Laura Zsigmond

Arabidopsis PPR40 Connects Abiotic Stress Responses to Mitochondrial Electron Transport

Oxidative respiration produces adenosine triphosphate through the mitochondrial electron transport system controlling the energy supply of plant cells. Here we describe a mitochondrial pentatricopeptide repeat (PPR) domain protein, PPR40, which provides a signaling link between mitochondrial electron transport and regulation of stress and hormonal responses in Arabidopsis (Arabidopsis thaliana). Insertion mutations inactivating PPR40 result in semidwarf growth habit and enhanced sensitivity to salt, abscisic acid, and oxidative stress. Genetic complementation by overexpression of PPR40 complementary DNA restores the ppr40 mutant phenotype to wild type. The PPR40 protein is localized in the mitochondria and found in association with Complex III of the electron transport system. In the ppr40-1 mutant the electron transport through Complex III is strongly reduced, whereas Complex IV is functional, indicating that PPR40 is important for the ubiqinol-cytochrome c oxidoreductase activity of Complex III. Enhanced stress sensitivity of the ppr40-1 mutant is accompanied by accumulation of reactive oxygen species, enhanced lipid peroxidation, higher superoxide dismutase activity, and altered activation of several stress-responsive genes including the alternative oxidase AOX1d. These results suggest a close link between regulation of oxidative respiration and environmental adaptation in Arabidopsis.

The Heat Shock Factor A4A Confers Salt Tolerance and Is Regulated by Oxidative Stress and the Mitogen-Activated Protein Kinases MPK3 and MPK6

An Arabidopsis Heat Shock Factor affects tolerance to salt as well as other abiotic stresses, forms homodimers dependent on the redox regulation, interacts with MAP kinases, and alters the expression of a large set of stress-induced genes. Heat shock factors (HSFs) are principal regulators of plant responses to several abiotic stresses. Here, we show that estradiol-dependent induction of HSFA4A confers enhanced tolerance to salt and oxidative agents, whereas inactivation of HSFA4A results in hypersensitivity to salt stress in Arabidopsis (Arabidopsis thaliana). Estradiol induction of HSFA4A in transgenic plants decreases, while the knockout hsfa4a mutation elevates hydrogen peroxide accumulation and lipid peroxidation. Overexpression of HSFA4A alters the transcription of a large set of genes regulated by oxidative stress. In yeast (Saccharomyces cerevisiae) two-hybrid and bimolecular fluorescence complementation assays, HSFA4A shows homomeric interaction, which is reduced by alanine replacement of three conserved cysteine residues. HSFA4A interacts with mitogen-activated protein kinases MPK3 and MPK6 in yeast and plant cells. MPK3 and MPK6 phosphorylate HSFA4A in vitro on three distinct sites, serine-309 being the major phosphorylation site. Activation of the MPK3 and MPK6 mitogen-activated protein kinase pathway led to the transcriptional activation of the HEAT SHOCK PROTEIN17.6A gene. In agreement that mutation of serine-309 to alanine strongly diminished phosphorylation of HSFA4A, it also strongly reduced the transcriptional activation of HEAT SHOCK PROTEIN17.6A. These data suggest that HSFA4A is a substrate of the MPK3/MPK6 signaling and that it regulates stress responses in Arabidopsis.

Gene Trapping with Firefly Luciferase in Arabidopsis. Tagging of Stress-Responsive Genes

To monitor the expression of T-DNA-tagged plant genes in vivo, a collection of 20,261 transgenic lines of Arabidopsis (Columbia-0) were generated with the promoter trap vector pTluc, which carries a promoterless firefly luc (luciferase) reporter gene linked to the right T-DNA border. By detection of bioluminescence in 3-week-old seedlings, 753 lines were identified showing constitutive, organ-specific, and stress-responsive luciferase expression patterns. To facilitate the identification of well-defined luciferase expression patterns, a pooled seed stock was established. Several lines showed sugar, salt, and abscisic acid (ABA)-inducible luciferase activity. Segregation analysis of 215 promoter trap lines indicated that about 50% of plants contained single insertions, whereas 40% carried two and 10% carried three or more T-DNA tags. Sequencing the T-DNA insert junctions isolated from 17 luciferase-expressing lines identified T-DNA tags in 5′- and 3′-transcribed domains and translational gene fusions generated by T-DNA insertions in exons and introns of Arabidopsis genes. Tissue specific expression of eight wild-type Arabidopsis genes was confirmed to be similar to the luminescence patterns observed in the corresponding luciferase-tagged lines. Here, we describe the characterization of a transcriptional luc reporter gene fusion with the WBC-type ABC transporter gene At1g17840. Expression of wild-type and luciferase-tagged At1g17840 alleles revealed similar induction by salt, glucose, and ABA treatments and gibberellin-mediated down-regulation of ABA-induced expression. These results illustrate that luciferase gene traps are well suited for monitoring the expression of stress-responsive Arabidopsis genes in vivo.

