Plant Physiology

Changes in intracellular calcium ion (Ca{superscript 2}) concentrations generate characteristic signatures that are decoded by specialized Ca{superscript 2}-binding proteins (CaBP). Although substantial progress has been made in understanding cytosolic calcium signaling pathways, calcium signaling within organelles, particularly chloroplasts, remains poorly understood, partly because only a few EF-hand CaBP have been identified in organelles. Here, we describe a novel EF-hand protein of 18 kDa, that was found to be associated with the chloroplast envelope and peroxisomal membrane and was therefore named OEF18 (ORGANELLAR EF-HAND PROTEIN OF 18 kDa). OEF18 has a very unusual structure, containing an N-terminal myristoylation site, followed by one EF-hand in the N-terminus facing to the cytosol, and a transmembrane domain in the C-terminus. OEF18 membrane-targeting was found to be mediated by ANKYRIN REPEAT-CONTAINING PROTEIN 2A (AKR2A) via the C-terminal transmembrane domain of OEF18. Furthermore, the EF-hand in OEF18 bound Ca{superscript 2} at a physiological concentration that led to a large protein conformational change, inducing oligomerization of the N-terminal part. We found that oef18 mutants accumulated less jasmonic acid (JA) and its bioactive conjugate JA-Ile, likely causing a defect in the insect herbivore response. Wild-type OEF18 complemented the herbivory phenotype of oef18 mutants, whereas an EF-hand point mutant lacking Ca{superscript 2}-binding capacity failed to restore the wild-type response. Furthermore, OEF18 was required for resistance to salt stress in combination with dark-induced senescence. Together, these results establish OEF18 as a previously unrecognized organellar Ca{superscript 2} sensor that couples Ca{superscript 2} perception to JA-mediated defense and abiotic stress responses in plants.

Acetic acid (AA), a volatile compound present in diverse microbial-derived biostimulants, enhances drought tolerance in plants. In Arabidopsis, soil-applied AA action has been linked to histone H4 acetylation and activation of jasmonate (JA) signalling. However, the mechanisms underlying AA action in crops of agronomic interest remain poorly understood. Here, we used an integrative approach to evaluate the effects of soil-applied AA on fruit yield, physiological performance, and leaf transcriptomic and proteomic profiles of tomato plants grown under optimal and suboptimal irrigation conditions (OIC and SOIC, respectively). While AA had little effect under OIC, it significantly enhanced fruit yield and photosynthesis under SOIC. Long-term water deficit triggered extensive transcriptomic and proteomic reprogramming, particularly affecting photosynthesis, RNA processing, protein biosynthesis-, modification- and homeostasis-related processes. Under SOIC, AA induced marked molecular changes that were not consistent with activation of canonical JA signaling pathways. Notably, only [~] 10% of the drought- or AA-responsive proteins were associated with corresponding transcript changes, highlighting a predominant role of regulatory layers beyond the transcriptional control to both long-term water deficit- and AA-induced protein remodeling. Strikingly, AA attenuated 47% and 35% of the transcriptomic and proteomic alterations induced by long-term water deficit, respectively. In addition, AA altered the abundance of numerous proteins that do not respond to drought, particularly ribosomal proteins and proteins involved in RNA processing. Collectively, our findings indicate that AA enhances tolerance to prolonged water deficit in tomato through mechanisms largely independent of canonical JA signaling and involving extensive downstream regulatory processes that partially mitigate stress-induced molecular reprogramming.

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.

