Over-expression of SOB5 suggests the involvement of a novel plant protein in cytokinin-mediated development

Cytokinins are a class of phytohormones that play a critical role in plant growth and development. sob5-D, an activation-tagging mutant, shows phenotypes typical of transgenic plants expressing the Agrobacterium tumefaciens isopentenyltransferase (ipt) gene that encodes the enzyme catalyzing the first step of cytokinin biosynthesis. The sob5-D mutant phenotypes are caused by over-expression of a novel gene, SOB5. Sequence analysis places SOB5 in a previously uncharacterized family of plant-specific proteins. A translational fusion between SOB5 and the green fluorescent protein reporter was localized in the cytoplasm as well as associated with the plasma membrane when transiently expressed in onion epidermal cells. Analysis of transgenic plants harboring an SOB5:SOB5-beta-glucuronidase (GUS) translational fusion under the control of the SOB5 promoter region showed GUS activity in vegetative tissues (hydathodes and trichomes of leaves, shoot meristems and roots) as well as in floral tissues (pistil tips, developing anthers and sepal vasculature). Cytokinin quantification analysis revealed that adult sob5-D plants accumulated higher levels of trans-zeatin riboside, trans-zeatin riboside monophosphate and isopentenyladenine 9-glucoside when compared to the wild-type. Consistent with this result, AtIPT3 and AtIPT7 were found to be up-regulated in a tissue-specific manner in sob5-D mutants. Physiological analysis of the sob5-D mutant demonstrated reduced responsiveness to exogenous cytokinin in both root-elongation and callus-formation assays. Taken together, our data suggest a role for the novel gene SOB5 in cytokinin-mediated plant development.     

Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants

A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5.     

RED AND FAR-RED INSENSITIVE 2, a RING-domain zinc finger protein, mediates phytochrome-controlled seedling deetiolation responses

Light is arguably the most important resource for plants, and an array of photosensory pigments enables plants to develop optimally in a broad range of ambient-light conditions. The red- and far-red-light-absorbing photosensory pigments or phytochromes (phy) regulate seedling deetiolation responses, photoperiodic flowering, and circadian rhythm. We have identified a long hypocotyl mutant under red and far-red light, rfi2-1 (red and far-red insensitive 2 to 1). rfi2-1 was also impaired in phytochrome-mediated end-of-day far-red light response, cotyledon expansion, far-red light block of greening, and light-induced expression of CHLOROPHYLL A/B BINDING PROTEIN 3 and CHALCONE SYNTHASE. Introduction of rfi2-1 mutation into phyB-9 or phyA-211 did not enhance or suppress the long hypocotyl phenotype of phyB-9 or phyA-211 under red or far-red light, respectively, and RFI2 likely functions downstream of phyB or phyA. RFI2 was identified through the segregation of two T-DNA insertions into different recombinant lines, genetic rescue, and phenotypic characterization of a second mutant allele rfi2-2. RFI2 encodes a protein with a C₃H₂C₃-type zinc finger or RING domain known to mediate protein-protein or protein-DNA interactions, and RFI2 is localized to the nucleus. RFI2 therefore reveals a signaling step that mediates phytochrome control of seedling deetiolation.     

Negative interference of endogenous phytochrome B with phytochrome A function in Arabidopsis

To study negative interactions between phytochromes, phytochrome B (phyB) overexpressor lines, the mutants phyA-201, phyB-4, phyB-5, phyD-1, phyA-201 phyB-5, phyA-201 phyD-1, and phyB-5 phyD-1 of Arabidopsis were used. Endogenous phyB, but not phytochrome D (phyD), partly suppressed phytochrome A (phyA)-dependent inhibition of hypocotyl elongation in far-red light (FR). Dichromatic irradiation demonstrated that the negative effect of phyB was largely independent of the photoequilibrium, i.e. far-red light absorbing form of phytochrome formation. Moreover, phyB-4, a mutant impaired in signal transduction, did not show a loss of inhibition of phyA by phyB. Overexpression of phyB, conversely, resulted in an enhanced inhibition of phyA function, even in the absence of supplementary carbohydrates. However, overexpression of a mutated phyB, which cannot incorporate the chromophore, had no detectable effect on phyA action. In addition to seedling growth, accumulation of anthocyanins in FR, another manifestation of the high irradiance response, was strongly influenced by phyB holoprotein. Induction of seed germination by FR, a very low fluence response, was suppressed by both endogenous phyB and phyD. In conclusion, we show that both classical response modes of phyA, high irradiance response, and very low fluence response are subject to an inhibitory action of phyB-like phytochromes. Possible mechanisms of the negative interference are discussed.     

