Reverse transcription quantitative real-time PCR (RT-qPCR) is esteemed for its precision and reliability, positioning it as the standard for evaluating gene expression. Selecting suitable reference genes is crucial for acquiring accurate data on target gene expression. However, identifying appropriate reference genes for specific rice tissues or growth conditions has been a challenge. To overcome this, we introduce the Rice Reference Genes (RRG) tool, which assists researchers in selecting reference genes for diverse experimental conditions in rice. This tool utilizes of 4,404 rice-derived RNA-seq datasets, categorized by five tissue types - leaf, root, seedling, panicle, and seed - and seven stress conditions (cold, disease, drought, heat, hormone, metal, and salt), along with corresponding control groups (mock). In this research, we employed the RRG web-based tool to identify candidate reference genes in rice leaves, roots, and seedlings exposed to salt and drought stress. These candidates were rigorously tested against conventionally established reference genes, confirming their accuracy and reliability. The RRG tool is designed to be user-friendly, allowing even those with limited experience to efficiently select optimal reference genes in rice with ease.

Plant membrane-bound prenyltransferases (PTs) catalyse the transfer of prenyl groups to acceptor substrates, phenols, using prenyl diphosphates as the donor substrate. The presence of prenyl residues in the reaction products, prenylated phenols, is key to the expression of a variety of physiological activities. Plant PTs generally exhibit high specificities for both substrate recognition and prenylation sites, while the molecular mechanism involved in these enzymatic properties is largely unknown. In this study, we performed a systematic biochemical analysis to elucidate the catalytic mechanism responsible for the reaction specificity of plant PTs. Using two representative PTs, PsPT1 and PsPT2, from parsnip (Pastinaca sativa, Apiaceae), which differ only in the regiospecificity of the prenylation site, we performed domain swapping and site-directed mutagenesis of these PTs, followed by detailed enzymatic analysis combined with three-dimensional modelling. As a result, we discovered the domains that control prenylation site specificity and further defined key amino acid residues responsible for the catalytic mechanism. In addition, we showed that the control mechanism of prenylation specificity revealed here is also highly conserved among coumarin-substrate PTs. These data suggest that the regulatory domain revealed here is commonly involved in prenylation regiospecificity in Apiaceae PTs.

Chlorophylls (Chls) are ubiquitous photosynthetic pigments with inherent potential to generate cytotoxic reactive oxygen species. Therefore, all phototrophs and any phagotrophs that attempt to digest phototrophic cells have presumably developed mechanisms to mitigate this phototoxicity. In aquatic environments, the Chls produced by the dominant producers, microalgae, are catabolized into nonphototoxic pigments, cyclopheophorbide enols (CPEs), either by microalga-feeding protists or autonomously, particularly by those carrying secondary chloroplasts during the dismantling of their chloroplasts. However, the biochemistry underpinning CPE-accumulating Chl catabolism (CACC) remains largely unexamined. To characterize the reactions in the transformation pathway and identify the pivotal enzyme for the formation of the seven-membered ring distinctive to CPEs, we conducted qualitative in vivo experiments using hemisynthetically prepared Chl derivatives in the cells of a euglenozoan algivorous (phycophagic) protist, Peranema trichophorum NIES-4660. We supplied polymer beads coated with Chl-b derivatives with their food cells, a unicellular red alga, Cyanidioschyzon merolae, which exclusively contains Chl-a. After administration of Chl-b or its free base with the beads, we detected a CPE derivative with a formyl group at the C7 position (cyclopheophorbide b-enol; cPPB-bE), clearly derived from the appended derivatives, and not from the Chl-a of the alga. In contrast, cPPB-bE was not detected when zinc- and copper-metalated Chls and C132-demethoxycarbonylated Chl-b were added, although the latter resulted in the generation of its demetalated free-base form. These results indicate that (1) pheophytins are the actual substrates of the cyclization enzyme and (2) cyclization proceeds after the enzymatic dechelation of the central magnesium of natural Chls.

