Interaction of ubiquitin-like protein SILENCING DEFECTIVE 2 with LIKE HETEROCHROMATIN PROTEIN 1 is required for regulation of anthocyanin biosynthesis in Arabidopsis thaliana in response to sucrose.

The regulatory mechanisms of anthocyanin biosynthesis have been well documented at the transcriptional and translational levels. By contrast, how anthocyanin biosynthesis is epigenetically regulated remains largely unknown. In this study, we employed genetic, molecular biology, and chromatin immunoprecipitation-quantitative polymerase chain reaction assays to identify a regulatory module essential for repressing the expression of genes involved in anthocyanin biosynthesis through chromatin remodeling. We found that SILENCING DEFECTIVE 2 (SDE2), which was previously identified as a negative regulator for sucrose-induced anthocyanin accumulation in Arabidopsis, is cleaved into N-terminal SDE2-UBL and C-terminal SDE2-C fragments at the first diglycine motif, and the cleaved SDE2-C, which can fully complement the sde2 mutant, is localized in the nucleus and physically interacts with LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) in vitro and in vivo. Genetic analyses showed that both SDE2 and LHP1 act as negative factors for anthocyanin biosynthesis. Consistently, immunoblot analysis revealed that the level of LHP1-bound histone H3 lysine 27 trimethylation (H3K27me3) significantly decreases in sde2 and lhp1 mutants, compared to wild-type (WT). In addition, we found that sugar can induce expression of SDE2 and LHP1, and enhance the level of the nucleus-localized SDE2-C. Taken together, our data suggest that the SDE2-C-LHP1 module is required for repression of gene expression through H3K27me3 modification during sugar-induced anthocyanin biosynthesis in Arabidopsis thaliana.

The chloroplast singlet oxygen-triggered biosynthesis of salicylic acid and jasmonic acid is mediated by EX1 and GUN1 in Arabidopsis.

Reactive oxygen species (ROS) and defence hormones like salicylic acid (SA) and jasmonic acid (JA) play pivotal roles in triggering cell death. However, the precise mechanism governing the interaction between ROS and SA/JA remains elusive. Recently, our research revealed that RNAi mutants with suppressed expression of PROGRAMMED CELL DEATH8 (PCD8) exhibit an overabundance of tetrapyrrole intermediates, particularly uroporphyrinogen III (Uro III), leading to the accumulation of singlet oxygen (1O2) during the transition from darkness to light, thereby instigating leaf necrosis. In this investigation, we uncovered that 1O2 stimulates biosynthesis of SA and JA, activating SA/JA signalling and the expression of responsive genes in PCD8 RNAi (pcd8) mutants. Introducing NahG or knocking out PAD4 or NPR1 significantly alleviates the cell death phenotype of pcd8 mutants, while coi1 partially mitigates the pcd8 phenotype. Further exploration revealed that EX1 and GUN1 can partially rescue the pcd8 phenotype by reducing the levels of Uro III and 1O2. Notably, mutations in EX1 mutations but not GUN1, substantially diminish SA content in pcd8 mutants compared to the wild type, implying that EX1 acts as the primary mediator of 1O2 signalling-mediated SA biosynthesis. Moreover, the triple ex1 gun1 pcd8 displays a phenotype similar to ex1. Overall, our findings underscore that the 1O2-induced cell death phenotype requires EX1/GUN1-mediated retrograde signalling in pcd8 mutants, providing novel insights into the interplay between ROS and SA/JA.

COBL7 is required for stomatal formation via regulation of cellulose deposition in Arabidopsis.

As a key regulator of plant photosynthesis, water use efficiency and immunity, stomata are specialized cellular structures that adopt defined shapes. However, our knowledge about the genetic players of stomatal pore formation and stomatal morphogenesis remains limited. Forward genetic screening, positional cloning, confocal and electron microscopy, physiological and pharmacological assays were employed for isolation and characterization of mutants and genes. We identified a mutant, dsm1, with impaired cytokinesis and deformed stomata. DSM1 is highly expressed in guard mother cells and guard cells, and encodes COBRA-LIKE 7 (COBL7), a plant-specific glycosylphosphatidylinositol (GPI)-anchored protein. COBRA-LIKE 7 and its closest homologue, COBL8, are first enriched on the forming cell plates during cytokinesis, and then their subcellular distribution and abundance change are correlated with the progressive stages of stomatal pore formation. Both COBL7 and COBL8 possess an ability to bind cellulose. Perturbing the expression of COBL7 and COBL8 leads to a decrease in cellulose content and inhibition of stomatal pore development. Moreover, we found that COBL7, COBL8 and CSLD5 have synergistic effects on stomatal development and plant growth. Our findings reveal that COBL7 plays a predominant and functionally redundant role with COBL8 in stomatal formation through regulating cellulose deposition and ventral wall modification in Arabidopsis.