O-Acetylation is the most abundant xylan decoration in eudicot plants and plays a critical role in determining xylan conformation and its interactions with cellulose and lignin, thereby contributing to secondary cell wall (SCW) integrity. In Arabidopsis, loss of the xylan O-acetyltransferase TBL29/ESK1 causes collapsed xylem and growth defects that can be suppressed by mutations in strigolactone (SL) biosynthesis genes such as MAX3. However, the molecular basis of this suppression remains unknown. Hypoacetylated xylan in tbl29 has a higher frequency of methyl glucuronic acid (MeGlcA) substituents, while the ratio of GlcA/MeGlcA is recovered in tbl29 max3. Furthermore, gene expression analyses reveal that the three xylan glucuronoxylan methyltransferases (GXM1/2/3) involved in xylan MeGlcA modification are upregulated in tbl29 SCWs but downregulated in tbl29 max3. Genetic analysis shows that the transcription factor MYC2 is required for max3-mediated suppression: the loss of MYC2 in tbl29 max3 prevents growth recovery and reverts GXM genes expression and xylan MeGlcA substitution levels. We propose a model where SL deficiency enhances MYC2 transcription, which in turn represses GXMs, thereby fine-tuning xylan methylation and re-establishing the MeGlcA/GlcA substitution balance under conditions of reduced O-acetylation. Our findings identify a MYC2-dependent regulatory module linking SL signalling to xylan methylation and reveal a genetically encoded compensatory mechanism that mitigates the consequences of defective xylan O-acetylation. More broadly, this work demonstrates that plants can preserve SCW function through adaptive remodelling of polysaccharide substitution patterns, highlighting an unexpected plasticity in SCW biosynthesis. Significance StatementSecondary cell wall integrity depends on the coordinated modification of xylan. We show that defects caused by reduced xylan O-acetylation can be alleviated through a strigolactone- and MYC2-dependent pathway that alters xylan methylglucuronidation. Rather than restoring the original wall composition, this mechanism appears to compensate for the loss of O-acetyl groups by remodelling polysaccharide substitution patterns to maintain cell wall function, revealing a new layer of plasticity in secondary wall biosynthesis.

To identify novel regulators of plant drought tolerance, we performed a forward genetic screen using thermal infrared imaging to detect alterations in leaf temperature, a proxy for transpiration. This screen identified AT-rich Interacting Domain Protein 4 (ARID4), a protein involved in chromatin remodeling, as a modulator of transpiration. arid4 loss-of-function mutants exhibited cooler leaves, increased water loss from detached leaves, and heightened susceptibility to drought relative to wild-type plants. Stomata of arid4 mutants were hyposensitive to abscisic acid (ABA), displaying reduced ABA-induced reactive oxygen species (ROS) production and impaired stomatal closure. Transcriptome profiling under drought revealed extensive misregulation of genes involved in stress-responses, redox homeostasis, iron acquisition, and photosynthesis. These findings indicate that ARID4 modulates the expression of key regulatory genes to maintain redox homeostasis and photosynthetic efficiency, thereby conferring drought stress tolerance.

O_LIPlants continuously adjust photosynthesis to balance growth and photoprotection under changing environmental conditions. Environmental fluctuations frequently impose a mismatch between energy production and CO2 assimilation. How photochemical reactions are regulated to maintain performance under these conditions remains a central question in plant biology. C_LIO_LIWe previously developed transplastomic tobacco (Fd1-OE plants) overexpressing ferredoxin (Fd) displaying enhanced photoprotection and growth penalties with a variegated leaf phenotype under greenhouse conditions. Here, we investigate how these plants respond to different growth irradiances using physiological, ultrastructural, and photosynthetic analyses, including PAM, gas exchange, and P700 absorbance measurements, and dynamic-light assays. C_LIO_LIFd1-OE plants progressively recovered growth, leaf phenotype and photosynthetic performance as growth irradiance increased, reaching near WT performance at 1400 mol m-{superscript 2} s-{superscript 1}. This enhanced adaptation to "high-light" was associated with a larger fraction of open PSII reaction centers and enhanced NPQ. Dynamic-light analyses further revealed faster plastoquinone (PQ) turnover, a more oxidized PQ pool and enhanced electron withdrawal downstream of PSI. C_LIO_LIOur results indicate that Fd overexpression redefines the balance between photochemistry and photoprotection. This adjustment shifts adaptation toward higher irradiance and enhances photosynthetic performance under changing light environments. Electron partitioning downstream of PSI emerges as a promising target to improve photosynthetic resilience. C_LI One sentence summaryOverexpression of Fd1 in tobacco plants adjusts photosynthesis/photoprotection trade off to enhance high-light adaptation

