Search

Chlorophyll degradation causes the release of phytol, which is converted into phytyl diphosphate (phytyl-PP) by phytol kinase (VITAMIN E PATHWAY GENE5 [VTE5]) and phytyl phosphate (phytyl-P) kinase (VTE6). The kinase pathway is important for tocopherol synthesis, as the Arabidopsis (Arabidopsis thaliana) vte5 mutant contains reduced levels of tocopherol. Arabidopsis harbors one paralog of VTE5, farnesol kinase (FOLK) involved in farnesol phosphorylation. Here, we demonstrate that VTE5 and FOLK harbor kinase activities for phytol, geranylgeraniol, and farnesol with different specificities. While the tocopherol content of the folk mutant is unchanged, vte5-2 folk plants completely lack tocopherol. Tocopherol deficiency in vte5-2 plants can be complemented by overexpression of FOLK, indicating that FOLK is an authentic gene of tocopherol synthesis. The vte5-2 folk plants contain only ∼40% of wild-type amounts of phylloquinone, demonstrating that VTE5 and FOLK both contribute in part to phylloquinone synthesis. Tocotrienol and menaquinone-4 were produced in vte5-2 folk plants after supplementation with homogentisate or 1,4-dihydroxy-2-naphthoic acid, respectively, indicating that their synthesis is independent of the VTE5/FOLK pathway. These results show that phytyl moieties for tocopherol synthesis are completely but, for phylloquinone production, only partially derived from geranylgeranyl-chlorophyll and phytol phosphorylation by VTE5 and FOLK., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Proper short- and long-term acclimation to different growth light intensities is essential for the survival and competitiveness of plants in the field. High light exposure is known to induce the down-regulation and photoinhibition of photosystem II (PSII) activity to reduce photo-oxidative stress. The xanthophyll zeaxanthin (Zx) serves central photoprotective functions in these processes. We have shown in recent work with different plant species (Arabidopsis, tobacco, spinach and pea) that photoinhibition of PSII and degradation of the PSII reaction center protein D1 is accompanied by the inactivation and degradation of zeaxanthin epoxidase (ZEP), which catalyzes the reconversion of Zx to violaxanthin. Different high light sensitivity of the above-mentioned species correlated with differential down-regulation of both PSII and ZEP activity. Applying light and electron microscopy, chlorophyll fluorescence, and protein and pigment analyses, we investigated the acclimation properties of these species to different growth light intensities with respect to the ability to adjust their photoprotective strategies. We show that the species differ in phenotypic plasticity in response to short- and long-term high light conditions at different morphological and physiological levels. However, the close co-regulation of PSII and ZEP activity remains a common feature in all species and under all conditions. This work supports species-specific acclimation strategies and properties in response to high light stress and underlines the central role of the xanthophyll Zx in photoprotection., (© 2023 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Despite serving as a major inorganic nitrogen source for plants, ammonium causes toxicity at elevated concentrations, inhibiting root elongation early on. While previous studies have shown that ammonium-inhibited root development relates to ammonium uptake and formation of reactive oxygen species (ROS) in roots, it remains unclear about the mechanisms underlying the repression of root growth and how plants cope with this inhibitory effect of ammonium. In this study, we demonstrate that ammonium-induced apoplastic acidification co-localizes with Fe precipitation and hydrogen peroxide (H 2 O 2 ) accumulation along the stele of the elongation and differentiation zone in root tips, indicating Fe-dependent ROS formation. By screening ammonium sensitivity in T-DNA insertion lines of ammonium-responsive genes, we identified PDX1.1, which is upregulated by ammonium in the root stele and whose product catalyzes de novo biosynthesis of vitamin B 6 . Root growth of pdx1.1 mutants is hypersensitive to ammonium, while chemical complementation or overexpression of PDX1.1 restores root elongation. This salvage strategy requires non-phosphorylated forms of vitamin B 6 that are able to quench ROS and rescue root growth from ammonium inhibition. Collectively, these results suggest that PDX1.1-mediated synthesis of non-phosphorylated B 6 vitamers acts as a primary strategy to protect roots from ammonium-dependent ROS formation., (Copyright © 2022 The Author. Published by Elsevier Inc. All rights reserved.)

With the notable exception of angiosperms, all phototrophs contain different sets of flavodiiron proteins that help to relieve the excess of excitation energy on the photosynthetic electron transport chain during adverse environmental conditions, presumably by reducing oxygen directly to water. Among them, the Flv2-Flv4 dimer is only found in β-cyanobacteria and induced by high light, supporting a role in stress protection. The possibility of a similar protective function in plants was assayed by expressing Synechocystis Flv2-Flv4 in chloroplasts of tobacco and Arabidopsis. Flv-expressing plants exhibited increased tolerance toward high irradiation, salinity, oxidants, and drought. Stress tolerance was reflected by better growth, preservation of photosynthetic activity, and membrane integrity. Metabolic profiling under drought showed enhanced accumulation of soluble sugars and amino acids in transgenic Arabidopsis and a remarkable shift of sucrose into starch, in line with metabolic responses of drought-tolerant genotypes. Our results indicate that the Flv2-Flv4 complex retains its stress protection activities when expressed in chloroplasts of angiosperm species by acting as an additional electron sink. The flv2-flv4 genes constitute a novel biotechnological tool to generate plants with increased tolerance to agronomically relevant stress conditions that represent a significant productivity constraint.

