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PubMed2001

The distribution of protochlorophyllide and chlorophyll within seedlings of the lip1 mutant of Pea.

The distribution of protochlorophyllide (Pchlide) and NADPH-Pchlide oxidoreductase (POR) was characterized in the epicotyls and roots of wild-type pea (Pisum sativum L. cv. Alaska) and lip1, a mutant with light-independent photomorphogenesis caused by a mutation in the COP1 locus. The upper part of the dark-grown lip1 mutant epicotyls had a high Pchlide content that decreased downward the organ. The elevated Pchlide level in lip1 seedlings was a result of the differentiation of more proplastids into Pchlide-containing plastids. The cortex cells in the lip1 epicotyl were filled with such plastids in contrast to the cortex cells of wild-type seedlings. The mutant also developed Pchlide-containing plastids in the roots, indicating the suppressing effect of the COP1 locus on development of plastids in the corresponding tissues in dark-grown wild-type plants. The distribution of Pchlide-containing plastids in dark-grown lip1 mutant stem and root was similar to the distribution of chloroplasts in irradiated wild-type plants. Both wild-type and lip1 epicotyls contained mostly short wavelength Pchlide fluorescing at 631 nm with only a small shoulder at 654 nm, which was transformed to a minute amount of chlorophyllide (Chlide) by flash irradiation. In contrast, with continuous irradiation a considerable amount of Chlide was formed especially in the lip1 epicotyls. Immunoblots indicated the presence of POR, as a 36 kDa band, in epicotyls of both dark-grown wild-type and lip1 mutant seedlings. However, lip1 stem tissue had a higher content of POR than the wild-type pea. The high content of POR was unexpected as lip1 lacked both the 654 nm fluorescing Pchlide form and the regular PLBs. In light, a significant amount of chlorophyll was formed also in the roots of the lip1 seedlings.

Arabidopsis Proteins↗
PubMed2002

Function of a plant stress-induced gene, HVA22. Synthetic enhancement screen with its yeast homolog reveals its role in vesicular traffic.

Expression of the barley (Hordeum vulgare) HVA22 gene is induced by environmental stresses, such as dehydration, salinity, and extreme temperatures, and by a plant stress hormone, abscisic acid. Genes sharing high level of sequence similarities with HVA22 exist in diverse eukaryotic organisms, including animals, plants, and fungi, but not in any prokaryotic organisms. The yeast (Saccharomyces cerevisiae) HVA22 homolog, Yop1p, has been shown to interact with the GTPase-interacting protein, Yip1p. Deletion of YOP1 led to only a modest reduction of the stationary phase titer at 37C. A synthetic enhancement mutant screen was performed in the yop1 deletion background to identify genes interacting with YOP1. The open reading frame YOR165W (renamed SEY1 for synthetic enhancement of YOP1) was identified as a YOP1-dependent complementation gene. The yeast SEY1 is a homolog of the Arabidopsis RHD3 gene whose mutations cause the accumulation of transport vesicles near the tips of defective root hairs. The yeast double mutant of yop1 and sey1 is defective in vesicular traffic as evidenced by the accumulation of transport vesicles and the decrease in invertase secretion. Based on these observations, we suggest that Yop1p/HVA22 regulates vesicular traffic in stressed cells either to facilitate membrane turnover, or to decrease unnecessary secretion.

Alleles↗
PubMed2001

The Arabidopsis dual-affinity nitrate transporter gene AtNRT1.1 (CHL1) is activated and functions in nascent organ development during vegetative and reproductive growth.

