Ariel Orellana

The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution

Rosaceae is the most important fruit-producing clade, and its key commercially relevant genera (Fragaria, Rosa, Rubus and Prunus) show broadly diverse growth habits, fruit types and compact diploid genomes. Peach, a diploid Prunus species, is one of the best genetically characterized deciduous trees. Here we describe the high-quality genome sequence of peach obtained from a completely homozygous genotype. We obtained a complete chromosome-scale assembly using Sanger whole-genome shotgun methods. We predicted 27,852 protein-coding genes, as well as noncoding RNAs. We investigated the path of peach domestication through whole-genome resequencing of 14 Prunus accessions. The analyses suggest major genetic bottlenecks that have substantially shaped peach genome diversity. Furthermore, comparative analyses showed that peach has not undergone recent whole-genome duplication, and even though the ancestral triplicated blocks in peach are fragmentary compared to those in grape, all seven paleosets of paralogs from the putative paleoancestor are detectable.

A rapid and efficient method for purifying high quality total RNA from peaches (Prunus persica) for functional genomics analyses

Prunus persica has been proposed as a genomic model for deciduous trees and the Rosaceae family. Optimized protocols for RNA isolation are necessary to further advance studies in this model species such that functional genomics analyses may be performed. Here we present an optimized protocol to rapidly and efficiently purify high quality total RNA from peach fruits (Prunus persica). Isolating high-quality RNA from fruit tissue is often difficult due to large quantities of polysaccharides and polyphenolic compounds that accumulate in this tissue and co-purify with the RNA. Here we demonstrate that a modified version of the method used to isolate RNA from pine trees and the woody plant Cinnamomun tenuipilum is ideal for isolating high quality RNA from the fruits of Prunus persica. This RNA may be used for many functional genomic based experiments such as RT-PCR and the construction of large-insert cDNA libraries.

ER-localized auxin transporter PIN8 regulates auxin homeostasis and male gametophyte development in Arabidopsis

Auxin is a key coordinative signal required for many aspects of plant development and its levels are controlled by auxin metabolism and intercellular auxin transport. Here we find that a member of PIN auxin transporter family, PIN8 is expressed in male gametophyte of Arabidopsis thaliana and has a crucial role in pollen development and functionality. Ectopic expression in sporophytic tissues establishes a role of PIN8 in regulating auxin homoeostasis and metabolism. PIN8 co-localizes with PIN5 to the endoplasmic reticulum (ER) where it acts as an auxin transporter. Genetic analyses reveal an antagonistic action of PIN5 and PIN8 in the regulation of intracellular auxin homoeostasis and gametophyte as well as sporophyte development. Our results reveal a role of the auxin transport in male gametophyte development in which the distinct actions of ER-localized PIN transporters regulate cellular auxin homoeostasis and maintain the auxin levels optimal for pollen development and pollen tube growth.

IRE1/bZIP60-mediated unfolded protein response plays distinct roles in plant immunity and abiotic stress responses

Endoplasmic reticulum (ER)-mediated protein secretion and quality control have been shown to play an important role in immune responses in both animals and plants. In mammals, the ER membrane-located IRE1 kinase/endoribonuclease, a key regulator of unfolded protein response (UPR), is required for plasma cell development to accommodate massive secretion of immunoglobulins. Plant cells can secrete the so-called pathogenesis-related (PR) proteins with antimicrobial activities upon pathogen challenge. However, whether IRE1 plays any role in plant immunity is not known. Arabidopsis thaliana has two copies of IRE1, IRE1a and IRE1b. Here, we show that both IRE1a and IRE1b are transcriptionally induced during chemically-induced ER stress, bacterial pathogen infection and treatment with the immune signal salicylic acid (SA). However, we found that IRE1a plays a predominant role in the secretion of PR proteins upon SA treatment. Consequently, the ire1a mutant plants show enhanced susceptibility to a bacterial pathogen and are deficient in establishing systemic acquired resistance (SAR), whereas ire1b is unaffected in these responses. We further demonstrate that the immune deficiency in ire1a is due to a defect in SA- and pathogen-triggered, IRE1-mediated cytoplasmic splicing of the bZIP60 mRNA, which encodes a transcription factor involved in the expression of UPR-responsive genes. Consistently, IRE1a is preferentially required for bZIP60 splicing upon pathogen infection, while IRE1b plays a major role in bZIP60 processing upon Tunicamycin (Tm)-induced stress. We also show that SA-dependent induction of UPR-responsive genes is altered in the bzip60 mutant resulting in a moderate susceptibility to a bacterial pathogen. These results indicate that the IRE1/bZIP60 branch of UPR is a part of the plant response to pathogens for which the two Arabidopsis IRE1 isoforms play only partially overlapping roles and that IRE1 has both bZIP60-dependent and bZIP60-independent functions in plant immunity.

