Integrative multi-omics analyses of date palm (Phoenix dactylifera) roots and leaves reveal how the halophyte land plant copes with sea water.

Date palm (Phoenix dactylifera L.) is able to grow and complete its life cycle while being rooted in highly saline soils. Which of the many well-known salt-tolerance strategies are combined to fine-tune this remarkable resilience is unknown. The precise location, whether in the shoot or the root, where these strategies are employed remains uncertain, leaving us unaware of how the various known salt-tolerance mechanisms are integrated to fine-tune this remarkable resilience. To address this shortcoming, we exposed date palm to a salt stress dose equivalent to seawater for up to 4 weeks and applied integrative multi-omics analyses followed by targeted metabolomics, hormone, and ion analyses. Integration of proteomic into transcriptomic data allowed a view beyond simple correlation, revealing a remarkably high degree of convergence between gene expression and protein abundance. This sheds a clear light on the acclimatization mechanisms employed, which depend on reprogramming of protein biosynthesis. For growth in highly saline habitats, date palm effectively combines various salt-tolerance mechanisms found in both halophytes and glycophytes: "avoidance" by efficient sodium and chloride exclusion at the roots, and "acclimation" by osmotic adjustment, reactive oxygen species scavenging in leaves, and remodeling of the ribosome-associated proteome in salt-exposed root cells. Combined efficiently as in P. dactylifera L., these sets of mechanisms seem to explain the palm's excellent salt stress tolerance.

Complementarity determining regions in SARS-CoV-2 hybrid immunity.

Pre-vaccination SARS-CoV-2 infection can boost protection elicited by COVID-19 vaccination and post-vaccination breakthrough SARS-CoV-2 infection can boost existing immunity conferred by COVID-19 vaccination. Such 'hybrid immunity' is effective against SARS-CoV-2 variants. In order to understand 'hybrid immunity' at the molecular level we studied the complementarity determining regions (CDR) of anti-RBD (receptor binding domain) antibodies isolated from individuals with 'hybrid immunity' as well as from 'naive' (not SARS-CoV-2 infected) vaccinated individuals. CDR analysis was done by liquid chromatography/mass spectrometry-mass spectrometry. Principal component analysis and partial least square differential analysis showed that COVID-19 vaccinated people share CDR profiles and that pre-vaccination SARS-CoV-2 infection or breakthrough infection further shape the CDR profile, with a CDR profile in hybrid immunity that clustered away from the CDR profile in vaccinated people without infection. Thus, our results show a CDR profile in hybrid immunity that is distinct from the vaccination-induced CDR profile.

Unravelling the complex story of intergenomic recombination in ABB allotriploid bananas.

BACKGROUND AND AIMS: Bananas (Musa spp.) are a major staple food for hundreds of millions of people in developing countries. The cultivated varieties are seedless and parthenocarpic clones of which the ancestral origin remains to be clarified. The most important cultivars are triploids with an AAA, AAB or ABB genome constitution, with A and B genomes provided by M. acuminata and M. balbisiana, respectively. Previous studies suggested that inter-genome recombinations were relatively common in banana cultivars and that triploids were more likely to have passed through an intermediate hybrid. In this study, we investigated the chromosome structure within the ABB group, composed of starchy cooking bananas that play an important role in food security. METHODS: Using SNP markers called from RADSeq data, we studied the chromosome structure of 36 ABB genotypes spanning defined taxonomic subgroups. To complement our understanding, we searched for similar events within nine AB hybrid genotypes. KEY RESULTS: Recurrent homologous exchanges (HEs), i.e. chromatin exchanges between A and B subgenomes, were unravelled with at least nine founding events (HE patterns) at the origin of ABB bananas prior to clonal diversification. Two independent founding events were found for Pisang Awak genotypes. Two HE patterns, corresponding to genotypes Pelipita and Klue Teparod, show an over-representation of B genome contribution. Three HE patterns mainly found in Indian accessions shared some recombined regions and two additional patterns did not correspond to any known subgroups. CONCLUSIONS: The discovery of the nine founding events allowed an investigation of the possible routes that led to the creation of the different subgroups, which resulted in new hypotheses. Based on our observations, we suggest different routes that gave rise to the current diversity in the ABB cultivars, routes involving primary AB hybrids, routes leading to shared HEs and routes leading to a B excess ratio. Genetic fluxes took place between M. acuminata and M. balbisiana, particularly in India, where these unbalanced AB hybrids and ABB allotriploids originated, and where cultivated M. balbisiana are abundant. The result of this study clarifies the classification of ABB cultivars, possibly leading to the revision of the classification of this subgroup.

