Gábor Rigó

Light-dependent induction of proline biosynthesis by abscisic acid and salt stress is inhibited by brassinosteroid in Arabidopsis.

Osmotic stress-induced accumulation of proline, an important protective osmolyte in higher plants, is dependent on the expression of Δ1-pyrroline-5-carboxylate synthase (P5CS) and proline dehydrogenase (PDH) enzymes that catalyze the rate-limiting steps of proline biosynthesis and degradation, respectively. Proline metabolism is modulated by differential regulation of organ specific expression of PDH and duplicated P5CS genes in Arabidopsis. Stimulation of proline synthesis by abscisic acid (ABA) and salt stress correlates with a striking activation of P5CS1 expression. By contrast, P5CS2 is only weakly induced, whereas PDH is inhibited to different extent by ABA and salt stress in shoots and roots of light-grown plants. Proline accumulation and light-dependent induction of P5CS1 by ABA and salt stress is inhibited in dark-adapted plants. During dark adaptation P5CS2 is also down-regulated, whereas PDH expression is significantly enhanced in shoots. The inhibitory effect of dark adaptation on P5CS1 is mimicked by the steroid hormone brassinolide. However, brassinolide fails to stimulate PDH, and inhibits P5CS2 only in shoots. Proline accumulation and induction of P5CS1 transcription are simultaneously enhanced in the ABA-hypersensitive prl1 and brassinosteroid-deficient det2 mutants, whereas P5CS2 shows enhanced induction by ABA and salt only in the det2 mutant. In comparison, the prl1 mutation reduces the basal level of PDH expression, whereas the det2 mutation enhances the inhibition of PDH by ABA. Regulation of P5CS1 expression thus appears to play a principal role in controlling proline accumulation stimulated by ABA and salt stress in Arabidopsis.

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.

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.

The arabidopsis ZINC FINGER PROTEIN3 interferes with abscisic acid and light signaling in seed germination and plant development

An Arabidopsis zinc finger protein acts as a negative regulator of ABA-suppressed seed germination and modulates plant development, fertility, and hypocotyl elongation under red light. Seed germination is controlled by environmental signals, including light and endogenous phytohormones. Abscisic acid (ABA) inhibits, whereas gibberellin promotes, germination and early seedling development, respectively. Here, we report that ZFP3, a nuclear C2H2 zinc finger protein, acts as a negative regulator of ABA suppression of seed germination in Arabidopsis (Arabidopsis thaliana). Accordingly, regulated overexpression of ZFP3 and the closely related ZFP1, ZFP4, ZFP6, and ZFP7 zinc finger factors confers ABA insensitivity to seed germination, while the zfp3 zfp4 double mutant displays enhanced ABA susceptibility. Reduced expression of several ABA-induced genes, such as RESPONSIVE TO ABSCISIC ACID18 and transcription factor ABSCISIC ACID-INSENSITIVE4 (ABI4), in ZFP3 overexpression seedlings suggests that ZFP3 negatively regulates ABA signaling. Analysis of ZFP3 overexpression plants revealed multiple phenotypic alterations, such as semidwarf growth habit, defects in fertility, and enhanced sensitivity of hypocotyl elongation to red but not to far-red or blue light. Analysis of genetic interactions with phytochrome and abi mutants indicates that ZFP3 enhances red light signaling by photoreceptors other than phytochrome A and additively increases ABA insensitivity conferred by the abi2, abi4, and abi5 mutations. These data support the conclusion that ZFP3 and the related ZFP subfamily of zinc finger factors regulate light and ABA responses during germination and early seedling development.

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.

Transformation using controlled cDNA overexpression system

The controlled cDNA overexpression system (COS) was developed to identify novel regulatory genes in model plants as well as in other species that might have a particular valuable trait. The COS system (Papdi et al. Plant Physiol 147:528-542, 2008) is composed of a random cDNA library prepared in a T-DNA plant expression vector, under the control of the estradiol-inducible XVE promoter. Large-scale genetic transformation of Arabidopsis thaliana generates a transgenic plant population with randomly inserted cDNA clones. Overexpression of the inserted cDNA can create selectable phenotypes, allowing the facile identification and cloning of the responsible genes. Here we describe protocols to create and use the COS system for diverse purposes in plant biology.

Gene mining in halophytes: functional identification of stress tolerance genes in Lepidium crassifolium

Extremophile plants are valuable sources of genes conferring tolerance traits, which can be explored to improve stress tolerance of crops. Lepidium crassifolium is a halophytic relative of the model plant Arabidopsis thaliana, and displays tolerance to salt, osmotic and oxidative stresses. We have employed the modified Conditional cDNA Overexpression System to transfer a cDNA library from L. crassifolium to the glycophyte A. thaliana. By screening for salt, osmotic and oxidative stress tolerance through in vitro growth assays and non-destructive chlorophyll fluorescence imaging, 20 Arabidopsis lines were identified with superior performance under restrictive conditions. Several cDNA inserts were cloned and confirmed to be responsible for the enhanced tolerance by analysing independent transgenic lines. Examples include full-length cDNAs encoding proteins with high homologies to GDSL-lipase/esterase or acyl CoA-binding protein or proteins without known function, which could confer tolerance to one or several stress conditions. Our results confirm that random gene transfer from stress tolerant to sensitive plant species is a valuable tool to discover novel genes with potential for biotechnological applications.

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.