Arabidopsis transcription factor TCP13 promotes shade avoidance syndrome-like responses by directly targeting a subset of shade-responsive gene promoters.

TCP13 belongs to a subgroup of TCP transcription factors implicated in the shade avoidance syndrome (SAS), but its exact role remains unclear. Here, we show that TCP13 promotes the SAS-like response by enhancing hypocotyl elongation and suppressing flavonoid biosynthesis as a part of the incoherent feed-forward loop in light signaling. Shade is known to promote the SAS by activating PHYTOCHROME-INTERACTING FACTOR (PIF)-auxin signaling in plants, but we found no evidence in a transcriptome analysis that TCP13 activates PIF-auxin signaling. Instead, TCP13 mimics shade by activating the expression of a subset of shade-inducible and cell elongation-promoting SAUR genes including SAUR19, by direct targeting of their promoters. We also found that TCP13 and PIF4, a molecular proxy for shade, repress the expression of flavonoid biosynthetic genes by directly targeting both shared and distinct sets of biosynthetic gene promoters. Together, our results indicate that TCP13 promotes the SAS-like response by directly targeting a subset of shade-responsive genes without activating the PIF-auxin signaling pathway.

Identification of GA20ox2 as a target of ATHB2 and TCP13 during shade response.

The shade avoidance syndrome (SAS) is a collective adaptive response of plants under shade highlighted by characteristic phenotypes such as hypocotyl elongation, which is largely mediated by concerted actions of auxin and GA. We identified ATHB2, a homeodomain-leucine zipper (HD-Zip) domain transcription factor known to be rapidly induced under shade condition, as a positive regulator of GA biosynthesis necessary for the SAS by transactivating the expression of GA20ox2, a key gene in the GA biosynthesis pathway. Based on promoter deletion analysis, EMSA and ChIP assay, ATHB2 appears to regulate the GA20ox2 expression as a direct binding target. We also found that the GA20ox2 expression is under negative control by TCP13, the effect of which can be suppressed by presence of ATHB2. Considering a rapid induction kinetics of ATHB2, this relationship between ATHB2 and TCP13 may allow ATHB2 to play a shade-specific activator for GA20ox by derepressing a pre-existing activity of TCP13.

Modeling-based identification of a Raptor-binding motif present in Arabidopsis ABA receptor PYL1.

By employing molecular modeling of interaction simulation combined with a confirmatory yeast two-hybrid analysis, we identified the Raptor-binding region in an ABA receptor PYL1 protein of Arabidopsis. The region was a part of the C-terminal alpha-helix structure of the protein within which a phenylalanine and an aspartate in the sequence of FADTV are predicted to form critical interactions with the Raptor. Although the sequence deviates a little from the plant TOS consensus that we previously identified and defined (FSD [V/I]F) from AtS6Ks and its orthologues as well as AtATG13, the modeling data indicate that the sequence and its neighboring area are structurally capable of establishing the interaction with the Raptor in the same mode as those of other TOS motif-containing structures. This finding provides a new insight into the understanding of plant TOS motif, based upon which a putative Raptor-binding region in TAP46, another TOR substrate, is proposed.

Identification of TCP13 as an Upstream Regulator of ATHB12 during Leaf Development.

Leaves grow by distinct phases controlled by gene regulatory networks including many transcription factors. Arabidopsis thaliana homeobox 12 (ATHB12) promotes leaf growth especially during the cell expansion phase. In this study, we identify TCP13, a member of the TCP transcription factor family, as an upstream inhibitor of ATHB12. Yeast one-hybrid screening using a 1.2-kb upstream region of ATHB12 resulted in the isolation of TCP13 as well as other transcription factors. Transgenic plants constitutively expressing TCP13 displays a significant reduction in leaf cell size especially during the cell expansion period, while repression of TCP13 and its paralogs (TCP5 and TCP17) result in enlarged leaf cells, indicating that TCP13 and its paralogs inhibit leaf development, mainly at the cell expansion phase. Its expression pattern during leaf expansion phase is opposite to ATHB12 expression. Consistently, the expression of ATHB12 and its downstream genes decreases when TCP13 was overexpressed, and increases when the expression of TCP13 and its paralogs is repressed. In chromatin immunoprecipitation assays using TCP13-GFP plants, a fragment of the ATHB12 upstream region that contains the consensus sequence for TCP binding is strongly enriched. Taken together, these findings indicate that TCP13 and its paralogs inhibit leaf growth by repressing ATHB12 expression.