The Use of Proteomics in Search of Allele-Specific Proteins in (Allo)polyploid Crops.

Most organisms are diploid, meaning they only have two copies of each chromosome (one set inherited from each parent). Polyploid organisms have more than two paired (homologous) sets of chromosomes. Many plant species are polyploid. Polyploid species cope better with stresses thanks to the redundancy in the chromosome copy number and dispose in this way a greater flexibility in gene expression. Allopolyploid species are polyploids that contain an alternative set of chromosomes by the cross of two (or more) species. Gene variants unique for a preferential phenotype are most probable candidate markers controlling the observed phenotype. Organ or tissue-specific silencing or overexpression of one parental homeolog is quite common. It is very challenging to find those tissue-specific gene variants. High-throughput proteomics is a successful method to discover them. This chapter proposes two possible workflows depending on the available resources and the knowledge of the species. An example is given for an AAB hybrid and an ABB hybrid. Allele-specific gene responses are picked up in this workflow as gene loci displaying genotype-specific differential expression that often have single amino acid polymorphisms. If the resources are sufficient, a genotype-specific mRNAseq database is recommended where a link is made to the allele-specific transcription levels. If the resources are limited, allele-specific proteins can be detected by the detection of genotype-specific peptides and the identification against existing genomics libraries of the parents.

The role of Arabidopsis ABA receptors from the PYR/PYL/RCAR family in stomatal acclimation and closure signal integration.

Stomata are microscopic pores found on the surfaces of leaves that act to control CO2 uptake and water loss. By integrating information derived from endogenous signals with cues from the surrounding environment, the guard cells, which surround the pore, 'set' the stomatal aperture to suit the prevailing conditions. Much research has concentrated on understanding the rapid intracellular changes that result in immediate changes to the stomatal aperture. In this study, we look instead at how stomata acclimate to longer timescale variations in their environment. We show that the closure-inducing signals abscisic acid (ABA), increased CO2, decreased relative air humidity and darkness each access a unique gene network made up of clusters (or modules) of common cellular processes. However, within these networks some gene clusters are shared amongst all four stimuli. All stimuli modulate the expression of members of the PYR/PYL/RCAR family of ABA receptors. However, they are modulated differentially in a stimulus-specific manner. Of the six members of the PYR/PYL/RCAR family expressed in guard cells, PYL2 is sufficient for guard cell ABA-induced responses, whereas in the responses to CO2, PYL4 and PYL5 are essential. Overall, our work shows the importance of ABA as a central regulator and integrator of long-term changes in stomatal behaviour, including sensitivity, elicited by external signals. Understanding this architecture may aid in breeding crops with improved water and nutrient efficiency.

Effect of paleopolyploidy and allopolyploidy on gene expression in banana.

BACKGROUND: Bananas (Musa spp.) are an important crop worldwide. Most modern cultivars resulted from a complex polyploidization history that comprised three whole genome duplications (WGDs) shaping the haploid Musa genome, followed by inter- and intra-specific crosses between Musa acuminata and M. balbisiana (A and B genome, respectively). Unresolved hybridizations finally led to banana diversification into several autotriploid (AAA) and allotriploid cultivars (AAB and ABB). Using transcriptomic data, we investigated the impact of the genome structure on gene expression patterns in roots of 12 different triploid genotypes covering AAA, AAB and ABB subgenome constitutions. RESULTS: We demonstrate that (i) there are different genome structures, (ii) expression patterns go beyond the predicted genomic groups, and (iii) the proportion of the B genome influences the gene expression. The presence of the B genome is associated with a higher expression of genes involved in flavonoid biosynthesis, fatty acid metabolism, amino sugar and nucleotide sugar metabolism and oxidative phosphorylation. There are cultivar-specific chromosome regions with biased B:A gene expression ratios that demonstrate homoeologous exchanges (HE) between A and B sub-genomes. In two cultivars, aneuploidy was detected. We identified 3674 genes with a different expression level between allotriploid and autotriploid with ~ 57% having recently duplicated copies (paralogous). We propose a Paralog Inclusive Expression (PIE) analysis that appears to be suitable for genomes still in a downsizing and fractionation process following whole genome duplications. Our approach allows highlighting the genes with a maximum likelihood to affect the plant phenotype. CONCLUSIONS: This study on banana is a good case to investigate the effects of alloploidy in crops. We conclude that allopolyploidy triggered changes in the genome structure of a crop and it clearly influences the gene.

