Arabidopsis ASYMMETRIC LEAVES2 and Nucleolar Factors Are Coordinately Involved in the Perinucleolar Patterning of AS2 Bodies and Leaf Development.

Arabidopsis ASYMMETRIC LEAVES2 (AS2) plays a key role in the formation of flat symmetric leaves. AS2 represses the expression of the abaxial gene ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3). AS2 interacts in vitro with the CGCCGC sequence in ETT/ARF3 exon 1. In cells of leaf primordia, AS2 localizes at peripheral regions of the nucleolus as two AS2 bodies, which are partially overlapped with chromocenters that contain condensed 45S ribosomal DNA repeats. AS2 contains the AS2/LOB domain, which consists of three sequences conserved in the AS2/LOB family: the zinc finger (ZF) motif, the ICG sequence including the conserved glycine residue, and the LZL motif. AS2 and the genes NUCLEOLIN1 (NUC1), RNA HELICASE10 (RH10), and ROOT INITIATION DEFECTIVE2 (RID2) that encode nucleolar proteins coordinately act as repressors against the expression of ETT/ARF3. Here, we examined the formation and patterning of AS2 bodies made from as2 mutants with amino acid substitutions in the ZF motif and the ICG sequence in cells of cotyledons and leaf primordia. Our results showed that the amino acid residues next to the cysteine residues in the ZF motif were essential for both the formation of AS2 bodies and the interaction with ETT/ARF3 DNA. The conserved glycine residue in the ICG sequence was required for the formation of AS2 bodies, but not for the DNA interaction. We also examined the effects of nuc1, rh10, and rid2 mutations, which alter the metabolism of rRNA intermediates and the morphology of the nucleolus, and showed that more than two AS2 bodies were observed in the nucleolus and at its periphery. These results suggested that the patterning of AS2 bodies is tightly linked to the morphology and functions of the nucleolus and the development of flat symmetric leaves in plants.

The formation of perinucleolar bodies is important for normal leaf development and requires the zinc-finger DNA-binding motif in Arabidopsis ASYMMETRIC LEAVES2.

In Arabidopsis, the ASYMMETRIC LEAVES2 (AS2) protein plays a key role in the formation of flat symmetric leaves via direct repression of the abaxial gene ETT/ARF3. AS2 encodes a plant-specific nuclear protein that contains the AS2/LOB domain, which includes a zinc-finger (ZF) motif that is conserved in the AS2/LOB family. We have shown that AS2 binds to the coding DNA of ETT/ARF3, which requires the ZF motif. AS2 is co-localized with AS1 in perinucleolar bodies (AS2 bodies). To identify the amino acid signals in AS2 required for formation of AS2 bodies and function(s) in leaf formation, we constructed recombinant DNAs that encoded mutant AS2 proteins fused to yellow fluorescent protein. We examined the subcellular localization of these proteins in cells of cotyledons and leaf primordia of transgenic plants and cultured cells. The amino acid signals essential for formation of AS2 bodies were located within and adjacent to the ZF motif. Mutant AS2 that failed to form AS2 bodies also failed to rescue the as2-1 mutation. Our results suggest the importance of the formation of AS2 bodies and the nature of interactions of AS2 with its target DNA and nucleolar factors including NUCLEOLIN1. The partial overlap of AS2 bodies with perinucleolar chromocenters with condensed ribosomal RNA genes implies a correlation between AS2 bodies and the chromatin state. Patterns of AS2 bodies in cells during interphase and mitosis in leaf primordia were distinct from those in cultured cells, suggesting that the formation and distribution of AS2 bodies are developmentally modulated in plants.

An Experimental System for Examining Phototropic Response of Gametophytic Shoots in the Moss Physcomitrella patens.

