Adenylates regulate Arabidopsis plastidial thioredoxin activities through the binding of a CBS domain protein.

Cystathionine-ß-synthase (CBS) domains are found in proteins of all living organisms and have been proposed to play a role as energy sensors regulating protein activities through their adenosyl ligand binding capacity. In plants, members of the CBSX protein family carry a stand-alone pair of CBS domains. In Arabidopsis (Arabidopsis thaliana), CBSX1 and CBSX2 are targeted to plastids where they have been proposed to regulate thioredoxins (TRXs). TRXs are ubiquitous cysteine thiol oxido-reductases involved in the redox-based regulation of numerous enzymatic activities as well as in the regeneration of thiol-dependent peroxidases. In Arabidopsis, 10 TRX isoforms have been identified in plastids and divided into five sub-types. Here, we show that CBSX2 specifically inhibits the activities of m-type TRXs toward two chloroplast TRX-related targets. By testing activation of NADP-malate dehydrogenase and reduction of 2-Cys peroxiredoxin, we found that TRXm1/2 inhibition by CBSX2 was alleviated in the presence of AMP or ATP. We also determined, by pull-down assays, a direct interaction of CBSX2 with reduced TRXm1 and m2 that was abolished in the presence of adenosyl ligands. In addition, we report that, compared with wild-type plants, the Arabidopsis T-DNA double mutant cbsx1 cbsx2 exhibits growth and chlorophyll accumulation defects in cold conditions, suggesting a function of plastidial CBSX proteins in plant stress adaptation. Together, our results show an energy-sensing regulation of plastid TRX m activities by CBSX, possibly allowing a feedback regulation of ATP homeostasis via activation of cyclic electron flow in the chloroplast, to maintain a high energy level for optimal growth.

Two interacting PPR proteins are major Arabidopsis editing factors in plastid and mitochondria.

RNA editing is converting hundreds of cytosines into uridines during organelle gene expression of land plants. The pentatricopeptide repeat (PPR) proteins are at the core of this posttranscriptional RNA modification. Even if a PPR protein defines the editing site, a DYW domain of the same or another PPR protein is believed to catalyze the deamination. To give insight into the organelle RNA editosome, we performed tandem affinity purification of the plastidial CHLOROPLAST BIOGENESIS 19 (CLB19) PPR editing factor. Two PPR proteins, dually targeted to mitochondria and chloroplasts, were identified as potential partners of CLB19. These two proteins, a P-type PPR and a member of a small PPR-DYW subfamily, were shown to interact in yeast. Insertional mutations resulted in embryo lethality that could be rescued by embryo-specific complementation. A transcriptome analysis of these complemented plants showed major editing defects in both organelles with a very high PPR type specificity, indicating that the two proteins are core members of E+-type PPR editosomes.

Responses of an adventitious fast-growing plant to photodynamic stress: comparative study of anionic and cationic porphyrin effect on Arabidopsis thaliana.

Antimicrobial photodynamic treatment (APDT) based on the use of a photosensitizer to produce reactive oxygen species (ROS) that induce cell death could be envisaged to fight against plant pathogens. For setting this strategy, we want to study how plants themselves respond to photodynamic treatment. In previous work we showed that tomato plantlets were able to resist photoactivated tetra (N-methylpyridyl) porphyrin (CP) or the zinc metalated form (CP-Zn). To enlarge our plant expertise related to exogenous porphyrins treatment and to further defend this approach, we studied how a weed like Arabidopsis thaliana responded to exogenous supply of anionic and cationic porphyrins. Both types of photosensitizers had no negative effect on seed germination and did not hamper the development etiolated Arabidopsis plantlet under dark conditions. Thus, post-emergence effects of porphyrin photoactivation on the development of 14 day-old in vitro Arabidopsis plantlet under light were observed. CP-Zn was the most efficient photosensitizer to kill Arabidopsis plantlets while anionic tetra (4-sulfonatophenyl) porphyrin only delayed their growth and development. Indeed only 7% of plantlets could be rescued after CP-Zn treatment. Furthermore, non-enzymatic and enzymatic defense components involved in detoxification of ROS generated by CP-Zn under illumination were downregulated or stable with the exception of sevenfold increase in proline content. As previously demonstrated in the literature for microbial agents and in the present work for Arabidopsis, CP-Zn was efficient enough to eradicate unwanted vegetation and plant pathogens without at the same time killing plants of agronomic interest such as tomato plantlets.

