Root Meristem Maintenance Research Articles

The Arabidopsis splicing factor PORCUPINE/SmE1 orchestrates temperature-dependent root development via auxin homeostasis maintenance.

Appropriate abiotic stress response is pivotal for plant survival and makes use of multiple signaling molecules and phytohormones to achieve specific and fast molecular adjustments. A multitude of studies has highlighted the role of alternative splicing in response to abiotic stress, including temperature, emphasizing the role of transcriptional regulation for stress response. Here we investigated the role of the core-splicing factor PORCUPINE (PCP) on temperature-dependent root development. We used marker lines and transcriptomic analyses to study the expression profiles of meristematic regulators and mitotic markers, and chemical treatments, as well as root hormone profiling to assess the effect of auxin signaling. The loss of PCP significantly alters RAM architecture in a temperature-dependent manner. Our results indicate that PCP modulates the expression of central meristematic regulators and is required to maintain appropriate levels of auxin in the RAM. We conclude that alternative pre-mRNA splicing is sensitive to moderate temperature fluctuations and contributes to root meristem maintenance, possibly through the regulation of phytohormone homeostasis and meristematic activity.

On salt stress, PLETHORA signaling maintains root meristems.

Salt stress is one of the unfavorable environmental factors to affect plants. Salinity represses root growth, resulting in reduced biomass of agricultural plants. Little is known about how plants maintain root growth to counteract salt stress. The AP2-domain transcription factors PLETHORA1/2 (PLT1/2) act as master regulators in root meristem maintenance in Arabidopsis. In this study, we report that the salt overly sensitive (SOS) pathway component SOS2 regulates PLT1/2 at the post-transcriptional level. Salt-activated SOS2 interacts and phosphorylates PLT1/2 through their conserved C-terminal motifs to stabilize PLT1/2, critical for root apical meristem maintenance under salt stress. The phospho-mimetic version of PLT1/2 restored meristem and primary root length reduction of sos2-2 and plt1-4 plt2-2 mutants on salt treatment. Moreover, SOS2-mediated PLT1/2 phosphorylation improves root growth recovery after salt stress alleviation. We identify a SOS2-PLT1/2 core protein module that is required for protecting primary root growth and meristem maintenance from salt stress.

AtHSPR functions in gibberellin-mediated primary root growth by interacting with KNAT5 and OFP1 in Arabidopsis.

AtHSPR forms a complex with KNAT5 and OFP1 to regulate primary root growth through GA-mediated root meristem activity. KNAT5-OFP1 functions as a negative regulator of AtHSPR in response to GA. Plant root growth is modulated by gibberellic acid (GA) signaling and depends on root meristem maintenance. ARABIDOPSIS THALIANA HEAT SHOCK PROTEIN-RELATED (AtHSPR) is a vital regulator of flowering time and salt stress tolerance. However, little is known about the role of AtHSPR in the regulation of primary root growth. Here, we report that athspr mutant exhibits a shorter primary root compared to wild type and that AtHSPR interacts with KNOTTED1-LIKE HOMEOBOX GENE 5 (KNAT5) and OVATE FAMILY PROTEIN 1 (OFP1). Genetic analysis showed that overexpression of KNAT5 or OFP1 caused a defect in primary root growth similar to that of the athspr mutant, but knockout of KNAT5 or OFP1 rescued the short root phenotype in the athspr mutant by altering root meristem activity. Further investigation revealed that KNAT5 interacts with OFP1 and that AtHSPR weakens the inhibition of GIBBERELLIN 20-OXIDASE 1 (GA20ox1) expression by the KNAT5-OFP1 complex. Moreover, root meristem cell proliferation and root elongation in 35S::KNAT5athspr and 35S::OFP1athspr seedlings were hypersensitive to GA3 treatment compared to the athspr mutant. Together, our results demonstrate that the AtHSPR-KNAT5-OFP1 module regulates root growth and development by impacting the expression of GA biosynthetic gene GA20ox1, which could be a way for plants to achieve plasticity in response to the environment.

The ratio of auxin to cytokinin controls leaf development and meristem initiation in Physcomitrium patens.

