Regulation of NB-LRR-type UNI and its related signaling pathway: Signaling crosstalk and methodology for quick identification of related factors

Activation of NB-LRR-related UNI proteins by uni-1D mutation, a gain-of-function mutation of the UNI gene, induces some pathogenesis-related responses and also affects morphology through modulation of meristem activities. In a recent study we reported that the uni-1D phenotypes require cooperative action of ERECTA (ER) receptor kinase family members in UNI-expressing cells, suggesting that an intracellular signaling crosstalk between ER-family-dependent and UNI-triggered signaling pathways plays a significant role in the phenotypes. Further we recently succeeded in the establishment of a methodology for rapid identification of factors involved in the UNI function. EMS-induced causal mutations that suppress the uni-1D phenotypes could be identified using whole-genome-sequencing technologies with much less labor compared with the conventional map-based cloning method that is generally time-consuming and labor-intensive. Thus it would be now possible to intensively identify factors that play significant roles in regulation of UNI proteins and/or UNI-related signaling pathways.Key words: NB-LRR, UNI, ERECTA, SGT1b, signaling crosstalk, mutation identification, EMS, whole genome sequencing, shoot apical meristem, axillary meristem     

RPT2a, a 26S proteasome AAA-ATPase, is directly involved in Arabidopsis CC-NBS-LRR protein uni-1D-induced signaling pathways

Arabidopsis semi-dominant uni-1D shows both constitutive defense responses and diverse morphological defects. In particular, uni-1D homozygote (uni-1D) mutants exhibit severe phenotypes including not only highly up-regulated pathogenesis related-1(PR-1) gene expression, but also lethality in the early stage of true leaf formation after germination. The gene responsible for the mutant encodes a coiled-coil-nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR)-type R protein that functions in the recognition of pathogen and the triggering of defense responses. However, the molecular basis of how uni-1D can induce these phenotypes was unknown. In this study, we isolated the regulatory particle triple-ATPase (RPT) subunits 2a and 2b, base components of the 19S regulatory particle in the 26S proteasome, as uni-1D-interacting proteins using yeast two-hybrid screening. Genetic studies showed that crossing with the rpt2a mutant reduces the level of uni-1D-induced PR-1 gene expression and suppresses the lethality of uni-1D, by leading to restoration of lost expression of the WUSCHEL gene, which functions to maintain meristem activity, in the shoot apical mersitem of uni-1D. These results suggest that RPT2a is a major interacting partner of uni-1D/UNI, and that the interaction between uni-1D and RPT2a is responsible for activating both morphology and defense signals.     

Constitutive activation of a CC-NB-LRR protein alters morphogenesis through the cytokinin pathway in Arabidopsis

Possible links between plant defense responses and morphogenesis have been postulated, but their molecular nature remains unknown. Here, we introduce the Arabidopsis semi-dominant mutant uni-1D with morphological defects. UNI encodes a coiled-coil nucleotide-binding leucine-rich-repeat protein that belongs to the disease resistance (R) protein family involved in pathogen recognition. The uni-1D mutation causes the constitutive activation of the protein, which is stabilized by the RAR1 function in a similar way as in other R proteins. The uni-1D mutation induces the upregulation of the Pathogenesis-related gene via the accumulation of salicylic acid, and evokes some of the morphological defects through the accumulation of cytokinin. The rin4 knock-down mutation, which causes the constitutive activation of two R proteins, RPS2 and RPM1, induces an upregulation of cytokinin-responsive genes and morphological defects similar to the uni-1D mutation, indicating that the constitutive activation of some R proteins alters morphogenesis through the cytokinin pathway. From these data, we propose that the modification of the cytokinin pathway might be involved in some R protein-mediated responses.     

SHEPHERD is the Arabidopsis GRP94 responsible for the formation of functional CLAVATA proteins

The Arabidopsis shepherd (shd) mutant shows expanded shoot apical meristems (SAM) and floral meristems (FM), disorganized root apical meristems, and defects in pollen tube elongation. We have discovered that SHD encodes an ortholog of GRP94, an ER-resident HSP90-like protein. Since the shd phenotypes in SAM and FM are similar to those of the clavata (clv) mutants, we have explored the possibility that CLV complex members could be SHD targets. The SAM and FM morphology of shd clv double mutants are indistinguishable from those of clv single mutants, and the wuschel (wus) mutation is completely epistatic to the shd mutation, indicating that SHD and CLV act in the same genetic pathway to suppress WUS function. Moreover, the effects of CLV3 overexpression that result in the elimination of SAM activity were abolished in the shd mutant, indicating that CLV function is dependent on SHD function. Therefore, we conclude that the SHD protein is required for the correct folding and/or complex formation of CLV proteins.     

