Brassinosteroids (BRs) are essential hormones for plant growth and development. Enzymes DET2 and CYP90 family are responsible for BR biosynthesis in seed plants. Yet, their roles in non-seed plants are unknown. Here, we report the first functional study of DET2 and all 4 CYP90 genes isolated from Selaginella moellendorfii. Sm89026 (SmCPD) belonged to a clade with CYP90A1 (CPD) and CYP90B1 (DWF4) while Sm182839, Sm233379 and Sm157387 formed a distinct clade with CYP90C1 (ROT3) and CYP90D1. SmDET2, SmCPD and Sm157387 were highly expressed in both leaves and strobili while Sm233379 was only highly expressed in the leaves but not strobili, implying their differential functions in a tissue-specific manner in S. moellendorfii. We showed that only SmDET2 and SmCPD completely rescued Arabidopsis det2 and cpd mutant phenotypes, respectively, suggestive of their conserved BR biosynthetic functions. However, neither SmCPD nor other CYP90 genes rescued any other cyp90 mutants. Yet overexpression of Sm233379 altered plant fertility and BR response, which means that Sm233379 is not an ortholog of any CYP90 genes in Arabidopsis but appears to have a BR function in the S. moellendorfii leaves. This function is likely turned off during the development of the strobili. Our results suggest a dramatic functional divergence of CYP90 family in the non-seed plants. While some of them are functionally similar to that of seed plants, the others may be functionally distinct from that of seed plants, shedding light for future exploration.
Citation: Xu W, Zheng B, Bai Q, Wu L, Liu Y, Wu G (2019) Functional study of the brassinosteroid biosynthetic genes from Selagnella moellendorfii in Arabidopsis. PLoS ONE 14(7): e0220038. https://doi.org/10.1371/journal.pone.0220038
Editor: Jin-Song Zhang, Institute of Genetics and Developmental Biology Chinese Academy of Sciences, CHINA
Received: April 4, 2019; Accepted: July 8, 2019; Published: July 25, 2019
Copyright: © 2019 Xu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files; information for all the genes used in this paper is included in S3 Table.
Funding: This work was supported by the Chinese National Foundation of Science grant numbers 31270324 and 31741014 to GW.
Competing interests: The authors have declared that no competing interests exist.
Brassinosteroids (BRs) are plant steroid hormones originally discovered in Brassica napus pollen , and later found in almost all plants examined . Up to now more than 70 BRs have been identified in plants . Impairing BR biosynthesis or signaling reduces plant growth and causes abnormal development, thereby limiting plant fertility and yield . Therefore, BRs play a broad role in plant growth and development.
At present, it is known that brassinolide (BL), the most active BR, is converted from castasterone (CS). CS is synthesized from campestanol (CN) through either the early C-6 oxidation and/or the late C-6 oxidation pathway(s) . The first reaction toward the BL is the conversion of campaesterol (CR) into CN, which is then converted into 6-deoxocathasterone (6-deoxoCT). This process has been demonstrated in cultured cells of Catharanthus rosenus. Through feeding test, 6-deoxoteasterone (6-deoxoTE) is detected as a major metabolite from 6-deoxoCT. At the end, 6-deoxoTE is finally converted into CS, BL, in that sequence (Fig 1). BR biosynthesis is achieved from CR to BL not by single but by parallel and highly branched pathways [6,7].
The Lycophytes are the earliest group of vascular plants entering onto the earth, playing a critical role in the evolution of plants. In view of this fact, the Selaginella moellendorfii, a species of Lycophytes, was taken as the material to study the function of BR biosynthesis genes. As reported previously, there are homologous genes involved in BR biosynthesis in Selaginella, which include DET2, Sm89026, Sm182839, Sm233379 and Sm157387 . However, whether they have biological functions has not been explored.
In this work, we demonstrate that SmDET2 can recover the det2 mutant and Sm89026 can rescue the cpd mutant. This implies that SmDET2 and Sm89026 are likely functional in S. moellendorfii. However, Sm182839, Sm233379 and Sm157387 clustered in the same clade with ROT3 cannot rescue cpd, rot3 or dwf4, but overexpression of Sm233379 does enhance a BR function and impact plant fertility. Additionally, we do not find ROT3 homologous gene in the monocots. Taken together, these results lead us to propose that there is similar BR biosynthesis pathway from S. moellendorfii to Arabidopsis thaliana, with SmDET2 and Sm89026 (SmCPD) playing a key role in Selaginella. Additionally, Sm233379 is involved in BR biosynthesis distinct from that of AtROT3 in the same clade. Yet, only future exploration can resolve the exact function of Sm233379, Sm182839 and Sm157387 in S. moellendorfii.
