Gerasimos Daras

Role of Lon1 protease in post-germinative growth and maintenance of mitochondrial function in Arabidopsis thaliana.

Maintenance of protein quality control and turnover is essential for cellular homeostasis. In plant organelles this biological process is predominantly performed by ATP-dependent proteases. Here, a genetic screen was performed that led to the identification of Arabidopsis thaliana Lon1 protease mutants that exhibit a post-embryonic growth retardation phenotype. Translational fusion to yellow fluorescent protein revealed AtLon1 subcellular localization in plant mitochondria, and the AtLon1 gene could complement the respiratory-deficient phenotype of the yeast PIM1 gene homolog. AtLon1 is highly expressed in rapidly growing plant organs of embryonic origin, including cotyledons and primary roots, and in inflorescences, which have increased mitochondria numbers per cell to fulfill their high energy requirements. In lon1 mutants, the expression of both mitochondrial and nuclear genes encoding respiratory proteins was normal. However, mitochondria isolated from lon1 mutants had a lower capacity for respiration of succinate and cytochrome c via complexes II and IV, respectively. Furthermore, the activity of key enzymes of the tricarboxylic acid (TCA) cycle was significantly reduced. Additionally, mitochondria in lon1 mutants had an aberrant morphology. These results shed light on the developmental mechanisms of selective proteolysis in plant mitochondria and suggest a critical role for AtLon1 protease in organelle biogenesis and seedling establishment.

Root gravitropism and root hair development constitute coupled developmental responses regulated by auxin homeostasis in the Arabidopsis root apex

Active polar transport establishes directional auxin flow and the generation of local auxin gradients implicated in plant responses and development. Auxin modulates gravitropism at the root tip and root hair morphogenesis at the differentiation zone. Genetic and biochemical analyses provide evidence for defective basipetal auxin transport in trh1 roots. The trh1, pin2, axr2 and aux1 mutants, and transgenic plants overexpressing PIN1, all showing impaired gravity response and root hair development, revealed ectopic PIN1 localization. The auxin antagonist hypaphorine blocked root hair elongation and caused moderate agravitropic root growth, also leading to PIN1 mislocalization. These results suggest that auxin imbalance leads to proximal and distal developmental defects in Arabidopsis root apex, associated with agravitropic root growth and root hair phenotype, respectively, providing evidence that these two auxin-regulated processes are coupled. Cell-specific subcellular localization of TRH1-YFP in stele and epidermis supports TRH1 engagement in auxin transport, and hence impaired function in trh1 causes dual defects of auxin imbalance. The interplay between intrinsic cues determining root epidermal cell fate through the TTG/GL2 pathway and environmental cues including abiotic stresses modulates root hair morphogenesis. As a consequence of auxin imbalance in Arabidopsis root apex, ectopic PIN1 mislocalization could be a risk aversion mechanism to trigger root developmental responses ensuring root growth plasticity.

The thanatos mutation in Arabidopsis thaliana cellulose synthase 3 (AtCesA3) has a dominant‐negative effect on cellulose synthesis and plant growth

Genetic functional analyses of mutants in plant genes encoding cellulose synthases (CesAs) have suggested that cellulose deposition requires the activity of multiple CesA proteins. Here, a genetic screen has led to the identification of thanatos (than), a semi-dominant mutant of Arabidopsis thaliana with impaired growth of seedlings. Homozygous seedlings of than germinate and grow but do not survive. In contrast to other CesA mutants, heterozygous plants are dwarfed and display a radially swollen root phenotype. Cellulose content is reduced by approximately one-fifth in heterozygous and by two-fifths in homozygous plants, showing gene-dosage dependence. Map-based cloning revealed an amino acid substitution (P578S) in the catalytic domain of the AtCesA3 gene, indicating a critical role for this residue in the structure and function of the cellulose synthase complex. Ab initio analysis of the AtCesA3 subdomain flanking the conserved proline residue predicted that the amino acid substitution to serine alters protein secondary structure in the catalytic domain. Gene dosage-dependent expression of the AtCesA3 mutant gene in wild-type A. thaliana plants resulted in a than dominant-negative phenotype. We propose that the incorporation of a mis-folded CesA3 subunit into the cellulose synthase complex may stall or prevent the formation of functional rosette complexes.