PNZIP Is a Novel Mesophyll-Specific cDNA That Is Regulated by Phytochrome and a Circadian Rhythm and Encodes a Protein with a Leucine Zipper Motif

Single, double, and triple null combinations of Arabidopsis mutants lacking the photoreceptors phytochrome (phy) A (phyA-201), phyB (phyB-5), and cryptochrome (cry) 1 (hy4-2.23n) were examined for de-etiolation responses in high-fluence red, far-red, blue, and broad-spectrum white light. Cotyledon unhooking, unfolding, and expansion, hypocotyl growth, and the accumulation of chlorophylls and anthocyanin in 5-d-old seedlings were measured under each light condition and in the dark. phyA was the major photoreceptor/effector for most far-red-light responses, although phyB and cry1 modulated anthocyanin accumulation in a phyA-dependent manner. phyB was the major photoreceptor in red light, although cry1 acted as a phyA/phyB-dependent modulator of chlorophyll accumulation under these conditions. All three photoreceptors contributed to most blue light deetiolation responses, either redundantly or additively; however, phyB acted as a modulator of cotyledon expansion dependent on the presence of cry1. As reported previously, flowering time in long days was promoted by phyA and inhibited by phyB, with each suppressing the other's effect. In addition to the effector/modulator relationships described above, measurements of hypocotyls from blue-light-grown seedlings demonstrated phytochrome activity in blue light and cry1 activity in a phyAphyB mutant background.     

Coupling of phytochrome B to the control of hypocotyl growth in Arabidopsis

Etiolated seedlings of the wild-type (WT) and of the phyB-1 mutant of Arabidopsis thaliana (L.) Heynh. were exposed to red-light (R) and far-red light (FR) treatments to characterize the action of phytochrome B on hypocotyl extension growth. A single R or FR pulse had no detectable effects on hypocotyl growth. After 24-h pre-treatment with continuous FR (FRc) a single R, compared to FR pulse inhibited (more than 70%) subsequent hypocotyl growth in the WT but not in the phyB-1 mutant. This effect of FRc was fluence-rate dependent and more efficient than continuous R (Rc) or hourly FR pulses of equal total fluence. Hypocotyl growth inhibition by Rc was larger in WT than phyB-1 seedlings when chlorophyll screening was reduced either by using broadband Rc (maximum emission 610 nm) or by using narrow-band Rc (658 nm) over short periods (24 h) or with seedlings bleached with Norflurazon. Hourly R or R + FR pulses had similar effects in WT and phyB-1 mutant etiolated seedlings. It is concluded that phytochrome B is not the only photoreceptor of Rc and that the action of phytochrome B is enhanced by a FRc high-irradiance reaction. Complementary experiments with the phyA-201 mutant indicate that this promotion of a phytochrome B-mediated response occurs via co-action with phytochrome A.Abbreviations D darkness - FR far-red light - FRc continuous FR - Pfr FR-absorbing form of phytochrome - HIR high-irradiance reaction - Pfr/P proportion of phytochrome as Pfr - phyA phytochrome A - phyB phytochrome B - R red light - Rc continuous R - WT wild-type I thank Professors R.E. Kendrick and M. Koornneef (Wageningen Agricultural University, The Netherlands) and Professor J. Chory (Salk Institute, Calif., USA) for their kind provision of the original WT and phyB-1 and phyA-201 seed, respectively. This work was financially supported by grants PID and PID-BID from CONICET, AG 040 from Universidad de Buenos Aires and A 12830/1-000019 from Fundación Antorchas.     

Inhibition of brassinosteroid biosynthesis by either a dwarf4 mutation or a brassinosteroid biosynthesis inhibitor rescues defects in tropic responses of hypocotyls in the arabidopsis mutant nonphototropic hypocotyl 4