LAX PANICLE2 of rice encodes a novel nuclear protein and regulates the formation of axillary meristems

Aerial architecture in higher plants is dependent on the activity of the shoot apical meristem (SAM) and axillary meristems (AMs). The SAM produces a main shoot and leaf primordia, while AMs are generated at the axils of leaf primordia and give rise to branches and flowers. Therefore, the formation of AMs is a critical step in the construction of plant architecture. Here, we characterized the rice (Oryza sativa) lax panicle2 (lax2) mutant, which has altered AM formation. LAX2 regulates the branching of the aboveground parts of a rice plant throughout plant development, except for the primary branch in the panicle. The lax2 mutant is similar to lax panicle1 (lax1) in that it lacks an AM in most of the lateral branching of the panicle and has a reduced number of AMs at the vegetative stage. The lax1 lax2 double mutant synergistically enhances the reduced-branching phenotype, indicating the presence of multiple pathways for branching. LAX2 encodes a nuclear protein that contains a plant-specific conserved domain and physically interacts with LAX1. We propose that LAX2 is a novel factor that acts together with LAX1 in rice to regulate the process of AM formation.     

The bHLH protein ROX acts in concert with RAX1 and LAS to modulate axillary meristem formation in Arabidopsis

During post-embryonic shoot development, new meristems are initiated in the axils of leaves. They produce secondary axes of growth that determine morphological plasticity and reproductive efficiency in higher plants. In this study, we describe the role of the bHLH-protein-encoding Arabidopsis gene REGULATOR OF AXILLARY MERISTEM FORMATION (ROX), which is the ortholog of the branching regulators LAX PANICLE1 (LAX1) in rice and barren stalk1 (ba1) in maize. rox mutants display compromised axillary bud formation during vegetative shoot development, and combination of rox mutants with mutations in RAX1 and LAS, two key regulators of axillary meristem initiation, enhances their branching defects. In contrast to lax1 and ba1, flower development is unaffected in rox mutants. Over-expression of ROX leads to formation of accessory side shoots. ROX mRNA accumulates at the adaxial boundary of leaf and flower primordia. However, in the vegetative phase, axillary meristems initiate after ROX expression has terminated, suggesting an indirect role for ROX in meristem formation. During vegetative development, ROX expression is dependent on RAX1 and LAS activity, and all three genes act in concert to modulate axillary meristem formation.     

The synergistic activation of FLOWERING LOCUS C by FRIGIDA and a new flowering gene AERIAL ROSETTE 1 underlies a novel morphology in Arabidopsis

The genetic changes underlying the diversification of plant forms represent a key question in understanding plant macroevolution. To understand the mechanisms leading to novel plant morphologies we investigated the Sy-0 ecotype of Arabidopsis that forms an enlarged basal rosette of leaves, develops aerial rosettes in the axils of cauline leaves, and exhibits inflorescence and floral reversion. Here we show that this heterochronic shift in reproductive development of all shoot meristems requires interaction between dominant alleles at AERIAL ROSETTE 1 (ART1), FRIGIDA (FRI), and FLOWERING LOCUS C (FLC) loci. ART1 is a new flowering gene that maps 14 cM proximal to FLC on chromosome V. ART1 activates FLC expression through a novel flowering pathway that is independent of FRI and independent of the autonomous and vernalization pathways. Synergistic activation of the floral repressor FLC by ART1 and FRI is required for delayed onset of reproductive development of all shoot meristems, leading to the Sy-0 phenotype. These results demonstrate that modulation in flowering-time genes is one of the mechanisms leading to morphological novelties.     

Axillary meristem development in Arabidopsis thaliana

Axillary shoot apical meristems initiate post-embryonically in the axils of leaves. Their developmental fate is a main determinant of the final plant body plan. In Arabidopsis, usually a single axillary meristem initiates in the leaf axil even though there is developmental potential for formation of multiple branches. While the wild-type plants rarely form multiple branches in the leaf axil, tfl1-2 plants regularly develop two or more branches in the axils of the rosette leaves. Axillary meristem formation in Arabidopsis occurs in two waves: an acropetal wave forms during plant vegetative development, and a basipetal wave forms during plant reproductive development. We report here the morphological and anatomical changes, and the STM expression pattern associated with the formation of axillary and accessory meristems during Arabidopsis vegetative development.     

