Exogenous application of the apocarotenoid retinaldehyde negatively regulates auxin-mediated root growth.

Root development is essential for plant survival. The lack of carotenoid biosynthesis in the phytoene desaturase 3 (pds3) mutant results in short primary roots (PR) and reduced lateral root (LR) formation. In this study, we showed that short-term inhibition of PDS by fluridone suppresses PR growth in WT, but to a lesser extent in auxin mutants of Arabidopsis (Arabidopsis thaliana). Such an inhibition of PDS activity increased endogenous indole-3-acetic acid (IAA) levels, promoted auxin signaling, and partially complemented the PR growth of an auxin deficient mutant, the YUCCA 3 5 7 8 9 quadruple mutant (yucQ). The exogenous application of retinaldehyde (retinal), an apocarotenoid derived from ß-carotene, complemented the fluridone-induced suppression of root growth, as well as the short roots of the pds3 mutant. Retinal also partially complemented the auxin-induced suppression of root growth. These results suggest that retinal may play a role in regulating root growth by modulating endogenous auxin levels.

Circadian Clock Controls Root Hair Elongation through Long-Distance Communication.

Plants adapt to periodic environmental changes, such as day and night, by using circadian clocks. Cell division and elongation are primary steps to adjust plant development according to their environments. In Arabidopsis, hypocotyl elongation has been studied as a representative model to understand how the circadian clock regulates cell elongation. However, it remains unknown whether similar phenomena exist in other organs, such as roots, where circadian clocks regulate physiological responses. Here, we show that root hair elongation is controlled by both light and the circadian clock. By developing machine-learning models to automatically analyze the images of root hairs, we found that genes encoding major components of the central oscillator, such as TIMING OF CAB EXPRESSION1 (TOC1) or CIRCADIAN CLOCK ASSOCIATED1 (CCA1), regulate the rhythmicity of root hair length. The partial illumination of light to either shoots or roots suggested that light received in shoots is mainly responsible for the generation of root hair rhythmicity. Furthermore, grafting experiments between wild-type (WT) and toc1 plants demonstrated that TOC1 in shoots is responsible for the generation of root hair rhythmicity. Our results illustrate the combinational effects of long-distance signaling and the circadian clock on the regulation of root hair length.

P-MIRU, a Polarized Multispectral Imaging System, Reveals Reflection Information on the Biological Surface.

Reflection light forms the core of our visual perception of the world. We can obtain vast information by examining reflection light from biological surfaces, including pigment composition and distribution, tissue structure and surface microstructure. However, because of the limitations in our visual system, the complete information in reflection light, which we term 'reflectome', cannot be fully exploited. For example, we may miss reflection light information outside our visible wavelengths. In addition, unlike insects, we have virtually no sensitivity to light polarization. We can detect non-chromatic information lurking in reflection light only with appropriate devices. Although previous studies have designed and developed systems for specialized uses supporting our visual systems, we still do not have a versatile, rapid, convenient and affordable system for analyzing broad aspects of reflection from biological surfaces. To overcome this situation, we developed P-MIRU, a novel multispectral and polarization imaging system for reflecting light from biological surfaces. The hardware and software of P-MIRU are open source and customizable and thus can be applied for virtually any research on biological surfaces. Furthermore, P-MIRU is a user-friendly system for biologists with no specialized programming or engineering knowledge. P-MIRU successfully visualized multispectral reflection in visible/non-visible wavelengths and simultaneously detected various surface phenotypes of spectral polarization. The P-MIRU system extends our visual ability and unveils information on biological surfaces.

ZEITLUPE enhances expression of PIF4 and YUC8 in the upper aerial parts of Arabidopsis seedlings to positively regulate hypocotyl elongation.

KEY MESSAGE: Microarray and genetic analyses reveal that ZTL induces the expression of genes related to auxin synthesis, thereby promoting hypocotyl elongation. ZTL is a blue-light receptor that possesses a light-oxygen-voltage-sensing (LOV) domain, an F-box motif, and a kelch repeat domain. ZTL promotes hypocotyl elongation under high temperature (28 °C) in Arabidopsis thaliana; however, the mechanism of this regulation is unknown. Here, we divided seedlings into hypocotyls and upper aerial parts, and performed microarray analyses. In hypocotyl, 1062 genes were down-regulated in ztl mutants (ztl-3 and ztl-105) compared with wild type; some of these genes encoded enzymes involved in cell wall modification, consistent with reduced hypocotyl elongation. In upper aerial parts, 1038 genes were down-regulated in the ztl mutants compared with wild type; these included genes involved in auxin synthesis and auxin response. Furthermore, the expression of the PHYTOCHROME INTERACTING FACTOR 4 (PIF4) gene, which encodes a transcription factor known to positively regulate YUCCA genes (YUCs), was also decreased in the ztl mutants. Genetic analysis revealed that overexpression of PIF4 and YUC8 could restore the suppressed hypocotyl length in the ztl mutants. Our results suggest that ZTL induces expression of YUC8 via PIF4 in upper aerial parts and promotes hypocotyl elongation.

