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Key Message: PAs varied greatly in leaves of different germplasm accessions in Lotus corniculatus and over-expression of LcMYB5 led to high PA accumulation in L. japonicus hairy roots. Proanthocyanidins (PAs) content in leaves is an important quality trait in forage species. The leaves of most forage crops accumulated no or little PAs, which makes it difficult to discover key genes involved in PA biosynthesis in the leaves. We found PAs content varied greatly in leaves of different germplasm accessions in Lotus corniculatus, which is one of the most agriculturally important forage crops. Through a combination of global transcriptional analysis, GO and KEGG analysis, and phylogenetic analysis, we discovered that LcMYB5 was strongly correlated with PA accumulation in leaves of L. corniculatus. The subcellular localization and transactivation activity assays demonstrated that LcMYB5 localized to the nucleus and acted as a transcriptional activator. Over-expression of the two homologs of LcMYB5 (LcMYB5a and LcMYB5b) in the L. japonicus hairy roots resulted in a particular high level of PAs. Global transcriptional analysis and qRT-PCR assays indicated that LcMYB5a and LcMYB5b up-regulated the transcript levels of many key PA pathway genes in the transgenic hairy roots, including structural genes (eg. CHS, F3H, LAR, ANR, and TT15) and regulatory genes (eg. TT8 and TTG1). Collectively, our data suggests that LcMYB5 independently regulates PA accumulation in the leaves of Lotus as a master regulator, which can be bioengineered for PAs production in the leaves of forage species., Competing Interests: Declarations Conflict of interest The authors have no conflicts of interest to declare. Data availability All data supporting the findings of this study are available within the paper and within its supplementary data published online., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: The extract from Metasequoia glyptostroboides Hu et Cheng, a rare and endangered species native to China, exhibits numerous biological and pharmacological activities. The species is recalcitrant to rooting during micropropagation, a challenge that has yet to be resolved. In this study, transcriptomic and hormonal analyses were conducted to appreciate the molecular mechanism of adventitious root (AR) formation in optimized rooting conditions., Results: The use of 2/5-strength Woody Plant Medium (WPM) significantly promoted AR formation of M. glyptostroboides shoots while the content of endogenous auxin, cytokinins and gibberellins (GAs) varied at different stages of AR formation. Transcriptomic analysis showed the significant up- or down-regulation of differentially expressed genes (DEGs) associated with plant hormone signal transduction and the phenylpropanoid biosynthesis pathway in response to 2/5-strength WPM. DEGs related to the biosynthesis of indole-3-acetic acid, cytokinins and GAs were identified. Transcript factors involved in 13 families were also revealed. A weighted gene co-expression network analysis indicated a strong correlation between hormones and genes involved in plant hormone signal transduction and the phenylpropanoid biosynthetic pathway., Conclusions: These results indicate that the AR-promoting potential of 2/5-strength WPM in M. glyptostroboides was due to complex interactions between hormones and the expression of genes related to plant hormone signal transduction and the phenylpropanoid biosynthetic pathway., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Main Conclusion: Association analysis identified 77 marker-trait associations (MTAs) for PUE traits, of which 10 were high-confidence MTAs. Candidate-gene mining and in-silico expression analysis identified 13 putative candidate genes for PUE traits. Bread wheat (Triticum aestivum L.) is a major cereal crop affected by phosphorus (P) deficiency, which affects root characteristics, plant biomass, and other attributes related to P-use efficiency (PUE). Understanding the genetic mechanisms of PUE traits helps in developing bread wheat cultivars that perform well in low-P environments. With this objective, we evaluated a bread wheat panel comprising 304 accessions for 14 PUE traits with high-throughput phenotyping under low-P and optimum-P treatments and observed a significant genetic variation among germplasm lines for studied traits. Genome-wide association study (GWAS) using 14,025 high-quality single-nucleotide polymorphisms identified 77 marker-trait associations (MTAs), of which 10 were chosen as high-confidence MTAs as they had > 10% phenotypic variation with logarithm of odds (LOD) scores of more than five. Candidate-gene (CG) mining from high-confidence MTAs identified 180 unique gene models, of which 78 were differentially expressed (DEGs) with at least twofold change in expression under low-P over optimum-P. Of the 78-DEGs, 13 were thought to be putative CGs as they exhibited functional relevance to PUE traits. These CGs mainly encode for important proteins and their products involved in regulating root system architecture, P uptake, transport, and utilization. Promoter analysis from 1500 bp upstream of gene start site for 13 putative CGs revealed the presence of light responsive, salicylic-acid responsive, gibberellic-acid (GA)-responsive, auxin-responsive, and cold responsive cis-regulatory elements. High-confidence MTAs and putative CGs identified in this study can be employed in breeding programs to improve PUE traits in bread wheat., Competing Interests: Declarations Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of interest. Ethical approval and consent to participate Not applicable. Consent to participate Not applicable. Consent for publication Not applicable., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Bacterial stem and root rot (BSRR) in sweetpotato caused by Dickeya dadantii is one of the ten major diseases of sweetpotatoes in China. However, the molecular mechanism underlying the resistance of sweetpotato to D. dadantii remains unclear. This study adopted a resistance identification assay that conformed Guangshu87 (GS87) as BSRR-resistant and Xinxiang (XX) as susceptible. Compared to XX, GS87 effectively prevented the invasion and dissemination of D. dadantii in planta. An RNA sequencing (RNA-seq) analysis identified 54,844 expressed unigenes between GS87 and XX at four different stages. Further, it revealed that GS87 was more able to regulate the expressions of more unigenes after the inoculation with D. dadantii, including resistance (R) and transcription factors (TF) genes. Moreover, content measurements of disease resistance-related phytohormones showed that both jasmonic acids (JAs) and salicylic acids (SAs) accumulated in D. dadantii-inoculated sweetpotatoes, and JAs may negatively regulate sweetpotato resistance against D. dadantii and accumulated faster than SAs. Meanwhile, determinations of ROS production rate and relevant enzymatic/non-enzymatic activity highlighted the vital roles of reactive oxygen species (ROS) and superoxide dismutase (SOD) in confering GS87 resistance against D. dadantii. Additionally, several hub genes with high connectivity were highlighted through Protein-Protein interaction (PPI) network analysis. In summary, the findings in this study contribute to the understanding of the different responses of resistant and susceptible sweetpotato cultivars to D. dadantii infection, and it also provide the first insight into the relevant candidate genes and phytohormones involved in the resistance of sweetpotato to D. dadantii., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Plasmodiophora brassicae, the causative agent of clubroot disease, establishes a long-lasting parasitic relationship with its host by inducing the expression of sugar transporters. Previous studies have indicated that most BrSWEET genes in Chinese cabbage are up-regulated upon infection with P. brassicae. However, the key BrSWEET genes responsive to P. brassicae have not been definitively identified. In this study, we selected five BrSWEET genes and conducted a functional analysis of them. These five BrSWEET genes showed a notable up-regulation in roots after P. brassicae inoculation. Furthermore, these BrSWEET proteins were localized to the plasma membrane. Yeast functional complementation assays confirmed transport activity for glucose, fructose, or sucrose in four BrSWEETs, with the exception of BrSWEET2a. Mutants and silenced plants of BrSWEET1a, -11a, and -12a showed lower clubroot disease severity compared to wild-type plants, while gain-of-function Arabidopsis thaliana plants overexpressing these three BrSWEET genes exhibited significantly higher disease incidence and severity. Our findings suggested that BrSWEET1a, BrSWEET11a, and BrSWEET12a play pivotal roles in P. brassicae-induced gall formation, shedding light on the role of sugar transporters in host-pathogen interactions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Protein phosphatase 2AB'' (PP2A B'') family subunits have calcium-binding EF-hand motifs, facilitating interaction with PP2A substrates. In Arabidopsis thaliana, the PP2A B'' family subunits consist of six members, AtB''α-ε and FASS. These subunits can interact with a basic leucine zipper transcription factor, VIP1, and its close homologs. Mechanical stress triggers PP2A-mediated dephosphorylation of VIP1 and its close homologs, leading to nuclear localization and gene upregulation to alleviate touch-induced root bending and leaf damage. However, the physiological roles of PP2A B'' family subunits in the mechanical stress response in Arabidopsis remain unclear. This study aims to characterize such roles. A quadruple knockout mutant with T-DNA insertions in AtB''α, AtB''β, AtB''γ, and AtB''δ was generated. atb''αβγδ mutants exhibited no significant damage upon brushing or touch-induced root bending compared to the wild type. Transcriptome analysis showed a significant decrease in the expression of CYP707A3, a gene potentially targeted by VIP1 that regulates abscisic acid (ABA) catabolism, in the atb''αβγδ mutant compared to wild type leaves. However, other genes, including XTH23, EXLA1, and CYP707A1, also VIP1 targets, exhibited similar induction in both brushed atb''αβγδ mutants and wild type leaves. We observed an enrichment of the CAMTA motif, CGCG(C/T) in the promoters of genes showing downregulated expression levels in brushed atb''αβγδ leaves compared to brushed wild type leaves. These findings suggest that PP2A B'' family subunits exhibit functional redundancy in the VIP1-dependent pathway but influence CAMTA-dependent gene expression under mechanical stress. Under calcium-deficient and ABA-supplemented conditions, growth of atb''αβγδ seedlings was retarded when compared to wild type and single knockout mutants, atb''γ and atb''δ, indicating a crucial role in plant development by modulating calcium or ABA signaling., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yoon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Key Message: Established an Agrobacterium-mediated hairy root transformation system for gene function analysis in Lathyrus sativus. Arabidopsis mutant complementation and genome editing in Lathyrus confirmed role of LsOCS in the oxalate metabolism. Grass pea (Lathyrus sativus) is a resilient legume cultivated for its protein-rich seeds and fodder. However, the presence of a naturally occurring neurotoxin, β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), which causes neurolathyrism, limits its extensive cultivation. This paper reports the in-planta characterization of oxalyl-CoA synthetase (OCS), an enzyme involved in oxalate metabolism and important in the oxalylating step leading to β-ODAP production in Lathyrus. For this, we used complementation experiments in an Arabidopsis OCS mutant. The LsOCS-complemented lines showed oxalate content similar to wild-type levels, and the analysis of seeds by field emission scanning electron microscope (FESEM) showed that the LsOCS-complemented lines were rescued from seed-coat defects found in the mutant seeds. We used genome editing of LsOCS in Lathyrus hairy roots to further characterize LsOCS function. The mutations in LsOCS resulted in the accumulation of oxalate in the hairy roots of Lathyrus, as observed in Arabidopsis mutants, but did not affect the ODAP levels. The hairy root genome editing system could serve as a rapid tool for functional studies of Lathyrus genes and optimizing the agronomic traits., Competing Interests: Declarations Conflict of interest No conflict of interest declared., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Increasing biological nitrogen (N) fixation (BNF) in maize production could reduce the environmental impacts of N fertilizer use, but reactive N in the rhizosphere of maize limits the BNF process. Using non-transgenic methods, we developed gene-edited strains of Klebsiella variicola (Kv137-2253) and Kosakonia sacchari (Ks6-5687) bacteria optimized for root-associated BNF and ammonium excretion in N-rich conditions. The aim of this research was to elucidate the mechanism of action of these strains. We present evidence from in vitro, in planta and field experiments that confirms that our genetic remodeling strategy derepresses BNF activity in N-rich systems and increases ammonium excretion by orders of magnitude above the respective wildtype strains. BNF is demonstrated in controlled environments by the transfer of labeled 15 N 2 gas from the rhizosphere to the chlorophyll of inoculated maize plants. This was corroborated in several 15 N isotope tracer field experiments where inoculation with the formulated, commercial-grade product derived from the gene-edited strains (PIVOT BIO PROVEN® 40) provided on average 21 kg N ha -1 to the plant by the VT-R1 growth stages. Data from small-plot and on-farm trials suggest that this technology can improve crop N status pre-flowering and has potential to mitigate the risk of yield loss associated with a reduction in synthetic N fertilizer inputs., Competing Interests: Declarations Competing interests Authors affiliated with Pivot Bio, Inc. declare a competing financial interest given that Pivot Bio, Inc. has obtained patents (World Intellectual Property Organization Patent No. WO2021221690A1) and is developing and commercializing products based on the microbial strains included in this study. All the remaining authors declare no conflict of interest., (© 2024. The Author(s).)

