Anibal Arce

Bio: Anibal Arce is an academic researcher from Pontifical Catholic University of Chile. The author has contributed to research in topics: Veraison & Auxin. The author has an hindex of 3, co-authored 7 publications receiving 63 citations. Previous affiliations of Anibal Arce include Millennium Science Initiative.

Omics Approaches for Understanding Grapevine Berry Development: Regulatory Networks Associated with Endogenous Processes and Environmental Responses.

Abstract: Grapevine fruit development is a dynamic process that can be divided into three stages: formation (I), lag (II), and ripening (III), in which physiological and biochemical changes occur, leading to cell differentiation and accumulation of different solutes. These stages can be positively or negatively affected by multiple environmental factors. During the last decade, efforts have been made to understand berry development from a global perspective. Special attention has been paid to transcriptional and metabolic networks associated with the control of grape berry development, and how external factors affect the ripening process. In this review, we focus on the integration of global approaches, including proteomics, metabolomics, and especially transcriptomics, to understand grape berry development. Several aspects will be considered, including seed development and the production of seedless fruits; veraison, at which anthocyanin accumulation begins in the berry skin of colored varieties; and hormonal regulation of berry development and signaling throughout ripening, focusing on the transcriptional regulation of hormone receptors, protein kinases, and genes related to secondary messenger sensing. Finally, berry responses to different environmental factors, including abiotic (temperature, water-related stress and UV-B radiation) and biotic (fungi and viruses) stresses, and how they can significantly modify both, development and composition of vine fruit, will be discussed. Until now, advances have been made due to the application of Omics tools at different molecular levels. However, the potential of these technologies should not be limited to the study of single-level questions; instead, data obtained by these platforms should be integrated to unravel the molecular aspects of grapevine development. Therefore, the current challenge is the generation of new tools that integrate large-scale data to assess new questions in this field, and to support agronomical practices.

Regulation of polar auxin transport in grapevine fruitlets (Vitis vinifera L.) and the proposed role of auxin homeostasis during fruit abscission

Abstract: Indole-3-acetic acid (IAA), the most abundant auxin, is a growth promoter hormone involved in several developmental processes Auxin homeostasis is very important to its function and this is achieved through the regulation of IAA biosynthesis, conjugation, degradation and transport In grapevine, IAA plays an essential role during initial stages of berry development, since it delays fruitlet abscission by reducing the ethylene sensitivity in the abscission zone For this reason, Continuous polar IAA transport to the pedicel is required This kind of transport is controlled by IAA, which regulates its own movement by modifying the expression and localization of PIN-FORMED (PIN) auxin efflux facilitators that localize asymmetrically within the cell On the other hand, the hormone gibberellin (GA) also activates the polar auxin transport by increasing PIN stability In Vitis vinifera, fruitlet abscission occurs during the first two to three weeks after flowering During this time, IAA and GA are present, however the role of these hormones in the control of polar auxin transport is unknown In this work, the use of radiolabeled IAA showed that auxin is basipetally transported during grapevine fruitlet abscission This observation was further supported by immunolocalization of putative VvPIN proteins that display a basipetal distribution in pericarp cells Polar auxin transport and transcripts of four putative VvPIN genes decreased in conjunction with increased abscission, and the inhibition of polar auxin transport resulted in fruit drop GA3 and IAA treatments reduced polar auxin transport, but only GA3 treatment decreased VvPIN transcript abundance When GA biosynthesis was blocked, IAA was capable to increase polar auxin transport, suggesting that its effect depends on GA content Finally, we observed significant changes in the content of several IAA-related compounds during the abscission period These results provide evidence that auxin homeostasis plays a central role during grapevine initial fruit development and that GA and IAA controls auxin homeostasis by reducing polar auxin transport

Accessible LAMP-Enabled Rapid Test (ALERT) for Detecting SARS-CoV-2.

