A novel aldo-keto reductase gene, OsAKR1, from rice confers higher tolerance to cadmium stress in rice by an in vivo reactive aldehyde detoxification.

Elevated levels of cadmium (Cd) have the ability to impede plant development. Aldo-keto reductases (AKRs) have been demonstrated in a number of plant species to improve tolerance to a variety of abiotic stresses by scavenging cytotoxic aldehydes; however, only a few AKRs have been identified to improve Cd tolerance. The OsAKR1 gene was extracted and identified from rice here. After being exposed to Cd, the expression of OsAKR1 dramatically rose in both roots and shoots, although more pronounced in roots. According to a subcellular localization experiment, the nucleus and cytoplasm are where OsAKR1 is primarily found. Mutants lacking OsAKR1 exhibited Cd sensitive phenotype than that of the wild-type (WT) Nipponbare (Nip), and osakr1 mutants exhibited reduced capacity to scavenge methylglyoxal (MG). Furthermore, osakr1 mutants exhibited considerably greater hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels, and increased catalase (CAT) activity in comparison to Nip. The expression of three isomeric forms of CAT was found to be considerably elevated in osakr1 mutants during Cd stress, as demonstrated by quantitative real-time PCR analysis, when compared to Nip. These results imply that OsAKR1 controlled rice's ability to withstand Cd by scavenging harmful aldehydes and turning on the reactive oxygen species (ROS) scavenging mechanism.

Assessing Temperature Responses in Roots.

Due to global warming, it is important to understand how plants respond to high ambient temperature. Plant growth responses to high ambient temperature are termed thermomophogenesis and have been explored for more than a decade. However, this was mostly focused on the above-ground part of plants, the shoot. In this chapter, we describe a simple method to assess root growth phenotype to high ambient temperatures. In principle, this protocol can be applied for any other treatments to test overall seedling growth.

Cytogenetic Bioindication in Root Meristems for Vitality Assessment of Trees.

Our method describes how to collect forest tree root tips in the field, to store them for transfer to the lab, to pretreat root tips in order to arrest cells in metaphase, fix root tips to preserve specific morphological organizations, to stain fixed root tips by Feulgen's Reaction in order to increase contrast, and to prepare the root meristem for analyzing mitotic stages and chromosomal aberrations via light microscopy. We further describe how to classify chromosomal abnormalities and quantify them via aberration indices.

Root Hair Imaging Using Confocal Microscopy.

Plant genetics plays a key role in determining root hair initiation and development. A complex network of genetic interactions therefore closely monitors and influences root hair phenotype and morphology. The significance of these genes can be studied by employing, for instance, loss-of-function mutants, overexpression plant lines, and fluorescently labeled constructs. Confocal laser scanning microscopy is a great tool to visually observe and document these morphological features. This chapter elaborates the techniques involved in handling of microscopic setup to acquire images displaying root hair distribution along the fully elongated zone of Arabidopsis thaliana roots. Additionally, we illustrate an approach to visualize early fate determination of epidermal cells in the root apical meristem, by describing a method for imaging YFP tagged transgenic plant lines.

Tomato root specialized metabolites evolved through gene duplication and regulatory divergence within a biosynthetic gene cluster.

Tremendous plant metabolic diversity arises from phylogenetically restricted specialized metabolic pathways. Specialized metabolites are synthesized in dedicated cells or tissues, with pathway genes sometimes colocalizing in biosynthetic gene clusters (BGCs). However, the mechanisms by which spatial expression patterns arise and the role of BGCs in pathway evolution remain underappreciated. In this study, we investigated the mechanisms driving acylsugar evolution in the Solanaceae. Previously thought to be restricted to glandular trichomes, acylsugars were recently found in cultivated tomato roots. We demonstrated that acylsugars in cultivated tomato roots and trichomes have different sugar cores, identified root-enriched paralogs of trichome acylsugar pathway genes, and characterized a key paralog required for root acylsugar biosynthesis, SlASAT1-LIKE (SlASAT1-L), which is nested within a previously reported trichome acylsugar BGC. Last, we provided evidence that ASAT1-L arose through duplication of its paralog, ASAT1, and was trichome-expressed before acquiring root-specific expression in the Solanum genus. Our results illuminate the genomic context and molecular mechanisms underpinning metabolic diversity in plants.

