CIN-like TCP13 is essential for plant growth regulation under dehydration stress.

KEY MESSAGE: A dehydration-inducible Arabidopsis CIN-like TCP gene, TCP13, acts as a key regulator of plant growth in leaves and roots under dehydration stress conditions. Plants modulate their shape and growth in response to environmental stress. However, regulatory mechanisms underlying the changes in shape and growth under environmental stress remain elusive. The CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family of transcription factors (TFs) are key regulators for limiting the growth of leaves through negative effect of auxin response. Here, we report that stress-inducible CIN-like TCP13 plays a key role in inducing morphological changes in leaves and growth regulation in leaves and roots that confer dehydration stress tolerance in Arabidopsis thaliana. Transgenic Arabidopsis plants overexpressing TCP13 (35Spro::TCP13OX) exhibited leaf rolling, and reduced leaf growth under osmotic stress. The 35Spro::TCP13OX transgenic leaves showed decreased water loss from leaves, and enhanced dehydration tolerance compared with their control counterparts. Plants overexpressing a chimeric repressor domain SRDX-fused TCP13 (TCP13pro::TCP13SRDX) showed severely serrated leaves and enhanced root growth. Transcriptome analysis of TCP13pro::TCP13SRDX transgenic plants revealed that TCP13 affects the expression of dehydration- and abscisic acid (ABA)-regulated genes. TCP13 is also required for the expression of dehydration-inducible auxin-regulated genes, INDOLE-3-ACETIC ACID5 (IAA5) and LATERAL ORGAN BOUNDARIES (LOB) DOMAIN 1 (LBD1). Furthermore, tcp13 knockout mutant plants showed ABA-insensitive root growth and reduced dehydration-inducible gene expression. Our findings provide new insight into the molecular mechanism of CIN-like TCP that is involved in both auxin and ABA response under dehydration stress.

The function of ETHYLENE RESPONSE FACTOR genes in the light-induced anthocyanin production of Arabidopsis thaliana leaves.

Plants grow under threats of environmental changes that could injure cellular viability and damage whole-plant physiology. To defend themselves against such threats, plants induce protective responses, including the production of defense molecules. The red/purple pigment anthocyanin is synthesized upon leaf and fruit development as well as environmental stimuli such as excess light exposure. Therefore, the anthocyanin biosynthesis is considered as a model signaling pathway of the integration of developmental and environmental responses. This integration is tightly regulated by transcription factors, but the integrative mode of these signaling pathways has received little attention. In this study, using an Arabidopsis mutant with mutation in two ETHYLENE RESPONSE FACTOR (ERF) genes, AtERF4 and AtERF8, we investigated the regulatory signaling pathway that leads to the production of anthocyanin in response to light. We detected the accumulation of anthocyanin in detached leaves after incubation on water under light illumination and intact leaves after being transferred into the strong light condition, suggesting that the photoinhibition mediated the production of anthocyanin. Our results demonstrated that the erf mutant decreased the rate and extent of the production of anthocyanin in association with changes of the transcript levels of anthocyanin-biosynthetic genes. As these ERF genes are known regulators of leaf senescence-the final stage of leaf development-we provide an insight into the ERF-mediated integration of two regulatory pathways of the light response and developmental age.

TCP4-dependent induction of CONSTANS transcription requires GIGANTEA in photoperiodic flowering in Arabidopsis.

Photoperiod is one of the most reliable environmental cues for plants to regulate flowering timing. In Arabidopsis thaliana, CONSTANS (CO) transcription factor plays a central role in regulating photoperiodic flowering. In contrast to posttranslational regulation of CO protein, still little was known about CO transcriptional regulation. Here we show that the CINCINNATA (CIN) clade of class II TEOSINTE BRANCHED 1/ CYCLOIDEA/ PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR (TCP) proteins act as CO activators. Our yeast one-hybrid analysis revealed that class II CIN-TCPs, including TCP4, bind to the CO promoter. TCP4 induces CO expression around dusk by directly associating with the CO promoter in vivo. In addition, TCP4 binds to another flowering regulator, GIGANTEA (GI), in the nucleus, and induces CO expression in a GI-dependent manner. The physical association of TCP4 with the CO promoter was reduced in the gi mutant, suggesting that GI may enhance the DNA-binding ability of TCP4. Our tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis identified all class II CIN-TCPs as the components of the in vivo TCP4 complex, and the gi mutant did not alter the composition of the TCP4 complex. Taken together, our results demonstrate a novel function of CIN-TCPs as photoperiodic flowering regulators, which may contribute to coordinating plant development with flowering regulation.

