Received: 26 April 2021 | Revised: 25 June 2021 | Accepted: 8 July 2021 DOI: 10.1111/acel.13446 ORIGINAL ARTICLE The intrinsic chaperone network of Arabidopsis stem cells confers protection against proteotoxic stress Ernesto Llamas1 | Salvador Torres-­Montilla2 | Hyun Ju Lee1 | María Victoria Barja2 | Elena Schlimgen1 | Nick Dunken3 | Prerana Wagle1 | Wolfgang Werr4 | Alga Zuccaro3 | Manuel Rodríguez-­Concepción2,5 | David Vilchez1,6,7 1 Cologne Excellence Cluster for Cellular Stress Responses in Aging-­A ssociated Abstract Diseases (CECAD), University of Cologne, The biological purpose of plant stem cells is to maintain themselves while providing Cologne, Germany 2 Centre for Research in Agricultural new pools of differentiated cells that form organs and rejuvenate or replace dam- Genomics (CRAG) CSIC-­IRTA-­UAB-­UB, aged tissues. Protein homeostasis or proteostasis is required for cell function and Campus UAB Bellaterra, Barcelona, Spain 3 viability. However, the link between proteostasis and plant stem cell identity remains Cluster of Excellence on Plant Sciences (CEPLAS), Institute for Plant Sciences, unknown. In contrast to their differentiated counterparts, we find that root stem cells University of Cologne, Cologne, Germany can prevent the accumulation of aggregated proteins even under proteotoxic stress 4 Developmental Biology, Biocenter, conditions such as heat stress or proteasome inhibition. Notably, root stem cells ex- University of Cologne, Cologne, Germany 5 Institute for Plant Molecular and Cell hibit enhanced expression of distinct chaperones that maintain proteome integrity. Biology (IBMCP) CSIC-­UPV, Valencia, Particularly, intrinsic high levels of the T-­complex protein-­1 ring complex/chaperonin Spain 6 containing TCP1 (TRiC/CCT) complex determine stem cell maintenance and their Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, remarkable ability to suppress protein aggregation. Overexpression of CCT8, a key Germany activator of TRiC/CCT assembly, is sufficient to ameliorate protein aggregation in dif- 7 Faculty of Medicine, University Hospital Cologne, Cologne, Germany ferentiated cells and confer resistance to proteotoxic stress in plants. Taken together, our results indicate that enhanced proteostasis mechanisms in stem cells could be an Correspondence David Vilchez, Cologne Excellence Cluster important requirement for plants to persist under extreme environmental conditions for Cellular Stress Responses in Aging-­ and reach extreme long ages. Thus, proteostasis of stem cells can provide insights to Associated Diseases (CECAD), University of Cologne, 50931 Cologne, Germany. design and breed plants tolerant to environmental challenges caused by the climate Email: dvilchez@uni-koeln.de change. Funding information KEYWORDS the European Research Council, Grant/ Award Number: 677427; Deutsche chaperones, heat stress, plant stem cells, protein aggregation, protein misfolding, proteostasis Forschungsgemeinschaft, Grant/Award Number: 390661388 1 | I NTRO D U C TI O N maintains the proper concentration, folding, and interactions of pro- teins from their synthesis through their degradation. As such, the Since proteins are involved in almost every biological process, pro- proteostasis network assures the integrity and quality of the pro- tein homeostasis (proteostasis) is an essential requirement for cell teome, preventing cell malfunction and death. However, aging as physiology and viability. A complex network of cellular pathways well as metabolic, environmental, and pathological conditions can This is an open access article under the terms of the Creat​ive Commo​ns Attri​bution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd. Aging Cell. 2021;20:e13446.  wileyonlinelibrary.com/journal/acel | 1 of 12 https://doi.org/10.1111/acel.13446 2 of 12 | LLAMAS et al. challenge the quality of the proteome in differentiated cells across to monitor protein aggregation under heat stress. In particular, we tissues (Hipp et al., 2019; Powers et al., 2009; Vilchez et al., 2014; focused on actin, one of the most abundant proteins in eukaryote Wong & Cuervo, 2010). cells which requires the assistance of chaperones for its proper fold- Unlike most animals, plants exhibit a continuous supply of new ing (Vallin & Grantham, 2019). We found that differentiated cells ex- and rejuvenated differentiated cells from the stem cell pools located hibit a pronounced aggregation of actin under heat stress, whereas in the root and shoot meristems (Weigel & Jurgens, 2002). For in- the stem cell region did not accumulate actin aggregates (Figure 1d). stance, the Sequoia tree contains stem cell reservoirs that can be ac- Together, our data suggest that plant stem cells have an enhanced tive for more than 2,000 years (Scheres, 2007). As sessile organisms ability to prevent protein misfolding and aggregation under stress unable to remove themselves from persistent stressful environments conditions compared with their differentiated counterparts. Stress (Heyman et al., 2014), this feature is particularly relevant to overcome conditions not only induced protein aggregation in differentiated ever-­changing conditions. Plant stem cells give rise to new organs or cells, but also impaired growth (Figure S3a–­b). However, plants re- rejuvenate and repair tissues, allowing the plant to persist amidst covered growth after removal of the stress, suggesting that stem variable and extreme conditions (Dijkwel & Lai, 2019). Thus, defining cells were able to restart their function to provide new pools of dif- molecular and cellular differences between stem cells and their dif- ferentiated cells after maintaining their proteostasis under stress ferentiated counterparts could shed light on how plants can live many (Figure S4). years even under variable environmental stress conditions (Dijkwel & Lai, 2019). In these lines, recent findings demonstrate that the shoot meristems of 200 years old oak trees are protected from the accumu- 2.2 | CCT subunits are upregulated in the root tip lation of deleterious mutations (Dijkwel & Lai, 2019; Schmid-­Siegert containing stem cells et al., 2017). Given the essential role of proteostasis for cell function and viability, here, we