Microfluidic Device for Simple Diagnosis of Plant Growth Condition by Detecting miRNAs from Filtered Plant Extracts.

Plants are exposed to a variety of environmental stress, and starvation of inorganic phosphorus can be a major constraint in crop production. In plants, in response to phosphate deficiency in soil, miR399, a type of microRNA (miRNA), is up-regulated. By detecting miR399, the early diagnosis of phosphorus deficiency stress in plants can be accomplished. However, general miRNA detection methods require complicated experimental manipulations. Therefore, simple and rapid miRNA detection methods are required for early plant nutritional diagnosis. For the simple detection of miR399, microfluidic technology is suitable for point-of-care applications because of its ability to detect target molecules in small amounts in a short time and with simple manipulation. In this study, we developed a microfluidic device to detect miRNAs from filtered plant extracts for the easy diagnosis of plant growth conditions. To fabricate the microfluidic device, verification of the amine-terminated glass as the basis of the device and the DNA probe immobilization method on the glass was conducted. In this device, the target miRNAs were detected by fluorescence of sandwich hybridization in a microfluidic channel. For plant stress diagnostics using a microfluidic device, we developed a protocol for miRNA detection by validating the sample preparation buffer, filtering, and signal amplification. Using this system, endogenous sly-miR399 in tomatoes, which is expressed in response to phosphorus deficiency, was detected before the appearance of stress symptoms. This early diagnosis system of plant growth conditions has a potential to improve food production and sustainability through cultivation management.

The Chaperone NASP Contributes to De Novo Deposition of the Centromeric Histone Variant CENH3 in Arabidopsis Early Embryogenesis.

The centromere is an essential chromosome region where the kinetochore is formed to control equal chromosome distribution during cell division. The centromere-specific histone H3 variant CENH3 (also called CENP-A) is a prerequisite for the kinetochore formation. Since CENH3 evolves rapidly, associated factors, including histone chaperones mediating the deposition of CENH3 on the centromere, are thought to act through species-specific amino-acid sequences. The functions and interaction networks of CENH3 and histone chaperons have been well-characterized in animals and yeasts. However, molecular mechanisms involved in recognition and deposition of CENH3 are still unclear in plants. Here, we used a swapping strategy between domains of CENH3 of Arabidopsis thaliana and the liverwort Marchantia polymorpha to identify specific regions of CENH3 involved in targeting the centromeres and interacting with the general histone H3 chaperone, NASP (nuclear autoantigenic sperm protein). CENH3's LoopN-α1 region was necessary and sufficient for the centromere targeting in cooperation with the α2 region and was involved in interaction with NASP in cooperation with αN, suggesting a species-specific CENH3 recognition. In addition, by generating an Arabidopsis nasp knockout mutant in the background of a fully fertile GFP-CENH3/cenh3-1 line, we found that NASP was implicated for de novo CENH3 deposition after fertilization and thus for early embryo development. Our results imply that the NASP mediates the supply of CENH3 in the context of the rapidly evolving centromere identity in land plants.

Plasma membrane damage limits replicative lifespan in yeast and induces premature senescence in human fibroblasts.

Plasma membrane damage (PMD) occurs in all cell types due to environmental perturbation and cell-autonomous activities. However, cellular outcomes of PMD remain largely unknown except for recovery or death. In this study, using budding yeast and normal human fibroblasts, we found that cellular senescence-stable cell cycle arrest contributing to organismal aging-is the long-term outcome of PMD. Our genetic screening using budding yeast unexpectedly identified a close genetic association between PMD response and replicative lifespan regulations. Furthermore, PMD limits replicative lifespan in budding yeast; upregulation of membrane repair factors ESCRT-III (SNF7) and AAA-ATPase (VPS4) extends it. In normal human fibroblasts, PMD induces premature senescence via the Ca2+-p53 axis but not the major senescence pathway, DNA damage response pathway. Transient upregulation of ESCRT-III (CHMP4B) suppressed PMD-dependent senescence. Together with mRNA sequencing results, our study highlights an underappreciated but ubiquitous senescent cell subtype: PMD-dependent senescent cells.