Effect of mutations in the PINHEAD gene of Arabidopsis on the formation of shoot apical meristems

The primary shoot apical meristem of angiosperm plants is formed during embryogenesis. Lateral shoot apical meristems arise postembryonically in the axils of leaves. Recessive mutations at the PINHEAD locus of Arabidopsis interfere with the ability of both the primary shoot apical meristem as well as lateral shoot apical meristems to form. However, adventitious shoot apical meristems can form in pinhead mutant seedlings from the axils of the cotyledons and also from cultred root explants. In this report, the phenotype of pinhead mutants is described, and a hypothesis for the role of the wild-type PINHEAD gene product in shoot meristem initiation is presented. © 1995 Wiley-Liss, Inc.     

Covalent heme binding to CYP4B1 via Glu310 and a carbocation porphyrin intermediate

Recently we found that CYP4B1, and several other members of the CYP4 family of enzymes, are covalently linked to their prosthetic heme group through an ester linkage. In the current study, we mutated a conserved CYP4 I-helix residue, E310 in rabbit CYP4B1, to glycine, alanine, and aspartate to examine the effect of these mutations on the extent of covalent heme binding and catalysis. All mutants expressed well in insect cells and were isolated as a mixture of monomeric and dimeric forms as determined by LC/ESI-MS of the intact proteins. Rates of metabolism decreased in the order E310 > A310 > G310 > D310, with the A310 and G310 mutants exhibiting alterations in regioselectivity for omega-1 and omega-2 hydroxylation of lauric acid, respectively. In marked contrast to the wild-type E310 enzyme, the G310, A310, and D310 mutants did not bind heme covalently. Uniquely, the acid-dissociable heme obtained from the D310 mutant contained an additional 16 amu relative to heme and exhibited the same chromatographic behavior as the monohydroxyheme species released upon base treatment of the covalently linked wild-type enzyme. Expression studies with H(2)(18)O demonstrated incorporation of the heavy isotope from the media into the monohydroxyheme isolated from the D310 mutant at a molar ratio of approximately 0.8:1. These data show (i) that E310 serves as the site of covalent attachment of heme to the protein backbone of rabbit CYP4B1; (ii) this I-helix glutamate residue influences substrate orientation in the active site of CYP4B1; and (iii) the mechanism of covalent heme attachment most likely involves a carbocation species located on the porphyrin.     

A mutant Arabidopsis heterotrimeric G-protein beta subunit affects leaf, flower, and fruit development.

A genetic screen was performed to find new mutants with an erecta (er) phenotype and to identify genes that may function with ER, a receptor-like kinase. These mutants were named elk (for erecta-like) and were placed into five complementation groups. We positionally cloned ELK4 and determined that it encodes AGB1, a putative heterotrimeric G-protein beta subunit. Therefore, elk4 was renamed agb1. agb1-1 plants express similar fruit phenotypes, as seen in er plants, but differ from er in that the stem is only slightly shorter than that in the wild type, the pedicel is slightly longer than that in the wild type, and the leaves are rounder than those in er mutants. Molecular analysis of agb1-1 indicates that it is likely a null allele. AGB1 mRNA is expressed in all tissues tested but is highest in the silique. Analysis of agb1-1 er double mutants suggests that AGB1 may function in an ER developmental pathway regulating silique width but that it functions in parallel pathways affecting silique length as well as leaf and stem development. The finding that AGB1 is involved in the control of organ shape suggests that heterotrimeric G-protein signaling is a developmental regulator in Arabidopsis.     

Auxin Depletion from the Leaf Axil Conditions Competence for Axillary Meristem Formation in Arabidopsis and Tomato

The enormous variation in architecture of flowering plants is based to a large extent on their ability to form new axes of growth throughout their life span. Secondary growth is initiated from groups of pluripotent cells, called meristems, which are established in the axils of leaves. Such meristems form lateral organs and develop into a side shoot or a flower, depending on the developmental status of the plant and environmental conditions. The phytohormone auxin is well known to play an important role in inhibiting the outgrowth of axillary buds, a phenomenon known as apical dominance. However, the role of auxin in the process of axillary meristem formation is largely unknown. In this study, we show in the model species Arabidopsis thaliana and tomato (Solanum lycopersicum) that auxin is depleted from leaf axils during vegetative development. Disruption of polar auxin transport compromises auxin depletion from the leaf axil and axillary meristem initiation. Ectopic auxin biosynthesis in leaf axils interferes with axillary meristem formation, whereas repression of auxin signaling in polar auxin transport mutants can largely rescue their branching defects. These results strongly suggest that depletion of auxin from leaf axils is a prerequisite for axillary meristem formation during vegetative development.     

