Late Embryogenesis Abundant Proteins Research Articles

Saussurea involucrata SiLEA5 Enhances Tolerance to Drought Stress in Solanum lycopersicum

Drought adversely affects plant growth, which leads to reduced crop yields and exacerbates food insecurity. Late embryogenesis abundant (LEA) proteins are crucial for plants’ responses to abiotic stresses. This research further investigates the role of SiLEA5 by utilizing transgenic tomatoes under drought stress. The expression of SiLEA5 was upregulated under drought and abscisic acid (ABA) treatment, resulting in decreased electrolyte leakage and malondialdehyde content, alongside increased levels of osmotic regulators and antioxidant enzyme activity. These biochemical alterations reduce oxidative damage and enhance drought resistance. qRT-PCR analysis revealed the upregulation of ABA signaling genes and key enzymes involved in proline biosynthesis (P5CS) and dehydrin (DHN) synthesis under drought stress. Additionally, overexpression of SiLEA5 increased the net photosynthetic rate (Pn) and fruit yield of tomatoes by regulating stomatal density and aperture. These findings suggest that SiLEA5 may be a potential target for improving drought tolerance in tomatoes and other crops.

Comprehensive identification of LEA protein family genes and functional analysis of MdLEA60 involved in abiotic stress responses in apple (Malus domestica).

Late embryogenesis abundant (LEA) proteins are important proteins that exists widely in many plants and contribute to physiological processes of plant stress resistance. Despite LEA proteins being identified in many plants, none have been reported in apple (Malus domestica) until this study. In this study, a total of 87 MdLEA proteins were identified in apple, and a comprehensive analysis was conducted to elucidate the functions of MdLEA proteins in response to abiotic stress. Results showed that they were classified into 7 groups and distributed on 16 chromosomes. Collineation analysis revealed that segmental duplication primarily drove the expansion of MdLEA genes. The MdLEA promoters were enriched with elements associated with various stress responses. Through transcriptome and qRT-PCR analysis, several MdLEA genes related to drought/salinity/cold were excavated, and MdLEA60 was selected for transgenic validation. The ectopic expression of MdLEA60 enhanced osmotic and extreme temperature tolerance in both prokaryotic and eukaryotic cells, providing stress resistance support via antioxidant protection. Overall, the comprehensive analyses and identification not only establish a basis for future investigation into the functional mechanism of MdLEA proteins but also provide potential candidate genes for apple resistance breeding optimization.

Identification of Picea mongolica LEA Gene Family Implicates PmLEA25 in Drought Resistance

Picea mongolica is a rare and valuable tree species in China, having high tolerance for drought, cold, and sand burial. The late embryogenesis abundant protein (LEA protein) is a crucial transcription factor that plays a key role in both plant embryonic development and stress response. LEA genes have, however, not yet been reported in P. mongolica. In this study, through the analysis of genome data from Picea abies and transcriptome data from P. mongolica, a total of 49 PmLEAs were discovered and categorized into eight subfamilies based on their Pfam domain and phylogenetic relationship. RNA-Seq research revealed that 37 PmLEAs were differentially expressed at various stages of embryonic development. Using qRT-PCR, we found that most PmLEAs responded strongly to drought stress, with genes in the same subfamily exhibiting identical expression patterns. In particular, PmLEA25 is the most highly induced by drought treatment. Furthermore, we heterologously transformed PmLEA25 into Arabidopsis. The overexpression of PmLEA25 remarkably increased the germination rate, root length, and antioxidant capacity in Arabidopsis under drought treatment, compared with WT. The results serve as a point of reference for gaining a deeper comprehension of the function of PmLEA25 in the molecular process of stress resistance in P. mongolica. Additionally, they offer significant genetic materials for the purpose of breeding stress-resistant spruce species.

Self-association and multimer formation in AtLEA4-5, a desiccation-induced intrinsically disordered protein from plants.