Broad Chain-Length Specificity of the Alkane-Forming Enzymes NoCER1A and NoCER3A/B in Nymphaea odorata.

Many terrestrial plants produce large quantities of alkanes for use in epicuticular wax and the pollen coat. However, their carbon chains must be long to be useful as fuel or as a petrochemical feedstock. Here, we focus on Nymphaea odorata, which produces relatively short alkanes in its anthers. We identified orthologs of the Arabidopsis alkane biosynthesis genes AtCER1 and AtCER3 in N. odorata and designated them NoCER1A, NoCER3A and NoCER3B. Expression analysis of NoCER1A and NoCER3A/B in Arabidopsis cer mutants revealed that the N. odorata enzymes cooperated with the Arabidopsis enzymes and that the NoCER1A produced shorter alkanes than AtCER1, regardless of which CER3 protein it interacted with. These results indicate that AtCER1 frequently uses a C30 substrate, whereas NoCER1A, NoCER3A/B and AtCER3 react with a broad range of substrate chain lengths. The incorporation of shorter alkanes disturbed the formation of wax crystals required for water-repellent activity in stems, suggesting that chain-length specificity is important for surface cleaning. Moreover, cultured tobacco cells expressing NoCER1A and NoCER3A/B effectively produced C19-C23 alkanes, indicating that the introduction of the two enzymes is sufficient to produce alkanes. Taken together, our findings suggest that these N. odorata enzymes may be useful for the biological production of alkanes of specific lengths. 3D modeling revealed that CER1s and CER3s share a similar structure that consists of N- and C-terminal domains, in which their predicted active sites are respectively located. We predicted the complex structure of both enzymes and found a cavity that connects their active sites.

HISTONE DEACETYLASE19 Controls Ovule Number Determination and Transmitting Tract Differentiation.

The gynoecium is critical for the reproduction of flowering plants as it contains the ovules and the tissues that foster pollen germination, growth, and guidance. These tissues, known as the reproductive tract (ReT), comprise the stigma, style, and transmitting tract (TT). The ReT and ovules originate from the carpel margin meristem (CMM) within the pistil. SHOOT MERISTEMLESS (STM) is a key transcription factor for meristem formation and maintenance. In all above-ground meristems, including the CMM, local STM downregulation is required for organ formation. However, how this downregulation is achieved in the CMM is unknown. Here, we have studied the role of HISTONE DEACETYLASE 19 (HDA19) in Arabidopsis (Arabidopsis thaliana) during ovule and ReT differentiation based on the observation that the hda19-3 mutant displays a reduced ovule number and fails to differentiate the TT properly. Fluorescence-activated cell sorting coupled with RNA-sequencing revealed that in the CMM of hda19-3 mutants, genes promoting organ development are downregulated while meristematic markers, including STM, are upregulated. HDA19 was essential to downregulate STM in the CMM, thereby allowing ovule formation and TT differentiation. STM is ectopically expressed in hda19-3 at intermediate stages of pistil development, and its downregulation by RNA interference alleviated the hda19-3 phenotype. Chromatin immunoprecipitation assays indicated that STM is a direct target of HDA19 during pistil development and that the transcription factor SEEDSTICK is also required to regulate STM via histone acetylation. Thus, we identified factors required for the downregulation of STM in the CMM, which is necessary for organogenesis and tissue differentiation.

The 33rd International Conference on Arabidopsis Research (ICAR2023).

The 33rd International Conference on Arabidopsis Research (ICAR2023) was held at Makuhari Messe International Conference Hall in Chiba prefecture from June 5 to 9, 2023. This annual conference, which rotates among hosts in North America, Asia-Oceania, and Europe, covers the full range of plant biology research involving Arabidopsis and other plant species. The conference hosted more than 1200 participants, including approximately 800 international attendees from 42 countries (or regions), and featured about 900 oral and poster presentations. Reflecting the conference theme, "Arabidopsis for Sustainable Development Goals (SDGs)," there were numerous exemplary presentations regarding basic plant science using Arabidopsis and translational research conducted to achieve SDGs by exploiting the knowledge gained from Arabidopsis to improve crop production. The conference concluded on a high note, with more than 99% of survey respondents expressing their general satisfaction with ICAR2023. This report aims to summarize the organization, objectives, and outcomes of the conference.

