Plant Cell Reports

Protein-protein interactions (PPIs) play a crucial role in enabling proteins to carry out their functions within various biological processes (Hui et al., 2003). Since the introduction of the yeast two-hybrid (Y2H) method for PPI detection in 1989 (Fields and Song, 1989), the identification of PPIs has become a significant focus in modern biological research. PPI goes beyond examining individual proteins, allowing researchers to establish a comprehensive network that regulates biological processes. Rice, as a key model organism in plant biological studies, has been at the forefront of PPI research. In 2008, prominent rice scientists in China called for concerted efforts to define a comprehensive protein-protein interaction network experimentally, which aimed to facilitate the prediction of the functional mechanisms operating throughout a plants lifecycle (Zhang et al., 2008). With efforts for 2 decades, the experimentally identified rice PPIs have reached over ten thousand. Several public databases have been established to systematically collate and store PPIs, including STRING (Szklarczyk et al., 2019), BioGRID (Oughtred et al., 2020), IntAct (del Toro et al., 2022), PRIN (Gu et al., 2011), RicePPINet (Liu et al., 2017) and RiceNet v2 (Lee et al., 2015). However, most PPI datasets in rice stem from computational predictions, while experiment-based rice PPI datasets are fragmented due to the lack of systematic profiling at the rice PPIome level, which largely hinders information sharing in the rice research community. To bridge this gap, we constructed the Port of Protein-Protein Interactomes (POPPIN; https://riceome.hzau.edu.cn/poppin/), an integrated database dedicated to sharing experimentally verified PPIs and functional clues in rice. Empowered by high-throughput PPIome profiling technologies and text mining assisted by a large language model (Huang et al., 2025; Liu et al., 2025), POPPIN currently has deposited over 150,451 pieces of rice PPI-related information. Additionally, POPPIN provides detailed protein information, including GO annotations, subcellular localizations, domains, trait ontology (TO) information, and hyperlinks to external biological databases. Through offering a user-friendly web interface for search and dynamic network visualization, POPPIN serves as the first large-scale, experiment-based database for searchable PPIs in rice, and has the potential to be extended to other species under this structural framework.

The barley yellow mosaic virus disease is one of the most important threats of barley production in Europe and Asia. Transmitted by the soil-borne plasmodiophorid Polymyxa graminis, there are no direct control options against the causal bymoviruses Barley Yellow Mosaic Virus (BaYMV) and Barley Mild Mosaic Virus (BaMMV). Resistance breeding is thus the only viable approach and has been very successful in the past, with the resistance-conferring alleles rym4 and rym5 of the EUKARYOTIC TRANSLATION INITIATION FACTOR 4E being used extensively in European winter barley breeding. However, virus strains have meanwhile overcome this resistance. Therefore, there is an urgent need for new sources of resistance. Genome editing with Cas endonucleases is a timely and promising approach in this respect. However, the small insertions and deletions that frequently arise during site-directed mutagenesis usually lead to the knockout of the target genes. In the case of EIF4E, loss-of-function is accompanied by significant yield reduction. Consequently, more precisely edited alleles with retained function are necessary for crop improvement. The present study represents the first application of base editing in barley plants, using the EIF4E gene as an example. Base exchanges were made at two positions in this gene using an nCas9-cytidine deaminase fusion, resulting in a total of 10 novel EIF4E alleles in addition to the introduction of a single nucleotide polymorphism that is part of rym4. Two of these newly generated alleles led to resistance upon BaMMV inoculation without adverse effects on yield, proving this approach promising to generate new material for resistance breeding.