Materials and methods
Phylogenetic and schematic analysis
The amino acid sequences of the enzymes involved in the BR biosynthesis were extracted from NCBI (https://www.ncbi.nlm.nih.gov/). Full-length protein sequences were aligned with ClustalX2. Phylogenetic trees were generated using the Neighbor-joining method with MEGA7 software.
Arabidopsis thaliana ecotype Columbia (Col-0) was used as the wild-type (WT) control. The BR-related mutants, cpd, rot3 and dwf4, transgenic SmCYP90 (Sm89026, Sm182839, Sm233379, Sm157387) lines, and overexpression lines (Sm89026-OX, Sm182839-OX, Sm233379-OX and Sm157387-OX) were all in the Col-0 background. Seeds were imbibed for 4 days at 4°C, then transplanted onto the soil. Plants were grown at 22°C with 70% humidity under a 16-h light (~120μmol.m-2.s-1)/8-h dark cycle.
Vector constructs and transgenic lines
Full-length gDNAs of DET2 and CYP90 genes without a stop codon were amplified by PCR using gene-specific primers (S1 Table) with 30 cycles. The amplified DNAs were then inserted into the plant expression vector (p35S-CHF3-GFP) individually to generate p35S:AtDET2-GFP, p35S:AtCPD-GFP, p35S:AtROT3-GFP, p35S:AtDWF4-GFP, p35S:SmDET2-GFP, p35S: Sm89026-GFP, p35S:Sm182839-GFP, p35S: Sm233379-GFP, and p35S: Sm157387-GFP. These constructs were then transferred into plants via Agrobacterium (GV3101)-mediated transformation using the floral dip method . The transformants were screened on 1/2 MS with 50 μg/mL kanamycin.
Hypocotyl growth analysis
Seeds were surface-sterilized and placed on half strength MS plates with 0.8% (w/v) agar, 1% (w/v) sucrose, with or without BRZ (brassinazole) with a concentration of 5 μM. The plates were cold treated at 4°C for two days to ensure uniform germination. Seeds were considered to begin germination after the plates were kept at 22°C for 24 h. Five DAG (days after germination) in the dark, seedlings were put on a transparent film and scanned to acquire images, which were then used to measure the hypocotyl length with ImageJ (http://rsb.info.nih.gov/ij/). For statistical comparisons, LSD (least significant difference) test was performed (p<0.01).
Light microscopy observation
Fresh buds approximately containing stage-8 anthers were fixed, dehydrated as described by Zhang et al., then stained with Alexander’s staining solution .
Total RNAs were extracted using a HiPure Plant RNA Mini Kit (Magen, R4151-02, China) according to the manufacturer’s protocol. First–strand cDNA was synthesized from 1μg of total RNA using M-MLV First Strand cDNA Synthesis Kit (Omega, TQ2501-02, Norcross, GA, USA). Semi-quantitative RT-PCR (PCR for genes of Arabidopsis thaliana ran 30 cycles, except for BAS1 that ran 32 cycles. PCR for genes of S. moellendorfii ran 31 cycles) analyses were performed using specific primers to study the expression levels. The primers used were listed in the S2 Table.
DET2 has a conserved function
Our results showed that, after 5 days of growth in total darkness, the det2 mutants were short with thick hypocotyls, and opened and expanded cotyledons (Fig 2C), consistent with previous reports . In order to determine the function of SmDET2, we expressed it under the control of CaMV 35S promoter. We found that it completely rescued the det2 mutant phenotypes. The transgenic plants showed normal adult phenotypes (Fig 2A), normal young plants (Fig 2B) and normal seedlings in the dark (Fig 2C). BRs activate the activities of the BES1 family transcription factors that downregulate the expression of BR biosynthetic genes, such as CPD and DWF4, and upregulate BR metabolic genes, such as BAS1 . Thus, this phenomenon has been used as a reliable marker for the presence of BRs or their signaling . Therefore, to more accurate estimate the role of SmDET2 in above transgenic plants, semi-quantitative RT-PCR with the total RNAs prepared from these seedlings was performed. We confirmed that SmDET2 rescued the det2 mutant phenotypes at a level of wild type plants (Fig 2D), which means that SmDET2 and DET2 have a conserved function. It is worth mentioning that the phenotypes of the expression of SmDET2 and AtDET2 in the det2 mutants were completely indistinguishable (Fig 2D). We thus conclude that SmDET2 and AtDET2 have very similar if not the same function.
