BRANCHED1 Research Articles

Class I TCP transcription factors TCP14 and TCP15 promote axillary branching in Arabidopsis by counteracting the action of Class II TCP BRANCHED1.

Shoot branching is determined by a balance between factors that promote axillary bud dormancy and factors that release buds from the quiescent state. The TCP family of transcription factors is classified into two classes, Class I and Class II, which usually play different roles. While the role of the Class II TCP BRANCHED1 (BRC1) in suppressing axillary bud development in Arabidopsis thaliana has been widely explored, the function of Class I TCPs in this process remains unknown. We analyzed the role of Class I TCP14 and TCP15 in axillary branch development in Arabidopsis through a series of genetic and molecular studies. In contrast to the increased branch number shown by brc1 mutants, tcp14 tcp15 plants exhibit a reduced number of branches compared with wild-type. Our findings provide evidence that TCP14 and TCP15 act by counteracting BRC1 function through two distinct mechanisms. First, they indirectly reduce BRC1 expression levels. Additionally, TCP15 directly interacts with BRC1 decoying it from chromatin and thereby preventing the transcriptional activation of a set of BRC1-dependent genes. We describe a molecular mechanism by which Class I TCPs physically antagonize the action of the Class II TCP BRC1, aligning with their opposite roles in axillary bud development.

MtTCP18 Regulates Plant Structure in Medicago truncatula.

Plant structure has a large influence on crop yield formation, with branching and plant height being the important factors that make it up. We identified a gene, MtTCP18, encoding a TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factor highly conserved with Arabidopsis gene BRC1 (BRANCHED1) in Medicago truncatula. Sequence analysis revealed that MtTCP18 included a conserved basic helix-loop-helix (BHLH) motif and R domain. Expression analysis showed that MtTCP18 was expressed in all organs examined, with relatively higher expression in pods and axillary buds. Subcellular localization analysis showed that MtTCP18 was localized in the nucleus and exhibited transcriptional activation activity. These results supported its role as a transcription factor. Meanwhile, we identified a homozygous mutant line (NF14875) with a mutation caused by Tnt1 insertion into MtTCP18. Mutant analysis showed that the mutation of MtTCP18 altered plant structure, with increased plant height and branch number. Moreover, we found that the expression of auxin early response genes was modulated in the mutant. Therefore, MtTCP18 may be a promising candidate gene for breeders to optimize plant structure for crop improvement.

Long noncoding RNA-mediated epigenetic regulation of auxin-related genes controls shade avoidance syndrome in Arabidopsis.

The long noncoding RNA (lncRNA) AUXIN-REGULATED PROMOTER LOOP (APOLO) recognizes a subset of target loci across the Arabidopsis thaliana genome by forming RNA-DNA hybrids (R-loops) and modulating local three-dimensional chromatin conformation. Here, we show that APOLO regulates shade avoidance syndrome by dynamically modulating expression of key factors. In response to far-red (FR) light, expression of APOLO anti-correlates with that of its target BRANCHED1 (BRC1), a master regulator of shoot branching in Arabidopsis thaliana. APOLO deregulation results in BRC1 transcriptional repression and an increase in the number of branches. Accumulation of APOLO transcription fine-tunes the formation of a repressive chromatin loop encompassing the BRC1 promoter, which normally occurs only in leaves and in a late response to far-red light treatment in axillary buds. In addition, our data reveal that APOLO participates in leaf hyponasty, in agreement with its previously reported role in the control of auxin homeostasis through direct modulation of auxin synthesis gene YUCCA2, and auxin efflux genes PID and WAG2. We show that direct application of APOLO RNA to leaves results in a rapid increase in auxin signaling that is associated with changes in the plant response to far-red light. Collectively, our data support the view that lncRNAs coordinate shade avoidance syndrome in A. thaliana, and reveal their potential as exogenous bioactive molecules. Deploying exogenous RNAs that modulate plant-environment interactions may therefore become a new tool for sustainable agriculture.

A gene regulatory network critical for axillary bud dormancy directly controlled by Arabidopsis BRANCHED1.

