Neutral invertase,hexokinase and mitochondrial ROS homeostasis: Emerging links between sugar metabolism,sugar signaling and ascorbate synthesis

Alkaline/neutral invertases (A/N-Invs) are unique to plants and photosynthetic bacteria. Although considerable advances have been made in our understanding of sucrose metabolic enzymes in plants, the function of A/N-Invs remained puzzling. In a recent study, we have analyzed the subcellullar localization of a cytosolic (At-A/N-InvG, At1g35580) and a mitochondrial (At-A/N-InvA, At1g56560) Arabidopsis A/N-Inv. Unexpectedly, At-A/N-InvA knockout plants showed a more severe growth defect than At-A/N-InvG knockout plants and a link between the two A/N-Invs and oxidative stress defense was found. Overexpression of At-A/N-InvA and At-A/N-InvG in leaf mesophyll protoplasts reduced the activity of the ascorbate peroxidase 2 (APX2) promoter, that was stimulated by hydrogen peroxide and abscisic acid. It is discussed here how sugars and ascorbate might contribute to mitochondrial reactive oxygen species homeostasis. We hypothesize that both mitochondrial and cytosolic A/N-Invs and mitochondria-associated hexokinases are key mediators, integrating metabolic and sugar signaling processes.Key words: ascorbate peroxidase, glucose, hexokinase, mitochondria, neutral invertase, oxidative stress, sucrose     

Control of plant growth and water loss by a lack of light-harvesting complexes in photosystem II in Arabidopsis thaliana ch1-1 mutant

The chlorinal-1 (ch1-1) mutant of Arabidopsis thaliana lacks the light-harvesting complexes in photosystem II (LHCII) due to deficiency of ability to synthesize chlorophyll (Chl) b. To investigate if a lack of LHCII affects plant growth and water loss, the Chl content, Chl fluorescence, glutathione (GSH) content, plant growth, water loss and stomatal aperture were measured using wild-type (WT) and ch1-1 mutant plants. The leaves of ch1-1 mutants accumulated significantly lower Chl content, Chl fluorescence and GSH content than WT plants. Plant growth and the leaf area of ch1-1 plants were also lower when compared to WT plants. The ch1-1 plant showed delayed flowering and higher a number of rosette leaves compared to the WT plants. The treatment of N-acetyl-cysteine increased Chl content and Chl fluorescence in leaves of both plants. Stomatal aperture was significantly lower in guard cells of the ch1-1 mutant than that of WT plants. Dark treatment increased stomatal closure which was corrected followed by the light treatment. Abscisic acid (ABA)-induced stomatal aperture was significantly lower in ch1-1 mutant than WT plants. Water loss through stomatal opening in ch1-1 plants was significantly lower than WT plants regardless of ABA treatment. This study suggests that a lack of LHCII might control plant growth and water loss in ch1-1 mutant of Arabidopsis thaliana.     

New insights on sucrose metabolism: evidence for an active A/N-Inv in chloroplasts uncovers a novel component of the intracellular carbon trafficking

The presence of sucrose (Suc) in plastids was questioned for several decades. Although it was reported some decades ago, neither Suc transporters nor Suc metabolizing enzymes were demonstrated to be active in those organelles. By biochemical, immunological, molecular and genetic approaches we show that alkaline/neutral invertases (A/N-Invs) are also localized in chloroplasts of spinach and Arabidopsis. A/N-Inv activity and polypeptide content were shown in protein extracts from intact chloroplasts. Moreover, we functionally characterized the Arabidopsis At-A/N-InvE gene coding for a chloroplast-targeted A/N-Inv. The At-A/N-InvE knockout plants displayed a lower total A/N-Inv activity in comparison with wild-type plants. Furthermore, neither A/N-Inv activity nor A/N-Inv polypeptides were detected in protein extracts prepared from chloroplasts of mutant plants. Also, the measurement of carbohydrate content, in leaves harvested either at the end of the day or at the end of the night period, revealed that the knockout plants showed a decrease in starch accumulation but no alteration in Suc levels. These are the first results demonstrating the presence of a functional A/N-Inv inside chloroplasts and its relation with carbon storage in Arabidopsis leaves. Taken together our data and recent reports, we conclude that the participation of A/N-Invs in the carbon flux between the cytosol and the plastids may be a general phenomenon in plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Two Alternatively Spliced Isoforms of the Arabidopsis SR45 Protein Have Distinct Roles during Normal Plant Development

