphys/article/185/2/503/6007478 by Universidad de Sevilla user on 06 April 2021 and ntrc mutants under regular light intensity (Figure 5, F and H). These data indicate that the redox status of some chloroplast proteins may be dependent on CYP38 activity and these proteins may overlap with Trx targets. We next evaluated photosynthetic performance of CYP38- deficient mutants. Similar to the ntrc mutant, here included for comparison, quantum yield of photosystem II (Y(II)) was diminished in cyp38 plants (Supplemental Figure S4D), indicating that both mutants had similarly affected the efficiency of light energy utilization. Interestingly, energy dissipation through nonphotochemical quenching (Y(NPQ)) was higher in ntrc than in the cyp38 mutants (Supplemental Figure S4E), whereas a substantial elevation of Y(NO), which is an indicator of nonregulated energy dissipation, was only observed in cyp38 (Supplemental Figure S4F). Overall, these photosynthetic parameters suggest that the cyp38 mutant does not only have reduced energy conversion efficiency, but is also inefficient in photodamage protection through heat dissipation. These data indicate that potential side reactions, such as excess Reactive Oxygen Species (ROS) production, may occur in cyp38, which coincides with previous findings (Wang et al., 2015). We hypothesized that the reduction in NADPH, rather than the accumulation of ROS, was the major driver of the cyp38 LR phenotype. This hypothesis is based on the phenotypic similarity of cyp38 mutants to low-light treated seedlings, which are expected to have decreased ROS levels due to the lower excitation energy being absorbed. Consistent with this hypothesis, adding diphenyleneiodonium chloride (DPI), which inhibits NADPH oxidase activity, partially rescues the LR defect in cyp38 under standard light conditions, as well as WT root growth under low-light conditions (Figure 5I). Sucrose, HY5 and b-cyclocitral-dependent signaling pathways likely regulate LR emergence independently from CYP38 Photosynthesis-derived sucrose serves as a long distance signal and resource to promote PR growth (Kircher and Schopfer, 2012). LR development can also be promoted by aerial uptake of sucrose from the media (Macgregor et al., 2008). Consistent with the reduced photosynthetic activity of cyp38 mutants, we found that early PR growth is highly dependent on supplementary sucrose in the media (Figure 6, A and B). We tested the effects of further increasing sucrose levels by first growing seedlings under our standard conditions (1% sucrose, 140 mEm –2 s –1 light), then transferring them to the media with additional sucrose (2% and 3%). Further elevating sucrose in the media promoted PR growth in cyp38-5, independent of lighting conditions, while WT responded only under low light (Figure 6, C and D). These data suggest that fixed carbon resources may be limiting for PR growth in cyp38-5 and WT under low-light conditions, and that both genotypes are able to respond when supplementary sucrose is provided in the media. In stark contrast, supplementary sucrose had no significant effect on LR density in cyp38-5 under standard or low light AB 0 2 4 6 WT cyp38-5 hy5 hy5/cyp38-5 LR density (#/cm) 0 1 2 3 4 LR density (#/cm) C E WT cyp38-5 hy5 hy5/cyp38-5 aa bb b aa c 0% 1% 0% 1% sucrose WT cyp38-5 F WT cyp38-5 0 1 2 3 4 5 PR length (cm) 01 01 % sucrose D aa b c WT c yp 38−5 PR length (cm) 123 123 % sucrose PR length (cm) 123 123 % sucrose WT c yp 38−5                                                                                                                  0 1 2 3 4 5 LR density (#/cm) 123 123 % sucrose 123 123 % sucrose                                                                          0 1 2 3 4 5 LR density (#/cm) WT c yp 38−5 WT c yp 38−5 GH ** n.s. n.s. n.s. 25 µE m-2 s-1 140 µE m-2 s-1 25 µE m-2 s-1 140 µE m-2 s-1                                                                                                                                   0 1 2 3                                                                                                                                             0 1 2 3 ** * *** * *** ** *** Figure 6 Sucrose and HY5 signaling are not necessary for CYP38-de- pendent LR emergence. A, Scanned images of 9-d-old WT and cyp38-5 seedlings on 0 and 1% sucrose medium. B, Quantifications of PR length comparing WT and cyp38-5 with 1% or without sucrose. Significantly different groups are indicated with letters (P50.05, pairwise ttest with Hochberg correction, n430). Cand D, Quantifications of PR growth comparing WT and cyp38-5 under different levels of sucrose in the growth media under regular, 140 mEm –2 s –1 (C) and low light, 25 mEm –2 s –1 (D) conditions (n.s. represents not significant, *P50.05, **P50.01, ***P50.001, pairwise ttest with Hochberg correction n419). Eand F, Quantifications of LR density comparing WT and cyp38-5 under different levels of sucrose in the growth media under regular (E) and low light (F) conditions (n.s. represents not significant, **P50.01, pairwise ttest with Hochberg correction, n419). Gand H, Quantifications of LR density comparing WT, cyp38-5,hy5,andhy5/cyp38-5 under regular light (G), and low light (H) conditions. 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