Panax japonicus, a traditional medicinal plant from the Araliaceae family, produces bioactive triterpenes with health benefits. In Traditional Chinese Medicine, its roots have been used, but the chemical basis of its medicinal use is unclear, particularly regarding the metabolism and regulation of triterpene saponin biosynthesis. This study employed an integrative approach using Ultra Performance Liquid Chromatography (UPLC) and transcriptome analysis. Our UPLC analysis showed that ginsenoside Ro and chikusetsusaponin IVa were mainly detected in P. japonicus root. Subsequently, a comparative transcriptome analysis of four P. japonicus tissues (roots, leaves, flowers and fruits) was conducted using Illumina sequencing. As a result, 90,985 unigenes were functionally annotated from a total of 211,650 assembled non-redundant transcripts. Among these, 42,829 unigenes were annotated in NR database. Tissue-specific gene analysis revealed that roots had the highest number of specifically expressed unigenes (11,832). The majority of these unigenes were associated with metabolic processes. Additionally, tissue expression patterns analysis for three common transcription factor families indicated that WRKY family genes showed a significantly root-specific expression pattern, potentially playing a role in triterpene saponin biosynthesis. Notably, we investigated the expression profiles of genes related to the biosynthesis of triterpene saponins and found that four genes, ACCT, HMGS, HMGR and SS, encoding key enzymes in triterpene saponins biosynthesis pathway, were primarily expressed in the root. Overall, our study provides a set of P. japonicus tissue transcriptome data, which will aid in the discovery of triterpene saponin biosynthetic genes and offers valuable genetic information for this medical plant., Competing Interests: Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Statement of permission for collecting Panax japonicus Experimental research and field studies on plants (either cultivated or wild), including the collection of plant material have complied with relevant institutional, national, and international guidelines and legislation. The experimental studies and collection of cultivated Panax japonicus plants were conducted in compliance with the collaboration agreements between the Biomedical Research Institute, Hubei University of Medicine (HBMU), and the Longwangzhai Rare Chinese Herbal Medicine Cultivation Farmers’ Cooperative (LRCC). Permission for the collection of P. japonicus was obtained through this collaboration. Voucher specimens of the plants used in this study have been deposited at the Biomedical Research Institute, HBMU, under the code number VofP-ZZS-2022. The identification of the plant material was confirmed by Prof. Yonghong Zhang at HBMU., (© 2024. The Author(s).)

Arbuscular mycorrhizal fungi (AMF) serve as both plant symbionts and allies in resisting pathogens and environmental stresses. Mycorrhizal colonization of plant roots can influence the outcomes of plant-pathogen interactions by enhancing specific host defense mechanisms. The transcriptional responses induced by AMF in virus-infected plants remain largely unexplored. In the presented study, we employed a comprehensive transcriptomic approach and qPCR to investigate the molecular determinants underlying the interaction between AMF and potato virus Y (PVY) in Solanum tuberosum L. Our primary goal was to identify the symbiosis- and defense-related determinants activated in mycorrhizal potatoes facing PVY. Through a comparative analysis of mRNA transcriptomes in experimental treatments comprising healthy and PVY-infected potatoes colonized by two AMF species, Rhizophagus regularis or Funneliformis mosseae, we unveiled the overexpression of genes associated with mycorrhiza, including nutrient exchange, lipid transfer, and cell wall remodeling. Furthermore, we identified several differentially expressed genes upregulated in all mycorrhizal treatments that encoded pathogenesis-related proteins involved in plant immune responses, thus verifying the bioprotective role of AMF. We investigated the relationship between mycorrhiza levels and PVY levels in potato leaves and roots. We found accumulation of the virus in the leaves of mycorrhizal plants, but our studies additionally showed a reduced PVY content in potato roots colonized by AMF, which has not been previously demonstrated. Furthermore, we observed that a virus-dependent reduction in nutrient exchange could occur in mycorrhizal roots in the presence of PVY. These findings provide an insights into the interplay between virus and AMF., Competing Interests: Declarations Competing interests The authors have no financial or non-financial interests to disclose., (© 2024. The Author(s).)