asked whether plant stem cells have enhanced The chaperome network is a key node of proteostasis to pre- proteostasis mechanisms to maintain their biological function. vent protein misfolding and aggregation (Brehme et al., 2014). In Arabidopsis, the chaperome network is formed by more than 300 chaperones and co-­chaperones that regulate protein folding and 2 | R E S U LT S aggregation under normal and stress conditions (Finka et al., 2011). To determine the molecular mechanisms that underlie the enhanced 2.1 | Root stem cells have increased ability to ability of stem cells to face proteotoxic stress, we analyzed avail- prevent accumulation of protein aggregates able RNA-­seq data from two different pools of root cells isolated by fluorescence-­activated cell sorting (FACS) (Wendrich et al., 2017). To assess the proteostasis capacity of root stem cells of Arabidopsis By comparing root cells from the proximal part (mostly stem cells) thaliana, we used ProteoStat, a dye that becomes highly fluores- and distal part (differentiated cells) respect to the QC (Figure 2a), cent when it binds to misfolded or aggregated proteins (Leeman we identified differentially expressed components of the chap- et al., 2018; Nakajima & Suzuki, 2013; Shen et al., 2011). First, we erome network such as distinct chaperones, co-­chaperones, and performed a ProteoStat staining comparing seedlings grown under foldases in stem cells (Figure 2b). Undifferentiated stem cells dis- either control or distinct proteotoxic stress conditions that is protea- played more and larger-­ magnitude transcriptional changes than some inhibition (i.e., MG-­132 treatment) and heat stress. In control their differentiated distal counterparts (Figure 2b, Data S1). Indeed, seedlings, we did not detect aggregated proteins in most of the cells almost half (49.4%) of the chaperome network was significantly up- (Figure 1a and Figure S1). The only exception was the cell population regulated (log2 fold change >2; p < 0.05) in stem cells (Figure 2b, forming the sloughing lateral root cap, a layer that is continuously Data S1). On the other hand, 35.9% of the chaperome was signifi- replaced through programmed cell death (Shi et al., 2018) (Figure 1a cantly downregulated (log2 fold change <−2; p < 0.05) in these cells and Figure S1). On the other hand, the seedlings subjected to proteo- (Figure 2b, Data S1). toxic stress exhibited high levels of aggregated proteins across dif- Remarkably, the transcriptomic analysis revealed that most ferentiated cells of the root (Figure 1a) and cotyledons (Figure S2a–­b). members (88.9%) of the heat shock protein (HSP) 60 family, also Damaged proteins in roots under stress showed a particular dis- known as chaperonins, were significantly increased in root stem tribution, as the stem cells next to the quiescent center (QC) had cells (Figure 2b, Data S1). Among them, all the subunits of the T-­ reduced amounts of protein aggregates compared with the rest of complex protein-­1 ring complex/chaperonin containing TCP1 (TRiC/ cells differentiating and expanding in opposite direction to the QC CCT) complex were upregulated in root stem cells (Figure 2c, Data (Figure 1a–­b). A closer magnification indicated that misfolded and S1). Since the TRiC/CCT complex is also highly expressed in mam- aggregated proteins can be gradually detected as the differentiation malian embryonic and adult stem cells (Noormohammadi et al., of cortical cells advances in opposite direction to the QC (Figure 1c). 2016; Vonk et al., 2020), we focused on this specific node of the Strikingly, cortical stem cells close to the QC showed undetectable chaperome network. First, we assessed the increased expression protein aggregates (Figure 1c). To further examine the increased pro- of CCT subunits by quantitative RT-­PCR (qPCR) analysis of samples teostasis ability of stem cells, we performed filter trap experiments manually isolated from the root tip containing mainly stem cells in LLAMAS et al. | 3 of 12 (a) (b) (c) (d) 22ºC DMSO 4 * Filter trap Diff. cell 37ºC 3 ProteoStat fluorescence Distal Control Relative 2 Proximal 1 Transition zone cell 37ºC SDS-resistant actin 0 aggregates 37ºC MG-132 4 **** Stem cell SDS-PAGE 3 ProteoStat fluorescence Proteotoxic stress 37ºC al im Relative tal ox 2 Dis Pr 1 Actin MG-132 0 Enlongation zone tat t rge tat t rge hs hs al tal oS oS ec ec Me Me im Meristematic zone Dis ote ote Ho Ho ox QC Pr Pr Pr F I G U R E 1 Proteotoxic stress causes differential protein aggregation in the distinct cell types of the root. (a) Representative images of wild-­t ype plant roots grown under control (22°C or 22°C + DMSO) or proteotoxic stress conditions stained with ProteoStat Aggresome Detection kit. ProteoStat (red, protein aggregates), Hoechst (blue, nuclei), and Merge (ProteoStat and Hoechst) images are shown. For heat stress assay, 4 days after germination (DAG) plants were transferred to 37°C for 2 days. For proteasome inhibition experiments, plants were germinated and grown in plates supplemented with 30 µM MG-­132 and analyzed at 6 DAG stage. White bars represent 100 µm. (b) Relative ProteoStat fluorescence levels comparing the area containing proximal (close to the QC) and distal cells from roots under proteotoxic stress (areas used for quantification are indicated with white dotted ovals in Figure 1a). Scatter plots represent mean ± s.e.m of four independent experiments. The statistical comparisons were made by two-­t ailed Student's t test for unpaired samples. p values: *p < 0.05, ****p < 0.0001. (c) Left side, scheme indicating the structure of the Arabidopsis root meristem. The QC is indicated in orange color, the meristematic zone in green, and the elongation zone in purple. Gray arrowhead indicates the transition zone, where cells leave the meristem and enter the elongation/differentiation zone. Right side, close examination of a representative MG-­132-­treated root showing differential protein aggregation in cortical cells dividing and differentiating in opposite direction to the QC. White bar indicates 50 µm. (d) Filter trap and SDS-­ PAGE analysis with antibody to actin of the areas containing proximal and distal cells from roots under proteotoxic stress (37°C for 2 days). The images are representative of two independent experiments comparison with the root base and the shoot, both of them con- levels of CCT7 correlated with higher amounts of the protein in the taining mainly differentiated cells (Figure 2d). The qPCR analysis of root tip (Figure 2h). In addition, the Plant eFP Viewer (bar.utoronto. root meristem maintenance marker genes (i.e., PLT2 (AT1G51190) ca), a bioinformatic tool that displays gene expression patterns, and TMO7 (AT1G74500)) confirmed that the distinct collected tis- indicates higher expression levels of all the CCT genes in the root sues contained different proportions of stem and differentiated stem cell niche (Figure S5a). This analysis also shows enhanced ex- cells (Figure 2e). Both marker genes showed a gradient of expres- pression of CCT genes in the meristematic zone enriched for stem sion with a maximum in the root stem cells (Durgaprasad et al., cells of the xylem when compared with vascular cells of the distal 2019; Wendrich et al., 2017), as we observed in root tip samples xylem that have undergone a gradual differentiation process along (Figure 2e). Likewise, qPCR analysis indicated that CCT transcripts the root (Figure S5b). Altogether, our data indicate that root plant are expressed at higher levels in the root tip compared with differ- stem cells have an intrinsic chaperome network characterized by entiated tissues (Figure 2f). To further support increased expres- enhanced levels of CCT subunits, a feature that could contribute to sion of CCT subunits in stem cells, we calculated the ratio between their enhanced ability to prevent protein aggregation under proteo- individual CCT and TMO7 transcripts levels obtained from each toxic stress. Importantly, CCT transcripts were not upregulated in isolated root tissues, and we found that CCT/TMO7 ratios were con- the root after heat stress treatment (Figure S6), suggesting that the sistently higher in root tips compared with the root base (Figure 2g). basal high expression of CCTs is sufficient to prevent proteostasis Importantly, Western blot analysis confirmed that the higher mRNA collapse under stress conditions. 4 of 12 | LLAMAS et al. (a) (b) Diff cells Stem cells Stem cells/ (c) Diff cells Stem cells (d) Diff cells Shoot Diff cells CCT1 1 2 Chaperonins Root base CCT2 0.5 1 CCT3 Expression Expression CCT4 0 0 CCT5 CCT6A 0.5 1 CCT6B DNAJ CCT7 1 Root tip proteins 2 CCT8 (e) 1.5 (f) 2.0 Root tip Root base Shoot Root tip Root base Shoot QC Relative mRNA levels Relative mRNA levels 1.5 1.0 Small HSPs 1.0 * Distal HSP70s 0.5 ** ** * * * * * ** ** 0.5 * HSP90s Stem cells ** *** 0.0 0.0 HSP90 co-chaperones T7 T1 T2 T3 T4 T5 T8 C A B T2 7 T6 O T6 C C C C C C C PL TM C C C C C C C C C HSP100s C (g) (h) HSP110s 2.0 Root tip Root base >10 e BAGs as ip tb tt t 1.5 oo oo 5 oo Fold change Sh Ratio CCT/TMO7 CYP-type R R PPIases 0 1.0 CCT7 QC FKBP-type 5 ** * PPIases 0.5 * *** * * ** ** < 10 Protein disulfide Actin 0.0 isomerases Proximal Replicates 1 2 3 1 2 3 T1 T2 T3 T8 T4 T5 T7 B A FC T6 T6 C C C C C C C Distal Proximal C C C C C C C C C C C F I G U R E 2 Chaperonin containing TCP1 subunits are upregulated in the root tip containing stem cells. (a) Diagram depicting cell populations in the proximal zone (stem cells) and distal zone (differentiated cells) respect to the QC of the root apical meristem. Data for RNA-­seq analysis were obtained from the indicated proximal and distal cells isolated by FACs (Wendrich et al., 2017). (b) Heatmap representing expression (fragments per kilobase of transcript per million, FKPM) and fold change expression of chaperome component transcripts comparing stem cells (proximal zone) with differentiated cells (distal zone) (log2 fold change (FC)>2, p < 0.05 was considered significant). The chaperome genes were divided into different groups: Chaperonins (HSP60s), DNAJ proteins, small HSPs, HSP70s, HSP90s, HSP90 co-­chaperones, HSP100s, HSP110s, BAGs, CYP-­t ype peptidyl-­proline isomerases (PPIases), FKBP-­t ype PPIases, and protein disulfide isomerase as defined by Finka et al., 2011. (c) Heatmap showing expression levels (FPKM) of all CCT subunits. Both FPKM heatmaps are row-­scaled. (d) Scheme depicting the different sections of wild-­t ype plants at 6 DAG. Isolated shoot (green), root base (blue), and root tip (purple) were used for RNA and protein extraction. (e) qPCR analysis of transcript levels of genes that are highly expressed in stem cells to regulate root meristem maintenance (mean ± s.e.m of 3 independent experiment). (f) qPCR analysis of transcript levels of the indicated CCT subunits relative to levels in the root tip (mean ± s.e.m of 3 independent experiment). (g) CCT/TMO7 ratio in root tip and root bases samples (mean ± s.e.m of 3 independent experiment). (h) Western blot analysis with antibody against CCT7 subunit. Actin is the loading control. The images are representative of three independent experiments. All the statistical comparisons in (e, f and g) between samples from root tip and differentiated cells were made by two-­t ailed Student's t test for unpaired samples. p value: *p < 0.05, **p < 0.01, ***p < 0.001 2.3 | Reduced levels of CCT trigger protein cct4-­2) or CCT7 (cct7-­1) led to slightly reduced expression, whereas aggregation in root stem cells and impairs stem insertions in transcribed but untranslated regions of CCT7 (cct7-­2) cell function and CCT8 (cct8-­2 and cct8-­4) caused a stronger reduction in tran- script levels (Figure S7b–­c). Thus, we classified the mutants as weak Intrigued by the intrinsic high levels of CCT subunits in root stem (cct4-­1, cct4-­2, and cct7-­1) and moderate loss of function (cct7-­2, cells, we asked whether TRiC/CCT determines stem cell function cct8-­2, and cct8-­4). While we did not observe root growth effects and viability. Since loss of a single CCT subunit is sufficient to impair in weak mutants, all the moderate mutant lines exhibited shorter the assembly and activity of the TRiC/CCT complex (Ahn et al., 2019; roots compared with wild-­t ype (WT) controls (Figure S8). In addi- Gonczy et al., 2000; Green et al., 2011; Lundin et al., 2008), we as- tion, we also examined the root length of mutants with alterations