Overexpression of the mitochondrial PPR40 gene improves salt tolerance in Arabidopsis.

Mitochondrial respiration is sensitive to environmental conditions and can be influenced by abiotic stress. Previously we described the Arabidopsis mitochondrial pentatricopeptide repeat domain protein PPR40, and showed that the stress hypersensitive ppr40-1 mutant is compromised in mitochondrial electron transport (Zsigmond et al., 2008) [20]. Overexpression of the PPR40 gene in Arabidopsis resulted in enhanced germination and superior plant growth in saline conditions. Respiration increased in PPR40 overexpressing plants during salt stress. Reduced amount of hydrogen peroxide, diminished lipid peroxidation, lower ascorbate peroxidase and superoxide dismutase activity accompanied salt tolerance. Proline accumulation was enhanced in the ppr40-1 mutant, but unaltered in the PPR40 overexpressing plants. Our data suggest that PPR40 can diminish the generation of reactive oxygen species by stabilizing the mitochondrial electron transport and protecting plants via reducing oxidative damage during stress.

Inactivation of Plasma Membrane–Localized CDPK-RELATED KINASE5 Decelerates PIN2 Exocytosis and Root Gravitropic Response in Arabidopsis

This work shows that CRK5, a plasma membrane–associated member of the Arabidopsis Ca2+/calmodulin-dependent kinase-related protein family, phosphorylates the hydrophilic loop of PIN2 and is required for proper polar localization of PIN2 in the transition zones of roots. Inactivation of CRK5 inhibits primary root elongation and delays gravitropic bending of roots and shoots. CRK5 is a member of the Arabidopsis thaliana Ca2+/calmodulin-dependent kinase-related kinase family. Here, we show that inactivation of CRK5 inhibits primary root elongation and delays gravitropic bending of shoots and roots. Reduced activity of the auxin-induced DR5–green fluorescent protein reporter suggests that auxin is depleted from crk5 root tips. However, no tip collapse is observed and the transcription of genes for auxin biosynthesis, AUXIN TRANSPORTER/AUXIN TRANSPORTER-LIKE PROTEIN (AUX/LAX) auxin influx, and PIN-FORMED (PIN) efflux carriers is unaffected by the crk5 mutation. Whereas AUX1, PIN1, PIN3, PIN4, and PIN7 display normal localization, PIN2 is depleted from apical membranes of epidermal cells and shows basal to apical relocalization in the cortex of the crk5 root transition zone. This, together with an increase in the number of crk5 lateral root primordia, suggests facilitated auxin efflux through the cortex toward the elongation zone. CRK5 is a plasma membrane–associated kinase that forms U-shaped patterns facing outer lateral walls of epidermis and cortex cells. Brefeldin inhibition of exocytosis stimulates CRK5 internalization into brefeldin bodies. CRK5 phosphorylates the hydrophilic loop of PIN2 in vitro, and PIN2 shows accelerated accumulation in brefeldin bodies in the crk5 mutant. Delayed gravitropic response of the crk5 mutant thus likely reflects defective phosphorylation of PIN2 and deceleration of its brefeldin-sensitive membrane recycling.

Full-length isoform sequencing reveals novel transcripts and substantial transcriptional overlaps in a herpesvirus

Whole transcriptome studies have become essential for understanding the complexity of genetic regulation. However, the conventionally applied short-read sequencing platforms cannot be used to reliably distinguish between many transcript isoforms. The Pacific Biosciences (PacBio) RS II platform is capable of reading long nucleic acid stretches in a single sequencing run. The pseudorabies virus (PRV) is an excellent system to study herpesvirus gene expression and potential interactions between the transcriptional units. In this work, non-amplified and amplified isoform sequencing protocols were used to characterize the poly(A+) fraction of the lytic transcriptome of PRV, with the aim of a complete transcriptional annotation of the viral genes. The analyses revealed a previously unrecognized complexity of the PRV transcriptome including the discovery of novel protein-coding and non-coding genes, novel mono- and polycistronic transcription units, as well as extensive transcriptional overlaps between neighboring and distal genes. This study identified non-coding transcripts overlapping all three replication origins of the PRV, which might play a role in the control of DNA synthesis. We additionally established the relative expression levels of gene products. Our investigations revealed that the whole PRV genome is utilized for transcription, including both DNA strands in all coding and intergenic regions. The genome-wide occurrence of transcript overlaps suggests a crosstalk between genes through a network formed by interacting transcriptional machineries with a potential function in the control of gene expression.