Recently, acyl plastoquinol (APQ) and plastoquinone-B (PQ-B), which are fatty acid esters of plastoquinol and plastoquinone-C respectively, have been identified as the major neutral lipids in cyanobacteria. In Synechocystis sp. PCC 6803, Slr2103 having homology with the eukaryotic enzyme for triacylglycerol (TAG) synthesis, diacylglycerol acyltransferase 2 (DGAT2), was identified as responsible for the synthesis of these plastoquinone-related lipids. On the other hand, TAG synthesis in cyanobacteria remains controversial due to the low accumulation level within cyanobacterial cells together with the high contamination level from the environment. In this study, to quantify more precisely and elucidate the relationship between the accumulation of neutral lipids and the presence or absence of DGAT2-like genes, plastoquinone-related lipids and TAG were analyzed directly from total lipids of six cyanobacterial species with different sets of genes encoding DGAT2-like proteins belonging to two distinct subclades. The results showed that the synthesis of these neutral lipids is highly dependent on clade A DGAT2-like proteins under the culture conditions used in this study, although accumulation level of TAG was quite low. In contrast to APQ highly abundant in saturated fatty acids, the fatty acid composition of TAG was species-specific and partly reflected the total lipid composition. Gloeobacter violaceus PCC 7421, which lacks a DGAT2-like gene, accumulated APQ with a high proportion of C18:0, suggesting APQ synthesis by an unidentified acyltransferase.

The emergence of unisexual flower is an important event during plant evolution. The molecular mechanism underlying the formation of unisexual flowers remains unclear in dioecious spinach. In this study, we identified the spinach MALE STERILITY1 gene, SpMS1, which serves as a masculine factor to regulate male fertility and sex reversion. Silencing SpMS1 led to stamen sterility in male flowers and the development of masculine traits in female flowers. Overexpression of SpMS1 in wild-type Arabidopsis resulted in sterile stamens and irregular pollen exine. Notably, ectopic expression of SpMS1 in Arabidopsis ms1 mutants restored pollen viability and flower fertility. Furthermore, our findings demonstrate that SpMS1 interacts with MADS-box transcription factor SpAP1 to regulate unisexual flower development. Thus, SpMS1 exhibits a conserved function in pollen fertility akin to bisexual flowers, while also acting as a key regulator of unisexual flower development in spinach. This study sheds light on the mechanism of sex differentiation in dioecious plants and also provides valuable insights for manipulating male sterility in plant breeding programs.

Seed vigour and longevity are intricate yet indispensable physiological traits for agricultural crops, as they play a crucial role in facilitating the successful emergence of seedlings and exert a substantial influence on crop productivity. Transcriptional regulation plays an important role in seed development, maturation, and desiccation tolerance, which are important attributes for seed vigour and longevity. Here, we have investigated the regulatory role of the seed-specific DNA binding with the One Finger (DOF) transcription factor and the RPBF (Rice P-box Binding Factor) in seed vigour. RPBF modulates the transcription of galactinol synthase and improves seed vigour. The promoter region of Galactinol synthase (GolS)-encoding genes from different species was enriched with DOF binding sites, and the expression levels of both RPBF and OsGolS were found to enhance during seed development. Furthermore, direct interaction of RPBF with OsGolS promoter has been demonstrated through multiple approaches: yeast one-hybrid (Y1H) assays, in planta promoter-GUS assays, dual luciferase assay, and in silico molecular docking. To assess functionality, Agrobacterium-mediated genetic transformation of rice was performed to generate the RNAi lines with reduced RPBF expression. In these RNAi lines, a reduction in both galactinol and raffinose content was observed. Since galactinol and raffinose are known contributors to seed vigour, the T2-transgenic lines were assessed for vigour and viability. For this, RNAi seeds were subjected to accelerated ageing by exposing them to high relative humidity and temperature, followed by scoring the germination and viability potential. Tetrazolium and seed germination assay revealed that the RNAi seeds were more sensitive to ageing compared to their wild-type and vector control counterparts. Collectively, this is the first report demonstrating that the DOF transcription factor RPBF controls the seed vigour through transcriptional regulation of galactinol synthase.

Plants exhibit shoot growth in the direction of the light source to facilitate photosynthesis, known as positive phototropism. In Arabidopsis hypocotyl phototropism, it is thought that a gradient of the signal intensity of the blue light photoreceptor phototropin1 (phot1) between the light-irradiated and shaded sides leads to the differential growth of hypocotyls. The intensity of phot1 signal is regulated not only by the protein kinase activity of phot1 but also by the phosphorylation status of the NONPHOTOTROPIC HYPOCOTYL3 (NPH3) protein, which has a dark form and a blue light form of the phosphorylation modification. Previous studies have shown that phot1 drives the forward reaction from the dark form to the blue light form of NPH3. However, the molecular mechanism underlying the reverse reaction remains unknown. Here, we show that protein phosphatase PP2C19 controls the reverse reaction that converts the blue light form of NPH3 to the dark form of NPH3. The PP2C19 protein possesses the PP2C domain, two cNMP-binding domains, and the protein kinase domain. Similar to phot1 and NPH3, PP2C19 localizes to the plasma membrane, and its PP2C domain is necessary and sufficient for PP2C19 function in hypocotyl phototropism. The pp2c19 mutants show abnormalities in second positive hypocotyl phototropism with a delay in the reverse reaction of NPH3 phosphorylation modification. The present study suggests that continuous blue light irradiation induces an equilibrium state of the reversible reaction of NPH3 phosphorylation, which acts as a phot1 signaling gradient with phot1 kinase activity to induce the second positive phototropism.