Shoot phototropism benefits growth and metabolism in land plants by enabling them to position their photosynthetic organs in favorable light conditions. Nonvascular land plants, like the ancestors of modern mosses, are believed to have been among the first plants to occupy the land. To understand the evolutional history of shoot phototropism in land plants, we have established a system for experimentally studying phototropism in gametophores of the moss Physcomitrella patens. Here we will describe the key points in our system, including obtaining etiolated gametophores, the light sources used for inducing bending, and the methods for evaluation of phototropic responses.

Quantitative Measurements of Curvature Along the Growth Axis in Tropic Responses Using Free Software Environments.

Tropic responses in plants have usually been studied by measuring changes in the deflection angle of the organ tip. However, the measurement of other geometric parameters, such as curvature along the entire length, may give us better understanding of tropic responses, particularly in shoots. Here, we describe methods for obtaining quantitative measurements of local curvature and other parameters based on digital images of bending Arabidopsis hypocotyls using the free software packages, ImageJ and R.

Correction to: Space-time analysis of gravitropism in etiolated Arabidopsis hypocotyls using bioluminescence imaging of the IAA19 promoter fusion with a destabilized luciferase reporter.

The article Space-time analysis of gravitropism in etiolated Arabidopsis hypocotyls using bioluminescence imaging of the IAA19 promoter fusion with a destabilized luciferase reporter, written by Kotaro T. Yamamoto, Masaaki K. Watahiki, Jun Matsuzaki, Soichirou Satoh and Hisayo Shimizu, was originally published electronically on the publisher's internet portal (currently SpringerLink) on 10 April 2017 without open access.

Space-time analysis of gravitropism in etiolated Arabidopsis hypocotyls using bioluminescence imaging of the IAA19 promoter fusion with a destabilized luciferase reporter.

Imaging analysis was carried out during the gravitropic response of etiolated Arabidopsis hypocotyls, using an IAA19 promoter fusion of destabilized luciferase as a probe. From the bright-field images we obtained the local deflection angle to the vertical, A, local curvature, C, and the partial derivative of C with respect to time, [Formula: see text]. These were determined every 19.9 µm along the curvilinear length of the hypocotyl, at ~10 min intervals over a period of ~6 h after turning hypocotyls through 90° to the horizontal. Similarly from the luminescence images we measured the luminescence intensity of the convex and concave flanks of the hypocotyl as well as along the median of the hypocotyl, to determine differential expression of auxin-inducible IAA19. Comparison of these parameters as a function of time and curvilinear length shows that the gravitropic response is composed of three successive elements: the first and second curving responses and a decurving response (autostraightening). The maximum of the first curving response occurs when A is 76° along the entire length of the hypocotyl, suggesting that A is the sole determinant in this response; in contrast, the decurving response is a function of both A and C, as predicted by the newly-proposed graviproprioception model (Bastien et al., Proc Natl Acad Sci USA 110:755-760, 2013). Further, differential expression of IAA19, with higher expression in the convex flank, is observed at A = 44°, and follows the Sachs' sine law. This also suggests that IAA19 is not involved in the first curving response. In summary, the gravitropic response of Arabidopsis hypocotyls consists of multiple elements that are each determined by separate principles.

A new temperature-insensitive allele of the Arabidopsis AXR6/CUL1 locus derived from a missense mutation in the C-terminal RBX1 binding region.

We isolated a new recessive allele at the AUXIN RESISTANT6/CULLIN1 (AXR6/CUL1) locus, axr6-101, from an EMS-mutagenized population of Arabidopsis thaliana, the Landsberg erecta ecotype. axr6-101 is auxin resistant and semi-dwarf similar to the other recessive axr6 mutants. The axr6-101 phenotype is caused by the E716K substitution of the CUL1 protein, which is likely to affect its ability to bind to the C-terminal RING domain of RING-box 1 (RBX1). The previously reported allele of AXR6, cul1-7, is caused by a substitution at T510 that binds to the N-terminal ß-strand of RBX1. Although cul1-7 shows temperature-sensitive phenotype, the axr6-101 phenotype is largely unaffected by temperature. axr6-101 may provide an important genetic resource for study of the structure-function relationship of the CUL1 protein.

