A genetically modified plant, plant part, or plant cell reduced for dopamine production without an accumulation of tyramine

Abstract

The present invention provides for a genetically modified plant, plant part, or plant cell comprising inhibition or reduced expression or knockouts of (a) one or more endogenous polyphenol oxidases (PPOs), and / or (b) one or more endogenous tyrosine decarboxylases (TyDCs). The genetically modified plant, plant part, or plant cell have a reduction of dopamine production without or essentially without an accumulation of tyramine.

Description

Attorney Docket: 2024-147-02 Lawrence Berkeley National LaboratoryA genetically modified plant, plant part, or plant cell reduced for dopamine production without an accumulation of tyramineInventors: Yezhang Ding, Trent R. Northen, Yi Zhai, John P. Vogel, Suzanne M. KosinaCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Ser. No. 63 / 744,089, filed January 10, 2025, both of which are hereby incorporated by reference.STATEMENT OF GO VERNMENTAL SUPPORT

[0002] The invention was made with government support under Contract Nos. DE-AC02-05CH11231 awarded by the U.S. Department of Energy. The government has certain rights in the invention.REFERENCE TO SEQUENCE. LISTING10003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on , 2026, is named “> ” and is > bytes in size.FIELD OF THE INVENTION

[0004] The present invention is in the field of production of dopamine in a plant.BACKGROUND OF THE INVENTION

[0005] Food browning causes significant losses for agricultural / food industries, especially in fruits such as bananas and apples where losses are estimated at 50%. Additionally, browning type reactions in food products such as wines and juices reduce shelflife and / or palatability. Current approaches to reduce browning in foods and food products focus on inhibition of enzymes, e g. addition of acids, sulfur dioxide, or enzymatic inhibitors.

[0006] Dopamine is a crucial neurotransmitter in humans, often termed the "feel-good" hormone due to its role in generating sensations of pleasure, satisfaction, and motivation (Mirenowicz and Schultz, 2022). It can spontaneously form neuromelanin through melanogenesis, which is a brown-to-black polymer in the brain (Hedges et al., 2020; KraincAttorney Docket: 2024-147-02 Lawrence Berkeley National Laboratoryet al., 2023). Many plants are also known to produce dopamine (Liu et al., 2020; Gomes et al., 2024), which plays a significant role in promoting plant disease resistance and enhancing plant tolerance against abiotic stresses (Liu et al., 2020; Ahmad et al., 2021). Recently, it was also shown that dopamine is present in root exudates of the model grass, Brachypodium distachyon (Novak et al., 2024), and can modulate soil microbial community structure (Ding et al., 2024). Additionally, dopamine is a precursor to various pharmaceutically valuable metabolites, such as phenethylisoquinoline alkaloids, benzylisoquinoline alkaloids, catecholamines, and phenylethylamines (Xu et al. 2019).

[0007] In humans, dopamine biosynthesis involves the hydroxylation of tyrosine to form L-4,5-dihydroxyphenylalanine (L-DOPA), which is subsequently decarboxylated by DOPA decarboxylase (DODC) to produce dopamine (Bromek et al., 2010). A minor pathway also exists, where tyrosine decarboxylases (TyDCs) first decarboxylate tyrosine to produce tyramine, which is then hydroxylated by cytochrome P450 enzymes to form dopamine (Bromek et al., 2010). Similar biosynthetic pathways have been proposed to exist in plants (Facchini et al., 2000; Liu et al., 2020). However, the enzymes responsible for the 3-hydroxylation of tyramine and / or tyrosine for dopamine biosynthesis remain unclear in plants (Xu et al., 2019).

[0008] Enzymatic browning is a biological process that significantly impacts food quality, particularly fruits and vegetables, reducing shelf-life and thereby leading to food waste. This process is primarily initiated by polyphenol oxidases (PPOs), which catalyze the oxidation of phenolic compounds. The resulting products undergo further non-enzymatic reactions, leading to the polymerization of these compounds into dark pigments known as melanin, often seen on cut apples, bananas, and other produce (Sui et al., 2023; Tilley et al., 2023). Enzymatic browning in food can be mitigated using traditional methods, such as lowering pH, reducing oxygen exposure, or applying chemical inhibitors, as well as advanced genomeediting techniques to knock down or knockout PPO genes (Sui et al., 2023; Tilley et al., 2023). However, understanding of the underlying mechanisms and genes involved could inform the development of foods less prone to browning.SUMMARY OF THE INVENTION

[0009] The present invention provides for a genetically modified plant, plant part, or plant cell comprising inhibition or reduced expression or knockouts of (a) one or more endogenousAttorney Docket: 2024-147-02 Lawrence Berkeley National Laboratorypolyphenol oxidases (PPOs), and / or (b) one or more endogenous tyrosine decarboxylases (TyDCs). In some embodiments, the genetically modified plant, plant part, or plant cell have a reduction of dopamine production without or essentially without an accumulation of tyramine.

[0010] In some embodiments, the plant part is fruit which takes a longer time to brown as compared to an unmodified fruit. In some embodiments, the fruit has an improved the quality, as a food for human consumption, in terms of shelflife and consumability as compared to an unmodified fruit. In some embodiments, the genetically modified plant, plant part, or plant cell wherein expression of one or more endogenous PPO genes are inhibited , tyramine synthesis.

[0011] In some embodiments, the genetically modified plant, plant part, or plant cell comprises inhibition or reduced expression or knockouts of Bdtydcl, Bdtydc2, Bdppol, and / or Bdppo3.

[0012] In plants, dopamine biosynthesis in plants proceeds primarily via tyramine as an intermediate, where TyDCs convert tyrosine into tyramine, which is then converted into dopamine by PPOs. The dopamine biosynthetic pathway is highly conserved across a wide range of plant species. Dopamine knockout mutants, including BdtydclBdtydc2, Bdppol, and BdppolBdppo3, exhibit a lack of dark brown coloration in root tissues after senescence or damage. Transient expression of the dopamine biosynthetic pathway in a plant, such as Nicotiana benlhamiana, recapitulates the browning phenotype. Dopamine oxidation, rather than PPO activity alone, directly contributes to tissue browning in plants.

[0013] In some embodiments, the genetically modified plant, plant part, or plant cell is gene editing to reduce food browning by targeting the PPO genes.

[0014] In some embodiments, the inhibition or reduced expression is by using RNA interference (RNAi) to silence the expression of the PPO gene(s) and / or TyDC(s), or using CRISPR-Cas9 gene editing to knock out the PPO gene(s) and / or TyDC(s). One of the advantages of using CRISPR-Cas9 gene editing is the reduced concern over genetically modified foods (GMOs).

[0015] The present invention provides for a dopamine pathway mutants have less browning as a result of tissue damage. Brachypodium dopamine pathway mutants (tydc2, tydc3, tydc2Attorney Docket: 2024-147-02 Lawrence Berkeley National Laboratorytydc3, ppol, ppo3, and / or ppol ppo3) are grown on agar plates for 7 days. Root tissues are harvested and damaged with metal beads beating for 30 seconds. 70% methanol is then added into the tubes. Extracts from roots with the fully functional dopamine pathway show a brown color after damage, while extracts from roots with the dysfunctional dopamine pathway are clear white. At 24 hours, the root tissues from the plants with the fully functional dopamine pathway became dark brown.

[0016] The present invention provides for two tydc2tydc3 double mutant alleles have the same growth rates as wildtype plants.

[0017] The present invention provides for an inhibition or knockout of PPOs without or essentially without an elevated level of the pathway intermediate tyramine. Elevated levels of tyramine can have negative effects on human health.

[0018] In some embodiments, the genetically modified plant, plant part, or plant cell is an apple, grape, tomato, potato, maize, banana, avocado, opium poppy, moringa, broad bean, or common bean, or any other plant category, genus or species described herein. In some embodiments, the plant part is a stem, leaf, fruit, seed, root, or the like, or any other plant part described herein.

[0019] The present invention provides for a method for constructing a genetically modified plant, plant part, or plant cell of the present invention.

[0020] In some embodiments, the plant, plant part, or plant cell has less melanin produced thereby resulting a reduced brown color.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The foregoing aspects and others will be readily appreciated by the skilled artisan from the following description of illustrative embodiments when read in conjunction with the accompanying drawings.

[0022] Fig. 1. Genetic mapping of candidate dopamine biosynthetic genes in B. distachyon. (A), Manhattan plot of a GWAS using the dopamine-to-tyramine ratio in B. distachyon roots as the mapping trait. The y-axis shows negative loglO-transformed P- values from the general linear model. The dashed line represents the 5% Bonferroni-corrected significance threshold (4,780,737 SNPs). (B), A regional Manhattan plot representing aAttorney Docket: 2024-147-02 Lawrence Berkeley National Laboratory‘zoomed-in’ view of the signal between 51.05 Mb and 51.15 Mb on Chr.2, and each dot representing a single SNP. (C), Genes located inside the mapping interval. (D), The transcript abundance of the three putative BdPPO derived from RNA-seq analyses of 2-week-old and 4-week-old Bd21-3 roots (Root_2 and Root_4) and shoots (Shoot_2 and Shoot_4) grown in a hydroponic system. (E), Three putative BdPPO genes are co-localized with two BdTyDC genes on Chromosome 2, which are approximately 784 kb apart. (F), The transcript abundance of BdTyDC 1 and BdTyDC2 derived from RNA-seq analyses of 2-week-old and 4-week-old Bd21-3 roots (Root_2 and Root_4) and shoots (Shoot_2 and Shoot_4) grown in a hydroponic system. Gene expression is given as fragments per kilobase of transcript per million mapped reads (FPKM). Error bars represent mean ± SEM (n = 3).