AtHMA1 Is a Thapsigargin-sensitive Ca2+/Heavy Metal Pump

The Arabidopsis thaliana AtHMA1 protein is a member of the PIB-ATPase family, which is implicated in heavy metal transport. However, sequence analysis reveals that AtHMA1 possesses a predicted stalk segment present in SERCA (sarcoplasmic/endoplasmic reticulum Ca2+ ATPase)-type pumps that is involved in inhibition by thapsigargin. To analyze the ion specificity of AtHMA1, we performed functional complementation assays using mutant yeast strains defective in Ca2+ homeostasis or heavy metal transport. The heterologous expression of AtHMA1 complemented the phenotype of both types of mutants and, interestingly, increased heavy metal tolerance of wild-type yeast. Biochemical analyses were performed to describe the activity of AtHMA1 in microsomal fractions isolated from complemented yeast. Zinc, copper, cadmium, and cobalt activate the ATPase activity of AtHMA1, which corroborates the results of metal tolerance assays. The outcome establishes the role of AtHMA1 in Cd2+ detoxification in yeast and suggests that this pump is able to transport other heavy metals ions. Further analyses were performed to typify the active Ca2+ transport mediated by AtHMA1. Ca2+ transport displayed high affinity with an apparent Km of 370 nm and a Vmax of 1.53 nmol mg–1 min–1. This activity was strongly inhibited by thapsigargin (IC50 = 16.74 nm), demonstrating the functionality of its SERCA-like stalk segment. In summary, these results demonstrate that AtHMA1 functions as a Ca2+/heavy metal pump. This protein is the first described plant P-type pump specifically inhibited by thapsigargin.

Nucleotide-sugar transporters: structure, function and roles in vivo

The glycosylation of glycoconjugates and the biosynthesis of polysaccharides depend on nucleotide-sugars which are the substrates for glycosyltransferases. A large proportion of these enzymes are located within the lumen of the Golgi apparatus as well as the endoplasmic reticulum, while many of the nucleotide-sugars are synthesized in the cytosol. Thus, nucleotide-sugars are translocated from the cytosol to the lumen of the Golgi apparatus and endoplasmic reticulum by multiple spanning domain proteins known as nucleotide-sugar transporters (NSTs). These proteins were first identified biochemically and some of them were cloned by complementation of mutants. Genome and expressed sequence tag sequencing allowed the identification of a number of sequences that may encode for NSTs in different organisms. The functional characterization of some of these genes has shown that some of them can be highly specific in their substrate specificity while others can utilize up to three different nucleotide-sugars containing the same nucleotide. Mutations in genes encoding for NSTs can lead to changes in development in Drosophila melanogaster or Caenorhabditis elegans, as well as alterations in the infectivity of Leishmania donovani. In humans, the mutation of a GDP-fucose transporter is responsible for an impaired immune response as well as retarded growth. These results suggest that, even though there appear to be a fair number of genes encoding for NSTs, they are not functionally redundant and seem to play specific roles in glycosylation.

Golgi transporters: opening the gate to cell wall polysaccharide biosynthesis

The synthesis of non-cellulosic polysaccharides occurs in the Golgi apparatus and requires a great number of metabolites (substrates and ions). Many enzymes use these substrates to add sugar residues to nascent polysaccharides chains or to introduce methyl and acetyl groups onto these polymers. Most of these metabolites are in the cytosol and their transport to the Golgi lumen is essential for proper polysaccharide biosynthesis. Different transporters activities have been described in Golgi membranes, but many more are thought to be present to provide all the substrates required for polysaccharide biosynthesis and to pump the ions for maintaining ionic homeostasis. Their functional analysis will help us to understand the role these transporters play in cell wall biosynthesis.