During seed maturation, plants may experience severe desiccation, leading to the accumulation of late embryogenesis abundant (LEA) proteins. These intrinsically disordered proteins also accumulate in plant tissues under water deficit. Functional roles of LEA proteins have been proposed based on in vitro studies, where monomers are considered as the functional units. However, the potential formation of homo-oligomers has been little explored. In this work, we investigated the potential self-association of Arabidopsis thaliana group 4 LEA proteins (AtLEA4) using in vitro and in vivo approaches. LEA4 proteins represent a compelling case of study due to their high conservation throughout the plant kingdom. This protein family is characterized by a conserved N-terminal region, with a high alpha-helix propensity and invitro protective activity, as compared to the highly disordered and low-conserved C-terminal region. Our findings revealed that full-length AtLEA4 proteins oligomerize and that both terminal regions are sufficient for self-association in vitro. However, the ability of both amino and carboxy regions of AtLEA4-5 to self-associate invivo is significantly lower than that of the entire protein. Using high-resolution and quantitative fluorescence microscopy, we were able to disclose the unreported ability of LEA proteins to form high-order oligomers in planta. Additionally, we found that high-order complexes require the simultaneous engagement of both terminal regions, indicating that the entire protein is needed to attain such structural organization. This research provides valuable insights into the self-association of LEA proteins in plants and emphasizes the role of protein oligomer formation.

Dehydrin Client Proteins Identified Using Phage Display Affinity Selected Libraries Processed With Paired-End Phage Sequencing

The LATE EMBRYOGENESIS ABUNDANT PROTEINs (LEAPs) are a class of noncatalytic, intrinsically disordered proteins with a malleable structure. Some LEAPs exhibit a protein and/or membrane binding capacity and LEAP binding to various targets has been positively correlated with abiotic stress tolerance. Regarding the LEAPs' presumptive role in protein protection, identifying client proteins (CtPs) to which LEAPs bind is one practicable means of revealing the mechanism by which they exert their function. To this end, we used phage display affinity selection to screen libraries derived from Arabidopsis thaliana seed mRNA with recombinant orthologous LEAPs from Arabidopsis and soybean (Glycine max). Subsequent high throughput sequencing of DNA from affinity-purified phage was performed to characterize the entire sub-population of phage retained by each LEAP orthologue. This entailed cataloging in-frame fusions, elimination of false positives, and aligning the hits on the CtP scaffold to reveal domains of respective CtPs that bound to orthologous LEAPs. This approach (Paired-end PhAge Sequencing, or PEPA-Seq) revealed a subpopulation of the proteome constituting the CtP repertoire in common between the two DHNs orthologues (LEA14 and GmPm12) compared to BSA (unrelated binding control). The veracity of LEAP:CtP binding for one of the CtPs (LEA14 and GmPM12 self-association) was independently assessed using temperature related intensity change (TRIC) analysis. Moreover, LEAP:CtP interactions for four other CtPs were confirmed in planta using bimolecular fluorescence complementation (BiFC) assays. The results provide insights into the involvement of the DHN Y-segments and K-domains in protein binding.

Comparative analysis of the transcriptomes from regenerated plants and root explants of endangered Oplopanax elatus.

Oplopanax elatus is a plant of therapeutic significance in oriental medicine; however, its mass cultivation is limited owing to the difficulties in propagating it from seeds. In this study, we investigated the transcriptome profiles and transcriptional regulatory factors expressed during plantlet regeneration from root tissues of the endangered O. elatus. The RNA-seq results for the control and regenerated plants cultured in liquid medium for 8weeks showed that the clean length of the control group was 11,901,667,912 and that of the 8-week sample was 10,115,155,171, indicating a clean value of 97% for both samples. The number of mapped paired-end reads was 63,922,480 for the control group and 54,146,902 for the 8-week sample. The number of genes for which at least one clean data point was mapped was 43,177 in the control group and 42,970 in the 8-week sample. The results of the differentially expressed gene analysis indicate that the number of upregulated genes in the 8-week sample was 158, and the number of downregulated genes was 424. Gene Ontology (GO) analysis of the upregulated genes revealed that GO terms were classified into 14 categories, and genes expressed in the biological process category occurred most frequently. GO terms of the downregulated genes were evenly distributed into two categories: biological process and molecular function. From the upregulated genes, eight reference genes with significant differences in expression were selected and analyzed using real-time PCR. The Oe38836 gene (late embryogenesis abundant protein M17-like isoform X1) showed the highest expression rate that was more than tenfold that of the control. Oe40610 (auxin-responsive protein SAUR21-like) and Oe07114 (glucose-1-phosphate adenyl transferase-like protein) genes showed expression levels that were increased eightfold relative to the control. The RNA sequencing (RNA-seq) results from the plants regenerated through liquid culture of O. elatus root tissue were confirmed using real-time PCR, indicating their reliability.

