Phenylalanine Suppresses Cell Death Caused by Loss of Fumarylacetoacetate Hydrolase in Arabidopsis

doi:10.21203/rs.3.rs-1221177/v1

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Phenylalanine Suppresses Cell Death Caused by Loss of Fumarylacetoacetate Hydrolase in Arabidopsis

https://doi.org/10.21203/rs.3.rs-1221177/v1

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Fumarylacetoacetate hydrolase (FAH) catalyzes the final step of Tyrosine (Tyr) degradation pathway essential to animals and the deficiency of FAH causes an inborn lethal disease. In plants, a role of this pathway was unknown until we found that mutation of Short-day Sensitive Cell Death1 (SSCD1), encoding Arabidopsis FAH, results in cell death under short day. Phenylalanine (Phe) could be converted to Tyr and then degraded in both animals and plants. Phe ingestion in animals worsens the disease caused by FAH defect. However, in this study we found that Phe represses cell death caused by FAH defect in plants. Phe treatment promoted chlorophyll biosynthesis and suppressed the up‑regulation of reactive oxygen species marker genes in the sscd1 mutant. Furthermore, the repression of sscd1 cell death by Phe could be reduced by α-aminooxi-β-phenylpropionic acid but increased by methyl jasmonate, which inhibits or activates Phe ammonia-lyase catalyzing the first step of phenylpropanoid pathway, respectively. In addition, we found that jasmonate signaling up‑regulates Phe ammonia-lyase 1 and mediates the methyl jasmonate enhanced repression of sscd1 cell death by Phe. These results uncovered the relation between chlorophyll biosynthesis, phenylpropanoid pathway and jasmonate signaling in regulating the cell death resulting from loss of FAH in plants.

Plant Physiology and Morphology

Plant Molecular Biology and Genetics

Tyrosine (Tyr)

Fumarylacetoacetate hydrolase (FAH)

Phenylalanine

cell death

Arabidopsis

Tyrosine (Tyr) degradation pathway includes the five-step enzymatic reactions, in which Tyr is first converted to 4-hydroxyphenylpyruvate by Tyr aminotransferase, then transformed into homogentisate by 4-hydroxyphenylpyruvate dioxygenase, next, homogentisate is catalyzed by homogentisate dioxygenase (HGO) to form maleylacetoacetate (MAA) that is isomerized by MAA isomerase (MAAI) to fumarylacetoacetate (FAA) and last hydrolyzed by fumarylacetoacetate hydrolase (FAH) to fumarate and acetoacetate^1,2. Tyr degradation is essential to animals, blockage of this pathway results in metabolic disorder diseases, in which the most severe disorder in humans is hereditary tyrosinemia type I (HT1), an inborn lethal disease caused by deficiency of FAH^3,4. Loss of FAH in HT1 patients results in the accumulation of Tyr degradation intermediates including FAA and MAA, and then both would undergo spontaneous reduction to succinylacetoacetate followed by spontaneous nonenzymatic decarboxylation to succinylacetone (SUAC), which is toxic to cells and tissues³. Although the homologous genes putatively encoding HGO, MAAI, and FAH were demonstrated to exist in plants^2,5, a role of Tyr degradation pathway in plants had been unclear until we cloned the short-day sensitive cell death 1 (SSCD1) gene encoding an Arabidopsis putative FAH⁶. Loss of FAH in the sscd1 mutant leads to spontaneous cell death under short-day conditions (SD)⁶ and the accumulation of SUAC⁷. SUAC inhibits an activity of δ-aminolevulinic acid dehydratase (ALAD) involved in Chlorophyll (Chl) biosynthesis, resulting in high production of protochlorophyllide (Pchlide), an intermediate of Chl biosynthesis, in the dark under SD and excessive accumulation of Pchlide induces the production of reactive oxygen species (ROS) upon light irradiation and thereby causes cell death⁸.

