- Research article
- Open Access
Glycolate oxidase-dependent H2O2 production regulates IAA biosynthesis in rice
BMC Plant Biology volume 21, Article number: 326 (2021)
Glycolate oxidase (GLO) is not only a key enzyme in photorespiration but also a major engine for H2O2 production in plants. Catalase (CAT)-dependent H2O2 decomposition has been previously reported to be involved in the regulation of IAA biosynthesis. However, it is still not known which mechanism contributed to the H2O2 production in IAA regulation.
In this study, we found that in glo mutants of rice, as H2O2 levels decreased IAA contents significantly increased, whereas high CO2 abolished the difference in H2O2 and IAA contents between glo mutants and WT. Further analyses showed that tryptophan (Trp, the precursor for IAA biosynthesis in the Trp-dependent biosynthetic pathway) also accumulated due to increased tryptophan synthetase β (TSB) activity. Moreover, expression of the genes involved in Trp-dependent IAA biosynthesis and IBA to IAA conversion were correspondingly up-regulated, further implicating that both pathways contribute to IAA biosynthesis as mediated by the GLO-dependent production of H2O2.
We investigated the function of GLO in IAA signaling in different levels from transcription, enzyme activities to metabolic levels. The results suggest that GLO-dependent H2O2 signaling, essentially via photorespiration, confers regulation over IAA biosynthesis in rice plants.
Photorespiration is the second-highest metabolic flux after photosynthesis in plants. It starts from the synthesis of 2-phosphoglycolate (2-PG) catalyzed by the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). 2-PG is immediately converted to glycolate in chloroplasts and transported to peroxisomes, where glycolate is detoxified into glycine by the glycolate/glyoxylate/glycine metabolic steps . Glycolate oxidase (GLO, EC 22.214.171.124) is a key enzyme for the glycolate-glyoxylate conversion during photorespiration, which catalyzes the oxidation of glycolate to generate glyoxylate and H2O2 . It has been estimated that the photorespiratory H2O2 produced by GLO may account for approximately 70% of the total H2O2 in C3 plants, thus making an important contribution to cellular redox status and participating in multiple H2O2-related processes . GLO activity was reported to be up-regulated in pea, cowpea, and tobacco under drought stress [3,4,5]. More importantly, GLO-derived H2O2 can mediate nonhost resistance and gene-for-gene-mediated resistance in Arabidopsis thaliana and Nicotiana benthamiana , and it is also involved in barley stripe mosaic virus infection in barley and basal defense against Pseudomonas syringae in tomato [7, 8]. In addition, initiation of the systemic acquired acclimation and formation of iron plaques on the surface of roots in plants is mediated by GLO-derived H2O2 [9, 10].
H2O2, as a typical reactive oxygen species, is not only biologically toxic but also serves as an important signaling molecule. Its homeostasis is elaborately regulated by the balance between the cellular generation and scavenging rates . Photorespiratory H2O2 is mainly scavenged by peroxisomal catalase (CAT, EC 126.96.36.199), and GLO and CAT usually act in concert to regulate intracellular photorespiratory H2O2 levels in plants . It has been observed that CAT-dependent H2O2 cross-talks with other phytohormone signaling pathways. For instance, ABA regulates the expression of peroxisomal CAT and hence the levels of H2O2 in water-stressed Arabidopsis . Another potential point of crosstalk is between the CAT-dependent H2O2 and IAA signaling pathway, which has recently gained much attention. Gao et al.  found that the absence of the photorespiratory CAT2 resulted in accumulation of the photorespiratory H2O2. Additionally, increased H2O2 suppressed the IAA synthesis in Arabidopsis. In turn, exogenous auxin activated IAA signaling to counteract photorespiratory H2O2-dependent cell death in Arabidopsis cat2 mutants . Yuan et al.  further demonstrated that the accumulated H2O2 in Arabidopsis resulting from CAT2 inhibition promoted the sulfenic acid modification of tryptophan synthetase β (TSB) subunit 1, subsequently decreasing TSB activity to repress the synthesis of the IAA precursor Trp. While CAT-dependent H2O2 decomposition intertwined with the IAA signaling pathway, the molecular details of this process remain scarce, and more notably, it is not clear whether GLO dependent H2O2 production plays a major role in IAA regulation.