(A) Phenotypes of WT, det2, AtDET2/det2 and SmDET2/det2 30 DAG (days after germination). Scale bar, 5 cm. (B) Phenotypes of 6-week plants grown under a long-day condition (16/8 h, light/dark). Scale bar, 5 cm. (C) hypocotyls 5-DAG dark-grown seedlings in 1/2 MS medium. Scale bar, 1 cm. (D) RNAs were prepared from wild-type (WT), det2 mutant (det2), and transgenic seedlings in det2 background grown in glass jars under white light for 10 days, semi-quantitative RT-PCR analysis of the transcripts of CDP, DWF4 and BAS1. To control equal loading of RNA samples, (At)ACT2 gene served as a control.
Phylogenetic analysis of CYP90 enzymes involved in BRs biosynthesis
Besides DET2, CPD, DWF4, ROT3 and CYP90D1 are also the critical enzymes that catalyze the important reactions in the later steps of BRs biosynthesis (Fig 1). Sm89026 was previously named SmCPD . However, protein sequence comparison and our phylogenetic analysis revealed that Sm89026 was in a clade with DWF4 sister with CPD while Sm182839, Sm233379 and Sm157387 formed a distinct clade with ROT3 and CYP90D1 (Fig 3) . These results suggest that either CPD or DWF4 is lost in S. moellendorfii (Fig 3). Thus, there is a need to determine whether Sm89026 functions as a CPD or a DWF4.
The phylogenic relationship of CYP90 enzymes in various plants and S. moellendorfii is shown. Full-length protein sequences were aligned with Clustal X2. A1, B1, C1 and D1 are short for CYP90A1, CYP90B1, CYP90C1 and CYP90D1, respectively. AthCYP51 served as an out-group. Bootstrap decimals were indicated at the branch points. The full names of the species used were showed in S3 Table.
CYP90 genes are differentially expressed in Selaginella moellendorfii
Gene expression patterns are major parameters of how genes function. Using semi-quantitative RT-PCR, we analyzed the expression level of CYP90 genes in a vegetative organ, leaf, and a reproductive organ, strobili, of S. moellendorfii. We demonstrated that both Sm89026 (SmCPD) and Sm157387 were highly expressed in both organs while Sm233379 was only significantly expressed in the leaves but not strobili. However, Sm182839 was under-expressed in both tissues (Fig 4). These results suggest an expression divergence in SmCYP90 genes. Together with their sequence divergence, this may indicate that SmCYP90 genes have distinct functions from each other.
CYP90 genes involved in BR biosynthesis in Selaginella moellendorfii
To ask whether SmCYP90 genes indeed encode enzymes involved in BR biosynthesis in S. moellendorfii, we cloned SmCYP90 genes under the control of 35S promoter and expressed them in wild type (WT) Arabidopsis plants. Most of these transgenic plants more or less resembled BR overproducing plants (Fig 5A–5D and 5L and S1 Fig) [12–13]. They showed longer petioles, larger rosette diameters, less rosette leaves and slender shoots (Fig 5A and 5C and S2 Fig). We further observed the elongated hypocotyls in all these over-expressing lines (Fig 5B and 5D) . Yet, they were all sensitive to BRZ (brassinozole) that specifically inhibits BR biosynthesis (Fig 5D and S3 Fig). Surprisingly, using upregulation of CPD and DWF4 and downregulation of BAS1 as the indicators of BR overproducing markers, only the transgenic plants of Sm89026 (SmCPD) and Sm233379 had undoubted BR overproducing phenotypes (Fig 5L). Nevertheless, an enhanced expression of BAS1 in Sm89026-OX and Sm233379-OX plants suggests a role of Sm89026 and Sm233379 in positive regulation of BR biosynthesis.