Strigolactones (SLs) are a group of plant hormones involved in many aspects of plant development and stress adaptation. Here, we investigated the drought response of a barley (Hordeum vulgare L.) mutant carrying a missense mutation in the gene encoding the SL-specific receptor HvD14. Our results clearly showed that hvd14.d mutant is hyper-sensitive to drought stress. This was illustrated by a lower leaf relative water content (RWC), impaired photosynthesis, disorganization of chloroplast structure, altered stomatal density and slower closure of stomata in response to drought in the mutant compared to the wild type parent cultivar Sebastian. Although the content of abscisic acid (ABA) and its derivatives remained unchanged in the mutant, significant differences in expression of genes related to ABA biosynthesis were observed. Moreover, hvd14.d was insensitive to ABA during seed germination. Analysis of Arabidopsis thaliana mutant atd14-1 also demonstrated that mutation in the SL receptor resulted in increased sensitivity to drought. Our results indicate that the drought-sensitive phenotype of barley SL mutant might be caused by a disturbed ABA metabolism and/or signalling pathways. These results together uncovered a link between SL signalling and ABA-dependent drought stress response in barley., (© 2020 John Wiley & Sons Ltd.)

Storage of meristematic tissue at ultra-low temperatures offers a mean to maintain valuable genetic resources from vegetatively reproduced plants. To reveal the biology underlying cryo-stress, shoot tips of the model plant Arabidopsis thaliana were subjected to a standard preservation procedure. A transcriptomic approach was taken to describe the subsequent cellular events which occurred. The cryoprotectant treatment induced the changes in the transcript levels of genes associated with RNA processing and primary metabolism. Explants of a mutant lacking a functional copy of the transcription factor WRKY22 were compromised for recovery. A number of putative downstream targets of WRKY22 were identified, some related to phytohormone-mediated defense, to the osmotic stress response, and to development. There were also alterations in the abundance of transcript produced by genes encoding photosynthesis-related proteins. The wrky22 mutant plants developed an open stomata phenotype in response to their exposure to the cryoprotectant solution. WRKY22 probably regulates a transcriptional network during cryo-stress, linking the explant's defense and osmotic stress responses to changes in its primary metabolism. A model is proposed linking WRKY53 and WRKY70 downstream of the action of WRKY22., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Key Message: We identify proteins that associate with the THO core complex, and show that the TEX1 and MOS11 components functionally interact, affecting mRNA export and splicing as well as plant development. TREX (TRanscription-EXport) is a multiprotein complex that plays a central role in the coordination of synthesis, processing and nuclear export of mRNAs. Using targeted proteomics, we identified proteins that associate with the THO core complex of Arabidopsis TREX. In addition to the RNA helicase UAP56 and the mRNA export factors ALY2-4 and MOS11 we detected interactions with the mRNA export complex TREX-2 and multiple spliceosomal components. Plants defective in the THO component TEX1 or in the mRNA export factor MOS11 (orthologue of human CIP29) are mildly affected. However, tex1 mos11 double-mutant plants show marked defects in vegetative and reproductive development. In tex1 plants, the levels of tasiRNAs are reduced, while miR173 levels are decreased in mos11 mutants. In nuclei of mos11 cells increased mRNA accumulation was observed, while no mRNA export defect was detected with tex1 cells. Nevertheless, in tex1 mos11 double-mutants, the mRNA export defect was clearly enhanced relative to mos11. The subnuclear distribution of TEX1 substantially overlaps with that of splicing-related SR proteins and in tex1 plants the ratio of certain alternative splicing events is altered. Our results demonstrate that Arabidopsis TEX1 and MOS11 are involved in distinct steps of the biogenesis of mRNAs and small RNAs, and that they interact regarding some aspects, but act independently in others.

Phosphatidylglycerol (PG) is the only phospholipid in the thylakoid membranes of chloroplasts of plants, and it is also found in extraplastidial membranes including mitochondria and the endoplasmic reticulum. Previous studies showed that lack of PG in the pgp1-2 mutant of Arabidopsis deficient in phosphatidylglycerophosphate (PGP) synthase strongly affects thylakoid biogenesis and photosynthetic activity. In the present study, the gene encoding the enzyme for the second step of PG synthesis, PGP phosphatase, was isolated based on sequence similarity to the yeast GEP4 and Chlamydomonas PGPP1 genes. The Arabidopsis AtPGPP1 protein localizes to chloroplasts and harbors PGP phosphatase activity with alkaline pH optimum and divalent cation requirement. Arabidopsis pgpp1-1 mutant plants contain reduced amounts of chlorophyll, but photosynthetic quantum yield remains unchanged. The absolute content of plastidial PG (34:4; total number of acyl carbons:number of double bonds) is reduced by about 1/3, demonstrating that AtPGPP1 is involved in the synthesis of plastidial PG. PGP 34:3, PGP 34:2 and PGP 34:1 lacking 16:1 accumulate in pgpp1-1, indicating that the desaturation of 16:0 to 16:1 by the FAD4 desaturase in the chloroplasts only occurs after PGP dephosphorylation., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