The Arabidopsis thaliana transcription factor BRANCHED1 (BRC1) plays a pivotal role in the control of shoot branching as it integrates environmental and endogenous signals that influence axillary bud growth. Despite its remarkable activity as a growth inhibitor, the mechanisms by which BRC1 promotes bud dormancy are largely unknown. We determined the genome-wide BRC1 binding sites in vivo and combined these with transcriptomic data and gene co-expression analyses to identify bona fide BRC1 direct targets. Next, we integrated multi-omics data to infer the BRC1 gene regulatory network (GRN) and used graph theory techniques to find network motifs that control the GRN dynamics. We generated an open online tool to interrogate this network. A group of BRC1 target genes encoding transcription factors (BTFs) orchestrate this intricate transcriptional network enriched in abscisic acid-related components. Promoter::β-GLUCURONIDASE transgenic lines confirmed that BTFs are expressed in axillary buds. Transient co-expression assays and studies in planta using mutant lines validated the role of BTFs in modulating the GRN and promoting bud dormancy. This knowledge provides access to the developmental mechanisms that regulate shoot branching and helps identify candidate genes to use as tools to adapt plant architecture and crop production to ever-changing environmental conditions.

A Study of the Molecular Regulatory Network of VcTCP18 during Blueberry Bud Dormancy.

BRANCHED1 (BRC1) is a crucial member of the TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) gene family and is well known for playing a central role in shoot branching by controlling buds' paradormancy. However, the expression characteristics and molecular regulatory mechanism of BRC1 during blueberry bud dormancy are unclear. To shed light on these topics, shoots of three blueberry cultivars with different chilling requirements (CRs) were decapitated in summer to induce paradormancy release and subjected to different levels of chilling in winter to induce endodormancy release. The results showed that the high-CR cultivar 'Chandler' had the strongest apical dominance among the three cultivars; additionally, the expression of VcTCP18, which is homologous to BRC1, was the highest under both the decapitation treatment and low-temperature treatment. The 'Emerald' cultivar, with a low CR, demonstrated the opposite trend. These findings suggest that VcTCP18 plays a negative regulatory role in bud break and that there may be a correlation between the CR and tree shape. Through yeast 1-hybrid (Y1H) assays, we finally screened 21 upstream regulatory genes, including eight transcription factors: zinc-finger homeodomain protein 1/4/5/9, MYB4, AP2-like ethylene-responsive transcription factor AINTEGUMENTA (ANT), ASIL2-like, and bHLH035. It was found that these upstream regulatory genes positively or negatively regulated the expression of VcTCP18 based on the transcriptome expression profile. In summary, this study enriched our understanding of the regulatory network of BRCl during bud dormancy and provided new insights into the function of BRC1.

HY5 functions as a systemic signal by integrating BRC1-dependent hormone signaling in tomato bud outgrowth

Light plays an important role in determining plant architecture, which greatly influences crop yield. However, the precise mechanisms by which light signaling regulates bud outgrowth remain to be identified. Here, we show that light regulates bud outgrowth via both HY5 and brassinosteroid (BR)-dependent pathways in tomato. Inactivation of the red-light photoreceptor PHYB, or deficiencies in PHYB or the blue-light photoreceptor CRY1a, inhibits bud outgrowth and leads to decreased accumulation of HY5 protein and increased transcript level of BRANCHED1 (BRC1), a central integrator of branching signals. HY5, functioning as a mobile systemic signal from leaves, promotes bud outgrowth by directly suppressing BRC1 transcript and activating the transcript of BR biosynthesis genes within the lateral buds in tomato. Furthermore, BRC1 prevents the accumulation of cytokinin (CK) and gibberellin (GA) by directly inhibiting the transcript of CK synthesis gene LOG4, while increasing the transcript levels of CK and GA degradation genes (CKX7, GA2ox4, and GA2ox5), leading to an arrest of bud outgrowth. Moreover, bud outgrowth occurs predominantly in the day due to the suppression of BRC1 transcript by HY5. These findings demonstrate that light-inducible HY5 acts as a systemic signaling factor in fine-tuning the bud outgrowth of tomato.

Precise A∙T to G∙C base editing in the allotetraploid rapeseed (Brassica napus L.) genome.