Involvement of TOR signaling motif in the regulation of plant autophagy.

In our previous studies, we have demonstrated that a stretch of amino-acid sequences identified from Arabidopsis ribosomal S6 kinase 1 (AtS6K1) provided a plant version of the TOS (TOR-signaling) motif, mediating the interaction with the Raptor protein in the TOR (Target of Rapamycin) kinase complex. Here we report the presence of same element in Arabidopsis Autophagy related-13 (AtATG13) protein, which is a key component of the plant autophagy response. Its composition is nearly identical to that found in the AtS6K1 in the five-amino-acid core sequence, and the presence of this five-amino-acid sequence was found to be essential for its interaction with the Raptor protein. A mutant AtATG13 protein lacking this five-amino-acid element conferred an elevated autophagy response and could not effectively phosphorylated by TOR kinase activity, demonstrating its role in mediating the TOR signaling to the components that carry it as a possible TOS motif. A ligand-binding simulation model using the MM-PBSA method indicates that both of the five-amino-acid sequence elements of AtS6K1 and AtATG13 have strong probability of making stable interface with the Raptor binding pocket, corroborating our proposition for this element as the plant TOS motif.

Molecular details of the Raptor-binding motif on Arabidopsis S6 kinase.

A putative raptor-binding fragment was identified from Arabidopsis S6 kinase 1 (AtS6K1) N-terminal domain in our previous study. Here, we report a further characterization of this fragment, which identified a 12-amino acid core element absolutely required for the interaction. Although the amino acid sequence of the element per se had no significant homology with the canonical consensus of the TOS (TOR-signaling) motif found in the mammalian TOR (target of rapamycin) kinase substrates, its overall sequence composition is similar to that of the TOS motif in that the acidic and non-polar amino acids residues are arranged in alternating fashion and having one or two of the bulky hydrophobic amino acid (F) buried in the interior. Substitution of this bulky residue completely abolished the binding of the fragment to AtRaptor1, as in the case of the mammalian TOS motif. Taken together with its position relative to the catalytic domain of the kinase, which also shows a resemblance with the TOS motif, these results appear to suggest that this core binding element in the N-terminus of AtS6K1 represents a plant version of the TOS motif.

Identification of the Raptor-binding motif on Arabidopsis S6 kinase and its use as a TOR signaling suppressor.

TOR (target of rapamycin) kinase signaling plays central role as a regulator of growth and proliferation in all eukaryotic cells and its key signaling components and effectors are also conserved in plants. Unlike the mammalian and yeast counterparts, however, we found through yeast two-hybrid analysis that multiple regions of the Arabidopsis Raptor (regulatory associated protein of TOR) are required for binding to its substrate. We also identified that a 44-amino acid region at the N-terminal end of Arabidopsis ribosomal S6 kinase 1 (AtS6K1) specifically interacted with AtRaptor1, indicating that this region may contain a functional equivalent of the TOS (TOR-Signaling) motif present in the mammalian TOR substrates. Transient over-expression of this 44-amino acid fragment in Arabidopsis protoplasts resulted in significant decrease in rDNA transcription, demonstrating a feasibility of developing a new plant-specific TOR signaling inhibitor based upon perturbation of the Raptor-substrate interaction.

Identification of nucleosome assembly protein 1 (NAP1) as an interacting partner of plant ribosomal protein S6 (RPS6) and a positive regulator of rDNA transcription.

The ribosomal protein S6 (RPS6) is a downstream component of the signaling mediated by the target of rapamycin (TOR) kinase that acts as a central regulator of the key metabolic processes, such as protein translation and ribosome biogenesis, in response to various environmental cues. In our previous study, we identified a novel role of plant RPS6, which negatively regulates rDNA transcription, forming a complex with a plant-specific histone deacetylase, AtHD2B. Here we report that the Arabidopsis RPS6 interacts additionally with a histone chaperone, nucleosome assembly protein 1(AtNAP1;1). The interaction does not appear to preclude the association of RPS6 with AtHD2B, as the AtNAP1 was also able to interact with AtHD2B as well as with an RPS6-AtHD2B fusion protein in the BiFC assay and pulldown experiment. Similar to a positive effect of the ribosomal S6 kinase 1 (AtS6K1) on rDNA transcription observed in this study, overexpression or down regulation of the AtNAP1;1 resulted in concomitant increase and decrease, respectively, in rDNA transcription suggesting a positive regulatory role played by AtNAP1 in plant rDNA transcription, possibly through derepression of the negative effect of the RPS6-AtHD2B complex.