Transient alkalinization of the leaf apoplast stiffens the cell wall during onset of chloride salinity in corn leaves.

During chloride salinity, the pH of the leaf apoplast (pHapo) transiently alkalizes. There is an ongoing debate about the physiological relevance of these stress-induced pHapo changes. Using proteomic analyses of expanding leaves of corn (Zea mays L.), we show that this transition in pHapo conveys functionality by (i) adjusting protein abundances and (ii) affecting the rheological properties of the cell wall. pHapo was monitored in planta via microscopy-based ratio imaging, and the leaf-proteomic response to the transient leaf apoplastic alkalinization was analyzed via ultra-high performance liquid chromatography-MS. This analysis identified 1459 proteins, of which 44 exhibited increased abundance specifically through the chloride-induced transient rise in pHapo These elevated protein abundances did not directly arise from high tissue concentrations of Cl- or Na+ but were due to changes in the pHapo Most of these proteins functioned in growth-relevant processes and in the synthesis of cell wall-building components such as arabinose. Measurements with a linear-variable differential transducer revealed that the transient alkalinization rigidified (i.e. stiffened) the cell wall during the onset of chloride salinity. A decrease in t-coumaric and t-ferulic acids indicates that the wall stiffening arises from cross-linkage to cell wall polymers. We conclude that the pH of the apoplast represents a dynamic factor that is mechanistically coupled to cellular responses to chloride stress. By hardening the wall, the increased pH abrogates wall loosening required for cell expansion and growth. We conclude that the transient alkalinization of the leaf apoplast is related to salinity-induced growth reduction.

Changes in the fine root proteome of Fagus sylvatica L. trees associated with P-deficiency and amelioration of P-deficiency.

Phosphorus is often the least available macronutrient in soil. Lack in phosphorus has detrimental effect on growth and biomass production of European Fagus sylvatica L., a major trees species in temperate forests. In contrast to leaf tissues, few studies have examined changes in the root system and no study has ever investigated the proteomic changes affected in beech roots by a lack in available phosphate (P). Here, we studied roots of young Fagus sylvatica L. trees in their native soils from two forests sites with contrasting availability of P: one P rich and P poor soil. To understand also the response to P fertilization, the trees were fertilized with triple superphosphate and the proteome of fine roots of all conditions was compared. Gel-free mass-spectrometry-based shotgun proteomics revealed that the proteome was differentially affected by diverging P availabilities. The proteomic changes that took place as the result of P fertilization were dependent on the supply level of P before the fertilization. When P was supplied to the P-rich soil proteins related to cell biogenesis exhibited increased abundances. Addition of P to soil that was strongly limited in P resulted in increased abundance of proteins associated with amino acid metabolism and transport. BIOLOGICAL SIGNIFICANCE: Beech (Fagus sylvatica L.) forests have a huge ecological and economic value across Europe. In recent years, however, these forest sites increasingly suffer under phosphorus (P) deficiency. As the consequence, growth and vitality of beech forests is impaired. For this reason, this study was conducted with the aim to identify and understand proteomic impairments and adjustments that evolve in the fine roots under both, a P deficiency and an amelioration thereof. For this, we analyzed (1) the fine root proteome of young beech trees grown (2) at two soil sites that contrast in their degree of availability P (low vs. high) in dependency (3) to a fertilization with P. This experiment revealed fundamental differences with respect to proteomic changes in dependency on the severity of P limitation and helped to identify processes that take place after amelioration of the deficiency. This information is useful to understand which physiological processes are impaired under P deficiency and, thus, impair growth. The fertilization experiment enabled to identify developmental processes that take place in fine roots when concentration of available P was increased. They are "cellular component organization and biogenesis" in the P rich soil and "synthesis of organonitrogen-containing compounds" in the P poor soil.