Plasmodesmata (PD) are microscopic pores connecting plant cells and enable cell-to-cell transport. Currently, little information is known about the molecular mechanisms regulating PD formation and development. To uncover components of PD development we made use of the 17 kDa movement protein (MP17) encoded by the Potato leafroll virus (PLRV). The protein is required for cell-to-cell movement of the virus and localises to complex PD. Forward genetic screening for Arabidopsis mutants with altered PD binding of MP17 revealed several mutant lines, while molecular genetics, biochemical and microscopic studies allowed further characterisation. Map-based cloning of one mutant revealed a point mutation in the choline transporter-like 1 (CHER1) protein, changing glycine 247 into glutamate. Mutation in CHER1 resulted in a starch excess phenotype and stunted growth. Ultrastructure analysis of shoot apical meristems, developing and fully developed leaves showed reduced PD numbers and the absence of complex PD in fully developed leaves. This indicates that cher1 mutants are impaired in PD formation and development. Global lipid profiling revealed only slight modifications in the overall lipid composition, however, altered composition of PD-associated lipids cannot be ruled out. Thus, cher1 is devoid of complex PD in developed leaves and provides insights into the formation of complex PD at the molecular level., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

The non-photochemical quenching of light energy as heat (NPQ) is an important photoprotective mechanism that is activated in plants when light absorption exceeds the capacity of light utilization in photosynthesis. The PsbS protein plays a central role in this process and is supposed to activate NPQ through specific, light-regulated interactions with photosystem (PS) II antenna proteins. However, NPQ-specific interaction partners of PsbS in the thylakoid membrane are still unknown. Here, we have determined the localization and protein interactions of PsbS in thylakoid membranes in the NPQ-inactive (dark) and NPQ-active (light) states. Our results corroborate a localization of PsbS in PSII supercomplexes and support the model that the light activation of NPQ is based on the monomerization of dimeric PsbS and a light-induced enhanced interaction of PsbS with Lhcb1, the major component of trimeric light-harvesting complexes in PSII.

The enzyme zeaxanthin epoxidase (ZEP) catalyzes the conversion of zeaxanthin to violaxanthin, a key reaction for ABA biosynthesis and the xanthophyll cycle. Both processes are important for acclimation to environmental stress conditions, in particular drought (ABA biosynthesis) and light (xanthophyll cycle) stress. Hence, both ZEP functions may require differential regulation to optimize plant fitness. The key to understanding the function of ZEP in both stress responses might lie in its spatial and temporal distribution in plant tissues. Therefore, we analyzed the distribution of ZEP in plant tissues and plastids under drought and light stress by use of a ZEP-specific antibody. In addition, we determined the pigment composition of the plant tissues and chloroplast membrane subcompartments in response to these stresses. The ZEP protein was detected in all plant tissues (except flowers) concomitant with xanthophylls. The highest levels of ZEP were present in leaf chloroplasts and root plastids. Within chloroplasts, ZEP was localized predominantly in the thylakoid membrane and stroma, while only a small fraction was bound by the envelope membrane. Light stress affected neither the accumulation nor the relative distribution of ZEP in chloroplasts, while drought stress led to an increase of ZEP in roots and to a degradation of ZEP in leaves. However, drought stress-induced increases in ABA were similar in both tissues. These data support a tissue- and stress-specific accumulation of the ZEP protein in accordance with its different functions in ABA biosynthesis and the xanthophyll cycle., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In plants, urea derives either from root uptake or protein degradation. Although large quantities of urea are released during senescence, urea is mainly seen as a short-lived nitrogen (N) catabolite serving urease-mediated hydrolysis to ammonium. Here, we investigated the roles of DUR3 and of urea in N remobilization. During natural leaf senescence urea concentrations and DUR3 transcript levels showed a parallel increase with senescence markers like ORE1 in a plant age- and leaf age-dependent manner. Deletion of DUR3 decreased urea accumulation in leaves, whereas the fraction of urea lost to the leaf apoplast was enhanced. Under natural and N deficiency-induced senescence DUR3 promoter activity was highest in the vasculature, but was also found in surrounding bundle sheath and mesophyll cells. An analysis of petiole exudates from wild-type leaves revealed that N from urea accounted for >13% of amino acid N. Urea export from senescent leaves further increased in ureG-2 deletion mutants lacking urease activity. In the dur3 ureG double insertion line the absence of DUR3 reduced urea export from leaf petioles. These results indicate that urea can serve as an early metabolic marker for leaf senescence, and that DUR3-mediated urea retrieval contributes to the retranslocation of N from urea during leaf senescence., (© 2014 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The heterodimeric complex SPT4/SPT5 is a transcript elongation factor (TEF) that directly interacts with RNA polymerase II (RNAPII) to regulate messenger RNA synthesis in the chromatin context. We provide biochemical evidence that in Arabidopsis, SPT4 occurs in a complex with SPT5, demonstrating that the SPT4/SPT5 complex is conserved in plants. Each subunit is encoded by two genes SPT4-1/2 and SPT5-1/2. A mutant affected in the tissue-specifically expressed SPT5-1 is viable, whereas inactivation of the generally expressed SPT5-2 is homozygous lethal. RNAi-mediated downregulation of SPT4 decreases cell proliferation and causes growth reduction and developmental defects. These plants display especially auxin signalling phenotypes. Consistently, auxin-related genes, most strikingly AUX/IAA genes, are downregulated in SPT4-RNAi plants that exhibit an enhanced auxin response. In Arabidopsis nuclei, SPT5 clearly localizes to the transcriptionally active euchromatin, and essentially co-localizes with transcribing RNAPII. Typical for TEFs, SPT5 is found over the entire transcription unit of RNAPII-transcribed genes. In SPT4-RNAi plants, elevated levels of RNAPII and SPT5 are detected within transcribed regions (including those of downregulated genes), indicating transcript elongation defects in these plants. Therefore, SPT4/SPT5 acts as a TEF in Arabidopsis, regulating transcription during the elongation stage with particular impact on the expression of certain auxin-related genes.

