Glutamine (Gln) is the first amino acid synthesized in nitrogen (N) assimilation in plants. Gln synthetase (GS), converting glutamate (Glu) and NH4+ into Gln at the expense of ATP, is one of the oldest enzymes in all life domains. Plants have multiple GS isoenzymes that work individually or cooperatively to ensure the Gln supply is sufficient for plant growth and development under various conditions. Gln is a building block for protein synthesis and an N-donor for the biosynthesis of amino acids, nucleic acids, amino sugars, and vitamin B coenzymes. Most reactions using Gln as an N-donor are catalyzed by Gln amidotransferase (GAT) that hydrolyzes Gln to Glu and transfers the amido group of Gln to an acceptor substrate. Several GAT domain-containing proteins of unknown function in the reference plant Arabidopsis thaliana suggest that some metabolic fates of Gln have yet to be identified in plants. In addition to metabolism, Gln signaling has emerged in recent years. The N regulatory protein PII senses Gln to regulate arginine biosynthesis in plants. Gln promotes somatic embryogenesis and shoot organogenesis with unknown mechanisms. Exogenous Gln has been implicated in activating stress and defense responses in plants. Likely, Gln signaling is responsible for some of the new Gln functions in plants.

Deep neural network (DNN) techniques, as an advanced machine learning framework, have allowed various image diagnoses in plants, which often achieve better prediction performance than human experts in each specific field. Notwithstanding, in plant biology, the application of deep neural networks is still mostly limited to rapid and effective phenotyping. Recent development of explainable CNN frameworks has allowed visualization of the features in the prediction by convolutional neural network (CNN), which potentially contributes to the understanding of physiological mechanisms in objective phenotypes. In this study, we propose an integration of explainable CNN and transcriptomic approach to make a physiological interpretation of a fruit internal disorder in persimmon, rapid over-softening. We constructed CNN models to accurately predict the fate to be rapid softening in persimmon cv. Soshu, only with photo images. The explainable CNNs, such as Grad-CAM and Guided Grad-CAM, visualized specific featured regions relevant to the prediction of rapid-softening, which would correspond to the premonitory symptoms in a fruit. Transcriptomic analyses to compare the featured regions of predicted rapid-softening and control fruits suggested that rapid softening is triggered by precocious ethylene signal-dependent cell wall modification, despite exhibiting no direct phenotypic changes. Further transcriptomic comparison between the featured and non-featured regions in predicted rapid-softening fruit suggested that premonitory symptoms reflected hypoxia and the related stress signals finally to induce ethylene signals. These results would provide a good example for the collaboration of image analysis and omics approaches in plant physiology, which uncovered a novel aspect of fruit premonitory reactions in the rapid softening fate.

The reflection light forms the core of our visual perception of the world. We can obtain vast information by examining reflection light from biological surfaces, including pigment composition and distribution, tissue structure, and surface microstructure. However, because of the limitations in our visual system, the complete information in reflection light, which we term "reflectome," cannot be fully exploited. For example, we may miss reflection light information outside our visible wavelengths. In addition, unlike insects, we have virtually no sensitivity to light polarization. We can detect non-chromatic information lurking in reflection light only with appropriate devices. Though previous studies have designed and developed systems for specialized uses supporting our visual systems, we still do not have a versatile, rapid, convenient, and affordable system for analyzing broad aspects of reflection from biological surfaces. To overcome this situation, we developed P-MIRU, a novel multi-spectral and polarization imaging system for reflecting light from biological surfaces. The hardware and software of P-MIRU are open-source and customizable and thus can be applied for virtually any research on biological surfaces. Furthermore, P-MIRU is a user-friendly system for biologists with no specialized programming or engineering knowledge. P-MIRU successfully visualized multi-spectral reflection in visible/non-visible wavelengths and simultaneously detected various surface phenotypes of spectral polarization. P-MIRU system extends our visual ability and unveils information on biological surfaces. (217/250 words).