Two principal growth regulators, cytokinins and ethylene, are known to interact in the regulation of plant growth. However, information about the underlying molecular mechanism and positional specificity of cytokinin/ethylene crosstalk in the control of root growth is scarce. We have identified the spatial specificity of cytokinin-regulated root elongation and root apical meristem (RAM) size, both of which we demonstrate to be dependent on ethylene biosynthesis. Upregulation of the cytokinin biosynthetic gene ISOPENTENYLTRANSFERASE (IPT) in proximal and peripheral tissues leads to both root and RAM shortening. By contrast, IPT activation in distal and inner tissues reduces RAM size while leaving the root length comparable to that of mock-treated controls. We show that cytokinins regulate two steps specific to ethylene biosynthesis: production of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) by ACC SYNTHASEs (ACSs) and its conversion to ethylene by ACC OXIDASEs (ACOs). We describe cytokinin- and ethylene-specific regulation controlling the activity of ACSs and ACOs that are spatially discrete along both proximo/distal and radial root axes. Using direct ethylene measurements, we identify ACO2, ACO3, and ACO4 as being responsible for ethylene biosynthesis and ethylene-regulated root and RAM shortening in cytokinin-treated Arabidopsis. Direct interaction between ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2), a member of the multistep phosphorelay cascade, and the C-terminal portion of ETHYLENE INSENSITIVE 2 (EIN2-C), a key regulator of canonical ethylene signaling, is involved in the cytokinin-induced, ethylene-mediated control of ACO4. We propose tight cooperation between cytokinin and ethylene signaling in the spatially specific regulation of ethylene biosynthesis as a key aspect of the hormonal control of root growth., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Rhizomania is the most destructive sugar beet disease in the world, and in recent years, it has widespread in most of the sugar beet growing areas in Iran. Since the control of this soil-borne disease is a difficult task, the use of resistant genotypes is known as the best measure against the disease. The ultimate goal of sugar beet breeders is to produce genotypes that can be used in both infected and non-infected fields without any reduction in terms of yield and quality. Twenty-one sugar beet genotypes along with four controls were evaluated in randomized complete block design with four replications in fields with natural infection to rhizomania in five research stations. Important sugar beet traits including root yield, sugar yield, sugar content, and white sugar yield were evaluated for two years (2021 and 2022). For all traits, location was the main source of variation that spanned 33 to 55% of the total sum of the square followed by the location×year×genotype accounted for 3-40% of the variation. Based on the results of analysis of variance, multivariate stability parameters were computed to evaluate the genotypes' stability. The first two principal components (IPCA1 and IPCA2) generated by GGE biplot contributed for 31.3 and 17.5% difference in genotype×environment interaction for root yield, respectively. According to the GGE biplot, genotypes RM-11 and RM-12 were identified as the winning genotypes across environments for both root yield and white sugar yield traits whereas AMMI model identified RM-14 and RM-9 (for root yield) and RM-1 (for white sugar yield) as best genotypes. Based on the ideal genotype ranking, RM-11 and RM-10 were the best performer with a high mean yield as well as stability in the studied environments. The biplot rendered using the weighted average of absolute scores (WAASB) and root yield and white sugar yield identified RM-11 and RM-9 as superior genotypes in terms of yield and stability. The selected genotypes can be used in breeding programs to transfer the disease resistance and cultivar development., (© 2024. The Author(s).)

Storage rots are a significant cause of postharvest losses for the sugar beet crop, however, intrinsic physiological and genetic factors that determine the susceptibility of roots to pathogen infection and disease development are unknown. Research, therefore, was carried out to evaluate the disease development in sugar beet roots caused by two common storage pathogens as a function of storage duration and storage temperature, and to identify changes in the expression of defense genes that may be influencing the root susceptibility to disease. To evaluate root susceptibility to disease, freshly harvested roots were inoculated with Botrytis cinerea or Penicillium vulpinum on the day of harvest or after 12, 40, or 120 d storage at 5 or 12 °C and the weight of rotted tissue present in the roots after incubation for 35 d after inoculation were determined. Disease susceptibility and progression to B. cinerea and P. vulpinum increased with storage duration with elevations in susceptibility occurring more rapidly to B. cinerea than P. vulpinum. Also, B. cinerea was more aggressive than P. vulpinum and caused greater rotting and tissue damage in postharvest sugar beet roots. Storage temperature had minimal effect on root susceptibility to these rot-causing pathogens. Changes in defense gene expression were determined by sequencing mRNA isolated from uninoculated roots that were similarly stored for 12, 40 or 120 d at 5 or 12 °C. As susceptibility to rot increased during storage, concurrent changes in defense-related gene expression were identified, including the differential expression of 425 pathogen receptor and 275 phytohormone signal transduction pathway-related genes. Furthermore, plant resistance and hormonal signaling genes that were significantly altered in expression coincident with the change in root susceptibility to storage rots were identified. Further investigation into the function of these genes may ultimately elucidate methods by which storage rot resistance in sugar beet roots may be improved in the future., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Main Conclusion: In the angiosperm root apical meristem, the holoploid DNA content is not directly related to cell cycle time. Instead, ploidy, chromosome number, and taxa emerge as key factors that influence this interaction. It is commonly considered that cell cycle time in the angiosperm root apical meristem is directly related to the holoploid DNA content, and this is one of the manifestations of the nucleotypic effect. In this paper, we significantly expanded the previously reported data on cell cycle time using the thymidine method and the rate-of-cell-production method and investigated the nucleotypic effect in different taxonomic groups, in diploids with varying numbers of chromosomes, and in species of different ploidy levels. The nucleotypic effect was most pronounced in diploids with a chromosome number of less than 17, especially in the orders Asparagales and Liliales. In diploids with 17 and a greater number of chromosomes, and in polyploids, the nucleotypic effect was only evident in these two orders and was practically absent in the rest of angiosperms, which have generally lower the holoploid DNA content and average DNA content per chromosome. Overall, our work demonstrates that in angiosperms the relationship between cell cycle time in the root apical meristem and the holoploid DNA content is complex and not homogeneous and it is not directly related to cell cycle time. Instead, ploidy, chromosome number, and taxa emerge as key factors that influence this interaction., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Main Conclusion: The dwarfing mechanism in apple clonal rootstocks is driven by complex interactions between anatomical, hormonal, genetic, and phenolic factors, offering potential for advanced genetic manipulation to optimize tree size and enhance orchard productivity. The widespread adoption of dwarfing rootstocks is pivotal to modern commercial apple (Malus × domestica Borkh) orchards due to their ability to control tree size, shorten the juvenile period, and enhance reproductive growth and overall productivity. The underlying mechanisms of rootstock-induced dwarfism are multifaceted and involve interactions between phenotypic, anatomical, genetic, and phytohormonal factors. This review consolidates current understanding, highlighting the importance of auxin (IAA), cytokinins (CKs), gibberellins (GAs), and abscisic acid (ABA) in mediating growth suppression through impaired transport and hormone signaling. The phenotypic impacts, including reduced root growth, shorter sylleptic shoots, and higher floral bud densities, are discussed alongside genetic loci such as Dw1, Dw2, and Dw3, and the influence of key genes/TFs like MdWRKY9, RGL, and PIN. Anatomically, dwarf rootstocks exhibit a higher bark-to-wood ratio and restricted hydraulic conductivity, which contribute to reduced scion vigour. Furthermore, the accumulation of phenolic compounds in the graft union of dwarfing rootstocks further modulates the growth inhibition. These insights lay the groundwork for advanced molecular breeding strategies, incorporating gene-editing technologies to improve dwarf rootstock development, providing avenues for enhanced orchard management and apple productivity., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Yellow lupine (Lupinus luteus L.) is a high-protein crop of considerable economic and ecological significance. It has the ability to fix atmospheric nitrogen in symbiosis with Rhizobium, enriching marginal soils with this essential nutrient and reducing the need for artificial fertilizers. Additionally, lupine produces seeds with a high protein content, making it valuable for animal feed production. However, drought negatively affects lupine development, its mutualistic relationship with bacteria, and overall yield. To understand how lupine responds to this stress, global transcriptome sequencing was conducted, along with in-depth biochemical, chromatography, and microscopy analyses of roots subjected to drought. The results presented here contribute to strategies aimed at mitigating the effects of water deficit on lupine growth and development., Results: Based on RNA-seq, drought-specific genes were identified and annotated to biological pathways involved in phytohormone biosynthesis/signaling, lipid metabolism, and redox homeostasis. Our findings indicate that drought-induced disruption of redox balance characterized by the upregulation of reactive oxygen species (ROS) scavenging enzymes, coincided with the accumulation of lipid-metabolizing enzymes, such as phospholipase D (PLD) and lipoxygenase (LOX). This disruption also led to modifications in lipid homeostasis, including increased levels of triacylglycerols (TAG) and free fatty acids (FFA), along with a decrease in polar lipid content. Additionally, the stress response involved alterations in the transcriptional regulation of the linolenic acid metabolism network, resulting in changes in the composition of fatty acids containing 18 carbons., Conclusion: The first comprehensive global transcriptomic profiles of lupine roots, combined with the identification of key stress-responsive molecules, represent a significant advancement in understanding lupine's responses to abiotic stress. The increased expression of the Δ12DESATURASE gene and enhanced PLD activity lead to higher level of linoleic acid (18:2), which is subsequently oxidized by LOX, resulting in membrane damage and malondialdehyde (MDA) accumulation. Oxidative stress elevates the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT), while the conversion of FFAs into TAGs provides protection against ROS. This research offers valuable molecular and biochemical candidates with significant potential to enhance drought tolerance . It enables innovative strategies in lupine breeding and crop improvement to address critical agricultural challenges., (© 2024. The Author(s).)