Molecular Mechanisms Underlying Freezing Tolerance in Plants: Implications for Cryopreservation.

Cryopreservation is a crucial technique for the long-term ex situ conservation of plant genetic resources, particularly in the context of global biodiversity decline. This process entails freezing biological material at ultra-low temperatures using liquid nitrogen, which effectively halts metabolic activities and preserves plant tissues over extended periods. Over the past seven decades, a plethora of techniques for cryopreserving plant materials have been developed. These include slow freezing, vitrification, encapsulation dehydration, encapsulation-vitrification, droplet vitrification, cryo-plates, and cryo-mesh techniques. A key challenge in the advancement of cryopreservation lies in our ability to understand the molecular processes underlying plant freezing tolerance. These mechanisms include cold acclimatization, the activation of cold-responsive genes through pathways such as the ICE-CBF-COR cascade, and the protective roles of transcription factors, non-coding RNAs, and epigenetic modifications. Furthermore, specialized proteins, such as antifreeze proteins (AFPs) and late embryogenesis abundant (LEA) proteins, play crucial roles in protecting plant cells during freezing and thawing. Despite its potential, cryopreservation faces significant challenges, particularly in standardizing protocols for a wide range of plant species, especially those from tropical and subtropical regions. This review highlights the importance of ongoing research and the integration of omics technologies to improve cryopreservation techniques, ensuring their effectiveness across diverse plant species and contributing to global efforts regarding biodiversity conservation.

Functional validation of mungbean LEA protein coding gene in bacterial expression system confers salt stress tolerance

Mungbean (Vigna radiata R. Wilczek) is a major tropical food grain legume that is widely cultivated in tropical part of the world. Mungbean like other plants, tolerate and survive limited water situation owing to expression of stress associated proteins that offers membrane stability and cell protection. Late Embryogenesis Abundant (LEA) proteins are among the group of low molecular weight proteins, that play diverse roles in stress protection in several species of plants and animals. A LEA protein coding gene VrLEA2 was isolated from mungbean and its role in stress tolerance has been demonstrated using a bacterial expression system. VrLEA2 gene isolated was of size 893 bp and characterized as a group 1 LEA protein based on the sequence signature motif with presence of hydrophilic domain and a characteristic 20-mer conserved amino acids motif. VrLEA2 gene was cloned into a bacterial expression vector, pET 28a (+), transformed into the E.coli BL21 (DE3) cells for recombinant protein expression and subsequently subjected to antibiotic selection with kanamycin. Functional validation of the VrLEA2 for salt stress tolerance with varied concentration of NaCl (0 mM to 600 mM) showed alteration in colony morphology and reduction in the number of colonies in control compared to the transformed cells demonstrating the improved survival rate of cells expressing VrLEA2 protein. These findings indicate the best use of bacterial expression system for functional validation of plant proteins under stressed environments.

Small LEA proteins mitigate air-water interface damage to fragile cryo-EM samples during plunge freezing

Air-water interface (AWI) interactions during cryo-electron microscopy (cryo-EM) sample preparation cause significant sample loss, hindering structural biology research. Organisms like nematodes and tardigrades produce Late Embryogenesis Abundant (LEA) proteins to withstand desiccation stress. Here we show that these LEA proteins, when used as additives during plunge freezing, effectively mitigate AWI damage to fragile multi-subunit molecular samples. The resulting high-resolution cryo-EM maps are comparable to or better than those obtained using existing AWI damage mitigation methods. Cryogenic electron tomography reveals that particles are localized at specific interfaces, suggesting LEA proteins form a barrier at the AWI. This interaction may explain the observed sample-dependent preferred orientation of particles. LEA proteins offer a simple, cost-effective, and adaptable approach for cryo-EM structural biologists to overcome AWI-related sample damage, potentially revitalizing challenging projects and advancing the field of structural biology.

