Complex plant responses to drought and heat stress under climate change.

Global climate change is predicted to result in increased yield losses of agricultural crops caused by environmental conditions. In particular, heat and drought stress are major factors that negatively affect plant development and reproduction, and previous studies have revealed how these stresses induce plant responses at physiological and molecular levels. Here, we provide a comprehensive overview of current knowledge concerning how drought, heat, and combinations of these stress conditions affect the status of plants, including crops, by affecting factors such as stomatal conductance, photosynthetic activity, cellular oxidative conditions, metabolomic profiles, and molecular signaling mechanisms. We further discuss stress-responsive regulatory factors such as transcription factors and signaling factors, which play critical roles in adaptation to both drought and heat stress conditions and potentially function as 'hubs' in drought and/or heat stress responses. Additionally, we present recent findings based on forward genetic approaches that reveal natural variations in agricultural crops that play critical roles in agricultural traits under drought and/or heat conditions. Finally, we provide an overview of the application of decades of study results to actual agricultural fields as a strategy to increase drought and/or heat stress tolerance. This review summarizes our current understanding of plant responses to drought, heat, and combinations of these stress conditions.

Methylomes as key features for predicting recombination in some plant species.

Knowing how chromosome recombination works is essential for plant breeding. It enables the design of crosses between different varieties to combine desirable traits and create new ones. This is because the meiotic crossovers between homologous chromatids are not purely random, and various strategies have been developed to describe and predict such exchange events. Recent studies have used methylation data to predict chromosomal recombination in rice using machine learning models. This approach proved successful due to the presence of a positive correlation between the CHH context cytosine methylation and recombination rates in rice chromosomes. This paper assesses the question if methylation can be used to predict recombination in four plant species: Arabidopsis, maize, sorghum, and tomato. The results indicate a positive association between CHH context methylation and recombination rates in certain plant species, with varying degrees of strength in their relationships. The CG and CHG methylation contexts show negative correlation with recombination. Methylation data was key effectively in predicting recombination in sorghum and tomato, with a mean determination coefficient of 0.65 ± 0.11 and 0.76 ± 0.05, respectively. In addition, the mean correlation values between predicted and experimental recombination rates were 0.83 ± 0.06 for sorghum and 0.90 ± 0.05 for tomato, confirming the significance of methylomes in both monocotyledonous and dicotyledonous species. The predictions for Arabidopsis and maize were not as accurate, likely due to the comparatively weaker relationships between methylation contexts and recombination, in contrast to sorghum and tomato, where stronger associations were observed. To enhance the accuracy of predictions, further evaluations using data sets closely related to each other might prove beneficial. In general, this methylome-based method holds great potential as a reliable strategy for predicting recombination rates in various plant species, offering valuable insights to breeders in their quest to develop novel and improved varieties.

Comprehensive benchmarking of software for mapping whole genome bisulfite data: from read alignment to DNA methylation analysis.

Whole genome bisulfite sequencing is currently at the forefront of epigenetic analysis, facilitating the nucleotide-level resolution of 5-methylcytosine (5mC) on a genome-wide scale. Specialized software have been developed to accommodate the unique difficulties in aligning such sequencing reads to a given reference, building on the knowledge acquired from model organisms such as human, or Arabidopsis thaliana. As the field of epigenetics expands its purview to non-model plant species, new challenges arise which bring into question the suitability of previously established tools. Herein, nine short-read aligners are evaluated: Bismark, BS-Seeker2, BSMAP, BWA-meth, ERNE-BS5, GEM3, GSNAP, Last and segemehl. Precision-recall of simulated alignments, in comparison to real sequencing data obtained from three natural accessions, reveals on-balance that BWA-meth and BSMAP are able to make the best use of the data during mapping. The influence of difficult-to-map regions, characterized by deviations in sequencing depth over repeat annotations, is evaluated in terms of the mean absolute deviation of the resulting methylation calls in comparison to a realistic methylome. Downstream methylation analysis is responsive to the handling of multi-mapping reads relative to mapping quality (MAPQ), and potentially susceptible to bias arising from the increased sequence complexity of densely methylated reads.

Interactomics in plant defence: progress and opportunities.