(A) Morphology of WT and overexpression of SmCYP90 genes mature plants grown 6-week after germination under a long-day condition (16/8 h, light/dark). Scale bar, 5 cm. (B) Morphology of the WT and lines with the overexpression of SmCYP90 seedlings grown on 1/2 MS medium 5 DAG in dark. Scale bar, 1.0 cm. (C) Morphology of WT and overexpression of SmCYP90 seedlings grown in soil 30 DAG under a long-day condition (16/8 h, light/dark). Scale bar, 3 cm. (D) Analysis of the length of hypocotyls in the seedlings of WT and lines with the overexpression of SmCYP90. Seedlings were grown on 1/2 MS medium in dark for 5 days. Values represented the mean of 30 measurements ± SD. Letters above each bar indicated a significant difference compared to the mock treatment. (E) Siliques of the WT. (F) A mature flower of the WT. Scale bar, 1 cm. (G) An anther of the WT. Scale bar, 50 μm. (H) The fertility of Sm233379-OX was largely reduced, which was indicated by the fact that most of siliques were completely or partially lacked of seeds. Scale bar, 1 cm. (I) The length of the stamens in Sm233379-OX was shorter than the stigma. (J) Anther staining examination revealed a reduction of viable pollen grains in Sm233379-OX anthers, compared to the WT. Scale bar, 50 μm. (K) Semi-quantitative RT-PCR analysis of the expression of SmCYP90 genes in 10-day-old seedlings of the transgenic lines was shown. ACT2 served as an internal control. (L) RNAs were prepared from seedlings grown in glass jars under white light for 10 days. Semi-quantative RT-PCRs were shown for the WT and transgenic lines of SmCYP90 genes (Sm89026, Sm182839, Sm233379 and Sm157387). ACT2 served as an internal control. Our analysis indicated that the CPD and DWF4 genes were down-regulated while BAS1 was up-regulated.
Intriguingly, the fertility of overexpression Sm233379 was dramatically reduced, compared to the WT (Fig 5A and 5H–5J, S4 Fig). Reduced fertility can be induced by many reasons, such as abnormal tapetum , impaired pollen tube-stigma interaction , failure of pollen tube integrity and sperm release , etc. We found that Sm233379-OX plants had little male fertility, and produced shorter siliques than WT plants. To further find the reason for the reduced fertility, we dissected the flower of Sm233379-OX, and found that the majority of transgenic plants with Sm233379-OX had fewer pollen grains with small size. As Sm233379 was only expressed in vegetative tissues of S. moellendorfii (Figs 4 and 5A), thus the fertility phenotypes could be a result of spatiotemporal mis-localization of certain BRs in the reproductive organs of the transgenic plants . Phenotypic differences in the transgenic lines of the overexpression of CYP90 genes suggest that the BR signaling in these plants might be altered. Therefore, we analyzed transcript levels of (At)CPD, (At)DWF4 and (At)BAS1 using semi-quantitative RT-PCRs in these transgenic plants and found that the transcript levels of all three genes were altered in SmCYP90-OX seedlings as compared with those of WT (Fig 5K and 5L).
Sm89026 is a functional equivalent of CPD in Selaginella moellendorfii
Among the transgenic lines of SmCYP90 family, only Sm89026/cpd, like AtCPD/cpd, had normal phenotypes as the WT, which led to the suggestion that Sm89026 can completely rescue the phenotypes of cpd (Fig 6A–6D), indicating that Sm89026 is functionally equivalent to the Arabidopsis CPD. To investigate if the morphological evidence for the BR biosynthesis was consistent with the indication at the molecular level, the expression of CPD, DWF4 and BAS1 was analyzed using semi-quantitative PCRs in WT, cpd and AtCPD/cpd and Sm89026/cpd seedlings. We found that the expression of BAS1 was significantly decreased in Sm89026/cpd plants, whereas the expression of CPD and DWF4 was slightly reduced (Fig 6E), supporting that Sm89026 functions as an Arabidopsis CPD.