The AtNRT1.1 (CHL1) transporter provides a primary mechanism for nitrate uptake in Arabidopsis and is expected to localize to the epidermis and cortex of the mature root, where the bulk of nitrate uptake occurs. Using fusions to GFP/GUS marker genes, we found CHL1 expression concentrated in the tips of primary and lateral roots, with very low signals in the epidermis and cortex. A time-course study showed that CHL1 is activated in the primary root tip early in seedling development and at the earliest stages of lateral root formation. Strong CHL1 expression also was found in shoots, concentrated in young leaves and developing flower buds but not in the shoot meristem. These expression patterns were confirmed by immunolocalization and led us to examine CHL1 function specifically in the growth of developing organs. chl1 mutants showed a reduction in the growth of nascent roots, stems, leaves, and flower buds. The growth of nascent primary roots was inhibited in the mutants even in the absence of added nitrate, whereas elongation of lateral root primordia was inhibited specifically at low nitrate and acidic pH. Interestingly, chl1 mutants also displayed a late-flowering phenotype. These results indicate that CHL1 is activated and functions in the growth of nascent organs in both shoots and roots during vegetative and reproductive growth.

Anion Transport Proteins↗
PubMed2002

Molecular genetic analysis of left-right handedness in plants.

Handedness in plant growth may be most familiar to us when we think of tendrils or twining plants, which generally form consistent right- or left-handed helices as they climb. The petals of several species are sometimes arranged like fan blades that twist in the same direction. Another less conspicuous example is 'circumnutation', the oscillating growth of axial organs, which alternates between a clockwise and an anti-clockwise direction. To unravel molecular components and cellular determinants of handedness, we screened Arabidopsis thaliana seedlings for helical growth mutants with fixed handedness. Recessive spiral1 and spiral2 mutants show right-handed helical growth in roots, hypocotyls, petioles and petals; semi-dominant lefty1 and lefty2 mutants show opposite left-handed growth in these organs. lefty mutations are epistatic to spiral mutations. Arabidopsis helical growth mutants with fixed handedness may be impaired in certain aspects of cortical microtubule functions, and characterization of the mutated genes should lead us to a better understanding of how microtubules function in left-right handedness in plants.

Genes, Plant↗
PubMed2002

Long-distance signaling in nodulation directed by a CLAVATA1-like receptor kinase.

Proliferation of legume nodule primordia is controlled by shoot-root signaling known as autoregulation of nodulation (AON). Mutants defective in AON show supernodulation and increased numbers of lateral roots. Here, we demonstrate that AON in soybean is controlled by the receptor-like protein kinase GmNARK (Glycine max nodule autoregulation receptor kinase), similar to Arabidopsis CLAVATA1 (CLV1). Whereas CLV1 functions in a protein complex controlling stem cell proliferation by short-distance signaling in shoot apices, GmNARK expression in the leaf has a major role in long-distance communication with nodule and lateral root primordia.

Amino Acid Sequence↗
PubMed1997

Structure and regulation of ferredoxin-dependent glutamase synthase from Arabidopsis thaliana. Cloning of cDNA expression in different tissues of wild-type and gltS mutant strains, and light induction.