Combinatorial control of meristem identity in maize inflorescences

The architecture of maize inflorescences, the male tassel and the female ear, is defined by a series of reiterative branching events. The inflorescence meristem initiates spikelet pair meristems. These in turn initiate spikelet meristems which finally produce the floret meristems. After initiating one meristem, the spikelet pair and spikelet meristem convert into spikelet and floret meristems, respectively. The phenotype of reversed germ orientation1 (rgo1) mutants is the production of an increased number of floret meristems by each spikelet meristem. The visible phenotypes include increased numbers of flowers in tassel and ear spikelets, disrupted rowing in the ear, fused kernels, and kernels with embryos facing the base of the ear, the opposite orientation observed in wild-type ears. rgo1 behaves as single recessive mutant. indeterminate spikelet1 (ids1) is an unlinked recessive mutant that has a similar phenotype to rgo1. Plants heterozygous for both rgo1 and ids1 exhibit nonallelic noncomplementation; these mutants fail to complement each other. Plants homozygous for both mutations have more severe phenotypes than either of the single mutants; the progression of meristem identities is retarded and sometimes even reversed. In addition, in rgo1; ids1 double mutants extra branching is observed in spikelet pair meristems, a meristem that is not affected by mutants of either gene individually. These data suggest a model for control of meristem identity and determinacy in which the progress through meristem identities is mediated by a dosage-sensitive pathway. This pathway is combinatorially controlled by at least two genes that have overlapping functions.     

Arabidopsis lonely guy (LOG) multiple mutants reveal a central role of the LOG-dependent pathway in cytokinin activation

Cytokinins are phytohormones that play key roles in the maintenance of stem cell activity in plants. Although alternative single-step and two-step activation pathways for cytokinin have been proposed, the significance of the single-step pathway which is catalyzed by LONELY GUY (LOG), is not fully understood. We analyzed the metabolic flow of cytokinin activation in Arabidopsis log multiple mutants using stable isotope-labeled tracers and characterized the mutants' morphological and developmental phenotypes. In tracer experiments, cytokinin activation was inhibited most pronouncedly by log7, while the other log mutations had cumulative effects. Although sextuple or lower-order mutants did not show drastic phenotypes in vegetative growth, the log1log2log3log4log5log7log8 septuple T-DNA insertion mutant in which the LOG-dependent pathway is impaired, displayed severe retardation of shoot and root growth with defects in the maintenance of the apical meristems. Detailed observation of the mutants showed that LOG7 was required for the maintenance of shoot apical meristem size. LOG7 was also suggested to play a role for normal primary root growth together with LOG3 and LOG4. These results suggest a dominant role of the single-step activation pathway mediated by LOGs for cytokinin production, and overlapping but differentiated functions of the members of the LOG gene family in growth and development.     

Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity

Cytokinins are hormones that regulate cell division and development. As a result of a lack of specific mutants and biochemical tools, it has not been possible to study the consequences of cytokinin deficiency. Cytokinin-deficient plants are expected to yield information about processes in which cytokinins are limiting and that, therefore, they might regulate. We have engineered transgenic Arabidopsis plants that overexpress individually six different members of the cytokinin oxidase/dehydrogenase (AtCKX) gene family and have undertaken a detailed phenotypic analysis. Transgenic plants had increased cytokinin breakdown (30 to 45% of wild-type cytokinin content) and reduced expression of the cytokinin reporter gene ARR5:GUS (beta-glucuronidase). Cytokinin deficiency resulted in diminished activity of the vegetative and floral shoot apical meristems and leaf primordia, indicating an absolute requirement for the hormone. By contrast, cytokinins are negative regulators of root growth and lateral root formation. We show that the increased growth of the primary root is linked to an enhanced meristematic cell number, suggesting that cytokinins control the exit of cells from the root meristem. Different AtCKX-green fluorescent protein fusion proteins were localized to the vacuoles or the endoplasmic reticulum and possibly to the extracellular space, indicating that subcellular compartmentation plays an important role in cytokinin biology. Analyses of promoter:GUS fusion genes showed differential expression of AtCKX genes during plant development, the activity being confined predominantly to zones of active growth. Our results are consistent with the hypothesis that cytokinins have central, but opposite, regulatory functions in root and shoot meristems and indicate that a fine-tuned control of catabolism plays an important role in ensuring the proper regulation of cytokinin functions.