Interactomics is a branch of systems biology that deals with the study of protein-protein interactions and how these interactions influence phenotypes. Identifying the interactomes involved during host-pathogen interaction events may bring us a step closer to deciphering the molecular mechanisms underlying plant defence. Here, we conducted a systematic review of plant interactomics studies over the last two decades and found that while a substantial progress has been made in the field, plant-pathogen interactomics remains a less-travelled route. As an effort to facilitate the progress in this field, we provide here a comprehensive research pipeline for an in planta plant-pathogen interactomics study that encompasses the in silico prediction step to the validation step, unconfined to model plants. We also highlight four challenges in plant-pathogen interactomics with plausible solution(s) for each.

Heterologous gene expression system for the production of hydrolyzable tannin intermediates in herbaceous model plants.

Aluminum toxicity is the main factor limiting the elongation of plant roots in acidic soil. The tree species Eucalyptus camaldulensis is considerably more resistant to aluminum than herbaceous model plants and crops. Hydrolyzable tannins (HTs) accumulating in E. camaldulensis roots can bind and detoxify the aluminum taken up by the roots. However, in herbaceous model plants, HTs do not accumulate and the genes involved in the HT biosynthetic pathway are largely unknown. The aim of this study was to establish a method for reconstituting the HT biosynthetic pathway in the HT non-accumulating model plant Nicotiana benthamiana. Four E. camaldulensis enzymes were transiently expressed in N. benthamiana leaves via Agrobacterium tumefaciens-mediated transformation. These enzymes included dehydroquinate dehydratase/shikimate dehydrogenases (EcDQD/SDH2 and EcDQD/SDH3), which catalyze the synthesis of gallic acid, the first intermediate of the HT biosynthetic pathway that branches off from the shikimate pathway. The others were UDP-glycosyltransferases (UGT84A25 and UGT84A26), which catalyze the conversion of gallic acid to ß-glucogallin, the second intermediate. The co-expression of the EcDQD/SDHs in transgenic N. benthamiana leaf regions promoted the synthesis of gallic acid. Moreover, the co-expression of the UGT84As in addition to the EcDQD/SDHs resulted in the biosynthesis of ß-glucogallin, the universal metabolic precursor of HTs. Thus, we successfully reconstituted a portion of the HT biosynthetic pathway in HT non-accumulating N. benthamiana plants. This heterologous gene expression system will be useful for co-expressing candidate genes involved in downstream reactions in the HT biosynthetic pathway and for clarifying their in planta functions.

Computational identification of receptor-like kinases "RLK" and receptor-like proteins "RLP" in legumes.

BACKGROUND: In plants, the plasma membrane is enclosed by the cell wall and anchors RLK and RLP proteins, which play a fundamental role in perception of developmental and environmental cues and are crucial in plant development and immunity. These plasma membrane receptors belong to large gene/protein families that are not easily classified computationally. This detailed analysis of these plasma membrane proteins brings a new source of information to the legume genetic, physiology and breeding research communities. RESULTS: A computational approach to identify and classify RLK and RLP proteins is presented. The strategy was evaluated using experimentally-validated RLK and RLP proteins and was determined to have a sensitivity of over 0.85, a specificity of 1.00, and a Matthews correlation coefficient of 0.91. The computational approach can be used to develop a detailed catalog of plasma membrane receptors (by type and domains) in several legume/crop species. The exclusive domains identified in legumes for RLKs are WaaY, APH Pkinase_C, LRR_2, and EGF, and for RLP are L-lectin LPRY and PAN_4. The RLK-nonRD and RLCK subclasses are also discovered by the methodology. In both classes, less than 20% of the total RLK predicted for each species belong to this class. Among the 10-species evaluated ~ 40% of the proteins in the kinome are RLKs. The exclusive legume domain combinations identified are B-Lectin/PR5K domains in G. max, M. truncatula, V. angularis, and V. unguiculata and a three-domain combination B-lectin/S-locus/WAK in C. cajan, M. truncatula, P. vulgaris, V. angularis. and V. unguiculata. CONCLUSIONS: The analysis suggests that about 2% of the proteins of each genome belong to the RLK family and less than 1% belong to RLP family. Domain diversity combinations are greater for RLKs compared with the RLP proteins and LRR domains, and the dual domain combination LRR/Malectin were the most frequent domain for both groups of plasma membrane receptors among legume and non-legume species. Legumes exclusively show Pkinase extracellular domains, and atypical domain combinations in RLK and RLP compared with the non-legumes evaluated. The computational logic approach is statistically well supported and can be used with the proteomes of other plant species.