A BLADE-ON-PETIOLE orthologue regulates corolla differentiation in the proximal region in Torenia fournieri.

The three-dimensional shape of a flower is integrated by morphogenesis along different axes. Differentiation along the petal proximodistal axis is tightly linked to the specification of pollinators; however, it is still unclear how a petal patterns this axis. The corolla of Torenia fournieri exhibits strong differentiation along the proximodistal axis, and we previously found a proximal regulator, TfALOG3, controlling corolla neck differentiation. Here, we report another gene, TfBOP2, which is predominantly expressed in the proximal region of the corolla. TfBOP2 mutants have shorter proximal corolla tubes and longer distal lobe, demonstrating its function as a proximal regulator. Arabidopsis BOPs mutant shows similar defects, favouring a shared role of BOPs homologues. Genetic analysis demonstrates the interaction between TfBOP2 and TfALOG3, and we further found that TfALOG3 physically interacts with TfBOP2 and can recruit TfBOP2 to the nuclear region. Our study favours a hypothetical shared BOP-ALOG complex that is recruited to regulate corolla differentiation in the proximal region axis of T. fournieri.

Reorganization of the ancestral sex-determining regions during the evolution of trioecy in Pleodorina starrii.

The coexistence of three sexual phenotypes (male, female and bisexual) in a single species, 'trioecy', is rarely found in diploid organisms such as flowering plants and invertebrates. However, trioecy in haploid organisms has only recently been reported in a green algal species, Pleodorina starrii. Here, we generated whole-genome data of the three sex phenotypes of P. starrii to reveal a reorganization of the ancestral sex-determining regions (SDRs) in the sex chromosomes: the male and bisexual phenotypes had the same "male SDR" with paralogous gene expansions of the male-determining gene MID, whereas the female phenotype had a "female SDR" with transposition of the female-specific gene FUS1 to autosomal regions. Although the male and bisexual sex phenotypes had the identical male SDR and harbored autosomal FUS1, MID and FUS1 expression during sexual reproduction differed between them. Thus, the coexistence of three sex phenotypes in P. starrii is possible.

Root-knot nematode modulates plant CLE3-CLV1 signaling as a long-distance signal for successful infection.

Plants use many long-distance and systemic signals to modulate growth and development, as well as respond to biotic and abiotic stresses. Parasitic nematodes infect host plant roots and cause severe damage to crop plants. However, the molecular mechanisms that regulate parasitic nematode infections are still unknown. Here, we show that plant parasitic root-knot nematodes (RKNs), Meloidogyne incognita, modulate the host CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (CLE)-CLV1 signaling module to promote the infection progression. Plants deficient in the CLE signaling pathway show enhanced RKN resistance, whereas CLE overexpression leads to increased susceptibility toward RKN. Grafting analysis shows that CLV1 expression in the shoot alone is sufficient to positively regulate RKN infection. Together with results from the split-root culture system, infection assays, and CLE3-CLV1 binding assays, we conclude that mobile root-derived CLE signals are perceived by CLV1 in the shoot, which subsequently produce systemic signals to promote gall formation and RKN reproduction.

Novel inhibitors of microtubule organization and phragmoplast formation in diverse plant species.