The apoplast is a primary location for interactions between plants and invasive pathogens and is the site where many pathogen-secreted effectors are perceived by cell surface immune receptors to activate defence responses. However, our understanding of apoplastic interactions remains limited because protein interactions are difficult to investigate in this harsh extracellular environment. TurboID-based proximity labelling (PL) has emerged as a powerful approach for studying protein interactions in plants, though its application has to date been restricted to intracellular proteins. Here, we designed and validated a TurboID-based PL strategy for investigating protein interactions in the leaf apoplast using the well-characterised interaction between the Phytophthora infestans elicitor INF1 and the receptor-like protein (RLP) REL in Nicotiana benthamiana. Transient expression of SP-INF1-TurboID (INF1-T) induced a cell death (CD) response comparable to that triggered by native INF1, demonstrating that fusion of the TurboID tag did not impair INF1 recognition by REL. Apoplastic localisation of both INF1-T and the control construct SP-eGFP-TurboID (eGFP-T) was confirmed, validating their suitability for PL experiments. Efficient TurboID-mediated biotinylation was achieved in the apoplast using co-infiltration of biotin, ATP and magnesium acetate. Streptavidin-HRP immunoblotting revealed distinct biotinylation profiles for INF1-T and eGFP-T. Furthermore, co-immunoprecipitation demonstrated specific biotinylation of REL by INF1-T, but not by eGFP-T, in wild-type, bak1 and sobir1/sobir1-like N. benthamiana. These findings demonstrate that TurboID-based PL is functional in the apoplast and provides a proof-of-concept for investigating elicitor-receptor interactions in this compartment.

Most transgene applications require high and sustained expression, particularly in stably transformed plants. Achieving optimal transgene performance, however, depends on the combined influence of multiple genetic and regulatory factors. In previous work, we systematically evaluated the contribution of different genetic elements to transient transgene expression and demonstrated that terminators are key determinants of transgene performance by reducing transcriptional read-through and preventing transgene silencing. Here, we extend these findings by investigating the roles of terminators and introns in the expression of transgenes in stably transformed plants. Our results show that optimal transgene performance arises from the complementary actions of these two elements. Terminator choice was a major determinant of transgene expression levels, whereas introns played a critical role in maintaining expression stability. We further demonstrate a strong relationship between transgene expression levels and small RNA accumulation and show that intron-containing endogenous genes are enriched among highly expressed and stress-responsive genes, suggesting that intron-mediated protection from silencing may facilitate higher levels of gene expression and have contributed to the emergence and evolutionary retention of intron-containing genes.

Background: Plant defensins are small cysteine-rich antimicrobial peptides. In Arabidopsis thaliana, the inducible Class I gene AtPDF1.2a is widely used as a transcriptional marker of jasmonate/ethylene immunity, whereas the constitutively expressed Class II members have received less functional attention. AtPDF2.2 was compared with AtPDF1.2a during infection of Arabidopsis by the vascular fungal pathogen Verticillium longisporum, the necrotrophic fungus Sclerotinia sclerotiorum and the bacterium Pseudomonas syringae pv. tomato DC3000. Results: Profiling of the AtPDF family showed that AtPDF2.2, together with the Class II genes AtPDF2.3 and AtPDF2.5, was significantly reduced during V. longisporum infection, whereas AtPDF1.2a was induced. A promoter-reporter line showed a corresponding decrease in AtPDF2.2 promoter activity during V. longisporum infection, strongest in infected roots. KD-pdf2.2 plants contained more V. longisporum DNA, whereas OE-PDF2.2 plants contained less. The same reciprocal disease pattern was observed during S. sclerotiorum and Pst DC3000 infection. Histochemical Prom::AtPDF2.2::GUS reporter signal decreased during S. sclerotiorum infection but increased following Pst DC3000 infection. Basal AtPDF2.2 expression was lower in JA and ET-signaling mutants and higher in the SA-deficient sid2 background, while the analyzed promoter lacked the GCC-box that mediates inducible AtPDF1.2a regulation. AtPDF1.2a overexpression reduced symptoms across the three pathosystems, but loss of AtPDF1.2a increased disease only during V. longisporum infection under the conditions tested and did not alter Pst DC3000 DNA abundance. AtPDF2.2 overexpression was not associated with reduced vegetative growth under the conditions tested, whereas AtPDF1.2a overexpression reduced rosette size and leaf number. Conclusions: AtPDF2.2 is a constitutively expressed Class II defensin whose expression level is associated with disease outcome across two fungal pathogens and a bacterium. Its promoter shows tissue-dependent regulation during V. longisporum infection, while histochemical reporter signal decreases during S. sclerotiorum infection and increases during Pst DC3000 infection. This regulatory behavior differs from that of the inducible reference gene AtPDF1.2a. The reciprocal disease phenotypes of the AtPDF2.2 transgenic backgrounds support a protective association that may involve direct antimicrobial activity, modulation of host responses or both. The absence of reduced vegetative growth in OE-PDF2.2 under the conditions tested supports further evaluation of PDF2-type genes in Brassica.