in sessed whether an unbalance in the expression of CCT subunits im- the prefoldin (PFD) complex (Grantham, 2020), a distinct molecular pairs stem cell activity and subsequent root growth. To this end, we chaperonin which is also highly expressed in stem cells (Figure 2b, performed a root growth screening among Arabidopsis T-­DNA mu- Data S1). Importantly, PFD and TRiC/CCT complex interact and act tants from genes encoding CCT subunits. We first isolated and char- together to facilitate folding of numerous proteins (Martin-­Benito acterized two insertion alleles for CCT4, CCT7, and CCT8 subunits et al., 2002). Similar to moderate cct7-­2, cct8-­2 and cct8-­4 mutants, (Figure S7a). T-­DNA insertions in the promoter of CCT4 (cct4-­1 and pfd3 and pfd5 mutants showed shorter roots compared with the WT LLAMAS et al. | 5 of 12 seedlings (Figure S8). Thus, our data indicate that TRiC/CCT along of stem cell maintenance. To this end, we compared the transcript with PFD complex contribute to proper maintenance of the meris- levels of PLT2 and TMO7 between WT, cct7-­2, and cct8-­2 plants. tem and subsequent continuous root growth. Notably, we found lower levels of PLT2 and TMO7 in the cct mutant Given that null mutations of CCT subunits are lethal in plants, seedlings (Figure S9). yeast, and mammals (Horwich et al., 2007; Xu et al., 2011), the We hypothesized that the imbalance in the TRiC/CCT complex of moderate loss-­of-­function mutants cct7-­2 and cct8-­2 provide an cct mutants could lead to aberrant protein aggregation in stem cells, invaluable mean to study proteostasis and stem cell maintenance. a process that impairs cell function. To assess this possibility, we used We further characterized these cct mutants and found that the root ProteoStat staining. Notably, we found increased protein aggrega- growth was impaired over time (Figure 3a). Moreover, mutations in tion in the stem cell niche of cct mutant plants compared with WT CCT genes led to a reduced number of cortical cells in the meristem, controls (Figure 3d–­e). Moreover, a closer examination of the cortical resulting in shorter meristems (Figure 3b–­c). Notably, cct7-­2 and stem cell region allowed us to detect fluorescence speckles in stem cct8-­2 mutant plants displayed a disordered stem cell niche with ab- cells, indicating protein aggregation (Figure 3f). Since actin is a major errant cellular organization and division planes in the columella root folding substrate of the TRiC/CCT complex (Vallin & Grantham, cap and the QC (Figure 3c). Prompted by these results, we tested 2019), we assessed actin aggregation in cct8-­2 plants. Notably, we whether downregulation of CCT levels alter expression of markers found that loss of cct8 function triggers actin aggregation (Figure 3g). (a) (b) (c) WT cct7-2 cct8-2 WT cct7-2 cct8-2 WT cct7-2 cct8-2 WT 50 60 50 40 40 cct7-2 Meristem cell number Root lenght (mm) 30 30 **** **** 20 20 **** **** cct8-2 10 **** 10 **** **** 0 0 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 DAG DAG (d) (e) (f) (g) Filter trap SDS-PAGE 4 Col-0 cct8-2 Aggregates Relative ProteoStat fluorescence Col-0 3 Col-0 Col-0 cct8-2 2 cct8-2 Actin cct8-2 1 SDS-resistant actin 0 aggregates ProteoStat Hoechst Merge Col-0 cct8-2 F I G U R E 3 Reduced expression of CCT triggers protein aggregation in root stem cells and impairs stem cell function. (a) Root length over time in wild-­t ype (WT), cct7-­2, and cct8-­2. DAG, days after germination (n = 30 roots from three independent experiments). (b) Meristem cell number over time in WT, cct7-­2, and cct8-­2 (n = 10 roots from three independent experiments). In (a–­b) staggered plot showing connecting lines to the means. (c) Representative images of propidium iodide (PI)-­stained roots show differences between the meristem size of Col-­0 WT and cct roots at 4 DAG. Green arrowheads indicate the junction between the meristematic and elongation zone. Lower panels show a magnification to the QC and columella cells. Scale bars represent 50 µm. (d) Representative images of ProteoStat staining of Col-­0 WT and cct8-­2 mutant at 6 DAG. Seedlings were grown under control conditions (22°C). Scale bar represents 50 µm. (e) Relative ProteoStat fluorescence levels within the area containing proximal cells (close to the QC) of cct8-­2 relative to Col-­0 control (mean ± s.e.m of 4 independent experiments). The areas used for quantification are indicated with white dotted ovals in Figure 3d. (f) Higher magnification of the stem cell niches of Col-­0 and cct8-­2. Scale bar represents 50 µm. (g) Filter trap and SDS-­PAGE analysis with antibody to actin of Col-­0 and cct8-­2 plants. The images are representative of two independent experiments. All the statistical comparisons in (a, b and e) were made by two-­t ailed Student's t test for unpaired samples. p value: ****p < 0.0001 6 of 12 | LLAMAS et al. Altogether, these data establish a link between proteostasis, TRiC/ maintain proteome integrity and cope with proteotoxic stress (Lopez-­ CCT complex activity, and root stem cell maintenance. Otin et al., 2013; Taylor & Dillin, 2011). However, mammalian em- bryonic stem cells rely on enhanced proteostasis mechanisms to replicate indefinitely while maintaining their undifferentiated state 2.4 | Overexpression of CCT8 confers resistance to and, therefore, are immortal in culture (Noormohammadi et al., 2016; proteotoxic stress in plants Vilchez et al., 2012; Vilchez et al., 2012). Since our results indicate that plant stem cells also exhibit enhanced proteostasis, we asked During organismal aging, differentiated cells of animals undergo a pro- whether mimicking root stem cell proteostasis in somatic tissues con- gressive decline in their proteostasis network, losing their ability to fers organismal protection to proteotoxic stress. For this purpose, we (a) (b) (c) (d) MG-132 Alive Dead 1.5 150 (% relativized to DMSO controls) 100 ProteoStat fluorescence Root lenght in MG- 13 2 Col-0 1.0 100 Percentage alive 75 35S:CCT8 13 Relative 35S:CCT8 12 50 0.5 50 25 0.0 0 0 Col-0 35S:CCT8 Col-0 35S:CCT8 Col-0 35S:CCT8 0 15 30 45 60 Time (hours) (e) (f) (g) (h) T8 Control Heat shock C S:C 400 l-0 8 8 8 8 Control Heat shock CT CT CT CT Co 35 kDa S:C S:C S:C