Cell division is essential for development and involves spindle assembly, chromosome separation, and cytokinesis. In plants, the genetic tools for controlling the events in cell division at the desired time are limited and ineffective owing to high redundancy and lethality. Therefore, we screened cell division-affecting compounds in Arabidopsis thaliana zygotes, whose cell division is traceable without time-lapse observations. We then determined the target events of the identified compounds using live-cell imaging of tobacco BY-2 cells. Subsequently, we isolated two compounds, PD-180970 and PP2, neither of which caused lethal damage. PD-180970 disrupted microtubule (MT) organization and, thus, nuclear separation, and PP2 blocked phragmoplast formation and impaired cytokinesis. Phosphoproteomic analysis showed that these compounds reduced the phosphorylation of diverse proteins, including MT-associated proteins (MAP70) and class II Kinesin-12. Moreover, these compounds were effective in multiple plant species, such as cucumber (Cucumis sativus) and moss (Physcomitrium patens). These properties make PD-180970 and PP2 useful tools for transiently controlling plant cell division at key manipulation nodes conserved across diverse plant species.

Poly(A) polymerase 1 contributes to competence acquisition of pollen tubes growing through the style in Arabidopsis thaliana.

Polyadenylation of mRNAs is critical for their export from the nucleus, stability, and efficient translation. The Arabidopsis thaliana genome encodes three isoforms of canonical nuclear poly(A) polymerase (PAPS) that redundantly polyadenylate the bulk of pre-mRNAs. However, previous studies have indicated that subsets of pre-mRNAs are preferentially polyadenylated by either PAPS1 or the other two isoforms. Such functional specialization raises the possibility of an additional level of gene-expression control in plants. Here we test this notion by studying the function of PAPS1 in pollen-tube growth and guidance. Pollen tubes growing through female tissue acquire the competence to find ovules efficiently and upregulate PAPS1 expression at the transcriptional, but not detectably at the protein level compared with in vitro grown pollen tubes. Using the temperature-sensitive paps1-1 allele we show that PAPS1 activity during pollen-tube growth is required for full acquisition of competence, resulting in inefficient fertilization by paps1-1 mutant pollen tubes. While these mutant pollen tubes grow almost at the wild-type rate, they are compromised in locating the micropyles of ovules. Previously identified competence-associated genes are less expressed in paps1-1 mutant than in wild-type pollen tubes. Estimating the poly(A) tail lengths of transcripts suggests that polyadenylation by PAPS1 is associated with reduced transcript abundance. Our results therefore suggest that PAPS1 plays a key role in the acquisition of competence and underline the importance of functional specialization between PAPS isoforms throughout different developmental stages.

Targeted expression of bgl23-D, a dominant-negative allele of ATCSLD5, affects cytokinesis of guard mother cells and exine formation of pollen in Arabidopsis thaliana.

MAIN CONCLUSION: Targeted expression of bgl23-D, a dominant-negative allele of ATCSLD5, is a useful genetic approach for functional analysis of ATCSLDs in specific cells and tissues in plants. Stomata are key cellular structures for gas and water exchange in plants and their development is influenced by several genes. We found the A. thaliana bagel23-D (bgl23-D) mutant showing abnormal bagel-shaped single guard cells. The bgl23-D was a novel dominant mutation in the A. thaliana cellulose synthase-like D5 (ATCSLD5) gene that was reported to function in the division of guard mother cells. The dominant character of bgl23-D was used to inhibit ATCSLD5 function in specific cells and tissues. Transgenic A. thaliana expressing bgl23-D cDNA with the promoter of stomata lineage genes, SDD1, MUTE, and FAMA, showed bagel-shaped stomata as observed in the bgl23-D mutant. Especially, the FAMA promoter exhibited a higher frequency of bagel-shaped stomata with severe cytokinesis defects. Expression of bgl23-D cDNA in the tapetum with SP11 promoter or in the anther with ATSP146 promoter induced defects in exine pattern and pollen shape, novel phenotypes that were not shown in the bgl23-D mutant. These results indicated that bgl23-D inhibited unknown ATCSLD(s) that exert the function of exine formation in the tapetum. Furthermore, transgenic A. thaliana expressing bgl23-D cDNA with SDD1, MUTE, and FAMA promoters showed enhanced rosette diameter and increased leaf growth. Taken together, these findings suggest that the bgl23-D mutation could be a helpful genetic tool for functional analysis of ATCSLDs and manipulating plant growth.