RNA mobility has emerged as a valuable component of RNA-based genome editing and DNA-free transformation technologies. However, experimental systems for rapidly evaluating RNA mobility remain limited, particularly in monocot species where grafting approaches are not feasible. Here, we developed immobilized versions of Foxtail Mosaic Virus (FoMV) and Tobacco Rattle Virus (TRV) with impaired systemic viral movement as generalizable platforms for transient expression and functional screening of mobile RNAs. A simple Nicotiana benthamiana leaf assay enabled direct visualization and molecular quantification of transcript mobility using fluorescent reporter fusions carrying seven previously described RNA mobility elements from dicot and monocot species. The platform consistently distinguished mobile elements displaying higher or lower frequencies of mobility across both viral systems, with T-RNA-like sequence (TLS), TLSgly and maize FLOWERING LOCUS T (FT) ortholog, ZCN19, and as well as ZCN16 displaying significantly higher frequencies of mobility compared to non-mobile element controls in FoMV and TRV, respectively. Translation of these findings to virus-induced genome editing demonstrated that mobile elements identified through the screening platform enhanced FoMV-mediated editing of PHYTOENE DESATURASE in Setaria viridis (SvPDS), with TLSgly increasing somatic editing frequencies approximately two-fold relative to sgRNA alone. Together, these results establish immobilized FoMV and TRV platforms as versatile screening tools for evaluating RNA mobility, optimizing RNA cargos for viral genome editing, and a scalable framework for engineering mobile RNAs and accelerating development of RNA-based technologies for functional genomics and crop improvement.

RNA-guided nucleases (RGNs), such as Cas9 from Streptococcus pyogenes (SpCas9), are widely used for plant genome editing. Previous surveys for off-targeting, the modification of unintended targets with similarity to the intended target, indicate high specificity of SpCas9 in plant cells. However, off-targeting has not been assessed for efficiency-optimized editing systems combined with extensive multiplexing, which might increase the likelihood of cleavage at unintended sites. We therefore analyzed Arabidopsis thaliana lines that had been extensively mutagenized using zCas9i and up to 29 gRNAs addressing >45 target sites over several rounds of editing. Genomes were sequenced by short- and long-read technologies, and genome-wide variants were catalogued. Our pipeline for variant calling reliably detected RGN-induced mutations at on-targets. When excluding these on-target modifications, variants were detected in edited lines at frequencies similar to those previously reported for spontaneous mutations. In further analyses, we did not find any evidence for an origin of these variants from RGN activity. Our data are thus consistent with high specificity of SpCas9. In contrast, we detected genomic reorganization events upon editing at two complex loci, RPP1 and RPP7, encompassing multiple homologous genes, and also identified an allele by WGS that had escaped detection by amplicon sequencing. We conclude that, while off-targets may efficiently be avoided by selection of specific gRNAs, on-target modifications may be more extensive than intended, especially at complex loci and/or during multiplexing.