[Uncovering the molecular mechanisms behind steroidal saponin accumulation in Liriope muscari (Decne.) Baily through transcriptome sequencing and bioinformatics analysis].

The leaves and roots of Liriope muscari (Decne.) Baily were subjected to high-throughput Illumina transcriptome sequencing. Bioinformatics analysis was used to investigate the enzyme genes and key transcription factors involved in regulating the accumulation of steroidal saponins, which are the main active ingredient in L. muscari. These analyses aimed to reveal the molecular mechanism behind steroidal saponin accumulation. The sequencing results of L. muscari revealed 31 enzymes, including AACT, CAS, DXS and DXR, that are involved in the synthesis of steroidal saponins. Among these enzymes, 16 were in the synthesis of terpenoid skeleton, 3 were involved in the synthesis of sesquiterpene and triterpene, and 12 were involved in the synthesis of steroidal compound. Differential gene expression identified 15 metabolic enzymes coded by 34 differentially expressed genes (DEGs) in the leaves and roots, which were associated with steroidal saponin synthesis. Further analysis using gene co-expression patterns showed that 14 metabolic enzymes coded by 31 DEGs were co-expressed. In addition, analysis using gene co-expression analysis and PlantTFDB's transcription factor analysis tool predicted the involvement of 8 transcription factors, including GAI, PIF4, PIL6, ERF8, SVP, LHCA4, NF-YB3 and DOF2.4, in regulating 6 metabolic enzymes such as DXS, DXR, HMGR, DHCR7, DHCR24, and CAS. These eight transcription factors were predicted to play important roles in regulating steroidal saponin accumulation in L. muscari. Promoter analysis of these transcription factors indicated that their main regulatory mechanisms involve processes such as abscisic acid response, drought-induction stress response and light response, especially abscisic acid responsive elements (ABRE) response and MYB binding site involved in drought-inducibility (MBS) response pathway. Furthermore, qRT-PCR analysis of these eight key transcription factors demonstrated their specific differences in the leaves and roots.

Callus Induction Followed by Regeneration and Hairy Root Induction in Common Buckwheat.

This section describes a set of methods for callus induction followed by the successful regeneration of whole plants and obtaining a culture of transgenic hairy roots from buckwheat plants (Fagopyrum esculentum Moench.). Callus induction and regeneration are key steps for many biotechnological, genetic, and breeding approaches, such as genetic modification, production of biologically active compounds, and propagation of valuable germplasm. Induction of hairy roots using Agrobacterium rhizogenes is also an important tool for functional gene research and plant genome modification. While many efforts were invested into the development of the corresponding protocols, they are not equally efficient for different cultivars. Here, we have tested and optimized the protocols of callus induction, regeneration, and transformation using A. rhizogenes for a set of cultivars of F. esculentum, including wild ancestor of cultivated buckwheat F. esculentum ssp. ancestrale and a self-pollinated accession KK8. The optimal medium for callus induction is Murashige-Skoog basal medium with 3% sucrose which includes hormones 2,4-dichlorophenoxyacetic acid 2 mg/L and kinetin 2 mg/L; for shoot initiation 6-benzylaminopurine 2 mg/L, kinetin 0.2 mg/L, and indole-3-acetic acid 0.2 mg/L; for shoot multiplication 6-benzylaminopurine 3 mg/L and indole-3-acetic acid 0.2 mg/L; and for root initiation half-strength Murashige-Skoog medium with 1.5% sucrose and indole-3-butyric acid 1 mg/L. A. rhizogenes R1000 strain proved to be the most efficient in inducing hairy roots in buckwheat and T-DNA transfer from binary vectors. Seedling explants cut at the root area and immersed in agrobacterium suspension, as well as prickling the cotyledonary area with agrobacteria dipped syringe needle, are the most labor-effective methods of infection, allowing to initiate hairy root growth in 100% of explants.

MaSMG7-Mediated Degradation of MaERF12 Facilitates Fusarium oxysporum f. sp. cubense Tropical Race 4 Infection in Musa acuminata.