Phototropism in gametophytic shoots of the moss Physcomitrella patens.

Shoot phototropism enables plants to position their photosynthetic organs in favorable light conditions and thus benefits growth and metabolism in land plants. To understand the evolution of this response, we established an experimental system to study phototropism in gametophores of the moss Physcomitrella patens. The phototropic response of gametophores occurs slowly; a clear response takes place more than 24 hours after the onset of unilateral light irradiation, likely due to the slow growth rate of gametophores. We also found that red and far-red light can induce phototropism, with blue light being less effective. These results suggest that plants used a broad range of light wavelengths as phototropic signals during the early evolution of land plants.

Negative phototropism is seen in Arabidopsis inflorescences when auxin signaling is reduced to a minimal level by an Aux/IAA dominant mutation, axr2.

Inflorescences of a dominant mutant of Arabidopsis Aux/IAA7, axr2, showed negative phototropism with a similar fluence response curve to the positive phototropism of wild-type stems. Application of a synthetic auxin, NAA, and an inhibitor of polar auxin transport, NPA, increased and decreased respectively the magnitude of the phototropic response in the wild type, while in axr2 application of NAA reduced the negative phototropic response and NPA had no effect. Decapitation of the apex induced a small negative phototropism in wild-type stems, and had no effect in axr2 plants. Inflorescences of the double mutants of auxin transporters, pgp1 pgp19, showed no phototropic response, while decapitation resulted in a negative phototropic response. These results suggest that negative phototropism can occur when the level of auxin or of auxin signaling is reduced to a minimal level, and that in plant axial organs the default phototropic response to unilateral blue light may be negative. Expression of axr2 protein by an endodermis-specific promoter resulted in agravitropism of inflorescences in a similar way to that of axr2, but phototropism was normal, confirming that the endodermis does not play a critical role in phototropism.

Light-dependent gravitropism and negative phototropism of inflorescence stems in a dominant Aux/IAA mutant of Arabidopsis thaliana, axr2.

Gravitropism and phototropism of the primary inflorescence stems were examined in a dominant Aux/IAA mutant of Arabidopsis, axr2/iaa7, which did not display either tropism in hypocotyls. axr2-1 stems completely lacked gravitropism in the dark but slowly regained it in light condition. Though wild-type stems showed positive phototropism, axr2 stems displayed negative phototropism with essentially the same light fluence-response curve as the wild type (WT). Application of 1-naphthaleneacetic acid-containing lanolin to the stem tips enhanced the positive phototropism of WT, and reduced the negative phototropism of axr2. Decapitation of stems caused a small negative phototropism in WT, but did not affect the negative phototropism of axr2. p-glycoprotein 1 (pgp1) pgp19 double mutants showed no phototropism, while decapitated double mutants exhibited negative phototropism. Expression of auxin-responsive IAA14/SLR, IAA19/MSG2 and SAUR50 genes was reduced in axr2 and pgp1 pgp19 stems relative to that of WT. These suggest that the phototropic response of stem is proportional to the auxin supply from the shoot apex, and that negative phototropism may be a basal response to unilateral blue-light irradiation when the levels of auxin or auxin signaling are reduced to the minimal level in the primary stems. In contrast, all of these treatments reduced or did not affect gravitropism in wild-type or axr2 stems. Tropic responses of the transgenic lines that expressed axr2-1 protein by the endodermis-specific promoter suggest that AXR2-dependent auxin response in the endodermis plays a more crucial role in gravitropism than in phototropism in stems but no significant roles in either tropism in hypocotyls.

Reduced phototropism in pks mutants may be due to altered auxin-regulated gene expression or reduced lateral auxin transport.