Rapeseed is an important source of oilseed crop in the world. Achieving genetic improvement has always been the major goal in rapeseed production. Single nucleotide substitution is the basis of most genetic variation underpinning important agronomic traits. Nowadays, Cas-base editing acts as an efficient tool to mediate single-base substitution at the target site. In this study, four adenine base editors (ABE) were modified to achieve adenosine base editing at different genome sites in allotetraploid Brassica napus. We designed 18 small guide RNAsto target phytoene desaturase (PDS), acetolactate synthase (ALS), CLAVATA3 (CLV3), CLV2, TRANSPARENT TESTA12 (TT12), carotenoid isomerase (CRTISO), designated de-etiolated-2 (DET2), BRANCHED1 (BRC1), zeaxanthin epoxidase (ZEP) genes, respectively. Among the four ABE systems, pBGE17 had the highest base-editing efficiency, with an average editing efficiency of 3.51%. Target sequencing results revealed that the editing window ranged from A5 to A8 of the protospacer-adjacent motif (PAM) sequence. Moreover, the ABEmax-nCas9NG system with NG PAM was developed, with a base-editing efficiency of 1.22%. These results revealed that ABE system developed in this study could efficiently induce A to G substitution and the ABE-nCas9NG system could broaden editing window in oilseed rape.

Brassica juncea BRC1-1 induced by SD negatively regulates flowering by directly interacting with BjuFT and BjuFUL promoter.

Flowering is crucial for sexual reproductive success in angiosperms. The core regulatory factors, such as FT, FUL, and SOC1, are responsible for promoting flowering. BRANCHED 1 (BRC1) is a TCP transcription factor gene that plays an important role in the regulation of branching and flowering in diverse plant species. However, the functions of BjuBRC1 in Brassica juncea are largely unknown. In this study, four homologs of BjuBRC1 were identified and the mechanism by which BjuBRC1 may function in the regulation of flowering time was investigated. Amino acid sequence analysis showed that BjuBRC1 contained a conserved TCP domain with two nuclear localization signals. A subcellular localization assay verified the nuclear localization of BjuBRC1. Expression analysis revealed that BjuBRC1-1 was induced by short days and was expressed abundantly in the leaf, flower, and floral bud but not in the root and stem in B. juncea. Overexpression of BjuBRC1-1 in the Arabidopsis brc1 mutant showed that BjuBRC1-1 delayed flowering time. Bimolecular fluorescent complementary and luciferase complementation assays showed that four BjuBRC1 proteins could interact with BjuFT in vivo. Notably, BjuBRC1 proteins formed heterodimers in vivo that may impact on their function of negatively regulating flowering time. Yeast one-hybrid, dual-luciferase reporter, and luciferase activity assays showed that BjuBRC1-1 could directly bind to the promoter of BjuFUL, but not BjuFT or BjuSOC1, to repress its expression. These results were supported by the reduced expression of AtFUL in transgenic Arabidopsis overexpressing BjuBRC1-1. Taken together, the results indicate that BjuBRC1 genes likely have a conserved function in the negative regulation of flowering in B. juncea.

Comparative transcriptomics identifies candidate genes involved in the evolutionary transition from dehiscent to indehiscent fruits in Lepidium (Brassicaceae)

BackgroundFruits are the seed-bearing structures of flowering plants and are highly diverse in terms of morphology, texture and maturation. Dehiscent fruits split open upon maturation to discharge their seeds while indehiscent fruits are dispersed as a whole. Indehiscent fruits evolved from dehiscent fruits several times independently in the crucifer family (Brassicaceae). The fruits of Lepidium appelianum, for example, are indehiscent while the fruits of the closely related L. campestre are dehiscent. Here, we investigate the molecular and genetic mechanisms underlying the evolutionary transition from dehiscent to indehiscent fruits using these two Lepidium species as model system.ResultsWe have sequenced the transcriptomes and small RNAs of floral buds, flowers and fruits of L. appelianum and L. campestre and analyzed differentially expressed genes (DEGs) and differently differentially expressed genes (DDEGs). DEGs are genes that show significantly different transcript levels in the same structures (buds, flowers and fruits) in different species, or in different structures in the same species. DDEGs are genes for which the change in expression level between two structures is significantly different in one species than in the other. Comparing the two species, the highest number of DEGs was found in flowers, followed by fruits and floral buds while the highest number of DDEGs was found in fruits versus flowers followed by flowers versus floral buds. Several gene ontology terms related to cell wall synthesis and degradation were overrepresented in different sets of DEGs highlighting the importance of these processes for fruit opening. Furthermore, the fruit valve identity genes FRUITFULL and YABBY3 were among the DEGs identified. Finally, the microRNA miR166 as well as the TCP transcription factors BRANCHED1 (BRC1) and TCP FAMILY TRANSCRIPTION FACTOR 4 (TCP4) were found to be DDEGs.ConclusionsOur study reveals differences in gene expression between dehiscent and indehiscent fruits and uncovers miR166, BRC1 and TCP4 as candidate genes for the evolutionary transition from dehiscent to indehiscent fruits in Lepidium.