Photosynthetic carbon assimilation is sensitive to heat stress, with a key component being the thermal sensitivity of Rubisco activase (RCA). One approach to increasing heat tolerance of photosynthesis is to increase the thermotolerance of RCA. Here, we have used a transgenic approach to express RCA{beta} from Agave tequilana (AtRCA) in Setaria viridis. A second line was created where the endogenous SvRCA{beta} was substituted for AtRCA{beta} through complementation of a null mutant, {triangleup}rcaB (AtRCA[bKO]). In vitro assays showed that Agave RCA readily activates Setaria Rubisco, and that its thermostability is higher than that of Setaria RCA. AtRCA[bKO] plants exhibited higher CO2 assimilation at 25{degrees}C compared to WT and AtRCA plants, although Rubisco content and activation did not change. After six hours of heat stress, AtRCA[bKO] plants retained higher CO2 assimilation rates than AtRCA, however, after 24 hours CO2 assimilation declined to similar levels in all lines, suggesting that RCA may not limit carbon assimilation at the conditions tested. Taken together, our results show that substitution of RCA from a CAM plant into a C4 model allows normal growth and supports slightly increased carbon assimilation under control and short-term heat conditions.

The rejection of self-incompatible (SI) Brassica pollen is mediated by three signaling branches that function in parallel in the stigma. The recognition of SI pollen by the stigma S-Receptor Kinase (SRK) results in activation of the ARM-Repeat-Containing 1 E3 ubiquitin ligase (ARC1) which mediates the degradation of compatibility factors, the FERONIA (FER) and ANJEA (ANJ) receptor kinases that induces ROS accumulation to inhibitory levels and the M Locus Protein Kinase (MLPK) which may also be connected to ROS production. Arabidopsis self-incompatibility is regulated by SRK as well, but the signaling events downstream of SRK following SI pollen perception are less well-understood. In this study, we evaluated the requirements of FER, ANJ and HERCULES RECEPTOR KINASE 1 (HERK1) for SI pollen rejection in the transgenic Arabidopsis thaliana SI-Col-0{psi} srka-1 line. The{psi} srka-1 T-DNA disrupting the expression of the endogenous{psi} SRKA gene was crossed into SI-Col-0 to prevent any potential SRK transgene silencing. T-DNA mutants for FER and ANJ/HERK1 were then crossed into the SI-Col-0{psi} srka-1 line. Using standard assays for pollen-stigma interactions, the SI phenotypes were assessed for the SI-Col-0 fer, SI-Col-0 anj-1 and SI-Col-0 anj-1 herk1-1 lines. Our results presented here indicated that FER and ANJ are not required in the stigma for Arabidopsis SI pollen rejection, further providing evidence for a divergence in the SI downstream signaling pathway in Arabidopsis.

Myoglobin, an oxygen-binding animal protein, was engineered in Pisum sativum (pea) to explore its potential as a food ingredient and balance the amino acid profile. In this study, minimal expression cassettes and binary vectors were used to express bovine myoglobin using particle gun and Agrobacterium-mediated transformation, respectively. Successful integration and expression of the myoglobin gene was achieved in P. sativum, with both methods yielding similar transformation efficiencies (~1%). Expression analysis of T2 seeds revealed that Agrobacterium-mediated transformation-derived transgenic lines that expressed myoglobin under the regulation of a Soybean 7S seed-specific promoter and Tobacco Etch Virus (TEV) translation enhancer and a chimeric Rb7MAR Terminator (Ps-BpRG13 events) consistently yielded the highest level of expression (0.32-1.57% of TSP), while transgenic lines with myoglobin expression under the regulation of a Soybean Phaseolin promoter and Rb7MAR Terminator (Ps-BpRG14 events) resulted in moderate levels of heterologous protein expression (0.13-0.83% TSP). Transgenic events with constitutive 2xCaMV35S promoter, TEV translation enhancer and Rb7MAR terminator (Ps-BpRG15 events) exhibited the lowest level of myoglobin expression (0.09-0.14% TSP). Co-bombardment of two minimal expression cassettes - one with myoglobin under the regulation of the Phaseolin promoter and Rb7MAR Terminator and the other with the nptII selectable marker under the regulation of a 2X constitutive CaMV35S promoter, TEV translational enhancer and TNOS Terminator, yielded lines that exhibited variable expression (0.03-0.77% TSP), with some events comparable in expression to Agrobacterium-derived Ps-pRG14 events. To the best of our knowledge, this is the first report of producing a heme-containing animal protein, myoglobin, in peas, with potential implications for sustainable production of food ingredients and nutritionally fortified and value-added plant products using molecular farming.