Generation of Triple-Transgenic Forsythia Cell Cultures as a Platform for the Efficient, Stable, and Sustainable Production of Lignans.

Sesamin is a furofuran lignan biosynthesized from the precursor lignan pinoresinol specifically in sesame seeds. This lignan is shown to exhibit anti-hypertensive activity, protect the liver from damages by ethanol and lipid oxidation, and reduce lung tumor growth. Despite rapidly elevating demand, plant sources of lignans are frequently limited because of the high cost of locating and collecting plants. Indeed, the acquisition of sesamin exclusively depends on the conventional extraction of particular Sesamum seeds. In this study, we have created the efficient, stable and sustainable sesamin production system using triple-transgenic Forsythia koreana cell suspension cultures, U18i-CPi-Fk. These transgenic cell cultures were generated by stably introducing an RNAi sequence against the pinoresinol-glucosylating enzyme, UGT71A18, into existing CPi-Fk cells, which had been created by introducing Sesamum indicum sesamin synthase (CYP81Q1) and an RNA interference (RNAi) sequence against pinoresinol/lariciresinol reductase (PLR) into F. koreanna cells. Compared to its transgenic prototype, U18i-CPi-Fk displayed 5-fold higher production of pinoresinol aglycone and 1.4-fold higher production of sesamin, respectively, while the wildtype cannot produce sesamin due to a lack of any intrinsic sesamin synthase. Moreover, red LED irradiation of U18i-CPi-Fk specifically resulted in 3.0-fold greater production in both pinoresinol aglycone and sesamin than production of these lignans under the dark condition, whereas pinoresinol production was decreased in the wildtype under red LED. Moreover, we developed a procedure for sodium alginate-based long-term storage of U18i-CPi-Fk in liquid nitrogen. Production of sesamin in U18i-CPi-Fk re-thawed after six-month cryopreservation was equivalent to that of non-cryopreserved U18i-CPi-Fk. These data warrant on-demand production of sesamin anytime and anywhere. Collectively, the present study provides evidence that U18i-CP-Fk is an unprecedented platform for efficient, stable, and sustainable production of sesamin, and shows that a transgenic and specific light-regulated Forsythia cell-based metabolic engineering is a promising strategy for the acquisition of rare and beneficial lignans.

Essences in metabolic engineering of lignan biosynthesis.

Lignans are structurally and functionally diverse phytochemicals biosynthesized in diverse plant species and have received wide attentions as leading compounds of novel drugs for tumor treatment and healthy diets to reduce of the risks of lifestyle-related non-communicable diseases. However, the lineage-specific distribution and the low-amount of production in natural plants, some of which are endangered species, hinder the efficient and stable production of beneficial lignans. Accordingly, the development of new procedures for lignan production is of keen interest. Recent marked advances in the molecular and functional characterization of lignan biosynthetic enzymes and endogenous and exogenous factors for lignan biosynthesis have suggested new methods for the metabolic engineering of lignan biosynthesis cascades leading to the efficient, sustainable, and stable lignan production in plants, including plant cell/organ cultures. Optimization of light conditions, utilization of a wide range of elicitor treatments, and construction of transiently gene-transfected or transgenic lignan-biosynthesizing plants are mainly being attempted. This review will present the basic and latest knowledge regarding metabolic engineering of lignans based on their biosynthetic pathways and biological activities, and the perspectives in lignan production via metabolic engineering.

The roles of ethylene and transcription factors in the regulation of onset of leaf senescence.