(A) Pictures from WT, cpd, AtCPD/cpd and Sm89026/cpd plants grown 6-week after germination. Scale bar, 5 cm. (B) Morphology of WT, cpd, AtCPD/cpd and SmCYP90 genes and seedlings grown 30 DAG. Scale bar, 3 cm. (C) Morphology of WT, cpd, AtCPD/cpd and Sm89026/cpd seedlings grown five DAG in dark on 1/2 MS medium with or without BRZ (brassinazole). Scale bar, 1.0 cm. (D) Analysis of the hypocotyl length of 5-DAG dark-grown WT, cpd, AtCPD/cpd and Sm89026/cpd. Values represented the mean of 30 measurements ± SD. Letters above each bar indicated a significant difference compared with the mock treatment. (E) Semi-quantitative PCR analysis expression of CPD, DWF4 and BAS1 in the 10-day-old WT, cpd, AtCPD/cpd and Sm89026/cpd. The AtACT2 gene served as a control.
No functional equivalent of DWF4 and ROT3 found in SmCYP90
Among the enzymes involved in BR biosynthesis in Arabidopsis, DWF4 catalyzes the rate-determining step , and DWF4 acts as a 22α-hydroxylase . The transgenic lines of SmCYP90 genes in dwf4 had smaller seedlings and shorter hypocotyl than WT (Fig 7A and 7B), indicating that SmCYP90 genes do not encode enzymes with an equivalent function to that of (At)DWF4. The DWF4 gene has been shown to encode a cytochrome P450 enzyme (CYP90B1) that only shares 43% amino acid sequence identity with CPD . Since Sm89026 could not rescue the DWF4, Sm89026 is not a functional equivalent of Arabidopsis DWF4 although Sm89026 was in a clade with DWF4 rather than with CPD (Fig 3).
(A) Morphology of WT, dwf4, AtDWF4/dwf4 and SmCYP90 transgenic seedlings grown 30 DAG in the light. Scale bar, 3 cm. (B) Morphology of WT, dwf4, AtDWF4/dwf4 and SmCYP90/rot3 genes transgenic seedlings grown on 1/2 MS medium 5 DAG in dark. Scale bar, 1.0 cm. (C) Morphology of WT, rot3, AtROT3/rot3 and SmCYP90/rot3 genes transgenic seedlings grown 30 DAG in the light. Scale bar, 3 cm. (D) Morphology of WT, rot3, AtROT3/rot3 and SmCYP90/rot3 transgenic seedlings grown on 1/2 MS medium 5 DAG in dark. Scale bar, 1.0 cm. (E) Comparison of the rosette with of WT, dwf4, AtDWF4/dwf4 and SmCYP90/rot3 transgenic seedlings grown 30 DAG in the light. (F) Comparison of the hypocotyl length of WT, dwf4, AtDWF4/dwf4 and SmCYP90/rot3 transgenic seedlings grown 5 DAG in the dark. (G) Comparison of the rosette with of WT, rot3, AtROT3/rot3 and SmCYP90/rot3 transgenic seedlings grown 30 DAG in the light. (H) Comparison of the hypocotyl length of WT, rot3, AtROT3/rot3 and SmCYP90 genes transgenic seedlings grown 5 DAG in the dark.
The polarized processes of cell elongation play a crucial role in morphogenesis of higher plants. The ROT3 gene encodes a cytochrome P450 (CYP90C1) with domains homologous to the regions of steroid hydroxylases of animals and plants, confirmed that the ROT3 gene controls polar elongation in leaf cells by an analysis of three rot3 mutants obtained from different mutagenesis experiments . The rot3 mutants exhibit short petioles  (Fig 7C), and a small ratio of length to width than that of the WT. No transgenic plants of SmCYP90 genes rescued the phenotypes of rot3 as being shown in the seedlings grown for 4 weeks (Fig 7C).
As a class of essential plants hormones, BRs play key roles in regulating a broad aspect of plant growth and development. BRs are biosynthesized from campesterol via a 5-alpha reductase and several cytochrome P450 (P450) catalyzed oxidative reactions . The BR biosynthetic and signaling pathways have been well characterized in Arabidopsis and other angiosperms, but our knowledge of these pathways is limited in other plant groups. Previously, it has been reported that the Lycophyte S. moellendorffii, an ancestral vascular plant, has physiological responses to the BRs and to the BR biosynthetic inhibitor, PCZ (propiconazole). This suggests that BRs are biosynthesized in Selaginella. Unfortunately, most BR intermediates found in Arabidopsis and rice were not detectable or only present at very low levels. So far, we do not know the biosynthetic process of BRs in Selaginella.
