Potassium transporter TRH1/KUP4 contributes to distinct auxin-mediated root system architecture responses.

In plants, auxin transport and development are tightly coupled, just as hormone and growth responses are intimately linked in multicellular systems. Here we provide insights into uncoupling this tight control by specifically targeting the expression of TINY ROOT HAIR 1 (TRH1), a member of plant high-affinity potassium (K+)/K+ uptake/K+ transporter (HAK/KUP/KT) transporters that facilitate K+ uptake by co-transporting protons, in Arabidopsis root cell files. Use of this system pinpointed specific root developmental responses to acropetal versus basipetal auxin transport. Loss of TRH1 function shows TRHs and defective root gravitropism, associated with auxin imbalance in the root apex. Cell file-specific expression of TRH1 in the central cylinder rescued trh1 root agravitropism, whereas positional TRH1 expression in peripheral cell layers, including epidermis and cortex, restored trh1 defects. Applying a system-level approach, the role of RAP2.11 and ROOT HAIR DEFECTIVE-LIKE 5 transcription factors (TFs) in root hair development was verified. Furthermore, ERF53 and WRKY51 TFs were overrepresented upon restoration of root gravitropism supporting involvement in gravitropic control. Auxin has a central role in shaping root system architecture by regulating multiple developmental processes. We reveal that TRH1 jointly modulates intracellular ionic gradients and cell-to-cell polar auxin transport to drive root epidermal cell differentiation and gravitropic response. Our results indicate the developmental importance of HAK/KUP/KT proton-coupled K+ transporters.

Clavibacter michiganensis Downregulates Photosynthesis and Modifies Monolignols Metabolism Revealing a Crosstalk with Tomato Immune Responses.

The gram-positive pathogenic bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial canker disease in tomato, affecting crop yield and fruit quality. To understand how tomato plants respond, the dynamic expression profile of host genes was analyzed upon Cmm infection. Symptoms of bacterial canker became evident from the third day. As the disease progressed, the bacterial population increased in planta, reaching the highest level at six days and remained constant till the twelfth day post inoculation. These two time points were selected for transcriptomics. A progressive down-regulation of key genes encoding for components of the photosynthetic apparatus was observed. Two temporally separated defense responses were observed, which were to an extent interdependent. During the primary response, genes of the phenylpropanoid pathway were diverted towards the synthesis of monolignols away from S-lignin. In dicots, lignin polymers mainly consist of G- and S-units, playing an important role in defense. The twist towards G-lignin enrichment is consistent with previous findings, highlighting a response to generate an early protective barrier and to achieve a tight interplay between lignin recomposition and the primary defense response mechanism. Upon progression of Cmm infection, the temporal deactivation of phenylpropanoids coincided with the upregulation of genes that belong in a secondary response mechanism, supporting an elegant reprogramming of the host transcriptome to establish a robust defense apparatus and suppress pathogen invasion. This high-throughput analysis reveals a dynamic reorganization of plant defense mechanisms upon bacterial infection to implement an array of barriers preventing pathogen invasion and spread.

Updating Insights into the Catalytic Domain Properties of Plant Cellulose synthase (CesA) and Cellulose synthase-like (Csl) Proteins.

The wall is the last frontier of a plant cell involved in modulating growth, development and defense against biotic stresses. Cellulose and additional polysaccharides of plant cell walls are the most abundant biopolymers on earth, having increased in economic value and thereby attracted significant interest in biotechnology. Cellulose biosynthesis constitutes a highly complicated process relying on the formation of cellulose synthase complexes. Cellulose synthase (CesA) and Cellulose synthase-like (Csl) genes encode enzymes that synthesize cellulose and most hemicellulosic polysaccharides. Arabidopsis and rice are invaluable genetic models and reliable representatives of land plants to comprehend cell wall synthesis. During the past two decades, enormous research progress has been made to understand the mechanisms of cellulose synthesis and construction of the plant cell wall. A plethora of cesa and csl mutants have been characterized, providing functional insights into individual protein isoforms. Recent structural studies have uncovered the mode of CesA assembly and the dynamics of cellulose production. Genetics and structural biology have generated new knowledge and have accelerated the pace of discovery in this field, ultimately opening perspectives towards cellulose synthesis manipulation. This review provides an overview of the major breakthroughs gathering previous and recent genetic and structural advancements, focusing on the function of CesA and Csl catalytic domain in plants.

Virus-Induced Gene Silencing in Olive Tree (Oleaceae).

Research on gene functions in non-model tree species is hampered by a number of difficulties such as time-consuming genetic transformation protocols and extended period for the production of healthy transformed offspring, among others. Virus-induced gene silencing (VIGS) is an alternative approach to transiently knock out an endogenous gene of interest (GOI) by the introduction of viral sequences encompassing a fragment of the GOI and to exploit the posttranscriptional gene silencing (PTGS) mechanism of the plant, thus triggering silencing of the GOI. Here we describe the successful application of Tobacco rattle virus (TRV)-mediated VIGS through agroinoculation of olive plantlets. This methodology is expected to serve as a fast tracking and powerful tool enabling researchers from diversified fields to perform functional genomic analyses in the olive tree.