The serine-arginine-rich (SR) proteins constitute a conserved family of pre-mRNA splicing factors. In Arabidopsis (Arabidopsis thaliana), they are encoded by 19 genes, most of which are themselves alternatively spliced. In the case of SR45, the use of alternative 3′ splice sites 21 nucleotides apart generates two alternatively spliced isoforms. Isoform 1 (SR45.1) has an insertion relative to isoform 2 (SR45.2) that replaces a single arginine with eight amino acids (TSPQRKTG). The biological implications of SR45 alternative splicing have been unclear. A previously described loss-of-function mutant affecting both isoforms, sr45-1, shows several developmental defects, including defects in petal development and root growth. We found that the SR45 promoter is highly active in regions with actively growing and dividing cells. We also tested the ability of each SR45 isoform to complement the sr45-1 mutant by overexpression of isoform-specific green fluorescent protein (GFP) fusion proteins. As expected, transgenic plants overexpressing either isoform displayed both nuclear speckles and GFP fluorescence throughout the nucleoplasm. We found that SR45.1-GFP complements the flower petal phenotype, but not the root growth phenotype. Conversely, SR45.2-GFP complements root growth but not floral morphology. Mutation of a predicted phosphorylation site within the alternatively spliced segment, SR45.1-S219A-GFP, does not affect complementation. However, a double mutation affecting both serine-219 and the adjacent threonine-218 (SR45.1-T218A + S219A-GFP) behaves like isoform 2, complementing the root but not the floral phenotype. In conclusion, our study provides evidence that the two alternatively spliced isoforms of SR45 have distinct biological functions.     

A novel mitochondrial DnaJ/Hsp40 family protein BIL2 promotes plant growth and resistance against environmental stress in brassinosteroid signaling

Plant steroid hormones, brassinosteroids, are essential for growth, development and responses to environmental stresses in plants. Although BR signaling proteins are localized in many organelles, i.e., the plasma membrane, nuclei, endoplasmic reticulum and vacuole, the details regarding the BR signaling pathway from perception at the cellular membrane receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) to nuclear events include several steps. Brz (Brz220) is a specific inhibitor of BR biosynthesis. In this study, we used Brz-mediated chemical genetics to identify Brz-insensitive-long hypocotyls 2-1D (bil2-1D). The BIL2 gene encodes a mitochondrial-localized DnaJ/Heat shock protein 40 (DnaJ/Hsp40) family, which is involved in protein folding. BIL2-overexpression plants (BIL2-OX) showed cell elongation under Brz treatment, increasing the growth of plant inflorescence and roots, the regulation of BR-responsive gene expression and suppression against the dwarfed BRI1-deficient mutant. BIL2-OX also showed resistance against the mitochondrial ATPase inhibitor oligomycin and higher levels of exogenous ATP compared with wild-type plants. BIL2 participates in resistance against salinity stress and strong light stress. Our results indicate that BIL2 induces cell elongation during BR signaling through the promotion of ATP synthesis in mitochondria.     

Characterization of Arabidopsis thaliana mutant ror-1 (roscovitine-resistant) and its utilization in understanding of the role of cytokinin N-glucosylation pathway in plants

Cytokinin analogue roscovitine exhibits a strong inhibitory effect on cytokinin N-glucosylation, one of the most important pathways of cytokinin inactivation in plants. Roscovitine-resistant mutant. (ror-1) was isolated using T-DNA tagged lines of Arabidopsis thaliana (L.) Heynh in order to find a gene putatively involved in cytokinin N-glucosylation. The amount of cytokinin N-glucosides of trans-zeatin- and isopentenyladenine-type was elevated by 20% in ror-1 mutant compared to WT. The cytokinin oxidase/dehydrogenase activity exhibited a mild elevation in ror-1 compared to WT in basal media. Additionally, ror-1 plants showed slightly enhanced resistance to exogenously supplied aromatic cytokinins (benzyladenine). Incubation with exogenous cytokinin (5 μM BA for 24 h) resulted in significant up-regulation of ROR-1 gene expression in ror-1 mutant. In silico analysis showed that ROR-1 gene encoded for a protein consisting of GRAM (Glycosyltransferases Rab-like GTPase activators and Myotubularins) and C2 domains. Here, we report on the role of ROR-1 gene in metabolism of bioactive cytokinins in the plants.     