Elucidation of the compatible interaction between banana and Meloidogyne incognita via high-throughput proteome profiling.

With a diverse host range, Meloidogyne incognita (root-knot nematode) is listed as one of the most economically important obligate parasites of agriculture. This nematode species establishes permanent feeding sites in plant root systems soon after infestation. A compatible host-nematode interaction triggers a cascade of morphological and physiological process disruptions of the host, leading to pathogenesis. Such disruption is reflected by altered gene expression in affected cells, detectable using molecular approaches. We employed a high-throughput proteomics approach to elucidate the events involved in a compatible banana- M. incognita interaction. This study serves as the first crucial step in developing natural banana resistance for the purpose of biological-based nematode management programme. We successfully profiled 114 Grand naine root proteins involved in the interaction with M. incognita at the 30th- and 60th- day after inoculation (dai). The abundance of proteins involved in fundamental biological processes, cellular component organisation and stress responses were significantly altered in inoculated root samples. In addition, the abundance of proteins in pathways associated with defence and giant cell maintenance in plants such as phenylpropanoid biosynthesis, glycolysis and citrate cycle were also implicated by the infestation.

Differential root transcriptomics in a polyploid non-model crop: the importance of respiration during osmotic stress.

To explore the transcriptomic global response to osmotic stress in roots, 18 mRNA-seq libraries were generated from three triploid banana genotypes grown under mild osmotic stress (5% PEG) and control conditions. Illumina sequencing produced 568 million high quality reads, of which 70-84% were mapped to the banana diploid reference genome. Using different uni- and multivariate statistics, 92 genes were commonly identified as differentially expressed in the three genotypes. Using our in house workflow to analyze GO enriched and underlying biochemical pathways, we present the general processes affected by mild osmotic stress in the root and focus subsequently on the most significantly overrepresented classes associated with: respiration, glycolysis and fermentation. We hypothesize that in fast growing and oxygen demanding tissues, mild osmotic stress leads to a lower energy level, which induces a metabolic shift towards (i) a higher oxidative respiration, (ii) alternative respiration and (iii) fermentation. To confirm the mRNA-seq results, a subset of twenty up-regulated transcripts were further analysed by RT-qPCR in an independent experiment at three different time points. The identification and annotation of this set of genes provides a valuable resource to understand the importance of energy sensing during mild osmotic stress.

A look behind the screens: characterization of the HSP70 family during osmotic stress in a non-model crop.

High-throughput molecular analysis is challenging in a non-model species. Proteomics has a great potential to characterize non-model species. We went beyond the level of simply 'identifying' the proteins of interest during osmotic stress experiments in an allopolyploid crop (in casu banana) and focus on an important stress family: the cytoplasmic HSP70s. HSP70s were already identified earlier in proteomics studies as an important player during stress but an insight into the different family members and their polymorphisms was lacking. One particular spot within a whole spot trail drew our attention: its abundance was significantly higher after osmotic stress. What distinguishes this spot from its brother and sister spots? To understand what was special about that particular spot, we characterized the whole spot family in roots and meristem cultures. Using an 2D-DIGE LC-MS/MS approach we were able to measure a proteotypic peptide for each paralog. From our data it is clear that the different paralogs have evolved over time and do not necessarily behave the same when subjected to a stress treatment. The presumable paralog that particularly reacted to the osmotic stress in roots and meristems is located on chromosome 2 and the promoter region contains a unique ABRE element. BIOLOGICAL SIGNIFICANCE: To our knowledge, this is the first time that a proteomics approach has led to the exploration of a protein family at the paralog and allelic variant level in a crop. Moreover we identified a specific osmotic responsive cytoplasmic HSP70 isoform, the HSP70 paralog 2, at the protein level. We have shown that the availability of genomic resources as well as the technique used for proteomics analysis are crucial in being able to go beyond the 'usual suspects'.