Crosstalk between auxin and cytokinin contributes to widespread developmental processes, including root and shoot meristem maintenance, phyllotaxy, and vascular patterning. However, our understanding of crosstalk between these hormones is limited primarily to angiosperms. The moss Physcomitrium patens (formerly Physcomitrella patens) is a powerful system for studying plant hormone function. Auxin and cytokinin play similar roles in regulating moss gametophore (shoot) architecture, to those in flowering plant shoots. However, auxin-cytokinin crosstalk is poorly understood in moss. Here we find that the ratio of auxin to cytokinin is an important determinant of development in P. patens, especially during leaf development and branch stem cell initiation. Addition of high levels of auxin to P. patens gametophores blocks leaf outgrowth. However, simultaneous addition of high levels of both auxin and cytokinin partially restores leaf outgrowth, suggesting that the ratio of these hormones is the predominant factor. Likewise, during branch initiation and outgrowth, chemical inhibition of auxin synthesis phenocopies cytokinin application. Finally, cytokinin-insensitive mutants resemble plants with altered auxin signaling and are hypersensitive to auxin. In summary, our results suggest that the ratio between auxin and cytokinin signaling is the basis for developmental decisions in the moss gametophore.

Bacterial-type ferroxidase tunes iron-dependent phosphate sensing during Arabidopsis root development

SummaryAccess to inorganic phosphate (Pi), a principal intermediate of energy and nucleotide metabolism, profoundly affects cellular activities and plant performance. In most soils, antagonistic Pi-metal interactions restrict Pi bioavailability, which guides local root development to maximize Pi interception. Growing root tips scout the essential but immobile mineral nutrient; however, the mechanisms monitoring external Pi status are unknown. Here, we show that Arabidopsis LOW PHOSPHATE ROOT 1 (LPR1), one key determinant of Fe-dependent Pi sensing in root meristems, encodes a novel ferroxidase of high substrate specificity and affinity (apparent KM ∼ 2 μM Fe2+). LPR1 typifies an ancient, Fe-oxidizing multicopper protein family that evolved early upon bacterial land colonization. The ancestor of streptophyte algae and embryophytes (land plants) acquired LPR1-type ferroxidase from soil bacteria via horizontal gene transfer, a hypothesis supported by phylogenomics, homology modeling, and biochemistry. Our molecular and kinetic data on LPR1 regulation indicate that Pi-dependent Fe substrate availability determines LPR1 activity and function. Guided by the metabolic lifestyle of extant sister bacterial genera, we propose that Arabidopsis LPR1 monitors subtle concentration differentials of external Fe availability as a Pi-dependent cue to adjust root meristem maintenance via Fe redox signaling and cell wall modification. We further hypothesize that the acquisition of bacterial LPR1-type ferroxidase by embryophyte progenitors facilitated the evolution of local Pi sensing and acquisition during plant terrestrialization.

Dissection of grasspea (Lathyrus sativus L.) root exoproteome reveals critical insights and novel proteins

The plant exoproteome is crucial because its constituents greatly influence plant phenotype by regulating physiological characteristics to adapt to environmental stresses. The root exudates constitute a dynamic aspect of plant exoproteome, as its molecular composition ensures a beneficial rhizosphere in a species-specific manner. We investigated the root exoproteome of grasspea, a stress-resilient pulse and identified 2861 non-redundant proteins, belonging to a myriad of functional classes, including root development, rhizosphere augmentation as well as defense functions against soil-borne pathogens. Significantly, we identified 1986 novel exoproteome constituents of grasspea, potentially involved in cell-to-cell communication and root meristem maintenance, among other critical roles. Sequence-based comparison revealed that grasspea shares less than 30 % of its exoproteome with the reports so far from model plants as well as crop species. Further, the exoproteome revealed 65 % proteins to be extracellular in nature and of these, 37 % constituents were predicted to follow unconventional protein secretion (UPS) mode. We validated the UPS for four stress-responsive proteins, which were otherwise predicted to follow classical protein secretion (CPS). Conclusively, we recognized not only the highest number of root exudate proteins, but also pinpointed novel signatures of dicot root exoproteome.

Plant signaling: Interplay of brassinosteroids and auxin in root meristems

The plant hormones brassinosteroids (BRs) and auxin interact to regulate root meristem size. A new study reveals dual and opposing roles for BRs in auxin biosynthesis and signaling output in the Arabidopsis root epidermis, demonstrating that epidermis-derived BR signaling is required for root meristem maintenance through its effect on auxin signaling.

Autophagy in plants: Physiological roles and post-translational regulation.