[0023] Fig. 2. Characterization of BdTyDCl and BdTyDC2. (A-B) Transient expression of these genes in N. benthamiana leaves. (A), Representative LC-MS / MS chromatograms of tyramine (m / z 138.0862-138.0962, [M+H]+) in N. benthamiana leaves transiently expressing BdTyDCl, BdTyDC2, or empty vector (EV). B, Representative LC-MS / MS chromatograms of13Ce-dopamine (m / z 143.0748-143.0848, [M+H]+) from in vitro assays. Total proteins were extracted from N. benthamiana leaves transiently expressing BdTyDCl, BdTyDC2, or EV and then incubated with a [ring-13C6]-labeled L-DOPA. (C-D) Comparison of dopamine levels in B. distachyon mutant lines. (C) Representative LC-MS / MS chromatograms of dopamine (m / z 154.0810-154.0910, [M+H]+) in roots (C) and shoots (D) of wildtype (WT) and the Bdtydc mutants, Bdtydc 1, Bdtydc2, and two alleles of Bdtydc lBdtydc2. Plants were grown in a hydroponic system for 4 weeks, and roots and shoots were harvested for metabolite analysis via LC-MS / MS.

[0024] Fig. 3. Characterization of three BdPPOs in dopamine biosynthesis. A, Representative LC-MS / MS chromatograms of a dopamine standard and dopamine (m / z 154.0810-154.0910, [M+H]+) in N. benthamiana leaves transiently co-expressing BdTyDC2 with BdPPO 1, BdPPO2, or BdPPO3. B, Proposed dopamine biosynthetic pathway in B. distachyon. (C, D) Representative LC-MS / MS chromatograms of dopamine (mtz 154.0810- 154.0910, [M+H]+) in the roots (C) and shoots (D) of wildtype (WT) wABdppo mutants, including Bdppo! -2, two alleles of Bdppo3, and two alleles of BdppolBdppo3. Plants were grown in a hydroponic system for 4 weeks, and roots and shoots were harvested for metabolite analysis using LC-MS / MS.

[0025] Fig. 4. Dopamine oxidation causes tissue browning in plants. (A) Root colorationAttorney Docket: 2024-147-02 Lawrence Berkeley National Laboratoryof eight- week-old Bd21-3 and dopamine pathway mutant plants grown on i MS plates. (B) The coloration of damaged roots (after 2 hours of air exposure) from three-week-old Bd21-3 and dopamine pathway mutant plants grown on / i MS plates. (C) Coloration of root extracts (after 2 hours of air exposure) from three-week-old Bd21-3 and dopamine pathway mutant plants grown on / i MS plates. The dopamine pathway mutants include Bdtydcl, Bdtydc2, two alleles of BdtydclBdtydc2, Bdppol-2, two alleles of Bdppo3, and two alleles of BdppolBdppo3. (D) Coloration of leaf extracts (after 2 hours of air exposure) from N. benthanmiana transiently expressing BdTyDCl, BdTyDC2, BdPPOl, BdPPO3, BdTyDCl + BdPPOl, BdTyDCl + BdPPO3, BdTyDC2 + BdPPOl, and BdTyDC2 + BdPPO3. An empty vector (EV) was used for the control.

[0026] Fig. 5. Conservation of the dopamine biosynthetic pathway in plants. (A) Numbers of homologous genes in the TyDC and PPO gene families across all flowering plants with sequenced genomes available on Phytozome, as well as Papaver somniferum, Musa acuminata, and Persea americana. Species highlighted in blue possess at least one TyDC-PPO gene pair located within 10 Mb on the same chromosome. Dark green bars indicate species known to produce dopamine. (B, C) Functional characterization of selected PPOs in dopamine production. PPOs from Zea mays (Zm), Papaver somniferum (Ps), Hordeum vulgare (Hv), Oryza sativa (Os), and Sorghum bicolor (Sb) were transiently coexpressed with BdTyDC2 in N. benthamiana leaves for five days. Dopamine levels (m!z 154.0810-154.0910, [M+H]+) were analyzed using LC-MS analysis. Error bars represent mean ± SEM (n = 3).

[0027] Fig. 6. Proposed dopamine biosynthetic pathway in plants.

[0028] Fig. SI. LC-MS total ion chromatograms of B. distachyon roots and shoots.Representative LC-MS / MS total ion chromatograms (TICs) show dopamine as one of the most abundant metabolites in both roots and shoots of four-week-old hydroponically-grown B. distachyon Bd21-3 plants.

[0029] Fig. S2. Quantification of tyramine in B. distachyon roots and shoots. Tyramine levels in B. distachyon Bd21-3 roots and shoots were measured based on an external calibration curve of a tyramine standard. Plants were grown in a hydroponic system for 4 weeks, and roots and shoots were harvested for metabolite analysis using LC-MS / MS. FW, fresh weight. Error bars represent mean ± SEM (n = 3).Attorney Docket: 2024-147-02 Lawrence Berkeley National Laboratory

[0030] Fig. S3. Metabolite-based genome-wide association studies. Manhattan plots showing metabolite-based GWASs using dopamine levels in roots (A) and shoots (B) and the dopamine-to-tyramine in shoots (C) as mapping traits. Negative loglO-transformed values from the general linear model are plotted on the y-axis with the dashed line indicating the 5% Bonferroni-corrected significance threshold (4,780,737 SNPs). The most statistically significant SNPs were identified on chromosome 2. (D), A regional Manhattan plot representing a ‘zoomed-in’ view of the signal between 51.05 Mb and 51.25 Mb on Chr.2 (Bd21-3 vl.l), and each dot representing a single SNP. The mapping interval overlaps with the region identified for the dopamine-to-tyramine ratio in roots (Fig. 1). Three putative BdPPO genes (indicated by red dashed lines) are located inside the mapping interval with BdPPOl (Bdi d21-3.2G0666400) in the first peak, BdPPO3 (BdiBd21-3.200668300) in the second, asx BdPPO2 (BdiBd21-3.2G0667800) between the two peaks. These findings suggest that BdPPOl and BdPPO 3 are likely involved in shoot dopamine production.

[0031] Fig. S4. Expression of genes in the mapping interval associated with the root dopamine-to-tyramine ratio. Eight genes were identified within the mapping region containing SNPs with negative loglO-transformed -values exceeding the significance threshold. The figure displays transcript abundance of these genes based on RNA-seq analyses of roots (Root_2 and Root_4) and shoots (Shoot_2 and Shoot_4) from 2-week-old and 4-week-old Bd21-3 plants grown in a hydroponic system. Gene expression is reported in fragments per kilobase of transcript per million mapped reads (FPKM). Error bars represent the mean ± SEM (n = 3). Among three genes with expression in roots, BdiBd21-3.2G0666400 encodes a putative polyphenol oxidase, which is implicated in dopamine biosynthesis.

[0032] Fig. S5. Amino acid sequence comparison of three B. distachyon PPOs. The predicted amino acid sequences of three B. distachyon polyphenol oxidases (PPOs) were aligned using the MUSCLE (codon) algorithm in MEGA7 (webpage for: megasoftware.net). The alignment was visualized with BIOEDIT (webpage for: mbio.ncsu.edu / BioEdit).Sequence identity analysis revealed that BdPPOl shares 52.1% and 78.9% protein sequence identity with BdPPO2 and BdPPO3, respectively, while BdPPO2 and BdPPO3 exhibit 51.0% identity to each other. The amino acid sequences for BdiBd21-3.2G0666400_PP01, BdiBd21-3.2G0667800_PP02, and BdiBd21-3.2G0668300_PP03, are SEQ IDNos:l-3, respectively.Attorney Docket: 2024-147-02 Lawrence Berkeley National Laboratory

[0033] Fig. S6. CRISPR / Cas9-induced mutations in BdTyDCl and BdBdTyDC2. (A), Gene structures of BdTyDCl (BdiBd21-3.2G0653800) and BdTyDC 2 BdiBd21-3.2G0654700) showing the positions of two guide RNAs (gRNAl and gRNA2) designed to target both BdTyDC genes in Bd21-3 (vl .1). B, Mutations induced by CRISPR / Cas9 at the two BdTyDC gene loci. The gRNA target sites are underlined in black, Protospacer adjacent motifs (PAMs) are highlighted in green, and red-highlighted nucleotides indicate insertions. The nucleotide sequences depicted herein for WT Bdtydc gRNAl, Bdtydcl gRNAl, Bdtydc2 gRNAl, WT Bdtydc gRNA2, Bdtydcl gRNA2, and Bdtydc2 gRNA2 are SEQ ID Nos:4-9„ respectively.

[0034] Fig. S7. Characterization of dopamine pathway gene mutants. Dopamine m / z 154.0810-154.0910, [M+H]+) and tyramine levels m / z 138.0862-138.0962, [M+H]+) in roots (A and B) and shoots (C and D) of Bd21-3 and dopamine pathway mutants, including Bdtydcl, Bdlydc2, two alleles of Bdtydc lBdtydc2, Bdppol-2, two alleles of Bdppo3, and two alleles of BdppolBdppo3. Plants were grown in a hydroponic system for 4 weeks, and roots and shoots were harvested for untargeted metabolite analysis using LC-MS / MS. Error bars represent mean ± SEM (n = 5).

[0035] Fig. S8. L-DOPA levels in dopamine pathway mutants. Representative LC-MS / MS chromatograms of an L-DOPA standard and L-DOPA (m / z 198.0751-198.0771, [M+H]+) in the roots of various B. distachyon genotypes. Plants were grown hydroponically for 4 weeks, and root samples were collected for LC-MS / MS analysis. At the expected retention time, L-DOPA was undetectable in the root extracts of all analyzed B. distachyon genotypes, including Bdtydcl, Bdtydc2, two alleles of Bdtydc lBdtydc2, Bdppol-2, two alleles of Bdppo3, and two alleles of BdppolBdppo3.

[0036] Fig. S9. BdPPOs lack activity for the 3-hydroxylation of tyrosine in vivo.Representative LC-MS / MS chromatograms of an L-DOPA standard and L-DOPA m / z 198.0751-198.0771, [M+H]+) in N. benthamiana leaves transiently expressing B. distachyon BdPPOl, BdPPO2, BdPPO3 or empty vector (EV). L-DOPA was not detectable in the leaf extracts transiently expressing any of the individual BdPPOs. These results suggest that none of the three BdPPOs exhibit enzymatic activity for catalyzing the 3-hydroxylation of tyrosine under the tested conditions.

[0037] Fig. S10. Sodium azide-induced mutations in BdPPOl and BAPPO2. (A, B) geneAttorney Docket: 2024-147-02 Lawrence Berkeley National Laboratorystructures of BdPPOl (BdiBd21-3.2G0666400) (A) and BdPPO2 (BdiBd21-3.2G0667800) (B) with the locations of mutations indicated by red arrows. In the NaN1435, BdPPOl contains a premature stop codon (W382*), while NaN1956 has a premature stop codon (E487*) in BdPPO2. Asterisks (*) represent stop codons.

