Planta

Ultraviolet-C (UV-C) radiation can be highly damaging to plants, yet its effects on gibberellin (GA) homeostasis are not well understood. In this study, we show that short daily UV-C pulse treatments (12 s, 1,200 J m-2) applied for seven days reduce plant height and delay flowering in Arabidopsis thaliana. Endogenous levels of the GA biosynthesis precursors GA12, GA53, GA15, and GA24, the bioactive GA4, and the GA catabolites GA34 and GA110 are all lower in UV-C treated plants than in untreated controls. These changes were accompanied by lower transcript levels of the GA biosynthesis genes KS, GA13ox1, GA20ox1, and GA3ox1, together with opposing changes in the expression of GA2ox genes. Exogenous GA4 restores growth in UV-C-treated plants, suggesting that reduced GA availability contributes to UV-C-induced growth inhibition. Consistent with this finding, the GA-signalling mutant gdella and the GA-biosynthesis mutants kao1 and kao2 show strongly reduced UV-C responses. Together, these findings highlight the importance of GA metabolism and signalling in the developmental response to repeated UV-C exposure, and suggest that exposure regimen influences the dynamics of UV-C-induced hormonal responses.

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

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

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

Following pollination, Arabidopsis pollen grains rapidly hydrate through the transfer of water from the stigma to the pollen. Several stigma regulators of pollen hydration have been identified, and the corresponding mutants generally support milder defects in wildtype Col-0 pollen hydration, signifying the involvement of other unidentified factors in this process. Here, we uncovered a role for the stigma-specific mechanosensitive channel gene, MscS-Like 7 (MSL7), in supporting pollen hydration. While the msl7 mutant stigmas were found to support reduced hydration of wildtype Col-0 pollen, the phenotype was quite mild and very similar to that observed for other published pollen hydration mutants. Thus, we conducted a detailed comparison of different pollen hydration mutants on the stigma side (receptor kinases, PIP aquaporins) and pollen (PCP-Bs, MLS8) to compare pollen hydration mutant phenotypes and look for any additive effects of combining different mutants. Overall, all combinations resulted in the same mild hydration defect with no additional reductions in pollen hydration and no impact on pollen germination. This is in contrast to that observed for self-incompatible (SI) pollen from a transgenic Arabidopsis SI Col-0 line which shows very little pollen hydration and no pollen germination as part of the SI pollen rejection response. Together, these findings suggest that the regulation of compatible pollen hydration is quite complex and that there are likely other unknown mechanisms involved. Key MessageThe same mild pollen hydration defect is observed across very different Arabidopsis stigma mutants and it does not prevent pollen germination and pollen tube growth.

Utricularia gibba L. is an aquatic carnivorous plant with a diverse set of capabilities. Reddening of traps frequently occurs in old in vitro cultures. While anthocyanins are often responsible for red coloration in plants, not every plant turns red. Stress factors like high light or excess sucrose have previously been shown to induce the formation of anthocyanins. Here, we hypothesized the red trap formation to be dependent on nutrient deprivation and tested nitrogen deprivation. The results suggest, that only in combination with light, nitrogen deficiency leads to the activation of the complete anthocyanin biosynthesis pathway and visible red coloration. However, in darkness, anthocyanin biosynthesis appears generally less active compared to light conditions and expression of most anthocyanin biosynthesis genes is not significantly upregulated under nitrogen deficiency.