Salinity tolerance is commonly associated with whole leaf Na exclusion and maintenance of K homeostasis, but whether spatial ion partitioning among functional leaf compartments contributes to stress adaptation remains unclear. Here, we investigated the relationship between bulk leaf and stomatal complex ionomes and gas exchange performance under salinity using two contrasting genotypes in both maize and faba bean crops. Maize generally maintained higher photosynthesis and stomatal conductance than faba bean under salt stress, which was associated with lower Na accumulation, stronger K retention and distinct ion partitioning patterns between bulk leaf tissue and the stomatal complex. Enrichment analysis revealed that stomatal complex ion composition provided information beyond bulk leaf ion concentrations, with Na and Cl- showing distinct distribution patterns associated with photosynthetic performance. Integrating physiological and ionomic traits further demonstrated that stomatal-complex ion traits captured additional variation in salinity responses. These findings identify the stomatal complex as a functionally distinct ionomic compartment and reveal compartment-specific ion partitioning as an important mechanism underlying species-specific salinity tolerance.

Salinity stress alters cellular ion homeostasis and photosynthetic activity, yet how guard cell chloroplast architecture contributes to salt adaptation remains poorly understood. Here, we investigated salt-induced chloroplast remodeling in guard cells of two faba bean genotypes, Fuego and Scoop, by integrating 3D chloroplast imaging, ion enrichment analysis and photosynthetic measurements. Salt stress induced distinct genotype-dependent changes in chloroplast morphology, with Fuego exhibiting pronounced chloroplast enlargement and reduced surface area-to-volume ratios under Na2SO and high NaCl, whereas Scoop showed treatment-dependent remodeling with larger chloroplasts under low NaCl and higher surface area-to-volume ratios under Na2SO and CaCl2. These structural responses were associated with differential Na and Cl partitioning at the stomatal complex surface. In Fuego, chloroplast size was negatively associated with photosynthetic rate, whereas Scoop showed positive relationships between chloroplast size and photosynthetic performance. Multivariate analysis further revealed coordinated associations among chloroplast architecture, ion enrichment and photosynthesis that distinguished the two genotypes under salinity. Our findings demonstrate that guard cell chloroplast remodeling is closely associated with genotype-specific salt responses and local ion partitioning. Integrating organelle structural plasticity with local ion homeostasis provides a spatially resolved perspective on the cellular basis of genotype-dependent salinity adaptation.

Tomato (Solanum lycopersicum) is classified as a day-neutral plant, whereas its wild relatives exhibit delayed flowering under long day (LD) conditions due to higher activity of the SELF-PRUNING 5G (SP5G). In Arabidopsis thaliana, CONSTANS (CO) activates FLOWERING LOCUS T (FT), a homolog of SP5G, but whether and how CO integrates with SP5G in tomato flowering was unclear. Here, we demonstrate that SlCOL1 (the tomato CO homolog) delays flowering by directly activating SP5G in a PHYTOCHROME B1 (PHYB1)-dependent manner. Importantly, genetic and molecular analyses combining a photoperiod-responsive tomato line carrying the wild SP5G allele from S. pennellii, together with SlCOL1 and flowering-pathway mutants, revealed synergistic crosstalk among the photoperiodic SlCOL1-SP5G module, age-dependent pathway (mediated mainly by the microRNA156-SlSBP module), gibberellin (GA) pathway, and SINGLE FLOWER TRUSS (SFT) pathway. Mechanistically, we show that SP5G forms a complex with miR156-targeted SlSBP13 to directly regulate SFT expression, and that GA may interfere with SP5G activity. Together, these findings revealed a coordinated network that integrates multiple flowering signals to modulate both shared and pathway-specific targets. Our findings provide a significant advance in understanding the molecular regulation of tomato flowering and offer promising avenues for breeding strategies optimized for diverse environmental conditions and latitudes.