The nonphototropic hypocotyl 4 (nph4)/auxin response factor 7 (arf7) mutant of Arabidopsis (Arabidopsis thaliana) is insensitive to auxin and has defects in hypocotyl tropism, hook formation, differential leaf growth, and lateral root formation. To understand an auxin-signaling pathway through NPH4, we carried out screening of suppressor mutants of nph4-103 and obtained a dwarf suppressor mutant, suppressor of nph4 (snp2). snp2 had short hypocotyls in the dark condition and dark green and round leaves, short petioles, and more lateral shoots than the wild type in the light condition. The snp2 phenotypes were rescued by adding brassinolide to the growth medium in both light and dark conditions. Genetic mapping, sequence analysis, and a complementation test indicated that snp2 was a weak allele of DWARF4 (DWF4), which functions in brassinosteroid (BR) biosynthesis. snp2, which was renamed dwf4-101, exhibited photo- and gravitropisms of hypocotyls similar to those of the wild type with a slightly faster response in gravitropism. dwf4-101 almost completely suppressed defects in both tropisms of nph4-103 hypocotyls and completely suppressed hyponastic growth of nph4-103 leaves. Treatment with brassinazole, an inhibitor of BR biosynthesis, also partially rescued the tropic defects in nph4-103. Hypocotyls of nph4-103 were auxin insensitive, whereas hypocotyls of dwf4-101 were more sensitive than those of the wild type. dwf4-101 nph4-103 hypocotyls were as sensitive as those of dwf4-101. Auxin inducibility of massugu 2 (MSG2)/IAA19 gene expression was reduced in nph4-103. mRNA level of MSG2 was reduced in dwf4-101 and dwf4-101 nph4-103, but both mutants exhibited greater auxin inducibility of MSG2 than the wild type. Taken together, dwf4-101 was epistatic to nph4-103. These results strongly suggest that BR deficiency suppresses nph4-103 defects in tropic responses of hypocotyls and differential growth of leaves and that BR negatively regulates tropic responses.     

Suppressor of <Emphasis Type="Italic">bri1-120</Emphasis> mutant allele revealed interrelated and independent actions of brassinosteroid and light signaling

Brassinosteroids (BRs) are plant hormones that affect diverse aspects of plant development. Various BR-biosynthetic or BR-signaling mutants contribute to BR functions and signaling events in many plant species. The BR receptor brassinosteroid-Insensitive 1 (BRI1) plays critical roles in BR signaling. We previously identified a weak bri1 mutant allele, bri1-120, that has a mutation site in the extracellular domain of BRI1. Here, genetic suppressor screening revealed that a PHYB gene mutation led to suppression of ethyl methanesulfonate (EMS)-mutagenized bri1-120. The morphology of bri1-120phyB-1 indicated that compact and rounded phenotypes of bri1-120 were suppressed. However, BR sensitivity of the bri1-120phyB-1 was only recovered in hypocotyl elongation, and overexpression of PHYB in bri1-120 did not enhance bri1-120 phenotypes. To further investigate the relationship between BR and light signalings, we examined the seed germination pattern and hypocotyl growth of bri1-120phyB-1 as compared to that of each single mutant under various light conditions. Seed germination in bri1-120phyB-1 was higher than in both the single mutants. Hypocotyl length in bri1-120phyB-1 was intermediate between that of bri1-120 and phyB-1, whereas sensitivity to red light in bri1-120phyB-1 remained the same as in phyB-1. These results suggest that BR and light signalings affect diverse cellular responses both together and independently, depending on the specific cellular processes.     

Functional interaction of cryptochrome 1 and phytochrome D

Arabidopsis thaliana wild-type and single, double and triple mutants lacking phytochrome A (phyA-201), phytochrome B (phyB-5), phytochrome D (phyD-1), phytochrome E (phyE-1), cryptochrome 1 (hy4-2.23n) and cryptochrome 2 (fha-1) were used to study the photoreceptor signal-transduction network. The inhibition of hypocotyl elongation was analysed using pulses of red light preceded by a pre-irradiation of white light. The interactions of phyA, phyB and cry1 have been studied in a series of previous papers. Here we focus on the signal transduction initiated by phyD. We observed that phyD can partly substitute for the loss of phyB. Specifically, in the phyB background, red pulses were only effective if both cry1 and phyD were present. The response to red pulses, enabled by the pre-irradiation of white light, was completely reversible by far-red light. Loss of reversibility occurred with an apparent half-life of 2 h, similar to the half-life of 3 h observed for the effect mediated by phyB. Furthermore, we could show that the response to an end-of-day far-red pulse in phyB depends on both phyD and cry1. In contrast to phyD, a functional interaction of phyE and cry1 could not be detected in Arabidopsis seedlings.     