The Arabidopsis abiotic stress-induced TSPO-related protein reduces cell-surface expression of the aquaporin PIP2;7 through protein-protein interactions and autophagic degradation.

The Arabidopsis thaliana multi-stress regulator TSPO is transiently induced by abiotic stresses. The final destination of this polytopic membrane protein is the Golgi apparatus, where its accumulation is strictly regulated, and TSPO is downregulated through a selective autophagic pathway. TSPO-related proteins regulate the physiology of the cell by generating functional protein complexes. A split-ubiquitin screen for potential TSPO interacting partners uncovered a plasma membrane aquaporin, PIP2;7. Pull-down assays and fluorescence imaging approaches revealed that TSPO physically interacts with PIP2;7 at the endoplasmic reticulum and Golgi membranes in planta. Intriguingly, constitutive expression of fluorescently tagged PIP2;7 in TSPO-overexpressing transgenic lines resulted in patchy distribution of the fluorescence, reminiscent of the pattern of constitutively expressed yellow fluorescent protein-TSPO in Arabidopsis. Mutational stabilization of TSPO or pharmacological inhibition of the autophagic pathway affected concomitantly the detected levels of PIP2;7, suggesting that the complex containing both proteins is degraded through the autophagic pathway. Coexpression of TSPO and PIP2;7 resulted in decreased levels of PIP2;7 in the plasma membrane and abolished the membrane water permeability mediated by transgenic PIP2;7. Taken together, these data support a physiological role for TSPO in regulating the cell-surface expression of PIP2;7 during abiotic stress conditions through protein-protein interaction and demonstrate an aquaporin regulatory mechanism involving TSPO.

The Arabidopsis TSPO-related protein is a stress and abscisic acid-regulated, endoplasmic reticulum-Golgi-localized membrane protein.

The Arabidopsis gene At2g47770 encodes a membrane-bound protein designated AtTSPO (Arabidopsis thaliana TSPO-related). AtTSPO is related to the bacterial outer membrane tryptophan-rich sensory protein (TspO) and the mammalian mitochondrial 18-kDa translocator protein (18 kDa TSPO), members of the group of TspO/MBR domain-containing membrane proteins. In this study we show that AtTSPO is mainly detected in dry seeds, but can be induced in vegetative tissues by osmotic or salt stress or abscisic acid (ABA) treatment, corroborating available transcriptome data. Using subcellular fractionation, immunocytochemistry and fluorescent protein tagging approaches we present evidence that AtTSPO is targeted to the secretory pathway in plants. Induced or constitutively expressed AtTSPO can be detected in the endoplasmic reticulum and the Golgi stacks of plant cells. AtTSPO tagged with fluorescent protein in transgenic plants (Arabidopsis and tobacco) was mainly detected in the Golgi stacks of leaf epidermal cells. Constitutive expression of AtTSPO resulted in increased sensitivity to NaCl, but not to osmotic stress, and in reduced greening of cultured Arabidopsis cells under light growing conditions. Transgenic Arabidopsis plants overexpressing AtTSPO were more sensitive to ABA-induced growth inhibition, indicating that constitutive expression of AtTSPO may enhance ABA sensitivity. AtTSPO is rapidly downregulated during seed imbibition, and the ABA-dependent induction in plant is transient. Downregulation of AtTSPO seems to be boosted by treatment with aminolevulinic acid. Taken together, these results suggest that AtTSPO is a highly regulated protein, induced by abiotic stress to modulate, at least in part, transient intracellular ABA-dependent stress perception and/or signalling.

The Arabidopsis thaliana trehalase is a plasma membrane-bound enzyme with extracellular activity.

The lack of trehalose accumulation in most plant species has been partly attributed to the presence of an active trehalase. Although trehalose synthesis enzymes are thought to be cytosolic, and previous studies have indicated that trehalase activity is extracellular, the exact location of the enzyme has not yet been established in plant cell. We present evidence that the yet uncharacterised full-length Arabidopsis trehalase is a plasma membrane-bound protein, probably anchored to the membrane through a predicted N-terminal membrane spanning domain. The full-length AtTRE1, when expressed in yeast can functionally substitute for the extracellularly active trehalase Ath1p, by sustaining the growth of an ath1 null mutant strain on trehalose and at pH 4.8. We further demonstrate that AtTRE1 expressed in yeast is plasma membrane-bound as in plant cell. In light of these findings, the regulation of plant cell endogenous trehalose by trehalase is discussed.

Synergistic enhancement of tolerance mechanisms in response to photoactivation of cationic tetra (N-methylpyridyl) porphyrins in tomato plantlets.