Based on ectopic expression and phenotypic complementation of BR biosynthetic mutants of Arabidopsis, we have studied the function of DET2 and CYP90 genes in non-seed plants using SmDET2, Sm89026, Sm182839, Sm233379 and Sm157387 isolated from S. moellendorfii. The results show that Sm89026 (SmCPD) belongs to a clade with CYP90A1 (CPD) and CYP90B1 (DWF4) while Sm182839, Sm233379 and Sm157387 forms a distinct clade with CYP90C1 (ROT3) and CYP90D1 (Fig 3). SmDET2, SmCPD and Sm1573872 are highly expressed in both leaves and strobili while Sm233379 is only highly expressed in the leaves but not in the strobili of S. moellendorfii (Fig 4), implying their differential functions. We show that only SmDET2 and SmCPD completely rescue det2 and cpd mutant phenotypes, respectively (Figs 1 and 6), suggestive of their conserved BR biosynthetic functions. However, neither SmCPD rescues any other cyp90 mutants, nor any other SmCYP90 genes rescue any cyp90 mutants. Yet, overexpression of Sm233379 alters plant fertility and the expression of BR biosynthetic and metabolic genes, markers of BR functions (Fig 5). Taken together, SmCPD and Sm233379 have a BR biosynthetic function. Furthermore, SmCPD is an equivalent of the Arabidopsis CPD, while Sm233379 has no equivalent in Arabidopsis and the function of the other two CYP90s remains for future exploration.
The activation of BRs signaling pathway depends on a series of signal transduction components. But the most important receptor, BRI1, does not exist in Selaginella, suggesting that there are different signal pathways in Selaginella and Arabidopsis. However, most BR biosynthetic genes and signal components share high similarity in between S. moellendorfii and Physcomitrella patens, a primitive terrestrial non-vascular plant, but P. patens does not respond to brassinolide . We infer that there is not conventional BR signal receptor to activate downstream transcription, although a relatively complete BR synthesis pathway exists, in Selaginella. This leads us to believe that castasterone precursor, the product of CPD, played a role as physiological active substance but not a hormone, as the responsive concentration is much higher in Selaginella than in Arabidopsis . Another possibility is that there may be a kind of completely unknown BR receptors in S. moellendorfii and P. patens, having a signaling pathway significantly distinguished from that of Arabidopsis.
The CPD encodes an enzyme having function in a key rate-limiting step, and cpd mutant shows extremely dwarf phenotypes with reduced fertility similar to the mutants of BR receptors, which is known as marker gene for estimating whether BR endogenous signal is strong or not according to CPD activity. Our results show that BR biosynthetic process in Selaginella is similar to that in Arabidopsis based on functional SmDET2 and SmCPD, and the critical BR synthetic products existed in other primitive terrestrial plants . These findings indicate that BR synthetic pathway has already appeared in early terrestrial plants before complete hormone-receptor signaling pathways arise (Fig 8). Together, our studies have been fruitful in identifying the function of putative genes involved in the biosynthesis of BR and analysis of differential expression, including SmDET2 and SmCYP90s. This would lay the foundation for studying on the mechanism of BR function and understanding the origin of BR signal from primitive vascular plants. Biochemical approaches are likely to play increasingly critical role in filling the gaps of synthesis from the product of SmDET2 to the substrates of SmCPD in future studies.
S1 Fig. Height of plants not significantly different from each other.
Data are presented as the mean ±SD.
S2 Fig. Radii of rosette leaves are not significantly different from each other.
Data are presented as the mean ±SD.
S3 Fig. Plants of SmCYP90 overexpression were still sensitive to BRZ like as WT.
Seedlings grown on 1/2MS medium 5 DAG in dark with/without 5μM BRZ. Scale bar, 1 cm.
S4 Fig. Most of siliques from Sm233379-OX plants were abnormal.
Scale bar, 2cm.
S2 Table. Primers for semi-quantitative RT-PCR.
The authors thank Qiang Wei and Xiaoxia Sun for technical assistance, and Xiaoping She for comments.
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