Plant and Cell Physiology

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.

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.

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

Old Yellow Enzymes (OYEs) are a widely distributed family of ene-reductases that were first described in a Saccharomyces cerevisiae ferment. In plants, cis-12-oxo-phytodienoic acid (cis-OPDA) reductase (OPR) is the best studied OYE. In Arabidopsis thaliana, the peroxisomal AtOPR3 was characterized as the major OPDA reductase, which generates 3-oxo-2-(2-pentenyl)-cyclopentane-1-octanoic acid in the jasmonic acid (JA) biosynthesis. In Atopr3 lines, only small amounts of JA are detectable after wounding. Here, we describe an OPR-like enzyme (named BnOPR) from the gram-positive Brevibacillus nitrificans. The sequence was identified in an early version of the Physcomitrium patens genome and is assumed to be a contamination by a bacterium growing in association with P. patens. In complementation experiments with an Atopr3 line, we demonstrate that expression of BnOPR, fused with a peroxisomal targeting signal, rescues the male infertile phenotype and increases JA and JA-Ile levels. The catalytic parameters of BnOPR were determined for a set of substrates, including cis-OPDA and prednisone. Interestingly, B. nitrificans, B. brevis, and Paenibacillus physcomitrellae were shown to have a positive effect on P. patens growth. HighlightThe bacterial enzyme BnOPR rescues the male infertile phenotype of Atopr3 plants.

Photosynthetic membranes undergo structural remodeling in response to environmental stress by altering fatty acid composition and desaturation levels. These changes, mediated by fatty acid desaturases (FADs), are essential for maintaining photosynthetic performance and adaptation. In this study, we demonstrate that copper-deficient Chlamydomonas reinhardtii cells upregulate the expression of the gene encoding stearoyl-ACP desaturase (SAD/FAB2). We propose that this four-fold induction reflects an increased physiological demand for its primary product, oleic acid (18:1{Delta}9), and its subsequent downstream derivatives. The sad mutants exhibit a significant reduction in 18:1{Delta}9 content compared to wild-type cells, which correlates with diminished growth rates. Although SAD abundance increases under Cu deficiency, loss of SAD strongly alters C18 fatty acid composition across Cu conditions, while the growth defect is most apparent under Cu-replete conditions. This suggests that SAD activity may be a limiting factor in copper-depleted environments, leading to slower growth and reduced 18:1{Delta}9 levels in the uncharged galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), both of which are critical for photosynthetic function. The desaturation reaction catalyzed by SAD requires molecular oxygen and electrons supplied by ferredoxin (Fd). Using reciprocal IP-MS, we identified FDX5 as a Cu-deficiency specific SAD interacting protein. However, fdx5 mutants retained wild-type fatty acid profiles, indicating that FDX5 is not strictly required for SAD-dependent lipid desaturation and that another ferredoxin, likely FDX1, can compensate.

Heavy metal-associated isoprenylated plant proteins (HIPPs) are encoded by large gene families, which have diversified specifically in vascular plants. Their physiological functions and molecular mode of activity are currently largely unknown. In this study, we characterize a group of phylogenetically closely related genes HIPP32, HIPP33, and HIPP34 in Arabidopsis thaliana, revealing their essential roles in controlling diverse developmental pathways. Through comprehensive genetic analyses, we demonstrate that these genes exhibit partially overlapping pleiotropic functions, influencing multiple aspects of plant growth such as embryogenesis, maintenance of apical meristems, root architecture, shoot branching, leaf morphogenesis and floral organ formation. Transcriptomic profiling of hipp mutants identified significant deregulation in several regulatory pathways involved in plant hormone responses, with a specific impact on auxin signaling processes. Interestingly, we show that the analyzed HIPP proteins localize very specifically to plasmodesmata, suggesting their potential function in regulating intercellular communication in shaping plant development.