S:C l-0 l-0 l-0 l-0 Number of aggregates 300 1048 Co Co Co Co 35 35 35 35 Stacking SDS- TriC/CCT gel resistant (1000KDa) Col-0 200 250 720 150 100 Native gel 100 analysis 75 480 (anti-CCT1) 0 50 242 35S:CCT8 l-0 8 l-0 8 CT CT Co Co 146 S:C S:C 37 35 35 66 Rubisco 25 Control Heat Holoenzyme kDa shock Soluble Insolube Soluble Insolube F I G U R E 4 Overexpression of CCT8 is sufficient to ameliorate protein aggregation in differentiated cells and confer resistance to proteotoxic stress in plants (a) Representative images of ProteoStat staining of 6 DAG Col-­0 and 35S:CCT8 germinated and grown in plates supplemented with 15 µM MG-­132. Scale bar represents 100 µm. (b) Relative ProteoStat fluorescence levels comparing the areas depicted with white dotted circles in Figure 4a. Graph represents mean ± s.e.m of four independent experiments. (c) Root lengths of MG-­132-­treated plants relative to their corresponding controls treated with DMSO. Statistical comparisons in (b–­c) were made by two-­t ailed Student's t test for unpaired samples. p value: **p < 0.01. (d) Heat shock survival assay of 35S:CCT8 plants and Col-­0 WT. 6 DAG seedlings grown at 22°C were subject to 45°C for 3 h (heat shock) and then shifted back to control conditions (22°C). Alive plants were scored. Dead plants were counted when they showed a complete pigment bleaching phenotype. Kaplan–­Meier survival data are representative of 2 independent experiments. All lines were grown in the same plate. 35S:CCT8 12 (log rank, p < 0.00138) and 35S:CCT8 13 (log rank, p < 0.00151) survive longer than control seedlings under heat stress. Col-­0 WT: median = 26, n = 76/108, 35S:CCT8 12: median = 29, n = 59/108. 35S:CCT8 13: median = 29. (e) Representative BlueSafe-­stained SDS-­PAGE gel of protein extracts from the soluble and insoluble fractions. Insoluble/ aggregated protein bands that show strong decreased in heat stress-­treated 35S:CCT8 plants compared with heat stress-­treated WT are indicated with orange arrowheads. SDS-­resistant aggregates retained on the stacking gel are also shown. (f) Plants from the heat shock assay were stained with ProteoStat and representative images are shown. Scale bar represents 10 µm. (g) Quantification of the number of aggregates stained with ProteoStat. Total number of aggregates were counted in the whole 63X captured image from the epidermis of the cotyledons (mean ± s.e.m from 3 independent experiments). (h) Representative images of two independent native gel electrophoresis of Col-­ 0 WT and 35S:CCT8 extracts collected from 14 DAG plants followed by immunoblotting with CCT1 antibody (right panel shows the TRiC/ CCT complex band with longer exposure time). Band corresponding to Rubisco holoenzyme after Ponceau staining is the loading control LLAMAS et al. | 7 of 12 overexpressed CCT8, a subunit that it is sufficient to increase TRiC/ our results indicate that root stem cells have an enhanced protein CCT assembly in both human cells and the roundworm Caenorhabditis folding capacity, providing them with a striking ability to cope with elegans (Noormohammadi et al., 2016). We generated two independ- proteotoxic stress compared with their differentiated counterparts. ent transgenic lines that express upregulated levels of CCT8 (Figure For instance, similar to animal stem cells (Noormohammadi et al., S10) and assessed resistance to proteotoxic stress. Notably, we found 2016; Thiruvalluvan et al., 2020; Vonk et al., 2020), we identified that CCT8-­overexpressing plants accumulate less aggregates in roots distinct components of the Arabidopsis chaperome upregulated in compared with wild-­type plants when both were treated with MG-­132 stem cells, such as DNAJs, HSPs, and peptidyl-­proline isomerases proteasome inhibitor (Figure 4a–­b), resulting in longer roots under (PPIases). Particularly, we found a strong enrichment in the levels of this deleterious condition (Figure 4c). In addition, stress-­challenged distinct members of the chaperonin family in plant stem cells, includ- transgenic lines displayed extra cell layers in the sloughing lateral ing all the CCT subunits of the TRiC/CCT complex. root cap that is normally subject to continuous cell death and replace- Plant and animal stem cells have unexpected similarities that ment (Figure S11a). This phenotype could be caused by the constitu- modulate their function (Sablowski, 2004). One classic paradigm is tive CCT8 expression during the columella differentiation process, in the conserved protein retinoblastoma (Rb), which is critical for cell which the levels of endogenous CCT8 transcript decay (Figure S11b–­c). division and differentiation of plant and animal stem cells (Sablowski, Besides proteasome inhibition, we also assessed the effects on heat 2004; Scheres, 2007). In addition, genome stability maintains a stress in the shoot of CCT8-­overexpressing plants. To this end, we healthy pool of stem cells in both plants and animals (Schmid-­Siegert performed a heat shock survival assay where 6 day after germination et al., 2017; Zheng, 2020). Recently, it has been reported that stem (DAG) seedlings were subjected to 3 h of 45°C heat shock and then cells of the shoot meristem are robustly protected from the accumu- shifted back to 22°C. We found that 35S:CCT8 plants exhibit increased lation of mutations in long-­lived trees (Schmid-­Siegert et al., 2017). survival after heat shock compared with wild-­type plants (Figure 4d). Besides cell division and genome stability, our results establish that Since heat shock triggers the accumulation of misfolded and aggre- both plant and animal stem cells rely on a powerful proteostasis ma- gated proteins that can cause cell death, enhanced survival after heat chinery for their function and maintenance (Lee et al., 2017). shock may be explained by reduced protein aggregation in 35S:CCT8 Whereas a collapse in the proteostasis of terminally differen- plants. In support of this hypothesis, soluble and insoluble protein tiated cells can lead to disease, human, and mouse stem cells ex- analysis as well as ProteoStat staining confirmed that 35S:CCT8 seed- hibit an enhanced ability to correct proteostatic deficiencies. For lings contained less protein aggregates after heat shock (Figure 4e–­g). instance, pluripotent and adult stem cells have increased proteo- By native gel analysis, we confirmed that TRiC/CCT complex assembly lytic systems and expression of distinct chaperones to prevent pro- was enhanced in plants upon CCT8 overexpression (Figure 4h). Thus, tein misfolding and aggregation (Leeman et al., 2018; Llamas et al., our data indicate that upregulation of CCT8 and subsequent assembly 2020; Noormohammadi et al., 2016, 2018; Prinsloo et al., 2009; of TRiC/CCT complex confers protection to heat stress by sustaining Thiruvalluvan et al., 2020; Vilchez et al., 2012, 2013; Vonk et al., the integrity of the proteome. 