Modern plant breeding relies heavily on the deployment of susceptibility and resistance genes to defend crops against diseases. The expression of these genes is usually regulated by transcription factors including members of the AP2/ERF family. While these factors are a vital component of the plant immune response, little is known of their specific roles in defense against Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) in banana plants. In this study, we discovered that MaERF12, a pathogen-induced ERF in bananas, acts as a resistance gene against Foc TR4. The yeast two-hybrid assays and protein-protein docking analyses verified the interaction between this gene and MaSMG7, which plays a role in nonsense-mediated RNA decay. The transient expression of MaERF12 in Nicotiana benthamiana was found to induce strong cell death, which could be inhibited by MaSMG7 during co-expression. Furthermore, the immunoblot analyses have revealed the potential degradation of MaERF12 by MaSMG7 through the 26S proteasome pathway. These findings demonstrate that MaSMG7 acts as a susceptibility factor and interferes with MaERF12 to facilitate Foc TR4 infection in banana plants. Our study provides novel insights into the biological functions of the MaERF12 as a resistance gene and MaSMG7 as a susceptibility gene in banana plants. Furthermore, the first discovery of interactions between MaERF12 and MaSMG7 could facilitate future research on disease resistance or susceptibility genes for the genetic improvement of bananas.

Are high-throughput root phenotyping platforms suitable for informing root system architecture models with genotype-specific parameters? An evaluation based on the root model ArchiSimple and a small panel of wheat cultivars.

Given the difficulties in accessing plant roots in situ, high-throughput root phenotyping (HTRP) platforms under controlled conditions have been developed to meet the growing demand for characterizing root system architecture (RSA) for genetic analyses. However, a proper evaluation of their capacity to provide the same estimates for strictly identical root traits across platforms has never been achieved. In this study, we performed such an evaluation based on six major parameters of the RSA model ArchiSimple, using a diversity panel of 14 bread wheat cultivars in two HTRP platforms that had different growth media and non-destructive imaging systems together with a conventional set-up that had a solid growth medium and destructive sampling. Significant effects of the experimental set-up were found for all the parameters and no significant correlations across the diversity panel among the three set-ups could be detected. Differences in temperature, irradiance, and/or the medium in which the plants were growing might partly explain both the differences in the parameter values across the experiments as well as the genotype × set-up interactions. Furthermore, the values and the rankings across genotypes of only a subset of parameters were conserved between contrasting growth stages. As the parameters chosen for our analysis are root traits that have strong impacts on RSA and are close to parameters used in a majority of RSA models, our results highlight the need to carefully consider both developmental and environmental drivers in root phenomics studies.

Papiliotrema flavescens, a plant growth-promoting fungus, alters root system architecture and induces systemic resistance through its volatile organic compounds in Arabidopsis.

The current trend in agricultural development is the establishment of sustainable agricultural systems. This involves utilizing and implementing eco-friendly biofertilizers and biocontrol agents as alternatives to conventional fertilizers and pesticides. A plant growth-promoting fungal strain, that could alter root system architecture and promote the growth of Arabidopsis seedlings in a non-contact manner by releasing volatile organic compounds (VOCs) was isolated in this study. 26S rDNA sequencing revealed that the strain was a yeast-like fungus, Papiliotrema flavescens. Analysis of plant growth-promoting traits revealed that the fungus could produce indole-3-acetic acid and ammonia and fix nitrogen. Transcriptome analysis in combination with inhibitor experiments revealed that P. flavescens VOCs triggered metabolic alterations, promoted auxin accumulation and distribution in the roots, and coordinated ethylene signaling, thus inhibiting primary root elongation and inducing lateral root formation in Arabidopsis. Additionally, transcriptome analysis and fungal infection experiments confirmed that pretreatment with P. flavescens stimulated the defense response of Arabidopsis to boost its resistance to the pathogenic fungus Botrytis cinerea. Solid-phase microextraction, which was followed by gas chromatography-mass spectrometry analysis, identified three VOCs (acetoin, naphthalene and indole) with significant plant growth-promoting attributes. Their roles were confirmed using further pharmacological experiments and upregulated expression of auxin- and ethylene-related genes. Our study serves as an essential reference for utilizing P. flavescens as a potential biological fertilizer and biocontrol agent.

Over-expression of the BnVIT-L2 gene improves the lateral root development and biofortification under iron stress.