Whole-mount RNA in situ hybridization technique in Torenia ovules.

The expression pattern of an interested gene at a cellular level provides strong evidence for its functions. RNA in situ hybridization has been proved to be a powerful tool in detecting the spatial-temporal expression pattern of a gene in various organisms. However, classical RNA in situ hybridization (ISH) technique is time-consuming and requires sophisticated sectioning skills. Therefore, we developed a method for whole-mount in situ hybridization (WISH) on ovules of Torenia fournieri, which is a model species in the study of plant reproduction. T. fournieri possesses ovules with protruding embryo sacs, making it easy to be observed and imaged through simple manipulation. To determine the effect of classical ISH and our newly established WISH, we detected the expression of a D-class gene, TfSTK3, using both methods. The expression patterns of TfSTK3 are similar in classical ISH and WISH, confirming reliability of the WISH method. Compared with WISH, classical ISH always leads to distorted embryo sacs, hence difficult to distinguish signals within the female gametophyte. To understand whether our WISH protocol also works well in detecting genes expressed within embryo sacs, we further examined the expression of a synergid-enriched candidate, TfPMEI1, and clearly observed specific signals within two synergid cells. To summarize, our WISH technique allows to visualize gene expression patterns in ovules of T. fournieri within one week and will benefit the field of plant reproduction in the future.

F-actin regulates the polarized secretion of pollen tube attractants in Arabidopsis synergid cells.

Pollen tube attraction is a key event of sexual reproduction in flowering plants. In the ovule, two synergid cells neighboring the egg cell control pollen tube arrival via the active secretion of attractant peptides such as AtLURE1 and XIUQIU from the filiform apparatus (FA) facing toward the micropyle. Distinctive cell polarity together with longitudinal F-actin and microtubules are hallmarks of the synergid cell in various species, though the functions of these cellular structures are unclear. In this study, we used genetic and pharmacological approaches to indicate the roles of cytoskeletal components in FA formation and pollen tube guidance in Arabidopsis thaliana. Genetic inhibition of microtubule formation reduced invaginations of the plasma membrane but did not abolish micropylar AtLURE1.2 accumulation. By contrast, the expression of a dominant-negative form of ACTIN8 induced disorganization of the FA and loss of polar AtLURE1.2 distribution toward the FA. Interestingly, after pollen tube reception, F-actin became unclear for a few hours in the persistent synergid cell, which may be involved in pausing and resuming pollen tube attraction during early polytubey block. Our data suggest that F-actin plays a central role in maintaining cell polarity and in mediating male-female communication in the synergid cell.

A novel function for the sperm adhesion protein IZUMO1 in cell-cell fusion.

Mammalian sperm-egg adhesion depends on the trans-interaction between the sperm-specific type I glycoprotein IZUMO1 and its oocyte-specific GPI-anchored receptor JUNO. However, the mechanisms and proteins (fusogens) that mediate the following step of gamete fusion remain unknown. Using live imaging and content mixing assays in a heterologous system and structure-guided mutagenesis, we unveil an unexpected function for IZUMO1 in cell-to-cell fusion. We show that IZUMO1 alone is sufficient to induce fusion, and that this ability is retained in a mutant unable to bind JUNO. On the other hand, a triple mutation in exposed aromatic residues prevents this fusogenic activity without impairing JUNO interaction. Our findings suggest a second function for IZUMO1 as a unilateral mouse gamete fusogen.

Non-cell-autonomous regulation of petal initiation in Arabidopsis thaliana.