Immunopurification of Golgi vesicles by magnetic sorting

We have designed a method that permits to isolate highly purified Golgi vesicles deprived of endoplasmic reticulum (ER), main contaminant of Golgi fractions. To this end, we prepared a rabbit polyclonal antibody against the cytosolic N-terminal oligopeptide of the enzyme heparan glucosaminyl N-deacetylase/N-sulphotransferase (HSST), a specific marker for Golgi apparatus. The Golgi localization of HSST was confirmed by indirect immunofluorescence microscopy. The antibody binding to Golgi vesicles was demonstrated by immunoelectronmicroscopy and allowed the immunopurification by magnetic sorting. Golgi vesicles subjected to purification by magnetic sorting showed the presence of HSST and p28, which is an integral membrane protein on the cis-Golgi also used as a specific Golgi marker. The purified material was devoid of calreticulin, a specific ER marker. This purification method will allow to improve studies requiring highly purified Golgi membranes such as identification of specific receptors and the electrophysiological characterization of Golgi membrane ion channels, which have been jeopardized up to now by ER membrane contamination.

The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis.

Significance Delivery of nucleotide sugar substrates into the Golgi apparatus and endoplasmic reticulum for processes such as cell wall biosynthesis and protein glycosylation is critical for plant growth and development. Plant genomes encode large families of uncharacterized nucleotide sugar transporters that are specifically presumed to deliver the diverse array of nucleotide sugars found in plants. This study has developed a novel approach that enabled functional characterization of six bifunctional UDP-rhamnose (Rha)/UDP-galactose (Gal) transporters from Arabidopsis. An analysis of loss-of-function and overexpression lines for two of these transporters identified biochemical alterations supporting their roles in the biosynthesis of Rha- and Gal-containing polysaccharides. Thus, cell wall polysaccharide biosynthesis in the Golgi apparatus of plants is likely also regulated by substrate transport mechanisms. Plant cells are surrounded by a cell wall that plays a key role in plant growth, structural integrity, and defense. The cell wall is a complex and diverse structure that is mainly composed of polysaccharides. The majority of noncellulosic cell wall polysaccharides are produced in the Golgi apparatus from nucleotide sugars that are predominantly synthesized in the cytosol. The transport of these nucleotide sugars from the cytosol into the Golgi lumen is a critical process for cell wall biosynthesis and is mediated by a family of nucleotide sugar transporters (NSTs). Numerous studies have sought to characterize substrate-specific transport by NSTs; however, the availability of certain substrates and a lack of robust methods have proven problematic. Consequently, we have developed a novel approach that combines reconstitution of NSTs into liposomes and the subsequent assessment of nucleotide sugar uptake by mass spectrometry. To address the limitation of substrate availability, we also developed a two-step reaction for the enzymatic synthesis of UDP–l-rhamnose (Rha) by expressing the two active domains of the Arabidopsis UDP–l-Rha synthase. The liposome approach and the newly synthesized substrates were used to analyze a clade of Arabidopsis NSTs, resulting in the identification and characterization of six bifunctional UDP–l-Rha/UDP–d-galactose (Gal) transporters (URGTs). Further analysis of loss-of-function and overexpression plants for two of these URGTs supported their roles in the transport of UDP–l-Rha and UDP–d-Gal for matrix polysaccharide biosynthesis.

Photosynthesis and metabolism interact during acclimation of Arabidopsis thaliana to high irradiance and sulphur depletion

Arabidopsis plants were exposed to high light or sulphur depletion alone or in combination for 6 d, and changes of photosynthetic parameters and metabolite abundances were quantified. Photosynthetic electron transport rates (ETRs) of plants exposed to sulphur depletion and high light decreased strongly at day 2 of the acclimation period. After 3 d of treatment, the photosynthetic capacity recovered in plants exposed to the combined stresses, indicating a short recovery time for re-adjustment of photosynthesis. However, at metabolic level, the stress combination had a profound effect on central metabolic pathways such as the tricarboxylic acid (TCA) cycle, glycolysis, pentose phosphate cycle and large parts of amino acid metabolism. Under these conditions, central metabolites, such as sugars and their phosphates, increased, while sulphur-containing compounds were decreased. Further differential responses were found for the stress indicator proline accumulating already at day 1 of the high-light regime, but in combination with sulphur depletion first declined and after a recovery phase reached a delayed elevated level. Other metabolites such as raffinose and putrescine seem to replace proline during the early combinatorial stress response and may act as alternative protectants. Our findings support the notion that plants integrate the selectively sensed stress factors in central metabolism.