Constitutive overexpression of phosphate (Pi) transporter family 1 often results in the accumulation of toxic levels of Pi, which causes growth retardation in plants. In contrast, we had previously reported that root epidermis-specific overexpression of the phosphate transporter TaPT2 in Arabidopsis leads to improved growth and Pi use efficiency. In the present study, we used promoters AtHKT1;1 and SKOR, which are predominantly expressed in the vascular bundle tissues, to overexpress TaPT2. Transgenic lines exhibited increased shoot growth compared to wild type plants under normal- and low-Pi conditions, along with elevated root Pi and total P content, and higher xylem sap Pi concentration, specifically under low-Pi conditions. This was attributed to moderate Pi accumulation in the xylem parenchyma cells, enhancing the Pi uploading capacity to the xylem. SKOR-TaPT2, however, did not complement pho1 mutant, which was defective in uploading Pi to the xylem. The transcriptional levels of VPT1 and VPT3, which are responsible for transporting excess Pi into a vacuole, were upregulated in SKOR promoter lines under normal-Pi conditions. Our results suggested that root vascular bundle-specific expression of TaPT2 is another promising strategy for increasing biomass production, Pi uptake, and Pi use efficiency while preventing growth retardation in transgenic plants., (© 2024. The Author(s).)

The involvement of nuclear factor Y (NF-Y) in transcriptional reprogramming during arbuscular mycorrhizal symbiosis has been demonstrated in several plant species. However, a comprehensive picture is lacking. We showed that the spatial expression of NF-YC3 was observed in cortical cells containing arbuscules via the cis-regulatory element GCC boxes. Moreover, the NF-YC3 promoter was transactivated by the combination of CYCLOPS and autoactive calcium and calmodulin-dependent kinase (CCaMK) via GCC boxes. Knockdown of NF-YC3 significantly reduced the abundance of all intraradical fungal structures and affected arbuscule size. BCP1, SbtM1, and WRI5a, whose expression associated with NF-YC3 levels, might be downstream of NF-YC3. NF-YC3 interacted with NF-YB3a, NF-YB5c, or NF-YB3b, in yeast (Saccharomyces cerevisiae) and in planta, and interacted with NF-YA3a in yeast. Spatial expression of 3 NF-YBs was observed in all cell layers of roots under both mock and mycorrhizal conditions. Simultaneous knockdown of 3 NF-YBs, but not individually, reduced the fungal colonization level, suggesting that there might be functional redundancy of NF-YBs to regulate AM symbiosis. Collectively, our data suggest that NF-YC3 and NF-YBs positively regulate AM symbiosis in tomato, and arbuscule-related NF-YC3 may be an important downstream gene of the common symbiosis signaling pathway., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Cold stress during early development limits maize (Zea mays L.) production in temperate zones. Low temperatures restrict root growth and reprogram gene expression. Here, we provide a systematic transcriptomic landscape of maize primary roots, their tissues, and cell types in response to cold stress. The epidermis exhibited a unique transcriptomic cold response, and genes involved in root hair formation were dynamically regulated in this cell type by cold. Consequently, activation of genes involved in root hair tip growth contributed to root hair recovery under moderate cold conditions. The maize root hair defective mutants roothair defective 5 (rth5) and roothair defective 6 (rth6) displayed enhanced cold tolerance with respect to primary root elongation. Furthermore, DEHYDRATION RESPONSE ELEMENT-BINDING PROTEIN 2.1 (DREB2.1) was the only member of the dreb subfamily of AP2/EREB transcription factor genes upregulated in primary root tissues and cell types but exclusively downregulated in root hairs upon cold stress. Plants overexpressing dreb2.1 significantly suppressed root hair elongation after moderate cold stress. Finally, the expression of rth3 was regulated by dreb2.1 under cold conditions, while rth6 transcription was regulated by DREB2.1 irrespective of the temperature regime. We demonstrated that dreb2.1 negatively regulates root hair plasticity at low temperatures by coordinating the expression of root hair defective genes in maize., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

EARLY NODULIN 93 (ENOD93) has been genetically associated with biological nitrogen fixation in legumes and nitrogen use efficiency in cereals, but its precise function is unknown. We show that hidden Markov models define ENOD93 as a homolog of the N-terminal domain of RESPIRATORY SUPERCOMPLEX FACTOR 2 (RCF2). RCF2 regulates cytochrome oxidase (CIV), influencing the generation of a mitochondrial proton motive force in yeast (Saccharomyces cerevisiae). Knockout of ENOD93 in Arabidopsis (Arabidopsis thaliana) causes a short root phenotype and early flowering. ENOD93 is associated with a protein complex the size of CIV in mitochondria, but neither CIV abundance nor its activity changed in ruptured organelles of enod93. However, a progressive loss of ADP-dependent respiration rate was observed in intact enod93 mitochondria, which could be recovered in complemented lines. Mitochondrial membrane potential was higher in enod93 in a CIV-dependent manner, but ATP synthesis and ADP depletion rates progressively decreased. The respiration rate of whole enod93 seedlings was elevated, and root ADP content was nearly double that in wild type without a change in ATP content. We propose that ENOD93 and HYPOXIA-INDUCED GENE DOMAIN 2 (HIGD2) are the functional equivalent of yeast RCF2 but have remained undiscovered in many eukaryotic lineages because they are encoded by 2 distinct genes., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Polar tip growth in plants occurs only in root hairs and pollen tubes. In particular, root hair growth is considered very important in the growth of plants, as it is critical for water and nutrient absorption. Polar tip growth is regulated by various factors, including plant hormones such as abscisic acid (ABA) and gibberellin (GA) and cell wall modifications. We aimed to elucidate the effects and mechanisms on tip growth of a novel gene containing the domain of unknown function (DUF) 3511. We found that Protein Involved in Tip Elongation (PITE) is involved in root hair development in rice (Oryza sativa L.). PITE protein was observed in the plasma membrane and cytoplasm of root hairs. Pite mutants generated by the CRISPR/Cas9 system showed a shorter root hair phenotype compared to the wild type. Through RNA sequencing and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis, we found that the expression of genes that affect cell wall rigidity and GA metabolism-related genes were differently regulated in pite mutants. PITE could interact with acyl transferase and haloacid dehalogenase-like hydrolase (HAD9) in the nucleus and cytoplasm. Our study suggests that PITEs containing the DUF3511 domain regulate root hair growth in rice by mediating the expression of genes that can regulate cell wall rigidity or cause changes in GA metabolism through interactors such as HAD9., (© 2024 Scandinavian Plant Physiology Society.)

Auxins play a critical role in several plant developmental processes and their endogenous levels are regulated at multiple levels. The enzymes of the GRETCHEN HAGEN 3 (GH3) protein family catalyze the conjugation of amino acids to indoleacetic acid (IAA), the major endogenous auxin. The GH3 proteins are encoded by multiple redundant genes in plant genomes, making it difficult to perform functional genetic studies to understand their role in auxin homeostasis. To address these challenges, we used a chemical approach that exploits the reaction mechanism of GH3 proteins to identify small molecule inhibitors of their activity from a defined chemical library. The study evaluated receptor-ligand complexes based on their binding energy and classified them accordingly. Docking algorithms were used to correct any deviations, resulting in a list of the most important inhibitory compounds for selected GH3 enzymes based on a normalized sum of energy. The study presents atomic details of protein-ligand interactions and quantifies the effect of several of the identified small molecule inhibitors on auxin-mediated root growth processes in Arabidopsis thaliana. The direct effect of these compounds on endogenous auxin levels was measured using appropriate auxin sensors and endogenous hormone measurements. Our study has identified novel compounds of the flavonoid biosynthetic pathway that are effective inhibitors of GH3 enzyme-mediated IAA conjugation. These compounds play a versatile role in hormone-regulated plant development and have potential applications in both basic research and agriculture., (© 2024 Scandinavian Plant Physiology Society.)