Ferredoxin (Fd)-dependent glutamate synthase is present in green leaves, etiolated leaves, shoots and roots of Arabidopsis thaliana (ecotype Columbia). In photosynthetic green leaves and shoots, Fd-dependent glutamate synthase accounts for more than 96% of the total glutamate synthase activity in vitro with the remaining activity derived from an enzyme that uses NADH as the electron donor. In etiolated leaves and roots, Fd-dependent glutamate synthase is 3-4-fold less active than in green leaves, but represents 70-85% of the total glutamate synthase activity in these tissues. Fd-dependent glutamate synthase is detected as a single peptide of 165 kDa on a western blot of green leaf and shoot tissues, and this Fd-dependent glutamate synthase polypeptide is 3-4-fold less abundant in etiolated leaves and roots. In these non-photosynthetic tissues, there is a higher activity of NADH-dependent glutamate synthase. The A. thaliana gltS mutant (strain CS254) contains only 1.7% and 17.5% of the wild-type Fd-dependent glutamate synthase activity in leaves and roots, respectively. Western blots indicate that the Fd-dependent glutamate synthase peptide of 165 kDa is absent from leaves and roots of the gltS mutant. In contrast, NADH-dependent glutamate synthase activity in leaves and roots is unaffected. During illumination of wild-type dark-grown leaves for 72 h, the levels of Fd-dependent glutamate synthase protein and its activity increased threefold to levels equivalent to those in green leaves. In contrast, NADH-dependent glutamate synthase activity decrease twofold during illumination. The complete nucleotide sequence of the complementary DNA for A. thaliana Fd-dependent glutamate synthase has been determined. Analysis of the amino acid sequence deduced from the complete cDNA sequence (5178 bp) has revealed that A. thaliana Fd-dependent glutamate synthase is synthesized as a 1648-amino-acid precursor protein (180090 Da) which consists of a 131-amino-acid transit peptide (14603 Da) and a 1517-amino-acid mature peptide (165487 Da). The A. thaliana Fd-dependent glutamate synthase has a high similarity to maize Fd-dependent glutamate synthase (83%) and to the analogous region of NADH-dependent glutamate synthase (42%) and NADPH-dependent glutamate synthases (40-43%) from different organisms. The A. thaliana Fd-dependent glutamate synthase contains the purF-type glutamine-amido-transfer domain as well as flavin and iron-sulfur-cluster-binding domains. The deduced primary structures of A. thaliana Fd-dependent glutamate synthase and of glutamate synthases from other organisms indicate that Fd-dependent glutamate synthase may have evolved from bacterial NADPH-dependent glutamate synthase. The cDNA hybridized to RNA of about 5.3 kb from different tissues of A. thaliana. A high steady-state level of Fd-dependent glutamate synthase mRNA is found in photosynthetic green leaves and shoots, and roots contain less mRNA for Fd-dependent glutamate synthase. In the gltS mutant, there are twofold and fourfold lower levels of Fd-dependent glutamate synthase mRNA in leaves and roots, respectively, relative to those in wild-type A. thaliana. Under continuous illumination of dark-grown leaves, the Fd-dependent glutamate synthase mRNA is induced twofold to a level equivalent to that in green leaves.

Amino Acid Sequence↗
PubMed2000

The axr6 mutants of Arabidopsis thaliana define a gene involved in auxin response and early development.

The indolic compound auxin regulates virtually every aspect of plant growth and development, but its role in embryogenesis and its molecular mechanism of action are not understood. We describe two mutants of Arabidopsis that define a novel gene called AUXIN-RESISTANT6 (AXR6) which maps to chromosome 4. Embryonic development of the homozygous axr6 mutants is disrupted by aberrant patterns of cell division, leading to defects in the cells of the suspensor, root and hypocotyl precursors, and provasculature. The homozygous axr6 mutants arrest growth soon after germination lacking a root and hypocotyl and with severe vascular pattern defects in their cotyledons. Whereas previously described mutants with similar developmental defects are completely recessive, axr6 heterozygotes display a variety of morphological and physiological alterations that are most consistent with a defect in auxin physiology or response. The AXR6 gene is likely to be important for auxin response throughout the plant, including early development.

Arabidopsis↗
PubMed2005

The promoter of the leghaemoglobin gene VfLb29: functional analysis and identification of modules necessary for its activation in the infected cells of root nodules and in the arbuscule-containing cells of mycorrhizal roots.

In this study the further characterization of the Vicia faba leghaemoglobin promoter pVfLb29 is presented that was previously shown to be specifically active in the infected cells of root nodules and in arbuscule-containing cells of mycorrhizal roots. Using promoter studies in transgenic hairy roots of the Pisum sativum mutant RisNod24, disabled in the formation of functional arbuscules, VfLb29 promoter activity is assigned to later stages of arbuscule development. In order to narrow down the regions containing cis-acting elements of pVfLb29, the activity of five VfLb29 promoter deletions (-797/-31 to -175/-31 in relation to the start codon) fused to the gusAint coding region were tested in transgenic V. hirsuta hairy roots. The results specify a promoter region ranging from position -410 to -326 (85 bp) as necessary for gus expression in arbuscule-containing cells, whereas this segment is not involved in the nodule-specific activity. Sequence analysis of the pVfLb29 fragment -410/-326 (85 bp) revealed sequence motifs previously shown to be cis-acting elements of diverse promoters. To investigate the autonomous function of pVfLb29 regions for activation in arbuscule-containing cells, different regions of pVfLb29 from positions -410 to -198 were used to prepare chimeric promoter constructs for trans-activation studies. These fragments alone did not activate the mycorrhiza inactive promoter of the Vicia faba leghaemoglobin gene VfLb3, showing that the activation of pVfLb29 in arbuscule-containing cells is governed by a complex regulatory system that requires at least two modules located between position -410 and -31 of the VfLb29 gene.