As an important engine for H2O2 production, GLO has various isoforms that are encoded by multi-gene families in plants. AtGOX1 and AtGOX2 are the major isoforms involved in basic photorespiration metabolism in Arabidopsis thaliana . We have earlier demonstrated that OsGLO1 and OsGLO4 are the dominant isoforms for photorespiration in rice [16, 17]. In this study, in order to understand whether and how GLO-dependent H2O2 modulates IAA signaling, various genetically-modified rice lines of GLO1 and GLO4 were generated. It was found that IAA levels in glo1 and glo4 knockout mutants were significantly up-regulated which was correlated with lower peroxisomal H2O2 levels. Further analyses showed that IAA and Trp contents were increased, and that TSB activity and the genes related to Trp-dependent IAA biosynthesis and IBA to IAA conversion were also up-regulated. The results suggest that GLO-dependent H2O2 production can regulate IAA biosynthesis in rice.
Rice GLO mutants exhibited photorespiration phenotypes as accompanied with decreased peroxisomal H2O2
The rice genome contains four GLO homologs, i.e., GLO1 (Os03g0786100), GLO3 (Os04g0623500), GLO4 (Os07g0152900), and GLO5 (Os07g0616500). GLO1 and GLO4 are the primary GLO genes that contribute to photorespiratory glycolate-glyoxylate metabolism [16, 17]. In this study, knockout and overexpression lines of GLO1 and GLO4 were generated in rice. Western blot analysis confirmed that GLO1 and GLO4 were absent in the glo1 and glo4 knockout mutants, respectively (Additional file 1A, B). GLO activities were decreased by approximately 55% and 30% in the glo1 and glo4 mutants, respectively. In GLO overexpression (GLOOE) lines, GLO activities were increased by 60–95% in GLO1OE lines and 40–55% in GLO4OE lines (Fig. 1A and Additional file 2A). Both seedlings of glo1 and glo4 knockout mutants showed dwarfish growth as compared with WT but recovered to normal stature when photorespiration was suppressed under high CO2 condition (Fig. 1B, C and D). The growth phenotypes of those overexpression lines have been described earlier in our previous study. Briefly, the growth of GLOOE lines was improved when GLO activities were increased by 60–100%, whereas reduced growth was noticed when GLO activity was increased over 150% . In addition, some downstream enzymes of GLO, such as SGAT and GGAT, were little altered in all GLO modified lines under both ambient and high CO2 conditions (Fig. 2 and Additional file 2). The CAT activity was suppressed by 30 to 40% under high CO2, but there was no difference in CAT activities between various GLO modified lines under ambient and high CO2 conditions (Fig. 2A and Additional file 2B). This is consistent with previous studies showing that GLO downstream steps in photorespiration are not significantly affected by GLO modification [19,20,21].
As the fastest pathway for H2O2 production, photorespiratory H2O2 is mainly generated by the GLO-catalyzed glycolate oxidation reaction [1, 2]. The absence of GLO1 and GLO4 can significantly decrease H2O2 levels in rice leaves under atmospheric condition (Fig. 3A, B), and this difference disappeared under high CO2 (Fig. 3B). No significant changes were observed in GLO1 and GLO4 overexpression lines under both atmospheric and high CO2 conditions (Additional file 3A, B). To further prove that the decreased H2O2 levels occur in peroxisomes, we quantified the transcript abundance of two previously identified peroxisome-specific H2O2-responsive genes, OsbHLH168 (Os01g0108600) and OsSAP17 (Os09g0385700) [22, 23], by qRT-PCR. As shown in Fig. 3C, transcript levels of both OsbHLH168 and OsSAP17 were significantly lowered in the glo1 and glo4 mutants under atmospheric condition. For GLO overexpression lines, the expression levels of these two genes were not significantly different (Additional file 3C). These results indicated that the decreased H2O2 levels occurred in peroxisomes, due to reduced GLO activities in glo1 and glo4 mutants.