2020). Interestingly, mimicking proteostasis of human pluripotent stem cells by ectopically increasing CCT8 in somatic tissues can suppress the aggregation of disease-­related proteins such as polyQ-­ 3 | DISCUSSION expanded huntingtin, the mutant protein underlying Huntington's disease (Noormohammadi et al., 2016). Besides CCT subunits, we The cells of all living organisms contain numerous proteins which are found that plant stem cells have increased levels of distinct PPIases at risk of misfolding and aggregation (Balchin et al., 2016; Knowles including cyclophilins (CYPs) and FK506-­binding proteins (FKBPs) et al., 2014). The accumulation of damaged proteins can lead to (Figure 2b). Importantly, CYPs and FKBPs facilitate the proper fold- defects in growth, decrease in yield, accelerated aging, and cellu- ing and prevent aggregation of human pathogenic proteins such as lar death (Planas-­Marques et al., 2016). A series of tightly regulated the prion protein (PrP) and presenilin 1, which is linked with familial and stringent mechanisms maintain proteostasis (Hipp et al., 2019; Alzheimer's disease (Cohen & Taraboulos, 2003; van de Hoef et al., Vilchez et al., 2014). However, aging, metabolic conditions, and en- 2013; Wang et al., 2005). Thus, the endogenous upregulation of vironmental challenges can overwhelm the proteostasis network. chaperonins and other proteostasis components such as PPIases For example, protein aggregation in aging neurons is linked with could be a conserved mechanism to increase protein folding activity several human neurodegenerative disorders such as Alzheimer's, and maintain proteome integrity, ensuring the survival of stem cells. Parkinson's, and Huntington's disease (Douglas & Dillin, 2010; Lee Moreover, due to the larger number of chaperone members in plants et al., 2017; Lopez-­Otin et al., 2013). Likewise, stress conditions compared to humans (Finka et al., 2011), we speculate that plant can induce proteostasis collapse and protein aggregation in plants chaperones could be used as therapeutic targets against human (Llamas et al., 2017; Nakajima & Suzuki, 2013; Pulido et al., 2016). pathogenic proteins to ameliorate neurodegenerative diseases. We found that maintaining low levels of protein aggregation during Animals exhibit a series of intracellular and cell-­nonautonomous stress conditions contribute to plant stem cell viability, ensuring tis- mechanisms that allow them to respond differently to acute stress sue rejuvenation by cellular replacement as well as continuous and (e.g., heat shock) or chronic stress ensued by aging or the expres- repetitive formation of new structures and organs. In these lines, sion of misfolded mutant proteins (Taylor et al., 2014). In animals 8 of 12 | LLAMAS et al. such as C. elegans, the ability to induce the heat shock response Interestingly, the TF2Network that predicts potential regulators of in specific tissues comes at the expense of the organismal ability sets of co-­expressed or functionally related genes in plants (Kulkarni to maintain proteostasis in the face of chronic proteotoxic stress et al., 2018), indicates that most of the CCT genes could be modu- (Prahlad & Morimoto, 2011; Volovik et al., 2014). In contrast to ani- lated by the zinc finger transcription factor GATA12 (AT5G25830) mals, plants cannot escape from temperature fluctuations in nature (Figure S12a). Similar to CCT genes, the expression of GATA12 is also and have evolved different strategies to cope with recurring envi- upregulated in stem cells when compared to differentiated cells ronmental stress. For instance, plants can be primed with moderate (Figure S12b–­c). Thus, it will be fascinating to examine in further heat stress, which triggers the activation of heat shock transcrip- studies whether GATA12 transcription factor modulates the high tion factors (HSFs) and subsequent upregulated expression of heat expression levels of CCT subunits. In these lines, it is important to shock proteins (HSPs). This prime response can last several days note that human embryonic stem cells also express higher amounts enabling plants to survive temperatures that otherwise would be of CCT subunits compared with their differentiated counterparts lethal for an unadapted plant (Baurle, 2016). However, when plants (Noormohammadi et al., 2016). Likewise, adult stem cells such as are exposed to unexpected long or chronic stress episodes, they mouse neural stem cells and C. elegans germline stem cells also have must have additional mechanisms to maximize survival. As sessile enhanced expression of CCT subunits (Noormohammadi et al., organisms, maintenance of plants stem cell reservoirs is critical to 2016; Vonk et al., 2020). The regulatory mechanisms underlying continuously supply new and rejuvenated differentiated cells in elevated CCT levels in these models remain unknown, but we spec- damaged organs or directly generate new organs to replace them ulate that specific transcriptions factors could cross evolutionary (Weigel & Jurgens, 2002). Thus, preventing protein misfolding and boundaries to promote high levels of CCT subunits and subsequent aggregation in plant stem cells before, during and after stress con- assembly of the TRiC/CCT complex in distinct stem cells of differ- ditions can be an important requirement for stem cell viability and ent species. function. As such, the enhanced proteostasis network of stem cells could allow the plant to keep growing during stress or regenerate after removal of the stress. This could be a potential explanation 4 | E