Phototropism allows plants to orient their photosynthetic organs towards the light. In Arabidopsis, phototropins 1 and 2 sense directional blue light such that phot1 triggers phototropism in response to low fluence rates, while both phot1 and phot2 mediate this response under higher light conditions. Phototropism results from asymmetric growth in the hypocotyl elongation zone that depends on an auxin gradient across the embryonic stem. How phototropin activation leads to this growth response is still poorly understood. Members of the phytochrome kinase substrate (PKS) family may act early in this pathway, because PKS1, PKS2 and PKS4 are needed for a normal phototropic response and they associate with phot1 in vivo. Here we show that PKS proteins are needed both for phot1- and phot2-mediated phototropism. The phototropic response is conditioned by the developmental asymmetry of dicotyledonous seedlings, such that there is a faster growth reorientation when cotyledons face away from the light compared with seedlings whose cotyledons face the light. The molecular basis for this developmental effect on phototropism is unknown; here we show that PKS proteins play a role at the interface between development and phototropism. Moreover, we present evidence for a role of PKS genes in hypocotyl gravi-reorientation that is independent of photoreceptors. pks mutants have normal levels of auxin and normal polar auxin transport, however they show altered expression patterns of auxin marker genes. This situation suggests that PKS proteins are involved in auxin signaling and/or lateral auxin redistribution.

AtIQM1, a novel calmodulin-binding protein, is involved in stomatal movement in Arabidopsis.

We recently identified a novel IQ motif-containing protein family, IQM, which shares sequence homology with a pea heavy metal-induced protein 6 and a ribosome inactivating protein, trichosanthin. Distinct expression patterns for each gene suggest that each IQM family member may play a different role in plant development and response to environmental cues. However functions of the IQM family members remain to be analyzed. IQM1 bound with calmodulin 5 (CaM5) in yeast two-hybrid assay via its IQ-motif. The CaM binding was Ca(2+)-independent in vitro, and was also observed in bimolecular fluorescence complementation analyses in onion epidermal cells. IQM1 was found to express strongly in guard cells and the cortex of roots. The T-DNA insertion mutants of IQM1 displayed a smaller stomatal aperture, a decreased water loss rate and a shorter primary root. Moreover, iqm1 did not change its stomatal aperture when treated with light, dark, ABA and chitin obviously. Microarray analyses showed that 243 and 28 genes were up- and down-regulated by more than twofold in iqm1-1, respectively. Interesting, 34 of 117 and 7 of 30 chitin-responsive transcriptional factor and ubiquitin ligase genes were up-regulated, respectively. Stomatal guard cells of iqm1-1 also showed enhanced expression of genes involved in production and signaling of reactive oxygen species (ROS). Consistently, increased ROS level was observed in the iqm1 guard cells.

Arabidopsis auxin response factor6 and 8 regulate jasmonic acid biosynthesis and floral organ development via repression of class 1 KNOX genes.

Two mutations in Arabidopsis thaliana, auxin response factor6 (arf6) and arf8, concomitantly delayed the elongation of floral organs and subsequently delayed the opening of flower buds. This phenotype is shared with the jasmonic acid (JA)-deficient mutant dad1, and, indeed, the JA level of arf6 arf8 flower buds was decreased. Among JA biosynthetic genes, the expression level of DAD1 (DEFECTIVE IN ANTHER DEHISCENCE1) was markedly decreased in the double mutant, suggesting that ARF6 and ARF8 are required for activation of DAD1 expression. The double mutant arf6 arf8 also showed other developmental defects in flowers, such as aberrant vascular patterning and lack of epidermal cell differentiation in petals. We found that class 1 KNOX genes were expressed ectopically in the developing floral organs of arf6 arf8, and mutations in any of the class 1 KNOX genes (knat2, knat6, bp and hemizygous stm) partially suppressed the defects in the double mutant. Furthermore, ectopic expression of the STM gene caused a phenotype similar to that of arf6 arf8, including the down-regulation of DAD1 expression. These results suggested that most defects in arf6 arf8 are attributable to abnormal expression of class 1 KNOX genes. The expression of AS1 and AS2 was not affected in arf6 arf8 flowers, and as1 and arf6 arf8 additively increased the expression of class 1 KNOX genes. We concluded that ARF6 and ARF8, in parallel with AS1 and AS2, repress the class 1 KNOX genes in developing floral organs to allow progression of the development of these organs.