The GLO-dependent H2O2 production regulated IAA and Trp levels
It was previously reported that the CAT-dependent H2O2 accumulation can suppress IAA synthesis in Arabidopsis [13, 15]. Since CAT and GLO may work cooperatively , it is of interest to further understand whether GLO is involved in the H2O2-IAA crosstalk. As the peroxisomal H2O2 levels were decreased (Fig. 3), the IAA contents were increased by 25–30% and 15–20% in the glo1 and glo4 knockout mutants, respectively (Fig. 4A). No obvious changes in IAA were observed in overexpression lines (Additional file 4A), in which H2O2 levels were unchanged (Additional file 3). Trp is the primary precursor for IAA biosynthesis in plants [24, 25], so we further determined if Trp contents were affected in various GLO genetically-modified rice lines. As shown in Fig. 4B, Trp content was about 6.5 µg g−1 FW in WT, which was increased to 8.5–10.0 µg g−1 FW in glo1 knockout mutants, and 8.0–8.5 µg g−1 FW in glo4 knockout mutants. No obvious changes were detected in the overexpression lines (Additional file 4B). Also, Trp contents were changed with a similar tendency as IAA. When GLO-dependent H2O2 production was suppressed under high CO2 condition, the differences in IAA and Trp contents in glo1 and glo4 knockout mutants vanished (Fig. 4A, B). The above results indicated that the reduced photorespiratory H2O2 resulting from the reduction of GLO activity was correlated with increased IAA and Trp contents in rice.
The regulation of IAA occurred at the biosynthetic level
Theoretically, IAA content may be regulated at the levels of biosynthesis and sequestration . The Trp-dependent pathway has been known to dominate IAA biosynthesis, so we first detected TSB activities that are responsible for the biosynthesis of the IAA precursor Trp. As shown in Fig. 5A, TSB activities were increased by 40% and 27% in glo1 and glo4 mutants, respectively. No changes were observed in GLO overexpression lines (Additional file 5). Correspondingly, transcripts of the two subunits of tryptophan synthase (β subunit 1, TSB1; α subunit 1, TSA1) were also significantly enhanced in glo mutants (Fig. 5B). Besides, some other downstream genes involved in the Trp-dependent IAA biosynthesis (Additional file 6), i.e. OsYUC2 (Os01g0732700), OsYUC5 (Os07g0437000), OsAO3 (Os03g0790700), and OsAMI1 (Os04g0118100), were still up-regulated in glo mutants (Fig. 5B).
The conversion of IBA to IAA occurr in the peroxisome, which could be intimately regulated by the redox state of peroxisomes [27, 28]. Hence, the expressions levels of those genes proposed to be involved in the conversion of IBA to IAA were determined (Additional file 6), i.e. OsIBR3 (Os07g0675133), OsECH2 (Os09g0544900), and OsIBR1 (Os09g0133200). These data showed that all of these genes were up-regulated in glo mutants (Fig. 5C).
Finally, the expression levels of various IAA-responsive/transport genes, which are known to be regulated in an IAA-dependent manner, were analyzed (Additional file 7). As expected, the genes involved in IAA responsive and transport processes, i.e. OsAIL5 (Os04g0653600), OsAIL7 (Os03g0313100), OsPBP1 (Os01g0783700), OsIAA26 (Os09g0527700), and OsLAZY1 (Os11g0490600), OsNS3 (Os10g0147400), OsNS4 (Os11g0169200) were transcriptionally up-regulated in glo mutants (Fig. 6A, B), and these genes may further function in multiple plant growth processes and stress responses. The above results collectively indicate that the GLO-dependent H2O2 production regulates IAA biosynthesis to perform various biological functions in rice.
