Plant and Cell Physiology

O_LISymbiosis between legumes and rhizobia is beneficial on nutrient-poor soils, as it enables the fixation of atmospheric N2. To establish this symbiosis, gene expression in both the host plant and the symbiont has to be regulated. To understand the underlying RNA-mediated regulation of host gene expression, we designed experiments to identify competing endogenous networks involving circular RNA, microRNA, and linear transcripts during symbiosis, using wt and symbiosis-deficient Lotus japonicus mutants with the rhizobium Mesorhizobium loti (M. loti). C_LIO_LICircRNA, miRNA, and linear transcripts were identified from Lotus japonicus wildtype and CCamK mutant (ccamk-13; snf-1) seedlings without inoculation or with M. loti inoculation using deep short-read sequencing with rRNA-depletion and random primers. C_LIO_LIDifferentially expressed miRNAs showed negative correlations to predicted target genes and may regulate symbiotic processes. The symbiosis essential iron-sensor LjnsRING/BRUTUS expresses a circRNA which was upregulated in symbiotic treatments. This circRNA may act as a target mimic and contribute to nodule longevity. CircRNAs are predicted to act predominantly as trans-regulatory molecules with similar frequencies in Arabidopsis thaliania, Oryza sativa, and Lotus japonicus. C_LIO_LIWe identified novel miRNAs, long noncoding RNAs, and circRNAs, and nominated several as potential new regulatory non-coding RNAs that may act as target mimics to stabilize genes and support symbiosis. C_LI SummarySymbiosis between Lotus japonicus and Mesorhizobium loti involves treatment-specific regulation of competing endogenous RNA networks involving circular RNA, miRNA, and linear transcripts.

Calreticulins are multifunctional proteins involved in calcium homeostasis, protein folding, and cellular signaling. In common bean (Phaseolus vulgaris), the molecular mechanisms that regulate infection and nodule development remain incompletely understood. The main objective of this study was to characterize the role of the calreticulin gene PvCRT08 during infection and nodulation processes. We first analyzed the calreticulin gene family in the P. vulgaris genome and identified three members, with PvCRT08 showing the highest transcript accumulation in roots and after inoculation with rhizobia. Spatial and temporal promoter analyses in transgenic composite bean roots revealed PvCRT08 activity in root hairs and in infected cells and vascular bundles of mature nodules. RNA interference (RNAi)-mediated PvCRT08 down-regulation in transgenic roots increased the number of infection threads and enhanced nitrogen fixation efficiency, leading to the formation of larger and more functional nodules, although total nodule number was unaffected. In contrast, overexpression of PvCRT08 impaired infection thread progression, reduced the expression of key nodulation marker genes (PvCyclin and PvNIN), decreased nodule number, and diminished nitrogen fixation capacity. These findings identify PvCRT08 as a key regulatory component of early infection events and nodule development in common bean. Furthermore, the study provides new insights into the molecular control of symbiotic efficiency and highlights PvCRT08 expression is critical to optimize the equilibrium between infection efficiency and nodule functionality.

Strigolactones (SLs) are an important class of plant hormones that play crucial roles in regulating plant branching, root architecture, and organ development. However, the regulatory mechanisms underlying the crosstalk between SLs and other plant hormones remain largely unclear, particularly regarding the key regulatory genes that integrate and coordinate multiple hormonal signaling pathways. In this study, secondary cup seedlings of the Pisang Awak banana cultivar Yufen 6 at the eight-leaf stage were used as experimental materials. The roots were treated with a nutrient solution containing 30 mol/L exogenous SLs, while a nutrient solution supplemented with water served as the control. Tissues near the corm growth point were collected at 0, 15, 30, 60, 90, and 120 days after treatment to measure corm weight, height, and diameter, and transcriptome sequencing was performed using the collected tissues. Differentially expressed genes (DEGs) at different treatment stages were identified, followed by Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses to systematically investigate the crosstalk between SLs and endogenous hormone metabolism and signaling during corm development in Pisang Awak banana. The results showed that SL treatment significantly inhibited the weight, height, and diameter of the corm. The regulatory effect of SLs on Pisang Awak banana corm development exhibited a clear temporal dynamic pattern, representing a gradual accumulation process that ultimately triggers key developmental transitions. The highest number of DEGs was detected at 15 days after treatment, including 3943 upregulated genes and 3704 downregulated genes, indicating that this stage represents a critical phase for SL response initiation. GO enrichment analysis revealed that the DEGs were mainly involved in metabolic processes, biological regulation, response to stimulus, and regulation of biological processes. KEGG pathway analysis indicated that these DEGs were significantly enriched in pathways related to plant hormone signal transduction, starch and sucrose metabolism, and secondary metabolite biosynthesis. Further analysis revealed that the crosstalk between SLs and multiple hormone metabolic and signaling pathways is mediated by the SPL15 gene, involving auxin (IAA), cytokinin (CTK), abscisic acid (ABA), brassinosteroids (BRs), gibberellins (GA), and jasmonic acid (JA) pathways. This study reveals the molecular mechanism by which SLs regulate Pisang Awak banana corm development through SPL15-mediated integration of multiple hormonal signals, providing new insights into the role of SLs in regulating the development of underground organs in banana.