[0038] Fig. Sil. CRISPR / Cas9-induced mutations in BdPPOl and BdPPO3. (A), Schematic representation of BdPPOl (BdiBd21-3.2G0666400) and BdPPO3 (BdiBd21-3.2G0668300), with locations of two guide RNAs. gRNAl was designed to target both genes in Bd21-3, while gRNA2 was designed to only target BdPPO3. (B), Mutations induced by CRISPR / Cas9 at the two gene loci, with gRNA target sites underlined in black. PAMs are marked in green. Red-highlighted nucleotides indicate insertions, and red dashes (-) represent nucleotide deletions. The nucleotide sequences depicted herein for WT Bdppol gRNAl, Bdppol-2 gRNAl, WT Bdppo3 gRNA2, Bdppo-1 gRNA2, Bdppo3-2 gRNA2, Bdppol gRNAl, an Bdppo3 gRNAl are SEQ ID Nos: 10-16,, respectively.

[0039] Fig. S12. Dopamine and tyramine levels in Bdppol-1. Dopamine (mlz 154.0810- 154.0910, [M+H]+) and tyramine levels (mlz 138.0862-138.0962, [M+H]+) in the roots (A and C) and shoots (B and D) of Bd21-3, wildtype sibling (WT-Sib), and Bdppol-1. Plants were grown in a hydroponic system for 4 weeks, and roots and shoots were harvested for untargeted metabolite analysis using LC-MS / MS. Error bars represent mean ± SEM (n = 3). Within the plot, different letters (a-b) represent significant differences (one-way ANOVA followed by Tukey’s test corrections for multiple comparisons; P < 0.05).

[0040] Fig. S13. Dopamine levels in Bdppo2. Dopamine levels (mlz 154.0810-154.0910, [M+H]+) in the roots (A) and shoots (B) of Bd21-3, wildtype sibling (WT-Sib), an Bdppol -1. Plants were grown in a hydroponic system for 4 weeks, and roots and shoots were harvested for untargeted metabolite analysis using LC-MS / MS. Error bars represent mean ± SEM (n = 3). Statistical significance was assessed using one-way ANOVA with Tukey’s test for multiple comparisons (P < 0.05).

[0041] Fig. S14. Root and shoot biomass of dopamine pathway mutants. Plants, including Bdlydcl. Bdlydc2. two alleles of BdlydclBdlydc2. Bdppol-2, two alleles of BdppoS, and two alleles of BdppolBdppo3 were grown in a hydroponic system. Root and shoot biomass were measured at week 4. Error bars represent mean ± SEM (n = 5). Within the plot, different letters (a-c) represent significant differences (one-way ANOVA followed by Tukey’s testAttorney Docket: 2024-147-02 Lawrence Berkeley National Laboratorycorrections for multiple comparisons; P < 0.05).

[0042] Fig. S15. Senescence-induced root browning in B. distachyon. Plants, including Bd21-3 and dopamine pathway mutants (Bdtydcl, Bdlydc2, two alleles of BdtydclBdtydc2, Bdppol-2, two alleles of Bdppo3, and two alleles of BdppolBdppo3') were grown on Vi MS plates for approximately 8 weeks before images were taken.

[0043] Fig. S16. Coloration of root extracts. Root extracts from three-week-old Bd21-3 and dopamine pathway mutant plants grown on i MS plates were exposed to air for 24 hours (A) and 1 week (B). The dopamine pathway mutants used here include Bdtydcl, Bdtydc2, two alleles of BdtydclBdtydc2, Bdppol-2, two alleles of Bdppo3, and two alleles of BdppolBdppo3.

[0044] Fig. S17. Autoxidation of dopamine-related compounds. Tyramine, L-DOPA, dopamine, norepinephrine, and epinephrine were dissolved in water at a concentration of 100 pM and exposed to air for 48 hours to monitor autoxidation.

[0045] Fig. S18. Tyramine and dopamine are not translocated in plants. Representative LC-MS / MS chromatograms of (A) tyramine (m / z 138.0862-138.0962, [M+H]+) and (B) dopamine (m / z 154.0810-154.0910, [M+H]+) in the roots and shoots of the BdtydclBdtydc2-1 plants, which were hydroponically grown for 3 weeks and then treated with 50 pM tyramine for two days and four days.

[0046] Fig. S19. Dopamine and tyramine levels in TyDC and PPO mutants.DETAILED DESCRIPTION OF THE INVENTION

[0047] Before the invention is described in detail, it is to be understood that, unless otherwise indicated, this invention is not limited to particular sequences, expression vectors, enzymes, host microorganisms, or processes, as such may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting.

[0048] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

[0049] The terms "optional" or "optionally" as used herein mean that the subsequentlyAttorney Docket: 2024-147-02 Lawrence Berkeley National Laboratorydescribed feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instances where a particular feature or structure is present and instances where the feature or structure is absent, or instances where the event or circumstance occurs and instances where it does not.

[0050] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0051] The term “about” refers to a value including 10% more than the stated value and 10% less than the stated value.

[0052] As used herein, the term "promoter" refers to a polynucleotide sequence capable of driving transcription of a DNA sequence in a cell. Thus, promoters used in the polynucleotide constructs of the invention include cis- and trans-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and / or rate of transcription of a gene. For example, a promoter can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on / off, regulate, modulate, etc.) gene transcription. Promoters are located 5' to the transcribed gene, and as used herein, include the sequence 5' from the translation start codon.

[0053] A "constitutive promoter" is one that is capable of initiating transcription in nearly all cell types, whereas a "cell type-specific promoter" initiates transcription only in one or a few particular cell types or groups of cells forming a tissue. In some embodiments, the promoterAttorney Docket: 2024-147-02 Lawrence Berkeley National Laboratoryis secondary cell wall-specific and / or fiber cell-specific. A "fiber cell-specific promoter" refers to a promoter that initiates substantially higher levels of transcription in fiber cells as compared to other non-fiber cells of the plant. A "secondary cell wall-specific promoter" refers to a promoter that initiates substantially higher levels of transcription in cell types that have secondary cell walls, e.g., lignified tissues such as vessels and fibers, which may be found in wood and bark cells of a tree, as well as other parts of plants such as the leaf stalk. In some embodiments, a promoter is fiber cell-specific or secondary cell wall-specific if the transcription levels initiated by the promoter in fiber cells or secondary cell walls, respectively, are at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, 000-fold higher or more as compared to the transcription levels initiated by the promoter in other tissues, resulting in the encoded protein substantially localized in plant cells that possess fiber cells or secondary cell wall, e.g., the stem of a plant. Nonlimiting examples of fiber cell and / or secondary cell wall specific promoters include the promoters directing expression of the genes IRX1, IRX3, IRX5, IRX7, IRX8, IRX9, IRX10, IRX14, NST1, NST2, NST3, MYB46, MYB58, MYB63, MYB83, MYB85, MYB103, PALI, PAL2, C3H, CcOAMT, CCR1, F5H, LAC4, LAC17, CADc, and CADd. See, e.g. Turner et al 1997; Meyer et al 1998; Jones et al 2001; Franke et al 2002; Ha et al 2002;Rohde et al 2004; Chen et al 2005; Stobout et al 2005; Brown et al 2005; Mitsuda et al 2005; Zhong et al 2006; Mitsuda et al 2007; Zhong et al 2007a, 2007b; Zhou et al 2009; Brown et al 2009; McCarthy et al 2009; Ko et al 2009; Wu et al 2010; Berthet et al 2011. In some embodiments, a promoter is substantially identical to a promoter from the lignin biosynthesis pathway. A promoter originated from one plant species may be used to direct gene expression in another plant species.

[0054] A polynucleotide or amino acid sequence is "heterologous" to an organism or a second polynucleotide or amino acid sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, when a polynucleotide encoding a polypeptide sequence is said to be operably linked to a heterologous promoter, it means that the polynucleotide coding sequence encoding the polypeptide is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence, e.g., from a different gene in the same species, or an allele from a different ecotype or variety, or a gene that is not naturally expressed in the target tissue).Attorney Docket: 2024-147-02 Lawrence Berkeley National Laboratory

[0055] The term "operably linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a DNA or RNA sequence if it stimulates or modulates the transcription of the DNA or RNA sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

[0056] The terms “host cell” of “host organism” is used herein to refer to a living biological cell that can be transformed via insertion of an expression vector.

[0057] The terms "expression vector" or "vector" refer to a compound and / or composition that transduces, transforms, or infects a host cell, thereby causing the cell to express nucleic acids and / or proteins other than those native to the cell, or in a manner not native to the cell. An "expression vector" contains a sequence of nucleic acids (ordinarily RNA or DNA) to be expressed by the host cell. Optionally, the expression vector also comprises materials to aid in achieving entry of the nucleic acid into the host cell, such as a virus, liposome, protein coating, or the like. The expression vectors contemplated for use in the present invention include those into which a nucleic acid sequence can be inserted, along with any preferred or required operational elements. Further, the expression vector must be one that can be transferred into a host cell and replicated therein. Particular expression vectors are plasmids, particularly those with restriction sites that have been well documented and that contain the operational elements preferred or required for transcription of the nucleic acid sequence. Such plasmids, as well as other expression vectors, are well known to those of ordinary skill in the art.

[0058] The terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs may be used that may have alternate backbones, comprising, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages (see Eckstein, OligonucleotidesAttorney Docket: 2024-147-02 Lawrence Berkeley National Laboratoryand Analogues: A Practical Approach, Oxford University Press); positive backbones; nonionic backbones, and non-ribose backbones. Thus, nucleic acids or polynucleotides may also include modified nucleotides that permit correct read-through by a polymerase."Polynucleotide sequence" or "nucleic acid sequence" includes both the sense and antisense strands of a nucleic acid as either individual single strands or in a duplex. As will be appreciated by those in the art, the depiction of a single strand also defines the sequence of the complementary strand; thus the sequences described herein also provide the complement of the sequence. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc.