In recent decades, information has become increasingly available indicating that photorespiration can influence various biological processes, e.g., carbon metabolism, energetics, nitrogen assimilation, and respiration [29,30,31]. Remarkably, photorespiration is a major source for H2O2 in photoautotrophic tissues, thereby making important contributions to cellular redox status and signaling [2, 11, 30]. Reports indicate that H2O2 is closely associated with IAA during plant development, and that IAA can facilitate H2O2 accumulation in the roots of maize, tomato, and Arabidopsis to regulate root growth and gravitropism [32,33,34], while IAA biosynthesis and signaling were inhibited by both exogenous and endogenous H2O2 in Arabidopsis seedlings [35, 36]. Nevertheless, how H2O2 production and signaling interact with IAA is still not well understood. Gao et al.  demonstrated that the CAT-dependent H2O2 was involved in the regulation of IAA, but no direct evidence was provided as to whether the H2O2 is derived from photorespiration, because CAT-dependent H2O2 may come from other sources in the peroxisome, such as fatty acid β-oxidation and dismutation of O2.− . By using GLO knockout mutants, we further demonstrated that the GLO-dependent H2O2 also regulated IAA levels in rice (Fig. 3 and Fig. 4A). However, overexpression of either GLO1 or GLO4 conferred no significant changes in H2O2 levels (Additional file 3), or on IAA contents (Additional file 4A). It has been similarly noticed that overexpression of GLO conferred no enhanced effects on photorespiratory flux and H2O2 production, considering that several photorespiratory enzymes (e.g. GLO, 2-phosphoglycolate phosphatase) are sufficiently high to manage the metabolic flux of photorespiration under normal conditions [18, 38] and further up-regulation is only beneficial under some stress environments [3,4,5, 8]. Overall, our present results suggest that GLO-dependent H2O2 regulates IAA levels in rice, not only supporting the results from the Arabidopsis cat2 mutants [13, 14], but also providing direct evidence that the CAT-dependent H2O2 ultimately comes from the GLO-catalyzed reaction, essentially via photorespiratory metabolism in plants.
Two IAA biosynthetic strategies exist in plants, namely, Trp-independent and Trp-dependent pathways. Indole-3-glycerol phosphate and indole are the likely precursors of Trp-independent IAA biosynthesis, but its complete biochemical pathway has not yet been elucidated. Trp is the precursor in the Trp-dependent IAA biosynthesis, and several pathways have been proposed for the IAA biosynthetic strategy, but molecular and genetic evidence of the key enzymes involved in such pathways are still not confirmed [24, 25]. Tryptophan synthetase β (TSB), which catalyzes Trp formation from Ser and indole, is a key limiting factor in Trp biosynthesis. It has also been documented that elevated H2O2 content in Arabidopsis leaves decreased TSB activity, thus reducing Trp and IAA levels [15, 39]. Our present study showed that the increased TSB activity (Fig. 5A) and up-regulated expression of genes responsible for the Trp-dependent IAA biosynthesis (Fig. 5B) were positively correlated with the increased levels of Trp and IAA in glo mutants (Fig. 4A, B). These lines of evidence suggest that the Trp-dependent pathway may have dominated in the IAA biosynthesis of rice plants that is regulated by GLO-dependent H2O2. In addition, modifications in IAA can alter its activity and sequester active IAA . For example, IBA is a chain-elongated form of IAA and functions as a pool for sequestering IAA, which may still be an efficient mechanism for IAA regulation in plants once IBA is converted to IAA [27, 40, 41]. Altering the pool of IAA derived from IBA is known to result in a set of developmental defects in Arabidopsis [42, 43]. IBA is likely converted to IAA through a β-oxidation pathway in the peroxisome [27, 40], where H2O2 might serve as a key regulator . Although we failed to accurately determine IBA contents due to technical difficulties [27, 44], the expression levels of the genes involved in the IBA to IAA conversion were shown to be significantly up-regulated in the glo mutants (Fig. 5C). Therefore, it is likely that the elevated IAA levels caused by decreased H2O2 levels in glo mutants may partially be contributed by the increased conversion of IBA to IAA.