Plants produce sugars by photosynthesis and use them for growth and development. Sugars are transported from source-to-sink organs via the phloem in the vasculature. It is well known that vascular development is precisely controlled by plant hormones and peptide hormones. However, the role of sugars in the regulation of vascular development is poorly understood. In this study, we examined the effects of sugars on vascular cell differentiation using a vascular cell induction system named Vascular cell Induction culture System Using Arabidopsis Leaves (VISUAL). We found that sucrose has the strongest inhibitory effect on xylem differentiation among several types of sugars. Transcriptome analysis revealed that sucrose suppresses xylem and phloem differentiation from cambial cells. Physiological and genetic analysis suggested that sucrose might function through the BES1 transcription factor, which is the central regulator of vascular cell differentiation. Conditional overexpression of cytosolic invertase led to a decrease in the number of cambium layers due to an imbalance between cell division and differentiation. Taken together, our results suggest that sucrose potentially acts as a signal that integrates environmental conditions with the developmental program.

In the anthers and ovaries of flowers, pollen grains and embryo sacs are produced with uniform cell compositions. This stable gametogenesis enables elaborate interactions between male and female gametophytes after pollination, forming the highly successful sexual reproduction system in flowering plants. As most ovules are fertilized with a single pollen tube, the resulting genome set in the embryo and endosperm is determined in a single pattern by independent fertilization of the egg cell and central cell by two sperm cells. However, if ovules receive four sperm cells from two pollen tubes, the expected options for genome sets in the developing seeds would more than double. In wild-type Arabidopsis thaliana plants, around 5% of ovules receive two pollen tubes. Recent studies have elucidated the abnormal fertilization in supernumerary pollen tubes and sperm cells related to polytubey, polyspermy, heterofertilization and fertilization recovery. Analyses of model plants have begun to uncover the mechanisms underlying this new pollen tube biology. Here, we review unusual fertilization phenomena and propose several breeding applications for flowering plants. These arguments contribute to the remodeling of plant reproduction, a challenging concept that alters typical plant fertilization by utilizing the current genetic toolbox.

Plant specialized metabolites (PSMs) are often stored as glycosides within cells and released from the roots with some chemical modifications. While isoflavones are known to function as symbiotic signals with rhizobia and to modulate the soybean rhizosphere microbiome, the underlying mechanisms of root-to-soil delivery are poorly understood. In addition to transporter-mediated secretion, the hydrolysis of isoflavone glycosides in the apoplast by an isoflavone conjugate-hydrolyzing β-glucosidase (ICHG) has been proposed but not yet verified. To clarify the role of ICHG in isoflavone supply to the rhizosphere, we have isolated two independent mutants defective in ICHG activity from a soybean high-density mutant library. In the root apoplastic fraction of ichg mutants, the isoflavone glycoside contents were significantly increased, while isoflavone aglycone contents were decreased, indicating that ICHG hydrolyzes isoflavone glycosides into aglycones in the root apoplast. When grown in a field, the lack of ICHG activity considerably reduced isoflavone aglycone contents in roots and the rhizosphere soil, although the transcriptomes showed no distinct differences between the ichg mutants and wild-types (WTs). Despite the change in isoflavone contents and composition of the root and rhizosphere of the mutants, root and rhizosphere bacterial communities were not distinctive from those of the WTs. Root bacterial communities and nodulation capacities of the ichg mutants did not differ from the WTs under nitrogen-deficient conditions either. Taken together, these results indicate that ICHG elevates the accumulation of isoflavones in the soybean rhizosphere but is not essential for isoflavone-mediated plant-microbe interactions.