In eukaryotes, autophagy helps maintain cellular homeostasis by degrading and recycling cytoplasmic materials via a tightly regulated pathway. Over the past few decades, significant progress has been made towards understanding the physiological functions and molecular regulation of autophagy in plant cells. Increasing evidence indicates that autophagy is essential for plant responses to several developmental and environmental cues, functioning in diverse processes such as senescence, male fertility, root meristem maintenance, responses to nutrient starvation, and biotic and abiotic stress. Recent studies have demonstrated that, similar to nonplant systems, the modulation of core proteins in the plant autophagy machinery by posttranslational modifications such as phosphorylation, ubiquitination, lipidation, S-sulfhydration, S-nitrosylation, and acetylation is widely involved in the initiation and progression of autophagy. Here, we provide an overview of the physiological roles and posttranslational regulation of autophagy in plants.

Inference of the gene regulatory network acting downstream of CROWN ROOTLESS1 in rice reveals a regulatory cascade linking genes involved in auxin signaling, crown root initiation, and root meristem specification and maintenance.

Crown roots (CRs) are essential components of the rice root system. Several genes involved in CR initiation or development have been identified but our knowledge about how they organize to form a gene regulatory network (GRN) is still limited. To characterize the regulatory cascades acting during CR formation, we used a systems biology approach to infer the GRN controlling CR formation downstream of CROWN ROOTLESS1 (CRL1), coding for an ASL (asymmetric leaves-2-like)/LBD (LOB domain) transcription factor necessary for CR initiation. A time-series transcriptomic dataset was generated after synchronized induction of CR formation by dexamethasone-mediated expression of CRL1 expression in a crl1 mutant background. This time series revealed three different genome expression phases during the early steps of CR formation and was further exploited to infer a GRN using a dedicated algorithm. The predicted GRN was confronted with experimental data and 72% of the inferred links were validated. Interestingly, this network revealed a regulatory cascade linking CRL1 to other genes involved in CR initiation, root meristem specification and maintenance, such as QUIESCENT-CENTER-SPECIFIC HOMEOBOX, and in auxin signalling. This predicted regulatory cascade was validated in vivo using transient activation assays. Thus, the CRL1-dependant GRN reflects major gene regulation events at play during CR formation and constitutes a valuable source of discovery to better understand this developmental process.

Quantitative Early Auxin Root Proteomics Identifies GAUT10, a Galacturonosyltransferase, as a Novel Regulator of Root Meristem Maintenance

Auxin induces rapid gene expression changes throughout root development. How auxin-induced transcriptional responses relate to changes in protein abundance is not well characterized. This report identifies early auxin responsive proteins in roots at 30 min and 2 h after hormone treatment using a quantitative proteomics approach in which 3,514 proteins were reliably quantified. A comparison of the >100 differentially expressed proteins at each the time point showed limited overlap, suggesting a dynamic and transient response to exogenous auxin. Several proteins with established roles in auxin-mediated root development exhibited altered abundance, providing support for this approach. While novel targeted proteomics assays demonstrate that all six auxin receptors remain stable in response to hormone. Additionally, 15 of the top responsive proteins display root and/or auxin response phenotypes, demonstrating the validity of these differentially expressed proteins. Auxin signaling in roots dictates proteome reprogramming of proteins enriched for several gene ontology terms, including transcription, translation, protein localization, thigmatropism, and cell wall modification. In addition, we identified auxin-regulated proteins that had not previously been implicated in auxin response. For example, genetic studies of the auxin responsive protein galacturonosyltransferase 10 demonstrate that this enzyme plays a key role in root development. Altogether these data complement and extend our understanding of auxin response beyond that provided by transcriptome studies and can be used to uncover novel proteins that may mediate root developmental programs.

OPENER Is a Nuclear Envelope and Mitochondria Localized Protein Required for Cell Cycle Progression in Arabidopsis.

Currently one-third of the proteins encoded by the Arabidopsis (Arabidopsis thaliana) genome are of unknown function. Some of these unknown proteins are likely to be involved in uncharacterized vital biological processes. Evolutionarily conserved single copy genes in flowering plants have been shown to be enriched in essential housekeeping functions. This together with publicly available gene expression data allows for a focused search for uncharacterized essential genes. Here we identify an essential single copy gene called OPENER (OPNR) in Arabidopsis. We show that OPNR is predominantly expressed in actively dividing cells and performs essential functions in seed development and root meristem maintenance. Cell cycle tracking using 5-ethynyl-2'-deoxyuridine staining and fluorescent cell cycle markers together with the increased size of nucleolus and nucleus in opnr mutants indicate that OPNR is required for cell cycle progression through the S or G2 phases. Intriguingly, OPNR localizes to the nuclear envelope and mitochondria. Furthermore, the nuclear envelope localization of OPNR is dependent on its interaction with nuclear inner membrane Sad1/UNC-84 (SUN) domain proteins SUN1 and SUN2. Taken together our results open a line of investigation into an evolutionarily conserved essential cellular process occurring in both the nuclear envelopes and mitochondria of dividing cells.