It is well known that IAA signaling controls numerous plant growth and development processes by regulating the expression of various IAA response factors/genes. Indeed, the expression of many IAA-responsive/transport genes (Additional file 7) was markedly up-regulated in glo1 and glo4 mutants (Fig. 6A, B). Our current data, in combination with the results from other studies [13, 15], strongly support that IAA levels can be regulated by either CAT or GLO in plants. IAA is a key phytohormone responding to various environmental stresses, and growth inhibition by decreased IAA levels is an important physiological strategy for plants in response to drought and pathogen infection [45, 46]. Meanwhile, GLO can be induced by drought, high temperature and pathogen infection [3, 5, 8], and these stresses can also result in salicylic acid (SA) accumulation in plants [47,48,49]. We have previously proposed a switch model, in which a physical association-dissociation of GLO and CAT, in response to stimuli such as SA, serves as a mechanism to modulate cellular H2O2 levels . More interestingly, it has been documented that SA could negatively regulate the synthesis and/or signaling of IAA, they act in a mutually antagonistic manner in plants [50,51,52,53], and a recent study further suggested that SA regulation of IAA is mediated by H2O2 . Therefore, we speculate that SA may modulate H2O2 through our proposed switch model, subsequently conferring regulation on IAA. Moreover, the regulation of H2O2 may occur in a fluctuating manner because the association-dissociation of GLO and CAT could take place dynamically and transiently in response to environmental stresses or stimuli, e.g., drought and pathogen infection. Therefore, we here further modified our proposed model, in which the switch-modulated H2O2 fluctuation confers biological functions via regulating IAA levels in plants (Fig. 7).
In this study, we found that decreases in GLO-dependent H2O2 levels were accompanied with IAA and Trp accumulation in glo rice mutants, whereas high CO2 was able to abolish the IAA difference. Subsequent analyses of transcript levels and enzyme activities of genes involved in IAA metabolism suggest that both Trp-dependent IAA biosynthesis and IBA to IAA conversion contribute to the increase of IAA contents. This data offer insights into how GLO-dependent H2O2, essentially via photorespiration, regulates IAA biosynthesis in plants. Furthermore, taken together with our previously reported association-dissociation mechanism of GLO and CAT, we further consider that H2O2 waves as regulated by SA via such a proposed mechanism may be an important point for crosstalk between SA and IAA. However, molecular details of this signalling pathway and its biological significance remain an important issue for our future research.
Plant materials, growth conditions, and treatments
Oryza sativa cv. Zhonghua 11 (japonica cultivar-group) preserved by our laboratory was used for the functional analyses and GLO transgenic line construction . Pre-germinated rice seeds were grown in Kimura B complete nutrient solution under natural conditions. After reaching the four-leaf stage, seedlings were transplanted, either being continuously grown in Kimura B complete nutrient solution in a plant growth chamber with a light cycle of 14 h light/10 h dark (30 °C / 25 °C, respectively) at 600 µmol photons m−2 s−1 on average, relative humidity 60%-80%, or grown in paddy fields under natural conditions. For high concentration CO2 treatment, pre-germinated rice seeds were cultured in Kimura B complete nutrient solution in a plant growth chamber (Percival E-41HO, USA) supplied with 3500 ppm CO2 [30, 54, 55].
Generation of genetically modified rice lines
Total RNA was extracted using the Plant RNA Purification Reagent (Magen Biotech), cDNA was prepared from 1 μg of total RNA using HiScript® II Reverse Transcriptase (Vazyme) as recommended. To generate GLO overexpression transgenic lines, each GLO sequence was cloned into the pYLox.5 vector using the ClonExpress Ultra One Step Cloning Kit (Vazyme) . For the generation of CRISPR-Cas9 knockout lines, specific targeting sequences of GLO1 and GLO4 (Additional file 8) were synthesized and cloned into the pYLCRISPR/Cas9Pubi vector . The constructed vectors were transformed into rice callus using Agrobacterium-mediated infection (strain EHA105). The T1 seeds from the positive T0 lines were germinated and then transplanted into soil to grow until the T2 seeds were harvested. After that, the T2 homozygous plants that originated from two independent lines were used for subsequent treatments and analyses.