The use of 2D-DIGE to understand the regeneration of somatic embryos in avocado.

Avocado embryogenic cell cultures can be classified into two groups based on their morphology when cultured on a medium containing auxin: somatic embryo (SE) and proembryonic masses (PEM) type cultures. The calli of SE-type cell lines are able to go through the maturation process, whereas the calli of PEM cell lines rarely mature. We have investigated four independent avocado cell cultures (two SE and two PEM). The aim of this study was to link the differential regeneration capacity of the four cell cultures to a proteomic pattern and to gain insight into the regeneration capacity. A 2D-DIGE analysis followed by a blind multivariate analysis was able to separate the two SE lines from the PEM lines indicating that the protein profiles of SE and PEM calli are different. Based on the variable importance, that is, the differential protein pattern, we hypothesize that the regeneration capacity in avocado is correlated to the ability to overcome the physicochemical stress stimuli associated with the in vitro culture. Our identical culture conditions do not seem to trigger an appropriate response in PEM lines.

The use of 2D-electrophoresis and de novo sequencing to characterize inter- and intra-cultivar protein polymorphisms in an allopolyploid crop.

Polyploidy and allopolyploidy have played an important role in the evolution of many plants and crops. Several techniques exist to characterize allopolyploid varieties. Analyzing the consequences of genomic reorganization at the gDNA level is a prerequisite but a better insight into the consequences for the phenotype is also primordial. As such, protein polymorphism analysis is important in understanding plant and crop biodiversity and is a driving force behind crop improvement. Our strategy to analyze protein isoforms and to detect possible gene silencing or deletion in bananas was based on protein analysis. Bananas are a good representative of a complex allopolyploid and important crop. We combined two-dimensional electrophoresis (2DE) and 2D DIGE with de novo MS/MS sequence determination to characterize a range of triploid varieties. Via Principal Component Analysis (PCA) and hierarchical clustering we were able to blindly classify the different varieties according to their presumed genome constitution. We report for the first time the application of an automated approach for the derivatization of peptides for facilitated MS/MS de novo sequence determination. We conclude that the proteome does not always correspond to the presumed genome formulae and that proteomics is a powerful tool to characterize varieties. The observations at the protein level provide good indications for a more complex genome structure and genomic rearrangement in some banana varieties.

Sugar-mediated acclimation: the importance of sucrose metabolism in meristems.

We have designed an in vitro experimental setup to study the role of sucrose in sugar-mediated acclimation of banana meristems using established highly proliferating meristem cultures. It is a first step toward the systems biology of a meristem and the understanding of how it can survive severe abiotic stress. Using the 2D-DIGE proteomic approach and a meristem-specific EST library, we describe the long-term acclimation response of banana meristems (after 2, 4, 8, and 14 days) and analyze the role of sucrose in this acclimation by setting up a control, a sorbitol, and a sucrose acclimation treatment over time. Sucrose synthase is the dominant enzyme for sucrose breakdown in meristem tissue, which is most likely related to its lower energy consumption. Metabolizing sucrose is of paramount importance to survive, but the uptake of sugar and its metabolism also drive respiration, which may result in limited oxygen levels. According to our data, a successful acclimation is correlated to an initial efficient uptake of sucrose and subsequently a reduced breakdown of sucrose and an induction of fermentation likely by a lack of oxygen.

Evaluation of chloroform/methanol extraction to facilitate the study of membrane proteins of non-model plants.

Membrane proteins are of great interest to plant physiologists because of their important function in many physiological processes. However, their study is hampered by their low abundance and poor solubility in aqueous buffers. Proteomics studies of non-model plants are generally restricted to gel-based methods. Unfortunately, all gel-based techniques for membrane proteomics lack resolving power. Therefore, a very stringent enrichment method is needed before protein separation. In this study, protein extraction in a mixture of chloroform and methanol in combination with gel electrophoresis is evaluated as a method to study membrane proteins in non-model plants. Benefits as well as disadvantages of the method are discussed. To demonstrate the pitfalls of working with non-model plants and to give a proof of principle, the method was first applied to whole leaves of the model plant Arabidopsis. Subsequently, a comparison with proteins extracted from leaves of the non-model plant, banana, was made. To estimate the tissue and organelle specificity of the method, it was also applied on banana meristems. Abundant membrane or lipid-associated proteins could be identified in both tissues, with the leaf extract yielding a higher number of membrane proteins.