Photoperiod sensitivity (PS) is a key biological response in plants as they adapt to specific environments. Rice (Oryza sativa L.) exhibits a clear PS, as it implements critical phase transition decisions based on PS signals. In this study, we identified a novel PS gene, JMJ706, that is expected to deliver photoperiod-related signals to the flowering-time regulatory network in a day-length-dependent manner. The JMJ706 mutants exhibit early flowering under LD and later flowering under SD compared to WT plants. The gene encodes an H3K9me2 demethylase, and under long-day (LD) conditions, its demethylase activity facilitates the expression of Grain number, Plant height, and Heading-date7 (Ghd7). Since Ghd7 is a floral repressor in LD, it promotes the vegetative phase by delaying flowering. Under short-day conditions (SD), H3K9me2 demethylase activity facilitates Early heading-date 1 (Ehd1) expression, and it acts as a floral accelerator by inducing Heading date 3 (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1). Furthermore, we propose that the daylength-dependent promotion of target genes (Ghd7 and Ehd1) occurs through demethylation of specific promoter regions at a crucial time window. In addition, JMJ706 may play an important role in regulating plant architecture, including plant height. The natural variation in JMJ706 alleles shows high frequencies across major rice subpopulations, suggesting that JMJ706 could play an important role in the geographical distribution and adaptation of rice cultivars. Our results may add a new layer to the rice flowering-time regulatory pathway, supporting regional adaptation and potential for future breeding.

Plants continuously develop shoot branches derived from axillary meristems. In rice (Oryza sativa), TILLERS ABSENT1 (TAB1), an ortholog of Arabidopsis WUSCHEL, plays an essential role in axillary meristem formation by promoting stem cell proliferation. Although several genes associated with TAB1 function have been identified, the molecular mechanisms underlying stem cell proliferation during axillary meristem formation remain poorly understood. Here we identify ABERRANT SPIKELET AND PANICLE1 (ASP1), a TOPLESS-like transcriptional corepressor, as a novel regulator of axillary meristem formation, and investigate downstream mechanisms regulated by TAB1 and ASP1. In asp1, the stem cell region was expanded, indicating that ASP1 negatively regulates stem cell proliferation. Notably, WOX4, a paralog of TAB1, was precociously expressed in asp1, possibly in association with expansion of the stem cell region. Genetic analysis further revealed that asp1 mutation rescued the loss of axillary meristems in tab1. Transcriptome analysis showed that several type-A RESPONSE REGULATOR (OsRR) genes, encoding negative regulators of cytokinin signaling, were upregulated in tab1 relative to wild type, asp1, and the tab1 asp1 double mutant. Consistently, fluorescence of the synthetic cytokinin reporter was absent during axillary meristem formation in tab1 but was detected in wild type and tab1 asp1. Moreover, overexpression of OsRR10 inhibited axillary meristem formation, phenocopying tab1. Collectively, these findings suggest that TAB1 activates cytokinin signaling by repressing type-A OsRR expression, whereas ASP1 negatively regulates cytokinin signaling by promoting the expression of these genes. Thus, rescue of the tab1 phenotype by asp1 mutation probably reflects restoration of cytokinin signaling.