Enzyme assays and protein measurement
GLO activity assay
GLO activity was detected in an enzyme coupled assay . Briefly, 0.1 g leaves were detached from the youngest fully expanded leaves (five-leaf stage) and homogenized in 1 mL of 50 mM PBS (pH 7.4) at 4 °C, the homogenates were centrifuged at 12 000 g for 20 min at 4 °C and the supernatants were used as enzyme extracts. The 1 mL reaction mixture containing 50 mM PBS (pH 7.8), 1 mM 4-amino-antipyrine, 0.1 mM FMN, 2 mM phenol, 5 units of horseradish peroxidase, and 5 mM glycolate. The reaction was started by adding enzyme and the absorbance at 520 nm was recorded at intervals of 5 s for 1 min. The protein content was determined using Coomassie brilliant blue G250.
TSB activity assay
TSB activity was measured according to Last et al. . All steps were performed at 4 °C, unless indicated otherwise. Plant extracts were prepared by grinding 1 g of leaves from five-leaf stage rice seedlings into a paste with a prechilled mortar and pestle containing 1.5 mL of 0.1 M K-phosphate (pH 8.2), 0.3 g quartz sand, and 0.1 g polyvinylpolypyrrolidone. Homogenates were then centrifuged twice at 12,000 g for 15 min and the supernatants were used as the enzyme extracts. The 1 mL reaction solution was prepared containing 60 mM L-serine, 0.2 mM indole, 80 mM potassium phosphate (pH8.2), 10 μg pyridoxal phosphate, and 0.4 mL plant extract with gentle agitation at 30 °C. The reaction was stopped by the addition of 0.1 mL of 0.2 M sodium hydroxide after 90 min. The residual indole was extracted into 4 mL of toluene by gentle vortexing (vigorous agitation created a permanent emulsion). After centrifugation for 15 min at 1500 g, 0.5 mL of the toluene layer was added to 2 mL ethanol and 1 mL Ehrlich’s Reagent (Sigma). The color could develop for 30 min at room temperature and the absorbance of the product was measured spectrophotometrically at 540 nm.
SGAT and GGAT activities assay
SGAT and GGAT activities were measured by detecting the reduction of glyoxylate according to Yu et al.  with some modifications. Briefly, 50 mg of leaves (five-leaf stage) were homogenized in 1 mL 50 mM K-phosphate (pH 7.4) at 4℃, and the homogenate was then centrifuged at 12 000 g and 4℃ for 15 min. The supernatant was used as enzyme extract. The 0.5 mL enzymatic reaction mixture containing 50 mM K-phosphate (pH7.4), 50 mM glyoxylate, 5.5 mM pyridoxal-5-phosphate and appropriate enzyme extract. The enzymatic reaction was started by adding 20 mM L-serine for SGAT or 20 mM L-glutamate for GGAT at 30℃ for 20 min. This enzymatic reaction was terminated by adding 0.1 mL 2 M HCl and neutralized with 0.1 mL 2 M NaOH. After that, 0.1 mL 0.33% phenylhydrazine hydrochloride was added to the mixture and incubated at 30℃ for 15 min. Finally, 0.5 mL HCl and 0.1 mL 1.65% potassium ferricyanide was added for color reaction, the red color produced was measured at 520 nm.
CAT activity assay
The CAT activity was detected using a UV spectrophotometer in a reaction mixture containing 50 mM PBS (pH 7.4), 25 mM H2O2 at 30℃. The consumption of H2O2 was measured at 240 nm and the CAT activity was calculated using the extinction coefficient for H2O2 of 43.6 M−1 cm−1 .
3, 3′-diaminobenzidine (DAB) staining for H2O2 detection
The leaf H2O2 abundance was determined in situ by DAB uptake methods . The youngest fully expanded leaves (10 cm, at the five-leaf stage) were detached, and the cut end was dipped into DAB solution (1 mg ml−1, pH 3.8) for 2 h in a growth chamber (light intensity 700 μmol m−2 s−1, temperature 25 °C, and relative humidity 60%). After that, the leaves were de-stained twice with ethanol and photographed.
Assay of H2O2 content
Endogenous H2O2 content in rice leaves (at five-leaf stage) were measured using an Amplex Red H2O2/peroxidase assay kit (Invitrogen, USA) . Briefly, 0.1 g leaves were detached and immediately ground in liquid nitrogen, and then the powder was extracted in 1 ml PBS (50 mM, pH 7.4) and centrifuged at 12 000 g for 15 min at 4℃. The supernatant was used to determine H2O2 levels.