[0059] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0060] In some embodiments, the present invention incorporated aspects and features described in U.S. Patent Application Ser. No. 63 / 620,067, filed Jan. 11, 2024, which is hereby incorporated by reference.

[0061] It is to be understood that, while the invention has been described in conjunction with the preferred specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0062] All patents, patent applications, and publications mentioned herein are herebyAttorney Docket: 2024-147-02 Lawrence Berkeley National Laboratoryincorporated by reference in their entireties.

[0063] The invention having been described, the following examples are offered to illustrate the subject invention by way of illustration, not by way of limitation.Example 1Plant dopamine biosynthetic pathway

[0064] Dopamine, an essential human neurotransmitter, also plays important roles in plantmicrobe interactions and abiotic stress tolerance in plants. Although dopamine biosynthesis is well-characterized in humans, it remains unclear in plants. Through metabolite-based genome-wide association studies, we identified two putative polyphenol oxidase genes for dopamine biosynthesis in Brachypodium distachyon, located in the same chromosomal region as two tyrosine decarboxylase genes. Biochemical and knockout mutant analyses revealed that dopamine biosynthesis proceeds primarily via tyramine as an intermediate, and dopamine oxidation leads to tissue browning in plants. Homolog searches and enzymatic assays suggest that the dopamine biosynthetic pathway is likely conserved across numerous plant species. This discovery provides valuable insights into plant dopamine biosynthesis with potential applications in reducing food browning, improving stress resilience, and bioengineering valuable compounds.

[0065] In this study, we present the identification of dopamine biosynthetic genes in B. distachyon, using a combination of metabolite-based genome-wide association studies (mGWAS), gene expression analysis, enzyme assays, and knockout mutant characterization. Our results indicate that unlike in humans, dopamine biosynthesis in plants proceeds primarily via tyramine as an intermediate, where TyDCs convert tyrosine into tyramine, which is then converted into dopamine by PPOs. Evidence from homologous searches and enzymatic assays suggests that the dopamine biosynthetic pathway is highly conserved across a wide range of plant species. Notably, we found that dopamine knockout mutants, including BdtydclBdtydc2, Bdppol , and BdppolBdppo3, exhibited a lack of dark brown coloration in root tissues after senescence or damage. Additionally, transient expression of the dopamine biosynthetic pathway in Nicotiana benthamiana recapitulated the browning phenotype. Together, these findings suggest that dopamine oxidation, rather than PPO activity alone, directly contributes to tissue browning in plants. The elucidation of the plant dopamine biosynthetic pathway offers new opportunities for development of crop plants withAttorney Docket: 2024-147-02 Lawrence Berkeley National Laboratoryreduced browning as well as production of valuable dopamine-derived compounds among other potential benefits.RESULTSIdentification of candidate genes in dopamine biosynthesis

[0066] Given the importance of dopamine in plants, we sought to identify the gene(s) responsible for its biosynthesis. AB. distachyon diversity panel, consisting of 100 natural accessions, was grown in a hydroponic system, and the roots and shoots of 4-week-old plants were harvested for metabolite analysis using LC-MS / MS. In addition to dopamine (Fig. SI), we measured high levels of tyramine and only detected trace levels of L-DOPA in B. distachyon (Fig. S2, Fig. 6).

[0067] To identify the gene(s) for dopamine biosynthesis, we conducted metabolite-based GWASs. When dopamine levels in roots or shoots were directly used as mapping traits, no significant single nucleotide polymorphisms (SNPs) were detected (Fig. S3, panels A and B). We reasoned that using ratios of dopamine to tyramine in roots or shoots could increase mapping sensitivity and found that when these were used as mapping traits, two overlapping peaks with significant SNPs were identified on chromosome 2 (Fig. 1, panels A and B; Fig. S3, panels C and D). Within the genetic mapping interval for the dopamine-to-tyramine ratio in roots, an examination of the Bd21-3 genome (vl.l) revealed eight genes, three of which showed dominant expression in roots, including a putative PPO-encoding gene, BdiBd21-3.2G0666400 (Fig. 1, panel C; Fig. S4). This evidence suggests that BdiBd21-3.2G0666400 is likely responsible for dopamine biosynthesis inB. distachyon roots. Interestingly, within the genetic mapping interval for the dopamine-to-tyramine ratio in shoots, three putative PPO-encoding genes were identified, BdiBd21-3.2G0666400, BdiBd21-3.2G0667800, and BdiBd21-3.2G0668300 (Fig. S3, panel D). However, SNPs within this interval were not significantly associated with the middle PPO-encoding gene, BdiBd21-3.2G0667800 (Fig. S3). The predicted protein encoded by BdiBd21-3.2G0666400 shares 52.1% and 78.9% sequence identity with the predicted proteins encoded by BdiBd21-3.2G0667800 and BdiBd21-3.2G0668300, respectively (Fig. S5). The three putative PPO-encoding genes were, therefore, named BdPPOl, BdPPO2, and BdPPO3 according to their physical order on the chromosome. Gene expression analysis revealed that BdPPOl and BdPPO2 were predominantly expressed in the roots of both 2-week-old and 4-week-old B. distachyonAttorney Docket: 2024-147-02 Lawrence Berkeley National LaboratoryBd21-3plants, whereas BdPPO3 was mainly expressed in the shoots of both 2-week-old and 4-week-old plants (Fig. 1, panel D).

[0068] Previous studies suggested that plant TyDCs are involved in dopamine biosynthesis (Facchini et al., 2000; Wang et al., 2021). Notably, we found that two known TyDC-encoding genes, BdiBd21-3.2G0653800 ax .BdiBd21-3.2G0654700 (Noda et al., 2015), are located within the same chromosomal region, approximately 829.0 kb and 783.6 kb upstream of BdPPOl, respectively (Bd21-3, vl.l) (Fig. 1, panel E), suggesting a loosely organized dopamine biosynthetic gene cluster in B. distachyon. The predicted proteins encoded by the two TyDC genes share a high degree of sequence identity (97.9%). We designated BdiBd21-3.2G0653800 s BdTyDCl n .BdiBd21-3.2G0654700 a BdTyDC2. Gene expression analysis revealed that BdTyDCl and BdTyDC2 were predominantly expressed in the roots of both 2-week-old and 4-week-old B. distachyon Bd21-3 plants (Fig. 1, panel F).

[0069] We next performed single nuclei RNA-seq (snRNA-seq) on roots and leaves of 8-day-old B. distachyon plants to determine whether candidate gene expression was limited to specific cell types and whether BdPPOs and BdTyDCs were co-expressed. After stringent filtering for high-quality cells, we constructed cell type-resolved gene expression atlases consisting of 9,700 root nuclei and 12,234 leaf nuclei. Leveraging published marker gene sets from maize, Sorghum bicolor, and rice (Zhang et al., 2021; Guillotin et al., 2023; He et al., 2024), we identified all major cell types in our atlases, including vasculature, ground cells, and epidermal cells of varying subtypes and developmental stages in roots, and vasculature, mesophyll, bundle sheath, and epidermis in leaves (Fig. S6, panels A and B). Consistent with our bulk RNA-seq data, we found that BdPPOl was predominantly expressed in roots while BdPPO3 was primarily expressed in leaves (Fig. 1, panels G and H). Notably, BdPPOl expression was specifically limited to developing cortex and exodermis cells, along with a subset of xylem cells expressing genes involved in secondary xylem differentiation (Fig. 1, panel G). In contrast, BdPPO2 exhibited a distinct expression pattern compared to BdPPOl (Fig. 1, panels G and H). BdPPO3 was weakly expressed in mature root cortex cells but strongly expressed in leaf mesophyll and epidermis (Fig. 1, panels G and H). Additionally, BdTyDCl and BdTyDC2 were predominantly expressed in roots, particularly in developing cortex and developing epidermal cells (Fig. 1, panels G and H). However, BdTyDCl was also expressed in leaf epidermal cells, while BdTyDC2 expression was completely absent in leaves (Fig. 1, panels G and H). Intriguingly, we observed that BdTyDCl and BdTyDC2 areAttorney Docket: 2024-147-02 Lawrence Berkeley National Laboratorythe top two genes co-expressed with BdPPOl in root), further supporting their involvement in root dopamine biosynthesis. Together, our results revealed that these candidate dopamine biosynthetic genes are spatially expressed not only in different tissues but also in distinct cell types within these tissues in B. distachyon.Characterization of BdTyDCs in dopamine biosynthesis

[0070] TyDCs are known to decarboxylate both tyrosine and L-DOPA (Facchini et al., 2000). BdTyDCl and BdTyDC2 were previously shown to exhibit L-tyrosine decarboxylation activity in yeast (Noda et al., 2015); however, their ability to decarboxylate L-DOPA remains unclear. To investigate the enzymatic functions of BdTyDCl and BdTyDC2, we transiently expressed these genes in N. benthamiana via Agrobacterium-mediated expression. We found that the expression of either BdTyDCl or BdTyDC2 led to tyramine production in the leaves of N. benthamiana (Fig. 2, panel A), confirming that both enzymes function as tyrosine decarboxylases. To determine whether BdTyDCl and BdTyDC2 also possess L-DOPA decarboxylase activity, we incubated [ring-13C6]-labeled L-DOPA with protein extracts from N. benthamiana leaves transiently expressing BdTyDCl or BdTyDC2. We found that both enzymes were capable of catalyzing the [ring-13C6]-labeled L-DOPA to the [ring-13C6]-labeled dopamine, indicative of L-DOPA decarboxylase activity (Fig. 2, panel B). Collectively, these results suggest that BdTyDCl and BdTyDC2 can use both tyrosine and L-DOPA as substrates, implying their involvement in dopamine biosynthesis.