A cytosolic thioredoxin acts as a molecular chaperone for peroxisome matrix proteins as well as antioxidant in peroxisome.

Thioredoxin (TRX) is a disulfide reductase present ubiquitously in all taxa and plays an important role as a regulator of cellular redox state. Recently, a redox-independent, chaperone function has also been reported for some thioredoxins. We previously identified nodulin-35, the subunit of soybean uricase, as an interacting target of a cytosolic soybean thioredoxin, GmTRX. Here we report the further characterization of the interaction, which turns out to be independent of the disulfide reductase function and results in the co-localization of GmTRX and nodulin-35 in peroxisomes, suggesting a possible function of GmTRX in peroxisomes. In addition, the chaperone function of GmTRX was demonstrated in in vitro molecular chaperone activity assays including the thermal denaturation assay and malate dehydrogenase aggregation assay. Our results demonstrate that the target of GmTRX is not only confined to the nodulin-35, but many other peroxisomal proteins, including catalase (AtCAT), transthyretin-like protein 1 (AtTTL1), and acyl-coenzyme A oxidase 4 (AtACX4), also interact with the GmTRX. Together with an increased uricase activity of nodulin-35 and reduced ROS accumulation observed in the presence of GmTRX in our results, especially under heat shock and oxidative stress conditions, it appears that GmTRX represents a novel thioredoxin that is co-localized to the peroxisomes, possibly providing functional integrity to peroxisomal proteins.

Arabidopsis thaliana homeobox 12 (ATHB12), a homeodomain-leucine zipper protein, regulates leaf growth by promoting cell expansion and endoreduplication.

Arabidopsis thaliana homeobox 12 (ATHB12), a homeodomain-leucine zipper class I (HD-Zip I) gene, is highly expressed in leaves and stems, and induced by abiotic stresses, but its role in development remains obscure. To understand its function during plant development, we studied the effects of loss and gain of function. Expression of ATHB12 fused to the EAR-motif repression domain (SRDX) - P35 S ::ATHB12SRDX (A12SRDX) and PATHB 12 ::ATHB12SRDX - slowed both leaf and root growth, while the growth of ATHB12-overexpressing seedlings (A12OX) was accelerated. Microscopic examination revealed changes in the size and number of leaf cells. Ploidy was reduced in A12SRDX plants, accompanied by decreased cell expansion and increased cell numbers. By contrast, cell size was increased in A12OX plants, along with increased ploidy and elevated expression of cell cycle switch 52s (CCS52s), which are positive regulators of endoreduplication, indicating that ATHB12 promotes leaf cell expansion and endoreduplication. Overexpression of ATHB12 led to decreased phosphorylation of Arabidopsis thaliana ribosomal protein S6 (AtRPS6), a regulator of cell growth. In addition, induction of ATHB12 in the presence of cycloheximide increased the expression of several genes related to cell expansion, such as EXPANSIN A10 (EXPA10) and DWARF4 (DWF4). Our findings strongly suggest that ATHB12 acts as a positive regulator of endoreduplication and cell growth during leaf development.

Ribosomal protein S6, a target of rapamycin, is involved in the regulation of rRNA genes by possible epigenetic changes in Arabidopsis.

The target of rapamycin (TOR) kinase pathway regulates various biological processes, including translation, synthesis of ribosomal proteins, and transcription of rRNA. The ribosomal protein S6 (RPS6) is one of the well known downstream components of the TOR pathway. Ribosomal proteins have been known to have diverse functions in regulating cellular metabolism as well as protein synthesis. So far, however, little is known about other possible role(s) of RPS6 in plants, besides being a component of the 40 S ribosomal subunit and acting as a target of TOR. Here, we report that RPS6 may have a novel function via interaction with histone deacetylase 2B (AtHD2B) that belongs to the plant-specific histone deacetylase HD2 family. RPS6 and AtHD2B were localized to the nucleolus. Co-expression of RPS6 and AtHD2B caused a change in the location of both RPS6 and AtHD2B to one or several nucleolar spots. ChIP analysis suggests that RPS6 directly interacts with the rRNA gene promoter. Protoplasts overexpressing both AtHD2B and RPS6 exhibited down-regulation of pre-18 S rRNA synthesis with a concomitant decrease in transcription of some of the ribosomal proteins, suggesting their direct role in ribosome biogenesis and plant development. This is consistent with the mutation in rps6b that results in reduction in 18 S rRNA transcription and decreased root growth. We propose that the interaction between RPS6 and AtHD2B brings about a change in the chromatin structure of rDNA and thus plays an important role in linking TOR signaling to rDNA transcription and ribosome biogenesis in plants.