Phenylacetic acid (PAA) is a naturally occurring auxin whose biosynthesis and function during plant regeneration remain poorly understood. PAA may be synthesized from phenylalanine via the CYP79A2-dependent phenylacetaldoxime pathway, in which benzyl cyanide/phenylacetonitrile (BnCN/PAN) is a potential intermediate and substrate for NITRILASE (NIT) enzymes. Here, we investigated whether BnCN/PAN promotes somatic embryogenesis (SE) through NIT-dependent PAA biosynthesis. Low concentrations of BnCN/PAN stimulated somatic embryo formation in Arabidopsis thaliana, whereas exogenous PAA also promoted embryogenic induction. Transcriptome profiling revealed that BnCN/PAN upregulated genes associated with SE, including key embryogenic regulators and EMBRYO DEFECTIVE genes. RNA-seq data further indicated enhanced auxin signalling, which was independently confirmed using the pDR5::GUS reporter line. Inhibition of NIT activity by heatin reduced the embryogenic competence of BnCN/PAN-treated explants, supporting the involvement of NIT enzymes in this response. Collectively, our findings provide the first evidence that BnCN/PAN promotes embryogenic transition and suggest that NIT functions in SE extend beyond their proposed role in indole-3-acetic acid biosynthesis. Summary statementThis study uncovers a previously unrecognized pathway regulating plant regeneration, providing new insights into how naturally occurring metabolites influence embryo formation.

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

Tomato production, yield and fruit quality face major challenges due to several factors, including the complex polygenic inheritance of agronomically relevant traits, biotic and abiotic stresses, and increasingly stringent regulations limiting the use of phytosanitary products. In this context, bioinoculants have emerged as a sustainable strategy capable of enhancing yield without compromising fruit quality, conferring protection against different stresses and exerting a minimal or no impact on environment and human health. In this study, we evaluated the effects and the underlying mechanisms by which Streptomyces sp. N2A, an actinobacteria isolated from soybean rhizosphere, promotes seed germination, vegetative growth and yield in tomato, without modifying fruit quality. The obtained results demonstrated that the bacterial treatment significantly improved seedlin[g]s emergence and growth and development in vegetative stage. At harvest, yield was also significantly enhanced, mainly driven by increased individual fruit weight, which was positively correlated with a thicker pericarp in fruits from N2A-treated plants. Transcriptional analysis during fruit development revealed a coordinated induction of auxin and cytokinin signaling pathways before and after anthesis, providing a hormonal framework that underlies the promotion of pericarp growth. This study provides evidence of the beneficial effect of inoculation with Streptomyces sp. N2A on tomato yield and constitutes the first report describing the modification of fruit morphology and expression of genes involved in phytohormonal modulation during early growth and development, induced by a plant growth-promoting Streptomyces.

Chloroplasts comprise photosynthesis and other important processes. Plants protect chloroplasts from stresses including Cd accumulation. Common terrestrial plants, excluders apply a set of mechanisms to restrict Cd penetration to chloroplasts. Removal of accumulated Cd from chloroplasts should also be a beneficial strategy. However, we do not know whether excluder plant species have ability to remove Cd from chloroplasts. We used barley as a common excluder plant species. To barley plants, we applied a model with two stable isotopes 111Cd and 114Cd to distinguish Cd accumulated earlier and later. A portion of Cd absorbed by roots continued translocation to shoot for some days after the external source of Cd was changed from one isotope to another. Chloroplasts acquired new portions of Cd and lost part of Cd accumulated earlier; a total Cd content remained rather unchanged. Cd loss from thylakoids was detected in vivo and in vitro. Cd loss from stroma and envelope was observed in vivo but not in vitro. Therefore, barley has at least two distinct mechanisms for Cd removal from chloroplasts: one from thylakoids and another from stroma. We hypothesized diverse chlorophagy pathways as a potential mechanism for Cd removal from chloroplasts. Cd accumulation by chloroplasts was mainly light-independent. In chloroplasts, Cd accumulated in vivo was tightly bound and mainly located in thylakoids. In vitro, chloroplasts from Cd-treated plants accumulated much less Cd than chloroplasts from untreated plants in a previous study. This implies reorganization of transport across chloroplast envelope membranes. HighlightsO_LICd was removed from thylakoids both in vivo and in vitro C_LIO_LICd was removed from stroma and envelope in vivo but not in vitro C_LIO_LIIn chloroplasts, Cd accumulated in vivo was tightly bound C_LIO_LICd accumulation by chloroplasts was mainly light-independent C_LIO_LIRoot barrier slowed down Cd translocation to shoot but not halted it C_LI