Cultivated strawberry HANABA TARANU: a tale of transcription factors, raging hormones, and asexual reproduction

Cultivated strawberry <scp><i>HANABA TARANU</i></scp>: a tale of transcription factors, raging hormones, and asexual reproduction

Shoot Branching Phenotyping in Arabidopsis and Tomato.

Shoot branching is an important trait that depends on the activity of axillary meristems and buds and their outgrowth into branches. It is remarkably plastic, being influenced by a number of external cues, such as light, temperature, soil nutrients, and mechanical manipulation. These are transduced into an internal hormone signaling network where auxin, cytokinins, and strigolactones play leading regulatory roles. Recently, sugars have also emerged as important signals promoting bud activation. These signals are in part integrated by the bud-specific growth repressor BRANCHED1 (BRC1).To understand how shoot branching is affected by particular growth conditions or in specific plant lines, it is necessary to count the number of branches and/or quantify other branch-related parameters. Here we describe how to perform such quantifications in Arabidopsis and in tomato.

Tissue-specific expression of barnase in tobacco delays axillary shoot development after topping.

Shoot branching is an important agronomic trait that determines plant architecture and affects crop productivity (Shen et al., 2019). Molecular signals from the shoot apical meristem (SAM) create a hormonal environment that integrates with the expression of axillary bud-specific repressors such as BRANCHED1 (BRC1) to inhibit axillary shoot formation (Wang et al., 2019 and references therein). The signal is eliminated by topping (SAM removal), enabling the formation of new shoots (suckers) from axillary buds (Figure 1a).

VvBRC inhibits shoot branching in grapevine

The BRANCHED1 (BRC1) gene has been reported as a key regulator of shoot branching in many model plants. However, its role in the shoot branching process of grapevine has not been described. In this present study, we isolated VvBRC, a member of the Teosinte branched 1/cycloidea/proliferating cell factor 1 (TCP) transcription factor family, from grapevine (Vitis vinifera L.). The VvBRC protein was localized in the nucleus. RT-qPCR analysis showed that VvBRC was primarily expressed in grapevine prompt and latent buds and stems. Moreover, VvBRC expression in grapevine prompt buds was suppressed in response to decapitation, and induced in response to hormones such as cytokinin and gibberellin. Heterologous overexpression of VvBRC inhibited plant shoot branching in transgenic Arabidopsis, and complementary expression restored the phenotype in mutant Arabidopsis thaliana. In addition, transcript levels of genes participating in ABA biosynthesis, such as NCED, and ABA signaling pathways, such as ABF3, ABI5, HB21, HB40, and HB53, were upregulated, whereas the transcript levels of genes involved in the cell cycle, such as CYCD3 and HISTONE H4, were downregulated in VvBRC-overexpressing plants. Collectively, these results suggest that VvBRC play critical and negative roles in grapevine shoot branching.

Brassinosteroid signaling integrates multiple pathways to release apical dominance in tomato

The control of apical dominance involves auxin, strigolactones (SLs), cytokinins (CKs), and sugars, but the mechanistic controls of this regulatory network are not fully understood. Here, we show that brassinosteroid (BR) promotes bud outgrowth in tomato through the direct transcriptional regulation of BRANCHED1 (BRC1) by the BR signaling component BRASSINAZOLE-RESISTANT1 (BZR1). Attenuated responses to the removal of the apical bud, the inhibition of auxin, SLs or gibberellin synthesis, or treatment with CK and sucrose, were observed in bud outgrowth and the levels of BRC1 transcripts in the BR-deficient or bzr1 mutants. Furthermore, the accumulation of BR and the dephosphorylated form of BZR1 were increased by apical bud removal, inhibition of auxin, and SLs synthesis or treatment with CK and sucrose. These responses were decreased in the DELLA-deficient mutant. In addition, CK accumulation was inhibited by auxin and SLs, and decreased in the DELLA-deficient mutant, but it was increased in response to sucrose treatment. CK promoted BR synthesis in axillary buds through the action of the type-B response regulator, RR10. Our results demonstrate that BR signaling integrates multiple pathways that control shoot branching. Local BR signaling in axillary buds is therefore a potential target for shaping plant architecture.

Functional Analysis of the teosinte branched 1 Gene in the Tetraploid Switchgrass (Panicum virgatum L.) by CRISPR/Cas9-Directed Mutagenesis.