Functional implication of ß-carotene hydroxylases in soybean nodulation.

Legume-Rhizobium spp. symbiosis requires signaling between the symbiotic partners and differential expression of plant genes during nodule development. Previously, we cloned a gene encoding a putative ß-carotene hydroxylase (GmBCH1) from soybean (Glycine max) whose expression increased during nodulation with Bradyrhizobium japonicum. In this work, we extended our study to three GmBCHs to examine their possible role(s) in nodule development, as they were additionally identified as nodule specific, along with the completion of the soybean genome. In situ hybridization revealed the expression of three GmBCHs (GmBCH1, GmBCH2, and GmBCH3) in the infected cells of root nodules, and their enzymatic activities were confirmed by functional assays in Escherichia coli. Localization of GmBCHs by transfecting Arabidopsis (Arabidopsis thaliana) protoplasts with green fluorescent protein fusions and by electron microscopic immunogold detection in soybean nodules indicated that GmBCH2 and GmBCH3 were present in plastids, while GmBCH1 appeared to be cytosolic. RNA interference of the GmBCHs severely impaired nitrogen fixation as well as nodule development. Surprisingly, we failed to detect zeaxanthin, a product of GmBCH, or any other carotenoids in nodules. Therefore, we examined the possibility that most of the carotenoids in nodules are converted or cleaved to other compounds. We detected the expression of some carotenoid cleavage dioxygenases (GmCCDs) in wild-type nodules and also a reduced amount of zeaxanthin in GmCCD8-expressing E. coli, suggesting cleavage of the carotenoid. In view of these findings, we propose that carotenoids such as zeaxanthin synthesized in root nodules are cleaved by GmCCDs, and we discuss the possible roles of the carotenoid cleavage products in nodulation.

RNA interference-mediated repression of S6 kinase 1 impairs root nodule development in soybean.

Symbiotic nodule formation on legume roots is characterized with a series of developmental reprograming in root tissues, including extensive proliferation of cortical cells. We examined a possible involvement of the target of rapamycin (TOR) pathway, a central regulator of cell growth and proliferation in animals and yeasts, during soybean nodule development. Our results show that transcription of both GmTOR and its key downstream effector, GmS6K1, are activated during nodulation, which is paralleled with higher kinase activities of these gene products as well. RNAi-mediated knockdown of GmS6K1 impaired the nodule development with severely reduced nodule weight and numbers. In addition, expression of a few nodulins including leghemoglobin was also decreased, and consequently nitrogen fixation was found to be reduced by half. Proteomic analysis of the GmS6K1-RNAi nodules identified glutamine synthetase (GS), an essential enzyme for nitrogen assimilation in nodules, as one of the proteins that are significantly down regulated. These results appear to provide solid evidence for a functional link between GmS6K1 and nodule development.

Possible dual regulatory circuits involving AtS6K1 in the regulation of plant cell cycle and growth.

The role of Arabidopsis S6 Kinase 1 (AtS6K1), a downstream target of TOR kinase, in controlling plant growth and ribosome biogenesis was characterized after generating transgenic plants expressing AtS6K1 under auxin-inducible promoter. Down regulation of selected cell cycle regulatory genes upon auxin treatment was observed in the transgenic plants, confirming the negative regulatory role of AtS6K1 in the plant cell cycle progression reported earlier. Callus tissues established from these transgenic plants grew to larger cell masses with more number of enlarged cells than untransformed control, demonstrating functional implication of AtS6K1 in the control of plant cell size. The observed negative correlation between the expression of AtS6K1 and the cell cycle regulatory genes, however, was completely reversed in protoplasts generated from the transgenic plants expressing AtS6K1, suggesting a possible existence of dual regulatory mechanism of the plant cell cycle regulation mediated by AtS6K1. An alternative method of kinase assay, termed "substrate-mediated kinase pull down", was employed to examine the additional phosphorylation on other domains of AtS6K1 and verified the phosphorylation of both amino- and carboxy-terminal domains, which is a novel finding regarding the phosphorylation target sites on plant S6Ks by upstream regulatory kinases. In addition, this kinase assay under the stress conditions revealed the salt- and sugar-dependencies of AtS6K1 phosphorylations.