Leaf senescence is the last stage of leaf development and is accompanied by cell death. In contrast to senescence in individual organisms that leads to death, leaf senescence is associated with dynamic processes that include the translocation of nutrients from old leaves to newly developing or storage tissues within the same plant. The onset of leaf senescence is largely regulated by age and internal and external stimuli, which include the plant hormone ethylene. Earlier studies have documented the important role of ethylene in the regulation of leaf senescence. The production of ethylene coincides with the onset of leaf senescence, whereas the application of ethylene to plants induces precocious leaf senescence. Recently, many studies have described the components of ethylene signaling and biosynthetic pathways that are involved in modulating the onset of leaf senescence. Particularly, transcription factors (TFs) integrate ethylene signals with those from environmental and developmental factors to accelerate or delay leaf senescence. This review aims to discuss the regulatory cascade involving ethylene and TFs in the regulation of onset of leaf senescence.

A regulatory cascade involving class II ETHYLENE RESPONSE FACTOR transcriptional repressors operates in the progression of leaf senescence.

Leaf senescence is the final process of leaf development that involves the mobilization of nutrients from old leaves to newly growing tissues. Despite the identification of several transcription factors involved in the regulation of this process, the mechanisms underlying the progression of leaf senescence are largely unknown. Herein, we describe the proteasome-mediated regulation of class II ETHYLENE RESPONSE FACTOR (ERF) transcriptional repressors and involvement of these factors in the progression of leaf senescence in Arabidopsis (Arabidopsis thaliana). Based on previous results showing that the tobacco (Nicotiana tabacum) ERF3 (NtERF3) specifically interacts with a ubiquitin-conjugating enzyme, we examined the stability of NtERF3 in vitro and confirmed its rapid degradation by plant protein extracts. Furthermore, NtERF3 accumulated in plants treated with a proteasome inhibitor. The Arabidopsis class II ERFs AtERF4 and AtERF8 were also regulated by the proteasome and increased with plant aging. Transgenic Arabidopsis plants with enhanced expression of NtERF3, AtERF4, or AtERF8 showed precocious leaf senescence. Our gene expression and chromatin immunoprecipitation analyses suggest that AtERF4 and AtERF8 targeted the EPITHIOSPECIFIER PROTEIN/EPITHIOSPECIFYING SENESCENCE REGULATOR gene and regulated the expression of many genes involved in the progression of leaf senescence. By contrast, an aterf4 aterf8 double mutant exhibited delayed leaf senescence. Our results provide insight into the important role of class II ERFs in the progression of leaf senescence.

Basic helix-loop-helix transcription factors and regulation of alkaloid biosynthesis.

Transcription factors of the basic Helix-Loop-Helix (bHLH) family play a central role in cell proliferation, determination, and differentiation. In plants, the regulatory functions of bHLHs in phenylpropanoid biosynthesis have been well established with regard to other interacting-proteins; i.e., MYB and WD40 repeat proteins. On the other hand, those in alkaloid biosynthesis are greatly limited due to the limited distribution of alkaloids in plant species. Recently, several groups have reported the regulatory functions of bHLH in alkaloid biosynthesis: novel CjbHLH1 in isoquinoline alkaloid biosynthesis in Coptis japonica, and Jasmonate-inducible MYC2-type bHLHs in nicotine-alkaloid biosynthesis in Nicotiana plants and indole alkaloid biosynthesis in Catharanthus roseus. We report here the JA-inducibility of CjbHLH1 and discuss the similarity and differences of non-MYC2-resemblant CjbHLH1 and MYC2-type bHLHs in nicotine and indole alkaloid biosynthesis.

Generation of serrated and wavy petals by inhibition of the activity of TCP transcription factors in Arabidopsis thaliana.

The final shape of shoot lateral organs, namely, leaves and flowers, is determined by coordinated growth after the initiation of primordia from shoot meristems in seed plants. This coordination is achieved by the complex action of many transcription factors, which include the TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) family. We have recently reported that CINCINNATA-like (CIN-like) TCP genes act dose-dependently to regulate the flat and smooth morphology of leaves in Arabidopsis thaliana. In contrast, the roles of CIN-like TCP genes in flower development are poorly understood. In this report, using multiple tcp mutants and transgenic plants in which the activity of CIN-like TCP transcription factors is dominantly inhibited, we found that these TCPs regulate the smooth and flat morphology of petals. Based on these findings, we discuss a possible strategy to generate a fringed morphology in floricultural plants.