Base Sequence↗
PubMed2005

The rib1 mutant of Arabidopsis has alterations in indole-3-butyric acid transport, hypocotyl elongation, and root architecture.

Polar transport of the auxin indole-3-butyric acid (IBA) has recently been shown to occur in Arabidopsis (Arabidopis thaliana) seedlings, yet the physiological importance of this process has yet to be fully resolved. Here we describe the first demonstration of altered IBA transport in an Arabidopsis mutant, and show that the resistant to IBA (rib1) mutation results in alterations in growth, development, and response to exogenous auxin consistent with an important physiological role for IBA transport. Both hypocotyl and root IBA basipetal transport are decreased in rib1 and root acropetal IBA transport is increased. While indole-3-acetic acid (IAA) transport levels are not different in rib1 compared to wild type, root acropetal IAA transport is insensitive to the IAA efflux inhibitor naphthylphthalamic acid in rib1, as is the dependent physiological process of lateral root formation. These observed changes in IBA transport are accompanied by altered rib1 phenotypes. Previously, rib1 roots were shown to be less sensitive to growth inhibition by IBA, but to have a wild-type response to IAA in root elongation. rib1 is also less sensitive to IBA in stimulation of lateral root formation and in hypocotyl elongation under most, but not all, light and sucrose conditions. rib1 has wild-type responses to IAA, except under one set of conditions, low light and 1.5% sucrose, in which both hypocotyl elongation and lateral root formation show altered IAA response. Taken together, our results support a model in which endogenous IBA influences wild-type seedling morphology. Modifications in IBA distribution in seedlings affect hypocotyl and root elongation, as well as lateral root formation.

Arabidopsis↗
PubMed2003

Five components of the ethylene-response pathway identified in a screen for weak ethylene-insensitive mutants in Arabidopsis.

Five ethylene-insensitive loci (wei1-wei5) were identified by using a low-dose screen for "weak" ethylene-insensitive mutants. wei1, wei2, and wei3 seedlings showed hormone insensitivity only in roots, whereas wei4 and wei5 displayed insensitivity in both roots and hypocotyls. The genes corresponding to wei1, wei4, and wei5 were isolated using a positional cloning approach. The wei1 mutant harbored a recessive mutation in TIR1, which encodes a component of the SCF protein ubiquitin ligase involved in the auxin response. wei4, a dominant mutant, resulted from a mutation in the ethylene receptor ERS, whereas wei5, a semidominant mutant, was caused by a mutation in the EIN3-related transcription factor gene EIL1. The simultaneous loss of functional WEI5EIL1 and EIN3 nearly completely abolished the ethylene response in etiolated seedlings, and adult plants were highly susceptible to infection by the necrotrophic fungal pathogen Botrytis cinerea. Moreover, wei5eil1 ein3 double mutants were able to fully suppress constitutive signaling caused by ctr1, suggesting a synergistic interaction among these gene products. Unlike previously known root ethylene-insensitive mutants, wei2 and wei3 were not affected in their response to auxin and showed a normal response to gravity. Genetic mapping studies indicate that wei2 and wei3 correspond to previously unidentified ethylene pathway genes that may control cell-elongation processes functioning at the intersection of the ethylene and auxin response pathways.

Alleles↗
PubMed2005

Chloroplast biogenesis 92: In situ screening for divinyl chlorophyll(ide) a reductase mutants by spectrofluorometry.