The Arabidopsis thaliana homeobox gene ATHB12 is involved in symptom development caused by geminivirus infection.

BACKGROUND: Geminiviruses are single-stranded DNA viruses that infect a number of monocotyledonous and dicotyledonous plants. Arabidopsis is susceptible to infection with the Curtovirus, Beet severe curly top virus (BSCTV). Infection of Arabidopsis with BSCTV causes severe symptoms characterized by stunting, leaf curling, and the development of abnormal inflorescence and root structures. BSCTV-induced symptom development requires the virus-encoded C4 protein which is thought to interact with specific plant-host proteins and disrupt signaling pathways important for controlling cell division and development. Very little is known about the specific plant regulatory factors that participate in BSCTV-induced symptom development. This study was conducted to identify specific transcription factors that are induced by BSCTV infection. METHODOLOGY/PRINCIPAL FINDINGS: Arabidopsis plants were inoculated with BSCTV and the induction of specific transcription factors was monitored using quantitative real-time polymerase chain reaction assays. We found that the ATHB12 and ATHB7 genes, members of the homeodomain-leucine zipper family of transcription factors previously shown to be induced by abscisic acid and water stress, are induced in symptomatic tissues of Arabidopsis inoculated with BSCTV. ATHB12 expression is correlated with an array of morphological abnormalities including leaf curling, stunting, and callus-like structures in infected Arabidopsis. Inoculation of plants with a BSCTV mutant with a defective c4 gene failed to induce ATHB12. Transgenic plants expressing the BSCTV C4 gene exhibited increased ATHB12 expression whereas BSCTV-infected ATHB12 knock-down plants developed milder symptoms and had lower ATHB12 expression compared to the wild-type plants. Reporter gene studies demonstrated that the ATHB12 promoter was responsive to BSCTV infection and the highest expression levels were observed in symptomatic tissues where cell cycle genes also were induced. CONCLUSIONS/SIGNIFICANCE: These results suggest that ATHB7 and ATHB12 may play an important role in the activation of the abnormal cell division associated with symptom development during geminivirus infection.

ATHB12, an ABA-inducible homeodomain-leucine zipper (HD-Zip) protein of Arabidopsis, negatively regulates the growth of the inflorescence stem by decreasing the expression of a gibberellin 20-oxidase gene.

Arabidopsis thaliana homeobox 12 (ATHB12) is rapidly induced by ABA and water stress. A T-DNA insertion mutant of ATHB12 with a reduced level of ATHB12 expression in stems had longer inflorescence stems and reduced sensitivity to ABA during germination. A high level of transcripts of gibberellin 20-oxidase 1 (GA20ox1), a key enzyme in the synthesis of gibberellins, was detected in athb12 stems, while transgenic lines overexpressing ATHB12 (A12OX) had a reduced level of GA20ox1 in stems. Consistent with these data, ABA treatment of wild-type plants resulted in decreased GA20ox1 expression whereas ABA treatment of the athb12 mutant gave rise to slightly decreased GA20ox1 expression. Retarded stem growth in 3-week-old A12OX plants was rescued by exogenous GA(9), but not by GA(12), and less GA(9) was detected in A12OX stems than in wild-type stems. These data imply that ATHB12 decreases GA20ox1 expression in stems. On the other hand, the stems of A12OX plants grew rapidly after the first 3 weeks, so that they were almost as high as wild-type plants at about 5 weeks after germination. We also found changes in the stems of transgenic plants overexpressing ATHB12, such as alterations of expression GA20ox and GA3ox genes, and of GA(4) levels, which appear to result from feedback regulation. Repression of GA20ox1 by ATHB12 was confirmed by transfection of leaf protoplasts. ABA-treated protoplasts also showed increased ATHB12 expression and reduced GA20ox1 expression. These findings all suggest that ATHB12 negatively regulates the expression of a GA 20-oxidase gene in inflorescence stems.

Identification of uricase as a potential target of plant thioredoxin: Implication in the regulation of nodule development.