Pepper fruit accumulated xanthophyll acyl esters during ripening. To characterize the genes involved, cDNAs for two putative xanthophyll acyltransferases named CaPYP1 and CaPYP1-like were cloned. The deduced amino acid sequences of CaPYP1 and CaPYP1-like had conserved hydrolase and acyltransferase domains. Phylogenetic analyses showed that both putative acyl transferases were conserved in Viridiplantae, suggesting important functions for both. CaPYP1 and CaPYP1-like GFP-fusion proteins were localized in the plastid when expressed in leaf tissue. Gene expression analyses indicated that both genes for CaPYP1 and CaPYP1-like were most expressed in ripening fruit (52 to 64 days after anthesis) and senescent leaf, CaPYP1 having relatively greater expression than CaPYP1-like. In virus-induced gene silencing experiments in pepper, xanthophyll esterification was greatly diminished when CaPYP1 was silenced with a simultaneous change in the ripening fruits color from dark red to bright red. In complementation tests, overexpression of CaPYP1 in a tomato mutant impaired for petal coloration and xanthophyll esterification, resulted in restoration of the petal color and the synthesis of both mono, diacyl and tri esters of xanthophylls. CaPYP1-like overexpression in the same genetic background resulted in the synthesis of relatively smaller amounts of xanthophyll monoesters. In an in vitro test, zeaxanthin was more sensitive to light than zeaxanthin dipalmitate but both were protected when triacylglycerol was mixed with it, suggesting that acyl moieties could improve xanthophyll stability. Together our results indicate that xanthophyll esterification during pepper fruit ripening is important for fruit color, xanthophyll accumulation and stability and is orchestrated by both CaPYP1 and CaPYP1-like with CaPYP1 playing a major role. HighlightRipening pepper fruit accumulates xanthophyll fatty acyl esters associated with nutritional quality and fruit color. The fruit expresses CaPYP1 and CaPYP1-like, two putative acyltransferases in their chromoplasts. In a tomato mutant impaired for xanthophyll esterification, transgenic expression of CaPYP1 promoted more xanthophyll esterification in ripe fruit than expressing CaPYP1-like.

Flower opening is a complex developmental process involving coordinated changes in cell proliferation and cell expansion. Although several regulators of flower opening have been identified, how transcriptional programs are coordinated with the cellular and metabolic changes underlying petal expansion immediately before flower opening remains incompletely understood. Japanese morning glory (Ipomoea nil) is a suitable model for investigating these processes because its flowers open synchronously at a predictable time. This study aimed to identify transcriptional regulators involved in petal development and flower opening in Japanese morning glory. Temporal analyses of petal growth, sugar metabolism, and gene expression revealed that petal development was driven by both cell proliferation and cell expansion until approximately 48 h before flower opening, whereas cell expansion predominated thereafter. Weighted gene co-expression network analysis identified two genes encoding R2R3-MYB subgroup 19 transcription factors, InMYB21A and InMYB21B, as candidate regulators associated with petal development. CRISPR/Cas9-mediated knockout analysis revealed a prominent role for InMYB21B, whose loss markedly impaired petal cell expansion and prevented flower opening. InMYB21B knockout also impaired stamen and pistil development, resulting in male and female sterility. Starch degradation and glucose accumulation were impaired in InMYB21B knockout petals. Transcriptome analysis revealed delayed transcriptomic progression during petal development and reduced expression of genes associated with starch degradation, sucrose metabolism, cell wall remodeling, and water transport. These findings identify InMYB21B as a key regulator of petal cell expansion and flower opening in Japanese morning glory and show that loss of InMYB21B disrupts both metabolic and transcriptomic progression during late petal development.