Ultra-high static magnetic field induces a change in the spectrum but not frequency of DNA spontaneous mutations in Arabidopsis thaliana.

Biological effects of magnetic fields have been extensively studied in plants, microorganisms and animals, and applications of magnetic fields in regulation of plant growth and phytoprotection is a promising field in sustainable agriculture. However, the effect of magnetic fields especially ultra-high static magnetic field (UHSMF) on genomic stability is largely unclear. Here, we investigated the mutagenicity of 24.5, 30.5 and 33.0 T UHSMFs with the gradient of 150, 95 and 0 T/m, respectively, via whole genome sequencing. Our results showed that 1 h exposure of Arabidopsis dried seeds to UHSMFs has no significant effect on the average rate of DNA mutations including single nucleotide variations and InDels (insertions and deletions) in comparison with the control, but 33.0 T and 24.5 T treatments lead to a significant change in the rate of nucleotide transitions and InDels longer than 3 bp, respectively, suggesting that both strength and gradient of UHSMF impact molecular spectrum of DNA mutations. We also found that the decreased transition rate in UHSMF groups is correlated with the upstream flanking sequences of G and C mutation sites. Furthermore, the germination rate of seeds exposed to 24.5 T SMF with -150 T/m gradient showed a significant decrease at 24 hours after sowing. Overall, our data lay a basis for precisely assessing the potential risk of UHSMF on DNA stability, and for elucidating molecular mechanism underlying gradient SMF-regulated biological processes in the future.

Arabidopsis pollen-specific glycerophosphodiester phosphodiesterase-like genes are essential for pollen tube tip growth.

In angiosperms, pollen tube growth is critical for double fertilization and seed formation. Many of the factors involved in pollen tube tip growth are unknown. Here, we report the roles of pollen-specific GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE-LIKE (GDPD-LIKE) genes in pollen tube tip growth. Arabidopsis thaliana GDPD-LIKE6 (AtGDPDL6) and AtGDPDL7 were specifically expressed in mature pollen grains and pollen tubes and green fluorescent protein (GFP)-AtGDPDL6 and GFP-AtGDPDL7 fusion proteins were enriched at the plasma membrane at the apex of forming pollen tubes. Atgdpdl6 Atgdpdl7 double mutants displayed severe sterility that was rescued by genetic complementation with AtGDPDL6 or AtGDPDL7. This sterility was associated with defective male gametophytic transmission. Atgdpdl6 Atgdpdl7 pollen tubes burst immediately after initiation of pollen germination in vitro and in vivo, consistent with the thin and fragile walls in their tips. Cellulose deposition was greatly reduced along the mutant pollen tube tip walls, and the localization of pollen-specific CELLULOSE SYNTHASE-LIKE D1 (CSLD1) and CSLD4 was impaired to the apex of mutant pollen tubes. A rice pollen-specific GDPD-LIKE protein also contributed to pollen tube tip growth, suggesting that members of this family have conserved functions in angiosperms. Thus, pollen-specific GDPD-LIKEs mediate pollen tube tip growth, possibly by modulating cellulose deposition in pollen tube walls.

PROGRAMMED CELL DEATH8 interacts with tetrapyrrole biosynthesis enzymes and ClpC1 to maintain homeostasis of tetrapyrrole metabolites in Arabidopsis.

Tetrapyrrole biosynthesis (TBS) is a dynamically and strictly regulated process. Disruptions in tetrapyrrole metabolism influence many aspects of plant physiology, including photosynthesis, programmed cell death (PCD), and retrograde signaling, thus affecting plant growth and development at multiple levels. However, the genetic and molecular basis of TBS is not fully understood. We report here PCD8, a newly identified thylakoid-localized protein encoded by an essential gene in Arabidopsis. PCD8 knockdown causes a necrotic phenotype due to excessive chloroplast damage. A burst of singlet oxygen that results from overaccumulated tetrapyrrole intermediates upon illumination is suggested to be responsible for cell death in the knockdown mutants. Genetic and biochemical analyses revealed that PCD8 interacts with ClpC1 and a number of TBS enzymes, such as HEMC, CHLD, and PORC of TBS. Taken together, our findings uncover the function of chloroplast-localized PCD8 and provide a new perspective to elucidate molecular mechanism of how TBS is finely regulated in plants.

