Plant Science

Background Strigolactones (SLs) regulate diverse aspects of plant development and have been implicated in promoting leaf senescence. However, senescence phenotypes associated with SL deficiency have not been consistently observed across species, suggesting that this function may be species- or context-dependent. Moreover, the contribution of endogenous SL biosynthesis to senescence in leafy vegetable crops remains unclear. Here, we investigated the role of the SL biosynthetic gene MORE AXILLARY GROWTH1 (MAX1) in dark-induced leaf senescence in lettuce (Lactuca sativa). Results We found that endogenous SL biosynthesis plays a major role in dark-induced senescence in lettuce. SL pathway genes were induced during dark storage, while exogenous GR24 accelerated senescence and lettuce MAX1 (LsMAX1) complemented the delayed-senescence phenotype of the Arabidopsis max1 mutant. Consistent with these findings, CRISPR/Cas9-generated Lsmax1 mutants exhibited a pronounced stay-green phenotype during prolonged darkness, accompanied by strongly reduced induction of key senescence-associated genes. Despite this delayed visible senescence, Lsmax1 retained a substantial transcriptional response to dark storage. Strikingly, loss of LsMAX1 did not simply weaken the wild-type senescence program, but redirected part of the response toward a distinct stress-associated transcriptional state that was largely absent from wild type. Loss of LsMAX1 did not affect vegetative rosette architecture, although increased branching emerged after bolting. Conclusions Our findings establish MAX1-dependent SL biosynthesis as an important regulator of leaf senescence in lettuce and reveal a role that extends beyond controlling the rate of senescence. Rather than simply delaying the wild-type program, loss of LsMAX1 alters the transcriptional trajectory of senescence, favoring an alternative stress-associated state during prolonged darkness. The strong stay-green phenotype without detectable changes to vegetative rosette architecture further highlights SL biosynthesis as a potential target for extending postharvest longevity in lettuce and other leafy crops.

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.

Accelerating photoprotective regulation to improve carbon assimilation is a promising strategy to increase crop productivity. Although rapid non-photochemical quenching (NPQ) relaxation has been validated as a target through metabolic engineering, it remains unclear whether conventional breeding has improved this trait. Here, we investigated whether more than a century of soybean breeding enhanced NPQ relaxation alongside light-saturated carbon assimilation and seed traits. We evaluated a historical panel of 24 soybean genotypes across vegetative and reproductive developmental stages by integrating NPQ relaxation, gas exchange parameters, xanthophyll-cycle pigment profiles, expression of key photoprotective genes (VDE, PsbS, and ZEP), seed number and seed weight. NPQ relaxation parameters were not consistently associated with genotype release year, seed number, or seed weight at either developmental stage. The only exception was the amplitude of the rapidly relaxing NPQ component (AqE), which was negatively correlated with all three variables during the reproductive stage. In contrast, genotype release year was positively associated with maximum net CO2 assimilation rate (Amax), maximum carboxylation rate of Rubisco (Vcmax), maximum electron transport rate (Jmax), seed number, and seed weight, while Amax and Vcmax were positively correlated with seed number and seed weight. These findings indicate that the greater photosynthetic capacity of modern genotypes was not accompanied by faster photoprotective response. Thus, photoprotective regulation has not kept pace with gains in photosynthetic capacity under field conditions. We conclude that rapid NPQ relaxation remains an important target for synchronizing photoprotection with the high photosynthetic capacity of modern soybean lines.

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.

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.

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.

Although the biosynthetic pathways of benzylisoquinoline alkaloids (BIAs) have been extensively investigated in several plant species, their transcriptional regulatory mechanisms remain only partially understood. Jasmonate (JA)-responsive group IX APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription factors (TFs) are well-known regulators of specialized plant metabolism, including the biosynthesis of various alkaloids. However, their specific roles in BIA biosynthesis remain largely elusive. Here, we isolated five novel group IX AP2/ERF TFs, designated Benzylisoquinoline alkaloid Jasmonate-responsive AP2/ERF (BJE1-5), from Coptis japonica. Phylogenetic analysis revealed that Benzylisoquinoline alkaloid Jasmonate-responsive AP2/ERF (BJE) proteins belong to subclades distinct from group IXa, which contains well-known AP2/ERF TFs involved in alkaloid biosynthesis. Transient expression analyses in C. japonica protoplasts demonstrated that certain BJEs, particularly CjBJE3 and CjBJE5, positively regulated BIA biosynthetic genes through a mutual regulatory network among BJE members. Moreover, CjBJE3 expression was regulated by CjbHLH1, a unique-type basic helix-loop-helix (bHLH) TF specific to BIA-producing plants. Furthermore, heterologous expression of CjBJE3 and CjBJE5 in cultured Eschscholzia californica cells significantly enhanced the overall BIA production, particularly by increasing end-product benzophenanthridine BIAs, highlighting several uncharacterized biosynthetic genes clustered in the genome. Our findings suggest that BIA-producing species have developed a specific regulatory network comprised of CjbHLH1 and BJE TFs, providing valuable clues for identifying novel biosynthetic enzymes.