Phenylalanine (Phe) is catalyzed by Phe hydroxylase to form Tyr and then degraded in animals, and dietary restriction of Tyr and Phe can improve the condition of HT1 patients^4,9. In plants, Phe could be also converted into Tyr and then via homogentisate, to plastoquinones and tocopherols or to degradation of the aromatic ring¹⁰, however, it can carry through phenylpropanoid metabolism to produce secondary metabolites¹⁰. Phenylpropanoids are precursors to flavonoids, isoflavonoids, cumarins, and stilbenes, which have important functions in plant defense against pathogens and other predators, as UV light protectants, and as regulatory molecules in signal transduction and communication with other organisms¹¹. In phenylpropanoid pathway, the first step is that Phe ammonia-lyase (PAL) catalyzes the deamination of Phe to give cinnamic acid^12,13. The α-aminooxi-β-phenylpropionic acid (AOPP) is an inhibitor of PAL, treatment with AOPP could reduce the activity of PAL^14–16. Jasmonates (JAs) including jasmonic acid, methyl jasmonate (MeJA), and other derivatives, are a basic class of plant hormones involved in plant growth, development, and responses to biotic and abiotic stresses^17–20. Treatment with MeJA could increase the activity of PAL in plants such as Chinese bayberry²¹, wheat²² and tuberose¹⁶. The PAL gene expression is responsive to a variety of environmental stimuli, including pathogen infection, wounding, nutrient depletion, UV irradiation, extreme temperatures, and other stress conditions^23–26.

To investigate whether the uptake of Phe in plants increases the cell death caused by loss of FAH like as that in animals, in this study, the sscd1 mutant was treated with Phe and it was found that the death of sscd1 seedlings was not increased but suppressed by Phe treatment. With further investigation, we found that Phe treatment promoted Chl biosynthesis and repressed the upregulation of ROS marker genes in the sscd1 mutant. Furthermore, AOPP treatment reduced the repression of sscd1 cell death by Phe. In addition, MeJA treatment enhanced the repression of sscd1 cell death by Phe as well as upregulated the PAL1 gene, both of which are mediated by JA signaling. Our work suggested that the effects of Phe on the cell death resulting from loss of FAH are different in plants and animals and uncovered the relation between Chl biosynthesis, phenylpropanoid pathway and JA signaling in regulating the cell death resulting from loss of FAH, which will help to further investigate the regulation of Tyr degradation, phenylpropanoid biosynthesis and the cell death in plants.

Phe treatment suppresses the death of sscd1 seedlings

Since Phe could be converted into Tyr and then degraded in plants¹⁰, we wondered whether Phe treatment promotes the death of sscd1 seedlings. To this end, the seeds of wild type and the sscd1 mutant were plated on MS medium without or with 0.1, 0.5, 1 and 2 mM Phe and grown under SD. Unexpectedly, the death of sscd1 seedlings treated with Phe was not increased, on the contrary, it was reduced (Fig. 1). When medium was supplemented with 0.1mM Phe, the death rate of sscd1 seedlings was slightly reduced compared to that without Phe, however, it was significantly reduced as concentrations of Phe was increased (Fig. 1a). For example, on the 7th day, more than 80% of sscd1 seedlings in medium without Phe were dead, however, when medium was supplemented with 0.5, 1 and 2 mM Phe, the rates of death seedlings were only about 70%, 30%, and 5%, respectively (Fig. 1a). The phenotype of seedlings shown in Fig. 1b clearly displayed that Phe treatment reduced the death of sscd1 seedlings. Similarly, when sscd1 seedlings grown in medium with Phe under LD were transferred to SD, the extent of cell death was obviously attenuated (Fig. 1c). All these results indicated that Phe treatment suppresses the death of sscd1 seedlings.

The upregulation of ROS marker genes in sscd1 could be repressed by Phe treatment

Previously, we have speculated that the production of ROS resulting from excessive accumulation of Pchlide causes the sscd1 cell death⁸. Because ROS marker genes such as ascorbate peroxidase 2 (APX2)²⁷, oxidative signal inducible 1 (OXI1)^28,29, bonzai1-associated protein 1 (BAP1) and transcription factor (ZP)³⁰ were up-regulated before an occurrence of cell death in the sscd1 mutant^8,31, we next investigated whether the repression of sscd1 cell death by Phe treatment is correlated with expression levels of these genes. The cell death of sscd1 seedlings which were transferred from LD to SD occurred on the 4th day⁶, so, the seedling transferred from LD to SD for 3 days growth was collected and expression levels of genes were assessed by an analysis of real-time quantitative PCR (RT-qPCR). As shown in Fig. 2, the expression levels of APX2, OXI1, BAP1 and ZP in the sscd1 mutant was significantly increased compared to that in wild type, however, they were clearly reduced after Phe treatment. Therefore, the up-regulation of ROS marker genes in sscd1 could be repressed by Phe treatment. Since the expression levels of ROS marker genes is positively correlated to the content of ROS^27,29,30, the repression of up-regulation of ROS marker genes in sscd1 by Phe treatment (Fig. 2) indicated less production of ROS after Phe treatment.