Role of the two PsaE isoforms on O2 reduction at photosystem I in Arabidopsis thaliana

Leaves of Arabidopsis thaliana plants grown in short days (8 h light) generate more reactive oxygen species in the light than leaves of plants grown in long days (16 h light). The importance of the two PsaE isoforms of photosystem I, PsaE1 and PsaE2, for O₂ reduction was studied in plants grown under these different growth regimes. In short day conditions a mutant affected in the amount of PsaE1 (psae1-1) reduced more efficiently O₂ than a mutant lacking PsaE2 (psae2-1) as shown by spin trapping EPR spectroscopy on leaves and by following the kinetics of P700⁺ reduction in isolated photosystem I. In short day conditions higher O₂ reduction protected photosystem II against photoinhibition in psae1-1. In contrast in long day conditions the presence of PsaE1 was clearly beneficial for photosynthetic electron transport and for the stability of the photosynthetic apparatus under photoinhibitory conditions. We conclude that the two PsaE isoforms have distinct functions and we propose that O₂ reduction at photosystem I is beneficial for the plant under certain environmental conditions.     

dhm1, an Arabidopsis mutant with increased sensitivity to alkamides shows tumorous shoot development and enhanced lateral root formation

The control of cell division by growth regulators is critical to proper shoot and root development. Alkamides belong to a class of small lipid amides involved in plant morphogenetic processes, from which N-isobutyl decanamide is one of the most active compounds identified. This work describes the isolation and characterization of an N-isobutyl decanamide-hypersensitive (dhm1) mutant of Arabidopsis (Arabidopsis thaliana). dhm1 seedlings grown in vitro develop disorganized tumorous tissue in petioles, leaves and stems. N-isobutyl decanamide treatment exacerbates the dhm1 phenotype resulting in widespread production of callus-like structures in the mutant. Together with these morphological alterations in shoot, dhm1 seedlings sustained increased lateral root formation and greater sensitivity to alkamides in the inhibition of primary root growth. The mutants also show reduced etiolation when grown in darkness. When grown in soil, adult dhm1 plants were characterized by reduced plant size, and decreased fertility. Genetic analysis indicated that the mutant phenotype segregates as a single recessive Mendelian trait. Developmental alterations in dhm1 were related to an enhanced expression of the cell division marker CycB1-uidA both in the shoot and root system, which correlated with altered expression of auxin and cytokinin responsive gene markers. Pharmacological inhibition of auxin transport decreased LR formation in WT and dhm1 seedlings in a similar manner, indicating that auxin transport is involved in the dhm1 root phenotype. These data show an important role of alkamide signaling in cell proliferation and plant architecture remodeling likely acting through the DHM1 protein.     

Deletion of an organellar peptidasome PreP affects early development in Arabidopsis thaliana

A novel peptidasome PreP is responsible for degradation of targeting peptides and other unstructured peptides in mitochondria and chloroplasts. Arabidopsis thaliana contains two PreP isoforms, AtPreP1, and AtPreP2. Here we have characterized single and double prep knockout mutants. Immunoblot analysis of atprep1 and atprep2 mutants showed that both isoforms are expressed in all tissues with the highest expression in flowers and siliques; additionally, AtPreP1 accumulated to a much higher level in comparison to AtPreP2. The atprep2 mutant behaved like wild type, whereas deletion of AtPreP1 resulted in slightly pale-green seedlings. Analysis of the atprep1 atprep2 double mutant revealed a chlorotic phenotype in true leaves with diminished chlorophyll a and b content, but unchanged Chl a/b ratio indicating a proportional decrease of both PSI and PSII complexes. Mitochondrial respiratory rates (state 3) were lower and the mitochondria were partially uncoupled. EM pictures on cross sections of the first true leaves showed aberrant chloroplasts, including less grana stacking and less starch granules. Mitochondria with extremely variable size could also be observed. At later developmental stages the plants appeared almost normal. However, all through the development there was a statistically significant decrease of ~40% in the accumulated biomass in the double mutant plants in comparison to wild type. In mitochondria, deletion of AtPreP was not compensated by activation of any peptidolytic activity, whereas chloroplast membranes contained a minor peptidolytic activity not related to AtPreP. In summary, the AtPreP peptidasome is required for efficient plant growth and organelle function particularly during early development.     

Poleta, Polzeta and Rev1 together are required for G to T transversion mutations induced by the (+)- and (-)-trans-anti-BPDE-N2-dG DNA adducts in yeast cells