Stem cells in the root and shoot apical meristem provide the descendant cells required for growth and development throughout the lifecycle of a plant. We found that mutations in the Arabidopsis MAINTENANCE OF MERISTEMS (MAIN) gene led to plants with distorted stem cell niches in which stem cells are not maintained and undergo premature differentiation or cell death. The malfunction of main meristems leads to short roots, mis-shaped leaves, reduced fertility and partial fasciation of stems. MAIN encodes a nuclear-localized protein and is a member of a so far uncharacterized plant-specific gene family. As main mutant plants are hypersensitive to DNA-damaging agents, expression of genes involved in DNA repair is induced and dead cells with damaged DNA accumulate in the mutant meristems, we propose that MAIN is required for meristem maintenance by sustaining genome integrity in stem cells and their descendants cells., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

Survival of plants at low temperature depends on mechanisms for limiting physiological damage and maintaining growth. We mapped the chs1-1 (chilling sensitive1-1) mutation in Arabidopsis accession Columbia to the TIR-NBS gene At1g17610. In chs1-1, a single amino acid exchange at the CHS1 N-terminus close to the conserved TIR domain creates a stable mutant protein that fails to protect leaves against chilling stress. The sequence of another TIR-NBS gene (At5g40090) named CHL1 (CHS1-like 1) is related to that of CHS1. Over-expression of CHS1 or CHL1 alleviates chilling damage and enhances plant growth at moderate (24°C) and chilling (13°C) temperatures, suggesting a role for both proteins in growth homeostasis. chs1-1 mutants show induced salicylic acid production and defense gene expression at 13°C, indicative of autoimmunity. Genetic analysis of chs1-1 in combination with defense pathway mutants shows that chs1-1 chilling sensitivity requires the TIR-NBS-LRR and basal resistance regulators encoded by EDS1 and PAD4 but not salicylic acid. By following the timing of metabolic, physiological and chloroplast ultrastructural changes in chs1-1 leaves during chilling, we have established that alterations in photosynthetic complexes and thylakoid membrane integrity precede leaf cell death measured by ion leakage. At 24°C, the chs1-1 mutant appears normal but produces a massive necrotic response to virulent Pseudomonas syringae pv. tomato infection, although this does not affect bacterial proliferation. Our results suggest that CHS1 acts at an intersection between temperature sensing and biotic stress pathway activation to maintain plant performance over a range of conditions., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

Heterosis-associated cellular and molecular processes were analyzed in seeds and seedlings of Arabidopsis thaliana accessions Col-0 and C24 and their heterotic hybrids. Microscopic examination revealed no advantages in terms of hybrid mature embryo organ sizes or cell numbers. Increased cotyledon sizes were detectable 4 days after sowing. Growth heterosis results from elevated cell sizes and numbers, and is well established at 10 days after sowing. The relative growth rates of hybrid seedlings were most enhanced between 3 and 4 days after sowing. Global metabolite profiling and targeted fatty acid analysis revealed maternal inheritance patterns for a large proportion of metabolites in the very early stages. During developmental progression, the distribution shifts to dominant, intermediate and heterotic patterns, with most changes occurring between 4 and 6 days after sowing. The highest incidence of heterotic patterns coincides with establishment of size differences at 4 days after sowing. In contrast, overall transcript patterns at 4, 6 and 10 days after sowing are characterized by intermediate to dominant patterns, with parental transcript levels showing the largest differences. Overall, the results suggest that, during early developmental stages, intermediate gene expression and higher metabolic activity in the hybrids compared to the parents lead to better resource efficiency, and therefore enhanced performance in the hybrids., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