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.

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.

Context. Barley production requires advanced knowledge of its response to changing environmental conditions in order to keep it competitive and sustainable. Aim. Advance in the understanding of barley phenology and foliar development under South American field conditions. Methods. 8 spring barley genotypes with differential phenology were studied in four field experiments under different temperature (through years and sowing dates) and photoperiod (through sowing dates) conditions. Time to anthesis, emergence to onset of stem elongation, stem elongation to anthesis, photoperiod response (PR) in these three traits, number of final leaves at anthesis (FLN) and phyllochron were measured. Key Results. Time to anthesis and its subphases were shorter in late plantings but under similar photoperiod, temperature increased them. Cultivars have differential responses but with magnitude interactions and not crossover ones. Cultivar effects defined PR with no interaction with year (temperature). Temperature and photoperiod affected FLN, phyllochron and their relationship with time to anthesis. Under the shorter photoperiod, FLN and phyllochron were negatively correlated, FLN was higher in the warmer year and positively correlated with time to anthesis while phyllochron was not affected by temperature and had no correlation with time to anthesis. Under longer photoperiod, phyllochron was higher in the warmer year and time to anthesis was positively correlated with both FLN and phyllochron. Conclusions. Cultivar basal thermal requirements and PR were consistent under the different studied conditions. Changes in temperature and photoperiod affected the relationship between time to anthesis, FLN and phyllochron suggesting that, although the three traits are arithmetically related, environmental conditions affect their balance.

Concurrent exposure to heat and drought stress compromises legume productivity, yet their combined effects are rarely quantified systematically. We compiled a database of 18 studies covering seven legume species. From these, we extracted 929 physiological, biochemical, and yield-related traits and calculated actual-to-additive ratios to classify heat-drought interactions as antagonistic (ratio < 1), additive (ratio = 1), or synergistic (ratio > 1). Additive heat-drought relationships accounted for 59 % of all classifiable observations, 37% relationships were antagonistic, and 4% synergistic. The relationship varied with species, genotype, trait, and experimental conditions highlighting the complexity of combined abiotic stress effects. The results challenge the common assumption that concurrent stresses invariably exacerbate damage and underscore the need for more realistic, quantitatively defined stress treatments as well as frameworks that integrate trait-level responses into predictive models of crop growth and development. Our synthesis provides a quantitative foundation to understand legume phenotypes under the increasingly frequent co-occurrence of heat and drought stress and identifies research areas where further work is needed to improve insight into combined stress responses. HighlightsO_LICombined heat and drought responses were mainly additive or antagonistic. C_LIO_LIEvidence is biased toward few legumes and controlled environments. C_LIO_LIField-based, multi-species studies are needed to identify adaptive traits. C_LI

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.

Kinase fusion proteins (KFPs) have emerged as an important group of immune receptors encoded by plant resistance genes. Here, we report a new type of gene pair that controls resistance of wheat to the blast fungus, Pyricularia oryzae. We cloned a fungal gene involved in avirulence of P. oryzae pathotype Eleusine on wheat and designated it PWT8. We also identified its corresponding resistance gene in wheat, and tentatively named it Rwt8. This resistance gene was located at the same locus as previously identified resistance genes Rwt3 and Rwt6. Molecular cloning revealed that Rwt3, Rwt6, and Rwt8 were the same gene consisting of an identical gene pair, one encoding an NLR and the other encoding a mixed lineage kinase-like (MLKL) protein. These two genes were closely linked in a head-to-head orientation and behaved as a single gene. This gene pair recognized three AVR genes, PWT3, PWT6, and PWT8, and was designated Rwt3.6.8. The distribution of Rwt3.6.8 in common wheat landraces suggested that the gene pair may have been a factor which the D genome provided to the genus Triticum to broaden its adaptability to various environments in the world, especially in Asia and Africa.