The functional implication of ATF6α in castration-resistant prostate cancer cells.

Stress in the endoplasmic reticulum (ER) may perturb proteostasis and activates the unfolded protein response (UPR). UPR activation is frequently observed in cancer cells and is believed to fuel cancer progression. Here, we report that one of the three UPR sensors, ATF6α, was associated with prostate cancer (PCa) development, while both genetic and pharmacological inhibition of ATF6α impaired the survival of castration-resistance PCa (CRPC) cells. Transcriptomic analyses identified the molecular pathways deregulated upon ATF6α depletion, and also discovered considerable disparity in global gene expression between ATF6α knockdown and Ceapin-A7 treatment. In addition, combined analyses of human CRPC bulk RNA-seq and single-cell RNA-seq (scRNA-seq) public datasets confirmed that CRPC tumors with higher ATF6α activity displayed higher androgen receptor (AR) activity, proliferative and neuroendocrine (NE) like phenotypes, as well as immunosuppressive features. Lastly, we identified a 14-gene set as ATF6α NE gene signature with encouraging prognostic power. In conclusion, our results indicate that ATF6α is correlated with PCa progression and is functionally relevant to CRPC cell survival. Both specificity and efficacy of ATF6α inhibitors require further refinement and evaluation.

Benzo[b]naphtho[1,2-d]thiophene Sulfoxides: Biomimetic Synthesis, Photophysical Properties, and Applications.

A practical synthesis of nonsymmetrical thiophene-fused aromatic systems has been developed that was inspired by the biodegradation of benzothiophene. For the first time, the photophysical properties of a series of π-conjugated benzo[b]naphtho[1,2-d]thiophene (BNT) sulfoxides were explored both in solution and in the solid state. The excellent fluorescence characteristics enable various applications of these compounds.

A transcriptional regulator that boosts grain yields and shortens the growth duration of rice.

Complex biological processes such as plant growth and development are often under the control of transcription factors that regulate the expression of large sets of genes and activate subordinate transcription factors in a cascade-like fashion. Here, by screening candidate photosynthesis-related transcription factors in rice, we identified a DREB (Dehydration Responsive Element Binding) family member, OsDREB1C, in which expression is induced by both light and low nitrogen status. We show that OsDREB1C drives functionally diverse transcriptional programs determining photosynthetic capacity, nitrogen utilization, and flowering time. Field trials with OsDREB1C-overexpressing rice revealed yield increases of 41.3 to 68.3% and, in addition, shortened growth duration, improved nitrogen use efficiency, and promoted efficient resource allocation, thus providing a strategy toward achieving much-needed increases in agricultural productivity.

Static Magnetic Field Inhibits Growth of Escherichia coli Colonies via Restriction of Carbon Source Utilization.

Magnetobiological effects on growth and virulence have been widely reported in Escherichia coli (E. coli). However, published results are quite varied and sometimes conflicting because the underlying mechanism remains unknown. Here, we reported that the application of 250 mT static magnetic field (SMF) significantly reduces the diameter of E. coli colony-forming units (CFUs) but has no impact on the number of CFUs. Transcriptomic analysis revealed that the inhibitory effect of SMF is attributed to differentially expressed genes (DEGs) primarily involved in carbon source utilization. Consistently, the addition of glycolate or glyoxylate to the culture media successfully restores the bacterial phenotype in SMF, and knockout mutants lacking glycolate oxidase are no longer sensitive to SMF. These results suggest that SMF treatment results in a decrease in glycolate oxidase activity. In addition, metabolomic assay showed that long-chain fatty acids (LCFA) accumulate while phosphatidylglycerol and middle-chain fatty acids decrease in the SMF-treated bacteria, suggesting that SMF inhibits LCFA degradation. Based on the published evidence together with ours derived from this study, we propose a model showing that free radicals generated by LCFA degradation are the primary target of SMF action, which triggers the bacterial oxidative stress response and ultimately leads to growth inhibition.

Expanding the Coverage of Metabolic Landscape in Cultivated Rice with Integrated Computational Approaches.