Genetic transformation of citrus is essential for functional genomics and trait improvement, yet remains limited by prolonged regeneration cycles, genotype-dependent responses, and inefficient screening of transformed tissues. Here, we established an Agrobacterium-mediated indirect organogenesis-based transformation system for Carrizo citrange and evaluated two visible reporter systems, the anthocyanin regulator ROSEA1 and the betalain biosynthetic cassette RUBY, for monitoring transformed tissues throughout regeneration. An optimized regeneration protocol enabled visible callus ini-tiation within 7 days following a 5-day pre-culture treatment and recovery of PCR-confirmed transgenic plantlets within 5 months. Both visible reporters were compatible with callus development and shoot regeneration throughout the indirect organogenesis workflow. During callus proliferation, visible pigmentation closely corresponded with GFP fluorescence, enabling rapid, non-destructive identification of transformed tissues. However, reporter performance diverged during shoot regeneration: RUBY maintained stable pigmentation throughout regeneration, whereas ROSEA1-associated pigmentation progressively declined despite continued GFP fluorescence. Consequently, RUBY exhibited a 4.7-fold higher pigmenta-tion-based detection rate than ROSEA1 at the shoot stage and showed closer agreement with GFP-based detection. Putative transgenic events were confirmed by PCR in independent lines of both constructs, together with GFP fluorescence in leaves and root tips. Together, these findings establish an efficient indirect organogenesis-based transformation platform for citrus and demonstrate that both ROSEA1 and RUBY are effective visual reporters during callus proliferation, whereas RUBY provides more reliable visual identification during shoot regeneration and plant recovery.

The CRISPR/Cas12a system has emerged as a highly useful tool for genomic editing due to its ability to process multiple guide RNAs (gRNAs) from a single promoter. In this study, we explored the potential of combinatorial CRISPR/Cas12a to excise targeted loci in the mammalian genome by inducing double-strand breaks (DSBs) upstream and downstream of targeted exons. We designed a library of gRNA pairs targeting introns flanking 54 exons of 18 essential genes and 17 exons of 10 nonessential genes, aiming to assess the efficiency of Cas12 in excising targeted loci. While single gRNAs directly targeting essential exons produced expected fitness defects in cells, pairs of guides targeting flanking intronic regions resulted in only trivial viability effects, suggesting that excision rarely occurred. Further analysis showed no meaningful correlation between gRNA efficiency, orientation, GC content, or cut site geometry and the success of locus excision. These findings indicate that, under the conditions tested, the Cas12a system does not yield locus excision at the efficiency required for sensitive negative selection assays.

Salinity is a major abiotic stress that severely limits plant growth and productivity, causing substantial yield losses. Despite barleys relative tolerance to salinity, the underlying physiological and molecular mechanisms remain incompletely understood. In this study, we employed an integrative approach combining agronomic, physiological, biochemical, and transcriptomic analyses to investigate salinity responses in the spring barley cultivar Giza 134 under both field and lysimeter-based conditions. Salinity stress significantly reduced growth and yield-related traits, with more pronounced effects observed under lysimeter-imposed salinity, reflecting higher stress intensity. These reductions were associated with impaired water status, altered leaf structural traits, and declines in photosynthetic pigment content. In contrast, proline accumulation increased, indicating activation of osmotic adjustment mechanisms. Salinity also disrupted ionic homeostasis, as evidenced by elevated Na+ levels, reduced K+ content, and an increased Na+/K+ ratio. Enhanced lipid peroxidation and elevated catalase and peroxidase activities suggested increased oxidative stress and activation of antioxidant defenses. Transcriptome profiling identified 4,298 differentially expressed genes, including 1,764 upregulated and 2,534 downregulated genes. Functional enrichment analyses revealed upregulation of pathways related to stress adaptation, redox regulation, and metabolic reprogramming, while genes associated with photosynthesis, ribosome biogenesis, and protein synthesis were strongly suppressed. Several novel stress-responsive genes involved in signaling, osmoprotection, antioxidant defense, and central metabolism were highly induced, supported by coordinated enrichment of cis-regulatory motifs in their promoter regions. Together, these findings provide a comprehensive physiological and molecular framework for salinity tolerance in Giza 134 and highlight candidate genes and pathways for breeding salt-resilient cultivars suited to saline-prone environments.