[0071] To further validate the functions of BdTyDCl and BdTyDC2 in dopamine biosynthesis, we generated knockout mutants for these genes using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing (Fig. S6). Knocking out BdTyDCl led to a 50% reduction in tyramine levels in roots and a 91% reduction in shoots, along with a decrease in dopamine levels by 26% in roots and 93% in shoots (Fig. 2, panel C and D; Fig. S7). In contrast, knocking out BdTyDC2 significantly reduced tyramine and dopamine levels in roots by 65% and 47%, respectively, but did not affect their levels in shoots (Fig. 2, panels C and D; Fig. S7). Furthermore, knocking out both BdTyDCl and BdTyDC2 resulted in a complete deficiency of tyramine and dopamine in both roots and shoots (Fig. 2 , panels C and D; Fig. S7). These findings suggest that BdTyDCl and BdTyDC2 play a redundant role in tyramine and dopamine biosynthesis in B. distachyon, with BdTyDCl having a more prominent role in shoots. Additionally, we observed that theAttorney Docket: 2024-147-02 Lawrence Berkeley National LaboratoryBdtydclBdtydc2 double mutant plants did not accumulate more L-DOPA in either roots or shoots as compared to wild-type plants (Fig. S8), suggesting that BdPPOs are not involved in the 3 -hydroxylation of tyrosine in B. distachyon. Taken together, our results suggest that dopamine is biosynthesized with tyramine as the intermediate in B. distachyon.Characterization of BdPPOs in dopamine biosynthesis

[0072] To confirm that BdPPOl, BdPPO2, and BdPPO3 lack tyrosinase or tyrosine hydroxylase activity in the 3 -hydroxylation of tyrosine, we transiently expressed each of these genes in N. benthamiana using d / Y>Ac / c7c / ' / / / / ?7-mediated expression. L-DOPA production was not detectable in N benthamiana leaves expressing BdPPOl, BdPPO2, or BdPPO3 (Fig. S9), suggesting that these BdPPOs don’t function in the 3 -hydroxylation of tyrosine. This finding aligns with the observation that the BdtydclBdtydc2 double mutant plants do not accumulate more L-DOPA than wild-type plants (Fig. S8). Next, we transiently co-expressed BdTyDC2 together with each of the three BdPPOs in N. benthamiana, and found that co-expression of BdTyDC2 with BdPPOl, BdPPO2, or BdPPO3 resulted in dopamine production (Fig. 3 panel A). Taken together, our results demonstrate that the three BdPPOs function in the 3 -hydroxylation of tyramine, rather than tyrosine, during dopamine biosynthesis (Fig. 3, panel B).

[0073] To further examine BdPPOl’s role in dopamine biosynthesis, we obtained two Bdppol knockout mutants: Bdppol-1 (NaN1435), identified from the publicly available sodium azide B. distachyon sequenced mutant population (webpage for: jgi.doe.gov / indexed-collection-of-brachy-mutants / ), and Bdppol -2. generated using CRISPR-Cas9 gene editing (Fig. S10, panel A, and SI 1). We found that both Bdppol mutant alleles exhibited a nearcomplete deficiency in root dopamine production, while dopamine levels in shoots remained similar to those in wild-type plants (Fig. 3, panels C and D; Fig. S12, panels A and B). In contrast, we found that knocking out BdPPO2 did not affect dopamine biosynthesis in both roots and shoots (Fig. S10, panel B; Fig. S13, panels A and B). These results suggest that dopamine production in# distachyon roots is predominantly catalyzed by BdPPOl .Additionally, we found that the Bdppol mutant plants accumulated approximately 10-fold more tyramine in roots (Fig. S7, panel B; Fig. S12, panel C), further supporting the hypothesis that# distachyon plants synthesize dopamine using tyramine as an intermediate (Fig. 3, panel B).Attorney Docket: 2024-147-02 Lawrence Berkeley National Laboratory

[0074] To investigate dopamine biosynthesis in shoots, we next generated Bdppo3 single and BdppolBdppo3 double knockout mutants using CRISPR-Cas9 gene editing (Fig. SI 1, panels A and B). The two Bdppo3 mutant alleles produced wild-type levels of dopamine in shoots, while the BdppolBdppo3 double mutant plants lacked the ability to produce dopamine in shoots (Fig. 3, panels C and D; Fig. S7), suggesting that BdPPOl and BdPPO3 play a redundant tole in shoot dopamine biosynthesis. Notably, we found that the dopamine levels in BdppolBdppo3 roots were significantly lower than those in Bdppol roots, suggesting that BdPPO3 contributes to root dopamine biosynthesis, albeit with a minor role (Fig. S7). Taken together, our results demonstrate that both BdPPOl and BdPPO3 are required for dopamine biosynthesis inB. distachyon, with BdPPOl having a predominant role in root dopamine production.Dopamine oxidation causes tissue browning in plants

[0075] To examine whether the dopamine pathway has an effect on plant growth, we grew these mutant plants in a hydroponic system. We did not observe any growth defects in the biomass of the roots and shoots of the 4-week-old dopamine-knockout plants compared with the wild type (Fig. S14, panels A and B). Interestingly, we observed that the root tissues of the dopamine knockout plants, including Bdtydc !Bdtydc2 , Bdppol, and BdppolBdppo3, did not turn dark brown after senescence or damage (Fig. 4, panels A and B). Moreover, we found that the root extracts of these dopamine-knockout plants did not turn brown after exposure to air for up to a week (Fig. 4, panel C; Fig. SI 5). Previous studies revealed that PPOs are responsible for the enzymatic browning of foods, particularly fruits and vegetables, where PPOs catalyze the oxidation of o-diphenols to o-quinones that further polymerize through non-enzymatic reactions to form brown pigments (Sui et al., 2023; Tilley et al., 2023). Since the browning phenotypes were not observed in the BdtydclBdtydc2 double mutant plants, and dopamine is known to undergo autoxidation at physiological pH to form brown polymeric products (Umek et al., 2018), we hypothesize that dopamine oxidation, rather than PPO activity alone, directly causes tissue browning in B. distachyon. To test our hypothesis, we transiently expressed the dopamine pathway in the leaves of N benthamiana. The extracts from leaves expressing the full dopamine pathway became brown, while those from leaves individually expressing either BdTyDCs or BdPPOs did not (Fig. 4, panel D). Additionally, we found that the extracts from the leaves of N benthamiana expressing the empty vector developed similar brown coloration after dopamine was added (Fig. 4, panel D).Attorney Docket: 2024-147-02 Lawrence Berkeley National LaboratoryTogether, these results strongly suggest that dopamine and its oxidation, rather than PPOs alone, are the direct cause of the observed tissue browning in plants.The conservation of the dopamine biosynthetic pathway in plants

[0076] To investigate the conservation of the dopamine biosynthetic pathway in plants, we searched for TyDC and PPO gene homologs across flowering plant species with genome sequences available on Phytozome. We found that all examined plant species have putative TyDC genes, whereas most plant species, except those in the Brassicaceae family, contain putative PPO genes (Fig. 5, panel A). Interestingly, 79 out of 121 (65%) plant species encoding both putative TyDCs and PPOs have at least one pair of them located within 10 Mb on the same chromosome (Fig. 5, panel A). Notably, several plant species prone to browning issues and known for producing high levels of dopamine in their fruits, including Musa acuminata (a species of banana), Per sea americana (avocado), and Malus domestica (apple) (Briguglio et al., 2018), were found to possess the dopamine biosynthetic gene homologs colocalized in the same chromosomal region (Fig. 5, panel A). The enzymatic activity of the TyDC family has been well-characterized in plants (Facchini et al., 2000; Wang et al., 2021); however, the activity of the PPO family remains underexplored in dopamine biosynthesis. To investigate the conserved function of PPOs in dopamine biosynthesis, we examined the activity of select PPOs from several plant species, including Zea mays, Oryza Saliva, Sorghum bicolor, Hordeum vulgare, and Papaver somniferum. Our findings revealed that at least one PPO from each select species has the capability to catalyze dopamine production when transiently co-expressed with BdTyDC2 in the leaves of N benthamiana (Fig. 5, panels B and C), indicating a conservation of the dopamine pathway in plants.CONCLUSIONS

[0077] We report the identification of genes responsible for dopamine biosynthesis in B. distachyon. These biosynthetic genes are localized in the chromosomal region but exhibit differential regulation in roots and shoots. Biochemical and genetic evidence supports that dopamine biosynthesis in this model grass proceeds through tyramine as an intermediate, which is different from the primary dopamine biosynthetic pathway in humans. Homology searches and enzymatic assays suggest that the identified dopamine biosynthetic pathway is likely conserved across many plant species, although some may use L-DOPA as an alternative intermediate.Attorney Docket: 2024-147-02 Lawrence Berkeley National Laboratory

[0078] PPO-mediated enzymatic browning affects the color quality of food, particularly fruits and vegetables (Sui et al., 2023). In this study, we observed that root tissues of dopamine knockout mutants, including BdtydclBdtydc2, Bdppol , and BdppolBdppo3, did not develop dark brown coloration after senescence or damage. Additionally, transient expression of the dopamine pathway (both TyDC and PPO) induced browning in leaf extracts of N. benthamiana. These findings demonstrate that dopamine oxidation, rather than PPO activity alone, directly causes tissue browning inB. distachyon. Our results further suggest that dopamine oxidation could contribute to browning in common foods such as bananas, avocados, and apples, as these species contain putative dopamine biosynthetic gene homologs and are known to accumulate high levels of dopamine. However, this hypothesis requires further experimental validation in these species. Currently, genome editing techniques, such as RNA interference (RNAi) and CRISPR / Cas9, are being used to prevent food browning by targeting PPO-encoding genes in various plants (Tilley et al., 2023). However, in this study, we found that knocking out PPOs resulted in the hyperaccumulation of the intermediate tyramine, which can have potentially dangerous hypertensive effects in humans (Rafehi et al., 2019). Our findings offer a new perspective for bioengineering efforts to reduce browning in plants by targeting alternative gene families, such as those encoding TyDCs, to minimize tyramine accumulation while reducing browning. This work also has broader implications for engineering the dopamine pathway to enhance plant stress resilience. Since dopamine is a potent antioxidant, bioengineering this pathway could help mitigate the damaging effects of reactive oxygen species (ROS) generated under stress conditions, such as drought and salinity (Liu et al., 2020). Additionally, the dopamine biosynthetic enzymes identified in this study could be utilized in microorganisms for the sustainable production of dopamine as a precursor for valuable pharmaceutical compounds.MATERIALS AND METHODSPlant materials and plant growth

[0079] AB. distachyon diversity panel, consisting of 100 natural genetic lines, was used in this study. B. distachyon seeds were sterilized in 70% (v / v) ethanol for 30 s and 5 min in 6% (w / v) sodium hypochlorite, followed by five washes with sterile water. The seeds were subsequently stratified on 1.5% (w / v) phytoagar plates containing 0.5 x Murashige & Skoog (0.5 x MS, MSP01, Caisson Laboratories, USA) and placed in the dark at 4°C for three days. The seeds were then germinated in a growth chamber with a temperature of 22°C,Attorney Docket: 2024-147-02 Lawrence Berkeley National Laboratoryphotosynthetic photon flux density at 150 pmol m’2s-1, and a 16 h light / 8 h dark photoperiod for two days.