Arabidopsis mutants resistant to S(+)-beta-methyl-alpha, beta-diaminopropionic acid, a cycad-derived glutamate receptor agonist

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that are the predominant neuroreceptors in the mammalian brain. Genes with high sequence similarity to animal iGluRs have been identified in Arabidopsis. To understand the role of Arabidopsis glutamate receptor-like (AtGLR) genes in plants, we have taken a pharmacological approach by examining the effects of BMAA [S(+)-beta-methyl-alpha, beta-diaminopropionic acid], a cycad-derived iGluR agonist, on Arabidopsis morphogenesis. When applied to Arabidopsis seedlings, BMAA caused a 2- to 3-fold increase in hypocotyl elongation and inhibited cotyledon opening during early seedling development. The effect of BMAA on hypocotyl elongation is light specific. Furthermore, BMAA effects on early morphogenesis of Arabidopsis can be reversed by the simultaneous application of glutamate, the native iGluR agonist in animals. To determine the targets of BMAA action in Arabidopsis, a genetic screen was devised to isolate Arabidopsis mutants with a BMAA insensitive morphology (bim). When grown in the light on BMAA, bim mutants exhibited short hypocotyls compared with wild type. bim mutants were grouped into three classes based on their morphology when grown in the dark in the absence of BMAA. Class-I bim mutants have a normal, etiolated morphology, similar to wild-type plants. Class-II bim mutants have shorter hypocotyls and closed cotyledons when grown in the dark. Class-III bim mutants have short hypocotyls and open cotyledons when grown in the dark, resembling the previously characterized constitutively photomorphogenic mutants (cop, det, fus, and shy). Further analysis of the bim mutants should help define whether plant-derived iGluR agonists target glutamate receptor signaling pathways in plants.     

The Histidine Kinase-Related Domain of Arabidopsis Phytochrome A Controls the Spectral Sensitivity and the Subcellular Distribution of the Photoreceptor

Phytochrome A (phyA) is the primary photoreceptor for sensing extremely low amounts of light and for mediating various far-red light-induced responses in higher plants. Translocation from the cytosol to the nucleus is an essential step in phyA signal transduction. EID1 (for EMPFINDLICHER IM DUNKELROTEN LICHT1) is an F-box protein that functions as a negative regulator in far-red light signaling downstream of the phyA in Arabidopsis (Arabidopsis thaliana). To identify factors involved in EID1-dependent light signal transduction, pools of ethylmethylsulfonate-treated eid1-3 seeds were screened for seedlings that suppress the hypersensitive phenotype of the mutant. The phenotype of the suppressor mutant presented here is caused by a missense mutation in the PHYA gene that leads to an amino acid transition in its histidine kinase-related domain. The novel phyA-402 allele alters the spectral sensitivity and the persistence of far-red light-induced high-irradiance responses. The strong eid1-3 suppressor phenotype of phyA-402 contrasts with the moderate phenotype observed when phyA-402 is introgressed into the wild-type background, which indicates that the mutation mainly alters functions in an EID1-dependent signaling cascade. The mutation specifically inhibits nuclear accumulation of the photoreceptor molecule upon red light irradiation, even though it still interacts with FHY1 (for far-red long hypocotyl 1) and FHL (for FHY1-like protein), two factors that are essential for nuclear accumulation of phyA. Degradation of the mutated phyA is unaltered even under light conditions that inhibit its nuclear accumulation, indicating that phyA degradation may occur mostly in the cytoplasm.     

Both phytochrome A and phytochrome B are required for the normal expression of phototropism in Arabidopsis thaliana seedlings

The role of phytochrome A (phyA) and phytochrome B (phyB) in phototropism was investigated by using the phytochrome-deficient mutants phyA-101 , phyB-1 and a phyA/phyB double mutant. The red-light-induced enhancement of phototropism, which is normally observed in wild-type seedlings, could not be detected in the phyA/phyB mutant at fluences of red light between 0.1 and 19 000 μmol m⁻². The loss of phyB has been shown to have no apparent effect on enhancement, while the loss of phyA resulted in a loss of enhancement only in the low fluence range (Janoudi et al. 1997). The conclusions of the aforementioned study can now be modified based on the current results which indicate that phototropic enhancement in the high fluence range is mediated by either phyA or phyB, and that other phytochromes have no role in enhancement. First positive phototropism was unaffected in phyA-101 and phyB-1 However, the magnitude of first positive phototropism in the phyA/phyB mutant was significantly lower than that of the wild-type Landsberg parent. Thus, the presence of either phyA or phyB is required for normal expression of first positive phototropism. The time threshold for second positive phototropism is unaltered in the phyA-101 and phyB mutants. However, the time threshold in the phyA/phyB mutant is about 2 h, approximately six times that of the wild type. Finally, the magnitude of second positive phototropism in both phyA-101 and phyB-1 is diminished in comparison with the wild-type response. Thus, phyA and phyB, acting independently or in combination, regulate the magnitude of phototropic curvature and the time threshold for second positive phototropism. We conclude that the presence of phyA and phyB is required, but not sufficient, for the expression of normal phototropism.     