In many flowering plants, petals initiate in alternate positions from first whorl sepals, suggesting possible signaling between sepal boundaries and petal initiation sites. PETAL LOSS (PTL) and RABBIT EARS (RBE) regulate petal initiation in Arabidopsis thaliana and their transcripts are expressed in sepal boundary and petal initiation sites, respectively, suggesting that PTL acts in a non-cell-autonomous manner. Here, we determined that cells expressing PTL and RBE fusion proteins did not overlap but were adjacent, confirming the non-cell-autonomous function of PTL. Genetic ablation of intersepal cells by expressing the diphtheria toxin-A chain gene driven by the PTL promoter resulted in flowers lacking petals, suggesting these cells are required for petal initiation. Transcriptome analysis combined with a PTL induction system revealed 42 genes that were upregulated under PTL activation, including UNUSUAL FLORAL ORGANS (UFO), which likely plays an important role in petal initiation. These findings suggest a molecular mechanism in which PTL indirectly regulates petal initiation and UFO mediates positional signaling between the sepal boundary and petal initiation sites.

Chemical synthesis of Torenia plant pollen tube attractant proteins by KAHA ligation.

The synthesis of secreted cysteine-rich proteins (CRPs) is a long-standing challenge due to protein aggregation and premature formation of inter- and intramolecular disulfide bonds. Chemical synthesis provides reduced CRPs with a higher purity, which is advantageous for folding and isolation. Herein, we report the chemical synthesis of pollen tube attractant CRPs Torenia fournieri LURE (TfLURE) and Torenia concolor LURE (TcLURE) and their chimeric analogues via α-ketoacid-hydroxylamine (KAHA) ligation. The bioactivity of chemically synthesized TfLURE protein was shown to be comparable to E. coli expressed recombinant protein through in vitro assay. The convergent protein synthesis approach is beneficial for preparing these small protein variants efficiently.

Live imaging-based assay for visualising species-specific interactions in gamete adhesion molecules.

Successful gamete fusion requires species-specific membrane adhesion. However, the interaction of adhesion molecules in gametes is difficult to study in real time through low-throughput microscopic observation. Therefore, we developed a live imaging-based adhesion molecule (LIAM) assay to study gamete adhesion molecule interactions in cultured cells. First, we modified a fusion assay previously established for fusogens introduced into cultured cells, and confirmed that our live imaging technique could visualise cell-cell fusion in the modified fusion assay. Next, instead of fusogen, we introduced adhesion molecules including a mammalian gamete adhesion molecule pair, IZUMO1 and JUNO, and detected their temporal accumulation at the contact interfaces of adjacent cells. Accumulated IZUMO1 or JUNO was partly translocated to the opposite cells as discrete spots; the mutation in amino acids required for their interaction impaired accumulation and translocation. By using the LIAM assay, we investigated the species specificity of IZUMO1 and JUNO of mouse, human, hamster, and pig in all combinations. IZUMO1 and JUNO accumulation and translocation were observed in conspecific, and some interspecific, combinations, suggesting potentially interchangeable combinations of IZUMO1 and JUNO from different species.

Quantification of Species-Preferential Micropylar Chemoattraction in Arabidopsis by Fluorescein Diacetate Staining of Pollen Tubes.

Sexual reproduction between males and females of the same species is essential for species maintenance. Ovular micropylar guidance, the last step of pollen tube guidance in angiosperms, contributes to species-preferential reproduction. Previous studies using semi-in vivo attraction assays showed that species-preferential attractant peptides are secreted from the ovule through its micropyle. However, conventional semi-in vivo assays usually depend on transgenic pollen tubes expressing a fluorescent protein to determine whether the tubes are attracted to the ovule to precisely penetrate the micropyle. Here, we found that fluorescein diacetate (FDA) staining was suitable for evaluating the micropylar guidance rate of non-transgenic pollen tubes in semi-in vivo conditions. Micropylar guidance was quantified for ovules and pollen tubes of Arabidopsis thaliana and Arabidopsis lyrata by combining FDA staining with modified semi-in vivo assays. Our results using the simple staining method showed that the ovules of each species secrete species-preferential attractants, and that pollen tubes respond more strongly to attractants of their own species compared with those of closely related species. LURE-type CRP810 attractant peptides were shown to be responsible for micropylar attraction of A. thaliana in the semi-in vivo assay. The POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 6 (PRK6) receptor for LURE1, as well as an unidentified receptor for other LURE-type attractants, are involved in the species-preferential response of these two Arabidopsis species.