Genome-scale metabolomics analysis is increasingly used for pathway and function discovery in the post-genomics era. The great potential offered by developed mass spectrometry (MS)-based technologies has been hindered, since only a small portion of detected metabolites were identifiable so far. To address the critical issue of low identification coverage in metabolomics, we adopted a deep metabolomics analysis strategy by integrating advanced algorithms and expanded reference databases. The experimental reference spectra and in silico reference spectra were adopted to facilitate the structural annotation. To further characterize the structure of metabolites, two approaches were incorporated into our strategy, i.e., structural motif search combined with neutral loss scanning and metabolite association network. Untargeted metabolomics analysis was performed on 150 rice cultivars using ultra-performance liquid chromatography coupled with quadrupole-Orbitrap MS. Consequently, a total of 1939 out of 4491 metabolite features in the MS/MS spectral tag (MS2T) library were annotated, representing an extension of annotation coverage by an order of magnitude in rice. The differential accumulation patterns of flavonoids between indica and japonica cultivars were revealed, especially O-sulfated flavonoids. A series of closely-related flavonolignans were characterized, adding further evidence for the crucial role of tricin-oligolignols in lignification. Our study provides an important protocol for exploring phytochemical diversity in other plant species.

A genetic screen in Arabidopsis reveals the identical roles for RBP45d and PRP39a in 5' cryptic splice site selection.

Cryptic splice sites in eukaryotic genome are generally dormant unless activated by mutation of authentic splice sites or related splicing factors. How cryptic splice sites are used remains unclear in plants. Here, we identified two cryptic splicing regulators, RBP45d and PRP39a that are homologs of yeast U1 auxiliary protein Nam8 and Prp39, respectively, via genetic screening for suppressors of the virescent sot5 mutant, which results from a point mutation at the 5' splice site (5' ss) of SOT5 intron 7. Loss-of-function mutations in RBP45d and PRP39a significantly increase the level of a cryptically spliced variant that encodes a mutated but functional sot5 protein, rescuing sot5 to the WT phenotype. We furtherly demonstrated that RBP45d and PRP39a interact with each other and also with the U1C, a core subunit of U1 snRNP. We found that RBP45d directly binds to the uridine (U)-rich RNA sequence downstream the 5' ss of SOT5 intron 7. However, other RBP45/47 members do not function redundantly with RBP45d, at least in regulation of cryptic splicing. Taken together, RBP45d promotes U1 snRNP to recognize the specific 5' ss via binding to intronic U-rich elements in plants.

The pentatricopeptide repeat protein EMB1270 interacts with CFM2 to splice specific group II introns in Arabidopsis chloroplasts.

Chloroplast biogenesis requires the coordinated expression of chloroplast and nuclear genes. Here, we show that EMB1270, a plastid-localized pentatricopeptide repeat (PPR) protein, is required for chloroplast biogenesis in Arabidopsis thaliana. Knockout of EMB1270 led to embryo arrest, whereas a mild knockdown mutant of EMB1270 displayed a virescent phenotype. Almost no photosynthetic proteins accumulated in the albino emb1270 knockout mutant. By contrast, in the emb1270 knockdown mutant, the levels of ClpP1 and photosystem I (PSI) subunits were significantly reduced, whereas the levels of photosystem II (PSII) subunits were normal. Furthermore, the splicing efficiencies of the clpP1.2, ycf3.1, ndhA, and ndhB plastid introns were dramatically reduced in both emb1270 mutants. RNA immunoprecipitation revealed that EMB1270 associated with these introns in vivo. In an RNA electrophoretic mobility shift assay (REMSA), a truncated EMB1270 protein containing the 11 N-terminal PPR motifs bound to the predicted sequences of the clpP1.2, ycf3.1, and ndhA introns. In addition, EMB1270 specifically interacted with CRM Family Member 2 (CFM2). Given that CFM2 is known to be required for splicing the same plastid RNAs, our results suggest that EMB1270 associates with CFM2 to facilitate the splicing of specific group II introns in Arabidopsis.

BIG3 and BIG5 Redundantly Mediate Vesicle Trafficking in Arabidopsis.