BackgroundCymbidium tortisepalum var. longibracteatum is a Class II nationally protected endangered plant in China with significant economic value. AimThis study aimed to establish an efficient in vitro flowering system and identify key hormonal factors regulating flower bud differentiation. MethodsOrthogonal experiments and hormone treatments were performed to evaluate the effects of TDZ, 6-BA, NAA, IBA, PP333, ABA, and GA3 on flower bud induction in tissue-cultured plantlets. ResultsTDZ was identified as the key initiator of flower bud differentiation, as no flower buds were induced in TDZ-free treatments. The optimal hormone combination was 1.0 mg{middle dot}L-1 6-BA + 0.4 mg{middle dot}L-1 TDZ + 0.8 mg{middle dot}L-1 NAA + 1.6 mg{middle dot}L-1 IBA, achieving a flower bud induction rate of 23.33% and a normal flower bud rate of 20.00%. PP333 inhibited flower bud differentiation but reduced malformation; at 0.2 mg{middle dot}L-1 PP333, the normal flower bud rate reached 20.00%. GA3 pretreatment resulted in a flower bud induction rate of 12.04%, whereas ABA pretreatment showed no significant promoting effect on flower bud induction. ConclusionsThis study provides an efficient in vitro flowering system and key hormonal parameters for shortening the breeding cycle and elucidating orchid flowering mechanisms.

Salinity stress affects rice productivity due to reduced growth and sodium ion toxicity. Previously, we identified a splice variant of RADIATION SENSITIVE23a (RAD23a) as the potential basis for variation in salt-tolerance in rice germplasm. RAD23 is a known moonlighting protein associated with protein degradation. To validate the role of RAD23a in salt stress response, we characterized gene edited mutant lines that targeted the UBL and UBA2 domains of this protein. Mutation in either domain promoted shoot growth under saline and control conditions. The mutants also differed from wildtype plants in Na and K accumulation in roots and shoots under salt stress. Transcriptome analysis of mutants versus wildtype showed differential transcript abundance of multiple inorganic phosphate (Pi) starvation related genes, including OsSPX2 and OsPHO2. As a result, mutants accumulate higher Pi compared to wildtype plants. The two allelic groups for RAD23a locus also differ in root and shoot phosphorus (P) content. Further, we show that RAD23a interacts with OsSPX2, a negative post-translational regulator of OsPHR2, the master regulator of Pi starvation response. Mutants have higher shoot growth and Pi levels under low Pi conditions, linking enhanced growth of mutants to increased Pi uptake. The UBA2 domain specific mutants have higher single grain weight and per plant grain weight than wildtype. In summary, we show that the RAD23a regulates differential growth, salt response and Pi uptake in rice in a domain-specific manner supporting the moonlighting roles of RAD23a in salt tolerance and phosphorus-dependent shoot growth.

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

Continuous-wave (CW) 450 nm blue diode-laser irradiation was evaluated as a physical seed-priming treatment for improving lettuce (Lactuca sativa L.) germination and early seedling establishment under reduced water availability. Dry seeds were irradiated at 50-250 mW for 2 min and at 100 mW for 0.5-10 min; each condition included three independent Petri-dish replicates with 100 seeds per replicate, and the replicates were monitored for 72 h. Laser treatment produced a dose-dependent biological response. The optimized condition, 100 mW for 2 min, increased final germination from approximately 65-70% to 90-95%, increased the germination speed index, and promoted root elongation more strongly than shoot elongation. Longer exposures reduced germination and seedling growth. Under reduced water availability (0.5-6 ml per Petri dish), laser-treated seeds germinated earlier, maintained final germination of 83-93% compared with 57-72% in controls, and produced better-developed seedlings. The relative benefit increased as water availability decreased, indicating that optimized blue laser priming partially compensated for low water supply during germination and early establishment. These results identify CW blue laser priming as a contactless, chemical-free approach for improving lettuce seed performance and early seedling vigor under limited water availability.