Chl biosynthesis could be promoted by Phe treatment

Since the sscd1 cell death is mediated by Chl biosynthetic pathway⁸, we next investigated whether treatment of Phe influences Chl biosynthesis. We first determined the content of Chl and found that it was increased after Phe treatment (Fig. 3a). Since HEMA1 and CHLH are two pivotal genes regulating Chl biosynthesis³², we next tested the expression of these genes by RT-qPCR. As we expected, the expression levels of HEMA1 and CHLH in both wild type and sscd1 were also increased after Phe treatment (Fig. 3b, c). Therefore, Phe treatment promotes Chl biosynthesis.

Repression of the sscd1 cell death by Phe could be reduced by AOPP and enhanced by MeJA

Phe is a precursor of phenylpropanoid biosynthesis and PAL catalyzes the first step of this pathway³³. If the repression of sscd1 cell death by Phe is related to phenylpropanoid biosynthesis, it would be changed by an inhibition or activation of PAL. To conform that, firstly, the sscd1 seedlings were treated with AOPP, a potent inhibitor of PAL¹⁵, on the basis of Phe treatment. As we expected, after treated with AOPP the death seedlings were clearly increased (Fig. 4a). For example, the death rate of 7-d-old sscd1 seedlings treated with 1 mM Phe was approximately 30%, however, when seedlings were treated with 1 mM Phe and 100 µM AOPP, it was increased to approximately 72% (Fig. 4b). These results indicated that the inhibition of PAL activity by AOPP could reduce repression of the sscd1 cell death by Phe.

Since the activity of PAL could be activated by MeJA¹⁶, we next investigated whether treatment with MeJA enhances the repression of sscd1 cell death by Phe. As shown in Fig. 5a, in the absence of Phe, treatment with 5 µM MeJA did not distinctly affect the phenotype of both wild type and sscd1 seedlings, however, when treated with 5 µM MeJA and 0.5 mM Phe the green seedlings of sscd1 obviously increased compared to those only treated with 0.5 mM Phe. The death rate of 7-d-old sscd1 seedlings treated with 0.5 mM Phe was approximately 64% whereas it was less than 40% once treated with 5 µM MeJA and 0.5 mM Phe (Fig. 5b). Therefore, the activation of PAL activity by MeJA could enhance repression of the sscd1 cell death by Phe.

Treatment with MeJA causes the COI1dependent upregulation of PAL1

In Arabidopsis, PAL is encoded by a small gene family including PAL1, PAL2, PAL3, and PAL4^26,34. We next investigated whether treatment with MeJA affects some or all of these genes’ expression, and if do, is it dependent on COI1¹⁷, a JA receptor of JA signaling³⁵? To this end, the seedlings of wild type and the coi1-2 mutant were treated with MeJA and the expression levels of PAL1, PAL2, PAL3, and PAL4 were assessed by an analysis of RT-qPCR. As shown in Fig. 6, after treatment of MeJA, the expression level of PAL1 was significantly increased in wild type but not in coi1-2. However, the expression levels of PAL2, PAL3, and PAL4 were not significantly altered in both wild type and coi1-2 after treated with MeJA (Fig. 6). These results suggested that treatment with MeJA upregulates PAL1, and this up-regulation is dependent on COI1.

Mutation of COI1 could eliminate MeJA-increased Phe repression of sscd1 cell death

Since treatment with MeJA enhanced the repression of sscd1 cell death by Phe (Fig. 5) as well as up-regulated PAL1 in dependence of COI1 (Fig. 6), we next investigated whether mutation of COI1 alters the effect of MeJA on Phe in inhibiting the sscd1 cell death. The seeds of the sscd1 single mutant and the sscd1coi1 double mutant were plated on medium added without or with 0.5 mM Phe or/and 5 µM MeJA and grown under SD, and then the death seedlings were counted. As shown in Fig. 7a, the death rate of sscd1coi1 seedlings was lower than that of sscd1, which is consistent with our previous study³¹. Phe treatment also significantly reduced the death seedlings of sscd1coi1 (Fig. 7a, right). However, treated with MeJA and Phe, not as in sscd1 (Fig. 7a, left), the death seedlings of sscd1coi1 were not significantly reduced compared to that treated with Phe (Fig. 7a, right). The phenotype of sscd1 and sscd1coi1 seedlings in Fig. 7b clearly displayed that MeJA treatment enhances the repression of seedlings death by Phe in sscd1 but not in sscd1coi1 (Fig. 7b, right). Therefore, mutation of COI1 could eliminate the MeJA-increased Phe repression of sscd1 cell death.