Vesicle trafficking plays an important role in delivering a diverse range of cargoes between different membranous systems in eukaryotes. It is well documented that the brefeldin A (BFA)-inhibited guanine nucleotide exchange factor (GEF), named BIG, regulates vesicle budding at the trans-Golgi network (TGN) and recycling endosomes through activating the ADP-ribosylation factor (ARFs). Among the five BIGs in Arabidopsis, BIG5 is characterized to mediate ARF-dependent trafficking at the plasma membrane or endosomes while the members from BIG1 to BIG4 (BIG1-BIG4) at the TGN in the secretory pathway. However, evidence is increasing to suggest that BIG5 can function redundantly with BIG1-BIG4 to regulate vesicular trafficking in response to various intra- and extra-cellular stimuli. In this study, our genetic analysis showed that BIG5 played an overlapping role at least with BIG3 in cell proliferation. To elucidate molecular mechanisms underlying the BIG5- and BIG3-regulated biological processes, we examined the effect of BIGs on expression patterns of the two transmembrane proteins, PINFORMED 2 (PIN2) epically localized in root epidermal cells and the regulator of G protein signaling 1 (RGS1) localized in the plasma membrane. Our data showed that the PIN2 polar distribution was slightly reduced in big3 big5 in the absence of BFA, and it was significantly reduced by the treatment of 0.1 µM BFA in big3 big5. Further analysis revealed that BFA bodies derived from the plasma membrane were only observed in wild type (WT), big3 and big5 cells, but not in the big3 big5 cells. These results indicate that BIG5 and BIG3 are functionally redundant in the endosome recycling pathway from the plasma membrane to TGN. On the other hand, the single BIG3 or BIG5 mutation had no effect on the plasma membrane expression of RGS1, whereas the double mutations in BIG3 and BIG5 led to a significant amount of RGS1 retained in the vesicle, indicating that BIG3 and BIG5 act redundantly in mediating protein trafficking. Furthermore, transmission electron microscopy assays showed that Golgi ultrastructure in big3 big5 cells was abnormal and similar to that in BFA-treated WT cells. Taken together, our data provide several new lines of evidence supporting that BIGs play a redundant role in vesicular trafficking and probably also in maintaining the Golgi structural integrity in Arabidopsis.

A new method for functional analysis of plastid EMBRYO-DEFECTIVE PPR genes by efficiently constructing cosuppression lines in Arabidopsis.

BACKGROUND: Pentatricopeptide-repeat proteins (PPRs) characterized by tandem arrays of a degenerate 35-amino-acid repeat (PPR motif) can bind a single strand RNA and regulate organelle gene expression at the post-transcriptional level, including RNA cleavage, splicing, editing and stability etc. PPRs are conserved in all eukaryotes and extremely expanded in higher plants. Many knockout mutants of PPR genes are embryonically lethal. These genes are named EMB PPRs and functional analysis of them is hindered by the difficulty in obtaining their knockout mutants. RESULTS: Here, we report a new method for functional analysis of plastid EMB PPRs by efficiently constructing their cosuppression lines in Arabidopsis. When we overexpressed a mutated full length or truncated coding sequence (CDS) of EMB PPRs, such as EMB2279, EMB2654 and EMB976 (all belong to the P family PPRs) in the wild-type (WT) background, a large portion of T1 plants displayed chlorosis phenotypes, which are similar to those of the weak allele mutants, knockdown lines or partially complementary lines. RT-PCR analysis showed that overexpression of the truncated EMB PPRs led to significant and specific downregulation of their corresponding endogenous mRNAs. However, when these EMB PPRs were overexpressed in the Post transcriptional Gene Silencing (PTGS) deficient mutant, RNA-dependent RNA polymerase 6 (rdr6), none of the T1 plants displayed chlorosis phenotypes. These results indicate that the chlorosis phenotype results from post transcriptional silencing of the corresponding endogenous gene (also known as sense cosuppression). CONCLUSIONS: Overexpression of an appropriately truncated EMB PPR CDS in WT leads to gene silencing in a RDR6-dependent manner, and this method can be employed to study the unknown function of EMB PPR genes. By this method, we showed that EMB976 is required for splicing of chloroplast clpP1 intron 2 and ycf3 intron 1.

Differential Transcription and Alternative Splicing in Cotton Underly Specialized Defense Responses Against Pests.

The green mirid bug (Apolygus lucorum) and the cotton bollworm (Helicoverpa armigera) are both preferred to live on cotton but cause different symptoms, suggesting specialized responses of cotton to the two insects. In this study, we investigated differential molecular mechanisms underlying cotton plant defenses against A. lucorum and H. armigera via transcriptomic analyses. At the transcription level, jasmonate (JA) signaling was dominated in defense against H. armigera whereas salicylic acid (SA) signaling was more significant in defense against A. lucorum. A set of pathogenesis-related (PR) genes and protease inhibitor genes were differentially induced by the two insects. Insect infestations also had an impact on alternative splicing (AS), which was altered more significantly by the H. armigera than A. lucorum. Interestingly, most differential AS (DAS) genes had no obvious change at the transcription level. GO analysis revealed that biological process termed "RNA splicing" and "cellular response to abiotic stimulus" were enriched only in DAS genes from the H. armigera infested samples. Furthermore, insect infestations induced the retained intron of GhJAZs transcripts, which produced a truncated protein lacking the intact Jas motif. Taken together, our data demonstrate that the specialized cotton response to different insects is regulated by gene transcription and AS as well.