Antimicrobial photodynamic treatment (APDT) is largely used in medical domain and could be envisaged as a farming practice against crop pathogens such as bacteria and fungi that generate drops in agricultural yields. Thus, as a prerequisite for this potential application, we studied the effect of water-soluble anionic (TPPS and Zn-TPPS) and cationic (TMPyP and Zn-TMPyP) porphyrins tested on tomato (Solanum lycopersicum) plantlets grown in vitro under a 16 h photoperiod. First of all, under dark conditions, none of the four porphyrins inhibited germination and induced cytotoxic effects on tomato plantlets as etiolated development was not altered. The consequences of porphyrin long-term photoactivation (14 days) were thus studied on in vitro-grown tomato plantlets at phenotypic and molecular levels. Cationic porphyrins especially Zn-TMPyP were the most efficient photosensitizers and dramatically altered growth without killing plantlets. Indeed, tomato plantlets were rescued after cationic porphyrins treatment. To gain insight, the different molecular ways implied in the plantlet tolerance to photoactivated Zn-TMPyP, lipid peroxidation, antioxidative molecules (total thiols, proline, ascorbate), and ROS detoxification enzymes were evaluated. In parallel to an increase in lipid peroxidation and hydrogen peroxide production, antioxidative molecules and enzymes (guaiacol peroxidase, catalase, and superoxide dismutase) were up-regulated in root apparatus in response to photoactivated Zn-TMPyP. This study showed that tomato plantlets could overcome the pressure triggered by photoactivated cationic porphyrin by activating antioxidative molecule and enzyme arsenal and confining Zn-TMPyP into cell wall and/or apoplasm, suggesting that APDT directed against tomato pathogens could be envisaged in the future.

ABA, porphyrins and plant TSPO-related protein.

We have shown that, unexpectedly, AtTSPO (Arabidopsis thaliana TSPO-related protein) is an endoplasmic reticulum and Golgi-localized membrane protein in plant cells.(1) This localization contrasts with that of mammalian 18-kDa translocator protein (at least for the mostly studied isoform, 18-kDa TSPO), a mitochondrial outer membrane protein (reviewed in ref. 2). Whereas the potential functions of 18-kDa TSPO are well documented, involved mainly in mitochondrial physiology,(2) and its interest as drugs target is been explored,(3) the roles of TSPO-related proteins in plant growth and development are yet to be specified. AtTSPO is expressed in dry seeds and can be induced in vegetative tissues by osmotic and salt stress or abscisic acid (ABA) treatment. Moreover, it was shown that the ABA-dependent induction is transient, and that boosting tetrapyrroles biosynthesis through 5-aminolevulinic acid (ALA) feeding enhanced downregulation of AtTSPO, suggesting an inherent post-translational regulation mechanism also involving ABA and likely porphyrins. We present additional evidence that ABA can help stabilize constitutively expressed AtTSPO and that ALA feeding to knockout mutant seeds, induces substantial germination delay. Here we discuss the possible link between ABA and tetrapyrroles in AtTSPO expression and post-translational regulation.

Expression patterns of LmAP2L1 and LmAP2L2 encoding two-APETALA2 domain proteins during somatic embryogenesis and germination of hybrid larch (Larix x marschlinsii).

Two APETALA2 domain transcription factors were characterized first in angiosperms, and, recently, in several gymnosperms. These proteins are involved in several processes, from flowering to embryogenesis in Arabidopsis thaliana. We extrapolated this result to hybrid larch (Larixxmarschlinsii Coaz) resulting from a cross between European (Larix decidua) and Japanese (Larix kaempferi) larches. Somatic embryogenesis is well described and controlled for this Pinaceae. We characterized two-AP2 domain genes: LmAP2L1 and LmAP2L2. Phylogenetic analysis confirmed that LmAP2L1 and LmAP2L2 were orthologous to Norway spruce PaAP2L1 and PaAP2L2 and that L1 forms appeared to be specific to Pinaceae. RT-PCR analysis showed that larch APETALA2 was differentially expressed during late somatic embryogenesis and during the first steps of germination. Whereas LmAP2L2 was constitutively expressed during this process, LmAP2L1 expression appeared only during late somatic embryogenesis, when embryos were able to germinate. Further, LmAP2L1 appeared to be the preferentially expressed form during embryo germination. Thus, LmAP2L1 seems to be a valuable molecular marker for hybrid larch late somatic embryogenesis and could play a role during post-embryonic development.