Benzo[a]pyrene is an important environmental mutagen and carcinogen. Its metabolism in cells yields the mutagenic, key ultimate carcinogen 7R,8S,9S,10R-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, (+)-anti-BPDE, which reacts via its 10-position with N²-dG in DNA to form the adduct (+)-trans-anti-BPDE-N²-dG. To gain molecular insights into BPDE-induced mutagenesis, we examined in vivo translesion synthesis and mutagenesis in yeast cells of a site-specific 10S (+)-trans-anti-BPDE-N²-dG adduct and the stereoisomeric 10R (−)-trans-anti-BPDE-N²-dG adduct. In wild-type cells, bypass products consisted of 76% C, 14% A and 7% G insertions opposite (+)-trans-anti-BPDE-N²-dG; and 89% C, 4% A and 4% G insertions opposite (−)-trans-anti-BPDE-N²-dG. Translesion synthesis was reduced by ~26–37% in rad30 mutant cells lacking Polη, but more deficient in rev1 and almost totally deficient in rev3 (lacking Polζ) mutants. C insertion opposite the lesion was reduced by ~24–33% in rad30 mutant cells, further reduced in rev1 mutant, and mostly disappeared in the rev3 mutant strain. The insertion of A was largely abolished in cells lacking either Polη, Polζ or Rev1. The insertion of G was not detected in either rev1 or rev3 mutant cells. The rad30 rev3 double mutant exhibited a similar phenotype as the single rev3 mutant with respect to translesion synthesis and mutagenesis. These results show that while the Polζ pathway is generally required for translesion synthesis and mutagenesis of the (+)- and (−)-trans-anti-BPDE-N²-dG DNA adducts, Polη, Polζ and Rev1 together are required for G→T transversion mutations, a major type of mutagenesis induced by these lesions. Based on biochemical and genetic results, we present mechanistic models of translesion synthesis of these two DNA adducts, involving both the one-polymerase one-step and two-polymerase two-step models.     

Effect of Cu content on the activity of Cu/ZnSOD1 in the Arabidopsis SUMO E3 ligase siz1 mutant

In a previous study, we found copper (Cu) accumulated to a higher level in the aerial parts of soil-grown plants of the SUMO E3 ligase siz1 mutant than in those of the wild-type. Here, we found that all superoxide dismutase (SOD) isoforms, such as FeSOD, MnSOD and different types of Cu/ZnSOD, were more active in the siz1 mutant than in the wild type under normal growth conditions. We further examined the expression and enzymatic activity of Cu/ZnSOD1 (CSD1) in shoots of the siz1 mutant under excess Cu. Shoot CSD1 protein level and activity were reduced in siz1 with excess Cu but induced in the wild type. SIZ1-dependent SUMOylation may be involved in maintaining CSD1 protein stability or repelling a feedback regulation under Cu stress.Key words: Cu/Zn SOD, CSD1, SUMO E3 ligase, SIZ1, Cu stress     

SOS2 promotes salt tolerance in part by interacting with the vacuolar H+-ATPase and upregulating its transport activity

The salt overly sensitive (SOS) pathway is critical for plant salt stress tolerance and has a key role in regulating ion transport under salt stress. To further investigate salt tolerance factors regulated by the SOS pathway, we expressed an N-terminal fusion of the improved tandem affinity purification tag to SOS2 (NTAP-SOS2) in sos2-2 mutant plants. Expression of NTAP-SOS2 rescued the salt tolerance defect of sos2-2 plants, indicating that the fusion protein was functional in vivo. Tandem affinity purification of NTAP-SOS2-containing protein complexes and subsequent liquid chromatography-tandem mass spectrometry analysis indicated that subunits A, B, C, E, and G of the peripheral cytoplasmic domain of the vacuolar H⁺-ATPase (V-ATPase) were present in a SOS2-containing protein complex. Parallel purification of samples from control and salt-stressed NTAP-SOS2/sos2-2 plants demonstrated that each of these V-ATPase subunits was more abundant in NTAP-SOS2 complexes isolated from salt-stressed plants, suggesting that the interaction may be enhanced by salt stress. Yeast two-hybrid analysis showed that SOS2 interacted directly with V-ATPase regulatory subunits B1 and B2. The importance of the SOS2 interaction with the V-ATPase was shown at the cellular level by reduced H⁺ transport activity of tonoplast vesicles isolated from sos2-2 cells relative to vesicles from wild-type cells. In addition, seedlings of the det3 mutant, which has reduced V-ATPase activity, were found to be severely salt sensitive. Our results suggest that regulation of V-ATPase activity is an additional key function of SOS2 in coordinating changes in ion transport during salt stress and in promoting salt tolerance.     

Loss of all three calreticulins, CRT1, CRT2 and CRT3, causes enhanced sensitivity to water stress in Arabidopsis

Key message

The calreticulin triple knockout mutant shows growth defects in response to abiotic stress.