Background: Arabidopsis DMP1 was discovered in a genome-wide screen for senescence-associated membrane proteins. DMP1 is a member of a novel plant-specific membrane protein family of unknown function. In rosette leaves DMP1 expression increases from very low background level several 100fold during senescence progression., Results: Expression of AtDMP1 fused to eGFP in Nicotiana benthamiana triggers a complex process of succeeding membrane remodeling events affecting the structure of the endoplasmic reticulum (ER) and the vacuole. Induction of spherical structures ("bulbs"), changes in the architecture of the ER from tubular to cisternal elements, expansion of smooth ER, formation of crystalloid ER, and emergence of vacuolar membrane sheets and foamy membrane structures inside the vacuole are proceeding in this order. In some cells it can be observed that the process culminates in cell death after breakdown of the entire ER network and the vacuole. The integrity of the plasma membrane, nucleus and Golgi vesicles are retained until this stage. In Arabidopsis thaliana plants expressing AtDMP1-eGFP by the 35S promoter massive ER and vacuole vesiculation is observed during the latest steps of leaf senescence, whereas earlier in development ER and vacuole morphology are not perturbed. Expression by the native DMP1 promoter visualizes formation of aggregates termed "boluses" in the ER membranes and vesiculation of the entire ER network, which precedes disintegration of the central vacuole during the latest stage of senescence in siliques, rosette and cauline leaves and in darkened rosette leaves. In roots tips, DMP1 is strongly expressed in the cortex undergoing vacuole biogenesis., Conclusions: Our data suggest that DMP1 is directly or indirectly involved in membrane fission during breakdown of the ER and the tonoplast during leaf senescence and in membrane fusion during vacuole biogenesis in roots. We propose that these properties of DMP1, exacerbated by transient overexpression, may cause or contribute to the dramatic membrane remodeling events which lead to cell death in infiltrated tobacco leaves.

Tetrapyrrole biosynthesis is controlled by multiple environmental and endogenous cues. Etiolated T-DNA insertion mutants were screened for red fluorescence as result of elevated levels of protochlorophyllide and four red fluorescent in the dark (rfd) mutants were isolated and identified. rfd3 and rfd4 belong to the group of photomorphogenic cop/det/fus mutants. rfd1 and rfd2 had genetic lesions in RIBA1 and FLU encoding the dual-functional protein GTP cyclohydrolase II/3,4-dihydroxy-2-butanone-4-phosphate synthase and a negative regulator of tetrapyrrole biosynthesis, respectively. RIBA1 catalyses the initial reaction of the metabolic pathway of riboflavin biosynthesis and rfd1 contains reduced contents of riboflavin and the flavo-coenzymes FMN and FAD. Transcriptome analysis of rfd1 revealed up-regulated genes encoding nucleus-localized factors involved in cytokinin signalling and numerous down-regulated LEA genes as well as an auxin-inducible GH3 gene. Alteration of cytokinin metabolism of rfd1was confirmed by elevated contents of active forms of cytokinin and stimulated expression of an ARR6::GUS reporter construct. An etiolated quadruple ckx (cytokinin oxidase) mutant with impaired cytokinin degradation as well as different knockout mutants for the negative AUX/IAA regulators shy2-101 (iaa3), axr2-1 (iaa7) and slr-1 (iaa14) showed also excessive protochlorophyllide accumulation. The transcript levels of CHLH and HEMA1 encoding Mg chelatase and glutamyl-tRNA reductase were increased in rfd1 and the AUX/IAA loss-of-function mutants. It is proposed that reduced riboflavin synthesis impairs the activity of the flavin-containing cytokinin oxidase, increases cytokinin contents and de-represses synthesis of 5-aminolevulinic acid of tetrapyrrole metabolism in darkness. As result of the mutant analyses, the antagonistic cytokinin and auxin signalling is required for a balanced tetrapyrrole biosynthesis in the dark.

5-Aminolevulinic acid (ALA) is the universal precursor for tetrapyrrole biosynthesis and is synthesized in plants in three enzymatic steps: ligation of glutamate (Glu) to tRNA(Glu) by glutamyl-tRNA synthetase, reduction of activated Glu to Glu-1-semialdehyde by glutamyl-tRNA reductase (GluTR), and transamination to ALA by Glu 1-semialdehyde aminotransferase. ALA formation controls the metabolic flow into the tetrapyrrole biosynthetic pathway. GluTR is proposed to be the key regulatory enzyme that is tightly controlled at transcriptional and posttranslational levels. We identified a GluTR binding protein (GluTRBP; previously called PROTON GRADIENT REGULATION7) that is localized in chloroplasts and part of a 300-kD protein complex in the thylakoid membrane. Although the protein does not modulate activity of ALA synthesis, the knockout of GluTRBP is lethal in Arabidopsis thaliana, whereas mutants expressing reduced levels of GluTRBP contain less heme. GluTRBP expression correlates with a function in heme biosynthesis. It is postulated that GluTRBP contributes to subcompartmentalized ALA biosynthesis by maintaining a portion of GluTR at the plastid membrane that funnels ALA into the heme biosynthetic pathway. These results regarding GluTRBP support a model of plant ALA synthesis that is organized in two separate ALA pools in the chloroplast to provide appropriate substrate amounts for balanced synthesis of heme and chlorophyll.