Enhanced activity of galactono-1,4-lactone dehydrogenase and ascorbate-glutathione cycle in mitochondria from complex III deficient Arabidopsis

The mitochondrial antioxidant homeostasis was investigated in Arabidopsis ppr40-1 mutant, which presents a block of electron flow at complex III. The activity of the ascorbate biosynthetic enzyme, L-galactono-1,4-lactone dehydrogenase (EC 1.3.2.3) (GLDH) was elevated in mitochondria isolated from mutant plants. In addition increased activities of the enzymes of Foyer-Halliwell-Asada cycle and elevated glutathione (GSH) level were observed in the mutant mitochondria. Lower ascorbate and ascorbate plus dehydroascorbate contents were detected at both cellular and mitochondrial level. Moreover, the more oxidized mitochondrial redox status of ascorbate in the ppr40-1 mutant indicated that neither the enhanced activity of GLDH nor Foyer-Halliwell-Asada cycle could compensate for the enhanced ascorbate consumption in the absence of a functional respiratory chain.

Regulation of ascorbate biosynthesis in green algae has evolved to enable rapid stress-induced response via the VTC2 gene encoding GDP-l-galactose phosphorylase

Ascorbate (vitamin C) plays essential roles in stress resistance, development, signaling, hormone biosynthesis and regulation of gene expression; however, little is known about its biosynthesis in algae. In order to provide experimental proof for the operation of the Smirnoff-Wheeler pathway described for higher plants and to gain more information on the regulation of ascorbate biosynthesis in Chlamydomonas reinhardtii, we targeted the VTC2 gene encoding GDP-l-galactose phosphorylase using artificial microRNAs. Ascorbate concentrations in VTC2 amiRNA lines were reduced to 10% showing that GDP-l-galactose phosphorylase plays a pivotal role in ascorbate biosynthesis. The VTC2 amiRNA lines also grow more slowly, have lower chlorophyll content, and are more susceptible to stress than the control strains. We also demonstrate that: expression of the VTC2 gene is rapidly induced by H2 O2 and 1 O2 resulting in a manifold increase in ascorbate content; in contrast to plants, there is no circadian regulation of ascorbate biosynthesis; photosynthesis is not required per se for ascorbate biosynthesis; and Chlamydomonas VTC2 lacks negative feedback regulation by ascorbate in the physiological concentration range. Our work demonstrates that ascorbate biosynthesis is also highly regulated in Chlamydomonas albeit via mechanisms distinct from those previously described in land plants.

Functional analysis of the Arabidopsis thaliana CDPK-Related Kinase family: AtCRK1 regulates responses to continuous light

The Calcium-Dependent Protein Kinase (CDPK)-Related Kinase family (CRKs) consists of eight members in Arabidopsis. Recently, AtCRK5 was shown to play a direct role in the regulation of root gravitropic response involving polar auxin transport (PAT). However, limited information is available about the function of the other AtCRK genes. Here, we report a comparative analysis of the Arabidopsis CRK genes, including transcription regulation, intracellular localization, and biological function. AtCRK transcripts were detectable in all organs tested and a considerable variation in transcript levels was detected among them. Most AtCRK proteins localized at the plasma membrane as revealed by microscopic analysis of 35S::cCRK-GFP (Green Fluorescence Protein) expressing plants or protoplasts. Interestingly, 35S::cCRK1-GFP and 35S::cCRK7-GFP had a dual localization pattern which was associated with plasma membrane and endomembrane structures, as well. Analysis of T-DNA insertion mutants revealed that AtCRK genes are important for root growth and control of gravitropic responses in roots and hypocotyls. While Atcrk mutants were indistinguishable from wild type plants in short days, Atcrk1-1 mutant had serious growth defects under continuous illumination. Semi-dwarf phenotype of Atcrk1-1 was accompanied with chlorophyll depletion, disturbed photosynthesis, accumulation of singlet oxygen, and enhanced cell death in photosynthetic tissues. AtCRK1 is therefore important to maintain cellular homeostasis during continuous illumination.