Transcriptome-level analysis of gene expressions in different tissues of tomato and key gene identifications during seed germination

Exploring the spatiotemporal expression patterns of genes is the foundation for studying their functions and molecular mechanisms. Genes with stable spatiotemporal expression patterns are often used as reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) and genes are significantly highly expressed in a specific tissue or stage often represent their unique biological functions. Tomato is an important economic crop and a model plant for scientific researches. However, the existing reference genes in tomatoes are not stable enough and researches on tissue-specific genes have been more focused on flowers and fruits, with less emphasis on studying the seed germination process. In this study, a total of 26 tissue/organ samples from the entire growth cycle of tomatoes were collected from Micro-Tom (Solanum lycopersicum cv. Micro-Tom). After RNA sequencing, a transcriptome database of the entire growth cycle of tomatoes was constructed. Solyc01g107870.3 gene was identified as a novel reference gene through systematic analysis and validated by qRT-PCR. Moreover, tissue-specific genes in seed and 8 days past germination (DPG) seedlings are screened. Among them, SlLEA16 gene belongs to late embryogenesis abundant protein (LEA) gene family, having subcellular localization in the nucleus and cytoplasm, and playing important roles in seed germination initiation. The above experimental results provide data support for studying tomato gene spatiotemporal expression patterns and can also serve as an experimental basis for investigating the deeper mechanisms of tomato seed germination processes.

Disordered proteins interact with the chemical environment to tune their protective function during drying.

The conformational ensemble and function of intrinsically disordered proteins (IDPs) are sensitive to their solution environment. The inherent malleability of disordered proteins combined with the exposure of their residues accounts for this sensitivity. One context in which IDPs play important roles that is concomitant with massive changes to the intracellular environment is during desiccation (extreme drying). The ability of organisms to survive desiccation has long been linked to the accumulation of high levels of cosolutes such as trehalose or sucrose as well as the enrichment of IDPs, such as late embryogenesis abundant (LEA) proteins or cytoplasmic abundant heat soluble (CAHS) proteins. Despite knowing that IDPs play important roles and are co-enriched alongside endogenous, species-specific cosolutes during desiccation, little is known mechanistically about how IDP-cosolute interactions influence desiccation tolerance. Here, we test the notion that the protective function of desiccation-related IDPs is enhanced through conformational changes induced by endogenous cosolutes. We find that desiccation-related IDPs derived from four different organisms spanning two LEA protein families and the CAHS protein family, synergize best with endogenous cosolutes during drying to promote desiccation protection. Yet the structural parameters of protective IDPs do not correlate with synergy for either CAHS or LEA proteins. We further demonstrate that for CAHS, but not LEA proteins, synergy is related to self-assembly and the formation of a gel. Our results suggest that functional synergy between IDPs and endogenous cosolutes is a convergent desiccation protection strategy seen among different IDP families and organisms, yet, the mechanisms underlying this synergy differ between IDP families.

Antioxidant Defense in the Toughest Animals on the Earth: Its Contribution to the Extreme Resistance of Tardigrades.

Tardigrades are unique among animals in their resistance to dehydration, mainly due to anhydrobiosis and tun formation. They are also very resistant to high-energy radiation, low and high temperatures, low and high pressure, and various chemical agents, Interestingly, they are resistant to ionizing radiation both in the hydrated and dehydrated states to a similar extent. They are able to survive in the cosmic space. Apparently, many mechanisms contribute to the resistance of tardigrades to harmful factors, including the presence of trehalose (though not common to all tardigrades), heat shock proteins, late embryogenesis-abundant proteins, tardigrade-unique proteins, DNA repair proteins, proteins directly protecting DNA (Dsup and TDR1), and efficient antioxidant system. Antioxidant enzymes and small-molecular-weight antioxidants are an important element in the tardigrade resistance. The levels and activities of many antioxidant proteins is elevated by anhydrobiosis and UV radiation; one explanation for their induction during dehydration is provided by the theory of "preparation for oxidative stress", which occurs during rehydration. Genes coding for some antioxidant proteins are expanded in tardigrades; some genes (especially those coding for catalases) were hypothesized to be of bacterial origin, acquired by horizontal gene transfer. An interesting antioxidant protein found in tardigrades is the new Mn-dependent peroxidase.

Transcriptome Analysis Reveals Genes and Pathways Associated with Drought Tolerance of Early Stages in Sweet Potato (Ipomoea batatas (L.) Lam.).