Plant hormones regulate almost every aspect of plant growth and development. KARRIKIN INSENSITIVE 2 (KAI2)-dependent signaling, which is thought to transduce signals derived from an unidentified ligand known as the KAI2 ligand (KL), regulates numerous traits, including seed germination in angiosperms and vegetative reproduction in bryophytes. The origin of KAI2 is believed to be ancient, and the evolution of its signaling pathways remains of significant interest. Ferns represent critical lineages for elucidating the evolution of land plant traits and growth mechanisms that enabled adaptation to terrestrial environments. Therefore, functional studies of key components of this pathway in ferns are essential for understanding the evolutionary trajectory of KAI2-dependent signaling during vascular plant diversification. However, experimental platforms for the CRISPR/Cas9 system, a powerful tool for investigating gene function, remain undeveloped in ferns. Here, we report an efficient CRISPR/Cas9 system based on ribozyme-gRNA-ribozyme (RGR) technology in the model fern, Ceratopteris richardii (C. richardii). We generated loss-of-function mutants of the KAI2 ortholog in C. richardii (CrKAI2), as well as the signaling components CrMAX2 and CrSMXL. We demonstrate that exogenous application of an artificial KL agonist increases the expression of KAI2-dependent signaling responsive genes in wild type plants; this response is abolished in Crkai2 mutants. These findings indicate that KAI2-dependent signaling is conserved in C. richardii. Furthermore, this study proposes an efficient CRISPR/Cas9 method that will facilitate genetic studies in ferns.

Oryza granulata Nees et Arn. ex Watt, a diploid wild rice (GG genome), possesses exceptional shade tolerance and is a key genetic resource for rice improvement. However, previous genome assemblies lacked continuity and completeness. Here we present a chromosome-scale reference genome of O. granulata using PacBio SMRT (113x), Hi-C (95x), and Illumina sequencing. The final assembly is ~764.24 Mb, with a scaffold N50 of ~59.32 Mb, and ~96.47% of the sequence anchored to 12 chromosomes. BUSCO completeness is ~98.6%. We annotated ~42,064 protein-coding genes, of which ~95.39% were functionally annotated, along with ~73.46% repetitive elements. The genome assembly and raw sequencing data are available at NGDC (PRJCA061980), NGDC GSA (CRA068332), and NGDC GWH (GWHISVE00000000.1). This high-quality genome will serve as a fundamental resource for evolutionary genomics, conservation biology, and breeding of shade-tolerant rice cultivars.

Microalgae are photosynthetic organisms capable of autotrophic growth. Their applicability in multiple industrial fields has been largely studied, thanks to their ability to fixate CO2 into high added value organic products like fatty acids and carotenoids. However, our understanding of the cellular signaling networks that control carbon flux and acclimation to environmental stress remains incomplete. In this study, we used the Chlamydomonas reinhardtii mutant strain vip1-1, which carries a loss-of-function mutation in the hexakisphosphate kinase re-sponsible for the synthesis of inositol pyrophosphates InsP7 and InsP8 (PP-InsPs), to investi-gate the role of these molecules during high-light acclimation. Our results indicate that PP-InsPs participate in the regulation of carbon storage in the form of starch and their deficiency increases TAGs levels in the algal cells. They also impact chloroplast-specific lipid remodeling by modifying membrane composition and fluidity through fatty acid desaturations and glycer-olipid composition. In addition, our findings suggest that PP-InsPs are involved in chloroplast-nucleus communication, where they coordinate transcriptional repression of photosynthesis associated nuclear genes (PhANGs), fatty acid desaturases and lipid synthases, contributing to cellular acclimation to high light. We also found that PP-InsPs modulating effect extended to protein synthesis and accumulation of Calvin-Benson-Bassham cycle intermediates. Therefore, we propose that PP-InsPs function as integratory molecules that balance carbon allocation between storage and structural pools, in response to environmental cues such as high light. These data uncover a novel function of PP-InsPs in high light acclimation and po-tentially in chloroplast-nucleus communication, providing new insights that may help engineering more resilient and efficient strains.