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.

YUCCA9-Mediated Auxin Biosynthesis and Polar Auxin Transport Synergistically Regulate Regeneration of Root Systems Following Root Cutting.

Recovery of the root system following physical damage is an essential issue for plant survival. An injured root system is able to regenerate by increases in lateral root (LR) number and acceleration of root growth. The horticultural technique of root pruning (root cutting) is an application of this response and is a common garden technique for controlling plant growth. Although root pruning is widely used, the molecular mechanisms underlying the subsequent changes in the root system are poorly understood. In this study, root pruning was employed as a model system to study the molecular mechanisms of root system regeneration. Notably, LR defects in wild-type plants treated with inhibitors of polar auxin transport (PAT) or in the auxin signaling mutant auxin/indole-3-acetic acid19/massugu2 were recovered by root pruning. Induction of IAA19 following root pruning indicates an enhancement of auxin signaling by root pruning. Endogenous levels of IAA increased after root pruning, and YUCCA9 was identified as the primary gene responsible. PAT-related genes were induced after root pruning, and the YUCCA inhibitor yucasin suppressed root regeneration in PAT-related mutants. Therefore, we demonstrate the crucial role of YUCCA9, along with other redundant YUCCA family genes, in the enhancement of auxin biosynthesis following root pruning. This further enhances auxin transport and activates downstream auxin signaling genes, and thus increases LR number.

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.

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.

UV-B-Induced CPD Photolyase Gene Expression is Regulated by UVR8-Dependent and -Independent Pathways in Arabidopsis.

Plants have evolved various mechanisms that protect against the harmful effects of UV-B radiation (280-315 nm) on growth and development. Cyclobutane pyrimidine dimer (CPD) photolyase, the repair enzyme for UV-B-induced CPDs, is essential for protecting cells from UV-B radiation. Expression of the CPD photolyase gene (PHR) is controlled by light with various wavelengths including UV-B, but the mechanisms of this regulation remain poorly understood. In this study, we investigated the regulation of PHR expression by light with various wavelengths, in particular low-fluence UV-B radiation (280 nm, 0.2 µmol m(-2) s(-1)), in Arabidopsis thaliana seedlings grown under light-dark cycles for 7 d and then adapted to the dark for 3 d. Low-fluence UV-B radiation induced CPDs but not reactive oxygen species. AtPHR expression was effectively induced by UV-B, UV-A (375 nm) and blue light. Expression induced by UV-A and blue light was predominantly regulated by the cryptochrome-dependent pathway, whereas phytochromes A and B played a minor but noticeable role. Expression induced by UV-B was predominantly regulated by the UVR8-dependent pathway. AtPHR expression was also mediated by a UVR8-independent pathway, which is correlated with CPD accumulation induced by UV-B radiation. These results indicate that Arabidopsis has evolved diverse mechanisms to regulate CPD photolyase expression by multiple photoreceptor signaling pathways, including UVR8-dependent and -independent pathways, as protection against harmful effects of UV-B radiation.

Periodic cytokinin responses in Lotus japonicus rhizobium infection and nodule development.

Host plants benefit from legume root nodule symbiosis with nitrogen-fixing bacteria under nitrogen-limiting conditions. In this interaction, the hosts must regulate nodule numbers and distribution patterns to control the degree of symbiosis and maintain root growth functions. The host response to symbiotic bacteria occurs discontinuously but repeatedly at the region behind the tip of the growing roots. Here, live-imaging and transcriptome analyses revealed oscillating host gene expression with approximately 6-hour intervals upon bacterial inoculation. Cytokinin response also exhibited a similar oscillation pattern. Cytokinin signaling is crucial to maintaining the periodicity, as observed in cytokinin receptor mutants displaying altered infection foci distribution. This periodic regulation influences the size of the root region responsive to bacteria, as well as the nodulation process progression.