Abstract: The coronavirus disease 2019 (COVID-19) pandemic has highlighted bottlenecks in large-scale, frequent testing of populations for infections. Polymerase chain reaction (PCR)-based diagnostic tests are expensive, reliant on centralized labs, can take days to deliver results, and are prone to backlogs and supply shortages. Antigen tests that bind and detect the surface proteins of a virus are rapid and scalable but suffer from high false negative rates. To address this problem, an inexpensive, simple, and robust 60-minute do-it-yourself (DIY) workflow to detect viral RNA from nasal swabs or saliva with high sensitivity (0.1 to 2 viral particles/μL) and specificity (>97% true negative rate) utilizing reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed. ALERT (Accessible LAMP-Enabled Rapid Test) incorporates the following features: (1) increased shelf-life and ambient temperature storage, compared to liquid reaction mixes, by using wax layers to isolate enzymes from other reagents; (2) improved specificity compared to other LAMP end-point reporting methods, by using sequence-specific QUASR (quenching of unincorporated amplification signal reporters); (3) increased sensitivity, compared to methods without purification through use of a magnetic wand to enable pipette-free concentration of sample RNA and cell debris removal; (4) quality control with a nasopharyngeal-specific mRNA target; and (5) co-detection of other respiratory viruses, such as influenza B, by multiplexing QUASR-modified RT-LAMP primer sets. The flexible nature of the ALERT workflow allows easy, at-home and point-of-care testing for individuals and higher-throughput processing for labs and hospitals. With minimal effort, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific primer sets can be swapped out for other targets to repurpose ALERT to detect other viruses, microorganisms, or nucleic acid-based markers.

Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts.

Abstract: Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been mainly implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Based on preliminary demonstrations of toehold sensors working on lysates, we describe the fast prototyping of RNA toehold switch-based sensors that can be produced locally and reduce the cost of sensors by two orders of magnitude. We demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize these lysates with a CRISPRi strategy to enhance the stability of linear DNAs by knocking-down genes responsible for linear DNA degradation. This enables the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toehold sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease.

Homebrew reagents for low cost RT-LAMP

Abstract: RT-LAMP (reverse transcription - Loop-mediated isothermal amplification) has gained popularity for the detection of SARS-CoV-2. The high specificity, sensitivity, simple protocols and potential to deliver results without the use of expensive equipment has made it an attractive alternative to RT-PCR. However, the high cost per reaction, the centralized manufacturing of required reagents and their distribution under cold chain shipping limits RT-LAMP's applicability in low-income settings. The preparation of assays using homebrew enzymes and buffers has emerged worldwide as a response to these limitations and potential shortages. Here, we describe the production of Moloney murine leukemia virus (M-MLV) Reverse Transcriptase and BstLF DNA polymerase for the local implementation of RT-LAMP reactions at low cost. These reagents compared favorably to commercial kits and optimum concentrations were defined in order to reduce time to threshold, increase ON/OFF range and minimize enzyme quantities per reaction. As a validation, we tested the performance of these reagents in the detection of SARS-CoV-2 from RNA extracted from clinical nasopharyngeal samples, obtaining high agreement between RT-LAMP and RT-PCR clinical results. The in-house preparation of these reactions results in an order of magnitude reduction in costs, and thus we provide protocols and DNA to enable the replication of these tests at other locations. These results contribute to the global effort of developing open and low cost diagnostics that enable technological autonomy and distributed capacities in viral surveillance.

Iso-Seq Allows Genome-Independent Transcriptome Profiling of Grape Berry Development

Abstract: Transcriptomics has been widely applied to study grape berry development. With few exceptions, transcriptomic studies in grape are performed using the available genome sequence, PN40024, as reference. However, differences in gene content among grape accessions, which contribute to phenotypic differences among cultivars, suggest that a single reference genome does not represent the species' entire gene space. Though whole genome assembly and annotation can reveal the relatively unique or "private" gene space of any particular cultivar, transcriptome reconstruction is a more rapid, less costly, and less computationally intensive strategy to accomplish the same goal. In this study, we used single molecule-real time sequencing (SMRT) to sequence full-length cDNA (Iso-Seq) and reconstruct the transcriptome of Cabernet Sauvignon berries during berry ripening. In addition, short reads from ripening berries were used to error-correct low-expression isoforms and to profile isoform expression. By comparing the annotated gene space of Cabernet Sauvignon to other grape cultivars, we demonstrate that the transcriptome reference built with Iso-Seq data represents most of the expressed genes in the grape berries and includes 1,501 cultivar-specific genes. Iso-Seq produced transcriptome profiles similar to those obtained after mapping on a complete genome reference. Together, these results justify the application of Iso-Seq to identify cultivar-specific genes and build a comprehensive reference for transcriptional profiling that circumvents the necessity of a genome reference with its associated costs and computational weight.