Here, we characterize a plastidial thioredoxin (TRX) isoform from Arabidopsis thaliana that defines a previously unknown branch of plastidial TRXs lying between x- and y-type TRXs and thus was named TRX z. An Arabidopsis knockout mutant of TRX z had a severe albino phenotype and was inhibited in chloroplast development. Quantitative real-time RT-PCR analysis of the mutant suggested that the expressions of genes that depend on a plastid-encoded RNA polymerase (PEP) were specifically decreased. Similar results were obtained upon virus-induced gene silencing (VIGS) of the TRX z ortholog in Nicotiana benthamiana. We found that two fructokinase-like proteins (FLN1 and FLN2), members of the pfkB-carbohydrate kinase family, were potential TRX z target proteins and identified conserved Cys residues mediating the FLN-TRX z interaction. VIGS in N. benthamiana and inducible RNA interference in Arabidopsis of FLNs also led to a repression of PEP-dependent gene transcription. Remarkably, recombinant FLNs displayed no detectable sugar-phosphorylating activity, and amino acid substitutions within the predicted active site imply that the FLNs have acquired a new function, which might be regulatory rather than metabolic. We were able to show that the FLN2 redox state changes in vivo during light/dark transitions and that this change is mediated by TRX z. Taken together, our data strongly suggest an important role for TRX z and both FLNs in the regulation of PEP-dependent transcription in chloroplasts.

The facilitates chromatin transcription (FACT) complex, consisting of the SSRP1 and SPT16 proteins, is a histone chaperone that assists the progression of transcribing RNA polymerase on chromatin templates by destabilizing nucleosomes. Here, we examined plants that harbour mutations in the genes encoding the subunits of Arabidopsis FACT. These experiments revealed that (i) SSRP1 is critical for plant viability, and (ii) plants with reduced amounts of SSRP1 and SPT16 display various defects in vegetative and reproductive development. Thus, mutant plants display an increased number of leaves and inflorescences, show early bolting, have abnormal flower and leaf architecture, and their seed production is severely affected. The early flowering of the mutant plants is associated with reduced expression of the floral repressor FLC in ssrp1 and spt16 plants. Compared to control plants, reduced amounts of FACT in mutant plants are detected at the FLC locus as well as at the locations of housekeeping genes (whose expression is not affected in the mutants), suggesting that expression of FLC is particularly sensitive to reduced FACT activity. Analysis of double mutants that are affected in the expression of both FACT subunits and factors catalysing the mono-ubiquitination of histone H2B (HUB1/2) demonstrates that they genetically interact to regulate various developmental processes (i.e. branching, leaf venation pattern, silique development) but independently regulate the growth of leaves and the induction of flowering.

The galactolipid digalactosyldiacylglycerol (DGD) is an abundant thylakoid lipid in chloroplasts. The introduction of the bacterial lipid glucosylgalactosyldiacylglycerol (GGD) from Chloroflexus aurantiacus into the DGD-deficient Arabidopsis (Arabidopsis thaliana) dgd1 mutant was previously shown to result in complementation of growth, but photosynthetic efficiency was only partially restored. Here, we demonstrate that GGD accumulation in the double mutant dgd1dgd2, which is totally devoid of DGD, also complements growth at normal and high-light conditions, but photosynthetic efficiency in the GGD-containing dgd1dgd2 line remains decreased. This is attributable to an increased susceptibility of photosystem II to photodamage, resulting in reduced photosystem II accumulation already at normal light intensities. The chloroplasts of dgd1 and dgd1dgd2 show alterations in thylakoid ultrastructure, a phenotype that is restored in the GGD-containing lines. These data suggest that the strong growth retardation of the DGD-deficient lines dgd1 and dgd1dgd2 can be primarily attributed to a decreased capacity for chloroplast membrane assembly and proliferation and, to a smaller extent, to photosynthetic deficiency. During phosphate limitation, GGD increases in plastidial and extraplastidial membranes of the transgenic lines to an extent similar to that of DGD in the wild type, indicating that synthesis and transport of the bacterial lipid (GGD) and of the authentic plant lipid (DGD) are subject to the same mechanisms of regulation.