During symbiotic nodule development in legume roots, early signaling events between host and rhizobia serve critical determinants for the proper onset of nodule morphogenesis, nitrogen fixation, and assimilation. Previously we isolated thioredoxin from soybean nodules as one of differentially expressed genes during nodulation and noted its positive role in nitrogen fixation. To identify the target proteins of thioredoxin in nodules, we used thioredoxin affinity chromatography followed by mass spectrometry. Nodulin-35, a subunit of uricase, was found to be a target of thioredoxin. Their interaction was confirmed by pull-down assay and by bimolecular fluorescent complementation. With an increased uricase activity observed also in the presence of thioredoxin, these results appear to implicate a novel role of thioredoxin in the regulation of enzyme activities involved in nodule development and nitrogen fixation.

An atypical soybean leucine-rich repeat receptor-like kinase, GmLRK1, may be involved in the regulation of cell elongation.

A leucine-rich repeat receptor-like kinase (LRR-RLK) encoded by one of the genes highly expressed in a specific stage of soybean seed development, referred to as GmLRK1, was identified and characterized. Examination of its kinase domain indicated that GmLRK1 may be a catalytically inactive atypical receptor kinase. An autophosphorylation assay confirmed that GmLRK1 is incapable of autophosphorylation in vitro. However, the phosphorylation of GmRLK1 could be induced after incubation with plant protein extracts, suggesting that some plant proteins may interact with GmLRK1 and phosphorylate the protein in vivo. Analyses of the expression profiles of GmLRK1 and its Arabidopsis ortholog At2g36570 revealed that they may be involved in regulation of more fundamental metabolic and/or developmental pathways, rather than a specialized developmental program such as seed development. Our results further indicate that the GmLRK1 and At2g36570 may play a role in the regulation of certain cellular processes that lead to cell elongation and expansion.

Increased expression of the F(1)F(o) ATP synthase in response to iron in heart mitochondria.

The objective of the present study was to identify mitochondrial components associated with the damage caused by iron to the rat heart. Decreased cell viability was assessed by increased presence of lactate dehydrogenase (LDH) in serum. To assess the functional integrity of mitochondria, Reactive Oxygen Species (ROS), the Respiratory Control Ratio (RCR), ATP and chelatable iron content were measured in the heart. Chelatable iron increased 15-fold in the mitochondria and ROS increased by 59%. Deterioration of mitochondrial function in the presence of iron was demonstrated by low RCR (46% decrease) and low ATP content (96% decrease). Using two dimensional gel electrophoresis (2DE), we identified alterations in 21 mitochondrial proteins triggered by iron overload. Significantly, expression of the alpha, beta, and d subunits of F(1)F(o) ATP synthase increased along with the loss of ATP. This suggests that the F(1)F(o) ATP synthase participates in iron metabolism.

Arabidopsis R2R3-MYB transcription factor AtMYB60 functions as a transcriptional repressor of anthocyanin biosynthesis in lettuce (Lactuca sativa).

The MYB transcription factors play important roles in the regulation of many secondary metabolites at the transcriptional level. We evaluated the possible roles of the Arabidopsis R2R3-MYB transcription factors in flavonoid biosynthesis because they are induced by UV-B irradiation but their associated phenotypes are largely unexplored. We isolated their genes by RACE-PCR, and performed transgenic approach and metabolite analyses in lettuce (Lactuca sativa). We found that one member of this protein family, AtMYB60, inhibits anthocyanin biosynthesis in the lettuce plant. Wild-type lettuce normally accumulates anthocyanin, predominantly cyanidin and traces of delphinidin, and develops a red pigmentation. However, the production and accumulation of anthocyanin pigments in AtMYB60-overexpressing lettuce was inhibited. Using RT-PCR analysis, we also identified the complete absence or reduction of dihydroflavonol 4-reductase (DFR) transcripts in AtMYB60- overexpressing lettuce (AtMYB60-117 and AtMYB60-112 lines). The correlation between the overexpression of AtMYB60 and the inhibition of anthocyanin accumulation suggests that the transcription factorAtMYB60 controls anthocyanin biosynthesis in the lettuce leaf. Clarification of the roles of the AtMYB60 transcription factor will facilitate further studies and provide genetic tools to better understand the regulation in plants of the genes controlled by the MYB-type transcription factors. Furthermore, the characterization of AtMYB60 has implications for the development of new varieties of lettuce and other commercially important plants with metabolic engineering approaches.