Brassinosteroid-Activated BRI1-EMS-SUPPRESSOR 1 Inhibits Flavonoid Biosynthesis and Coordinates Growth and UV-B Stress Responses in Plants.

UV-B light is a potential stress factor in plants, but how plants coordinate growth and UV-B stress responses is not well understood. Here, we report that brassinosteroid (BR) signaling inhibits UV-B stress responses in Arabidopsis (Arabidopsis thaliana) and various crops by controlling flavonol biosynthesis. We further demonstrate that BRI1-EMS-SUPPRESSOR 1 (BES1) mediates the tradeoff between plant growth and UV-B defense responses. BES1, a master transcription factor involved in BR signaling, represses the expression of transcription factor genes MYB11, MYB12, and MYB111, which activate flavonol biosynthesis. BES1 directly binds to the promoters of these MYBs in a BR-enhanced manner to repress their expression, thereby reducing flavonol accumulation. However, exposure to broadband UV-B down-regulates BES1 expression, thus promoting flavonol accumulation. These findings demonstrate that BR-activated BES1 not only promotes growth but also inhibits flavonoid biosynthesis. UV-B stress suppresses the expression of BES1 to allocate energy to flavonoid biosynthesis and UV-B stress responses, allowing plants to switch from growth to UV-B stress responses in a timely manner.

SPLICING FACTOR1 Is Important in Chloroplast Development under Cold Stress.

RNA SPLICING FACTOR1 (SF1) is responsible for recognizing the branch point site (BPS) sequence in introns and is critical for pre-mRNA splicing. In Arabidopsis (Arabidopsis thaliana), splicing factor1 (AtSF1) has been shown to retain the conserved function, but it is unexpected that null atsf1 mutants are viable. Here, we identified an allele of atsf1, named suppressor of thf1-4 (sot4), from suppressor screening for leaf variegation of thylakoid formation1 The sot4 mutant resulting from the G-to-R mutation at the highly conserved 198th amino acid residue within the functionally unknown domain exhibits leaf virescence associated with less accumulation of mature plastid ribosomal RNA, particularly under cold stress. Interestingly, the same point mutation in yeast Saccharomyces cerevisiae MUD synthetic-lethal 5p (SF1/Msl5p) also causes hypersensitivity to coldness and a low splicing activity for the introns with suboptimal BPS sequences. Transcriptomic profiling and reverse-transcription quantitative PCR analyses showed that expression of many genes were up- or downregulated in atsf1 via insufficient intron splicing. Our search for a BPS consensus from the retained introns in atsf1 transcriptomes, combined with RNA electrophoresis mobility shift assays, revealed that AtSF1 directly binds to the BPS consensus containing 5'-CU(U/A)AU-3'. Taken together, our data provide insight into a role for AtSF1 in regulating intron splicing efficiency, which helps plants acclimate to coldness.

Arogenate dehydratases can modulate the levels of phenylacetic acid in Arabidopsis.

Phenylacetic acid (PAA) is one type of natural auxin and widely exists in plants. Previous biochemical studies demonstrate that PAA in plants is synthesized from phenylalanine (Phe) via phenylpyruvate (PPA), but the PAA biosynthetic genes and its regulation remain unknown. In this article, we show that the AROGENATE DEHYDRATASE (ADT) family, which catalyzes the conversion of arogenate to Phe, can modulate the levels of PAA in Arabidopsis. We found that overexpression of ADT4 or ADT5 remarkably increased the amounts of PAA. Due to an increase in PAA levels, ADT4ox and ADT5ox plants can partially restore the auxin-deficient phenotypes caused by treatments with an inhibitor of the biosynthesis of indole-3-acetic acid (IAA), a main auxin in plants. In contrast, the levels of PAA were significantly reduced in adt multiple knockout mutants. Moreover, the levels of PPA are substantially increased in ADT4 or ADT5 overexpression plants but reduced in adt multiple knockout mutants, suggesting that PPA is a key intermediate of PAA biosynthesis. These results provide an evidence that members of the ADT family of Arabidopsis can modulate PAA level via the PPA-dependent pathway.