[0080] The culture vessels (PTL-100™, PhytoTech Labs) were rinsed five times with MilliQ water and autoclaved. Two seedlings of each B. distachyon line were placed onto a floating plastic holder and then transferred to each vessel with 40 mL of 0.5 x MS. Plants were grown in a 16 h light / 8 h dark regime at 22 °C and 50% relative humidity with 150 pmol m'2s_1illumination for four weeks. The culture vessels without plants, filled with the growth medium, were incubated in the same conditions as the controls. At week four, root and shoot tissues were harvested and stored at -80°C for further analysis. The sterility of the hydroponics setup was examined by plating 50 pL medium on Luria Broth (LB, Sigma-Aldrich) solid plates, followed by seven-day incubation at 30°C.

[0081] For snRNA-seq experiments, B. distachyon Bd21 seeds were surface sterilized by soaking in 75% ethanol for 1 minute and 50% bleach for 8 minutes, 25% bleach with 0.2% Triton X-100 (Sigma-Aldrich 93443) for 8 minutes, and then rinsed with sterile water 5 times. After sterilization, seeds were planted vertically on agar plates containing 3 mM Ca(NOs)2, 1.5 mMMgSCU, 1.25 mMNFLFLPCU, 1 mMKCl, and lx micronutrients (MS Salts 100x, MSP18-10LT), in 0.8% agar with pH 5.7. Plants were grown in a Percival chamber under conditions of 22°C with 158 pmol m'2s_1illumination on a cycle of 16 h light / 8 h dark.Metabolite analysis and identification with LC-MS / MS

[0082] B. distachyon root and shoot tissues were harvested from 4-week-old hydroponically grown plants and stored at -80°C for further analysis. Before extraction, tissues (approximately 0.1 g fresh weight) were ground into powder using bead milling in a Biospec Minibeadbeater-96. 500 pL of extraction buffer (70% methanol, methanol: H2O, 70:30, v / v) was then added to the tissue powders for metabolite extraction overnight at 4°C. The samples were subsequently centrifuged for 10 min at 15,000 g. The supernatants were dried in a speed vac, resuspended with 150 pL pure methanol, and filtered with 0.22-pm polyvinylidene difluoride microcentrifuge filtration devices (Pall Corporation, NY, USA) (10,000g for 5 min at 10°C). Filtrates were used for metabolite analysis. Polar metabolites were separated using hydrophilic interaction chromatography (HILIC) and detected on a Thermo Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer. Briefly, an Infinity Lab Poroshell 120Attorney Docket: 2024-147-02 Lawrence Berkeley National LaboratoryHILIC-Z, 2.1x150 mtn, 2.7 um column equipped on an Agilent 1290 HPLC stack was used for separations. Data were collected using data-dependent MS2 acquisition to select the top two most intense ions not previously fragmented within 7 seconds. Internal and external standards were used for quality control purposes. Using the conditions defined above, dopamine (Sigma-Aldrich) and tyramine (Sigma-Aldrich) were identified by comparing the retention times (min) and MS2 spectra with those of standards.Metabolite-based genome-wide association studies (mGWASs)

[0083] Previously, we observed genetic variation in dopamine levels in root exudates of ten B. distachyon lines (Ding et al., 2024). To map the gene(s) involved in dopamine biosynthesis, aB. distachyon diversity panel, consisting of 100 lines, was grown in a hydroponic system for four weeks, and roots and shoots were harvested for metabolite analysis. Metabolite-based GWASs (mGWASs) were performed using the ion intensity of dopamine (m / z, 137.0546-137.0646, fragment ion, [M+H]+) and the ratios of dopamine (m / z, 137.0546-137.0646, fragment ion, [M+H]+) to tyramine (m / z, 121.0598-121.0698, fragment ion, [M+H]+) in roots and shoots as mapping traits. Genotypic data (the reference genome Bd21-3 vl.l) from a genotyping-by-sequencing (GBS) strategy (Gordon et al., 2020; Scarlett et al., 2023) with less than 20% missing genotypes and a >15% minor allele frequency was used to generate 4,780,736 final SNP (single nucleotide polymorphism) markers. mGWASs were initially conducted using the General Linear Model (GLM) in TASSEL 5.0 (webpage for: tassel.bitbucket.io / ), with final analyses conducted using the R package GAPIT (Lipka et al., 2012). Manhattan plots were constructed in the R package qqman (vO.1.4) (webpage for: cran.r-project.org / web / packages / qqman) (Turner, 2018).RNA isolation and bulk RNA-seq analyses

[0084] Root and shoot tissues were harvested from hydroponically grown B. distachyon Bd21-3 plants at weeks 2 and 4. Total RNA was isolated with a NucleoSpin® RNA Plant Kit (Takara Bio USA) according to the manufacturer’s protocol. Three biological replicates were performed using different plants. RNA quality was assessed based on RNA integrity number using an Agilent Bioanalyzer.

[0085] RNA-seq library construction and sequencing were performed by Novogene.Messenger RNA (mRNA) was isolated from total RNA using poly-T oligo-attached magnetic beads. Following fragmentation, the first strand of cDNA was synthesized using randomAttorney Docket: 2024-147-02 Lawrence Berkeley National Laboratoryhexamer primers, and the second strand of cDNA was then synthesized using either dUTP for directional library preparation or dTTP for non-directional library preparation. The libraries were assessed for quantification using Qubit and real-time PCR, and the size distribution was analyzed with a Bioanalyzer. The quantified libraries were pooled and sequenced on Illumina platforms, based on the effective library concentration and desired data yield. Clustering of the index-coded samples was carried out according to the manufacturer’s instructions. Once cluster generation was complete, the libraries were sequenced on an Illumina platform, producing paired-end reads. The raw reads in fastq format were initially processed using Perl scripts. During this step, clean reads were obtained by removing reads containing adapters, reads with poly-N sequences, and low-quality reads from the raw data. Simultaneously, Q20, Q30, and GC content of the clean data were calculated. Paired-end clean reads were then aligned to the reference genome (B. distachyon B21-3 vl.l) using Hisat2 (v2.0.5) (Kim et al., 2015). FeatureCounts (vl.5.0-p3) was used to count the reads numbers mapped to each gene (Liao et al., 2014). The FPKM (Fragments Per Kilobase of transcript sequence per Million base pairs) for each gene was then calculated based on the gene’s length and the number of reads mapped to it using HTSeq (vO.6.1) (Anders et al., 2015).Single-nuclei RNA-seq and Analyses

[0086] Leaf and root samples for snRNA-seq were harvested from 8-day-old plants at the two-leaf stage. For root samples, sections of 2-4 cm in length beginning from the primary root tips were harvested. For leaf samples, the entire leaf including a small section of coleoptile was harvested. Tissue samples were harvested at 2.5 hours after the start of the day cycle, immediately flash- frozen in liquid nitrogen, and then stored at -80°C for approximately 2.5 months before nuclei isolation. Nuclei isolation from tissues was performed as follows:

[0087] Buffer 1 (lysis buffer) consisted of 0.275 M sorbitol (Sigma-Aldrich S6021), 0.1% Triton X-100 (Sigma-Aldrich 93443), 0.01 M MgCh (Ambion AM9530G), lx protease inhibitor cocktail (Sigma-Aldrich 4693132001), 0.3 U / pL RNase inhibitor (Roche 03335399001), and 1 mMDTT (Teknova D9750).

[0088] Buffer 2 (wash and resuspension buffer) consisted of 1 x phosphate buffer saline (without Mg and Ca, Coming, 21-040-CM), Spermidine trihydrochloride 0.5mM (S2501-25G), Spermine tetrahydrochloride O.lmM (S1141-10G), 0.4 U / pL RNase inhibitor, and 1Attorney Docket: 2024-147-02 Lawrence Berkeley National LaboratorymM DTT.

[0089] All nuclei isolation steps were carried out in a cold room at 4°C. For each sample, 20-30 mg frozen tissue was chopped for 3 minutes using a razor blade on a glass plate with 100-200 pL buffer 1, then transferred to a petri dish with 1.5 mL cold buffer 1 and allowed to rest on ice with gentle shaking for 2 minutes. The samples were then transferred to a 48-well filter plate (25 pm, 4 mL, Agilent, 201003-100), which was pre-wet with 1 mL cold buffer 1 before it was placed on the receiving plate attached to a QIAvac96 (Qiagen). Pressure was maintained below 100 bar during filtration. The filtered nuclei solution was centrifuged at 500 g for 10 minutes at 4°C to pellet the nuclei, after which the supernatant was discarded. The pellet was resuspended by gentle flicking and pipetting, washed with 2 mL cold buffer 2, centrifuged at 500 g for 5 minutes, and the supernatant was discarded, after which the pellet was again resuspended in 100 pL cold buffer 2. Nuclei quality and quantity were assessed by flow cytometry (BD Accuri C6 plus), for which they were stained by propidium iodide (Sigma-Aldrich P4864) added to a final concentration of 50 pg / mL. The nuclei suspension was then diluted to a concentration of 200-500 nuclei / pL using cold buffer 2.