Climate oscillations in the Quaternary forced species to major latitudinal or altitudinal range shifts. It has been suggested that adaptation concomitant with range shifts plays key roles in species responses during climate oscillations, but the role of selection for local adaptation to climatic changes remains largely unexplored. Here, we investigated population structure, demographic history and signatures of climate-driven selection based on genome-wide polymorphism data of 141 Japanese Arabidopsis halleri individuals, with European ones as outgroups. Coalescent-based analyses suggested a genetic differentiation between Japanese subpopulations since the Last Glacial Period (LGP), which would have contributed to shaping the current pattern of population structure. Population demographic analysis revealed the population size fluctuations in the LGP, which were particularly prominent since the subpopulations started to diverge (∼50, 000 years ago). The ecological niche modeling predicted the geographic or distribution range shifts from southern coastal regions to northern coastal and mountainous areas, possibly in association with the population size fluctuations. Through genome-wide association analyses of bioclimatic variables and selection scans, we investigated whether climate-associated loci are enriched in the extreme tails of selection scans, and demonstrated the prevailing signatures of selection, particularly toward a warmer climate in southern subpopulations and a drier environment in northern subpopulations, which may have taken place during or after the LGP. Our study highlights the importance of integrating climate associations, selection scans and population demographic analyses for identifying genomic signatures of population-specific adaptation, which would also help us predict the evolutionary responses to future climate changes.

The synthesis and assembly of functioning photosynthetic complexes in chloroplasts developing from etioplasts during the de-etiolation of angiosperm seedlings are imperative for the plant's autotrophic lifestyle. This study compared de-etiolation process under monochromatic red or blue light of equal photon flux density during a 24-hour illumination period of etiolated Arabidopsis seedlings. The aim was to elucidate the impact of these light wavelength on the etioplast-to-chloroplast transformation and the initiation of light-dependent photosynthetic reactions. Both treatments lead to the formation of functional young chloroplasts; however, the etioplast-to-chloroplast transition and the assembly of photosynthetic complexes occurred unevenly, with individual steps tuned by red or blue light. Ultrastructural analysis suggested faster prolamellar bodies disassembly under blue light, while low temperature fluorescence studies indicated a slower transformation of protochlorophyllide to chlorophyllide, and chlorophyll a, under these conditions. Red light further promoted the synthesis of chlorophyll b and LHCII antenna proteins. However, the efficiency of antennae in dissipating excess absorbed energy was higher for seedlings de-etiolated under blue light; the maximum quantum yield of the photosystem II reached 0.81 after 24-hour de-etiolation, equivalent to mature plants. Blue light seemed to enhance the development of well-functioning photosystems (I and II) and antennae. These findings are important for gaining a deeper understanding of photoreceptor regulation of de-etiolation and for utilizing selected light regimes to improve crop yield.

Extreme environments and plants thriving in them, known as extremophytes, offer promising platforms for studying the diverse adaptive mechanisms that have evolved in plants. However, research on adaptation to extreme environments is still limited to those environments where model species or their relative can survive. Fumarole fields, an extreme environment often overlooked, are characterized by multi-hazardous abiotic stressors, including atmospheric contamination (high concentration of H2S, SO2, and CO2), high soil temperature (~60℃), and strong soil acidification (pH=2-3). These conditions make fumarole fields a rich source for studying stress tolerance mechanisms in plants. In this review, we highlight the recent ecological, physiological, and genomic advances involved in fumarole field adaptation, and discuss the forward avenues. The studies outlined in this paper demonstrate that the extreme levels of abiotic stressors found in fumarole fields make them unparalleled field laboratories for studying the unknown stress tolerance mechanisms, warranting further genomic assessments. Some studies succeeded in identifying genes associated with fumarole field adaptation and shedding light on evolutionary implications; however, they have also encountered challenges such as limited genome resources and high genetic differentiation from related species and/or neighboring populations. To overcome such difficulties, we propose integrating ecophysiological and genomic approaches, drawing from the recent studies in other extreme environments. We expect that further studies in the fumarole fields will contribute to broadening our general knowledge of the limits of life.