Changes in growth kinetics of stamen filaments cause inefficient pollination in massugu2, an auxin insensitive, dominant mutant of Arabidopsis thaliana.

We investigated the physiological and molecular basis of lower fecundity of massugu2 (msg2), which is a dominant mutant of an auxin primary response gene, IAA19, in Arabidopsis thaliana. By measuring the length of all stamens and pistils in inflorescences and the reference growth rate of pistils, we constructed growth curves of pistils and stamens between stages 12 and 15 of flower development. Pistil growth was found to consist of a single exponential growth, while stamen growth consisted of three exponential phases. During the second exponential phase, the growth rate of stamen filaments was approximately 10 times greater than the growth rates in the other two phases. Consequently, stamens whose growth was initially retarded grew longer than the pistil, putting pollen grains on the stigma. msg2-1 stamens, on the other hand, exhibited a less obvious growth increase, resulting in less frequent contact between anthers and stigma. MSG2 was expressed in the stamen filaments and its expression almost coincided with the second growth phase. Stamen filaments appeared to elongate by cell elongation rather than cell division in the epidermal cell file. Considering that MSG2 is likely to be a direct target of the auxin F-box receptors, MSG2 may be one of the master genes that control the transient growth increase of stamen filaments.

Phytochrome-mediated growth inhibition of seminal roots in rice seedlings.

In rice (Oryza sativa) seedlings, continuous white-light irradiation inhibited the growth of seminal roots but promoted the growth of crown roots. In this study, we examined the mechanisms of photoinhibition of seminal root growth. Photoinhibition occurred in the absence of nitrogen but increased with increasing nitrogen concentrations. In the presence of nitrogen, photoinhibition was correlated with coiling of the root tips. The seminal roots were most photosensitive 48-72 h after germination during the 7-day period after germination. White-light irradiation for at least 6 h was required for photoinhibition, and the Bunsen-Roscoe law of reciprocity was not observed. Experiments with phytochrome mutants showed that far-red light was perceived exclusively by phyA, red light was perceived by both phyA and phyB, and phyC had little or no role in growth inhibition or coiling of the seminal roots. These results also suggest that other blue-light photoreceptors are involved in growth inhibition of the seminal roots. Fluence-response curve analyses showed that phyA and phyB control very low-fluence response and low-fluence response, respectively, in the seminal roots. This was essentially the same as the growth inhibition previously observed at the late stage of coleoptile development (80 h after germination). The photoperceptive site for the root growth inhibition appeared to be the roots themselves. All three phytochrome species of rice were detected immunochemically in roots.

Fluorescence cross-correlation spectroscopy of plant proteins.

Fluorescence cross-correlation spectroscopy (FCCS) is a technique that physically evaluates the molecular interaction between two fluorophore-tagged molecules such as proteins and oligonucleotides in a quantitative manner. Because it simply makes use of the coincidental movement of two molecules, it could avoid the complexity that sometimes occurs in other fluorescent techniques such as fluorescence resonance energy transfer. The present chapter describes procedures for FCCS of plant proteins expressed and measured in HeLa cells. Determination in plant cells is also mentioned briefly.

Overexpression of the non-canonical Aux/IAA genes causes auxin-related aberrant phenotypes in Arabidopsis.