Peptide hormone signaling coordinates plant growth and osmotic stress responses, yet how the transition between these responses is regulated remains poorly understood. Here, we investigated the function of the rice PLANT PEPTIDES CONTAINING SULFATED TYROSINE 8 (OsPSY8) peptide in osmotic stress responses. OsPSY8 was predominantly expressed in root tissues under non-stress conditions, with preferential expression in lateral roots where it promoted root growth. Osmotic stress rapidly reduced OsPSY8 expression in roots through the OsWRKY24 transcription factor. Loss-of-function ospsy8 mutants exhibited enhanced osmotic stress tolerance, whereas OsPSY8 overexpression increased osmotic stress susceptibility. Transcriptomic analyses revealed that disruption of OsPSY8 activated stress-responsive pathways, including those associated with lignin biosynthesis, compatible solute production, cell wall remodeling, and reactive oxygen species (ROS) scavenging, and was accompanied by increased lignin accumulation in roots. In contrast, overexpression of OsPSY8 resulted in maintenance of growth-associated transcriptional programs while suppressing stress-responsive pathways under osmotic stress. Together, these findings identify OsPSY8 as an important regulator of the transition from growth to stress adaptation in rice and suggest that stress-induced repression of PSY signaling is required to disengage growth programs and activate adaptive responses during osmotic stress. Significance StatementCrop survival during drought depends on the ability to transition from growth to stress adaptation. Plant peptide hormones have emerged as important regulators of this critical transition, highlighting the importance of investigating their roles and potential for improving crop resilience. We show that a rice peptide hormone regulates this transition. Under non-stress conditions, this peptide hormone, predominantly expressed in rice roots, promotes root growth while suppressing stress responses. During osmotic stress, expression of the peptide hormone decreases, resulting in activation of stress-responsive pathways, such as lignin biosynthesis and reactive oxygen species scavenging. These findings demonstrate that a peptide hormone coordinates the balance between growth and stress adaptation in rice, with broader implications for understanding and improving crop resilience.

The plant circadian clock governs the precise regulation of plant developmental and environmental responses within a 24 h photoperiod. Consisting of a series of interconnected transcription factor moderated feedback loops, each operating at specific times of day within the photoperiod, more than 30% of Arabidopsis genes show circadian regulation. REVEILLE (RVE) genes exhibit highest expression in the afternoon, and act as activators of afternoon-expressed clock genes, including TOC1 and PRR5. Specifically, RVE4, 6 and 8 have been identified as regulators of both plant growth and development, along with temperature responses. Previous work using Arabidopsis has implicated soybean RVE8-like proteins in drought responses, however, there remains a lack of understanding of how these RVEs control abiotic stress responses, particularly relating to proteome-level regulation. Here, using quantitative proteomics, we resolve how the rve4 6 8 proteome responds to osmotic and salt stress at end-of-day and end-of-night timepoints. Our results indicate that rve4 6 8 plants have substantially altered regulation of proteins related to osmotic stress, photosynthesis and phenylpropanoid metabolism, particularly at end-of-day. Further, we resolve new drought-responsive targets impacted by the loss of RVE4, 6, and 8 that are involved in vesicle transport, fatty acid metabolism and abscission. Overall, our data provide a new resource for understanding how RVE8-like proteins impact plant drought-like responses, offering opportunities for future chronoculture-informed breeding of climate-resilient crops.

Accurately modeling carbon (C) allocation is essential for predicting crop yield and the performance of new cultivars in various environments. Most crop models allocate C empirically, using fixed partitioning tables or harvest indices that prescribe allocation without representing the underlying physiology, limiting their predictive power under novel conditions. A mechanistic alternative, in which C allocation emerges from local utilization and transport, could instead respond dynamically to environmental changes, source-sink perturbations, and organ-level trait modifications. To achieve this design, we integrated a utilization-transport-resistance (UTR) allocation model into the Soybean-BioCro crop growth modeling framework. We calibrated and validated the model using organ biomass data from two soybean cultivars grown at two CO2 levels over eight seasons, achieving accuracy comparable to partitioning-based models while predicting more reasonable carbon allocation fractions. Further, the UTR-BioCro model predicted leaf and stem total nonstructural carbohydrate concentrations with reasonable accuracy compared to experimental measurements across the 2022 growing season. A local sensitivity analysis of the model parameters indicated that the onset of reproductive growth influenced yield more strongly than utilization or transport parameters suggesting the timing of this transition as a potential target for crop improvement. Finally, the UTR-BioCro model reproduced yield responses to source-sink perturbations including shading and pod removal, and captured the qualitative response to defoliation without requiring scenario-specific tuning as most partitioning approaches require. By grounding C allocation in physiological mechanisms, this work provides a foundation for predicting crop responses across diverse environments and engineered traits, supporting crop improvement for a changing environment.

