Peng Zhou

The phytohormone abscisic acid (ABA) is rapidly induced in roots exposed to aluminium (Al) stress, yet the mechanisms by which it modulates Al‐detoxification genes remain elusive. Here we uncover a hierarchical ABA‐responsive regulatory network that orchestrates a Late Embryogenesis Abundant (LEA)_2 protein MsLEA2, which modulates Al tolerance in alfalfa ( Medicago sativa L.). Using yeast one‐hybrid (Y1H) assays, luciferase complementation imaging (LCI), mutagenesis, and electrophoretic mobility shift assays (EMSA), we show that three antagonistic bZIP transcription factors—MsABF2 (activator), MsDPBF3 (hyper‐activator) and MsGBF1 (repressor)—dynamically regulate MsLEA2 expression through spatially distinct ABRE motifs, enabling precise transcriptional control. Functional analyses reveal that MsLEA2 acts as a multifunctional hub, that promotes (i) oxalate exudation via interactions with aquaporins (MsPIP2;1, MsPIP2;2, MsTIP1;1), (ii) reactive oxygen species (ROS) scavenging, and (iii) cell wall remodelling through binding to UDP‐xylose synthase (MsUXS1). Golgi‐targeted MsLEA2 facilitates apoplastic Al exclusion, a mechanism distinct from its cytoplasmic homologue AtLEA14, underscoring the importance of subcellular compartmentalisation in Al detoxification. Our findings establish LEA_2 proteins as pivotal components of secretory‐pathway‐mediated Al tolerance and provide actionable targets for engineering crops resilient to acidic soils.

Alfalfa (Medicago sativa L.) is a globally important forage crop. Identification of enhancer cis-regulatory elements (CREs) in high-expression gene promoters is essential for precise gene regulation and molecular breeding. This study aimed to systematically screen and functionally validate enhancer CREs in alfalfa. Twenty constitutively highly expressed genes (coefficient of variation < 0.2) were identified from leaf, stem, and root tissues of 15-day-old alfalfa seedlings (nine RNA-seq libraries, n = 3 per tissue). Promoter regions (2,000 bp upstream to 500 bp downstream of the transcription start site) were extracted and analyzed using PlantCARE, FIMO, and Fisher's exact test (adjusted p < 0.05). Ten candidate enhancer CREs (M1–M10) were identified, and an 89-bp fragment containing a CAAT box from the MsEF1-α promoter, named M11, was selected for functional validation. Recombinant promoter-LUC vectors containing M1, M2, M7, and M11 were constructed and transiently expressed in Nicotiana benthamiana. Luciferase (LUC) activity was quantified to assess enhancer function. Deletion of M11 from the MsEF1-α promoter reduced LUC activity by approximately 55%, whereas insertion of M11 into the MsLEA2 promoter increased activity by approximately 183%. M1 and M7 exhibited no significant enhancer activity, while M2 showed unstable enhancing effects. M11 is a robust and promoter-independent enhancer CRE in alfalfa, providing a valuable tool for gene regulation and molecular breeding.

Root zone restriction (RZR) technology optimizes plant growth and quality. However, the fleshy root system of Paeonia ostii exhibits sensitivity to spatial constraints, and research on the plasticity of its root architecture and adaptation mechanisms remains inadequate. This study provides a functional analysis of biomass allocation and root architectural responses to the root-zone restriction (RZR) in P. ostii, comparing three container volumes (8.5, 17, and 34 L). While the total biomass increased with root zone volume (e.g., shoot biomass rose from 9.30 g to 59.94 g), RZR induced a 44.8% increase in root-to-shoot ratio, indicating carbon reallocation to enhance belowground resource acquisition. The principal component analysis identified root biomass, volume, and surface area as key plasticity drivers. Optimal root efficiency occurred at 26.09–28.23 L, where root length and tip/fork numbers peaked. Mechanistically, RZR elevated superoxide dismutase (SOD) activity by 49.74% but reduced catalase (CAT) by 74.24%, disrupting H₂O₂ homeostasis. Concurrently, auxin transporter genes (PIN1, AUX1) were upregulated, promoting root elongation and lateral branching through auxin redistribution. We hypothesize that ROS–auxin crosstalk mediates architectural reconfiguration to mitigate spatial stress, with thickened roots enhancing structural stability in restricted environments. The study underscores the need to optimize root zone volume in woody species cultivation, providing thresholds (e.g., >28 L for mature plants) to balance biomass yield and physiological costs in horticultural management.