Stylosanthes is an important forage legume in tropical areas with strong resistance to aluminum (Al) toxicity, though knowledge of mechanisms underlying this resistance remains fragmentary. We found that border-like cells (BLCs) were constitutively produced surrounding the root tips of all 54 examined Stylosanthes guianensis genotypes, but not the Stylosanthes viscose genotype TF0140. In genotypic comparisons under Al conditions, the S. guianensis genotype RY#2 retained significantly more Al in BLCs and thereby showed higher relative root growth than TF0140. Formation of BLCs accompanied changes in cell wall pectin epitopes and differential expression of genes involved in pectin metabolism, including a polygalacturonase (SgPG1). The expression pattern of SgPG1 was consistent with the formation of BLCs in both RY#2 and TF0140. SgPG1 was localized in cell walls and exhibited high activities mediating demethyl-esterified homogalacturonan degradation. Overexpressing SgPG1 changed cell wall pectin epitopes, enhanced BLCs production, and Al resistance in both Arabidopsis and Stylosanthes hairy roots. Furthermore, combining protein-DNA binding assays in vitro and in vivo, a bHLH transcription factor SgbHLH19 was demonstrated to be the upstream regulator of SgPG1. Our study demonstrates that S. guianensis Al resistance mainly relies on BLCs, whose formation involves cell wall pectin epitope modification by SgPG1., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Boron (B) is an important limiting factor for plant growth and yield in saline soils, but the underlying molecular mechanisms remain poorly understood. In this study, we found that appropriate B supply obviously complemented rapeseed (Brassica napus L.) growth under salinity accompanied by higher biomass production and less reactive oxygen species accumulation. Determination of Na + content in shoots and roots indicated that B significantly repressed root-to-shoot Na + translocation, and non-invasive micro-tests of root xylem sap demonstrated that B increased xylem Na + unloading in the roots of rapeseed plants under salinity. Comparative transcriptomic profiling revealed that B strongly upregulated BnaHKT1s expression, especially BnaA2.HKT1, in rapeseed roots exposed to salinity. In situ hybridizations analysis showed that BnaA2.HKT1 was significantly induced in root stelar tissues by high B (HB) under salinity. Green fluorescent protein and yeast heterologous expression showed that BnaA2.HKT1 functioned as a plasma membrane-localized Na + transporter. Knockout of BnaA2.HKT1 by CRISPR/Cas9 resulted in hypersensitive of rapeseed plants to salinity even under HB condition, with higher shoot Na + accumulation and lower biomass production. By contrast, overexpression of BnaA2.HKT1 ameliorated salinity-induced growth inhibition under B deficiency and salinity. Overall, our results proposed that B functioned as a positive regulator for the rapeseed growth and seed production under salt stress through facilitating BnaA2.HKT1-mediated root xylem Na + unloading. This study may also provide an alternative strategy for the improvement of crop growth and development in saline soils., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Radish (Raphanus sativus L.) is one of the most important root vegetable crops worldwide. However, gene function exploration and germplasm innovation still face tremendous challenges due to its extremely low transformation efficiency. Here, an economic and highly efficient genetic transformation method for radish was explored by Agrobacterium rhizogenes-mediated transformation with the help of combining special developmental regulator (DR) genes and the visual identification reporter. Firstly, the RUBY gene, a betalain biosynthesis system, could result in a visual red-violet color used as a convenient and effective reporter for monitoring transgenic hairy roots screening of radish. However, the hairy roots-to-shoots conversion system of radish still stands as a barrier to the obtainment of whole transgenic plants, although different hormone combinations and various culture conditions were tried. Following, two DR genes including Wuschel2 (Wus2) and isopentenyl transferase (ipt), as well as their combination Wus2-ipt were introduced for the shoot regeneration capacity improvement. The results showed that the transgenic shoots could be directly generated without externally supplying any hormones in the presence of a Wus2-ipt combination. Then, Wus2-ipt along with the RUBY reporter was employed to establish an efficient genetic transformation system of radish. Moreover, this system was applied in generating gene-edited radish plants and the phytoene desaturase (RsPDS) gene was effectively knockout through albino phenotype observation and sequencing analysis. These findings have the potential to be widely applied in genetic transformation and genome-editing genetic improvement of other vegetable species., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Stem cells in plant meristems are kept undifferentiated by signals from surrounding cells and provide the basis for continuous organ formation. In the stem cell organizer of the Arabidopsis thaliana root, the quiescent centre (QC), the WOX5 transcription factor, functions as a central hub in regulating columella stem cell (CSC) homoeostasis. However, the processes mediating WOX5 function are only poorly understood. Here we identify the transcription factor HAN as a central mediator of WOX5-regulated stem cell maintenance. HAN is required for mitotic quiescence of QC and CSC maintenance and is sufficient to induce ectopic stem cells. WOX5 and HAN repress transcription of the differentiation factor gene CDF4 in a coherent feed-forward loop (cFFL), one output of which is the expression of the auxin biosynthesis gene TAA1 and maintenance of auxin response maxima in the organizer. These findings and mathematical modelling provide a mechanistic framework for WOX5 function in the root stem cell niche., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Cytokinin is central to coordinating plant adaptation to environmental stresses. Here, we first demonstrated the involvement of cytokinin in Arabidopsis responses to arsenite [As(III)] stress. As(III) treatment reduced cytokinin contents, while cytokinin treatment repressed further primary root growth in Arabidopsis plants under As(III) stress. Subsequently, we revealed that the cytokinin signaling members ARR1 and ARR12, the type-B ARABIDOPSIS RESPONSE REGULATORs, participate in cytokinin signaling-mediated As(III) responses in plants as negative regulators. A comprehensive transcriptome analysis of the arr1 and arr12 single and arr1,12 double mutants was then performed to decipher the cytokinin signaling-mediated mechanisms underlying plant As(III) stress adaptation. Results revealed important roles for ARR1 and ARR12 in ion transport, nutrient responses, and secondary metabolite accumulation. Furthermore, using hierarchical clustering and regulatory network analyses, we identified two NODULIN 26-LIKE INTRINSIC PROTEIN (NIP)-encoding genes, NIP1;1 and NIP6;1, potentially involved in ARR1/12-mediated As(III) uptake and transport in Arabidopsis. By analyzing various combinations of arr and nip mutants, including high-order triple and quadruple mutants, we demonstrated that ARR1 and ARR12 redundantly function as negative regulators of As(III) tolerance by acting upstream of NIP1;1 and NIP6;1 to modulate their function in arsenic accumulation. ChIP-qPCR, EMSA, and transient dual-LUC reporter assays revealed that ARR1 and ARR12 transcriptionally activate the expression of NIP1;1 and NIP6;1 by directly binding to their promoters and upregulating their expression, leading to increased arsenic accumulation under As(III) stress. These findings collectively provide insights into cytokinin signaling-mediated plant adaptation to excessive As(III), contributing to the development of crops with low arsenic accumulation., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

NAR2 (Nitrate assimilation related protein) is a protein chaperone involved in transporting nitrate across membranes. However, the expression pattern and function of NAR2 genes in wheat are still largely unknown. Here, we cloned two TaNAR2 genes (TaNAR2.1 and TaNAR2.2). To assess and compare the functional differences of TaNAR2.1 and TaNAR2.2, we analyzed the subcellular localization and expression pattern of the two genes in wheat under low nitrogen (LN) and high nitrogen (HN) conditions, as well as the nitrate influx and root system architecture of TaNAR2.1 and TaNAR2.2 overexpression wheat under LN and HN. Additionally, we investigated the effects of TaNAR2.1 and TaNAR2.2 overexpression on the growth phenotype, nitrogen uptake and yield of wheat throughout the growth period. There are significant differences in the expression patterns and functions of TaNAR2.1 and TaNAR2.2. TaNAR2.1 is located in the cytoplasm, nucleus and the plasma membrane, whereas TaNAR2.2 is a cytoplasm-specific protein. TaNAR2.1 appears to exhibit larger changes in expression levels and a higher capacity for nitrate influx than TaNAR2.2 under external nitrate supply. Overexpression of TaNAR2.1 significantly improves grain nitrogen use efficiency and increases grain yield, whereas overexpression of TaNAR2.2 enhances vegetative and reproductive growth of wheat roots. These findings indicate that TaNAR2.1 plays a crucial role in wheat nitrogen accumulation and yield, while TaNAR2.2 is pivotal for wheat root growth., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

Nitrogen levels and distribution in the rhizosphere strongly regulate the root architecture. Nitrate is an essential nutrient and an important signaling molecule for plant growth and development. Hydroponic experiments were conducted to investigate the differences in endodermal suberization in tobacco (Nicotiana tabacum L.) roots at three nitrate levels. Nitrogen accumulation was detected in the roots, shoots, and xylem sap. Nitrate influx on the root surface was also measured using the non-invasive self-referencing microsensor technique (SRMT). RNA-Seq analysis was performed to identify the genes related to endodermal suberization, nitrate transport, and endogenous abscisic acid (ABA) biosynthesis. The results showed that root length, root-shoot ratio, nitrate influx on the root surface, and NiA and NRT2.4 genes were regulated to maintain the nitrogen nutrient supply in tobacco under low nitrate conditions. Low nitrate levels enhanced root endodermal suberization and hence reduced the apoplastic transport pathway, and genes from the KCS, FAR, PAS2, and CYP86 families were upregulated. The results of exogenous fluridone, an ABA biosynthesis inhibitor, indicated that suberization of the tobacco root endodermis had no relevance to radial nitrate transport and accumulation. However, ABA enhances suberization, relating to ABA biosynthesis genes in the CCD family and degradation gene ABA8ox1., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