FAH catalyzes the final step of Tyr degradation pathway and the deficiency of FAH in animals causes an inborn lethal disease, which was named as HT1 in humans^1,3. Phe could be converted to Tyr and then degraded in animals, the dietary restriction of Tyr as well as Phe can improve the condition of HT1 patients^4,9. In plants, the SSCD1 gene encodes the Arabidopsis FAH, mutation of SSCD1 results in spontaneous cell death under SD⁶. Like as in animals, Phe could be also converted into Tyr in plants and then degraded¹⁰. However, in our study the death of sscd1 seedlings was not increased but repressed by Phe treatment (Fig. 1). So, why would Phe treatment repress the cell death resulting from loss of FAH in plants?

Previously, we demonstrated that the sscd1 cell death is mediated by Chl biosynthesis⁸. The inhibition of the ALAD activity by SUAC in the sscd1 mutant influences Chl biosynthesis resulting in impairment of feedback inhibition of Chl biosynthesis from the light–dark transition under SD, which activates Chl biosynthesis and accumulation of Pchlide in the dark, and then upon re-illumination the excessive accumulation of Pchlide induces the mass production of ROS and thereby causes cell death⁸. Thus, it is the excess ROS that induces the sscd1 cell death. In this study, treatment of sscd1 seedlings with Phe distinctly repressed the up-regulation of ROS marker genes including APX2, OXI1, ZP and BAP1 (Fig. 2), which indicated that ROS is reduced by Phe treatment.

In Chl biosynthetic pathway there are two pivotal control points mainly regulated by HEMA1 and CHLH respectively^32,36−38. In our study, after treatment with Phe, both the content of Chl and the expression levels of HEMA1 and CHLH were increased (Fig. 3), suggesting that Phe treatment could promote Chl biosynthesis. The increase in Chl biosynthesis would restore to some extent the feedback inhibition of Chl biosynthesis in the sscd1 mutant from the light–dark transition, as a result, reducing the accumulation of Pchlide in the dark and the production of ROS after subsequent exposure to light.

In plants, Phe could be metabolized through the phenylpropanoid pathway to produce secondary metabolites, which plays an important role in plant against stress including UV-light, drought and pathogen attack^{10,11,26,39−41}. PAL catalyzes the first step of the phenylpropanoid pathway, which is a key step in phenylpropanoid biosynthesis^13,34,42. The activity of PAL could be inhibited by AOPP and promoted by MeJA¹⁶. Treatment with AOPP prevents the increase in resistance to B. cinerea due to the application of external Phe⁴¹. In our study, the repression of sscd1 seedlings death by Phe was reduced by AOPP treatment (Fig. 4), however, it was enhanced by MeJA (Fig. 5), which suggested that the suppression of sscd1 cell death by Phe is related to the phenylpropanoid pathway. Since the secondary metabolites produced by Phe metabolism through the phenylpropanoid pathway have antioxidant function⁴¹, ROS could be also reduced by the metabolism of Phe through the phenylpropanoid pathway, which should be another important cause for the repression of sscd1 cell death by Phe.

Recently, we found that JA signaling is involved in the sscd1 cell death³¹. In the sscd1 mutant, the accumulation of SUAC results in the generation of ROS, which induces cell death as well as JA synthesis³¹. JA up-regulates Tyr degradation pathway, producing more SUAC, which promotes cell death³¹. Once JA signaling is broken by mutation of COI1 encoding a JA receptor³⁵, the up-regulation of Tyr degradation pathway by JA is eliminated, reducing the production of SUAC, as a result, the sscd1 cell death is repressed³¹. In this study, MeJA treatment markedly increased the expression level of PAL1 in wild type but not in the coi1-2 mutant (Fig. 6), indicating that JA signaling up-regulates the phenylpropanoid pathway. The repression of sscd1 cell death by Phe could be enhanced by MeJA treatment in the sscd1 single mutant (Figs. 5 and 7) but not in the sscd1coi1 double mutant (Fig. 7), which suggested that MeJA treatment enhances Phe inhibition of the sscd1 cell death through JA signaling. Therefore, JA has a dual regulatory effect on the sscd1 cell death. On the one hand, JA up-regulates the Tyr degradation pathway, promoting the sscd1 cell death, on the other hand, JA up-regulates the phenylpropanoid pathway, inhibiting the sscd1 cell death. For this reason, the death of sscd1 seedlings was not increased by MeJA treatment, it seemed to decrease slightly (Figs. 5 and 7), which suggested that the effect of MeJA treatment on the sscd1 cell death through phenylpropanoid pathway might be greater than that through Tyr degradation pathway.