Conjugates of 3-phenyllactic acid and tryptophan enhance root-promoting activity without adverse effects in Vigna angularis.

3-Phenyllactic acid (PLA) is a common secondary product of Lactobacillus sp. and promotes adventitious-root formation in Azuki beans (Vigna angularis). Root promotion activity of PLA is synergistically enhanced by tryptophan (Trp). In this study, stereoisomers of PLA and Trp amide conjugates and their alkyl esters were synthesized to investigate the structure-activity relationships on root-promotion activity. The rooting activity of D-PLA-L-Trp conjugate shows more than 40 times higher than that of the mixture of D-PLA and L-Trp. Modification of PLA-Trp with ethyl ester showed the highest activity at 3,400 times of a mixture of D-PLA and L-Trp. However, L-or D-PLA-D-Trp conjugate and the isopropyl ester of PLA-Trp conjugates, both lost the root promotion activity and implicated that a requirement for steric structure for PLA related root promotion mechanism. Unlike auxin substances, which are commonly used as rooting agents that displayed high activity in low concentrations, PLA-Trp ethyl ester exhibited far less phytotoxicity at high concentration of 1 mM, despite its high rooting activity. Innovation of PLA-Trp ethyl ester may be expected for agricultural aspects with low environmental impact.

3-Phenyllactic acid is converted to phenylacetic acid and induces auxin-responsive root growth in Arabidopsis plants.

Many microorganisms have been reported to produce compounds that promote plant growth and are thought to be involved in the establishment and maintenance of symbiotic relationships. 3-Phenyllactic acid (PLA) produced by lactic acid bacteria was previously shown to promote root growth in adzuki cuttings. However, the mode of action of PLA as a root-promoting substance had not been clarified. The present study therefore investigated the relationship between PLA and auxin. PLA was found to inhibit primary root elongation and to increase lateral root density in wild-type Arabidopsis, but not in an auxin signaling mutant. In addition, PLA induced IAA19 promoter fused ß-glucuronidase gene expression, suggesting that PLA exhibits auxin-like activity. The inability of PLA to promote degradation of Auxin/Indole-3-Acetic Acid protein in a yeast heterologous reconstitution system indicated that PLA may not a ligand of auxin receptor. Using of a synthetic PLA labeled with stable isotope showed that exogenously applied PLA was converted to phenylacetic acid (PAA), an endogenous auxin, in both adzuki and Arabidopsis. Taken together, these results suggest that exogenous PLA promotes auxin signaling by conversion to PAA, thereby regulating root growth in plants.

A PSTAIRE-type cyclin-dependent kinase controls light responses in land plants.

Light is a critical signal perceived by plants to adapt their growth rate and direction. Although many signaling components have been studied, how plants respond to constantly fluctuating light remains underexplored. Here, we showed that in the moss Physcomitrium (Physcomitrella) patens, the PSTAIRE-type cyclin-dependent kinase PpCDKA is dispensable for growth. Instead, PpCDKA and its homolog in Arabidopsis thaliana control light-induced tropisms and chloroplast movements by probably influencing the cytoskeleton organization independently of the cell cycle. In addition, lower PpCDKA kinase activity was required to elicit light responses relative to cell cycle regulation. Thus, our study suggests that plant CDKAs may have been co-opted to control multiple light responses, and owing to the bistable switch properties of PSTAIRE-type CDKs, the noncanonical functions are widely conserved for eukaryotic environmental adaptation.

3-Phenyllactic acid, a root-promoting substance isolated from Bokashi fertilizer, exhibits synergistic effects with tryptophan.

Bokashi fertilizer, an organic fertilizer made of plant residue, has been used in Japan not only to fertilize plants but to regulate their growth. Lactic acid bacteria have been found to play an important role in the fermentation process of Bokashi, but the relationship between these bacteria and plant growth activity has not been clarified. Using the adzuki rooting assay, this study identified 3-phenyllactic acid (PLA) produced by lactic acid bacteria as a root promoting compound in Bokashi. PLA showed synergistic effect with tryptophan, but no stem elongation activity. Lactic acid bacteria produced equal quantities of the L- and D-forms of PLA, which have similar root promoting activity. PLA did not significantly affect the amount of endogenous indole-3-acetic acid (IAA), although the chemical structure of PLA is highly similar to that of L-2-aminooxy-3-phenypropionic acid (L-AOPP), which inhibits IAA biosynthesis. These results indicate that the root promoting activity of PLA is not simply due to its increase in the amount of active auxin.

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.

Systems, variation, individuality and plant hormones.