The vacuolar iron transporter (VIT) family is responsible for absorbing and storing iron ions in vacuoles. Here, the BnVIT-L2 gene from Brassica napus has been cloned for the first time and was found to be expressed in multiple tissues and organs, induced by iron stress. The BnVIT-L2 protein is located in vacuolar membranes and has the ability to bind both iron and other bivalent metal ions. Over-expression of the BnVIT-L2 gene increased lateral root number and main root length, as well as chlorophyll and iron content in transgenic Arabidopsis plants (BnVIT-L2/At) exposed to iron stress, compared to wild type Col-0. Furthermore, over-expression of this gene improved the adaptability of transgenic B. napus plants (BnVIT-L2-OE) under iron stress. The regulation of plant tolerance under iron stress by BnVIT-L2 gene may involve in the signal of reactive oxygen species (ROS), as suggested by Ribosome profiling sequencing (Ribo-seq). This study provides a reference for investigating plant growth and biofortification under iron stress through the BnVIT-L2 gene.

Perfluorooctanesulfonic Acid Alters the Plant's Phosphate Transport Gene Network and Exhibits Antagonistic Effects on the Phosphate Uptake.

Per- and polyfluoroalkyl substances (PFASs), with significant health risks to humans and wildlife, bioaccumulate in plants. However, the mechanisms underlying plant uptake remain poorly understood. This study deployed transcriptomic analysis coupled with genetic and physiological studies using Arabidopsis to investigate how plants respond to perfluorooctanesulfonic acid (PFOS), a long-chain PFAS. We observed increased expressions of genes involved in plant uptake and transport of phosphorus, an essential plant nutrient, suggesting intertwined uptake and transport processes of phosphorus and PFOS. Furthermore, PFOS-altered response differed from the phosphorus deficiency response, disrupting phosphorus metabolism to increase phosphate transporter (PHT) transcript. Interestingly, pht1;2 and pht1;8 mutants showed reduced sensitivity to PFOS compared to that of the wild type, implying an important role of phosphate transporters in PFOS sensing. Furthermore, PFOS accumulated less in the shoots of the pht1;8 mutant, indicating the involvement of PHT1;8 protein in translocating PFOS from roots to shoots. Supplementing phosphate improved plant's tolerance to PFOS and reduced PFOS uptake, suggesting that manipulating the phosphate source in PFOS-contaminated soils may be a promising strategy for minimizing PFOS uptake by edible crops or promoting PFOS uptake during phytoremediation. This study highlighted the critical role of phosphate sensing and transport system in the uptake and translocation of PFOS in plants.

Heritable microbiome variation is correlated with source environment in locally adapted maize varieties.

Beneficial interactions with microorganisms are pivotal for crop performance and resilience. However, it remains unclear how heritable the microbiome is with respect to the host plant genotype and to what extent host genetic mechanisms can modulate plant-microbiota interactions in the face of environmental stresses. Here we surveyed 3,168 root and rhizosphere microbiome samples from 129 accessions of locally adapted Zea, sourced from diverse habitats and grown under control and different stress conditions. We quantified stress treatment and host genotype effects on the microbiome. Plant genotype and source environment were predictive of microbiome abundance. Genome-wide association analysis identified host genetic variants linked to both rhizosphere microbiome abundance and source environment. We identified transposon insertions in a candidate gene linked to both the abundance of a keystone bacterium Massilia in our controlled experiments and total soil nitrogen in the source environment. Isolation and controlled inoculation of Massilia alone can contribute to root development, whole-plant biomass production and adaptation to low nitrogen availability. We conclude that locally adapted maize varieties exert patterns of genetic control on their root and rhizosphere microbiomes that follow variation in their home environments, consistent with a role in tolerance to prevailing stress.

Characterization of SgALMT genes reveals the function of SgALMT2 in conferring aluminum tolerance in Stylosanthes guianensis through the mediation of malate exudation.