Elevated temperature has been shown to compromise salicylic acid (SA)-mediated immunity in plants. The Arabidopsis thaliana accession C24 retains constitutively elevated SA and resistance to the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) at elevated temperature. C24 exhibits reduced biomass compared to that of a commonly studied accession, Col-0, in which SA-mediated immunity is compromised at elevated temperature. Neither the genetic basis of temperature-resilient immunity (TRI) nor the apparent growth-defense tradeoff in C24 is known. Here, we show that a Col-0 x C24 recombinant inbred line (RIL) population resolves TRI to a chromosome 5 locus accounting for most of the mapped genetic variance. This locus (named TRI hereinafter) coincides with a hotspot of structural rearrangement between the two accessions and includes a calcium-sensor gene (CBL9) and several NLR-type paralogs found only in C24. Consistent with a calcium-dependent signaling component, C24 mounts an elevated cytosolic Ca{superscript 2} response to Pst DC3000. Surprisingly, across the RIL population, disease resistance and biomass are only weakly correlated, with some lines exhibiting both large biomass and high pathogen resistance. These results show that temperature-resilient disease resistance is not only genetically tractable in C24 but also can be uncoupled from biomass cost. The TRI locus in C24 therefore encodes a natural mechanism(s) of temperature-resilient immunity with the growth-defense tradeoff resolved.

Tropospheric ozone (O) is a major atmospheric pollutant that affects plant carbon metabolism, redox homeostasis, and secondary metabolism, including the biosynthesis and emission of biogenic volatile organic compounds (BVOCs). However, the contribution of BVOCs to O3 tolerance, particularly in tropical woody species, remains poorly understood. Here, we investigated whether acute O exposure (cumulative AOT40 of 3497.82 ppb h) induces alterations in photosynthetic performance, redox homeostasis, and BVOC partitioning in Eugenia uniflora. We evaluated gas exchange, photosynthetic pigments, ascorbate and glutathione pools, emitted BVOCs, modeled intercellular BVOC concentrations, and the relative carbon cost associated with BVOC emissions. O exposure significantly increased net CO2 assimilation without affecting stomatal conductance, transpiration, leaf water status, or chlorophyll concentrations, indicating maintenance of photosynthetic performance. Carotenoid concentrations and total glutathione decreased, whereas glutathione redox status was maintained. O induced marked compound-specific changes in BVOC composition and partitioning. Several monoterpenes appeared exclusively under O exposure, {gamma}-elemene emission increased significantly, and the relative distribution of individual BVOCs between the modeled intercellular and emitted pools was altered. These findings show that the response of E. uniflora to acute O exposure was characterized by interplay among carbon assimilation, glutathione redox regulation, and BVOC partitioning rather than by increased total volatile emission. Enhanced carbon assimilation occurred without additional carbon loss through BVOC release, while changes in the modeled intercellular pool indicate that part of the volatile response remained within the leaf. Our findings highlight BVOC partitioning as an important dimension of the plant response to oxidative stress and demonstrate that emission measurements alone may not fully capture the fate and potential physiological role of volatile carbon under O exposure. O_FIG O_LINKSMALLFIG WIDTH=178 HEIGHT=200 SRC="FIGDIR/small/743946v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@653af1org.highwire.dtl.DTLVardef@ca5forg.highwire.dtl.DTLVardef@1e641bforg.highwire.dtl.DTLVardef@1e68fae_HPS_FORMAT_FIGEXP M_FIG C_FIG BVOC Partitioning Contributes to Oxidative Stress Defence Under Acute O Exposure