A comparative analysis of the <Emphasis Type="Italic">Arabidopsis</Emphasis> mutant <Emphasis Type="Italic">amp1-1</Emphasis> and a novel weak <Emphasis Type="Italic">amp1</Emphasis> allele reveals new functions of the AMP1 protein

Ethylene and gibberellins have a synergistic stimulatory effect on hypocotyl elongation of light-grown Arabidopsis thaliana (L.) Heynh. seedlings. A screen for mutants with decreased response to these hormones led to the isolation of a novel allele (amp1-7) of the ALTERED MERISTEM PROGRAM (AMP) 1 locus. The amp1-7 allele contains a missense mutation causing a phenotype, which is weaker than that of the amp1-1 mutant that carries a nonsense mutation. The mutant phenotype prompted the hypothesis that AMP1 is involved in ethylene and GA signalling pathways or in a parallel pathway-controlling cell and hypocotyl elongation and cellular organization. Amp1 mutants contain higher zeatin concentrations causing enlargement of the apical meristem, which was confirmed by cytokinin application to wild type seedlings. Light grown amp1 seedlings have shorter hypocotyls than wild type; however, application of cytokinins promotes hypocotyl elongation of both Col-0 and amp1. We suggest that in amp1 mutants either zeatin overproduction or its action is strictly localized. Nelson J. M. Saibo and Wim H. Vriezen contributed equally to this work.     

SPINDLY and GIGANTEA interact and act in Arabidopsis thaliana pathways involved in light responses, flowering, and rhythms in cotyledon movements

SPINDLY (SPY) is a negative regulator of gibberellin signaling in Arabidopsis thaliana that also functions in previously undefined pathways. The N terminus of SPY contains a protein-protein interaction domain consisting of 10 tetratricopeptide repeats (TPRs). GIGANTEA (GI) was recovered from a yeast two-hybrid screen for proteins that interact with the TPR domain. GI and SPY also interacted in Escherichia coli and in vitro pull-down assays. The phenotypes of spy and spy-4 gi-2 plants support the hypothesis that SPY functions with GI in pathways controlling flowering, circadian cotyledon movements, and hypocotyl elongation. GI acts in the long-day flowering pathway upstream of CONSTANS (CO) and FLOWERING LOCUS T (FT). Loss of GI function causes late flowering and reduces CO and FT RNA levels. Consistent with SPY functioning in the long-day flowering pathway upstream of CO, spy-4 partially suppressed the reduced abundance of CO and FT RNA and the late flowering of gi-2 plants. Like gi, spy affects the free-running period of cotyledon movements. The free-running period was lengthened in spy-4 mutants and shortened in plants that overexpress SPY under the control of the 35S promoter of Cauliflower mosaic virus. When grown under red light, gi-2 plants have a long hypocotyl. This hypocotyl phenotype was suppressed in spy-4 gi-2 double mutants. Additionally, dark-grown and far-red-light-grown spy-4 seedlings were found to have short and long hypocotyls, respectively. The different hypocotyl length phenotypes of spy-4 seedlings grown under different light conditions are consistent with SPY acting in the GA pathway to inhibit hypocotyl elongation and also acting as a light-regulated promoter of elongation.     

The RHG Gene Is Involved in Root and Hypocotyl Gravitropism in Arabidopsis thaliana

In higher plants, shoots show negative gravitropism and rootsshow positive gravitropism. To elucidate the molecular mechanismsof root and hypocotyl gravitropism, we segregated the secondmutation from the original phyB-1 mutant line which impairedboth root and hypocotyl gravitropism and characterized thisnovel mutation named rhg (for root and hyzypocotyl gravitropism).The rhg is a single recessive nuclear mutation and it is mappedon the lower part of the chromosome 1. Analyses on the gravitropicresponses of the rhg mutant indicate that root and hypocotylgravitropism are severely impaired but inflorescence stem gravitropismis not affected by the rhg mutation. In the rhg mutant seedlings,amyloplasts (statoliths for gravity-perception) were presentin the presumptive statocytes of roots and hypocotyls. Phototropismby roots and hypocotyls was not impaired in the rhg mutant.These results suggest that the RHG gene product probably actson the gravity-perception and/or the gravity-signal transductionin root and hypocotyl gravitropism. This is the first reportabout the genetic locus specifically involved in both root andhypocotyl gravitropism but not inflorescence stem gravitropism,supporting our hypothesis that the mechanisms of gravitropismare genetically different between hypocotyls and inflorescencestems. (Received March 11, 1997; Accepted April 17, 1997)