Blurring the boundaries between cereal crops and model plants.

The cereal crops rice (Oryza sativa), maize (Zea mays ssp. mays) and wheat (Triticum aestivum) provide half of the food eaten by humankind. However, understanding their biology has proved challenging due to their large size, long lifecycle and large genomes. The model plant Arabidopsis thaliana avoids these practical problems and has provided fundamental understanding of plant biology, however not all of this knowledge is directly transferrable to cereals. Recent developments in gene editing, speed breeding and genome assembly techniques mean that the challenges associated with working with the major cereal crops can be overcome. Resources such as mutant collections and genome sequences are now available for these crops, making them attractive experimental systems with which to make discoveries that are directly applicable to increasing crop production.

Development of an efficient root transgenic system for pigeon pea and its application to other important economically plants.

For non-model plants, functional characterization of genes is still hampered by lack of efficient stable transformation procedures. Here, we report a simple, fast and efficient transformation technique with Agrobacterium rhizogenes for generating stable transgenic roots in living plants to facilitate functional studies in vivo. We showed that injection of A. rhizogenes into stems of various plant species lead to stable transgenic root generation, which can sustain plant growth after the original, non-transgenic roots were cut off. A transformation system was established for pigeon pea, a major woody food crop, after optimizing the selection of A. rhizogenes strains, bacterium concentration, injection position and seedling age. RT-PCR and fluorescence observation indicated a transgenic root induction efficiency of about 39% in pigeon pea. Furthermore, induction of hairy roots was achieved in nine out of twelve tested economically important plants at an efficiency of 15-39%. As proof of concept, bimolecular fluorescence complementation (BiFC) assay was applied to test the interaction between CcCIPK14 and CcCBL1/2 in pigeon pea. Additionally, ectopic expression of the bZIP transcription factor MdHY5 from apple confirmed the utility of the transformation technique for engineering anthocyanin synthesis in roots. Taken together, we show that this method allows fast in vivo studies of gene function in a wide range of plant species.

Application of in-house virtual protein database performed in genomic-proteomic combined research on heavy-metal stressed onion roots.

OBJECTIVES: To establish an in-house virtual protein database that can be employed in proteomic research on non-model plants. RESULTS: A total of 87,430 unigenes were obtained through transcriptome sequencing from onion roots. Of these, 24,305 unigenes were annotated and their nucleotide sequences of coding regions were translated into amino acid sequences. The corresponding 24,305 amino acid sequences were considered as an in-house virtual protein database. Thirty-two protein spots with significant differential abundance were selected. Their MS data were submitted to a restriction enzyme map which was converted from the in-house virtual protein database. A total of 27 proteins were finally matched. CONCLUSIONS: The in-house protein database is a feasible and innovative strategy for proteomic research on non-model plants.

Rediscovering Chara as a model organism for molecular and evo-devo studies.

Chara has been used as a model for decades in the field of plant physiology, enabling the investigation of fundamental physiological processes. In electrophysiological studies, Chara has been utilized thanks to its large internodal cells that can be easily manipulated. Additionally, Chara played a pioneering role in elucidating the presence and function of the cytoskeleton in cytoplasmic streaming, predating similar findings in terrestrial plants. Its representation considerably declined following the establishment and routine application of genetic transformation techniques in Arabidopsis. Nevertheless, the recent surge in evo-devo studies can be attributed to the whole genome sequencing of the Chara braunii, which has shed light on ancestral traits prevalent in land plants. Surprisingly, the Chara braunii genome encompasses numerous genes that were previously regarded as exclusive to land plants, suggesting their acquisition prior to the colonization of terrestrial habitats. This review summarizes the established methods used to study Chara, while incorporating recent molecular data, to showcase its renewed importance as a model organism in advancing plant evolutionary developmental biology.

RNA-Seq Data Analysis: A Practical Guide for Model and Non-Model Organisms.