High pH saline-alkali stress, mainly NaHCO 3 , limited the development of animal husbandry in Songnen Plain. Ion imbalance and reactive oxygen species (ROS) metabolism disorder caused by saline-alkali stress inhibited plant growth. In this study, we compared the differences in ion absorption, transport and ROS metabolism between saline-tolerant alfalfa (ZD) and saline-sensitive alfalfa (ZM) under NaHCO 3 stress using physiology and transcripomics techniques. WGCNA analysis identified key genes associated with NaHCO 3 stress-induced changes. NaHCO 3 stress inhibited the absorption of K + and Mg 2+ , but activated Ca 2+ signal. Furthermore, ZD maintained higher K + , Mg 2+ and Ca 2+ contents and the K + /Na + ratio than ZM, this is mainly related to the higher expression of proteins or channel-encoding genes involved in ion absorption and transport in ZD. Antioxidant enzyme systems can be activated in response to NaHCO 3 stress. Peroxidase (EC 1.11.1.6), catalase (EC 1.11.1.7) and glutathione transferase (EC 2.5.1.18) activities were higher in ZD than ZM, and most genes encoding the relevant enzymes also demonstrated a stronger up-regulation trend in ZD. Although NaHCO 3 stress inhibited Trx-Prx pathway, ZD related enzymes and their genes were also inhibited less than ZM. WGCNA results identified many genes involved in ion absorption, transport and antioxidant systems that play an important role in NaHCO 3 stress adaptation. Collectively, ZD has the stronger ion homeostasis regulation and ROS scavenging ability, so it's more resistant to NaHCO 3 . The results provide theoretical guidance for further understanding of the molecular mechanism of NaHCO 3 resistance and provide potential genes for research to improve saline-alkali tolerance in alfalfa., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Actin cytoskeleton and reactive oxygen species are principal determinants of root hair polarity and tip growth. Loss of function in RESPIRATORY BURST OXIDASE HOMOLOG C/ROOT HAIR DEFECTIVE 2 (AtRBOHC/RHD2), an NADPH oxidase emitting superoxide to the apoplast, and in ACTIN 2, a vegetative actin isovariant, in rhd2-1 and der1-3 mutants, respectively, lead to similar defects in root hair formation and elongation Since early endosome-mediated polar localization of AtRBOHC/RHD2 depends on actin cytoskeleton, comparing the proteome-wide consequences of both mutations might be of eminent interest. Therefore, we employed a differential proteomic analysis of Arabidopsis rhd2-1 and der1-3 mutants. Both mutants exhibited substantial alterations in abundances of stress-related proteins. Notably, plasma membrane (PM)-localized PIP aquaporins showed contrasting abundance patterns in the mutants compared to wild-types. Drought-responsive proteins were mostly downregulated in rhd2-1 but upregulated in der1-3. Proteomic data suggest that opposite to der1-3, altered vesicular transport in rhd2-1 mutant likely contributes to the deregulation of PM-localized proteins, including PIPs. Moreover, lattice light sheet microscopy revealed reduced actin dynamics in rhd2-1 roots, a finding contrasting with previous reports on der1-3 mutant. Phenotypic experiments demonstrated a drought stress susceptibility in rhd2-1 and resistance in der1-3. Thus, mutations in AtRBOHC/RHD2 and ACTIN2 cause similar root hair defects, but they differently affect the actin cytoskeleton and vesicular transport. Reduced actin dynamics in rhd2-1 mutant is accompanied by alteration of vesicular transport proteins abundance, likely leading to altered protein delivery to PM, including aquaporins, thereby significantly affecting drought stress responses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Aluminium (Al) stress is the second-leading abiotic stress on crops. An improved understanding of the response mechanisms of plants to Al stress will provide scientific guidance for enhancing the crops' tolerance to Al stress. In this study, Al stress (50-200 μM AlCl 3 ) caused visible damage to broad bean (Vicia faba L.) roots rather than shoots, which was attributed to Al accumulation and distribution in different tissues. Root transcriptomic analysis revealed that Al stress altered cell wall properties by downregulating lignin synthesis and several xyloglucan endotransglucosylase/hydrolase-, expansin- and peroxidase (POD)-encoding genes, which likely weakened cell extensibility to inhibit root growth. Additionally, Al stress impeded reactive oxygen species scavenging pathways involving POD activity and flavonoid biosynthesis, leading to oxidative damage characterised by malondialdehyde accumulation. These results indicate that optimising cell wall properties and/or enhancing antioxidant processes are crucial for alleviating Al toxicity to broad beans. Interestingly, exogenous application (500 and 1000 μM) of the flavonoid apigenin effectively alleviated Al toxicity in broad bean roots by partially improving the total antioxidant capacity of the roots. This study contributes to understanding the interaction between plants and Al and provides new strategies to alleviate Al toxicity in crops., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Plant root development depends on signaling pathways responding to external and internal signals. In this study we demonstrate the involvement of the Lotus japonicus LjNPF4.6 gene in the ABA and nitrate root responding pathways. LjNPF4.6 expression in roots is induced by external application of both nitrate and ABA. LjNPF4.6 promoter activity is spatially localized in epidermal cell layer and vascular bundle structures with the latter pattern being controlled by externally applied ABA. LjNPF4.6 cRNA injection achieves both nitrate and ABA uptake in Xenopus laevis oocytes and the analyses of L. japonicus knock-out insertion mutants confirmed the role played by LjNPF4.6 in root nitrate uptake. The phenotypic characterization of the Ljnpf4.6 plants indicates the role played by LjNPF4.6 in the root program development in response to exogenously applied nitrate and ABA. Based on the presented data, the mode of action of this transporter is discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