X PE R I M E NTA L PRO C E D U R E S to why CCT genes, as many other chaperonins, are constitutively highly expressed in plant stem cells (Figure 2) and why their expres- 4.1 | Plant material, constructs, and growth sion is independent of the heat stress response (Figure S6). In these conditions lines, we speculate that the enhanced proteostasis of plant stem cells contribute to the extreme ages that distinct plants are able All the Arabidopsis thaliana lines used in this work are in to achieve, even when they are continuously exposed to abiotic Columbia-­0 (Col-­0) ecotype. Wild-­t ype, and loss-­of-­function mu- stresses such as high temperature (Weigel & Jurgens, 2002). While tants cct4-­1 (SALKSEQ_069998.1), cct4-­2 (SALKSEQ_076214.0), very few mammals, including humans, can live more than 100 years, cct7-­1 (SALKSEQ_135744.2), cct7-­2 (SALK_099986), cct8-­2 plants like the bristlecone pine and the giant sequoia are able to (SALKSEQ_082168)(Xu et al., 2011), cct8-­4 (SALKSEQ_137802), live over a 1,000 years (Dijkwel & Lai, 2019; Heyman et al., 2014; pfd3 (Rodriguez-­Milla & Salinas, 2009), and pfd5 (Rodriguez-­Milla & Scheres, 2007), probably due to their stem cell pools (Heyman Salinas, 2009) were used in this study. T-­DNA mutants were geno- et al., 2014). Remarkably, we found that mimicking the proteostasis typed by PCR. All seeds were surface-­sterilized and germinated network of stem cells in somatic tissues by increasing TRiC/CCT on solid 0.5X Murashige and Skoog (MS) medium without sucrose assembly is sufficient to prevent protein aggregation in differenti- neither vitamins, and plants were incubated in a growth chamber ated cells and increase organismal survival under stress conditions. at 22°C (or otherwise indicated in the figure) under long-­day condi- However, further studies will be required to assess whether this tions as previously described (Pulido et al., 2013). When indicated upregulated ability to cope with acute heat stress could eventually in the figure, medium was supplemented with MG-­132 (Sigma). For jeopardize proteostasis under chronic stress as it occurs in animals. the root growth analysis, we used the software MyRoot to semi-­ Nevertheless, our results indicate that rewiring proteostasis mech- automatically measure the root length from 6 day after germination anisms in crop plants could have an important agronomic value to (DAG) seedlings grew in vertical agar plates (Betegon-­Putze et al., maintain yield in the face of unpredictable daily temperature fluc- 2019). For qPCR, filter traps and Western blot analysis of the root tuations due to the global climate change. tip, base root and shoot, seeds were germinated on vertical plates Given that CCT subunits are expressed constitutively under with a sterile mesh (SefarNitex 03-­100/44) placed on the medium. normal growth conditions in eukaryotes (Simmer et al., 2003), an 6 DAG plants were dissected manually with a surgical blade for intriguing question raised by our results is how plant stem cells posterior RNA and protein extraction. For the generation of the maintain elevated levels of CCT subunits compared with differen- 35S:CCT8 lines, the full-­ length coding sequence of Arabidopsis tiated cells. Similar to our findings, previous studies in yeast have CCT8 (AT3G03960) was cloned under the 35S promoter using the demonstrated that, unlike most chaperones, the expression of CCT entry vector pDONR207 and the destination vector pGWB502. genes is not induced by heat shock and associated transcription fac- Arabidopsis plants were transformed by the floral dip method tors such as HSFs (Somer et al., 2002; Ursic & Culbertson, 1992). (Clough & Bent, 1998). LLAMAS et al. | 9 of 12 4.2 | Analysis of RNA-­seq data The pellet was washed with fresh buffer and centrifuged again. The obtained pellet fraction was then resuspended in denaturing TKMES We analyzed publicly available RNA-­Seq data from two different pool buffer and centrifuged again to collect the supernatant as the insoluble of cells isolated by FACS from Arabidopsis roots of the lines PUB25 fraction. Protein concentration from each fraction was determined with and SPT. We used the RNA-­sequencing samples PUB25 proximal 1–­3 Pierce Coomassie Plus (Bradford) Protein-­Assay (Thermo Scientific). (corresponding to mainly stem cells) and SPT distal 1–­3 (corresponding to differentiated vascular cells) (Wendrich et al., 2017) to compare the expression profile of the Arabidopsis chaperome as defined by Finka 4.5 | Filter trap assay et al., 2011. RNA-­seq data were analyzed using a QuickNGS pipe- line (Wagle et al., 2015). This workflow system provided a basic read Whole seedlings, root base cells, and root tips containing stem cells quality check using FastQC (version 0.10.1) and read statistics using were recollected and grinded in liquid N2. Protein extracts were ob- SAMtools (version 0.1.19). The basic data processing of the Quick-­ tained with native lysis buffer (300 mM NaCl, 100 mM Hepes pH 7.4, NGS pipeline consists of a splicing-­aware alignment using Tophat2 2 mM EDTA, 2% Triton X-­100) supplemented with 1X plant protease (version 2.0.10) followed by reference-­guided transcriptome reassem- inhibitor (Sigma). Cellular debris was removed with 80,000 g centrif- bly with Cufflinks2 (version 2.1.1). The QuickNGS pipeline calculated ugation for 10 min at 4°C. Supernatant was recollected and protein read count means, fold change, and p values with DEseq2 and gene concentration determined with Pierce Coomassie Plus (Bradford) expression for the individual samples with Cufflinks2 (version 2.1.1) as Protein-­A ssay (Thermo Scientific). A cellulose acetate membrane fil- fragments per kilobase of transcript per million (FPKMs), in both cases ter (GE Healthcare Life Sciences) was placed in a slot blot apparatus using genomic annotation from the Ensembl database, version 32. All (Bio-­Rad) coupled to a vacuum system. Membrane was equilibrated data preprocessing and visualization were done with R version 3.2.2 with 3 washes with equilibration buffer (native buffer supplemented and Bioconductor version 3.0. Differential expressed genes (DEGs) with 0.5% SDS). 