Processing of ribosomal RNA (rRNA) is essential for ribosome biogenesis, translation, plant development, and stress adaptation. Ribosome processing factor-2 (RPF2), which plays a role in the later stages of rRNA maturation, interacts with ribosomal protein L10A (RPL10A). RPF2 overexpression in Arabidopsis and Nicotiana benthamiana showed enhanced plant growth and trichome development due to increased gibberellic acid (GA) levels. Conversely, RPF2-silenced and mutant plants had a dwarf phenotype, reduced stomatal apertures, and decreased glucosinolate accumulation. RPF2 silenced and mutant plants also showed compromised nonhost disease resistance, whereas RPF2 overexpression lines exhibited enhanced disease resistance to both host and nonhost pathogens. RPL10A and RPF2 overexpression lines were sensitive to abscisic acid (ABA) and tolerant to drought, which is attributed to their unique roles in translation regulation. Despite having larger stomatal apertures, RPF2 overexpression plants displayed low pathogen multiplication rates and reduced water loss, indicating independent resistance mechanisms associated with ribosomal functions in translation regulation. Although both RPL10A and RPF2 proteins interact with each other and are involved in translation regulation, proteomic analysis suggests that they regulate the translation of distinct sets of genes during pathogen or drought stress. These findings indicate that RPF2 and RPL10A play independent roles in the regulation of unique protein translation.

Chilling stress induces photosystem I (PSI) photoinhibition in chilling-sensitive cucumber, in which insufficient activity of the chloroplast NADH dehydrogenase-like complex (NDH) leads to PSI over-reduction and damage. However, it is not yet clear whether these findings can be generalized to other species or what the molecular mechanism underlying impaired NDH function is. In this study, we first examined whether NDH is essential for PSI protection under chilling stress using an NDH-deficient rice mutant. Compared with wild-type plants, the NDH-deficient mutant exhibited enhanced PSI over-reduction and pronounced PSI photoinhibition under chilling stress. In contrast, rice plants expressing flavodiiron protein (FLV), which functions as an alternative electron acceptor downstream of PSI, did not exhibit PSI photoinhibition under chilling stress, demonstrating that electron sink capacity of NDH is important for PSI protection under chilling stress. Furthermore, analysis of the factors responsible for NDH dysfunction under chilling stress in cucumber revealed that chilling stress destabilizes the PSI-NDH supercomplex, leading to NDH monomerization and a consequent loss of NDH activity. This NDH monomerization is likely attributable to chilling-induced damage to the light-harvesting complex Lhca, which mediates the association between PSI and NDH. Together, these results indicate that NDH is essential for protecting PSI from photoinhibition under chilling stress in both rice and cucumber, and that chilling-induced destabilization of the PSI-NDH supercomplex represents a key molecular mechanism underlying PSI over-reduction and photoinhibition.

Symbiotic nitrogen fixation (SNF) relies on aerobic respiration, yet the key enzyme, nitrogenase, is extremely oxygen labile. Leghemoglobin (Lb) resolves this "oxygen paradox" by buffering and facilitating O2 transport. However, the dynamic regulation of Lb during nodule development remains poorly understood. Earlier results from our laboratory demonstrated that site-specific serine phosphorylation of Lb reduces its oxygen sequestration capacity. Here, we investigated the spatio-temporal regulation of Lb with the progress of rhizobial load during SNF. Fluorescence immunohistochemistry (FIHC) using anti-Lb antibody revealed that its localization gradually shifted from the plasma membrane to the cytoplasm of infected cells as nodules mature. Using phospho-peptide (Lb) specific antibodies, we found that serine phosphorylation triggers this translocation. Furthermore, FIHC in conjunction with immunoprecipitation followed by immunoblotting with phospho- and non-phospho-peptide specific antibodies demonstrated that the non-phosphorylated form is detectable as early as 9 dpi, whereas the phosphorylated forms were first detected at 11 dpi and progressively accumulated during nodule maturation. This spatio-temporal transition coincides with increasing rhizobial colonization and is accompanied by a decline in the non-phosphorylated pool. Therefore, the increased cytoplasmic pool of phosphorylated Lb, which exhibits reduced oxygen sequestration capacity, likely functions in promoting oxygen transport to sustain elevated rhizobial respiration. Together, these findings demonstrate that site-specific serine phosphorylation represents one of the key regulatory mechanisms linking Lb localization dynamics with progression of rhizobial infection, thereby contributing to the maintenance of oxygen homeostasis during SNF.