The yield of sweet potato [Ipomoea batatas (L.) Lam] can be easily threatened by drought stress. Typically, early stages like the seedling stage and tuber-root expansion stage are more vulnerable to drought stress. In this study, a highly drought-tolerant sweet potato cultivar "WanSu 63" was subjected to drought stress at both the seedling stage (15 days after transplanting, 15 DAT) and the tuber-root expansion stage (45 DAT). Twenty-four cDNA libraries were constructed from leaf segments and root tissues at 15 and 45 DAT for Next-Generation Sequencing. A total of 663, 063, and 218 clean reads were obtained and then aligned to the reference genome with a total mapped ratio greater than 82.73%. A sum of 7119, 8811, 5463, and 930 differentially expressed genes were identified from leaves in 15 days (L15), roots in 15 days (R15), leaves in 45 days (L45), and roots in 45 days (R45), respectively, in drought stress versus control. It was found that genes encoding heat shock proteins, sporamin, LEA protein dehydrin, ABA signaling pathway protein gene NCED1, as well as a group of receptor-like protein kinases genes were enriched in differentially expressed genes. ABA content was significantly higher in drought-treated tissues than in the control. The sweet potato biomass declined sharply to nearly one-quarter after drought stress. In conclusion, this study is the first to identify the differentially expressed drought-responsive genes and signaling pathways in the leaves and roots of sweet potato at the seedling and root expansion stages. The results provide potential resources for drought resistance breeding of sweet potato.

Differential Gene Expression in Contrasting Common Bean Cultivars for Drought Tolerance during an Extended Dry Period.

Common beans (Phaseolus vulgaris L.), besides being an important source of nutrients such as iron, magnesium, and protein, are crucial for food security, especially in developing countries. Common bean cultivation areas commonly face production challenges due to drought occurrences, mainly during the reproductive period. Dry spells last approximately 20 days, enough time to compromise production. Hence, it is crucial to understand the genetic and molecular mechanisms that confer drought tolerance to improve common bean cultivars' adaptation to drought. Sixty six RNASeq libraries, generated from tolerant and sensitive cultivars in drought time sourced from the R5 phenological stage at 0 to 20 days of water deficit were sequenced, generated over 1.5 billion reads, that aligned to 62,524 transcripts originating from a reference transcriptome, as well as 6673 transcripts obtained via de novo assembly. Differentially expressed transcripts were functionally annotated, revealing a variety of genes associated with molecular functions such as oxidoreductase and transferase activity, as well as biological processes related to stress response and signaling. The presence of regulatory genes involved in signaling cascades and transcriptional control was also highlighted, for example, LEA proteins and dehydrins associated with dehydration protection, and transcription factors such as WRKY, MYB, and NAC, which modulate plant response to water deficit. Additionally, genes related to membrane and protein protection, as well as water and ion uptake and transport, were identified, including aquaporins, RING-type E3 ubiquitin transferases, antioxidant enzymes such as GSTs and CYPs, and thioredoxins. This study highlights the complexity of plant response to water scarcity, focusing on the functional diversity of the genes involved and their participation in the biological processes essential for plant adaptation to water stress. The identification of regulatory and cell protection genes offers promising prospects for genetic improvement aiming at the production of common bean varieties more resistant to drought. These findings have the potential to drive sustainable agriculture, providing valuable insights to ensure food security in a context of climate change.

Genome-wide and functional analysis of late embryogenesis abundant (LEA) genes during dormancy and sprouting periods of kernel consumption apricots (P. armeniaca L. × P. sibirica L.)