Tillering is an important biomass yield component trait in switchgrass (Panicum virgatum L.). Teosinte branched 1 (tb1)/Branched 1 (BRC1) gene is a known regulator for tillering/branching in several plant species; however, its role on tillering in switchgrass remains unknown. Here, we report physiological and molecular characterization of mutants created by CRISPR/Cas9. We successfully obtained nonchimeric Pvtb1a and Pvtb1b mutants from chimeric T0 mutants using nodal culture. The biallelic Pvtb1a-Pvtb1b mutant plants produced significantly more tillers and higher fresh weight biomass than the wild-type plants. The increased tiller number in the mutant plants resulted primarily from hastened outgrowth of lower axillary buds. Increased tillers were also observed in transgene-free BC1 monoallelic mutants for either Pvtb1a-Pvtb1b or Pvtb1b gene alone, suggesting Pvtb1 genes act in a dosage-dependent manner. Transcriptome analysis showed 831 genes were differentially expressed in the Pvtb1a-Pvtb1b double knockdown mutant. Gene Ontology analysis revealed downregulation of Pvtb1 genes affected multiple biological processes, including transcription, flower development, cell differentiation, and stress/defense responses in edited plants. This study demonstrates that Pvtb1 genes play a pivotal role in tiller production as a negative regulator in switchgrass and provides opportunities for further research aiming to elucidate the molecular pathway regulating tillering in switchgrass.

Antagonistic regulation of axillary bud outgrowth by the BRANCHED genes in tobacco

As a key integrator of shoot branching, BRANCHED 1 (BRC1) coordinates and is orchestrated by endogenous and environmental signals involved in the regulation of axillary bud outgrowth. In the present study, we characterized the regulatory roles of five BRC gene members in tobacco (Nicotiana tabacum L.) using CRISPR site-directed mutagenesis and overexpression assays. It was shown that lateral branching was negatively regulated by NtBRC1A-1, 1B-1, and 1B-2, but was unexpectedly promoted by NtBRC2A. Suppression of bud growth may be attained by direct binding of NtBRCs to the Tassels Replace Upper Ears 1 (TRU1) genes. It was speculated that NtBRC2A probably confers a dominant negative effect by interfering with the branching-inhibitory BRC1 genes. Our results suggested that highly homologous gene family members may function antagonistically in the same signaling pathway. However, the molecular mechanism underlying NtBRC2A-mediated outgrowth of axillary buds needs to be further addressed. KEY MESSAGE: Axillary bud outgrowth in general is negatively regulated by the BRANCHED gene. Here we show that the BRANCHED genes play opposing regulatory roles in tobacco lateral branching.

Regulation by sugar and hormone signaling of the growth of Brassica napus L. axillary buds at the transcriptome level

Branching is an important factor affecting the yield of Brassica napus L. Generally, however, field-grown B. napus cannot develop branches from the middle and lower parts of the plant, although axillary buds are present in these parts. To explore the cause of this phenomenon at the mRNA level, we analyzed buds from the top, upper, middle and lower parts of B. napus by RNA-SEq. In total, 27,992 expressed genes were found, 479 of which were considered to be closely related to axillary bud development. Genes related to the regulation of sugar signaling and plant hormone signal transduction were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Seven types of transcription factors, including phytochrome-interacting factors (PIFs), homeobox 1 (HB-1), light sensitive hypocotyls 4 (LSH4), BRANCHED 1 (BRC1), WRKY DNA-binding proteins (WRKYs) and Transducin/WD40 repeat-like superfamily proteins (WD40s), were enriched. The soluble sugar content in the axillary buds was assessed and found to differ significantly among the four regions of the plant; in short, the soluble sugar content decreased from top to bottom. In this study, we provide a transcriptional profile of axillary buds from different parts of B. napus plants; these data will be important for future studies on the growth and development of axillary buds.

Branching Regulator BRC1 Mediates Photoperiodic Control of Seasonal Growth in Hybrid Aspen