GOLDEN2-like (GLK) transcription factors are key regulators of chloroplast differentiation and photosynthetic gene expression. The classical yellow mutation in Japanese morning glory (Ipomoea nil) produces yellowish-green leaves, whereas an unstable allele, yellow-mutable, produces green somatic sectors on a yellowish-green background. The gene responsible for these mutations was identified as InGLK, which encodes a GOLDEN2-like transcription factor. The stable yellow mutant carried a 4-bp frameshift insertion in InGLK, whereas two yellow-mutable lines carried the Tpn1-family transposon Tpn12 in intron 5. Excision of Tpn12 in germinal revertants left short footprints and restored the green leaf phenotype. Genome searches identified InGLK as the sole GLK gene in I. nil. Pigment analysis of green somatic reversion sectors and yellowish-green background areas showed that most of the measured photosynthetic pigments were significantly reduced in the yellowish-green background, whereas the chlorophyll a/b ratio was unchanged. Chloroplasts in the yellowish-green tissue retained thylakoid-like membranes and starch granule-like structures but had less distinct grana-like stacks and sparse stromal lamellae-like structures. Wild-type-like chloroplast ultrastructure was restored in germinal revertants. These findings show that loss of function of a single-copy GLK gene broadly reduces photosynthetic pigment accumulation and alters chloroplast internal membrane organization. The yellow mutants of I. nil therefore provide a genetic system for examining non-redundant GLK function.

Arabidopsis Leucine-Rich Repeat-Malectin Receptor Kinase (LRR-MAL RK) genes have been previously implicated in the early stages of pollen-pistil interactions to support compatible pollen. One member, Receptor Kinase in Flowers 1 (RKF1), has been associated with roles in the stigma to support pollen hydration as well as pollen tube growth. To better understand the function of RKF1 in these processes, a yeast two-hybrid screen was conducted with the RKF1 cytosolic kinase domain. Two positive interactors identified from this screen were the Group VII Ethylene Response Factors (ERFVIIs), RELATED TO APETALA 2.12 (RAP2.12) and RAP2.3. Their putative roles in pollen-pistil interactions were investigated using the quintuple erfvii mutant, and novel pistil-mediated pollen tube callose deposition phenotypes were uncovered during the pollen tube growth stage. Loss of seven LRR-MAL RKs including RKF1 in the pistil was previously found to cause an unusual phenotype where shorter callose plugs were deposited in wildtype pollen tubes compared to that seen in wildtype Col-0 pistils. Contrary to this, wildtype pollen tubes growing through the quintuple erfvii mutant pistil deposited callose plugs that were more elongated than that seen in wildtype Col-0 pistils. Further analyses with the proteolysis 6 (prt6) mutant and RAP2.12 rescue constructs were consistent with these phenotypes providing support that RKF1 is a negative regulator of RAP2.12 and RAP2.3 in the pistil during pollen tube growth.