In conclusion, although Phe can be degraded through Tyr degradation pathway, unlike in animals, Phe treatment does not increase the cell death resulting from loss of FAH in plants, instead, it represses the cell death. A possible mechanism for the repression of sscd1 cell death by Phe treatment can be described as follows (Fig. 8). Loss of FAH in the sscd1 mutant results in a decline of Chl biosynthesis, which impairs the feedback inhibition of Chl biosynthesis from light–dark transition under SD, leading to the accumulation of ROS and then cell death. Phe treatment, on the one hand, promotes Chl biosynthesis, increasing the feedback inhibition of Chl biosynthesis from light–dark transition under SD, and on the other hand, activates phenylpropanoid pathway, both of which reduce ROS and subsequent cell death. In addition, in the sscd1 mutant ROS induces cell death as well as JA synthesis. JA signaling up-regulates Tyr degradation pathway, promoting the sscd1 cell death, however, it also up-regulates PAL1 that activates phenylpropanoid pathway, repressing the sscd1 cell death. Since the effect of MeJA treatment on the sscd1 cell death through phenylpropanoid pathway might be greater than that through Tyr degradation pathway, the repression of sscd1 cell death by Phe could be enhanced by MeJA treatment.

Plant Materials and Growth Conditions

Arabidopsis thaliana L. ecotype Columbia-0 (Col-0) was obtained from the Arabidopsis Biological Resource Center (ABRC; Ohio State University, Columbus, OH, USA) and the mutants used in this study are in Col-0 background. The sscd1 mutant⁶ was isolated by Han et al (2013) in our laboratory. The coi1-2 mutant⁴³ was kindly provided by Professor Xie (Tsinghua University, Beijing, China) and the sscd1coi1 double mutant³¹ was generated through a cross of sscd1 with coi1-2 by Zhou et al (2020) in our laboratory. Experimental research on plants including the collection of plant material was performed in accordance with relevant institutional, national, and international guidelines and legislation.

Seeds were surface sterilized with 20% (v/v) chlorine bleach containing 0.1% (v/v) Triton X-100 for 10 min and washed three to five times with sterile water, then plated on Murashige & Skoog medium supplemented with 1% (m/v) sucrose and 0.7% (w/v) agar (pH 5.8) (MS). The different concentrations of Phe (SIGMA) were added in MS medium. Plates were chilled at 4 °C in darkness for 3 days and then transferred to a growth chamber with LD (16-h light/8-h dark) or SD (8-h light/16-h dark) under 150 μmol photons m⁻² s⁻¹, controlled temperature (22±2 °C).

For RT-qPCR analysis (Figs. 2, 3b) and determination of Chl content (Fig. 3a), One-week-old seedlings growing under LD were transplanted onto MS added without or with Phe and grown under LD for an additional 1 week’s growth, and then transferred to SD for three days growth. The seedlings were harvested at the end of the third day's light for the determination of Chl content or harvested at 2 hours light after three days for RT-qPCR analysis.

Determination of the dead seedlings

A dead seedling is one for which all leaves were completely bleached. The rate of seedling death was calculated as the percentage of dead seedlings. The total number of seedlings counted was approximately 100, and the experiment was performed in three independent biological repeats.

RT-qPCR analysis

Total RNA was isolated using TRIZOL reagent (Life Technologies, https://www.thermofisher.com/us/en/home/brands/life-technologies.html). After incubation with DNase I (RNase Free, Thermo Fisher Scientific, https://www.thermofisher.com/) at 37°C for 30 min and then at 65°C for 10 min to remove genomic DNA, the RNA concentration and purity were measured spectrophotometrically using OD260/OD280 and OD260/OD230 ratios (ND-1000, NanoDrop, THERMO FISHER SCIENTIFIC). Complementary DNA was synthesized from the mixture of oligo-dT primers and random primers using a ReverTraAce qPCR RT kit (perfect real time) according to the manufacturer’s instructions (Toyobo, http://www.toyobo-global.com/).