3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1 (PDK1), a conserved master regulator of AGC kinases, is encoded by two redundant genes in Arabidopsis thaliana, PDK1 and PDK2. pdk1 pdk2 mutants exhibit a broad range of defects, including apolar or arrested pollen tube growth, a phenotype also observed in agc1.5 agc1.7 mutants. Pollen-specific expression of constitutively active AGC1.5 in pdk1 pdk2 restores polar pollen tube growth, indicating that PDK1 functions upstream of redundant AGC1.5/AGC1.7 signaling in this process. In contrast, the PDK1 splice variant PDK1S0, lacking the phospholipid-binding PH domain, cannot restore polar pollen tube growth. Our results indicate a key role for the phospholipid PI(4,5)P2 in recruiting PDK1 through its PH domain to establish polar pollen tube growth, as PI(4,5)P2 marks the pollen germination initiation site together with PDK1, it forms a dome at the plasma-membrane of the pollen tube tip beneath which PDK1 remains largely cytosolic and exhibits reciprocal feedback regulation with the PDK1-AGC1.5/1.7 kinases. Defects in endocytosis and actin organization further support that phospholipid-dependent PDK1-AGC signaling maintains pollen tube growth polarity.

Fruit development is a typical angiosperm feature that greatly facilitates seed dispersal. Despite extensive studies on the gene regulatory network underlying pod shattering in the dry Arabidopsis fruit and the ripening process in the fleshy tomato fruit, it is yet unclear if a conserved regulatory network acts in early fruit development. Here, we investigated the roles of Petunia x hybrida (petunia) FRUITFULL (FUL), SHATTERPROOF (SHP) and APETALA 2 (AP2) homologs, three types of transcription factors repeatedly associated with fruit development and/or ripening. Petunia is closely related to tomato but produces dry dehiscent fruits like Arabidopsis. Our functional analysis revealed that the three petunia FUL-like genes, PETUNIA FLOWERING GENE (PFG), FLORAL BINDING PROTEIN 26 (FBP26) and FBP29, redundantly regulate endocarp development. They promote the formation of regularly shaped inner endocarp cells, probably via auxin/brassinosteroid signalling and cell wall modification. Furthermore, we discovered that the SHP-like gene FLORAL BINDING PROTEIN 6 (FBP6) has an opposite role, promoting more mesocarp-shaped endocarp cells, indicating that the FUL-like and SHP-like genes act antagonistically in early pericarp development. Finally, we show that the AP2-like genes REPRESSOR OF B-FUNCTION 1 (ROB1), ROB2 and ROB3 are crucial factors in petunia fruit development. rob1 rob2 rob3 mutants completely fail to dehisce and show major defects in pericarp patterning. The ROB transcription factors repress the activity of the FUL-like genes, and have, together with FBP6, an opposite effect on auxin and brassinosteroid signalling genes. Our study suggests that a module consisting of antagonistically acting TFs, including co-orthologs of AP2, FUL and SHP, regulates early pericarp patterning, at least partially via auxin and brassinosteroids.

Improving rice (Oryza sativa L.) yield requires a balanced enhancement of both sink size and source capacity. While many QTLs for sink size have been identified, only a few are known for source capacity, which is essential for achieving high yield. Here we identified qHP10 as a major QTL for increased photosynthetic rate by using chromosome segment substitution lines derived from a cross between the high-yielding indica cultivar Takanari and the average-yielding japonica cultivar Koshihikari. High-resolution mapping combined with CRISPR/Cas9-induced mutagenesis revealed that the causative gene underlying qHP10 is Mitogen-Activated Protein Kinase 4 (OsMPK4). A near-isogenic line carrying the OsMPK4Takanari allele (NIL-OsMPK4) had a 15-25% higher photosynthetic rate than Koshihikari. NIL-OsMPK4 also had higher stomatal conductance than Koshihikari but similar stomatal pore size and density, indicating that increased stomatal aperture increases photosynthetic rate. This enhancement is likely attributable to the down-regulation of OsMPK4 expression, which increases stomatal conductance and thus promotes CO2 uptake. Our findings demonstrate that OsMPK4 is a promising genetic target for increasing source capacity and, potentially, rice yield through molecular breeding. (175 words)