Chlorophyll biosynthetic heterogeneity is rooted mainly in parallel divinyl (DV) and monovinyl (MV) biosynthetic routes interconnected by 4-vinyl reductases (4VRs) that convert DV tetrapyrroles to MV tetrapyrroles by conversion of the vinyl group at position 4 of the macrocycle to ethyl. What is not clear at this stage is whether the various 4VR activities are catalyzed by one enzyme of broad specificity or by a family of enzymes encoded by one gene or multiple genes with each enzyme having narrow specificity. Additional research is needed to identify the various regulatory components of 4-vinyl reduction. In this undertaking, Arabidopsis mutants that accumulate DV chlorophyllide a and/or DV chlorophyll [Chl(ide)] a are likely to provide an appropriate resource. Because the Arabidopsis genome has been completely sequenced, the best strategy for identifying 4VR and/or putative regulatory 4VR genes is to screen Arabidopsis Chl mutants for DV Chl(ide) a accumulation. In wild-type Arabidopsis, a DV plant species, only MV chlorophyllide (Chlide) a is detectable. However in Chl mutants lacking 4VR activity, DV Chl(ide) a may accumulate in addition to MV Chl(ide) a. In the current work, an in situ assay of DV Chl(ide) a accumulation, suitable for screening a large number of mutants lacking 4-vinyl Chlide a reductase activity with minimal experimental handling, is described. The assay involves homogenization of the tissues in Tris-HCl:glycerol buffer and the recording of Soret excitation spectra at 77K. DV Chlide a formation is detected by a Soret excitation shoulder at 459 nm over a wide range of DV Chlide a/MV Chl a ratios. The DV Chlide a shoulder became undetectable at DV Chlide a/MV Chl a ratios less than 0.049, that is, at a DV Chlide a content of less than 5%.

Chloroplasts↗
PubMed2002

The POLARIS gene of Arabidopsis encodes a predicted peptide required for correct root growth and leaf vascular patterning.

The POLARIS (PLS) gene of Arabidopsis was identified as a promoter trap transgenic line, showing beta-glucuronidase fusion gene expression predominantly in the embryonic and seedling root, with low expression in aerial parts. Cloning of the PLS locus revealed that the promoter trap T-DNA had inserted into a short open reading frame (ORF). Rapid amplification of cDNA ends PCR, RNA gel blot analysis, and RNase protection assays showed that the PLS ORF is located within a short ( approximately 500 nucleotides) auxin-inducible transcript and encodes a predicted polypeptide of 36 amino acid residues. pls mutants exhibit a short-root phenotype and reduced vascularization of leaves. pls roots are hyperresponsive to exogenous cytokinins and show increased expression of the cytokinin-inducible gene ARR5/IBC6 compared with the wild type. pls seedlings also are less responsive to the growth-inhibitory effects of exogenous auxin and show reduced expression of the auxin-inducible gene IAA1 compared with the wild type. The PLS peptide-encoding region of the cDNA partially complements the pls mutation and requires the PLS ORF ATG for activity, demonstrating the functionality of the peptide-encoding ORF. Ectopic expression of the PLS ORF reduces root growth inhibition by exogenous cytokinins and increases leaf vascularization. We propose that PLS is required for correct auxin-cytokinin homeostasis to modulate root growth and leaf vascular patterning.

Amino Acid Sequence↗
PubMed2006

Whole-genome analysis of the SHORT-ROOT developmental pathway in Arabidopsis.

Stem cell function during organogenesis is a key issue in developmental biology. The transcription factor SHORT-ROOT (SHR) is a critical component in a developmental pathway regulating both the specification of the root stem cell niche and the differentiation potential of a subset of stem cells in the Arabidopsis root. To obtain a comprehensive view of the SHR pathway, we used a statistical method called meta-analysis to combine the results of several microarray experiments measuring the changes in global expression profiles after modulating SHR activity. Meta-analysis was first used to identify the direct targets of SHR by combining results from an inducible form of SHR driven by its endogenous promoter, ectopic expression, followed by cell sorting and comparisons of mutant to wild-type roots. Eight putative direct targets of SHR were identified, all with expression patterns encompassing subsets of the native SHR expression domain. Further evidence for direct regulation by SHR came from binding of SHR in vivo to the promoter regions of four of the eight putative targets. A new role for SHR in the vascular cylinder was predicted from the expression pattern of several direct targets and confirmed with independent markers. The meta-analysis approach was then used to perform a global survey of the SHR indirect targets. Our analysis suggests that the SHR pathway regulates root development not only through a large transcription regulatory network but also through hormonal pathways and signaling pathways using receptor-like kinases. Taken together, our results not only identify the first nodes in the SHR pathway and a new function for SHR in the development of the vascular tissue but also reveal the global architecture of this developmental pathway.