Aluminum (Al) toxicity is the major constraint on plant growth and productivity in acidic soils. An adaptive mechanism to enhance Al tolerance in plants is mediated malate exudation from roots through the involvement of ALMT (Al-activated malate transporter) channels. The underlying Al tolerance mechanisms of stylo (Stylosanthes guianensis), an important tropical legume that exhibits superior Al tolerance, remain largely unknown, and knowledge of the potential contribution of ALMT genes to Al detoxification in stylo is limited. In this study, stylo root growth was inhibited by Al toxicity, accompanied by increases in malate and citrate exudation from roots. A total of 11 ALMT genes were subsequently identified in the stylo genome and named SgALMT1 to SgALMT11. Diverse responses to metal stresses were observed for these SgALMT genes in stylo roots. Among them, the expressions of 6 out of the 11 SgALMTs were upregulated by Al toxicity. SgALMT2, a root-specific and Al-activated gene, was selected for functional characterization. Subcellular localization analysis revealed that the SgALMT2 protein is localized to the plasma membrane. The function of SgALMT2 in mediating malate release was confirmed by analysis of the malate exudation rate from transgenic composite stylo plants overexpressing SgALMT2. Furthermore, overexpression of SgALMT2 led to increased root growth in transgenic stylo plants treated with Al through decreased Al accumulation in roots. Taken together, the results of this study suggest that malate secretion mediated by SgALMT2 contributes to the ability of stylo to cope with Al toxicity.

Molecular Techniques for Root-Knot Nematode Identification.

Among plant-parasitic nematodes, root-knot nematodes (RKN), Meloidogyne spp., are the most important parasite infecting economically important crops globally and causing severe losses in crop production. The use of efficient nematode control methods against these parasites depends upon their correct detection in roots and soil samples. Currently, the use of integrated identification methods, including biochemical, molecular, and morphological-based characters, is preferred. But the techniques using morphology and phylogenetic analysis are time-consuming and not suitable for routine analysis. They have only been used for studies of cryptic species, which were identified using integrative taxonomy. Here we describe the enzymatic and molecular-based methods that have successfully been used in Brazil for more than 25 years in the Nematology Lab at Embrapa Genetic Resources and Biotechnology for routine analysis. This technique is a combination of isozyme esterase profiling and molecular markers, with the aim of having a rapid and correct diagnosis of Meloidogyne spp. populations from field and greenhouse.

Near-gapless and haplotype-resolved apple genomes provide insights into the genetic basis of rootstock-induced dwarfing.

Dwarfing rootstocks have transformed the production of cultivated apples; however, the genetic basis of rootstock-induced dwarfing remains largely unclear. We have assembled chromosome-level, near-gapless and haplotype-resolved genomes for the popular dwarfing rootstock 'M9', the semi-vigorous rootstock 'MM106' and 'Fuji', one of the most commonly grown apple cultivars. The apple orthologue of auxin response factor 3 (MdARF3) is in the Dw1 region of 'M9', the major locus for rootstock-induced dwarfing. Comparing 'M9' and 'MM106' genomes revealed a 9,723-bp allele-specific long terminal repeat retrotransposon/gypsy insertion, DwTE, located upstream of MdARF3. DwTE is cosegregated with the dwarfing trait in two segregating populations, suggesting its prospective utility in future dwarfing rootstock breeding. In addition, our pipeline discovered mobile mRNAs that may contribute to the development of dwarfed scion architecture. Our research provides valuable genomic resources and applicable methodology, which have the potential to accelerate breeding dwarfing rootstocks for apple and other perennial woody fruit trees.

Comparative Transcriptome Analysis Reveals the Genes and Pathways Related to Wheat Root Hair Length.

Tube-like outgrowths from root epidermal cells, known as root hairs, enhance water and nutrient absorption, facilitate microbial interactions, and contribute to plant anchorage by expanding the root surface area. Genetically regulated and strongly influenced by environmental conditions, longer root hairs generally enhance water and nutrient absorption, correlating with increased stress resistance. Wheat, a globally predominant crop pivotal for human nutrition, necessitates the identification of long root hair genotypes and their regulatory genes to enhance nutrient capture and yield potential. This study focused on 261 wheat samples of diverse genotypes during germination, revealing noticeable disparities in the length of the root hair among the genotypes. Notably, two long root hair genotypes (W106 and W136) and two short root hair genotypes (W90 and W100) were identified. Transcriptome sequencing resulted in the development of 12 root cDNA libraries, unveiling 1180 shared differentially expressed genes (DEGs). Further analyses, including GO function annotation, KEGG enrichment, MapMan metabolic pathway analysis, and protein-protein interaction (PPI) network prediction, underscored the upregulation of root hair length regulatory genes in the long root hair genotypes. These included genes are associated with GA and BA hormone signaling pathways, FRS/FRF and bHLH transcription factors, phenylpropanoid, lignin, lignan secondary metabolic pathways, the peroxidase gene for maintaining ROS steady state, and the ankyrin gene with diverse biological functions. This study contributes valuable insights into modulating the length of wheat root hair and identifies candidate genes for the genetic improvement of wheat root traits.