Cessation of growth as winter approaches is a key adaptive trait for survival of perennial plants, such as long-lived trees native to boreal and temperate regions [1, 2]. The timing of growth cessation in these plants is controlled by photoperiodic cues. As shown recently, perception of growth-repressive short photoperiod (SP) mediated via components of circadian clock results indownregulation of the tree ortholog of Arabidopsis flowering regulator FLOWERING LOCUS T(FT), FT2[3, 4]. Downregulation of FT2 results in suppression of downstream components LAP1 (orthologousto the Arabidopsis floral meristem identity gene APETALA1) and AIL1 (orthologous to AINTEGUMENTA in Arabidopsis), culminating in induction of growth cessation and bud set [5-7]. Results presented here reveal that, in addition to the CO/FT pathway, a photoperiodically controlled negative feedback loop involving a tree ortholog of Arabidopsis BRANCHED1 (BRC1) (a member of TEOSINTE BRANCHED 1, CYCLOIDEA, PCF family), LAP1, and FT2 participates in regulation of seasonal growth in the model tree hybrid aspen. In growth-promotive long photoperiod, LAP1 suppresses expression of BRC1, but upon perception of growth-repressive SP, downregulation of LAP1 de-represses expression of its downstream target BRC1. BRC1 physically interacts with FT2, and BRC1-FT interaction further reinforces the effect of SP and triggers growth cessation by antagonizing FT action. Accordingly, BRC1 gain and loss of function result in early and retarded growth cessation responses to SP, respectively. Thus, these results reveal a regulatory feedback loop that reinforces responses to SP and induction of seasonal growth cessation.

CsBRC1 inhibits axillary bud outgrowth by directly repressing the auxin efflux carrier CsPIN3 in cucumber

Shoot branching is an important agronomic trait that directly determines plant architecture and affects crop productivity. To promote crop yield and quality, axillary branches need to be manually removed during cucumber production for fresh market and thus are undesirable. Auxin is well known as the primary signal imposing for apical dominance and acts as a repressor for lateral bud outgrowth indirectly. The TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family gene BRANCHED1 (BRC1) has been shown to be the central integrator for multiple environmental and developmental factors that functions locally to inhibit shoot branching. However, the direct molecular link between auxin and BRC1 remains elusive. Here we find that cucumber BRANCHED1 (CsBRC1) is expressed in axillary buds and displays a higher expression level in cultivated cucumber than in its wild ancestor. Knockdown of CsBRC1 by RNAi leads to increased bud outgrowth and reduced auxin accumulation in buds. We further show that CsBRC1 directly binds to the auxin efflux carrier PIN-FORMED (CsPIN3) and negatively regulates its expression in vitro and in vivo. Elevated expression of CsPIN3 driven by the CsBRC1 promoter results in highly branched cucumber with decreased auxin levels in lateral buds. Therefore, our data suggest that CsBRC1 inhibits lateral bud outgrowth by direct suppression of CsPIN3 functioning and thus auxin accumulation in axillary buds in cucumber, providing a strategy to breed for cultivars with varying degrees of shoot branching grown in different cucumber production systems.

Posttranscriptional Regulation of RhBRC1 (Rosa hybrida BRANCHED1) in Response to Sugars is Mediated via its Own 3' Untranslated Region, with a Potential Role of RhPUF4 (Pumilio RNA-Binding Protein Family).

The shoot branching pattern is a determining phenotypic trait throughout plant development. During shoot branching, BRANCHED1 (BRC1) plays a master regulator role in bud outgrowth, and its transcript levels are regulated by various exogenous and endogenous factors. RhBRC1 (the homologous gene of BRC1 in Rosa hybrida) is a main branching regulator whose posttranscriptional regulation in response to sugar was investigated through its 3′UTR. Transformed Rosa calluses containing a construction composed of the CaMV35S promoter, the green fluorescent protein (GFP) reporter gene, and the 3′UTR of RhBRC1 (P35S:GFP::3′UTRRhBRC1) were obtained and treated with various combinations of sugars and with sugar metabolism effectors. The results showed a major role of the 3′UTR of RhBRC1 in response to sugars, involving glycolysis/the tricarboxylic acid cycle (TCA) and the oxidative pentose phosphate pathway (OPPP). In Rosa vegetative buds, sequence analysis of the RhBRC1 3′UTR identified six binding motifs specific to the Pumilio/FBF RNA-binding protein family (PUF) and probably involved in posttranscriptional regulation. RhPUF4 was highly expressed in the buds of decapitated plants and in response to sugar availability in in-vitro-cultured buds. RhPUF4 was found to be close to AtPUM2, which encodes an Arabidopsis PUF protein. In addition, sugar-dependent upregulation of RhPUF4 was also found in Rosa calluses. RhPUF4 expression was especially dependent on the OPPP, supporting its role in OPPP-dependent posttranscriptional regulation of RhBRC1. These findings indicate that the 3′UTR sequence could be an important target in the molecular regulatory network of RhBRC1 and pave the way for investigating new aspects of RhBRC1 regulation.