The AAA-ATPase MIDASIN 1 Functions in Ribosome Biogenesis and Is Essential for Embryo and Root Development.

Ribosome biogenesis is an orchestrated process that relies on many assembly factors. The AAA-ATPase Midasin 1 (Mdn1) functions as a ribosome assembly factor in yeast (Saccharomyces cerevisiae), but the roles of MDN1 in Arabidopsis (Arabidopsis thaliana) are poorly understood. Here, we showed that the Arabidopsis null mutant of MDN1 is embryo-lethal. Using the weak mutant mdn1-1, which maintains viability, we found that MDN1 is critical for the regular pattern of auxin maxima in the globular embryo and functions in root meristem maintenance. By detecting the subcellular distribution of ribosome proteins, we noted that mdn1-1 impairs nuclear export of the pre-60S ribosomal particle. The processing of ribosomal precusor RNAs, including 35S, 27SB, and 20S, is also affected in this mutant. MDN1 physically interacts with PESCADILLO2 (PES2), an essential assembly factor of the 60S ribosome, and the observed mislocalization of PES2 in mdn1-1 further implied that MDN1 plays an indispensable role in 60S ribosome biogenesis. Therefore, the observed hypersensitivity of mdn1-1 to a eukaryotic translation inhibitor and high-sugar conditions might be associated with the defect in ribosome biogenesis. Overall, this work establishes a role of Arabidopsis MDN1 in ribosome biogenesis, which agrees with its roles in embryogenesis and root development.

Locally Sourced: Auxin Biosynthesis and Transport in the Root Meristem

Localized maxima of the plant hormone auxin are crucial to root development and meristem maintenance. In this issue of Developmental Cell, Brumos etal. used elegant genetic and grafting experiments to distinguish between the contributions of local and distal auxin sources to auxin maxima generation and root meristem maintenance.

Local Auxin Biosynthesis Is a Key Regulator of Plant Development

Auxin is a major phytohormone that controls numerous aspects of plant development and coordinates plant responses to the environment. Morphogenic gradients of auxin govern cell fate decisions and underlie plant phenotypic plasticity. Polar auxin transport plays a central role in auxin maxima generation. The discovery of the exquisite spatiotemporal expression patterns of auxin biosynthesis genes of the WEI8/TAR and YUC families suggested that local auxin production may contribute to the formation of auxin maxima. Herein, we systematically addressed the role of local auxin biosynthesis in plant development and responses to the stress phytohormone ethylene by manipulating spatiotemporal patterns of WEI8. Our study revealed that local auxin biosynthesis and transport act synergistically and are individually dispensable for root meristem maintenance. In contrast, flower fertility and root responses to ethylene require local auxin production that cannot be fully compensated for by transport in the generation of morphogenic auxin maxima.

Autophagy regulates glucose-mediated root meristem activity by modulating ROS production in Arabidopsis