BackgroundRAB Guanine Nucleotide Dissociation Inhibitors (RAB GDIs) are important vesicle transport regulators in eukaryotes, participating in the functional cycle of RAB GTPases by stabilizing their non-active GDP-conformation. AimsWe address the importance of the three Arabidopsis thaliana RAB GDI paralogs by genetic and developmental analyses and put these results into the seed plants evolution context. MethodsWe use methods of genetics, microscopy and phylogenetics. ResultsOur genetic analyses of Arabidopsis T-DNA insertional mutants confirm recent CRISPR alleles data indicating lethality of double gdi1 gdi2 mutants, and our microscopic data point to embryo development arrest in double mutant seeds. We also confirm the involvement of GDI2 and GDI3 in pollen tube growth. Moreover, our data show that GDI1 also contributes to proper pollen function. Our phylogenetic analysis reveals independent diversification of RAB GDIs in Gymnosperms and Angiosperms, with early specialization of an Angiosperm reproduction-and gametophyte-related clade. ConclusionsIn Arabidopsis, RAB GDI1 and 2 are important for the vegetative growth while RAB GDI2 and 3 are vital for reproduction. Evolution of the RAB GDI family reflects the evolution of seed plants. HighlightsRAB GDIs are vital for plant growth and reproduction and act redundantly. Even the low-transcribed RAB GDI1 isoform contributes to the proper pollen function. Two RAB GDI clades evolved in early Angiosperms.

Photosynthetic electron transport is mediated by several protein supercomplexes that are spatially arranged in the thylakoid membranes of chloroplasts. The chloroplast NADH dehydrogenase-like (NDH) complex is part of the photosynthetic alternative electron transport (AET) chain, which reduces the plastoquinone (PQ) pool using reduced ferredoxin as a substrate. This NDH complex is associated with photosystem I (PSI) and mediates a portion of AET in stroma lamellae, whereas photosystem II (PSII) is concentrated in grana stacks. This study presents the findings regarding post-illumination chlorophyll fluorescence increase (PIFI), a protein crucial for regulating AET via the NDH pathway. A marked increase in NDH activity and a reduction in the PQ pool in the dark were observed in PIFI-deficient mutant strains (g-pifi) generated by genome editing. Blue native PAGE analysis indicated that PIFI was associated with the NDH-PSI supercomplex in the wild type, and the NDH complex was dissociated from PSI in the g-pifi mutants. Additionally, the g-pifi mutants exhibited a decrease in the maximum quantum yield of PSII (Fv/Fm). Notably, Fv/Fm was restored in a double mutant harboring both g-pifi and NDH-deficient pnsl1 mutations, demonstrating that deregulated NDH activity in g-pifi causes downregulation of PSII efficiency. However, the lower Fv/Fm was not observed in a mutant lacking thioredoxin m4 (trxm4), which showed deregulated NDH activity but maintained the NDH-PSI supercomplex. These data suggest that PIFI stabilizes the NDH-PSI supercomplex and maintains the spatial localization of PQ reduction via AET in thylakoid membranes, which is essential for the proper functioning of PSII.

Starch synthesis in wheat endosperm involves the initiation of large A-type starch granules during early grain development, followed by small B-type granules in later grain development. It is established that MAR-BINDING FILAMENT-LIKE PROTEIN 1 (MFP1) plays an important role in granule initiation in Arabidopsis chloroplasts, but how it influences A- and B-type initiations in wheat amyloplasts is not known. We discovered that due to a gene duplication in cereals, wheat contains two MFP1 paralogs, MFP1.1 and MFP1.2, which are both expressed in the developing endosperm. We generated a series of durum wheat mutants defective in all homoeologs of either MFP1.1 or MFP1.2, or both. While starch granule size distributions and granule morphology of mfp1.1 and mfp1.2 mutants were identical to those of the wild-type, the mfp1.1 mfp1.2 mutants had fewer, but larger B-type granules - suggesting that the two paralogs play redundant roles in B-type granule initiation. Consistent with this, both paralogs interacted with B-GRANULE CONTENT 1 (BGC1), a key protein required for proper B-type granule initiation in wheat, and both paralogs could partially complement defects in starch initiation in the Arabidopsis mfp1 mutant. Our work demonstrates that MFP1 is required for establishing correct starch granule number in non-photosynthetic amyloplasts, but its role in wheat is limited to B-type granule initiation. One-sentence summaryWheat has two MFP1 paralogs that interact with the granule initiation protein, BGC1 and influence B-type granule initiation in non-photosynthetic amyloplasts of endosperm.