Plants make diverse metabolites to outcompete neighboring organisms for space and resources. Some of these toxic metabolites broadly disrupt conserved molecular mechanisms, such as protein biosynthesis. Nonproteogenic amino acids (NPAAs) are a structurally diverse class of metabolites that interfere with protein biosynthesis. The proline (Pro) analog azetidine-2-carboxylic acid (Aze) inhibits plant growth through misincorporation during protein biosynthesis. However, it is unknown if a cascade of downstream stress responses is triggered following Aze misincorporation. Here, we investigate the morphological and stress responses in Arabidopsis grown on Aze. Investigation of root morphological responses show not only reduced root growth, but increased root branching following growth on Aze. Altered root morphology is coupled with a reduced gravitropic response. Aboveground organs were also affected by Aze, including reduced chlorophyll content, reduced photosynthetic efficiency, and increased anthocyanin content. We then tested whether Aze induces reactive oxygen species (ROS) accumulation using multiple approaches and observed both immediate and sustained accumulation of general ROS and H2O2 following treatment with Aze. When plants were grown on Aze supplemented with Pro, ROS levels were restored to normal levels, suggesting that reducing misincorporation events results in less downstream stress responses. In summary, we find that following Aze treatment a cascade of downstream stress responses is induced that exacerbates the effects of toxic NPAAs. This study sheds light on the mechanism of action of NPAAs and provides information on the downstream consequences of translational errors.

Stomata are the pores on plant surface, and these tiny pores are responsible for the flow of gas between plants and atmosphere. Currently, what effects of the broad and continuous increase in stomatal density achieved via genetic engineering on plant growth and development remain poorly understood. The 9 Arabidopsis transgenic lines with increased stomatal density were acquired through overexpressing FSTOMAGEN (the homologs of STOMAGEN, which are in Flaveria). The intermediate stomatal density (SD) lines exhibited increased trend in biomass. Compared with the lines with low SD, the biomass of Arabidopsis lines with intermediate SD (484 mm-2) significantly increased. There was a positive and significant correlation between biomass and relative water content. Across these transgenic lines, only during the earlier phase of growth, the leaf area exhibited a gradually increased trend as stomatal density increased, and there was both a significant linear relationship between SD and leaf growth rate and a strong linear relationship between SD and leaf area. In contrast, a clear relationship during the later phase wasnt observed. Under lower growth light intensity, there was an increased trend of biomass from other lines to the lines with intermediate SD, and the photosynthetic rate and stomatal conductance of the intermediate line were significantly increased. This study reveals plant-growth alterations that correspond to broad and near-continuous increases in stomatal density achieved via genetic engineering. Our study sheds light on the prerequisites for elevated stomatal density achieved via genetic engineering to promote plant growth.

Applications of tissue culture are critical for Cannabis sativa L. (cannabis), supporting clonal propagation, germplasm preservation, pathogen elimination, among other biotechnological applications. However, extensive genetic diversity associated with cannabis results in highly variable responses to in vitro conditioning, and no consensus basal media formulation exists to support reproducible micropropagation across genotypes. To address these limitations, a hybridized ensemble-NSGA-II approach was employed for concurrent optimization of individual media components to create a species specific, cultivar inclusive basal salt formulation for cannabis micropropagation. The resulting PHJ media represents a unique formulation that overcomes recalcitrance across a wide array of cannabis cultivars, facilitating improved growth and uniformity for the nine cultivars used in its development and validation. These results remain consistent from explant initiation through multiple rounds of subculture. The ability of PHJ to overcome genotypic recalcitrance is telling of its potential applicability with an array of plant species beyond cannabis. Additionally, robust performance both with and without plant growth regulators underscores the plausible use of PHJ for diverse applications beyond standard micropropagation. Ultimately, this cultivar-inclusive basal medium demonstrates utility for both scientific research and industrial-scale operations.