ABSTRACTGlucose produced from photosynthesis is a key nutrient signal regulating root meristem activity in plants; however, the underlying mechanisms remain poorly understood. Here, we show that, by modulating reactive oxygen species (ROS) levels, the conserved macroautophagy/autophagy degradation pathway contributes to glucose-regulated root meristem maintenance. In Arabidopsis thaliana roots, a short exposure to elevated glucose temporarily suppresses constitutive autophagosome formation. The autophagy-defective autophagy-related gene (atg) mutants have enhanced tolerance to glucose, established downstream of the glucose sensors, and accumulate less glucose-induced ROS in the root tips. Moreover, the enhanced root meristem activities in the atg mutants are associated with improved auxin gradients and auxin responses. By acting with AT4G39850/ABCD1 (ATP-binding cassette D1; Formerly PXA1/peroxisomal ABC transporter 1), autophagy plays an indispensable role in the glucose-promoted degradation of root peroxisomes, and the atg mutant phenotype is partially rescued by the overexpression of ABCD1. Together, our findings suggest that autophagy is an essential mechanism for glucose-mediated maintenance of the root meristem.Abbreviation: ABA: abscisic acid; ABCD1: ATP-binding cassette D1; ABO: ABA overly sensitive; AsA: ascorbic acid; ATG: autophagy related; CFP: cyan fluorescent protein; Co-IP: co-immunoprecipitation; DAB: 3’,3’-diaininobenzidine; DCFH-DA: 2’,7’-dichlorodihydrofluorescin diacetate; DR5: a synthetic auxin response element consists of tandem direct repeats of 11 bp that included the auxin-responsive TGTCTC element; DZ: differentiation zone; EZ, elongation zone; GFP, green fluorescent protein; GSH, glutathione; GUS: β-glucuronidase; HXK1: hexokinase 1; H2O2: hydrogen peroxide; IAA: indole-3-acetic acid; IBA: indole-3-butyric acid; KIN10/11: SNF1 kinase homolog 10/11; MDC: monodansylcadaverine; MS: Murashige and Skoog; MZ: meristem zone; NBT: nitroblue tetrazolium; NPA: 1-N-naphtylphthalamic acid; OxIAA: 2-oxindole-3-acetic acid; PIN: PIN-FORMED; PLT: PLETHORA; QC: quiescent center; RGS1: Regulator of G-protein signaling 1; ROS: reactive oxygen species; SCR: SCARECROW; SHR, SHORT-ROOT; SKL: Ser-Lys-Leu; SnRK1: SNF1-related kinase 1; TOR: target of rapamycin; UPB1: UPBEAT1; WOX5: WUSCHEL related homeobox 5; Y2H: yeast two-hybrid; YFP: yellow fluorescent protein

Regulation of the stability of RGF1 receptor by the ubiquitin-specific proteases UBP12/UBP13 is critical for root meristem maintenance

ROOT MERISTEM GROWTH FACTOR (RGF) 1 is an important peptide hormone that regulates root growth. Upon binding to its receptor, RGFR1, RGF1 regulates the expression of two transcription factors, PLETHORA 1 and 2 (PLT1/2), to influence root meristem development. Here, we show that the ubiquitin-specific proteases UBP12 and UBP13 are positive regulators of root meristem development and that UBP13 interacts directly with RGF1 receptor (RGFR1) and its close homolog RGFR2. The ubp12,13 double-mutant root is completely insensitive to exogenous applied RGF1. Consistent with this result, RGF1-induced ubiquitination and turnover of RGFR1 protein were accelerated in ubp12,13-mutant plants but were delayed in transgenic plants overexpressing UBP13 Genetic analysis showed that PLT2 or RGFR1 overexpression partially rescued the short-root phenotype and the reduced cortical root meristem cell number in ubp12,13 plants. Together, our results demonstrate that UBP12/13 are regulators of the RGF1-RGFR1-PLT1/2 signaling pathway and that UBP12/13 can counteract RGF1-induced RGFR1 ubiquitination, stabilize RGFR1, and maintain root cell sensitivity to RGF1.

Sirtinol, a Sir2 protein inhibitor, affects stem cell maintenance and root development in Arabidopsis thaliana by modulating auxin-cytokinin signaling components

In Arabidopsis thaliana, besides several key transcription factors and chromatin modifiers, phytohormones auxin and cytokinin play pivotal role in shoot and root meristem maintenance, and lateral root (LR) development. Sirtinol, a chemical inhibitor of Sir2 proteins, is known to promote some auxin induced phenotypes in Arabidopsis. However, its effect on plant stem cell maintenance or organ formation remained unaddressed. Here we show that sirtinol affects meristem maintenance by altering the expression of key stem cell regulators, cell division and differentiation by modulating both auxin and cytokinin signaling in Arabidopsis thaliana. The expression of shoot stem cell niche related genes WUSCHEL (WUS) and CLAVATA3 (CLV3) was upregulated, whereas SHOOT MERISTEMLESS (STM) was downregulated in sirtinol treated seedlings. The expression level and domain of key root stem cell regulators PLETHORA (PLTs) and WUS-Related Homeobox 5 (WOX5) were altered in sirtinol treated roots. Sirtinol affects LR development by disturbing proper auxin transport and maxima formation, similar to 2,4-dichlorophenoxyacetic acid (2,4-D). Sirtinol also affects LR formation by altering cytokinin biosynthesis and signaling genes in roots. Therefore, sirtinol affects shoot and root growth, meristem maintenance and LR development by altering the expression of cytokinin-auxin signaling components, and regulators of stem cells, meristems, and LRs.

Q&A: How does peptide signaling direct plant development?