Abstract

The endoplasmic reticulum (ER) is an essential organelle that is responsible for the folding and maturation of proteins. During ER stress, unfolded protein aggregates accumulate in the cell, leading to the unfolded protein response (UPR). The UPR up-regulates the expression of ER-stress-responsive genes encoding calreticulin (CRT), an ER-localized Ca²⁺-binding protein. To understand the function of plant CRTs, we generated a triple knockout mutant, t123, which lacks CRT1, CRT2 and CRT3 and examined the roles of calreticulins in abiotic stress tolerance. A triple knockout mutant increased sensitivity to water stress which implies that calreticulins are involved in the Arabidopsis response to water stress. We identified that the cyclophilin AtCYP21-2, which is located in the ER, was specifically enhanced in the t123 mutants. Seed germination of the atcyp21-1 mutant was retarded by water stress. Taken together, these results suggest that regulatory proteins that serve to protect plants from water stress are folded properly in part with the help of calreticulins. The AtCYP21-2 may also participate in this protein-folding process in association with calreticulins.     

A mutation in a coproporphyrinogen III oxidase gene confers growth inhibition, enhanced powdery mildew resistance and powdery mildew-induced cell death in Arabidopsis

Key message

A gene encoding a coproporphyrinogen III oxidase mediates disease resistance in plants by the salicylic acid pathway.

Abstract

A number of genes that regulate powdery mildew resistance have been identified in Arabidopsis, such as ENHANCED DISEASE RESISTANCE 1 to 3 (EDR1 to 3). To further study the molecular interactions between the powdery mildew pathogen and Arabidopsis, we isolated and characterized a mutant that exhibited enhanced resistance to powdery mildew. The mutant also showed dramatic powdery mildew-induced cell death as well as growth defects and early senescence in the absence of pathogens. We identified the affected gene by map-based cloning and found that the gene encodes a coproporphyrinogen III oxidase, a key enzyme in the tetrapyrrole biosynthesis pathway, previously known as LESION INITIATION 2 (LIN2). Therefore, we designated the mutant lin2-2. Further studies revealed that the lin2-2 mutant also displayed enhanced resistance to Hyaloperonospora arabidopsidis (H.a.) Noco2. Genetic analysis showed that the lin2-2-mediated disease resistance and spontaneous cell death were dependent on PHYTOALEXIN DEFICIENT 4 (PAD4), SALICYLIC ACID INDUCTION-DEFICIENT 2 (SID2), and NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1), which are all involved in salicylic acid signaling. Furthermore, the relative expression levels of defense-related genes were induced after powdery mildew infection in the lin2-2 mutant. These data indicated that LIN2 plays an important role in cell death control and defense responses in plants.     

Altered ARA2 (RABA1a) expression in Arabidopsis reveals the involvement of a Rab/YPT family member in auxin-mediated responses

Ras super family proteins serve as molecular switches regulating many different cellular processes. However, given the large number of family members, sequence information has provided little insight into the function of individual proteins. This study examined phenotypic alterations in an Arabidopsis ara2 mutant, in which a Ras super family member-encoding gene is disrupted by a T-DNA insertion. Although one mutant line (Salk_013811) was hypersensitive to auxin, its T-DNA insertion was in the 5′-UTR of ARA2. Thus, we examined a true ARA2 knock-out mutant (Salk_077747) which contains an insertion in the first exon of ARA2. We found that ARA2 expression is responsive to auxin and at low concentrations, ara2 mutant plants exhibit increased numbers of lateral roots. ARA2 overexpression causes plants to exhibit hypersensitivity to auxin, due to altered expression of auxin-responsive genes, and these plants exhibited reduced numbers of lateral roots. A GFP-ARA2 fusion protein localized to the endosomes, suggesting that ARA2 may play a role in vesicle trafficking of components involved in polar auxin transport. Taken together, these results show that ARA2 is an essential component of a pathway that couples auxin signaling to plant growth and development.     

Lack of digalactosyldiacylglycerol increases the sensitivity of Synechocystis sp. PCC 6803 to high light stress

The physiological role of digalactosyldiacylglycerol (DGDG) in photosynthesis was examined using a dgdA mutant of Synechocystis sp. PCC 6803 that is defective in the biosynthesis of DGDG. The dgdA mutant cells showed normal growth under low light (LL) conditions. However, their growth was retarded under high light (HL) conditions and under Ca²⁺- and/or Cl⁻-limited conditions compared to wild-type cells. The retardation in growth of the mutant cells was recovered by exogenous supply of DGDG in the growth medium. The dgdA mutant showed increased sensitivity to photoinhibition. Although both photodamage and repair processes of photosynthesis were affected, the repair process was more severely affected than the photodamage process, suggesting that DGDG plays an important role in the photosynthetic repair cycle.