Arabidopsis↗
PubMed1989

Reduced numbers of calcitonin gene-related peptide-(CGRP-) and tachykinin-immunoreactive sensory neurones associated with greater enkephalin immunoreactivity in the dorsal horn of a mutant rat with hereditary sensory neuropathy.

The mutilated foot rat is a mutant with autosomal recessive sensory neuropathy and frequent mutilation of the hindlimbs. Decreased numbers of dorsal root ganglion cells and diminished sensitivity to painful stimuli are characteristics of these animals. By use of immunocytochemistry, changes in the distributions of peptides involved in sensory and/or autonomic regulation, i.e., calcitonin gene-related peptide (CGRP), tachykinins, enkephalin and neuropeptide Y in spinal cord, dorsal root ganglia and skin of these animals, were studied. In comparison with normal litter-mate controls, the dorsal horn of mutilated foot rats contained substantially fewer CGRP- and tachykinin-immunoreactive fibres but more fibres immunoreactive for enkephalin. Many enkephalin-immunoreactive cell bodies were also found in the dorsal horn of the mutants, by contrast none were visible in control animals. Neuropeptide Y immunoreactivity was, however, unchanged in the spinal cord of the mutants. In the dorsal root ganglia of the mutants, the number of CGRP- or tachykinin-immunoreactive cells and their proportion to total neuronal numbers were significantly less in comparison with normal controls. The diameter range of CGRP- and tachykinin-immunoreactive cells shifted from small (15-25 microns) to medium size (25-45 microns) as revealed by frequency distribution histograms. The skin from the affected fore- and hindlimbs of the mutant rats, in keeping with fewer CGRP- and tachykinin-immunoreactive cells in the dorsal root ganglia, contained substantially less fibres immunoreactive for CGRP and tachykinins; a difference that was not seen in skin of unaffected areas (whiskers and snout). By contrast, neuropeptide Y-immunoreactive fibres showed a normal distribution around blood vessels and sweat glands of mutilated foot rats. The data suggest that diminished pain perception in the mutilated foot rat is related to loss of peptide-containing sensory neurones. Furthermore, the intraspinal increase of enkephalinergic neurons in the dorsal horn, concomitant with the decreased number of primary sensory neurones, may also play a contributory rôle in reducing pain thresholds.

Animals↗
PubMed1997

Analysis of SCARECROW expression using a rapid system for assessing transgene expression in Arabidopsis roots.

Although the introduction of foreign genes into Arabidopsis has become routine, the production of transgenic Arabidopsis plants still requires several months. A transgene expression system (TES) has been developed that allows characterization of gene expression patterns and the effects of foreign genes in the Arabidopsis root in 2-4 weeks. The method is based on regeneration of stably transformed roots directly from callus tissue. TES has been used to study the expression of the SCARECROW gene, which is involved in establishing radial patterning in the root. The 2.5 kb region directly upstream of the SCARECROW coding region was found to be sufficient to confer cell-type specific expression. Furthermore, this promoter is active in the scr mutant background, indicating that factors essential for cell-type specific expression are present even in the absence of correct radial patterning. Finally, TES was used to demonstrate that the SCARECROW gene under control of this promoter complements the root organization defect of the scr mutant. These experiments demonstrate the utility of the TES system for studying gene expression in roots in wild-type and mutant backgrounds and for molecular complementation of root mutant phenotypes. It is possible that the method will also be applicable to other organs.

Arabidopsis↗
PubMed2001

sas1, an Arabidopsis mutant overaccumulating sodium in the shoot, shows deficiency in the control of the root radial transport of sodium.