The Arabidopsis mutants lacking the pathogen-inducible callose synthase POWDERY MILDEW RESISTANCE 4 (PMR4)/GLUCAN SYNTHASE-LIKE 5 (GSL5) exhibit enhanced disease resistance accompanied by reduced growth, spontaneous necrosis, and premature senescence. This autoimmune phenotype of pmr4 is associated with a high accumulation of N-hydroxypipecolic acid (NHP) and a moderate increase in salicylic acid. We previously reported three suppressors of pmr4 (spm), PAD4 and the NHP biosynthetic genes ALD1 and FMO1, whose loss-of-function mutations abolish NHP accumulation and completely suppress the growth defects of pmr4, demonstrating that NHP is critical to the autoimmune growth phenotype. Here, we characterize a fourth suppressor, spm4, which similarly fully suppresses the growth defects of pmr4. Map-based cloning and molecular genetic analyses revealed that spm4 carries a mutation in FLOWERING LOCUS D (FLD). Reintroduction of a wild-type FLD allele into the pmr4 spm4 background restored both NHP accumulation and the characteristic growth defects of pmr4. Conversely, independent FLD loss-of-function alleles generated by CRISPR/Cas-mediated genome editing suppressed NHP accumulation and rescued the autoimmune growth defects of pmr4, confirming that FLD is required for manifestation of the pmr4 phenotype. Beyond its established role in flowering-time regulation, FLD has been implicated in plant immunity, particularly in systemic acquired resistance (SAR), although the mechanistic basis of its immune function remains poorly understood. Our findings identify FLD as an essential positive regulator of NHP accumulation, thereby providing a mechanistic link between FLD-dependent epigenetic regulation, NHP-mediated signaling, and SAR.

Abscisic acid (ABA) plays a critical role in regulating plant responses to abiotic stress by modulating various physiological processes through complex molecular networks. TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORS (TCP) transcription factors are known developmental regulators, but their roles in ABA signaling and abiotic stress responses remain poorly understood. We addressed this question by analyzing the closely related Arabidopsis thaliana class I TCPs TCP14 and TCP15 using a variety of genetic and molecular approaches. We found that TCP14 and TCP15 negatively influence ABA and salt stress responses. They function by directly activating genes encoding negative regulators of ABA signaling, such as ABA-INSENSITIVE FIVE-BINDING PROTEIN 2 (AFP2), thereby suppressing the expression of ABA-INSENSITIVE 5 (ABI5) and downstream ABA-responsive genes to inhibit ABA responses under non-stressful conditions. Notably, the TCPs are targets of ABA-mediated regulation, as ABA negatively affects TCP14 and TCP15 protein abundance, providing a mechanism to limit TCP-dependent transcriptional growth responses and de-repress ABA-signaling pathways during times of stress. We propose that the antagonistic interplay between class I TCPs and ABA may serve to fine-tune plant growth and stress responses according to environmental conditions, positioning TCP14 and TCP15 as crucial players in balancing plant developmental progression and stress adaptation.

Root phenotypic plasticity is often proposed to improve crop performance under stress, yet it remains unclear how much plasticity is beneficial and whether adaptive responses require changes across many traits or adjustments in few specific traits. Using public data of 6,500 field-grown maize and barley plants, this study examined the extent and distribution of root plasticity, and when it is associated with yield stability. We quantified root plasticity across nine anatomical and architectural traits using complementary statistical models and applied a feature-discovery framework to identify the drought-associated optimal integrated phenotypes and determine whether plasticity toward these phenotypes improved yield stability. More plasticity did not mean greater yield stability. Neither the number of plastic traits nor the magnitude of plastic responses predicted yield stability. Rather, we identified species-specific high-yielding, stable integrated phenotypes defined by distinct trait configurations. Critically, genotypes whose plastic responses moved their root phenotype toward these targets achieved greater yield stability, whereas movement away from them was associated with lower stability. Root plasticity is adaptive when it shifts root phenotypes towards an optimal integrated phenotype. These findings show that the value of plasticity depends on the trajectory of phenotypic change rather than its magnitude alone.

Topologically associating domains (TADs) are essential structural and functional modules of the genome that play a crucial role in regulating gene expression. In this study, we systematically investigated the conservation and evolution of TADs in five closely related crops, including maize, sorghum, coix, foxtail millet and broomcorn millet. Our results show that 74% of TAD boundaries are conserved between two inbred maize lines, B73 and Mo17, and that approximately 50% or more of TAD boundaries are conserved across different crop species. TAD number remains relatively stable in the face of changes in genome size. However, the length of TADs varies depending on genome size. Furthermore, we found that large-scale transposable element expansion leads to TAD expansion, while chromosomal inversions lead to TAD fusion and the formation of new TAD boundaries. Frequent chromatin interactions between subgenome chromosomes occur after whole-genome duplication. Moreover, we also found that crossovers are enriched at TAD boundaries in maize, indicating the importance of TADs as a fundamental unit during species evolution. Overall, our study provides insights into the conservation and evolution of TADs in crop genomes and their roles in genome organization and function.