Alternative Transcription Initiation and the AUG Context Configuration Control Dual-Organellar Targeting and Functional Competence of Arabidopsis Lon1 Protease

Cellular homeostasis relies on components of protein quality control including chaperones and proteases. In bacteria and eukaryotic organelles, Lon proteases play a critical role in removing irreparably damaged proteins and thereby preventing the accumulation of deleterious degradation-resistant aggregates. Gene expression, live-cell imaging, immunobiochemical, and functional complementation approaches provide conclusive evidence for Lon1 dual-targeting to chloroplasts and mitochondria. Dual-organellar deposition of Lon1 isoforms depends on both transcriptional regulation and alternative translation initiation via leaky ribosome scanning from the first AUG sequence context that deviates extensively from the optimum Kozak consensus. Organelle-specific Lon1 targeting results in partial complementation of Arabidopsis lon1-1 mutants, whereas full complementation is solely accomplished by dual-organellar targeting. Both the optimal and non-optimal AUG sequence contexts are functional in yeast and facilitate leaky ribosome scanning complementing the pim1 phenotype when the mitochondrial presequence is used. Bioinformatic search identified a limited number of Arabidopsis genes with Lon1-type dual-targeting sequence organization. Lon4, the paralog of Lon1, has an ambiguous presequence likely evolved from the twin presequences of an ancestral Lon1-like gene, generating a single dual-targeted protein isoform. We postulate that Lon1 and its subfunctional paralog Lon4 evolved complementary subsets of transcriptional and posttranscriptional regulatory components responsive to environmental cues for dual-organellar targeting.

The multifaceted role of Lon proteolysis in seedling establishment and maintenance of plant organelle function: living from protein destruction

Intracellular selective proteolysis is an important post-translational regulatory mechanism maintaining protein quality control by removing defective, damaged or even deleterious protein aggregates. The ATP-dependent Lon protease is a key component of protein quality control that is highly conserved across the kingdoms of living organisms. Major advancements have been made in bacteria and in non-plant organisms to understand the role of Lon in protection against protein oxidation, ageing and neurodegenerative diseases. This review presents the progress currently made in plants. The Lon gene family in Arabidopsis consists of four members that produce distinct protein isoforms localized in several organelles. Lon1 and Lon4 that potentially originate from a recent gene duplication event are dual-targeted to mitochondria and chloroplasts through distinct mechanisms revealing divergent evolution. Arabidopsis mutant analysis showed that mitochondria and peroxisomes biogenesis or maintenance of function is modulated by Lon1 and Lon2, respectively. Consequently, the lack of Lon selective proteolysis leading to growth retardation and impaired seedling establishment can be attributed to defects in the oil reserve mobilization pathway. The current progress in Arabidopsis research uncovers the role of Lon in the proteome homeostasis of plant organelles and stimulates biotechnology scenarios of plant tolerance against harsh abiotic conditions because of climate instability.

Differential responsiveness of cortical microtubule orientation to suppression of cell expansion among the developmental zones of Arabidopsis thaliana root apex.

Τhe bidirectional relationship between cortical microtubule orientation and cell wall structure has been extensively studied in elongating cells. Nevertheless, the possible interplay between microtubules and cell wall elements in meristematic cells still remains elusive. Herein, the impact of cellulose synthesis inhibition and suppressed cell elongation on cortical microtubule orientation was assessed throughout the developmental zones of Arabidopsis thaliana root apex by whole-mount tubulin immunolabeling and confocal microscopy. Apart from the wild-type, thanatos and pom2-4 mutants of Cellulose SynthaseA3 and Cellulose Synthase Interacting1, respectively, were studied. Pharmacological and mechanical approaches inhibiting cell expansion were also applied. Cortical microtubules of untreated wild-type roots were predominantly transverse in the meristematic, transition and elongation root zones. Cellulose-deficient mutants, chemical inhibition of cell expansion, or growth in soil resulted in microtubule reorientation in the elongation zone, wherein cell length was significantly decreased. Combinatorial genetic and chemical suppression of cell expansion extended microtubule reorientation to the transition zone. According to the results, transverse cortical microtubule orientation is established in the meristematic root zone, persisting upon inhibition of cell expansion. Microtubule reorientation in the elongation zone could be attributed to conditional suppression of cell elongation. The differential responsiveness of microtubule orientation to genetic and environmental cues is most likely associated with distinct biophysical traits of the cells among each developmental root zone.

Mitochondria biogenesis via Lon1 selective proteolysis: Who dares to live for ever?

Quality control of proteins in eukaryotic organelles is predominantly maintained by members of the ATP-dependent proteases. Even though numerous biological analyses have shed light on the functional implications of such proteases, their involvement in developmental processes of multicellular organisms has not been determined. We recently identified two lon1 mutant alleles, both missing the carboxy terminal proteolytic domain, that show post-embryonic growth retardation resulting in delayed seedling establishment. In this addendum, we enlighten the role of Lon1 selective proteolysis in plant mitochondria biogenesis, a prerequisite for post-embryonic development and growth. In contrast to the weak lon1-2 allele, the polypeptide encoded by the strong lon1-1 allele carries the sensor- and substrate-discrimination domain allowing substrate recognition and binding. This type of molecular recognition hinders further degradation by the complementary Lon-independent proteolytic machineries resulting in an extra deleterious accumulation of protein aggregates into lon1-1 mitochondria. The most challenging and informative task will be to identify the recognition motifs on the Lon protein substrates and elucidate the molecular events that control plant mitochondrial differentiation.