The ability of seeds to survive until dormancy recedes and the germination requirements are met is an adaptive strategy. Proteomics improves our understanding of the mechanisms that control the adaptation to environmental heterogeneity. In this study, we investigated two European beech populations from different habitats that differed in dormancy and germination traits. We found that the populations exhibited different germination strategies, which were reflected in coordinated but quantitatively different proteomic reprogramming. The Miekinia population exhibited stronger accumulation of proteins involved in nucleotide sugar biosynthesis, S-adenosylmethionine metabolism, and flavonoid biosynthesis. Enhanced nucleotide sugar biosynthesis indicates more intensive cell wall remodelling and carbohydrate metabolism, which support embryo growth and faster germination. Increased S-adenosylmethionine metabolism suggests the epigenetic and hormonal regulation of germination differences between populations. Higher flavonoid biosynthesis indicates an enhanced antioxidant capacity associated with environmental protection. In contrast, the Wisa population showed stronger accumulation of proteins involved in RNA processing, suggesting tighter post-transcriptional regulation and proteome reorganization during germination. Consistent with its deeper dormancy and later germination, the Wisa population appears to rely more on RNA-level regulation, whereas the Miekinia population prioritizes metabolic activation. These contrasting proteomic profiles likely reflect population-specific physiological strategies associated with dormancy depth and adaptation to different climatic conditions. HighlightProteomic reprogramming reveals population-specific germination strategies in European beech, linking dormancy depth with contrasting metabolic activation and RNA-level regulation during the transition from dormancy to germination.

Drought stress severely limits tree growth and survival, a problem intensifying under climate change due to the rising frequency of extreme weather events. Identifying genetic variants that modulate drought stress responses and enhance resilience is therefore of great interest for developing climate-adapted trees. While the MADS-box transcription factor AGL16 has been implicated in drought responses and stomatal regulation in herbaceous species, the function of its homolog in woody perennials remains largely unexplored. Here, we investigate the role of PtaAGL16 in the poplar hybrid Populus tremula x P. alba. We generated CRISPR/Cas9 knockout lines of PtaAGL16 in the poplar clone INRA 717-1B4. Plants were subjected to a severe drought stress regime followed by a re-watering recovery phase. Morphological and physiological traits, including growth, stomatal conductance, shoot vitality, and stomatal density, together with stress-associated metabolites and drought-responsive gene expression, were analyzed. All agl16 knockout lines exhibited significantly reduced drought-induced damage compared to the wild-type. While the wild-type suffered extensive defoliation and persistent stem necrosis, the knockout lines retained higher shoot vitality and resumed growth after re-watering, including the formation of new leaves. Consequently, vitality in the knockout lines reached up to 86%, compared to only 34% in the wild-type. This improved resilience was accompanied by reduced persistent oxidative membrane damage, as indicated by lower MDA levels after recovery. These findings indicate that the disruption of PtaAGL16 is associated with enhanced drought resilience in a woody perennial species and highlight its potential as a genetic target for improving climate adaptation in fast-growing forest trees.

Although the KINETOCHORE NULL2 (KNL2) protein is an essential inner centromeri c protein that is crucially important for assembly and functioning of kinetochores, our understanding of its organization, dynamics and function of distinct isoforms in the cells of plant species undergoing mitosis/meiosis is far from complete. In this study, we identified and characterized two KNL2.1/KNL2.2 genes in cowpea. GUS reporter constructs and qRT-PCR revealed that the expression profiles of both genes were variable across organs, with the highest expression in leaves and roots. Using an EYFP gene fusion coupled with immunostaining, it was demonstrated that both KNL2 variants colocalized at centromeres in a cell-cycle-dependent manner. The CRISPR/Cas9 technique was used to generate various in-frame deletion and out-of-frame knock-out knl2 mutants. Single- and double-gene knock-out mutants were generated, and the effects of mutations on plant development and seed setting were analyzed. The results are discussed both with respect to the roles of these proteins in kinetochore assembly and in the context of using KNL2 genes for in vivo production of haploids in cowpea. Significance statementThis study identifies two paralogous KNL2 genes in cowpea and reveals their functional redundancy during centromere assembly and essential role in seed development. These findings expand our knowledge of kinetochore dynamics and provide a basis for exploring the evolutionary diversification of centromeric proteins in legumes.