[0090] Single nuclei were partitioned and barcoded on a BD Rhapsody system (BD Rhapsody HT Xpress, BD Rhapsody Scanner), using the BD Rhapsody WTA V3 kit and cartridge, targeting 10,000-25,000 B. distachyon nuclei for each of the four samples (two replicate root samples, two replicate leaf samples). Note that samples were multiplexed on the same lane of the cartridge with samples from several other plant species, which were resolved independently by mapping to their respective reference genomes after sequencing. Library creation was carried out following the manufacturer’s protocol. Sequencing was performed on an Illumina NovaSeq X plus 25B flow cell, using 2x150 bp paired-end sequencing and targeting an average of at least 50,000 reads (25,000 fragments) per nucleus. The resulting sequences were pre-processed by filtering out ribosomal RNA using BBTools v38.96 bbduk.sh (BBMap) to remove read pairs with 31-mers in read 2 matching common ribosomal 31-mers. Remaining reads were processed with the BD Rhapsody Sequence Analysis Pipeline v2.2.1 (webpage for: bitbucket.org / CRSwDev / cwl / src / master / ), supplied with a combined-species reference genome that included the Bd21 v3.2 reference genome (webpage for: phytozome-next.jgi.doe.gov / sorghumpan / info / Bdistachyon_v3_2) for B. distachyon. Separately, rRNA-filtered reads were also mapped to the same reference with STARsolo (Kaminow et al., 2021) in Velocyto mode (‘— soloFeatures Velocyto’) in order toAttorney Docket: 2024-147-02 Lawrence Berkeley National Laboratorydetermine splice rates for each cell barcode. Additional QC metrics were compiled by running the R package diem (Alvarez et al., 2020) to assign a ‘debris score’ to each cell barcode, and by fitting a model to the proportion of each species contributing UMIs to each cell barcode (Baumgart et al., 2024) in order to identify putative empty wells and estimate ambient RNA levels. UMIs mapping the organellar genomes were quantified and removed from the analysis, and then cell barcodes were then filtered to those with at least 500 UMIs, at least 250 genes, 90% non-organelle UMIs, 5% unspliced UMIs, debris score <=2, and estimated ambient RNA rate <50%. Count matrices for all remaining cell barcodes were loaded into Seurat v5.0.0 (Hao et al., 2024), and SCTransform v2 normalization was performed, followed by an initial clustering using Seurat’s RunPCA (with 5,000 variable genes and 30 PCs), FindNeighbors, and FindClusters (resolution 0.6). Next, DoubletFinder (McGinnis et al., 2019) was run and cells with a doublet score >0.4 were removed. Finally, the two replicates from each tissue were merged into final tissue-specific atlases, which were then re-normalized with SCT before performing a final PCA (30 PCs) and clustering (resolution = 1.0). Cell type labels were assigned to each cluster in each atlas by first examining the expression of a small number of FISH-validated Sorghum bicolor, maize, and rice orthologs from previously published studies (Guillotin et al., 2023; Zhang et al., 2021). Additional cell types were identified using larger sets of previously computed marker genes from the same studies as well as scPlantDB (He et al., 2024). Final cell type labels were compiled from these two sources, with additional manual annotation based on literature support for data-derived markers when there were discrepancies or no significant matches.5’ RACE cDNA library construction and gene cloning

[0091] Total RNA was isolated from roots and shoots of 4-week-old hydroponically grown Bd21-3 plants using a NucleoSpin® RNA Plant Kit (Takara Bio USA) according to the manufacturer’s protocol. Total RNA, approximately 2 pg, was subjected to TURBO DNA-free treatment (Ambion) and used for the construction of a 5' rapid amplification of cDNA ends (RACE) cDNA library with the SMART er RACE 573' Kit (Clontech) in accordance with the manufacturer’s protocol. Genes with full-length open reading frames (ORFs) were amplified using gene-specific oligonucleotides. For HgroZ>actera / m-mediated transient expression in N. benlhamiana. full-length open reading frames, including BdTyDCl, BdTyDC2, BdPPOl, BdPPO2, and BdPPO3, were cloned from cDNA libraries into the expression vector pLIFE33 using the Uracil-Specific-Excision-Reaction (USER) method withAttorney Docket: 2024-147-02 Lawrence Berkeley National LaboratoryUSER cloning-specific primers (Geu-Flores et al., 2007). The homologous genes of B. distachyon PPO1 from other species, including ZmPPOl (Zea mays), PsPPOs (PsPPOl, PsPPO2, PsPPO3, and PsPPO4, Papaver somniferum), HvPPOs (HvPPOl and HvPPO2, Hordeum vulgare), OsPPOl (Oryza saliva). and SbPPOs (SbPPOl and SbPPO2, Sorghum bicolor), were synthesized (Twist Biosciences) and subcloned into pLIFE33.Transient expression in N. benthamiana

[0092] For transient expression in N. benthamiana, full-length open reading frames cloned into the pLIFE33 vector were introduced into Agrobacterium tumefaciens strain GV3101. The transformed bacterial cells were cultured at 28°C for 24 hours in LB liquid medium (Sigma-Aldrich) supplemented with 50 mg / L kanamycin, 30 mg / L gentamicin, and 50 mg / L rifampicin. The cells were then harvested and resuspended to a final ODeoo of 0.8 in a 10 mM MES buffer containing 10 mM MgCh. Equal volumes of the various cultures, along with the silencing suppressor strain Pl 9, were mixed and infiltrated into the newly expanded leaves of 6-week-old N. benthamiana plants using a needleless syringe. Five days after infiltration, the Agrobacterium-mCAix^QA leaves were harvested for metabolite analysis using LC-MS / MS.In vitro enzymatic assay

[0093] BdTyDCs and BdPPOs were transiently expressed in TV. benthamiana.Agrobaclerium-m' Cx v&iQ N. benthamiana leaf tissues were harvested 2 days post-infiltration. Aliquots of approximately 100 mg FW tissues were ground to a fine powder in liquid nitrogen using bead milling, followed by resuspension in 200 pL of protein extraction buffer [0.1 M phosphate buffer (pH 6.2), 0.1% Triton X-100, and protease inhibitors: 50 pg / mL TPCK, 50 pg / mL TLCK, and 0.6 mM PMSF). Crude extracts were incubated at 4°C for 10 min with shaking and centrifuged at 15,000 g for 15 min at 4°C to remove cell debris. The soluble proteins were buffer-exchanged into a phosphate buffer (0.1 M, pH 6.2) using 10 kDa Amicon Ultra- 15 centrifugal filter units (Millipore) to remove small molecules. To test if TyDCl and TyDC2 have DOPA decarboxylase activity, the filtered protein extracts (approximately 100 pL) were individually incubated with a [ring-13C6]-labeled L-DOPA at a final concentration of 10 pM for 30 min at room temperature. To examine if BdPPOs function in the 3 -hydroxylation of tyramine, the filtered protein extracts (approximately 100 pL) from Agrobacterium-m?A\x^QA tobacco leaves of BdPPOs were incubated with tyramine at 10 pM for 30 min at room temperature. The reactions were stopped by adding 0.2 mL of aAttorney Docket: 2024-147-02 Lawrence Berkeley National Laboratorymetabolite extraction buffer (95% methanol and 5% 1 M hydrochloric acid). The mixtures were further filtered using 0.22-pm polyvinylidene difluoride microcentrifuge filtration devices (Pall Corporation, NY, USA) by centrifuging at 10,000 g for 5 min at 10°C. The filtrates were analyzed using hydrophilic interaction chromatography (HILIC) on a Thermo Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer.Identification of the sodium-azide-induced Bdppol-1 and Bdppo2 mutants

[0094] Seeds of Bdppol-1 (NaN1435, containing a premature stop codon, W382*, in BdPPOT) w\ dppo2 (NaN1956, containing a premature stop codon, E487*, in BdPPO2) were obtained from the publicly available sodium azide-induced B. distachyon sequenced mutant population (webpage for: jgi.doe.gov / indexed-collection-of-brachy-mutants / ). The mutations were verified by sequencing PCR products amplified with gene-specific primers.Creation of the dopamine pathway mutants using CRISPR / Cas9

[0095] Dopamine biosynthetic gene mutants were generated using CRISPR / Cas9 gene editing. Two gRNAs were designed to simultaneously target BdTyDCl and BdTyDC2, resulting in single or double knockouts. For the creation of Bdppol, Bdppo3, and BdppolBdppo3 mutants, one gRNA was designed to target both BdPPOl and BdPPO3 and another to target BdPPO3. Oligonucleotides were phosphorylated and annealed using T4 polynucleotide kinase (New England Biolabs), followed by ligation into the CRISPR destination vector JD633 (Addgene) that was linearized by Aarl (New England Biolabs) using a Golden Gate Reaction. All constructs were sequence-verified and introduced individually into tumefaciens strain AGL1 via electroporation. The transformed bacterial cells were cultured at 28°C for 48 hours on LB agar plates (Sigma-Aldrich) supplemented with 50 mg / L kanamycin, 50 mg / L carbenicillin, and 25 mg / L rifampicin.

[0096] B. distachyon (Bd21-3) transformation was performed following an established protocol (Bragg et al., 2015). Briefly, plants were grown in a walk-in growth chamber under a long-day photoperiod (16 h light / 8 h dark) at 22°C during the day and 26°C at night.Immature seeds were isolated from spikes of approximately 7-week-old plants and sterilized using 0.6% (v / v) sodium hypochlorite and 0.1% Triton X-100 for 4 minutes. After sterilization, seeds were washed 5 times with sterile water to remove residual chemicals. Immature embryos were isolated under a stereomicroscope and placed on a callus induction medium (CIM) containing Linsmaier and Skoog basal medium (PhytoTech Labs) andAttorney Docket: 2024-147-02 Lawrence Berkeley National Laboratorysucrose. Callus induction was carried out in the dark at 28°C for 4 weeks, with calli subcultured twice to promote embryogenesis. Prior to transformation, Agrobacterium carrying the CRISPR / Cas9 construct was streaked onto LB plates and incubated overnight at 28°C. Bacterial cells were then harvested by scrapping off the plates, resuspended in liquid CIM to an ODeoo of 0.6, and used to inoculate the embryogenic calli. The calli were immersed in the bacterial suspension for 5 minutes, briefly dried on filter paper, and incubated in the dark at 22°C for 3 days. Following co-culture, the calli were transferred to selective CIM plates containing 40 mg / L hygromycin and 300 mg / L Timentin (PhytoTech Labs) and incubated at 28°C for 10 days under a 16 h light / 8 h dark photoperiod and 150 pmol m2s ' illumination. Selected calli were then transferred to a regeneration medium containing MS basal medium with vitamins, maltose, 40 mg / L hygromycin, and 300 mg / L Timentin. Regeneration was conducted at 28°C under the same light and humidity conditions for 2-4 weeks. Once shoots were fully developed, regenerated plants were transferred to a rooting medium containing MS basal medium with vitamins and sucrose and incubated at 28°C for 2 weeks. Plants with well-developed roots were transferred to soil for further growth.