O_LIResearch on the functions and molecular mechanisms of long non-coding RNAs (lncRNAs) involved in regulating plant freezing tolerance is still in its infancy. Our previous research work identified that lncRNA DROUGHT INDUCED LNCRNA (DRIR) regulates gene expressions in Arabidopsis. However, the underlying molecular mechanism is still unknown. C_LIO_LIThis study demonstrates that lncRNA DRIR regulates plant freezing tolerance by affecting alternative splicing patterns of pre-mRNAs. C_LIO_LIThrough chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-MS), we identified two DRIR interacting proteins: CP29A and CP29B. We showed that the drirD mutant, which exhibits elevated DRIR expression and DRIR overexpression lines showed increased sensitivity to freezing stress, whereas DRIR RNAi lines were more tolerant to the stress. CP29A and CP29B bind to nuclear transcripts and, together with DRIR, regulate pre-mRNA alternative splicing under freezing stress. Notably, DRIR induces the relocalization of CP29A and CP29B to autophagosomes, leading to autophagy-mediated protein degradation. C_LIO_LICollectively, our findings elucidate the molecular mechanism by which DRIR influences the autophagy-based degradation of its binding proteins CP29A and CP29B, thereby regulating plant freezing tolerance by altering the alternative splicing patterns of pre-mRNAs, providing novel insights into the functions and mechanisms of lncRNAs in plants adapting to freezing environments. C_LI

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.

The TOR kinase is an important and conserved signaling hub in plants, as in other eukaryotes. However, the identification of the TOR pathway components and regulators in plants is still fragmentary. Using a genetic screen based on altered sensitivity to TOR inhibitors, we have selected an Arabidopsis mutant that develops leaf ectopic cell clusters of enlarged cells in a TOR-inhibition dependent manner. We have named this mutant loki (Localized growth depending on TOR Kinase Inhibition) and identified the causal mutation in a gene coding for the Arabidopsis homolog of the yeast Rav1 protein. This protein serves as the scaffold for the RAVE complex (Regulator of the ATPase of Vacuolar and Endosomal membranes), which regulates the V-ATPase activity in yeasts and animals. The overall V-ATPase activity is decreased in loki mutants and consistently the endosomal pH is increased. However, the vacuolar pH was found to be unaffected by this mutation. Interestingly, the det3 mutant, which is affected in the C subunit of the V-ATPase, also develops similar cell clusters. Finally, transcriptomic and metabolic analyses revealed that many pathways are affected by both the loki and det3 mutations, including cell wall integrity. This study establishes a new connection between the V-ATPase and the central TOR kinase in plants.

Plant regeneration is a major determinant of transformation and genome-editing efficiency, yet the endogenous regulatory networks controlling regenerative competence in horticultural crops remain incompletely understood. The miR319-TCP module regulates multiple developmental processes in plants, but its function in lettuce regeneration has not been defined. Here, we performed a genome-wide analysis of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) gene family in lettuce (Lactuca sativa). Thirty-three LsTCP genes were identified and classified into Class I/PCF, Class II/CIN, and Class II/CYC/TB1 groups. Five CIN-class genes, LsTCP2, LsTCP3, LsTCP4, LsTCP10, and LsTCP24, were predicted as high-confidence miR319 targets and supported by degradome-based cleavage evidence. MIR319-overexpression (OX319) explants showed enhanced de novo shoot regeneration, with 94.5% regeneration efficiency and 1.92 shoots per explant, whereas STTM-miR319 suppression (S319) explants showed reduced regeneration, with 28.5% regeneration efficiency and 0.36 shoots per explant. These phenotypes were associated with altered expression of several miR319-targeted CIN-TCP genes, particularly LsTCP4, LsTCP10, and LsTCP24. Disruption of LsTCP4 increased regeneration efficiency to 91.4% and shoot production to 2.05 shoots per explant, resembling the regeneration-enhancing effect of miR319 overexpression. In contrast, disruption of the non-target CIN gene LsTCP17 did not significantly affect regeneration under the tested conditions. Together, these results identify LsTCP4 as a key miR319-responsive negative regulator of de novo shoot regeneration and highlight miR319-mediated repression of LsTCP4 as a potential endogenous strategy for improving lettuce regeneration.