Late embryogenesis abundant (LEA) proteins play a crucial role in protecting cells from stress, making them potential contributors to abiotic stress tolerance. This study focuses on apricot (P. armeniaca L. × P. sibirica L.), where a comprehensive genome-wide analysis identified 54 LEA genes, categorized into eight subgroups based on phylogenetic relationships. Synteny analysis revealed 14 collinear blocks containing LEA genes between P. armeniaca × P. sibirica and Arabidopsis thaliana, with an additional 9 collinear blocks identified between P. armeniaca × P. sibirica and poplar. Examination of gene structure and conserved motifs indicated that these subgroups exhibit consistent exon-intron patterns and shared motifs. The expansion and duplication of LEA genes in P. armeniaca × P. sibirica were driven by whole-genome duplication (WGD), segmental duplication, and tandem duplication events. Expression analysis, utilizing RNA-seq data and quantitative real-time RT-PCR (qRT-PCR), indicated induction of PasLEA2-20, PasLEA3-2, PasLEA6-1, Pasdehydrin-3, and Pasdehydrin-5 in flower buds during dormancy and sprouting phases. Coexpression network analysis linked LEA genes with 15 cold-resistance genes. Remarkably, during the four developmental stages of flower buds in P. armeniaca × P. sibirica - physiological dormancy, ecological dormancy, sprouting period, and germination stage - the expression patterns of all PasLEAs coexpressed with cold stress-related genes remained consistent. Protein-protein interaction networks, established using Arabidopsis orthologs, emphasized connections between PasLEA proteins and cold resistance pathways. Overexpression of certain LEA genes in yeast and Arabidopsis conferred advantages under cold stress, including increased pod length, reduced bolting time and flowering time, improved survival and seed setting rates, elevated proline accumulation, and enhanced antioxidative enzymatic activities. Furthermore, these overexpressed plants exhibited upregulation of genes related to flower development and cold resistance. The Y1H assay confirmed that PasGBF4 and PasDOF3.5 act as upstream regulatory factors by binding to the promoter region of PasLEA3-2. PasDOF2.4, PasDnaJ2, and PasAP2 were also found to bind to the promoter of Pasdehydrin-3, regulating the expression levels of downstream genes. This comprehensive study explores the evolutionary relationships among PasLEA genes, protein interactions, and functional analyses during various stages of dormancy and sprouting in P. armeniaca × P. sibirica. It offers potential targets for enhancing cold resistance and manipulating flower bud dormancy in this apricot hybrid.

HcLEA113, a late embryogenesis abundant protein gene, positively regulates drought-stress responses in kenaf.

Late embryogenesis abundant (LEA) proteins have been widely recognized for their role in various abiotic stress responses in higher plants. Nevertheless, the specific mechanism responsible for the function of LEA proteins in plants has not yet been explored. This research involved the isolation and characterization of HcLEA113 from kenaf, revealing a significant increase in its expression in response to drought stress. When HcLEA113 was introduced into yeast, it resulted in an improved survival rate under drought conditions. Furthermore, the overexpression of HcLEA113 in tobacco plants led to enhanced tolerance to drought stress. Specifically, HcLEA113-OE plants exhibited higher germination rates, longer root lengths, greater chlorophyll content, and higher relative water content under drought stress compared to wild-type (WT) plants, while their relative conductivity was significantly lower than that of WT plants. Further physiological measurements revealed that the proline content, soluble sugars, and antioxidant activities of WT and HcLEA113-OE tobacco leaves increased significantly under drought stress, with greater changes in HcLEA113-OE plants than WT. The increase in hydrogen peroxide (H2O2), superoxide anions (O2 -), and malondialdehyde (MDA) content was significantly lower in HcLEA113-OE lines than in WT plants. Additionally, HcLEA113-OE plants can activate reactive oxygen species (ROS)- and osmotic-related genes in response to drought stress. On the other hand, silencing the HcLEA113 gene through virus-induced gene silencing (VIGS) in kenaf plants led to notable growth suppression when exposed to drought conditions, manifesting as decreased plant height and dry weight. Meanwhile, antioxidant enzymes' activity significantly decreased and the ROS content increased. This study offers valuable insights for future research on the genetic engineering of drought resistance in plants.

The superior allele LEA12OR in wild rice enhances salt tolerance and yield.

Soil salinity has negative impacts on food security and sustainable agriculture. Ion homeostasis, osmotic adjustment and reactive oxygen species scavenging are the main approaches utilized by rice to resist salt stress. Breeding rice cultivars with high salt tolerance (ST) and yield is a significant challenge due to the lack of elite alleles conferring ST. Here, we report that the elite allele LEA12OR, which encodes a late embryogenesis abundant (LEA) protein from the wild rice Oryza rufipogon Griff., improves osmotic adjustment and increases yield under salt stress. Mechanistically, LEA12OR, as the early regulator of the LEA12OR-OsSAPK10-OsbZIP86-OsNCED3 functional module, maintains the kinase stability of OsSAPK10 under salt stress, thereby conferring ST by promoting abscisic acid biosynthesis and accumulation in rice. The superior allele LEA12OR provides a new avenue for improving ST and yield via the application of LEA12OR in current rice through molecular breeding and genome editing.