[0097] Leaves from TO transgenic plants were collected for genotyping using the Phire Plant Direct PCR kit (Thermo Scientific). Mutations in the target gene were confirmed with genespecific primers. Homozygous mutant plants were identified in the T1 generation by genotyping the target gene. To prevent sustained off-target effects, T1 homozygous mutants were further genotyped to confirm the absence of the Cas9-containing transgene by PCR amplification of the selectable marker gene (hygromycin B phosphotransferase). All sequencing was performed using Sanger sequencing at the UC Berkeley DNA Sequencing Facility (Berkeley, CA).Comparative gene homolog analyses

[0098] Amino acid sequences corresponding to the genes BdiBd21-3.2G0666400 (BdPPOP), BdiBd21-3.2G0667800 (BdPPO2 BdiBd21-3.2G0668300 (BdPPOS), BdiBd21-3.2G0653800 (BdTyDC 7), ax .BdiBd21-3.2G0654700 (BdTyDC2)' inB. distachyon Bd21-3 vl.l (webpage for: phytozome-next.jgi.doe.gov / info / BdistachyonBd21_3_vl_l) were searched using DIAMOND (v2.0.152, Buchfink et al., 2021) with default parameters to identify homologous sequences in the protein databases of Mesangiospermae genomes available on Phytozome (Goodstein et al., 2012). Additionally, proteomes of PapaverAttorney Docket: 2024-147-02 Lawrence Berkeley National Laboratorysomniferum, Musa acuminata, and Persea americana were downloaded from NCBI (accession numbers GCF_003573695.1, GCF_000313855.2, and GCA_029852735, respectively) and included in the search. For species with multiple representatives on Phytozome, such as different cultivars or genome versions, the number of distinct hits for each query protein was averaged across all genome representatives. Homologous hits were considered to be colocalized in the same chromosomal region if the genomic coordinates of at least one pair of genes corresponding to hits from the PPO and TyDC families were located within 10 Mb on the same chromosome. Plant species known to produce dopamine were indicated in Fig. 5, panel A (Liu et al., 2020; Novak et al., 2024; Gomes et al., 2024).Sequence analysis and phylogenetic tree construction

[0099] Sequence analysis and phylogenetic tree construction were conducted. Protein sequence alignments, derived from GenBank IDs, were performed using Clustal W as implemented in the BioEdit software package (webpage for: mbio.ncsu.edu / BioEdit / bioedit.html). Maximum-likelihood phylogenetic trees were constructed using MEGA7 (webpage for: megasoftware.net / megabeta.php) with bootstrap values calculated from 1,000 iterations.Statistical analysis

[0100] Statistical analyses were conducted using JMP Pro v.13.0 (SAS Institute) and Prism v.9.0 (GraphPad). One-way ANOVA was performed to evaluate statistical differences. Tukey tests were used to correct for multiple comparisons between control and treatment groups. Student’s unpaired two-tailed / -tests were conducted for pairwise comparisons. P < 0.05 was considered to be statistically significant.

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Briguglio M, Dell'Osso B, Panzica G, Malgaroli A, Banfi G, Zanaboni Dina C, Galentino R, Porta M. Dietary Neurotransmitters: A Narrative Review on Current Knowledge.Attorney Docket: 2024-147-02 Lawrence Berkeley National LaboratoryNutrients. 2018 May 10; 10(5):591.35. Araji S, Grammer TA, Gertzen R, Anderson SD, Mikulic-Petkovsek M, Veberic R, Phu ML, Solar A, Leslie CA, Dandekar AM, Escobar MA. Novel roles for the polyphenol oxidase enzyme in secondary metabolism and the regulation of cell death in walnut. Plant Phy siol . 2014 Mar; 164(3 ): 1191 -203.36. Tilley A, McHenry MP, McHenry JA, Solah V, Bayliss K. Enzymatic browning: The role of substrates in polyphenol oxidase mediated browning. Curr Res Food Sci. 2023 Oct 20;7: 100623.37. Rafehi M, Faltraco F, Matthaei J, Prukop T, Jensen O, Grytzmann A, Biome FG, Berger RG, Krings U, Vormfelde SV, Tzvetkov MV, Brockmoller J. Highly Variable Pharmacokinetics of Tyramine in Humans and Polymorphisms in OCTI, CYP2D6, and MAO-A. Front Pharmacol. 2019 Oct 30; 10: 1297.38. Facchini PJ, Huber-Allanach KL, Tari LW. Plant aromatic L-amino acid decarboxylases:evolution, biochemistry, regulation, and metabolic engineering applications.Phytochemistry. 2000 May;54(2): 121-38.39. Li Y, Wang Y, Huang L, Chen C, An N, Zheng X. Identification and Functional Characterization of Tyrosine Decarboxylase from Rehmannia glutinosa. Molecules. 2022 Mar 1;27(5):1634.40. Luaces P, Sanchez R, Exposito J, Perez-Pulido AJ, Perez AG, Sanz C. Functional and Physiological Characterization of Tyrosine Decarboxylases from Olea europaea L.Involved in the Synthesis of the Main Phenolics in Olive Fruit and Virgin Olive Oil. Int J Mol Sci. 2024 Oct 10;25(20): 10892.41. Zhang J, Sun X. Recent advances in polyphenol oxidase-mediated plant stress responses.Phytochemistry. 2021 Jan;181:112588.42. Cole B, Bergmann D, Blaby-Haas CE, Blaby IK, Bouchard KE, Brady SM, Ciobanu D, Coleman-Derr D, Leiboff S, Mortimer JC, Nobori T, Rhee SY, Schmutz J, Simmons BA, Singh AK, Sinha N, Vogel JP, OMalley RC, Visel A, Dickel DE. Plant single-cell solutions for energy and the environment. Commun Biol. 2021 Aug 12;4(1):962.Attorney Docket: 2024-147-02 Lawrence Berkeley National Laboratory43. Ding et al. Insights into the role of dopamine in rhizosphere microbiome assembly.bioRxiv, (2024).44. Novak V, Andeer PF, Bowen BP, Ding Y, Zhalnina K, Hofmockel KS, Tomaka C, Harwood TV, van Winden MCM, Golini AN, Kosina SM, Northen TR. Reproducible growth of Brachypodium in EcoFAB 2.0 reveals that nitrogen form and starvation modulate root exudation. Sci Adv. 2024 Jan 5;10(l):eadg7888.45. Li F, Yu Y, Wang Q, Yuan J, Wang P, Fan X. Polymerization of dopamine catalyzed by laccase: Comparison of enzymatic and conventional methods. 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[0102] Further references include: webpage for: ncbi.nlm.nih.gov / pmc / articles / PMC5986471 / ; webpage for: onlinelibrary.wiley.com / doi / full / 10.llll / tpj.13835; Odjakova M., Hadjiivanova C. Animal neurotransmitter substances in plants. Bulg. J. Plant Physiol. 1997;23:94-102; and Feldman J.M., Lee E.M., Castleberry C.A. Catecholamine and serotonin content of foods: Effect on urinary excretion of homovanillic and 5-hydroxyindoleacetic acid. J. Am. DietAssoc. 1987;87:1031-1035.

[0103] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

Attorney Docket: 2024-147-02 Lawrence Berkeley National LaboratoryWhat is claimed is:

1. A genetically modified plant, plant part, or plant cell comprising inhibition or reduced expressi on or knockouts of (a) one or more endogenous polyphenol oxidases (PPOs), and / or (b) one or more endogenous tyrosine decarboxylases (TyDCs).

2. The genetically modified plant, plant part, or plant cell of claim 1, wherein the genetically modified plant, plant part, or plant cell has a reduction of dopamine production without or essentially without an accumulation of tyramine compared to a corresponding unmodified plant, plant part, or plant cell.

3. The genetically modified plant, plant part, or plant cell of claim 1, wherein the genetically modified plant, plant part, or plant cell wherein expression of one or more endogenous PPO genes are inhibited, tyramine synthesis.

4. The genetically modified plant, plant part, or plant cell of claim 1, wherein the genetically modified plant, plant part, or plant cell comprises inhibition or reduced expression or knockouts of Bdtydcl, Bdlydc2. Bdppol , and / or Bdppo3.

5. The genetically modified plant, plant part, or plant cell of claim 1, wherein the genetically modified plant, plant part, or plant cell is gene editing to reduce food browning by targeting the PPO genes.

6. The genetically modified plant, plant part, or plant cell of claim 5, wherein the inhibition or reduced expression is by using RNA interference (RNAi) to silence the expression of the PPO gene(s) and / or TyDC(s), or using CRISPR-Cas9 gene editing to knock out the PPO gene(s) and / or TyDC(s).

7. The genetically modified plant, plant part, or plant cell of claim 1, comprising a tydc2 tydc3 double mutant alleles and having about the same growth rate as a corresponding unmodified modified plant, plant part, or plant cell.

8. The genetically modified plant, plant part, or plant cell of claim 1, the genetically modified plant, plant part, or plant cell is an apple, grape, tomato, potato, maize, banana, avocado, opium poppy, moringa, broad bean, or common bean.

9. The genetically modified plant, plant part, or plant cell of claim 1, wherein the plantAttorney Docket: 2024-147-02 Lawrence Berkeley National Laboratorypart is a stem, leaf, fruit, seed, or root.

10. The genetically modified plant, plant part, or plant cell of claim 1, wherein the plant, plant part, or plant cell has less melanin produced thereby resulting a reduced brown color.

11. A genetically modified fruit comprising inhibition or reduced expression or knockouts of (a) one or more endogenous polyphenol oxidases (PPOs), and / or (b) one or more endogenous tyrosine decarboxylases (TyDCs), wherein the genetically modified fruit which takes a longer time to brown as compared to an unmodified fruit.

12. The genetically modified fruit of claim 11, wherein the fruit has one or more an improved quality, as a food for human consumption, in terms of shelflife and consumability as compared to the unmodified fruit.

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