Degradation of Aux/IAA proteins which are triggered by the ubiquitin ligase complex containing the auxin F-box receptors (AFBs), is thought to be the primary reaction of auxin signaling. Upon auxin perception, AFBs bind domain II of Aux/IAA proteins that is conserved in most of the 29 family members in Arabidopsis. However, IAA20 and IAA30 lack domain II. Furthermore, IAA31, which forms a single clade with IAA20 and IAA30 in Aux/IAA protein family, has a partially conserved domain II, which contains an amino acid substitution that would cause a dominant mutation of Aux/IAA genes. It has been shown that the half-lives of these proteins are much longer than those of the canonical Aux/IAA proteins. We generated overexpression lines (OXs) of IAA20, IAA30 and IAA31 by the use of cauliflower mosaic virus 35S promoter to better understand the molecular function of atypical Aux/IAA proteins in Arabidopsis. OXs of the three genes exhibited similar auxin-related aberrant phenotypes, with IAA20 OX showing the most severe defects: Some of them showed a semi-dwarf phenotype; gravitropic growth orientation was often affected in hypocotyl and root; vasculature of cotyledons was malformed; the primary root stopped growing soon after germination because of collapse of root apical meristem. IAA 20 and IAA30 were early auxin inducible, but IAA31 was not. These results showed that the wild-type genes of the three Aux/IAAs could disturb auxin physiology when ectopically overexpressed.

What's the physiological role of domain II-less Aux/IAA proteins?

Arabidopsis has Aux/IAA proteins that lack domain II which is a binding site for the TIR1 auxin receptor. These proteins have been shown to be more stable than the canonical Aux/IAA proteins. We investigated the phenotypes of overexpression lines of domain II-less Aux/IAA proteins, IAA20 and IAA30, by the use of 35S promoter. The transgenic lines showed many aberrant phenotypes in auxin physiology. The most conspicuous phenotype was collapse of the root apical meristem as occurred in plt1 plt2 double mutants. Because IAA20 and IAA30 were early auxin-inducible and were expressed in the root apical meristem, they may have a physiological role in maintaining the stem cell niche of root, by keeping the activity of MP/ARF5 and NPH4/ARF7 at an acceptable level. On the other hand, domain-II less Aux/IAA proteins are not present in balck cottonwood tree or grapevine, suggesting wide diversification of Aux/IAA proteins in higher plants.

Specificity and similarity of functions of the Aux/IAA genes in auxin signaling of Arabidopsis revealed by promoter-exchange experiments among MSG2/IAA19, AXR2/IAA7, and SLR/IAA14.

As indicated by various and some overlapped phenotypes of the dominant mutants, the Aux/IAA genes of Arabidopsis (Arabidopsis thaliana) concomitantly exhibit a functional similarity and differentiation. To evaluate the contributions of their expression patterns determined by promoter activity and molecular properties of their gene products to Aux/IAA function, we examined phenotypes of transgenic plants expressing the green fluorescent protein (GFP)-tagged msg2-1/iaa19, axr2-1/iaa7, or slr-1/iaa14 cDNA by the MSG2 or AXR2 promoter. When driven by the MSG2 promoter (pMSG2), each GFP-tagged cDNA caused the msg2-1 phenotype, that is, the wild-type stature in the mature-plant stage, long and straight hypocotyls in the dark, reduced lateral root formation, relatively mild agravitropic traits in hypocotyls, and a normal gravitropic response in roots. However, development of one or two cotyledonary primordia was often arrested in embryogenesis of the pMSG2::axr2-1::GFP and pMSG2::slr-1::GFP plants, resulting in monocotyledonary or no cotyledonary seedlings. Such defects in embryogenesis were never seen in pMSG2::msg2-1::GFP or the msg2-1, axr2-1, or slr-1 mutant. The MSG2 promoter-GUS staining showed that expression of MSG2 started specifically in cotyledonary primordia of the triangular-stage embryos. When driven by the AXR2 promoter (pAXR2), each GFP-tagged mutant cDNA caused, in principle, aberrant aboveground phenotypes of the corresponding dominant mutant. However, either the axr2-1::GFP or slr-1::GFP cDNA brought about dwarf, agravitropic stems almost identical to those of axr2-1, and the pAXR2::msg2-1::GFP and pAXR2::slr-1::GFP hypocotyls exhibited complete loss of gravitropism as did axr2-1. These results showed functional differences among the msg2-1, axr2-1, and slr-1 proteins, though some phenotypes were determined by the promoter activity.