A recessive mutation of Arabidopsis designated sas1 (for sodium overaccumulation in shoot) that was mapped to the bottom of chromosome III resulted in a two- to sevenfold overaccumulation of Na(+) in shoots compared with wild-type plants. sas1 is a pleiotropic mutation that also caused severe growth reduction. The impact of NaCl stress on growth was similar for sas1 and wild-type plants; however, with regard to survival, sas1 plants displayed increased sensitivity to NaCl and LiCl treatments compared with wild-type plants. sas1 mutants overaccumulated Na(+) and its toxic structural analog Li(+), but not K(+), Mg(2)+, or Ca(2)+. Sodium accumulated preferentially over K(+) in a similar manner for sas1 and wild-type plants. Sodium overaccumulation occurred in all of the aerial organs of intact sas1 plants but not in roots. Sodium-treated leaf fragments or calli displayed similar Na(+) accumulation levels for sas1 and wild-type tissues. This suggested that the sas1 mutation impaired Na(+) long-distance transport from roots to shoots. The transpiration stream was similar in sas1 and wild-type plants, whereas the Na(+) concentration in the xylem sap of sas1 plants was 5.5-fold higher than that of wild-type plants. These results suggest that the sas1 mutation disrupts control of the radial transport of Na(+) from the soil solution to the xylem vessels.

Arabidopsis↗
PubMed2001

Chloroplast protein translocon components atToc159 and atToc33 are not essential for chloroplast biogenesis in guard cells and root cells.

Protein import into chloroplasts is mediated by a protein import apparatus located in the chloroplast envelope. Previous results indicate that there may be multiple import complexes in Arabidopsis. To gain further insight into the nature of this multiplicity, we analyzed the Arabidopsis ppi1 and ppi2 mutants, which are null mutants of the atToc33 and atToc159 translocon proteins, respectively. In the ppi2 mutant, in contrast to the extremely defective plastids in mesophyll cells, chloroplasts in guard cells still contained starch granules and thylakoid membranes. The morphology of root plastids in both mutants was similar to that in wild type. After prolonged light treatments, root plastids of both mutants and the wild type differentiated into chloroplasts. Enzymatic assays indicated that the activity of a plastid enzyme was reduced only in leaves but not in roots. These results indicated that both the ppi1 and ppi2 mutants had functional root and guard cell plastids. Therefore, we propose that import complexes are cell type specific rather than substrate or plastid specific.

Arabidopsis↗
PubMed2004

The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine in Arabidopsis.

Despite its importance in a variety of plant defense responses, our understanding of how jasmonic acid (JA) functions at the biochemical level is limited. Several amino acid conjugates of JA were tested for their ability to complement the JA-insensitive Arabidopsis thaliana mutant jar1-1. Unlike free JA, JA-Ile inhibited root growth in jar1-1 to the same extent as in the wild type, whereas JA-Val, JA-Leu, and JA-Phe were ineffective inhibitors in both genotypes. Thin-layer chromatography and gas chromatography-mass spectrometry (GC-MS) analysis of products produced in vitro by recombinant JAR1 demonstrated that this enzyme forms JA-amido conjugates with several amino acids, including JA-Ile. JA-Val, -Leu, -Ile, and -Phe were each quantified in Arabidopsis seedlings by GC-MS. JA-Ile was found at 29.6 pmole g(-1) fresh weight (FW) in the wild type but was more than sevenfold lower in two jar1 alleles. JA-Leu, -Val, and -Phe were present at only low levels in both genotypes. Expression of wild-type JAR1 in transgenic jar1-1 plants restored sensitivity to JA and elevated JA-Ile to the same level as in the wild type. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) conjugated to JA was also found in plant tissue at 18.4 pmole g(-1) FW. JA-ACC was determined not be an effective jasmonate root inhibitor, and surprisingly, was twofold higher in the mutants than in the wild type. This suggests that another JA-conjugating enzyme(s) is present in Arabidopsis. Synthesis of JA-ACC might provide a mechanism to coregulate the availability of JA and ACC for conversion to the active hormones JA-Ile and ethylene, respectively. We conclude that JAR1 is a JA-amino synthetase that is required to activate JA for optimal signaling in Arabidopsis. Plant hormone activation by conjugation to amino acids and the enzymes involved in their formation were previously unknown.

Amino Acid Motifs↗