TCP transcription factors regulate the activities of ASYMMETRIC LEAVES1 and miR164, as well as the auxin response, during differentiation of leaves in Arabidopsis.

Coordination of the maintenance of the undifferentiated fate of cells in the shoot meristem and the promotion of cellular differentiation in plant organs is essential for the development of plant shoots. CINCINNATA-like (CIN-like) TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) transcription factors are involved in this coordination via the negative regulation of CUP-SHAPED COTYLEDON (CUC) genes, which regulate the formation of shoot meristems and the specification of organ boundaries. However, the molecular mechanism of the action of CIN-like TCPs is poorly understood. We show here that TCP3, a model of CIN-like TCPs of Arabidopsis thaliana, directly activates the expression of genes for miR164, ASYMMETRIC LEAVES1 (AS1), INDOLE-3-ACETIC ACID3/SHORT HYPOCOTYL2 (IAA3/SHY2), and SMALL AUXIN UP RNA (SAUR) proteins. Gain of function of these genes suppressed the formation of shoot meristems and resulted in the fusion of cotyledons, whereas their loss of function induced ectopic expression of CUC genes in leaves. Our results indicate that miR164, AS1, IAA3/SHY2, and SAUR partially but cooperatively suppress the expression of CUC genes. Since CIN-like TCP genes were revealed to act dose dependently in the differentiation of leaves, we propose that evolutionarily diverse CIN-like TCPs have important roles in the signaling pathways that generate different leaf forms, without having any lethal effects on shoots.

Functional analysis of Arabidopsis ethylene-responsive element binding protein conferring resistance to Bax and abiotic stress-induced plant cell death.

Arabidopsis (Arabidopsis thaliana) ethylene-responsive element binding protein (AtEBP) gene was isolated as a suppressor of Bax-induced cell death by functional screening in yeast (Saccharomyces cerevisiae). To further examine the cell death suppressive action of AtEBP in plant cells, we established transgenic tobacco (Nicotiana tabacum) plants overexpressing AtEBP as well as transgenic tobacco plants ectopically expressing mouse Bax protein under a dexamethasone-inducible promoter. We prepared the crosses of the selective lines of each transgenic plant, which were evaluated in terms of cell death suppression activity. Results indicate that AtEBP suppressed Bax-induced cell death in tobacco plants, an action also associated with a lowered level of ion leakage. Furthermore, tobacco Bright Yellow-2 cells overexpressing AtEBP conferred resistance to hydrogen peroxide (H(2)O(2)) and heat treatments. AtEBP protein localized in the nucleus and functioned as an in vivo transcription activator as confirmed in transient assays and experiments using stable transgenic system. Up-regulation of defense genes was observed in transgenic Arabidopsis plants overexpressing AtEBP. Based on the analysis of mRNA accumulation in ethylene-related mutants, the position of AtEBP in signaling pathway is presented.

Isolation of tobacco ubiquitin-conjugating enzyme cDNA in a yeast two-hybrid system with tobacco ERF3 as bait and its characterization of specific interaction.

Tobacco ETHYLENE-RESPONSIVE FACTOR3 (ERF3) is a member of the ERF-domain transcription factors and has a transcriptional repressor activity, whereas other ERF proteins show activation activity. To understand the regulation of ERF3-repressor activity, protein(s) were screened which interact with ERF3 in a yeast two-hybrid system. A partial sequence (B8) of NtUBC2, a tobacco ubiquitin-conjugating enzyme was isolated. This B8 specifically interacted with ERF3 in the yeast two-hybrid system. Further analyses revealed that the region unique to ERF3 interacted with B8. The physiological functions of NtUBC2 and the stability of ERF3 are discussed in relation to the regulation of the repression activity of ERF3.