Potassium transporter TRH1 subunits assemble regulating root-hair elongation autonomously from the cell fate determination pathway.

Trichoblasts of trh1 plants form root-hair initiation sites that fail to undergo tip growth resulting in a tiny root-hair phenotype. TRH1 belongs to Arabidopsis KT/KUP/HAK potassium transporter family controlling root-hair growth and gravitropism. Double mutant combinations between trh1 and root-hair mutants affecting cell fate or root-hair initiation exhibited additive phenotypes, suggesting that TRH1 acts independently and developmentally downstream of root-hair initiation. Bimolecular Fluorescence Complementation (BiFC), upon TRH1-YFP(C) and TRH1-YFP(N) co-transformation into tobacco epidermal cells, led to fluorescence emission indicative of TRH1 subunit homodimerization. Yeast two-hybrid analysis revealed two types of interactions. The hydrophilic segment between the second and the third transmembrane domain extending from residues Q105 to T141 is competent for a relatively weak interaction, whereas the region at the C-terminal beyond the last transmembrane domain, extending from amino acids R565 to A729, strongly self-interacts. These domains likely facilitate the co-assembly of TRH1 subunits forming an active K(+) transport system within cellular membrane structures. The results support the role of TRH1 acting as a convergence point between the developmental root-hair pathway and the environmental/hormonal signaling pathway to preserve auxin homeostasis ensuring plant adaptation in changing environments.

Evolution and significance of the Lon gene family in Arabidopsis organelle biogenesis and energy metabolism

Lon is the first identified ATP-dependent protease highly conserved across all kingdoms. Model plant species Arabidopsis thaliana has a small Lon gene family of four members. Although these genes share common structural features, they have distinct properties in terms of gene expression profile, subcellular targeting and substrate recognition motifs. This supports the notion that their functions under different environmental conditions are not necessarily redundant. This article intends to unravel the biological role of Lon proteases in energy metabolism and plant growth through an evolutionary perspective. Given that plants are sessile organisms exposed to diverse environmental conditions and plant organelles are semi-autonomous, it is tempting to suggest that Lon genes in Arabidopsis are paralogs. Adaptive evolution through repetitive gene duplication events of a single archaic gene led to Lon genes with complementing sets of subfunctions providing to the organism rapid adaptability for canonical development under different environmental conditions. Lon1 function is adequately characterized being involved in mitochondrial biogenesis, modulating carbon metabolism, oxidative phosphorylation and energy supply, all prerequisites for seed germination and seedling establishment. Lon is not a stand-alone proteolytic machine in plant organelles. Lon in association with other nuclear-encoded ATP-dependent proteases builds up an elegant nevertheless, tight interconnected circuit. This circuitry channels properly and accurately, proteostasis and protein quality control among the distinct subcellular compartments namely mitochondria, chloroplasts, and peroxisomes.

Oleosin di-or tri-meric fusions with GFP undergo correct targeting and provide advantages for recombinant protein production.

Plant oleosins are small proteins embedded within the phospholipid monolayer separating the triacylglycerol storage site of embryo-located oilbodies from the cytoplasm of oilseed cells. The potential of oleosins to act as carriers for recombinant proteins foreign to plant cells has been well established. Using this approach, the recombinant polypeptide is accumulated in oilbodies as a fusion with oleosin. DNA constructs having tandemly arranged oleosins followed by GFP or flanked by oleosins were used to transform Arabidopsis plants. In all cases the green fluorescence revealed that the fusion polypeptide had a native conformation and the recombinant proteins were correctly targeted to seed oilbodies. Mobilization of lipids was not retarded when using homo-dimer or -trimer oleosin fusions, since seed production, germination rates and seedling establishment were similar among all constructs, and comparable to wild-type Arabidopsis plants. Plant physiology and growth of recombinant lines were similar to wild-type plants. The construct specifying two oleosins flanking the GFP polypeptide revealed interesting properties regarding both the accumulation and the relative stability of the oilbody protein assembly. Although expression levels varied among transgenic lines, those transgenes accumulated significantly higher levels of fusion proteins as compared to previously reported values obtained by a single-oleosin configuration, reaching up to 2.3% of the total embryo proteins. These results shows that the expression cassettes comprising three oleosin molecules in frame to the GFP molecule or two oleosins flanking the GFP could be advantageous over the single-oleosin configuration for higher production and better commercialization of this plant biotechnological platform without jeopardizing plant vigour and physiology or oilbody stability.