Coordination among cell growth, chloroplast expansion, and organelle genome dynamics is fundamental to algal physiology, yet its regulation remains unclear. We used time-resolved single-cell analyses to examine scaling relationships among cell size, chloroplast volume, nuclear dynamics, and nucleoid organization in Desmodesmus communis and Chlamydomonas reinhardtii under normal conditions and after inhibition of chloroplast DNA replication with nalidixic acid (NAL). Under control conditions, both species showed coordinated scaling among cell, chloroplast, and nuclear size, while nucleoid dynamics were driven mainly by changes in number. NAL disrupted these relationships in a species- and time-dependent manner. In C. reinhardtii, prolonged treatment uncoupled chloroplast and nuclear growth from cell expansion and led to fewer, enlarged nucleoids, consistent with impaired replication. In contrast, D. communis largely maintained coordinated scaling, with effects mainly limited to reduced nucleoid proliferation and delayed division. Temporal analyses indicated that NAL primarily affected nucleoid replication and segregation, with secondary consequences for chloroplast growth and cell-cycle progression. These findings identify chloroplast genome dynamics as a regulatory link between organelle growth and cell division.

Copper (Cu) is an essential micronutrient whose bioavailability is strongly affected by soil physicochemical properties. During evolution, plants have developed mechanisms to flexibly adjust their metabolism to Cu status. Superoxide dismutases (SODs), including Cu/ZnSOD1 (CSD1) and FeSOD1 (FSD1), are key antioxidant enzymes regulated in Cu dependent manner in Arabidopsis thaliana. Examination of CSD1 cellular distribution and activity revealed that CSD1 is a nuclear and cytosolic SOD whose abundance and activity respond to Cu availability inversely to FSD1. Combined microscopic and biochemical analyses of Cu-dependent dynamics revealed that, unlike FSD1, CSD1 localization in guard cells (GCs) remains independent of Cu availability. CSD1 escapes miR398-mediated regulation in GCs through a cell type-specific splice variant (CSD1.2) that carries an altered miR398-binding site. In silico analyses indicate that this mechanism is also present in crop species. Functionally, the csd1 mutant showed reduced sensitivity to abscisic acid (ABA)-induced stomatal closure, a phenotype rescued by reintroducing CSD1. Biochemical and reactive oxygen species (ROS) level analyses indicate that CSD1.2 most likely acts independently of its canonical enzymatic activity in GCs and functions upstream of the ROS burst in the ABA signaling pathway. Together, we present a novel, cell-type-specific mechanism that safeguards ABA-driven stomatal closure under fluctuating Cu supply.

Under natural growth conditions, plants are not usually exposed to the high-energy ultraviolet C range (UV-C, 100-280 nm) of the solar spectrum, as this is absorbed by the ozone layer. However, low doses of UV-C radiation can trigger stress responses in plants. Nevertheless, it is not yet fully understood how UV-C light affects plant development at the hormonal level. Here we show that a single one-min UV-C light pulse (20 W/m2) alters gibberellin (GA) homeostasis in Arabidopsis in two phases: initially, the level of GA12 - a key precursor of the final part of gibberellin biosynthesis - is reduced. Consistent with this, the transcript levels of the CPS, KS and KAO2 genes, which encode enzymes involved in the initial parts of gibberellin biosynthesis, decrease. The level of the plant hormone GA4 also decreases initially, probably due to the reduced GA12 precursor levels. However, in a second phase, the endogenous GA4 levels rise in UV-C treated plants relative to control plants. This increase leads to an early onset of flowering, as well as increased growth and fertility, in UV-C-treated Arabidopsis plants. The GA signalling mutant gdella does not exibit wild-type phenotypic responses to UV-C treatment, indicating that GA signalling is essential for the UV-C response. To further narrow down the responsible steps in the GA-signalling pathway, we tested the kao1 and kao2 mutants, which are both impaired in early gibberellin biosynthesis. Neither mutant displays phenotypic responses to the UV-C treatment, indicating that both genes are required for mediating the UV-C response. In contrast, the quintuple 2-oxidase mutant C19--2oxqM exhibits responses to UV-C treatment similar to the wild-type, suggesting that the five catabolic 2-oxidases that act on C19-GAs play a negligible role in regulation GA-hormone levels for growth and development in this case. HighlightUV-C pulse triggers biphasic gibberellin dynamics, delaying early development but ultimately enhancing growth and fertility in Arabidopsis thaliana.