Recent Advances in Hormonal Regulation and Cross-Talk during Non-Climacteric Fruit Development and Ripening

Abstract: Fleshy fruits are characterized by having a developmentally and genetically controlled, highly intricate ripening process, leading to dramatic modifications in fruit size, texture, color, flavor, and aroma. Climacteric fruits such as tomato, pear, banana, and melon show a ripening-associated increase in respiration and ethylene production and these processes are well-documented. In contrast, the hormonal mechanism of fruit development and ripening in non-climacteric fruit, such as strawberry, grape, raspberry, and citrus, is not well characterized. However, recent studies have shown that non-climacteric fruit development and ripening, involves the coordinated action of different hormones, such as abscisic acid (ABA), auxin, gibberellins, ethylene, and others. In this review, we discuss and evaluate the recent research findings concerning the hormonal regulation of non-climacteric fruit development and ripening and their cross-talk by taking grape, strawberry, and raspberry as reference fruit species.

Insights Into the Genetic Basis of Blueberry Fruit-Related Traits Using Diploid and Polyploid Models in a GWAS Context

Abstract: Polyploidization is an ancient and recurrent process in plant evolution, impacting the diversification of natural populations and plant breeding strategies. Polyploidization occurs in many important crops; however, its effects on inheritance of many agronomic traits are still poorly understood compared with diploid species. Higher levels of allelic dosage or more complex interactions between alleles could affect the phenotype expression. Hence, the present study aimed to dissect the genetic basis of fruit-related traits in autotetraploid blueberries and identify candidate genes affecting phenotypic variation. We performed a genome-wide association study (GWAS) assuming diploid and tetraploid inheritance, encompassing distinct models of gene action (additive, general, different orders of allelic interaction and the corresponding diploidized models). A total of 1,575 southern highbush blueberry individuals from a breeding population of 117 full-sib families were genotyped using sequence capture and next-generation sequencing, and evaluated for eight fruit-related traits. For the diploid allele calling, 77,496 SNPs were detected; while 80,591 SNPs were obtained in tetraploid, with a high degree of overlap (95%) between them. A linear mixed model that accounted for population and family structure was used for the GWAS analyses. By modeling tetraploid genotypes, we detected 15 SNPs significantly associated with five fruit-related traits. Alternatively, seven significant SNPs were detected for only two traits using diploid genotypes, with two SNPs overlapping with the tetraploid scenario. Our results showed that the importance of tetraploid models varied by trait and that the use of diploid models has hindered the detection of SNP-trait associations and, consequently, the genetic architecture of some commercially important traits in autotetraploid species. Furthermore, 14 SNPs co-localized with candidate genes, five of which lead to non-synonymous amino acid changes. The potential functional significance of these SNPs is discussed.

ABA-mediated responses to water deficit separate grapevine genotypes by their genetic background

Abstract: ABA-mediated processes are involved in plant responses to water deficit, especially the control of stomatal opening. However in grapevine it is not known if these processes participate in the phenotypic variation in drought adaptation existing between genotypes. To elucidate this question, the response to short-term water-deficit was analysed in roots and shoots of nine Vitis genotypes differing in their drought adaptation in the field. The transcript abundance of 12 genes involved in ABA biosynthesis, catabolism, and signalling were monitored, together with physiological and metabolic parameters related to ABA and its role in controlling plant transpiration. Although transpiration and ABA responses were well-conserved among the genotypes, multifactorial analyses separated Vitis vinifera varieties and V. berlandieri x V. rupestris hybrids (all considered drought tolerant) from the other genotypes studied. Generally, V. vinifera varieties, followed by V. berlandieri x V. rupestris hybrids, displayed more pronounced responses to water-deficit in comparison to the other genotypes. However, changes in transcript abundance in roots were more pronounced for Vitis hybrids than V. vinifera genotypes. Changes in the expression of the cornerstone ABA biosynthetic gene VviNCED1, and the ABA transcriptional regulator VviABF1, were associated with the response of V. vinifera genotypes, while changes in VviNCED2 abundance were associated with the response of other Vitis genotypes. In contrast, the ABA RCAR receptors were not identified as key components of the genotypic variability of water-deficit responses. Interestingly, the expression of VviSnRK2.6 (an AtOST1 ortholog) was constitutively lower in roots and leaves of V. vinifera genotypes and higher in roots of V. berlandieri x V. rupestris hybrids. This study highlights that Vitis genotypes exhibiting different levels of drought adaptation differ in key steps involved in ABA metabolism and signalling; both under well-watered conditions and in response to water-deficit. In addition, it supports that adaptation may be related to various mechanisms related or not to ABA responses.

Detection of SARS-CoV-2 RNA using RT-LAMP and molecular beacons.

Abstract: Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection. Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in “single pot” reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes. LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.