Most proteins targeted to the organelles of endosymbiotic origin are encoded in the nuclear genome, placing them under the regulatory dominance of the nucleus. For photosynthetic eukaryotes, nuclear-encoded chloroplast proteins arise via two routes: First, genes of cyanobacterial origin were relocated to the nucleus through endosymbiotic gene transfer (EGT). Second, proteins of eukaryotic origin emerged to support chloroplast function and structure. These proteins are reimported into the chloroplast via an import machinery. Reversing the transfer of such genes from the nucleus to the chloroplast genome may offer insights into chloroplast regulation and evolution. In this study, we established a highly efficient and accessible electroporation protocol for chloroplast transformation in the green alga Chlamydomonas reinhardtii, and used it to reverse-transfer two nuclear-encoded genes encoding proteins arising via the two routes described above: the cyanobacteria-derived chloroplast division protein FtsZ1 and the Rubisco-linker EPYC1 of eukaryotic origin. Regardless of origin, both chloroplast-encoded FtsZ1 and EPYC1 showed proper localization and functionality comparable to their nuclear-encoded counterparts. Together, our study provides a robust protocol for chloroplast transformation, a platform for investigating the evolutionary drivers of EGT, and a foundation for advancing chloroplast bioengineering. SIGNIFICANCE STATEMENTO_LIEndosymbiotic gene transfer has resulted in the mass migration of genes from the chloroplast genome to the nuclear genome. Reversing the gene transfer could reveal the evolutionary significance of genome partitioning. C_LIO_LIUsing the green alga Chlamydomonas reinhardtii, this study developed an efficient, electroporation-based protocol for chloroplast transformation. Relocating the genes encoding two chloroplast-targeted proteins, FTSZ1 and EPYC1, to the chloroplast genome showed that the proteins maintained normal localization and function. C_LIO_LIThe established transformation protocol facilitates systematic testing of reverse gene transfer to elucidate the potential evolutionary advantages of genome partitioning and opens new avenues for chloroplast bioengineering. C_LI

Aquaporins at the plant plasmalemma are divided into two highly conserved subclasses, PLASMA MEMBRANE INTINSIC PROTEINs 1 (PIP1) and PIP2. Arabidopsis thaliana encodes five PIP1 and eight PIP2 isoforms. Individual loss-of-function mutants had been employed for functional analyses. Here, we observe that the pip2;1 pip2;2 pip2;4 pip2;6 pip2;7 quintuple mutant defective of major PIP2 isoforms concomitantly leads to a strongly reduced PIP1 protein level. Lower order mutants pip2;1 pip2;2 and pip2;1 pip2;2 pip2;7 still harbor only 60% and 20% residual PIP1, respectively. This repression is established post-translationally, since neither PIP1s steady-state transcripts nor polysome-associated PIP1 mRNAs are suppressed by pip2;1 pip2;2 pip2;7. Thus, the two major pathways operating in eukaryotes for removal of aberrant proteins, ubiquitin proteasome system (UPS)-dependent ER-associated degradation (ERAD) and autophagy/vacuole-linked degradation, were assessed. Introgression of atg7 blocking autophagy-mediated degradation does not affect the PIP1 protein level of pip2;1 pip2;2 pip2;7. In contrast, introgression of ERAD loss-of-function mutations hrd1A hrd1B and dln1 into pip2;1 pip2;2 pip2;7 partially stabilizes its PIP1 protein level. PIP1 accumulates intracellularly upon pharmacological inhibition of proteasomal degradation by MG132. Nevertheless, the lack of a full PIP1 recovery by these means suggests the flexible operation of parallel ERAD components or unknown pathways. In conclusion, the essential dependence of PIP1 expression on PIP2 isoforms intrinsically interconnects the two PIP subclades at the protein level and will thereby affect their mutual functions. Significance statementPlasma membrane intrinsic proteins constituting the most homogenous plant aquaporin family are nonetheless split into two highly conserved subfamilies, PIP1 and PIP2. The loss of major Arabidopsis PIP2 isoforms does not lead to compensation by PIP1 members, but rather to PIP1s concomitant, post-translational repression. This dependence of PIP1 isoforms inevitably ties the two PIP subfamilies and their function.