Glutathione peroxidases (GPXs; EC 1.11.1.9) are key enzymes of the antioxidant network in plants and animals. In order to investigate the role of antioxidant systems in plant chloroplasts, we generated Arabidopsis (Arabidopsis thaliana) transgenic lines that are depleted specifically in chloroplastic (cp) forms of GPX1 and GPX7. We show that reduced cpGPX expression, either in transgenic lines with lower total cpGPX expression (GPX1 and GPX7) or in a gpx7 insertion mutant, leads to compromised photooxidative stress tolerance but increased basal resistance to virulent bacteria. Depletion of both GPX1 and GPX7 expression also caused alterations in leaf cell and chloroplast morphology. Leaf tissues were characterized by shorter and more rounded palisade cells, irregular spongy mesophyll cells, and larger intercellular air spaces compared with the wild type. Chloroplasts had larger and more abundant starch grains than in wild-type and gpx7 mutant plants. Constitutively reduced cpGPX expression also led to higher foliar ascorbic acid, glutathione, and salicylic acid levels in plants exposed to higher light intensities. Our results suggest partially overlapping functions of GPX1 and GPX7. The data further point to specific changes in the chloroplast ascorbate-glutathione cycle due to reduced cpGPX expression, initiating reactive oxygen species and salicylic acid pathways that affect leaf development, light acclimation, basal defense, and cell death programs. Thus, cpGPXs regulate cellular photooxidative tolerance and immune responses.

Transcript elongation factor TFIIS promotes efficient transcription by RNA polymerase II, since it assists in bypassing blocks during mRNA synthesis. While yeast cells lacking TFIIS are viable, inactivation of mouse TFIIS causes embryonic lethality. Here, we have identified a protein encoded in the Arabidopsis genome that displays a marked sequence similarity to TFIIS of other organisms, primarily within domains II and III in the C-terminal part of the protein. TFIIS is widely expressed in Arabidopsis, and a green fluorescent protein-TFIIS fusion protein localises specifically to the cell nucleus. When expressed in yeast cells lacking the endogenous TFIIS, Arabidopsis TFIIS partially complements the sensitivity of mutant cells to the nucleotide analog 6-azauridine, which is a typical characteristic of transcript elongation factors. We have characterised Arabidopsis lines harbouring T-DNA insertions in the coding sequence of TFIIS. Plants homozygous for T-DNA insertions are viable, and genomewide transcript profiling revealed that compared to control plants, a relatively small number of genes are differentially expressed in mutant plants. TFIIS(-/-) plants display essentially normal development, but they flower slightly earlier than control plants and show clearly reduced seed dormancy. Plants with RNAi-mediated knockdown of TFIIS expression also are affected in seed dormancy. Therefore, TFIIS plays a critical role in Arabidopsis seed development.

Many metabolic reactions in the endoplasmic reticulum (ER) require high levels of energy in the form of ATP, which is important for cell viability. Here, we report on an adenine nucleotide transporter residing in the ER membranes of Arabidopsis thaliana (ER-ANT1). Functional integration of ER-ANT1 in the cytoplasmic membrane of intact Escherichia coli cells reveals a high specificity for an ATP/ADP antiport. Immunodetection in transgenic ER-ANT1-C-MYC-tag Arabidopsis plants and immunogold labeling of wild-type pollen grain tissue using a peptide-specific antiserum reveal the localization of this carrier in ER membranes. Transgenic ER-ANT1-promoter-beta-glucuronidase Arabidopsis lines show high expression in ER-active tissues (i.e., pollen, seeds, root tips, apical meristems, or vascular bundles). Two independent ER-ANT1 Arabidopsis knockout lines indicate a high physiological relevance of ER-ANT1 for ATP transport into the plant ER (e.g., disruption of ER-ANT1 results in a drastic retardation of plant growth and impaired root and seed development). In these ER-ANT1 knockout lines, the expression levels of several genes encoding ER proteins that are dependent on a sufficient ATP supply (i.e., BiP [for luminal binding protein] chaperones, calreticulin chaperones, Ca2+-dependent protein kinase, and SEC61) are substantially decreased.

When grown in short day conditions and at low light, leaves of Arabidopsis plants with mutations in the genes encoding two plastidial ATP/ADP transporters (so-called null mutants) spontaneously develop necrotic lesions. Under these conditions, the mutants also display light-induced accumulation of H(2)O(2) and constitutive expression of genes for copper/zinc superoxide dismutase 2 and ascorbate peroxidase 1. In the light phase, null mutants accumulate high levels of phototoxic protoporphyrin IX but have only slightly reduced levels of Mg protoporphyrin IX. The physiological changes are associated with reduced magnesium-chelatase activity. Since the expression of genes encoding any of the three subunits of magnesium-chelatase is similar in wild type and null mutants, decreased enzyme activity is probably due to post-translational modification which might be due to limited availability of ATP in plastids during the night. Surprisingly, the formation of necrotic lesions was absent when null mutants were grown either in long days and low light intensity or in short days and high light intensity. We ascribe the lack of lesion phenotype to increased nocturnal ATP supply due to glycolytic degradation of starch which may lead to additional substrate-level phosphorylation in the stroma. Thus, nocturnal import of ATP into chloroplasts represents a crucial, previously unknown process that is required for controlled chlorophyll biosynthesis and for preventing photooxidative damage.