Appropriate abiotic stress response is pivotal for plant survival and makes use of multiple signaling molecules and phytohormones to achieve specific and fast molecular adjustments. A multitude of studies has highlighted the role of alternative splicing in response to abiotic stress, including temperature, emphasizing the role of transcriptional regulation for stress response. Here we investigated the role of the core-splicing factor PORCUPINE (PCP) on temperature-dependent root development. We used marker lines and transcriptomic analyses to study the expression profiles of meristematic regulators and mitotic markers, and chemical treatments, as well as root hormone profiling to assess the effect of auxin signaling. The loss of PCP significantly alters RAM architecture in a temperature-dependent manner. Our results indicate that PCP modulates the expression of central meristematic regulators and is required to maintain appropriate levels of auxin in the RAM. We conclude that alternative pre-mRNA splicing is sensitive to moderate temperature fluctuations and contributes to root meristem maintenance, possibly through the regulation of phytohormone homeostasis and meristematic activity., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Plant root system is significantly influenced by high soil levels of ammonium nitrogen, leading to reduced root elongation and enhanced lateral root branching. In Arabidopsis, these processes have been reported to be mediated by phytohormones and their downstream signaling pathways, while the controlling mechanisms remain elusive in crops. Through a transcriptome analysis of roots subjected to high/low ammonium treatments, we identified a cytokinin oxidase/dehydrogenase encoding gene, CKX3, whose expression is induced by high ammonium. Knocking out CKX3 and its homologue CKX8 results in shorter seminal roots, fewer lateral roots, and reduced sensitivity to high ammonium. Endogenous cytokinin levels are elevated by high ammonium or in ckx3 mutants. Cytokinin application results in shorter seminal roots and fewer lateral roots in wild-type, mimicking the root responses of ckx3 mutants to high ammonium. Furthermore, CKX3 is transcriptionally activated by type-B RR25 and RR26, and ckx3 mutants have reduced auxin content and signaling in roots under low ammonium. This study identified RR25/26-CKX3-cytokinin as a signal module that mediates root responses to external ammonium by modulating of auxin signaling in the root meristem and lateral root primordium. This highlights the critical role of cytokinin metabolism in regulating rice root development in response to ammonium., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Node Inception (NIN) plays a crucial role in legume symbiosis by participating in both infection and nodule formation processes. However, its specific function in mungbean (Vigna radiata) remains poorly understood. This study aimed to functionally characterize the VrNIN1 gene in mungbean through an enhanced hairy root transformation approach. Examination of proVrNIN1: GUS hairy roots via GUS staining indicated the expression of VrNIN1 in later root promodia, nodule primordia, and nodules. Phenotypic evaluation revealed that overexpression or silencing of VrNIN1 led to a significant reduction in nodule numbers in hairy roots compared to controls. Additionally, interaction between VrNIN1 and VrNNC1 was confirmed through yeast two-hybrid, luciferase complementation and Co-immunoprecipitation assays. VrNNC1 expression was observed in the vascular bundle and cortex of roots and root nodules, where it notably suppressed nodule formation in transgenic hairy roots. Furthermore, gene expression analysis demonstrated the involvement of VrNIN1 and VrNNC1 in regulating root nodulation by modulating the expression of VrRIC1 and VrEDOD40. This study not only optimized the genetic transformation system for hairy roots in mungbean, but also provided mechanistic insights into the regulatory role of VrNIN1 in root nodule symbiosis in mungbean., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Arabidopsis thaliana synthesizes various medicinal compounds, and serves as a model plant for medicinal plant research. Single-cell transcriptomics technologies are essential for understanding the developmental trajectory of plant roots, facilitating the analysis of synthesis and accumulation patterns of medicinal compounds in different cell subpopulations. Although methods for interpreting single-cell transcriptomics data are rapidly advancing in Arabidopsis, challenges remain in precisely annotating cell identity due to the lack of marker genes for certain cell types. In this work, we trained a machine learning system, AtML, using sequencing datasets from six cell subpopulations, comprising a total of 6000 cells, to predict Arabidopsis root cell stages and identify biomarkers through complete model interpretability. Performance testing using an external dataset revealed that AtML achieved 96.50% accuracy and 96.51% recall. Through the interpretability provided by AtML, our model identified 160 important marker genes, contributing to the understanding of cell type annotations. In conclusion, we trained AtML to efficiently identify Arabidopsis root cell stages, providing a new tool for elucidating the mechanisms of medicinal compound accumulation in Arabidopsis roots., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The formation and development of storage roots is the most important physiological process in sweetpotato production. Sucrose transporters (SUTs) regulate sucrose transport from source to sink organs and play important roles in growth and development of plants. However, whether SUTs involved in sweetpotato storage roots formation is so far unknown. In this study, we show that IbSUT1, a SUT, is localized to the plasma membrane. Overexpression of IbSUT1 in sweetpotato promotes the sucrose efflux rate from leaves, leading to increased sucrose levels in roots, thus induces lignin deposition in the stele, which inhibits the storage roots formation and compromises the yield. Heterologous expression of IbSUT1 in Arabidopsis significantly increases the sucrose accumulation and promotes lignification in the inflorescence stems. RNA-seq and biochemical analysis further demonstrated that IbMYB1 negatively regulates the expression of IbSUT1. Overexpression of IbMYB1 in Arabidopsis reduces the sucrose accumulation and lignification degree in the inflorescence stems. Moreover, co-overexpression of IbMYB1 and IbSUT1 restores the phenotype of lignin over-deposition in Arabidopsis. Collectively, our results reveal that IbSUT1 regulates source-sink sucrose transport and participates in the formation of sweetpotato storage roots and highlight the potential application of IbSUT1 in improving sweetpotato yield in the future., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Isoxaben is a pre-emergent herbicide used to control broadleaf weeds. While the phytotoxic mechanism is not completely understood, isoxaben interferes with cellulose synthesis. Certain mutations in cellulose synthase complex proteins can confer isoxaben tolerance; however, these mutations can cause compromised cellulose synthesis and perturbed plant growth, rendering them unsuitable as herbicide tolerance traits. We conducted a genetic screen to identify new genes associated with isoxaben tolerance by screening a selection of Arabidopsis thaliana T-DNA mutants. We found that mutations in a FERREDOXIN-NADP(+) OXIDOREDUCTASE-LIKE (FNRL) gene enhanced tolerance to isoxaben, exhibited as a reduction in primary root stunting, reactive oxygen species accumulation and ectopic lignification. The fnrl mutant did not exhibit a reduction in cellulose levels following exposure to isoxaben, indicating that FNRL operates upstream of isoxaben-induced cellulose inhibition. In line with these results, transcriptomic analysis revealed a highly reduced response to isoxaben treatment in fnrl mutant roots. The fnrl mutants displayed constitutively induced mitochondrial retrograde signalling, and the observed isoxaben tolerance is partially dependent on the transcription factor ANAC017, a key regulator of mitochondrial retrograde signalling. Moreover, FNRL is highly conserved across all plant lineages, implying conservation of its function. Notably, fnrl mutants did not show a growth penalty in shoots, making FNRL a promising target for biotechnological applications in breeding isoxaben tolerance in crops., (© 2024 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Nitrate is a nutrient and signal that regulates gene expression. The nitrate response has been extensively characterized at the organism, organ, and cell-type-specific levels, but intracellular mRNA dynamics remain unexplored. To characterize nuclear and cytoplasmic transcriptome dynamics in response to nitrate, we performed a time-course expression analysis after nitrate treatment in isolated nuclei, cytoplasm, and whole roots. We identified 402 differentially localized transcripts (DLTs) in response to nitrate treatment. Induced DLT genes showed rapid and transient recruitment of the RNA polymerase II, together with an increase in the mRNA turnover rates. DLTs code for genes involved in metabolic processes, localization, and response to stimulus indicating DLTs include genes with relevant functions for the nitrate response that have not been previously identified. Using single-molecule RNA FISH, we observed early nuclear accumulation of the NITRATE REDUCTASE 1 (NIA1) transcripts in their transcription sites. We found that transcription of NIA1, a gene showing delayed cytoplasmic accumulation, is rapidly and transiently activated; however, its transcripts become unstable when they reach the cytoplasm. Our study reveals the dynamic localization of mRNAs between the nucleus and cytoplasm as an emerging feature in the temporal control of gene expression in response to nitrate treatment in Arabidopsis roots., (© 2024 John Wiley & Sons Ltd.)

To understand whether domestication had an impact on susceptibility and responsiveness to arbuscular mycorrhizal fungi (AMF) in tomato (Solanum lycopersicum), we investigated two tomato cultivars ("M82" and "Moneymaker") and a panel of wild relatives including S. neorickii, S. habrochaites and S. pennellii encompassing the whole Lycopersicon clade. Most genotypes revealed good AM colonisation levels when inoculated with the AMF Funneliformis mosseae. By contrast, both S. pennellii accessions analysed showed a very low colonisation, but with normal arbuscule morphology, and a negative response in terms of root and shoot biomass. This behaviour was independent of fungal identity and environmental conditions. Genomic and transcriptomic analyses revealed in S. pennellii the lack of genes identified within QTLs for AM colonisation, a limited transcriptional reprogramming upon mycorrhization and a differential regulation of strigolactones and AM-related genes compared to tomato. Donor plants experiments indicated that the AMF could represent a cost for S. pennellii: F. mosseae could extensively colonise the root only when it was part of a mycorrhizal network, but a higher mycorrhization led to a higher inhibition of plant growth. These results suggest that genetics and functional traits of S. pennellii are responsible for the limited extent of AMF colonisation., (© 2024 The Author(s). Plant, Cell & Environment published by John Wiley & Sons Ltd.)

The domestication process progressively differentiated wild relatives from modern cultivars, thus impacting plant-associated microorganisms. Endophytic bacterial communities play vital roles in plant growth, development, and health, which contribute to the crop's sustainable development. However, how plant domestication impacts endophytic bacterial communities and relevant root exudates in wheat remains unclear. First, we have observed that the domestication process increased the root endophytic microbial community diversity of wheat while decreasing functional diversity. Second, domestication decreased the endophytic bacterial co-occurrence network stability, and it did significantly alter the abundances of core microorganisms or potential probiotics. Third, untargeted LC-MS metabolomics revealed that domestication significantly altered the metabolite profiles, and the abundances of various root exudates released were significantly correlated with keystone taxa including the Chryseobacterium, Massilia, and Lechevalieria. Moreover, we found that root exudates, especially L-tyrosine promote the growth of plant-beneficial bacteria, such as Chryseobacterium. Additionally, with L-tyrosine and Chryseobacterium colonized in the roots, the growth of wild wheat's roots was significantly promoted, while no notable effect could be found in the domesticated cultivars. Overall, this study suggested that wild wheat as a key germplasm material, and its native endophytic microbes may serve as a resource for engineering crop microbiomes to improve the morphological and physiological traits of crops in widely distributed poor soils., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