Shoot branching is an important biological trait affecting alfalfa (Medicago sativa L.) production, but its development is complicated and the mechanism is not fully clear. In the present study, pectin acetylesterase 12 (MsPAE12) and NAM/ATAF/CUC-domain transcription factor gene (MsNAC73) were isolated from alfalfa. MsPAE12 was highly expressed in shoot apexes, and MsNAC73 was found to be a key transcriptional repressor of MsPAE12 by directly binding to salicylic acid (SA) and jasmonic acid (JA) elements in the MsPAE12 promoter. The biological functions of MsPAE12 and MsNAC73 were studied through overexpression (OE) and down-expression (RNAi) of the 2 genes in alfalfa. The numbers of shoot branches increased in MsPAE12-OE lines but decreased in MsPAE12-RNAi and MsNAC73-OE plants, which was negatively related to their indole-3-acetic acid (IAA) accumulation in shoot apexes. Furthermore, the contents of acetic acid (AA) in shoot apexes decreased in MsPAE12-OE plants but increased in MsPAE12-RNAi and MsNAC73-OE plants. The changes of AA contents were positively related to the expression of TRYPTOPHAN AMINOTRANSFERASE 1 (MsTAA1), TRYPTOPHAN AMINOTRANSFERASE-RELATED 2 (MsTAR2), and YUCCA flavin monooxygenase (MsYUCC4) and the contents of tryptophan (Trp), indole-3-pyruvic acid (IPA), and IAA in shoot apexes of MsPAE12-OE, MsPAE12-RNAi, and MsNAC73-OE plants. Exogenous application of AA to wild type (WT) and MsPAE12-OE plants increased Trp, IPA, and IAA contents and decreased branch number. Exogenous IAA suppressed shoot branching in MsPAE12-OE plants, but exogenous IAA inhibitors increased shoot branching in MsPAE12-RNAi plants. These results indicate that the MsNAC73-MsPAE12 module regulates auxin-modulated shoot branching via affecting AA accumulation in shoot apexes of alfalfa.

The toxicity of aluminum (Al) in acidic soils poses a significant limitation to crop productivity. In this study, we found a notable increase in DnaJ (HSP40) expression in the roots of Al-tolerant alfalfa (WL-525HQ), which we named MsDjB4. Transient conversion assays of tobacco leaf epidermal cells showed that MsDjB4 was targeted to the membrane system including Endoplasmic Reticulum (ER), Golgi, and plasma membrane. We overexpressed (MsDjB4-OE) and suppressed (MsDjB4-RNAi) MsDjB4 in alfalfa hairy roots and found that MsDjB4-OE lines exhibited significantly better tolerance to Al stress compared to wild-type and RNAi hairy roots. Specifically, MsDjB4-OE lines had longer root length, more lateral roots, and lower Al content compared to wild-type and RNAi lines. Furthermore, MsDjB4-OE lines showed lower levels of lipid peroxidation and ROS, as well as higher activity of antioxidant enzymes SOD, CAT, and POD compared to wild-type and RNAi lines under Al stress. Moreover, MsDjB4-OE lines had higher soluble protein content compared to wild-type and RNAi lines after Al treatment. These findings provide evidence that MsDjB4 contributes to the improved tolerance of alfalfa to Al stress by facilitating protein synthesis and enhancing antioxidant capacity.

Editorial: The role of stress proteins in plants under abiotic stress

Late embryonic development abundant proteins (LEAs) are a large family of proteins commonly existing in plants. LEA_2 is the largest subfamily in the LEA, it plays an important role in plant resistance to abiotic stress. In order to explore the characteristics of LEA_2 gene family members in alfalfa (Medicago sativa L.), 155 members of LEA_2 (MsLEA_2) family were identified from alfalfa genome. Bioinformatics analysis was conducted from the aspects of phylogenetic relationship, chromosome distribution, chromosome colinearity, physical and chemical properties, motif composition, exon-intron structure, cis-element and so on. Expression profiles of MsLEA_2 gene were obtained based on Real-time fluorescent quantitative PCR (qRT-PCR) analysis and previous RNA-seq data under aluminum (Al) stress. Bioinformatics results were shown that the MsLEA_2 genes are distributed on all 32 chromosomes. Among them, 85 genes were present in the gene clusters, accounting for 54.83%, and chromosome Chr7.3 carries the largest number of MsLEA_2 (19 LEA_2 genes on Chr7.3). Chr7.3 has a unique structure of MsLEA_2 distribution, which reveals a possible special role of Chr7.3 in ensuring the function of MsLEA_2. Transcriptional structure analysis revealed that the number of exons in each gene varies from 1 to 3, and introns varies from 0 to 2. Cis-element analysis identified that the promoter region of MsLEA_2 is rich in ABRE, MBS, LTR, and MeJARE, indicating MsLEA_2 has stress resistance potential under abiotic stress. RNA-seq data and qRT-PCR analyses showed that most of the MsLEA_2 members were up-regulated when alfalfa exposed to Al stress. This study revealed that phylogenetic relationship and possible function of LEA_ 2 gene in alfalfa, which were helpful for the functional analysis of LEA_ 2 proteins in the future and provided a new theoretical basis for improving Al tolerance of alfalfa.