Quantification of IAA
For IAA quantification, 1 g of leave material was detached from the youngest fully expanded leaves (five-leaf stage) and immediately frozen in liquid nitrogen. The extraction and quantification of endogenous IAA were conducted according to the manufacturer’s instructions (Wuhan Metware Biotechnology Co., Ltd., Wuhan, China) . Briefly, 50 mg of frozen leaf material was ground into a powder and extracted with methanol/water/formic acid (15:4:1, V/V/V). The combined extracts were evaporated to dryness under a stream of nitrogen gas, were reconstituted in 80% methanol (V/V), and filtered (PTFE, 0.22 μm, Anpel) before LC–MS/MS analysis. The quantification of IAA was conducted using an ultra-performance liquid chromatography-tandem mass spectrometry (LC–MS/MS) system (UPLC, Shim-pack UFLC SHIMADZU CBM30A system, Kyoto, Japan; MS, Applied Biosystems, Foster City, CA). The content of IAA was determined using an external standard method and was expressed as ng/g fresh weight (FW). Three biological replications were performed per sample.
Determination of Trp content
Measurement of Trp content was based on a method reported previously with some modifications . The youngest fully expanded leaves were detached and frozen in liquid nitrogen immediately for subsequent analysis. Harvested leaves were sampled at 0.5 g each and homogenized in 3 mL of 2% (w/v) sulphosalicylic acid. After incubating at 25 °C for 2 h, the homogenates were centrifuged at 12 000 g for 20 min, and then the supernatants were filtered through a 0.22 μm nylon membrane. Trp contents in the filtrates were determined with a high-speed automatic amino acid analyzer (Hitachi 835–50, Tokyo, Japan).
Real-time quantitative PCR (qRT-PCR) analysis
The total RNA and cDNA were prepared as described above. To quantify the expression levels of genes related to peroxisomal H2O2 production and IAA metabolism in leaves of various GLO transgenic rice lines and WT, qRT-PCR analysis was performed on a Bio-Rad CFX96 apparatus with SYBR Green I dye (Vazyme). PCR was carried out in 96-well plates using the following program: denaturation for 5 min at 95 °C, followed by 40 cycles of denaturation for 10 s at 95 °C and incorporative annealing and extension for 30 s at 60 °C. The primers used for qRT-PCR were designed on a dedicated website (https://biodb.swu.edu.cn/qprimerdb/?tds-ourcetag=s_pcqq_aiomsg). The data were normalized to the amplification of the OsActin1 gene (Os03g0718100). All experiments were performed with three biological and three technical replicates per biological replicate. The primer sequences used in this paper are presented in Supporting Information (Additional files 9, 10).
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information files; the datasets and accession numbers used during the current study (Os03g0786100, Os04g0623500, Os07g0152900, Os07g0616500, Os01g0108600, Os09g0385700, Os01g0732700, Os07g0437000, Os03g0790700, Os04g0118100, Os07g0675133, Os09g0544900, Os09g0133200, Os04g0653600, Os03g0313100, Os01g0783700, Os09g0527700, Os11g0490600, Os10g0147400, Os11g0169200, Os03g0718100) are available in the Rice Annotation Project (RAP) repository (https://rapdb.dna.affrc.go.jp/index.html).
Tryptophan synthetase β
Real-time quantitative PCR
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The authors would like to thank Prof. Yao-Guang Liu (College of Life Sciences, South China Agricultural University) for providing the pYLox.5 and pYLCRISPR/Cas9 vectors.
This work was supported by the Major Program of Guangdong Basic and Applied Research (2019B030302006), the National Natural Science Foundation of China (32070265, 31770256), and the Natural Science Foundation of Guangdong Province (2019A1515011438). The funding bodies had no role in the design of this study and collection, analysis, and interpretation of data and in writing the manuscript.
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Li, X., Liao, M., Huang, J. et al. Glycolate oxidase-dependent H2O2 production regulates IAA biosynthesis in rice. BMC Plant Biol 21, 326 (2021). https://doi.org/10.1186/s12870-021-03112-4
- Glycolate oxidase