LEFKOTHEA Regulates Nuclear and Chloroplast mRNA Splicing in Plants.

Eukaryotic organisms accomplish the removal of introns to produce mature mRNAs through splicing. Nuclear and organelle splicing mechanisms are distinctively executed by spliceosome and group II intron complex, respectively. Here, we show that LEFKOTHEA, a nuclear encoded RNA-binding protein, participates in chloroplast group II intron and nuclear pre-mRNA splicing. Transiently optimized LEFKOTHEA nuclear activity is fundamental for plant growth, whereas the loss of function abruptly arrests embryogenesis. Nucleocytoplasmic partitioning and chloroplast allocation are efficiently balanced via functional motifs in LEFKOTHEA polypeptide. In the context of nuclear-chloroplast coevolution, our results provide a strong paradigm of the convergence of RNA maturation mechanisms in the nucleus and chloroplasts to coordinately regulate gene expression and effectively control plant growth.

Detection of RNA-protein interactions using a highly sensitive non-radioactive electrophoretic mobility shift assay.

Electrophoretic mobility shift assay (EMSA) is a sensitive technique useful in the identification and characterization of protein interactors with nucleic acids. This assay provides an efficient method to study DNA or RNA binding proteins and to identify nucleic acid substrates. The specific interaction plays important roles in many biological processes such as transcription, translation, splicing, and global gene expression. In this article, we have modified the EMSA technique and developed a non-radioactive straightforward method to study and determine RNA-protein interactions. The labeling of target RNAs by 3'-end biotinylation and the detection of biotin reactivity to streptavidin-conjugated horseradish peroxidase is a highly sensitive approach capable to detect the formation of RNA-protein complexes. Overall, we provide a complete technical guide useful to determine in vitro RNA-protein interactions and analyze RNA target specificity.

Comprehensive analysis of Lon proteases in plants highlights independent gene duplication events.

The degradation of damaged proteins is essential for cell viability. Lon is a highly conserved ATP-dependent serine-lysine protease that maintains proteostasis. We performed a comparative genome-wide analysis to determine the evolutionary history of Lon proteases. Prokaryotes and unicellular eukaryotes retained a single Lon copy, whereas multicellular eukaryotes acquired a peroxisomal copy, in addition to the mitochondrial gene, to sustain the evolution of higher order organ structures. Land plants developed small Lon gene families. Despite the Lon2 peroxisomal paralog, Lon genes triplicated in the Arabidopsis lineage through sequential evolutionary events including whole-genome and tandem duplications. The retention of Lon1, Lon4, and Lon3 triplicates relied on their differential and even contrasting expression patterns, distinct subcellular targeting mechanisms, and functional divergence. Lon1 seems similar to the pre-duplication ancestral gene unit, whereas the duplication of Lon3 and Lon4 is evolutionarily recent. In the wider context of plant evolution, papaya is the only genome with a single ancestral Lon1-type gene. The evolutionary trend among plants is to acquire Lon copies with ambiguous pre-sequences for dual-targeting to mitochondria and chloroplasts, and a substrate recognition domain that deviates from the ancestral Lon1 type. Lon genes constitute a paradigm of dynamic evolution contributing to understanding the functional fate of gene duplicates.

Trichome patterning control involves TTG1 interaction with SPL transcription factors.

Epidermal cell differentiation is a paramount and conserved process among plants. In Arabidopsis, a ternary complex formed by MYB, bHLH transcription factors and TTG1 modulates unicellular trichome morphogenesis. The formation of multicellular glandular trichomes of the xerophytic shrub Cistus creticus that accumulate labdane-type diterpenes, has attained much attention renowned for its medicinal properties. Here, we show that C. creticus TTG1 (CcTTG1) interacts with the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPLA/B) proteins, putative homologs of AtSPL4/5 that in turn interact with AtTTG1. These interactions occur between proteins from evolutionarily distant species supporting the conserved function of TTG1-SPL complex. Overexpression of AtSPL4 and AtSPL5 decreased the expression of GLABRA2 (AtGL2), the major regulator of trichome morphogenesis, resulting in trichome reduction on the adaxial surface of cauline leaves, thereby illuminating the significance of TTG1-SPLs interactions in trichome formation control. AtGL2 and AtSPL4 have opposite expression patterns during early stages of leaf development. We postulate an antagonistic effect between SPLs and the heterogeneous MYB-bHLH factors binding to TTG1. Hence, the SPLs potentially rearrange the complex, attenuating its transcriptional activity to control trichome distribution.

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.

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.