To promote disease resistance, plant nucleotide-binding, leucine-rich repeat (NLR) immune receptors often require paired co-receptors. In many cases, paired NLRs comprise one NLR to perceive effectors (the sensor) and another NLR to execute cell death (the helper). However, NLRs can also pair with sensor kinase fusion protein (KFP) receptors, but whether non-NLR components within such pairs can execute cell death, remains unclear. Here, we investigate the mechanism of an immune receptor pair comprising the wheat NLR Rwt3.6.8 NLR (R3NLR) and an MLKL protein, Rwt3.6.8 associated kinase (R3AK). Using Nicotiana benthamiana transient expression assays we confirmed that both R3NLR and R3AK are required for cell death in response to blast pathogen effectors PWT3, PWT6 or PWT8. Through mutational analysis we show the 4-helical bundle (4HB) domain of R3AK is required to execute cell death and R3AK can be made auto-active by perturbing the kinase catalytic active site. Activation of R3AK is also associated with a shift to a higher oligomeric state. Furthermore, as the NLR R3NLR is not actively involved in the execution of cell death we hypothesise that R3AK acts as a helper. A phylogenetic analysis indicates widespread distribution of this paired configuration in Poales. Together, this study establishes a novel resistance mechanism involving a non-canonical NLR/MLKL system. Significance StatementHere we investigate the mechanism of a novel plant immune receptor pair from wheat, R3NLR/R3AK. A nucleotide-binding, leucine-rich repeat (NLR) receptor and a mixed lineage kinase like (MLKL) protein are both required to mediate resistance to blast pathogen effector proteins PWT3, PWT6 and PWT8. Adopting a mutagenesis approach, we show that the MLKL protein executes cell death through its N-terminal 4-helical bundle domain, and this is associated with a shift to a higher oligomeric state. Mutations of conserved sequence motifs in the NLR support its role as a sensor, although effector interactions have not yet been observed. This study reveals a plant immune receptor pair that functions via a putative NLR sensor paired to a cell death executing MLKL protein.

O_LIThis study investigated whether PEN2/BGLU26 has uniquely evolved as an indole glucosinolate-hydrolysing myrosinase required for Arabidopsis thaliana pre-invasive immunity, or whether related myrosinases can replace its function when targeted to the same subcellular context. C_LIO_LIPEN2-homologous and other selected myrosinases from A. thaliana and Brassica rapa were expressed in the pen2-2 mutant background using a PEN2-like targeting strategy. The resulting lines were assessed by gene expression, protein accumulation, metabolite analysis and pathogen resistance assays. In parallel, targeted mutagenesis, structural comparison and phylogenetic analysis were used to examine molecular and evolutionary features of PEN2-related myrosinases. C_LIO_LIAtBGLU27 and BrBABG.a, but not AtBGLU18, AtBGLU23 or AtBGLU28, partially restored indole glucosinolate hydrolysis and resistance to Colletotrichum tropicale in pen2-2. Unlike AtPEN2, both enzymes acted mainly constitutively and showed distinct substrate preferences. PEN2, BGLU27 and BABG proteins lacked conserved post-translational modification sites, including residues associated with a conserved disulfide bond. Restoring this disulfide bond in AtPEN2 abolished its activity. C_LIO_LIPEN2-related myrosinases form an evolutionarily distinct BGLU lineage associated with indole glucosinolate metabolism in Brassicales. Loss of the conserved disulfide bond appears to be required for PEN2 activity, whereas additional PEN2-specific regulatory features are needed for pathogen-triggered, rather than constitutive, glucosinolate metabolism. C_LI

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.

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

Cassava (Manihot esculenta Crantz) is a major staple crop across tropical and subtropical regions. Despite advances in genomic selection, delayed, non-flowering, and asynchronous flowering remain key bottlenecks in breeding programs. To better understand the molecular basis of flowering-time variation, we performed RNA sequencing across three genotypes with contrasting flowering phenotypes (early, late, and non-flowering) sampled at three developmental stages under contrasting light regimes in field conditions (natural light and three-hour night-break with white light). Comparative transcriptomic analysis revealed distinct gene expression patterns associated with flowering responses. Genotype comparisons with no light supplementation revealed stage-specific enrichment of biological processes. Light supplementation was associated with changes in the expression of key components of photoperiodic and circadian regulation, as well as pathways involved in flowering-time integration and hormone and sugar-related signaling. These findings suggest that coordinated changes across multiple biological pathways regulate flowering behavior in cassava. The candidate genes and expression patterns reported provide a foundation for functional studies and advance our understanding of molecular mechanisms governing flowering-time regulation in cassava.

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.

Blue light using Flavin (BLUF) proteins are microbial photoreceptors that are involved in various physiological responses. Their occurrence and biochemical properties in fungi remain poorly understood. Here, we investigated a putative BLUF photoreceptor from the corn-smut fungus Mycosarcoma maydis (MmBLUF). Domain analysis, multiple sequence alignment of BLUF core regions, and structural modelling indicated conserved canonical BLUF fold and flavin-pocket residues. However, when heterologously expressed, UV-visible and fluorescence spectroscopy revealed different spectral behaviour than canonical BLUF protein. Further, we tested the role of extended N-terminus in modulation of chromophore binding by expressing N-terminus truncated protein variants. Our results suggest that the unusual spectral behaviour is not linked to the truncation construct (extended N-terminus), which also showed similar spectral features, indicating that the extended N-terminus is unlikely to account for an unusual photodynamics characteristics. Our findings support MmBLUF as a structurally conserved putative fungal BLUF-like photoreceptor with different photochemical properties. Further studies are required to establish its chromophore identity, photocycle and function of this unusual BLUF-like domain from fungal system.