A significant part of the communication between plant cells is mediated by signaling peptides and their corresponding plasma membrane-localized receptor-like kinases. This communication mechanism serves as a key regulatory unit for coordination of plant growth and development. In the past years more peptide–receptor signaling pathways have been shown to regulate developmental processes, such as shoot and root meristem maintenance, seed formation, and floral abscission. More detailed understanding of the processes behind this regulation might also be helpful to increase the yield of crop plants.

Shared and distinct functions of the pseudokinase CORYNE (CRN) in shoot and root stem cell maintenance of Arabidopsis

Stem cell maintenance in plants depends on the activity of small secreted signaling peptides of the CLAVATA3/EMBRYO SURROUNDING REGION (CLE) family, which, in the shoot, act through at least three kinds of receptor complexes, CLAVATA1 (CLV1) homomers, CLAVATA2 (CLV2) / CORYNE (CRN) heteromers, and CLV1/CLV2/CRN multimers. In the root, the CLV2/CRN receptor complexes function in the proximal meristem to transmit signals from the CLE peptide CLE40. While CLV1 consists of an extracellular receptor domain and an intracellular kinase domain, CLV2, a leucine-rich repeat (LRR) receptor-like protein, and CRN, a protein kinase, have to interact to form a receptor-kinase complex. The kinase domain of CRN has been reported to be catalytically inactive, and it is not yet known how the CLV2/CRN complex can relay the perceived signal into the cells, and whether the kinase domain is necessary for signal transduction at all. In this study we show that the kinase domain of CRN is actively involved in CLV3 signal transduction in the shoot apical meristem of Arabidopsis, but it is dispensable for CRN protein function in root meristem maintenance. Hence, we provide an example of a catalytically inactive pseudokinase that is involved in two homologous pathways, but functions in distinctively different ways in each of them.

WOX3 in the scene: intimacy with hormones.

How do WOX genes function to regulate key developmental programs in plants? We’re aware of their love affair with hormones, but lack good evidence of direct cause-and-effect relationships linking transcriptional activity and hormonal changes. In this issue of Journal of Experimental Botany (pages 1677–1687), Cho et al. provide that evidence for rice OsWOX3A and gibberellic acid. The name WOX stands for WUSCHEL-related homeobox, named after the founding member of the group, Arabidopsis WUSCHEL (WUS) (Mayer et al., 1998). WOX transcription factors are plant-specific, homeodomain-containing transcriptional regulators known to function in several key plant developmental programs including embryo development, root and shoot apical meristem maintenance, lateral root and crown root development, tillering and vegetative growth, leaf blade development, vascular patterning, inflorescence development, floral organ development and seed development. A major outstanding question is how WOX genes function to regulate these important processes. WOX genes do appear to have a love affair with hormones since auxin, cytokinin, abscisic acid (ABA) and gibberellic acid (GA) have been implicated in their actions. However, most of these associations were inferred based on gene expression changes and fluorescent markers signaling changes in the activity of the corresponding hormones. Direct cause-and-effect relationships that demonstrate transcriptional activity of WOX genes linked to measured steady-state level hormonal changes, with classical reversal of effects with hormone applications, have not been well established. Now, Cho et al. (2016) have demonstrated that rice OsWOX3A is induced by GA and directly binds to the promoter of ent-kaurenoic acid oxidase (KAO), an enzyme involved in GA biosynthesis, repressing its activity. Transgenic rice plants overexpressing OsWOX3A became dwarf (Fig. 1), suggesting a defect in GA biosynthesis or signaling. By quantifying endogenous GA intermediates, Cho and colleagues were able to show that GA20 and GA1 levels decrease in these plants. Although in vivo data were not provided for the binding of OsWOX3A to the KAO promoter and other potential OsWOX3A binding sites in the GA pathway are still unclear, the induction of OsWOX3A by GA3 treatment, reversion of the dwarf phenotype in OsWOX3A transgenic plants by GA application, the measurement of GA intermediates and the interaction of OsWOX3A with the KAO promoter using yeast one-hybrid and EMSA assays are powerful pieces of evidence that together speak loudly for the involvement of OsWOX3A in gibberellin negative-feedback regulation. But then what is the contribution of GA to the Oswox3a (nal2/3) mutant phenotypes? What other hormones are involved in generating the pleiotropic defects? Fig. 1. OsWOX3A-OX transgenic plants in the field. Courtesy of Prof. Nam-Chon Paek.