Proteome analysis of non-model plants: a challenging but powerful approach.

Biological research has focused in the past on model organisms and most of the functional genomics studies in the field of plant sciences are still performed on model species or species that are characterized to a great extent. However, numerous non-model plants are essential as food, feed, or energy resource. Some features and processes are unique to these plant species or families and cannot be approached via a model plant. The power of all proteomic and transcriptomic methods, that is, high-throughput identification of candidate gene products, tends to be lost in non-model species due to the lack of genomic information or due to the sequence divergence to a related model organism. Nevertheless, a proteomics approach has a great potential to study non-model species. This work reviews non-model plants from a proteomic angle and provides an outline of the problems encountered when initiating the proteome analysis of a non-model organism. The review tackles problems associated with (i) sample preparation, (ii) the analysis and interpretation of a complex data set, (iii) the protein identification via MS, and (iv) data management and integration. We will illustrate the power of 2DE for non-model plants in combination with multivariate data analysis and MS/MS identification and will evaluate possible alternatives.

Functional genomics in a non-model crop: transcriptomics or proteomics?

There is no question that protein- and RNA-based measurements are complementary, but which approach has the highest return in the case of a non-model crop and what is the correlation between mRNA and proteins? We describe and evaluate in detail the advantages and pitfalls of both a proteomics and a transcriptomics approach. The information on the abundance of transcripts was obtained by serial analysis of gene expression (SAGE), while information on the abundance of proteins was obtained via two-dimensional gel electrophoresis.

Finding the significant markers: statistical analysis of proteomic data.

After separation through two-dimensional gel electrophoresis (2DE), several hundreds of individual protein abundances can be quantified in a cell population or sample tissue. Both a good experimental setup and a valid statistical approach are essential to get insight into the data and to draw correct conclusions. High-throughput 2DE proteomics yield complex and large datasets with a huge disproportion between the hundreds of variables and the restricted number of replicates. However, the most commonly used statistical tests have been designed to cope with a high number of replicates and a restricted number of variables. There is some inconsistency in the proteomics community related to the use of statistics. Two approaches of data analysis can be distinguished: exploratory data analysis and confirmatory data analysis. Currently, most proteomic data are analyzed with the emphasis on confirmatory analysis and do not take into account the exploratory data analysis. This chapter gives an overview of the typical statistical exploratory and confirmatory tools available and suggests case-specific guidelines for a reliable statistical approach that can be used for 2DE analysis. Examples are given for an experimental setup based on classical staining methods as well as for the more advanced difference gel electrophoresis.

Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis.

This study focuses on the specific problems of protein extraction from recalcitrant plant tissues and evaluates several methods to bypass them. Sample preparation is a critical step in a two-dimensional gel electrophoresis proteome approach and is absolutely essential for good results. We evaluated four methods: the classical trichloroacetic acid (TCA)/acetone precipitation, TCA/acetone precipitation and fractionation, an alternative based on fractionation and without precipitation, and phenol extraction methanol/ammonium acetate precipitation. We optimized the phenol extraction protocol for small amounts of tissue, which is essential when the study material is limited. The protocol was optimized for banana (Musa spp.) and was subsequently applied to two other plant species: apple (Malus domestica L.) and potato (Solanum tuberosum L.). Banana (Musa spp.) is a good representative of a "difficult" plant species since it contains many interfering metabolites. Only classical TCA/acetone precipitation and phenol extraction methods proved useful as standard methods. Both methods are associated with a minor but reproducible loss of proteins. Every extraction method and the subsequent analytical procedure have their physicochemical limitations; both methods should be investigated before selecting an appropriate protocol. The study, which is presented in this paper, is useful for guiding the experimental setup of many other nonmodel species, containing various interfering elements.