Understanding plant responses to developmental and environmental cues is crucial for studying morphological divergence and local adaptation. Gene expression changes, governed by cis-regulatory modules (CRMs) including enhancers, are a major source of plant phenotypic variation. However, while genome-wide approaches have revealed thousands of putative enhancers in mammals, far fewer have been identified and functionally characterized in plants. This review provides an overview of how enhancers function to control gene regulation, methods to predict DNA sequences that may have enhancer activity, methods utilized to functionally validate enhancers, and the current knowledge of enhancers in plants, including how they impact plant development, response to environment, and evolutionary adaptation.

Ubiquitination is a reversible post-translational modification involving the attachment of ubiquitin, a 76-amino acid protein conserved among eukaryotes. The protein "ubiquitin" was named after it was found to be ubiquitously expressed in cells. Ubiquitination was first identified as a post-translational modification that mediates energy-consuming protein degradation by the proteasome. After half a century, the manifold functions of ubiquitin are widely recognized to play key roles in diverse molecular pathways and physiological processes. Compared to humans, the number of enzymes related to ubiquitination is almost twice as high in plant species such as Arabidopsis and rice, suggesting that this modification plays a critical role in many aspects of plant physiology including development and environmental stress responses. Here, we summarize and discuss recent knowledge of ubiquitination focusing on the regulation of membrane trafficking in plants. Ubiquitination of plasma membrane localized proteins often leads to endocytosis and vacuolar targeting. In addition to cargo proteins, ubiquitination of membrane trafficking regulators regulates the morphodynamics of the endomembrane system. Thus, throughout this review, we focus on the physiological responses regulated by ubiquitination and their underlying mechanisms to clarify what is already known and what would be interesting to investigate in the future.

Calcium sensor proteins play important roles by detecting changes in intracellular calcium and relaying that information onto downstream targets through protein-protein interaction. Very little is known about calcium sensors from plant species that predate land colonization and the evolution of embryophytes. Here, we examined the genome of the multicellular algae, Chara braunii, for orthologs to the evolutionarily-conserved calcium sensor calmodulin (CaM), and for CaM-like proteins (CMLs). We identified one CaM and eight CML isoforms which range in size from 16.4 to 21.3 kDa and are predicted to have between two to four calcium-binding (EF-hand) domains. Using recombinant protein, we tested whether CbCaM and CbCMLs1-7 possess biochemical properties of typical calcium sensors. CbCaM and the CbCMLs all displayed high-affinity calcium binding with estimated global KD,app values in the physiological µM range. In response to calcium binding, CbCaM and the CbCMLs exhibited varying degrees of increase in exposed hydrophobicity, suggesting different calcium-induced conformational changes occur among isoforms. We found many examples of putative CaM targets encoded in the C. braunii genome and explored the ability of CbCaM and CbCMLs to interact in planta with a representative putative target, a C. braunii CaM-binding transcription factor (CbCAMTA1). CbCaM, CbCML2, and CbCML4 associated with the C-terminal region of CbCAMTA1. Collectively, our data support the hypothesis that complex calcium signaling and sensing networks involving CaM and CMLs evolved early in the green lineage. Similarly, it seems likely that calcium-mediated regulation of transcription occurs in C. braunii via CAMTAs and is an ancient trait predating embryophytic emergence.

The symbiosis between legumes and nitrogen-fixing bacteria (rhizobia) is instrumental in sustaining the nitrogen cycle and providing fixed nitrogen to the food chain. Both partners must maintain an efficient nutrient exchange to ensure a successful symbiosis. This mini-review highlights the intricate phosphate and iron uptake and homeostasis processes taking place in legumes during their interactions with rhizobia. The coordination of transport and homeostasis of these nutrients in host plants and rhizobia ensures an efficient nitrogen fixation process and nutrient use. We discuss the genetic machinery controlling the uptake and homeostasis of these nutrients in the absence of rhizobia and in symbiotic conditions with this soil bacteria. We also highlight the genetic impact of the availability of phosphate and iron to coordinate the activation of the genetic programs that allow legumes to engage in symbiosis with rhizobia. Finally, we discuss how the transcription factor Phosphate Starvation Response (PHR) might be a crucial genetic element to integrate the nitrogen, iron, and phosphate plant's needs while interacting with rhizobia. Understanding the coordination of the iron and phosphate uptake and homeostasis can lead us to better harness the ecological benefits of the legume-rhizobia symbiosis, even under adverse environmental conditions.