Iron (Fe) is an essential micronutrient for plant growth, and the hormone auxin is a key regulator of developmental processes, including root gravitropism. Here, we investigated the molecular mechanisms underlying the crosstalk between iron nutrition and auxin-mediated root growth in Arabidopsis thaliana. Phenotypic analysis revealed that iron deficiency strongly shaped root system architecture and root gravitropism, and these phenotypes were exacerbated in the iron uptake mutant irt1-1. Genetic analysis revealed that iron deficiency did not aggravate the gravitropic defect of the pin2 mutant, eir1-4, suggesting that iron availability modulates root gravitropism through a PIN2-dependent pathway. Further transcriptomic analysis confirmed that iron deficiency significantly altered the expression of numerous genes related to the auxin pathway, providing molecular evidence for the observed physiological connection. Collectively, this study revealed that iron availability regulates root gravitropic growth by modulating PIN-mediated auxin transport and distribution, providing insights into how plants integrate nutritional cues with developmental programs. Graphical abstract A brief descriptionIron modulates auxin transport and root tip distribution by regulating PIN2 protein, thereby mediating root gravitropism in Arabidopsis. Public summaryO_LIIron nutrition specifically regulates root gravitropism and architecture in Arabidopsis. C_LIO_LIIron deficiency disrupts local auxin homeostasis in root tips and impairs asymmetric distribution in response to gravity. C_LIO_LIIron deficiency stress significantly reduces the abundance of PIN2 protein in root tip cells and disrupts its polar localization pattern on the plasma membrane, thereby precisely modulating polar auxin transport by interfering with the vesicle trafficking and recycling efficiency of PIN2. C_LIO_LIRNA-seq results showed that iron deficiency induced differential expression of multiple auxin-related genes, indicating that iron nutrition affects root development through the auxin pathway. C_LI

Previously, the chitin receptor-interacting protein kinase LIK1 (LysM receptor kinase 1/CERK1-interacting kinase) was shown to play an important role in regulating chitin signaling and plant defense. A limited proteolysis proteomics study revealed several LIK1-derived peptides that showed differential abundance between ATP-treated and mock-treated Arabidopsis samples, suggesting a possible involvement of LIK1 in extracellular ATP (eATP) signaling. To explore this possibility, LIK1 mutants were obtained and examined for their response to ATP. The results showed that mutations in LIK1 significantly reduced the expression of eATP-responsive genes. In addition, LIK1 was found to interact with the eATP receptor P2K1 and to be phosphorylated by it. The LIK1 protein was localized to the plasma membrane and its gene expression appeared to be ubiquitous. Collectively, these findings indicate that LIK1 not only contributes to chitin signaling but also participates in eATP signaling, highlighting its potential role as a shared component in multiple signaling pathways to regulate plant responses to diverse internal and external cues.

The RETINOBLASTOMA-RELATED (RBR) protein in plants functions as a cell-cycle inhibitor, regulating cell numbers in developing organs and establishing cellular quiescence during growth. Although the role of RBR counterparts in animals also involves regulating cell size, this potential function remains unexplored in plants. We investigated transgenic Arabidopsis plants with altered RBR levels and observed corresponding changes in cell size from embryogenesis through organ development. In addition, stomatal meristemoid cells with reduced RBR levels divided beyond the size threshold, whereas elevated RBR levels increased their size. RBR stimulated terminal differentiation in the stomatal lineage by inducing MUTE and CYCLIN D5;1 expression, whereas reduced RBR levels maintained asymmetric divisions through high SPEECHLESS and CYCLIN D3;1 expression. Interestingly, the cell proliferation-dependent phosphorylation of RBR at the conserved 911Ser site positively correlated with RBR protein levels in the transgenic lines and aligned with the effect of RBR on cell size. This study discusses the potential link between RBRs control of cell proliferation and cell size, providing new insights into the coordinated regulation of plant development.