The plant hormone gibberellin (GA) is known to modulate various aspects of plant cell differentiation and development. The current model of GA-mediated regulation is based on a de-repressible system and includes specific protein modification and degradation. HRT, a zinc finger protein from barley has been shown to have GA-dependent transcriptional repressing activity on the seed-specific alpha-amylase promoter [Raventos, D., Skriver, K., Schlein, M., Karnahl, K., Rogers, S.W., Rogers, J.C. and Mundy, J. 1998. J. Biol. Chem. 273: 23313-23320]. Here we report the characterization of a dicot homologue from Brassica napus (BnET) and provide evidence for its role in GA response modulation suggesting that this could be a conserved feature of this gene family. When BnET is ectopically expressed in either Arabidopsis or tobacco the phenotypes include dwarfism due to shorter internodes and late flowering, reduced germination rate, increased anthocyanin content and reduced xylem lignification as a marker for terminal cell differentiation. Transient expression in protoplasts supports the notion that this most likely is due to a transcriptional repression of GA controlled genes. Finally, histological analysis showed that in contrast to other GA deficient mutants the shorter internodes were due to fewer but not smaller cells, suggesting a function of BnET in GA-mediated cell division control.

Guard cell chloroplasts are unable to perform significant photosynthetic CO2 fixation via Rubisco. Therefore, guard cells depend on carbon supply from adjacent cells even during the light period. Due to their reversible turgor changes, this import cannot be mediated by plasmodesmata. Nevertheless, guard cells of several plants were shown to use extracellular sugars or to accumulate sucrose as an osmoticum that drives water influx to increase stomatal aperture. This paper describes the first localization of a guard cell-specific Arabidopsis sugar transporter involved in carbon acquisition of these symplastically isolated cells. Expression of the AtSTP1 H+-monosacharide symporter gene in guard cells was demonstrated by in situ hybridization and by immunolocalization with an AtSTP1-specific antiserum. Additional RNase protection analyses revealed a strong increase of AtSTP1 expression in the dark and a transient, diurnally regulated increase during the photoperiod around midday. This transient increase in AtSTP1 expression correlates in time with the described guard cell-specific accumulation of sucrose. Our data suggest a function of AtSTP1 in monosaccharide import into guard cells during the night and a possible role in osmoregulation during the day.

Plants, in contrast to other eukaryotes, possess not only homologs of subunit A (AtSPO11-1, 2, 3) but also of subunit B (AtTOP6B) of the archaebacterial topoisomerase VI. AtTOP6B and AtSPO11-3 are strongly expressed in somatic tissue of Arabidopsis and are able to interact with each other in vitro. A T-DNA insertion in AtTOP6B results in deficient cell proliferation; plants stop growing at the rosette stage, have small crinkled leaves, and die about 4 weeks after germination. Cultured root cells die after a limited number of cell divisions. The mitotic index of the root meristems is strongly reduced. Flow cytometric analysis demonstrates that endoreplication in mutant plants is stopped at the 8C stage; the last cycle is not completed in most cases. Mutant plants show a significant increase in nuclear DNA strand breaks. A T-DNA insertion mutant of AtSPO11-3 has a phenotype that is almost to that of AtTOP6B and the double mutant. Thus, both genes seem to act in vivo as subunits of a functional entity. A loss of this function most likely results in a defect in DNA replication, leading directly, or via the activation of a DNA damage checkpoint, to an arrest of cell division and endoreduplication. The dependence on an archaebacterial topoisomerase VI homolog distinguishes plants from the other eukaryotic kingdoms.

The cDNA corresponding to the open reading frame T17M13.3 from Arabidopsis chromosome II was isolated and the encoded protein was characterized as a member of a subgroup of higher plant sucrose transporters. The AtSUC3 (Arabidopsis thaliana sucrose transporter 3) open reading frame encodes a protein with 594 amino acid residues, being 81 and 82 residues longer than the previously described Arabidopsis sucrose carriers AtSUC1 and AtSUC2. About 50 of these additional amino acids are part of an extended cytoplasmic loop separating the N-terminal from the C-terminal half of the protein. For functional characterization the AtSUC3 cDNA was expressed in baker's yeast. Substrate specificities, energy dependence and K(m) values of the recombinant protein were determined. Removal of the enlarged cytoplasmic loop and expression of the truncated cDNA caused no detectable change in the kinetic properties of the protein, suggesting a transport-independent function for this cytoplasmic domain. Immunolocalization with an AtSUC3-specific antiserum identified the protein in a cell layer separating the phloem from the mesophyll and in a single, subepidermal cell layer of the carpels that is important for pod dehiscence. These localizations suggest a possible role of AtSUC3 in the funnelling of sucrose from the mesophyll towards the phloem, and possibly in pod shatter.