RNA sequencing (RNA-seq) has emerged as a powerful tool for assessing genome-wide gene expression, revolutionizing various fields of biology. However, analyzing large RNA-seq datasets can be challenging, especially for students or researchers lacking bioinformatics experience. To address these challenges, we present a comprehensive guide to provide step-by-step workflows for analyzing RNA-seq data, from raw reads to functional enrichment analysis, starting with considerations for experimental design. This is designed to aid students and researchers working with any organism, irrespective of whether an assembled genome is available. Within this guide, we employ various recognized bioinformatics tools to navigate the landscape of RNA-seq analysis and discuss the advantages and disadvantages of different tools for the same task. Our protocol focuses on clarity, reproducibility, and practicality to enable users to navigate the complexities of RNA-seq data analysis easily and gain valuable biological insights from the datasets. Additionally, all scripts and a sample dataset are available in a GitHub repository to facilitate the implementation of the analysis pipeline. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Analysis of data from a model plant with an available reference genome Basic Protocol 2: Gene ontology enrichment analysis Basic Protocol 3: De novo assembly of data from non-model plants.

An Introduction to Plant Cell, Tissue, and Organ Culture: Current Status and Perspectives.

Plant cell, tissue, and organ cultures (PCTOC) have been used as experimental systems in basic research, allowing gene function demonstration through gene overexpression or repression and investigating the processes involved in embryogenesis and organogenesis or those related to the potential production of secondary metabolites, among others. On the other hand, PCTOC has also been applied at the commercial level for the vegetative multiplication (micropropagation) of diverse plant species, mainly ornamentals but also horticultural crops such as potato or fruit and tree species, and to produce high-quality disease-free plants. Moreover, PCTOC protocols are important auxiliary systems in crop breeding crops to generate pure lines (homozygous) to produce hybrids for the obtention of polyploid plants with higher yields or better performance. PCTOC has been utilized to preserve and conserve the germplasm of different crops or threatened species. Plant genetic improvement through genetic engineering and genome editing has been only possible thanks to the establishment of efficient in vitro plant regeneration protocols. Different companies currently focus on commercializing plant secondary metabolites with interesting biological activities using in vitro PCTOC. The impact of omics on PCTOC is discussed.

Expression and Functional Studies of Leaf, Floral, and Fruit Developmental Genes in Non-model Species.

Researchers working on evolutionary developmental plant biology are inclined to choose non-model taxa to address how specific features have been acquired during ontogeny and fixed during phylogeny. In this chapter we describe methods to extract RNA, to assemble de-novo transcriptomes, to isolate orthologous genes within gene families, and to evaluate expression and function of target genes. We have successfully optimized these protocols for non-model plant species including ferns, gymnosperms, and a large assortment of angiosperms. In the latter, we have ranged a large number of families including Aristolochiaceae, Apodanthaceae, Chloranthaceae, Orchidaceae, Papaveraceae, Rubiaceae, Solanaceae, and Tropaeolaceae.

Genomic Approaches for the Study of Flower Development in Floriculture Crops.

The advances in genomics and bioinformatics have made possible the study in non-model plants of phenotypes associated to flower development. Floriculture crops are an interesting source of traits associated to flower development such as the transition between zygomorphic and actinomorphic flowers or the production of flowers with double and triple corollas. In this chapter, we summarize the material and methods for the use of floriculture crops to study flower development using genomic tools, from the sequencing and assembly of a reference genome to QTL and RNA-Seq analysis to search candidate genes associated to specific traits.

Target capture and genome skimming for plant diversity studies.

Recent technological advances in long-read high-throughput sequencing and assembly methods have facilitated the generation of annotated chromosome-scale whole-genome sequence data for evolutionary studies; however, generating such data can still be difficult for many plant species. For example, obtaining high-molecular-weight DNA is typically impossible for samples in historical herbarium collections, which often have degraded DNA. The need to fast-freeze newly collected living samples to conserve high-quality DNA can be complicated when plants are only found in remote areas. Therefore, short-read reduced-genome representations, such as target capture and genome skimming, remain important for evolutionary studies. Here, we review the pros and cons of each technique for non-model plant taxa. We provide guidance related to logistics, budget, the genomic resources previously available for the target clade, and the nature of the study. Furthermore, we assess the available bioinformatic analyses, detailing best practices and pitfalls, and suggest pathways to combine newly generated data with legacy data. Finally, we explore the possible downstream analyses allowed by the type of data generated using each technique. We provide a practical guide to help researchers make the best-informed choice regarding reduced genome representation for evolutionary studies of non-model plants in cases where whole-genome sequencing remains impractical.