RT-qPCR was performed in 96-well blocks using a SYBR qPCR mix (Roche, https://lifescience.roche.com/) with a Bio-Rad CFX Connect^TMReal-Time PCR detection system (http://www.biorad.com/) following the manufacturer’s instructions. The RT-qPCR amplifications were performed under the following conditions: initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. The primers of genes tested by RT-qPCR are listed in Table 1, and ACTIN2 was used as an internal control. The gene expression for each sample was calculated on three analytical replicates, and the relative expression was quantified using the 2^-△△Ct method. The experiment was performed in three independent biological repeats. The significance of differences between datasets was evaluated using the two-tailed Student’s t-test.

Determination of Chl content

The content of Chl was determined referring to the method described by Lichtenthaler⁴⁴ (1987). Weighed segments of frozen crushed material (about 0.04 g) were homogenized in 1 mL 80% acetone and stood for 5 to 6 hours, then centrifuged for 10 min at 5000 rpm at 4°C and assayed spectrophotometrically at 663 nm and 645 nm. Result calculation: C_(mg/g)= (17.32 A₆₆₃+7.18 A₆₄₅)/m_(g). The experiment was performed in three independent biological repeats.

AOPP treatment

Seeds were germinated on MS added with 1 mM Phe (SIGMA) and grown under SD. On the third day seedlings were sprayed with 100 μM AOPP (Wako) or ddH₂O (as a control) once a day for 5 days, then the rate of death seedlings was counted, and the seedlings were photographed. The experiment was performed in three independent biological repeats.

MeJA treatment

For determination of dead seedlings (Figs. 5 and 7), seeds were germinated on MS added without or with 0.5 mM Phe and/or 5 μM MeJA (SIGMA) and grown under SD for 6-8 days. For RT-qPCR analysis (Fig. 6), about two-week-old seedlings growing under LD were transferred to SD and on the fourth day the seedlings were sprayed with 100 μM MeJA or ddH₂O (as a control). After MeJA treatment for one day the seedlings were harvested and used for RT-qPCR analysis. The experiment was performed in three independent biological repeats.

Acknowledgements

This work was supported by grants from the Program for Key Basic Research of the Ministry of Science and Technology of China (2014CB160308) and the National Science Foundation of China (30671121 and 31571802).

Author contribution statement

C.R. conceived, designed and supervised the experiments; Y.J. and T.Z. performed the experiments; Q.Z. and H.Y. provided technical assistance to Y.J.; Y.J., Q.Z. and C.R. analyzed the data; Y.J. wrote the article with contributions from all authors; and C.R. supervised, modified and complemented the writing.

Competing interests

The authors declare no competing interests.

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Table 1 Primers of genes tested by real-time quantitative PCRs

Gene	Forward primer	Reverse primer
APX2 (AT3G09640)	5'-ACAAAGTTGAGCCACCTCCT-3'	5'-AAGGTGTGTCCACCAGACAA-3'
OXI1 (AT3G25250)	5'-GTTGAGGAAATCAAGGGTCATG-3'	5'-TGGACGATATTCTCCACATCC-3'
ZP (AT5G04340)	5'-TACGAAGGAAAGAACGGAGGC-3'	5'-GGTATCGGCGGTATGTTGAGG-3'
CHLH (AT5G13630)	5'-CAGCCAACATCAGTCTTGCT-3'	5'-ACCTGCTTCTTCTCAGCCAT-3'
BAP1 (AT3G61190)	5'-ATCGGATCCCACCAGAGATTACGG-3'	5'-AATCTCGGCCTCCACAAACCAG-3'
HEMA1 (AT1G58290)	5'-GTTGCTGCCAACAAAGAAGA-3'	5'-AATCCCTCCATGCTTCAAAC-3'
PAL1 (AT2G37040)	5'-TTTTGGTGCTACTTCTCATCG-3'	5'-CTTGTTTCTTTCGTGCTTCC-3'
PAL2 (AT2G37040)	5'-GTGCTACTTCTCACCGGAGA-3'	5'-TATTCCGGCGTTCAAAAATC-3'
PAL3 (AT5G04230)	5'-CAACCAAACGCAACAGCA-3'	5'-CTCCAGGTGGCTCCCTTTTA-3'
PAL4 (AT3G10340)	5′-GGTGCACTTCAAAATGAGCT-3′	5′-CAACGTGTGTGACGTGTCC-3'
ACTIN2 (AT3G18780)	5′-AGCACTTGCACCAAGCAGCATG-3′	5′-ACGATTCCTGGACCTGCCTCATC-3′

No competing interests reported.

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Phenylalanine Suppresses Cell Death Caused by Loss of Fumarylacetoacetate Hydrolase in Arabidopsis

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