Heavy-metal accumulation in cereal grains is becoming critical for European food safety, regulation and plant breeding. In the EU, Cd maximum levels in certain foodstuffs have been revised, including lowering or establishing limits for relevant food categories, while new maximum levels for nickel (Ni) have recently been introduced for several foodstuffs, including cereal categories, with limits for oats and selected cereals applying from 2026. Together, these developments create an urgent need to identify genetic and physiological mechanisms that reduce Cd and Ni accumulation in cereal grains while maintaining crop quality and productivity. Against this regulatory and food-safety background, our broader RNA-seq experiment investigates early transcriptional responses to Cd and Ni in oat F2 segregants contrasting for metal accumulation. The full dataset includes low- and high-accumulating segregants, roots and developing caryopses sampled at 3 h and 7 h after treatment. In the present pilot analysis, we focus on the Cd response in developing caryopses of the low-Cd accumulating segregant AS131 to identify candidate processes associated with reduced grain Cd accumulation. The strongest transcriptional responses were not dominated by canonical Cd-detoxification pathways. At 3 h after Cd exposure, differentially expressed transcripts were mainly associated with cell-wall functions, endosperm transfer-cell-specific PR60 proteins, DUF239-containing proteins and cysteine proteinase inhibitors, whereas several dehydration-, pathogen-, defence-, cell-wall-loosening- and ROS- related genes were repressed. By 7 h, the response suggested a shift towards homeostatic acclimation, with induction of TIP2 aquaporins, thiamine thiazole synthases, EF-Tu proteins, coatomer-related genes and carbohydrate metabolism-associated genes, together with repression of LEA/SMP/dehydrin genes, FRO7-like genes, EF-hand calcium-binding proteins and stress-regulatory transcription factors. Pathway-level analyses were broadly consistent with these transcript-level patterns, highlighting structural, nucleosome-associated, translation-related, metabolic and developmental processes. Several Cd-responsive transcripts were also associated with broader abiotic-stress responses, suggesting recruitment of shared stress-regulatory modules rather than Cd-specific detoxification pathways alone. Overall, these results support a working hypothesis in which low Cd accumulation in developing oat grain may involve regulation of solute-transfer interfaces, cellular protection, intracellular homeostasis, trafficking pathways and caryopsis developmental programmes. These findings provide candidate processes for future comparison with high-Cd accumulating segregants, root tissues and Ni responses in the broader dataset.

Cannabis is well known for its pungent, skunk-like aroma. Recent chemical studies have identified prenylated and C6 volatile sulfur compounds as contributors to its skunky and citrus-like aromas, but the pathways that produce these compounds remain unknown. This gap limits efforts to explain variation in sulfur-aroma traits and to selectively enhance or reduce those traits. To address this gap, we used the known chemistry of sulfur-containing volatiles in Cannabis and characterized sulfur and volatile biosynthetic pathways in other plant species to select candidate enzyme groups. Because the GMO cultivar is anecdotally associated with a pronounced sulfurous aroma, reference protein sequences and profile hidden Markov models were used to search its version 1 (v1) primary high-confidence protein set of 55,790 sequences. These searches recovered 975 unique proteins. Sequence screening retained 941 candidates across 20 reporting categories; 939 contained all expected domains, while the two candidates assigned to the methionine gamma-lyase (MGL)-nearest category had no category-specific expected-domain rule. The largest reporting category comprised 359 proteins containing a cytochrome P450 domain, recovered through a search motivated by cytochrome P450 family 74 (CYP74) enzymes involved in oxylipin and plant volatile formation. Thirteen of these proteins were also recovered by at least one full-length CYP74 reference search. Other large reporting categories included 218 sugar-transferase, 83 glutathione-transferase, and 61 alcohol dehydrogenase candidates. Comparison with the Cannabis Expression Atlas linked 168 candidates to 128 annotated genes through 100%-identity amino-acid matches spanning at least 80% of each GMO v1 candidate protein. Twenty-nine genes were tissue-specific, including 13 root-specific and 6 trichome-specific genes. These results define candidates for biochemical testing and direct searches for additional enzymes acting upstream and downstream in Cannabis sulfur-volatile pathways.

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.