The Arabidopsis oligopeptide transporter AtOPT6 is reportedly involved in the long-distance transport of thiol compounds into sink organs. In the present study, transgenic Arabidopsis lines overexpressing AtOPT6 under the control of a phloem-specific promoter, sucrose-proton symporter 2 (pSUC2), were analyzed for thiol and cadmium (Cd) distribution during the reproductive stage, both with and without Cd exposure. Phloem specific AtOPT6-overexpressing lines did not exhibit an evident impact on bolting time. In the absence of Cd exposure, these transgenic lines showed significantly enhanced transport of endogenous glutathione into siliques, accompanied by a reduction in the glutathione content of flowers and roots during the reproductive stage. Additionally, exposure of the roots of the phloem specific AtOPT6-overexpressing lines to Cd altered the distribution of thiol compounds, resulting in an increase in the content of phytochelatins in sink organs, contributing to a significant elevation of Cd contents in reproductive sink. Our findings confirm the crucial role of AtOPT6 in unloading phytochelatin-Cd conjugates from the phloem into sink organ., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Transformation of plants using wild strains of agrobacteria is termed natural transformation and is not covered by GMO legislation in e.g. European Union and Japan. In the current study, offspring lines (A11 and B3) of Rhizobium rhizogenes naturally transformed oilseed rape (Brassica napus) were randomly selected to characterize the morphological traits, and analyze the implications of such morphological changes on plant drought resilience. It was found that the introduction of Ri-genes altered the biomass partitioning to above- and under-ground parts of oilseed rape plants. Compared to the wild type (WT), the A11 and B3 lines exhibited 1.2-4.0 folds lower leaf and stem dry weight, leaf area and plant height, but had 1.3-5.8 folds greater root dry weight, root length and root surface area, resulting in a significantly enhanced root: shoot dry mass ratio and root surface area: leaf area ratio. In addition, the introduction of Ri-genes conferred reduced stomatal pore aperture and increased stomatal density in the B3 line, and increased leaf thickness in A11 line, which could benefit plant drought resilience. Finally, the modulations in morphological traits as a consequence of transformation with Ri-genes are discussed concerning resilience in water-limited conditions. These findings reveal the potential of natural transformation with R. rhizogenes for drought-targeted breeding in crops., Competing Interests: Declaration of Competing Interest No conflict of interest declared, (Copyright © 2024 Elsevier B.V. All rights reserved.)

Chromosome numbers and morphology are important characteristics of a species and its evolution. Root tips are the most commonly used tissue as a source of actively dividing cells for chromosome visualization in plants. Previously, rapidly growing root tips were collected from germinating kernels or from seedlings growing in pots or fields. However, the use of adventitious roots (ARs) derived from aerial tissue as meristems for chromosome visualization has always been overlooked. Here, we successfully induced ARs in 12 materials that were investigated, with the exception of Sorghum nitidum . Using ARs meristem we obtained high-quality chromosome spreads for Morus alba , Broussonetia papyrifera , Lolium multiflorum , Sorghum sudanense , S . propinquum , S . bicolor × S . sudanense , Zea mays , Z . mexicana , Glycine max , Medicago sativa , and Brassica napus . The results reported here demonstrate that layering is an alternative and effective method for producing meristematic cells for high-quality chromosome preparation in plant species producing ARs. For species that produce ARs by layering, this protocol is particularly valuable for the development of cost-effective and high-throughput non-invasive cytogenetic studies.

Sweet potato ( Ipomoea batatas ) is an important food crop that plays a pivotal role in preserving worldwide food security. Due to its polyploid genome, high heterogeneity, and phenotypic plasticity, sweet potato genetic characterization and breeding is challenging. Genome-wide association studies (GWASs) can provide important resources for breeders to improve breeding efficiency and effectiveness. GWASpoly was used to identify 28 single nucleotide polymorphisms (SNPs), comprising 21 unique genetic loci, associated with sweet potato storage root traits including dry matter (4 loci), subjective flesh color (5 loci), flesh hue angle (3 loci), and subjective skin color and skin hue angle (9 loci), in 384 accessions from the USDA sweet potato germplasm collection. The I. batatas 'Beauregard' and I. trifida reference genomes were utilized to identify candidate genes located within 100 kb from the SNPs that may affect the storage traits of dry matter, flesh color, and skin color. These candidate genes include transcription factors (especially Myb, bHLH, and WRKY family members), metabolite transporters, and metabolic enzymes and associated proteins involved in starch, carotenoid, and anthocyanin synthesis. A greater understanding of the genetic loci underlying sweet potato storage root traits will enable marker-assisted breeding of new varieties with desired traits. This study not only reinforces previous research findings on genes associated with dry matter and β-carotene content but also introduces novel genetic loci linked to these traits as well as other root characteristics.

Clubroot disease caused by the infection of Plasmodiophora brassicae is widespread in China, and significantly reduces the yield of Chinese cabbage (Brassica rapa L. ssp. pekinensis). However, the resistance mechanism of Chinese cabbage against clubroot disease is still unclear. It is important to exploit the key genes that response to early infection of P. brassicae. In this study, it was found that zoospores were firstly invaded hair roots on the 8th day after inoculating with 1 × 10 7 spores/mL P. brassicae. Transcriptome analysis found that the early interaction between Chinese cabbage and P. brassicae caused the significant expression change of some defense genes, such as NBS-LRRs and pathogenesis-related genes, etc. The above results were verified by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Otherwise, peroxidase (POD) salicylic acid (SA) and jasmonic acid (JA) were also found to be important signal molecules in the resistance to clubroot disease in Chinese cabbage. This study provides important clues for understanding the resistance mechanism of Chinese cabbage against clubroot disease., (© 2024. The Author(s).)

During development, most cells experience a progressive restriction of fate that ultimately results in a fully differentiated mature state. Understanding more about the gene expression patterns that underlie developmental programs can inform engineering efforts for new or optimized forms. Here, we present a four-state integrase-based recorder of gene expression history and demonstrate its use in tracking gene expression events in Arabidopsis thaliana in two developmental contexts: lateral root initiation and stomatal differentiation. The recorder uses two serine integrases to mediate sequential DNA recombination events, resulting in step-wise, history-dependent switching between expression of fluorescent reporters. By using promoters that express at different times along each of the two differentiation pathways to drive integrase expression, we tie fluorescent status to an ordered progression of gene expression along the developmental trajectory. In one snapshot of a mature tissue, our recorder is able to reveal past gene expression with single cell resolution. In this way, we are able to capture heterogeneity in stomatal development, confirming the existence of two alternate paths of differentiation., (© 2024. The Author(s).)

Sugarcane (Saccharum spp.) growth is regulated by intricate gene networks and hormone secretions, positively correlating with sugarcane yield. There is a rising interest in exploring how the candidate genes found in sugarcane respond to plant growth. In this study, we simulated a typical growth environment to obtain accurate phenotypic data and screened for potential genes associated with plant growth through transcriptomics. Compared to Saccharum GuiTang 42, the other variety Saccharum GuiTang 44 exhibited earlier germination, a higher emergence rate, thicker pseudostems, taller plants, and a more extensive root system. The middle buds formed the greatest number of roots, followed by the lower and upper buds. Indole-3-acetic acid (IAA) and jasmonic acid effectively promoted bud development, while abscisic acid and trans-zeatin exhibited negative correlations with sugarcane bud growth. Transcriptome data from the upper, middle, and lower buds revealed 24,158 differentially expressed genes in all three comparisons, with MAPK signaling emerging as a critical pathway. The photosynthesis-antenna protein pathway is vital for middle and lower bud development during root germination. Lastly, key gene modules related to differences in hormone content between the two varieties were defined through weighted correlation network analysis and identified. The module significantly associated with IAA was enriched in pathways such as Proteasome and Protein processing in the endoplasmic reticulum, and the upregulation of key genes involved in this gene module had a highly significant positive correlation with bud outgrowth combined with IAA secretion. In conclusion, we have elucidated the pathways of hormones during sugarcane growth and the interactions between IAA and critical genes. These in-depth findings may guide modern sugarcane breeding., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Key Message: Based on transport inhibition and genome-wide analysis, 123 ABC transporters of Euphorbia lathyris were identified, and it was found that the PDR family members ElABCG39 mediated ingenol efflux. Identification of ingenol biosynthetic enzymes and transporters in plant is fundamental to realize its biosynthesis in chassis cells. At present, several key enzymes of the ingenol biosynthesis pathway have been identified, while the mechanisms governing the accumulation or transport of ingenol to distinct plant tissue compartments remain elusive. In this study, transport inhibition analyses were performed, along with genome-wide identification of 123 genes encoding ABC proteins in Euphorbia lathyris L., eventually discovering that a PDR transporter ElABCG39 mediates ingenol transmembrane transport and is localized on the plasma membrane. Expression of this protein in yeast AD1-8 promoted the transmembrane efflux of ingenol with strong substrate specificity. Furthermore, in ElABCG39 RNAi transgenic hairy roots, ingenol transmembrane efflux was significantly reduced and hairy root growth was inhibited. The discovery of the first Euphorbia macrocyclic diterpene transporter ElABCG39 has not only further improved the ingenane diterpenoid biosynthesis regulatory network, but also provided a new key element for ingenol production in chassis cells., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)