Scaling-up to understand tree-pathogen interactions: A steep, tough climb or a walk in the park?

Plants have proficient tools that allow them to survive interactions with pathogens. Upon attack, they respond with specific countermeasures, which are controlled by the immune system. However, defences can fail and this failure exposes plants to fast-spreading devastation. Trees face similar challenges to other plants and their immune system allows them to mount defences against pathogens. However, their slow growth, longevity, woodiness, and size can make trees a challenging system to study. Here, we review scientific successes in plant systems, highlight the key challenges and describe the enormous opportunities for pathology research in trees. We discuss the benefits that scaling-up our understanding on tree-pathogen interactions can provide in the fight against plant pathogenic threats.

Pangenome Analysis of Plant Transcripts and Coding Sequences.

The pangenome of a species is the sum of the genomes of its individuals. As coding sequences often represent only a small fraction of each genome, analyzing the pangene set can be a cost-effective strategy for plants with large genomes or highly heterozygous species. Here, we describe a step-by-step protocol to analyze plant pangene sets with the software GET_HOMOLOGUES-EST . After a short introduction, where the main concepts are illustrated, the remaining sections cover the installation and typical operations required to analyze and annotate pantranscriptomes and gene sets of plants. The recipes include instructions on how to call core and accessory genes, how to compute a presence-absence pangenome matrix, and how to identify and analyze private genes, present only in some genotypes. Downstream phylogenetic analyses are also discussed.

Scripting Analyses of Genomes in Ensembl Plants.

Ensembl Plants ( http://plants.ensembl.org ) offers genome-scale information for plants, with four releases per year. As of release 47 (April 2020) it features 79 species and includes genome sequence, gene models, and functional annotation. Comparative analyses help reconstruct the evolutionary history of gene families, genomes, and components of polyploid genomes. Some species have gene expression baseline reports or variation across genotypes. While the data can be accessed through the Ensembl genome browser, here we review specifically how our plant genomes can be interrogated programmatically and the data downloaded in bulk. These access routes are generally consistent across Ensembl for other non-plant species, including plant pathogens, pests, and pollinators.

Desert-adapted fungal endophytes induce salinity and drought stress resistance in model crops.

Abiotic stresses are among the most damaging and ever-increasing threats to crop production worldwide. Utilizing extreme-habitat-adapted symbiotic microorganisms is a well-known strategy to mitigate the destructive effects of abiotic stresses on agricultural products. Here, we show the effects of the inoculation of halotolerant endophytic fungi recovered from desert plants on drought and salinity stress tolerance in two model agricultural plants A Periconia and two Neocamarosporium species were selected for this study after an in vitro halotolerant assay. Then, a random block design with three factors including fungi, salinity, and drought treatments was used to investigate the ability of these endophytes to induce stress resistance in tomato and cucumber plants. Physiological markers including proline content and activities of superoxide dismutase, catalase and peroxidase enzymes; as well as growth parameters and chlorophyll contents were assessed in all model plants. Fungal symbiosis increased chlorophyll concentration and plant growth, under all levels of salinity and drought stress. In model plants associated with P. macrospinosa significant increase in proline content and antioxidant enzymatic activities was observed under all levels of the salinity and drought stresses compared to the endophyte-free plants, while plants associated with the two Neocamarosporium species, indicated significant increasing proline content and antioxidant enzymatic activities only in high levels of the salinity and drought stresses. Our findings provide novel insights into the eco-physiological mechanisms of halotolerant fungal endophyte-mediated drought and salinity stress tolerance in cucumber and tomato plants, which signify the prospective applications of arid and saline habitat adapted endophytes in agricultural systems.

To-Do and Not-To-Do in Model Studies of the Uptake, Fate and Metabolism of Metal-Containing Nanoparticles in Plants.

Due to the increasing release of metal-containing nanoparticles into the environment, the investigation of their interactions with plants has become a hot topic for many research fields. However, the obtention of reliable data requires a careful design of experimental model studies. The behavior of nanoparticles has to be comprehensively investigated; their stability in growth media, bioaccumulation and characterization of their physicochemical forms taken-up by plants, identification of the species created following their dissolution/oxidation, and finally, their localization within plant tissues. On the basis of their strong expertise, the authors present guidelines for studies of interactions between metal-containing nanoparticles and plants.