Common bermudagrass (Cynodon dactylon) is a highly resilient and cosmopolitan grass widely used for turf, forage, and soil stabilization. Although its genome has been sequenced, little study has focused on characterizing genes underlying its resilience, including the NAC transcription factor family, which is well known for its physiological and stress-related functions. This study aimed to systematically characterize NAC TF genes in the bermudagrass genome and assess their potential roles in abiotic stress tolerance. A total of 237 CdNAC genes were identified and phylogenetically classified into 14 groups, including 40 members in the NAM/NAC1 class, which is associated with plant growth and development, and 23 members in the SNAC class, which is associated with stress responses. Tissue-specific RNA-seq analysis indicated that about one-fourth of CdNAC genes were expressed across all tissues, whereas 13 genes showed relatively higher expression in roots and 9 in inflorescence, suggesting both essential and specialized functions. Stress-responsive expression profiling revealed that 35 CdNAC genes were upregulated in response to drought, 43 to heat, 10 to salt, and 42 to submergence stress. Notably, CdNAC122, 149, and 155, the members of SNAC class, were consistently upregulated across all stress conditions, while others exhibited stress-specific expression, such as CdNAC37, 130, 145, and 199 in drought, CdNAC7, 12, 18, and 29 in heat, CdNAC46 and 151 in salt, and CdNAC9 and 31 in submergence. In contrast, 53 genes were downregulated during different stresses, with most belonging to NAM/NAC1, TERN, or OsNAC7 classes, possibly reflecting suppression of photosynthesis and development-related processes under stress. These results provide the first comprehensive characterization of CdNAC genes, reveal their distinct regulatory roles in abiotic stress responses, and establish a foundation for future functional validation and applications in breeding of stress-resilient bermudagrass.

The establishment of aphid-plant interaction involves the secretion of a salivary MIF protein. Morphological analyses revealed that aphid MpMIF1 prevents plant cell death, protects organelles from stress, and may promote plant cellular recovery. Co-expression of aphid MpMIF1 and the cell death inducer Npp1 revealed that MpMIF1 modulates autophagy-related genes ATG7/BECLIN1, impair plant senescence regulator ATAF1 and regulate apoptosis-like via Caspase-3-like activity. This effect on multiple-cell death pathways helps to maintain cellular homeostasis during aphid infection. Investigations on DNA Damage Response (DDR) signaling pathways demonstrated that aphid MpMIF1 reduces {gamma}H2A.X phosphorylation, maintains activity of the DNA repair protein RAD51 and stabilizes cell cycle checkpoint expression WEE1 under genotoxic stress. Therefore, MpMIF1 actively participates to the maintenance of a functional DDR. Finally, we showed that aphid MpMIF1 physically interacts with SOG1, a functional analog of animal p53 and central regulator of DDR, cell cycle arrest and programmed cell death in plants. These findings establish MpMIF1 as a key regulator of plant cell death during aphid-plant interactions and highlight its potential as a biotechnological tool for protecting major crops against aphid infection.

RAP2.6, an AP2/ERF transcription factor (TF), regulates plant stress responses; however, its role in floral transition remains unexplored. Here, we evaluated RAP2.6s role in flowering and the associated transcriptional changes in Arabidopsis thaliana under long-day conditions. RAP2.6-overexpressing line showed early flowering with fewer rosette leaves, whereas rap2.6-1 mutant flowered later, had more rosette leaves, and higher expression of the floral repressor FLOWERING LOCUS C (FLC). Early flowering in the overexpressing line was accompanied by transcriptional activation of the floral integrators GIGANTEA (GI), FLOWERING LOCUS T (FT), and COSTANS (CO), potentially through RAP2.6 interaction with GCC/DRE cis-regulatory elements. RAP2.6-mediated floral transition depended on nitric oxide (NO), with flowering time largely varying based on NO bioactivity. RAP2.6 was found to be a downstream regulator of Arabidopsis S-NITROSOGLUTATHIONE REDUCTASE 1 (GSNOR1) in controlling S-nitrosothiol (SNO) levels, flowering time, and silique formation. The NITRIC OXIDE-ASSOCIATED 1 (NOA1)-dependent reduction in NO levels abolished early flowering in 35S::RAP2.6 plants without affecting silique formation. Furthermore, enhanced cytokinin sensitivity and upregulation of cytokinin biosynthetic genes suggest cytokinin involvement in RAP2.6-mediated flowering. Together, these findings highlight the crucial role of RAP2.6 in regulating flowering time by integrating redox and hormonal signaling to coordinate reproductive development in A. thaliana.