Multi-scale characterisation of cold response reveals immediate and long-term impacts on cell physiology up to seed composition in sunflower.

Early sowing can help summer crops escape drought and can mitigate the impacts of climate change on them. However, it exposes them to cold stress during initial developmental stages, which has both immediate and long-term effects on development and physiology. To understand how early night-chilling stress impacts plant development and yield, we studied the reference sunflower line XRQ under controlled, semi-controlled and field conditions. We performed high-throughput imaging of the whole plant parts and obtained physiological and transcriptomic data from leaves, hypocotyls and roots. We observed morphological reductions in early stages under field and controlled conditions, with a decrease in root development, an increase in reactive oxygen species content in leaves and changes in lipid composition in hypocotyls. A long-term increase in leaf chlorophyll suggests a stress memory mechanism that was supported by transcriptomic induction of histone coding genes. We highlighted DEGs related to cold acclimation such as chaperone, heat shock and late embryogenesis abundant proteins. We identified genes in hypocotyls involved in lipid, cutin, suberin and phenylalanine ammonia lyase biosynthesis and ROS scavenging. This comprehensive study describes new phenotyping methods and candidate genes to understand phenotypic plasticity better in response to chilling and study stress memory in sunflower.

Construction of a Membrane Yeast Two-Hybrid Library and Screening of MsPYR1-Like Interacting Proteins in Malus sieversii.

To investigate the biological effects of the ABA receptor pyrabactin resistance 1-like (PYR1-like) in Malus sieversii seeds, the proteins interacting with MsPYR1-like were screened by the membrane yeast two-hybrid library based on the split-ubiquitin system, and to construct the bait vector pBT3-SUC-PYR1 for Malus sieversii cDNA library, which had no self-activating effect on the yeast cells of the pPR3-N membrane yeast two-hybrid library. The library titer assay showed that it could meet the requirements for membrane yeast two-hybrid library screening. After sequencing, GenBank database blast, and yeast rotary validation, 28 candidate proteins interacting with MsPYR1-like were obtained, including ribosomal proteins, late embryogenesis abundant proteins, F-actin-capping proteins, phytochrome-interacting proteins, low-temperature-inducible 65kDa protein-like, senescence-associated, PP2C and SnRK2 family members, and unknown proteins. Gene ontology analysis of the interaction proteins was related to plant hormone response and negative regulation of seed germination, overexpression of MsPYR1-like in Arabidopsis negatively regulates seed germination, and the study of the biological roles of MsPYR1-like interacting proteins lays the foundation for revealing the lifting of seed dormancy in Malus sieversii.

Genome-wide characterization of LEA gene family reveals a positive role of BnaA.LEA6.a in freezing tolerance in rapeseed (Brassica napus L.)

BackgroundFreezing stress is one of the major abiotic stresses that causes extensive damage to plants. LEA (Late embryogenesis abundant) proteins play a crucial role in plant growth, development, and abiotic stress. However, there is limited research on the function of LEA genes in low-temperature stress in Brassica napus (rapeseed).ResultsTotal 306 potential LEA genes were identified in B. rapa (79), B. oleracea (79) and B. napus (148) and divided into eight subgroups. LEA genes of the same subgroup had similar gene structures and predicted subcellular locations. Cis-regulatory elements analysis showed that the promoters of BnaLEA genes rich in cis-regulatory elements related to various abiotic stresses. Additionally, RNA-seq and real-time PCR results indicated that the majority of BnaLEA family members were highly expressed in senescent tissues of rapeseed, especially during late stages of seed maturation, and most BnaLEA genes can be induced by salt and osmotic stress. Interestingly, the BnaA.LEA6.a and BnaC.LEA6.a genes were highly expressed across different vegetative and reproductive organs during different development stages, and showed strong responses to salt, osmotic, and cold stress, particularly freezing stress. Further analysis showed that overexpression of BnaA.LEA6.a increased the freezing tolerance in rapeseed, as evidenced by lower relative electrical leakage and higher survival rates compared to the wild-type (WT) under freezing treatment.ConclusionThis study is of great significance for understanding the functions of BnaLEA genes in freezing tolerance in rapeseed and offers an ideal candidate gene (BnaA.LEA6.a) for molecular breeding of freezing-tolerant rapeseed cultivars.