200 µg of protein extract was supplemented with calculated by DESeq2 were filtered using p value <0.05 cutoff of sig- SDS at a final concentration of 0.5% and loaded and filter through nificance, and log2 fold change >2 and log2 fold change <−2 cutoffs of the membrane. Then, the membrane was washed with 0.2% SDS. relative expression for upregulated and downregulated DEGs, respec- Retained aggregates of actin were detected using anti-­actin anti- tively. Heatmaps were created using "pheatmap" package. body [1:5,000] (Agrisera, AS132640). Extracts were also analyzed by SDS-­PAGE to determine total actin levels. 4.3 | Western blot analysis 4.6 | Blue native gel immunoblotting of TRiC/ Total protein extracts were obtained from Arabidopsis lyophilized CCT complex powder. The powder was resuspended on ice-­cold TKMES homoge- nization buffer (100 mM Tricine-­potassium hydroxide pH 7.5, 10 mM Whole Arabidopsis seedlings were frozen in liquid nitrogen and KCl, 1 mM MgCl2, 1 mM EDTA, and 10% [w/v] Sucrose) supplemented ground to powder using a mortar and pestle. The frozen powder was with 0.2% (v/v) Triton X-­100, 1mM DTT, 100 µg/ml PMSF, 3 µg/ collected in lysis buffer (50 mM Tris-­ HCl [pH 7.5], 1 mM dithiothrei- ml E64, and 1X plant protease inhibitor (Sigma). The resuspended tol and 10% glycerol supplemented with 1X plant protease inhibitor sample was centrifuged at 10,000 ×g for 10 min at 4°C and the su- [Sigma]). After centrifugation 10,000 ×g for 10 min at 4°C, the su- pernatant recovered for a second step of centrifugation. Protein pernatant was collected and protein concentration was determined. concentration was determined using the kit Pierce Coomassie Plus 90 µg of total protein was run on a 3–­13% gel in deep blue cath- (Bradford) Protein-­ A ssay (Thermo Scientific). Approximately 40–­ ode buffer (50 mM Tricine, 7.5 mM Imidazole and 0.02% Coomassie 50 µg of total protein was separated by SDS–­PAGE, transferred G250) at 4°C for 3 h at 100 V and then exchange deep blue cath- to nitrocellulose membrane, and subjected to immunoblotting. ode buffer to slightly blue cathode buffer (50 mM Tricine, 7.5 mM The following antibodies were used anti-­CCT7 [1:1,000] (Abcam, Imidazole, and 0.002% Coomassie G250) and run at 100 V overnight. ab170861) and anti-­Actin [1:5000] (Agrisera, AS132640). Proteins were then transferred to a polyvinylidene difluoride mem- brane at 400 mV for 3 h by semi-­dry blotting. For loading control, the membrane was stained with Ponceau S. Western blot analysis 4.4 | Separation of soluble and insoluble was performed with a monoclonal antibody against CCT1 [1:1,000] protein extracts (Abcam, ab109126). For the separation of soluble and insoluble (with protein aggregates) fractions, native protein extracts were obtained using a buffer con- 4.7 | Gene expression analysis taining 100 mM Tris-­HCl pH 7.9, 10 mM MgCl2, 1% (v/v) glycerol, and 1X plant protease inhibitor (Sigma). After centrifugation for 10 min Total RNA was extracted from plant tissue, using the Maxwell 16 at 10,000 ×g, the supernatant was collected as the soluble fraction. LEV Plant RNA Kit (Promega). RNA was quantified using a NanoDrop 10 of 12 | LLAMAS et al. (Thermo Scientific). cDNA was synthetized using the kit NZY First-­ root analysis and measurement. We are grateful to J. Salinas for Strand cDNA Synthesis Kit (nzytech). SybrGreen real-­time quantita- providing the pfd3 and pfd5 seeds. Anti-­Actin antibody was a gift tive PCR experiments were performed with a 1:20 dilution of cDNA from J.F. Martínez-­García. pGWB502 was a gift from T. Nakagawa using a CFC384 Real-­Time System (Bio-­Rad). Data were analyzed (Addgene plasmid 74844). We thank the Salk Institute Genomic with the comparative 2ΔΔCt method using the geometric mean of Analysis Laboratory and NASC for providing the sequence indexed Ef1α and PP2A as housekeeping genes. See Table S1 for qPCR prim- Arabidopsis T-­DNA insertion mutants. We thank the CRAG (M. ers used in this work. Amenós) and CECAD (A. Schauss, C. Jungst) imaging facilities for their support in confocal microscopy. Open Access funding enabled and organized by Projekt DEAL. 4.8 | Confocal microscopy and fluorescence quantification C O N FL I C T O F I N T E R E S T The authors declare no competing interests. Confocal microscopy images were taken either with FV1000 Confocal Laser-­ scanning Microscope (Olympus) or a Meta 710 Confocal AU T H O R C O N T R I B U T I O N S Microscope with laser ablation 266 nm (Zeiss). All images were ac- E.L. designed and performed most of the experiments, analyzed the quired using the same parameters between experiments. For the de- data, and wrote the paper. D.V. planned and supervised the project tection of aggregated proteins, we used the ProteoStat Aggresome and wrote the paper. S.T.M., M.V.B., E.S., and N.D. contributed to detection kit (Enzo Life Sciences). Seedlings were stained according some of the experiments. H.J.L performed TRiC/CCT assembly to the manufacturer's instructions. Briefly, seedlings were collected assay. S.T.M. and P.W. analyzed the RNA-­seq data. A.Z. and M.R.C and were fixed in 4% formaldehyde solution for 30 min at room tem- interpreted and discussed the results. W.W., A.Z., and M.R.C pro- perature. Formaldehyde solution was removed and seedlings were vided reagents and equipment for the research. washed twice with 1X PBS. Then, seedlings were incubated with per- meabilizing solution (0.5% Triton X-­100, 3 mM EDTA, pH 8.0) with DATA AVA I L A B I L I T Y S TAT E M E N T gently shaking for 30 min at 4°C. Seedlings were washed twice with There is no restriction on data availability. All data needed to evalu- 1X PBS. Then, plants were incubated with 1X PBS supplemented with ate the conclusions in the paper are present in the paper and/or the 1 µl/ml of ProteoStat and 1 µl/ml Hoechst 33342 (nuclear staining) Appendix S1. for 30 min at room temperature. Finally, seedlings were washed twice with 1X PBS and mounted on a slide. Quantification of ProteoStat ORCID fluorescence was performed with ImageJ software. David Vilchez https://orcid.org/0000-0002-0801-0743 REFERENCES 4.9 | Heat shock survival assay Ahn, H. K., Yoon, J. T., Choi, I., Kim, S., Lee, H. S., & Pai, H. S. (2019). 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