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.

Vacuoleless Gametophytes (VLG) is a DC1 domain-containing protein initially characterized as essential for the development of both female and male gametophytes in Arabidopsis thaliana. In addition, VLG regulates stamen development through the involvement in lignin and jasmonic acid biosynthesis pathways. In this work, we report that VLG is also involved in salt stress tolerance in A. thaliana. Under salt stress, VLG-knock-down plants exhibited reduced germination, root elongation, biomass accumulation, photosynthetic pigment content, along with diminished expression of key salt-responsive genes. Conversely, these plants accumulated higher anthocyanins, and reactive oxygen species (H2O2 and O2-) compared to wilt type, indicating impaired oxidative stress control. In contrast, VLG-overexpressing plants showed a salt stress resistant phenotype with enhanced biomass and increased expression of salt-responsive genes under saline conditions. Together, these findings uncover an unexpected role for VLG as a positive regulator of salt tolerance, expanding the functional scope of DC1 domain proteins beyond reproductive development and providing new insights into plant mechanisms of abiotic stress resilience.

Leaf senescence directly affects lettuce quality and postharvest shelf life, but the regulatory roles of miR319-targeted and non-target CIN-TCP transcription factors remain unclear. Here, we examined whether the miR319-TCP module controls lettuce leaf senescence through separable genetic branches. MIR319 overexpression delayed dark-induced senescence, whereas STTM-mediated miR319 suppression accelerated chlorophyll loss, photosynthetic decline, and senescence-marker activation. Disruption of the miR319-targeted gene LsTCP4 phenocopied MIR319 overexpression, supporting LsTCP4 as a pro-senescence factor downstream of miR319. We further found that the miR319 non-target CIN gene LsTCP17 also promoted senescence, as tcp17 leaves retained more chlorophyll than wild type during dark treatment. Genetic combinations showed that tcp17 enhanced chlorophyll retention in the OX319 background and partially rescued the accelerated senescence phenotype of S319, indicating that LsTCP17 acts through a route separable from the miR319-targeted branch. Together, these results reveal a split CIN-TCP architecture in which miR319-targeted LsTCP4 and non-target LsTCP17 provide parallel pro-senescence inputs, offering a genetic framework for targeted improvement of lettuce quality.

High humidity greatly influences plant growth and development and triggers adaptive physiological responses such as leaf hyponasty (elongation of leaf petiole and upward leaf movement). A recent study identified Cyclic Nucleotide-Gated Ion Channels 2 and 4 (CNGC2/4)-mediated Ca2+ influx and Calmodulin Binding Transcription Activators 2 and 3 (CAMTA2/3)-mediated transcription as essential for high humidity response in Arabidopsis, but the upstream regulators that control these pathways remain unknown. Here, we show that the receptor-like kinase FERONIA and its co-receptor LORELEI-LIKE GPI-ANCHORED PROTEIN1 (LLG1) are required for a large portion of high humidity-associated Arabidopsis transcriptomic changes, including CNGC2, CAMTA-regulated genes, and cell wall remodeling genes, and for high humidity-induced leaf hyponasty. High humidity triggers a previously uncharacterized petiole-localized Ca2+ waves that precede hyponastic leaf movement. The petiole-localized Ca2+ signals were significantly altered in the fer-4 mutant. Thus, FERONIA is a key regulator of plant responses to extracellular high humidity. Highlights FERONIA plays a prominent role in transcriptomic responses to high humidity FERONIA is required for high humidity-induced leaf hyponasty High humidity induces petiole calcium waves FERONIA is required for normal petiole calcium waves in response to high humidity