The phytohormone salicylic acid (SA) regulates plant responses to various types of environmental stress, particularly pathogen infections. We previously revealed that the benzyl alcohol O-benzoyltransferase HSR201 was required for pathogen signal-induced SA synthesis, and its overexpression together with NtCNL, encoding a cinnamate-coenzyme A ligase, was sufficient for the production of significant amounts of SA in tobacco. We herein examined the subcellular localization of HSR201 and found that it fused to a yellow fluorescent protein localized in peroxisomes. Most peroxisomal matrix proteins possess peroxisomal targeting signal type-1 (PTS1) located at the extreme C terminus or PTS2 located at the N terminus; however, a bioinformatics analysis failed to identify similar signals for HSR201. Deletion and mutation analyses of HSR201 identified one essential (extreme C-terminal Leu46°) and three important (Ile455, Ile456 and Ala459) amino acid residues for its peroxisomal localization. The virus-induced gene silencing (VIGS) of PEX5, a PTS1 receptor, but not PEX7, a PTS2 receptor, compromised the peroxisomal targeting of HSR201 in Nicotiana benthamiana. When overexpressed with NtCNL, HSR201 mutants with reduced or non-peroxisomal targeting induced lower SA levels than the wild type; however, these mutations did not affect the protein stability or activity of HSR201. VIGS of the HSR201 homolog compromised pathogen signal-induced SA accumulation in N. benthamiana, which was complemented by the HSR201 wild type, but not the mutant with non-peroxisomal targeting. These results suggest that the peroxisomal localization of HSR201 is mediated by a non-canonical PTS1 and required for SA biosynthesis.

Mitochondria play a crucial role in eukaryotic organisms, housing their own genome with genes vital for oxidative phosphorylation. Coordination between nuclear and mitochondrial genomes is pivotal for organelle gene expression. Splicing, editing and processing of mitochondrial transcripts are regulated by nuclear-encoded factors. Splicing efficiency (SEf) of the many group II introns present in plant mitochondrial genes is critical for mitochondrial function since a splicing defect or splicing deficiency can severely impact plant growth and development. This study investigates SEf in free-living and holoparasitic plants, focusing on 25 group II introns from 15 angiosperm species. Our comparative analyses reveal distinctive splicing patterns with holoparasites exhibiting significantly lower SEf, potentially linked to their unique evolutionary trajectory. Given the preponderance of horizontal gene transfer (HGT) in parasitic plants, we investigated the effect of HGT on SEf, such as the presence of foreign introns or foreign nuclear-encoded splicing factors. Contrary to expectations, the SEf reductions do not correlate with HGT events, suggesting that other factors are at play, such as the loss of photosynthesis or the transition to a holoparasitic lifestyle. The findings of this study broaden our understanding of the molecular evolution in parasitic plants and shed light on the multifaceted factors influencing organelle gene expression.

Shoot branching is a critical determinant of plant architecture and a key factor affecting crop yield. The shoot branching involves two main processes: axillary meristem formation and subsequent bud outgrowth. While considerable progress has been made in elucidating the genetic mechanisms underlying the latter process, our understanding of the former process remains limited. Rice FINE CULM1 (FC1), which is an ortholog of teosinte branched1 in maize (Zea mays) and BRANCHED1/2 in Arabidopsis (Arabidopsis thaliana), is known to act in the latter process by repressing bud outgrowth. In this study, we found that FC1 also plays a role in the former process, i.e. axillary meristem formation, in rice. This study was triggered by our unexpected observation that fc1 mutation suppresses the loss of axillary meristems in the loss-of-function mutant of the rice WUSCHEL gene TILLERS ABSENT1 (TAB1). In tab1 fc1, unlike in tab1, both stem cells and undifferentiated cells were maintained during axillary meristem formation, similar to the wild type. Morphological analysis showed that axillary meristem formation was accelerated in fc1, compared to the wild type. Consistent with this, cell proliferation was more active in the region containing stem cells and undifferentiated cells during axillary meristem formation in fc1 than in the wild type. Taken altogether, these findings suggest that FC1 negatively regulates axillary meristem formation by mildly repressing cell proliferation during this process.