Inter-individual variation in plants and particularly in hormone content, figures strongly in evolution and behaviour. Homo sapiens and Arabidopsis exhibit similar and substantial phenotypic and molecular variation. Whereas there is a very substantial degree of hormone variation in mankind, reports of inter-individual variation in plant hormone content are virtually absent but are likely to be as large if not larger than that in mankind. Reasons for this absence are discussed. Using an example of inter-individual variation in ethylene content in ripening, the article shows how biological time is compressed by hormones. It further resolves an old issue of very wide hormone dose response that result directly from negative regulation in hormone (and light) transduction. Negative regulation is used because of inter-individual variability in hormone synthesis, receptors and ancillary proteins, a consequence of substantial genomic and environmental variation. Somatic mosaics have been reported for several plant tissues and these too contribute to tissue variation and wide variation in hormone response. The article concludes by examining what variation exists in gravitropic responses. There are multiple sensing systems of gravity vectors and multiple routes towards curvature. These are an aspect of the need for reliability in both inter-individual variation and unpredictable environments. Plant hormone inter-individuality is a new area for research and is likely to change appreciation of the mechanisms that underpin individual behaviour.

Arabidopsis RPT2a, 19S proteasome subunit, regulates gene silencing via DNA methylation.

The ubiquitin/proteasome pathway plays a crucial role in many biological processes. Here we report a novel role for the Arabidopsis 19S proteasome subunit RPT2a in regulating gene activity at the transcriptional level via DNA methylation. Knockout mutation of the RPT2a gene did not alter global protein levels; however, the transcriptional activities of reporter transgenes were severely reduced compared to those in the wild type. This transcriptional gene silencing (TGS) was observed for transgenes under control of either the constitutive CaMV 35S promoter or the cold-inducible RD29A promoter. Bisulfite sequencing analysis revealed that both the transgene and endogenous RD29A promoter regions were hypermethylated at CG and non-CG contexts in the rpt2a mutant. Moreover, the TGS of transgenes driven by the CaMV 35S promoters was released by treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine, but not by application of the inhibitor of histone deacetylase Trichostatin A. Genetic crosses with the DNA methyltransferase met1 single or drm1drm2cmt3 triple mutants also resulted in a release of CaMV 35S transgene TGS in the rpt2a mutant background. Increased methylation was also found at transposon sequences, suggesting that the 19S proteasome containing AtRPT2a negatively regulates TGS at transgenes and at specific endogenous genes through DNA methylation.

What Makes each Aux/IAA Gene Unique in its Gene Family, Expression Pattern or Properties of the Gene Product?

In the auxin signal transduction, two protein families, Aux/IAAs and auxin response factors, play a crucial role just downstream of auxin F-box receptors. Distinct and overlapping phenotypes of the dominant Aux/IAA mutants suggest some functional differentiation of the Aux/IAA genes in auxin signaling. Taking advantage of unique phenotypes of the msg2/iaa19 mutants, we carried out promoter-exchange experiments, where cDNA of the msg2, axr2/iaa7 or slr/iaa14 gene was driven by the MSG2 or AXR2 promoter. The cDNAs were translationally fused to the green fluorescent protein gene to measure levels of expressed protein. Results showed that many abnormal phenotypes of the dominant Aux/IAA mutants were governed by their promoter activity, but some were dependent on their gene products. The latter result highlights the possible importance of Aux/IAA protein level controled by auxin F-box receptors.

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.

Overexpression of the RADICAL-INDUCED CELL DEATH1 (RCD1) gene of Arabidopsis causes weak rcd1 phenotype with compromised oxidative-stress responses.

rcd1 is a mutant of Arabidopsis thaliana that is more resistant to methyl viologen, but more sensitive to ozone than the wild type. rcd1-2 is caused by a single nucleotide substitution that results in a premature stop codon at Trp-332. The rcd1-2 mRNA level does not change significantly with the mutation. Since overexpression of rcd1-1 cDNA has been shown to bring about an rcd1-like phenotype, we created and examined the overexpression lines of RCD1 by the use of the cauliflower mosaic virus 35S promoter. The transgenic lines exhibited a weak rcd1-like phenotype, although no resistance to methyl viologen was observed. Further, they fully complemented the aberrant rcd1-2 phenotype. Subcellular localization of RCD1 was examined by transiently expressing green fluorescent protein (GFP) fused with RCD1 in onion epidermal cells. GFP signals are observed as aggregated foci in the inner nuclear matrix-like region.