OsMYB58 Negatively Regulates Plant Growth and Development by Regulating Phosphate Homeostasis.

Phosphate (Pi) starvation is a critical factor limiting crop growth, development, and productivity. Rice (Oryza sativa) R2R3-MYB transcription factors function in the transcriptional regulation of plant responses to various abiotic stresses and micronutrient deprivation, but little is known about their roles in Pi starvation signaling and Pi homeostasis. Here, we identified the R2R3-MYB transcription factor gene OsMYB58, which shares high sequence similarity with AtMYB58. OsMYB58 expression was induced more strongly by Pi starvation than by other micronutrient deficiencies. Overexpressing OsMYB58 in Arabidopsis thaliana and rice inhibited plant growth and development under Pi-deficient conditions. In addition, the overexpression of OsMYB58 in plants exposed to Pi deficiency strongly affected root development, including seminal root, lateral root, and root hair formation. Overexpressing OsMYB58 strongly decreased the expression of the rice microRNAs OsmiR399a and OsmiR399j. By contrast, overexpressing OsMYB58 strongly increased the expression of rice PHOSPHATE 2 (OsPHO2), whose expression is repressed by miR399 during Pi starvation signaling. OsMYB58 functions as a transcriptional repressor of the expression of its target genes, as determined by a transcriptional activity assay. These results demonstrate that OsMYB58 negatively regulates OsmiR399-dependent Pi starvation signaling by enhancing OsmiR399s expression.

The single-cell transcriptome program of nodule development cellular lineages in Medicago truncatula.

Legumes establish a symbiotic relationship with nitrogen-fixing rhizobia by developing nodules. Nodules are modified lateral roots that undergo changes in their cellular development in response to bacteria, but the transcriptional reprogramming that occurs in these root cells remains largely uncharacterized. Here, we describe the cell-type-specific transcriptome response of Medicago truncatula roots to rhizobia during early nodule development in the wild-type genotype Jemalong A17, complemented with a hypernodulating mutant (sunn-4) to expand the cell population responding to infection and subsequent biological inferences. The analysis identifies epidermal root hair and stele sub-cell types associated with a symbiotic response to infection and regulation of nodule proliferation. Trajectory inference shows cortex-derived cell lineages differentiating to form the nodule primordia and, posteriorly, its meristem, while modulating the regulation of phytohormone-related genes. Gene regulatory analysis of the cell transcriptomes identifies new regulators of nodulation, including STYLISH 4, for which the function is validated.

Molecular mechanism of drought resistance in soybean roots revealed using physiological and multi-omics analyses.

Soybeans are one of the most cultivated crops worldwide and drought can seriously affect their growth and development. Many studies have elucidated the mechanisms through which soybean leaves respond to drought; however, little is known about these mechanisms in roots. We used two soybean varieties with different drought tolerances to study the morphological, physiological, and molecular response mechanisms of the root system to drought stress in seedlings. We found that drought stress led to a significant decrease in the root traits and an increase in antioxidant enzyme activity in the two varieties. Drought-resistant varieties accumulate large amounts of flavonoids and phenolic acids at the metabolic level, which causes variations in drought resistance. Additionally, differences in gene expression and drought-resistance pathways between the two varieties were clarified using transcriptome analysis. Through a multi-omics joint analysis, phenylpropanoid and isoflavonoid biosynthesis were identified as the core drought resistance pathways in soybean roots. Candidate genes and marker metabolites affecting drought resistance were identified. The phenylpropanoid pathway confers drought tolerance to roots by maintaining a high level of POD activity and mediates the biosynthesis of various secondary drought-resistant metabolites to resist drought stress. This study provides useful data for investigating plant root drought responses and offers theoretical support for plant breeding for drought resistance.