Phosphate (Pi) deficiency leads to the induction of purple acid phosphatases (PAPs) in plants, which dephosphorylate organic phosphorus (P) complexes in the rhizosphere and intracellular compartments to release Pi. In this study, we demonstrate that OsPAP3b belongs to group III low-molecular weight PAP and is low Pi-responsive, preferentially in roots. The expression of OsPAP3b is negatively regulated with Pi resupply. Interestingly, OsPAP3b was found to be dual localized to the nucleus and secretome. Furthermore, OsPAP3b is transcriptionally regulated by OsPHR2 as substantiated by DNA-protein binding assay. Through in vitro biochemical assays, we further demonstrate that OsPAP3b is a functional acid phosphatase (APase) with broad substrate specificity. The overexpression (OE) of OsPAP3b in rice led to increased secreted APase activity and improved mineralization of organic P sources, which resulted in better growth of transgenics compared to the wild type when grown on organic P as an exogenous P substrate. Under Pi deprivation, OsPAP3b knock-down and knock-out lines showed no significant changes in total P content and dry biomass. However, the expression of other phosphate starvation-induced genes and the levels of metabolites were found to be altered in the OE and knock-down lines. In addition, in vitro pull-down assay revealed multiple putative interacting proteins of OsPAP3b. Our data collectively suggest that OsPAP3b can aid in organic P utilization in rice. The APase isoform behavior and nuclear localization indicate its additional role, possibly in stress signaling. Considering its important roles, OsPAP3b could be a potential target for improving low Pi adaptation in rice.

Autophagy in plants is regulated by diverse signaling cascades in response to environmental changes. Fine-tuning of its activity is critical for the maintenance of cellular homeostasis under basal and stressed conditions. In this study, we compared the Arabidopsis autophagy-related (ATG) system transcriptionally under inorganic phosphate (Pi) deficiency versus nitrogen deficiency and showed that most ATG genes are only moderately upregulated by Pi starvation, with relatively stronger induction of AtATG8f and AtATG8h among the AtATG8 family. We found that Pi shortage increased the formation of GFP-ATG8f-labeled autophagic structures and the autophagic flux in the differential zone of the Arabidopsis root. However, the proteolytic cleavage of GFP-ATG8f and the vacuolar degradation of endogenous ATG8 proteins indicated that Pi limitation does not drastically alter the autophagic flux in the whole roots, implying a cell type-dependent regulation of autophagic activities. At the organismal level, the Arabidopsis atg mutants exhibited decreased shoot Pi concentrations and smaller meristem sizes under Pi sufficiency. Under Pi limitation, these mutants showed enhanced Pi uptake and impaired root cell division and expansion. Despite a reduced steady-state level of several PHOSPHATE TRANSPORTER 1s (PHT1s) in the atg root, cycloheximide treatment analysis suggested that the protein stability of PHT1;1/2/3 is comparable in the Pi-replete wild type and atg5-1. By contrast, the degradation of PHT1;1/2/3 is enhanced in the Pi-deplete atg5-1. Our findings reveal that both basal autophagy and Pi starvation-induced autophagy are required for the maintenance of Pi homeostasis and may modulate the expression of PHT1s through different mechanisms.

Acetylation is one of the most common chemical modifications found on a variety of molecules ranging from metabolites to proteins. Although numerous chloroplast proteins have been shown to be acetylated, the role of acetylation in the regulation of chloroplast functions has remained mainly enigmatic. The chloroplast acetylation machinery in Arabidopsis thaliana consists of eight General control non-repressible 5 (GCN5)-related N-acetyltransferase (GNAT)-family enzymes that catalyze both N-terminal and lysine acetylation of proteins. Additionally, two plastid GNATs have also been reported to be involved in the biosynthesis of melatonin. Here, we have characterized six plastid GNATs (GNAT1, GNAT2, GNAT4, GNAT6, GNAT7 and GNAT10) using a reverse genetics approach with an emphasis on the metabolomes and photosynthesis of the knock-out plants. Our results reveal the impact of GNAT enzymes on the accumulation of chloroplast-related compounds, such as oxylipins and ascorbate, and the GNAT enzymes also affect the accumulation of amino acids and their derivatives. Specifically, the amount of acetylated arginine and proline was significantly decreased in the gnat2 and gnat7 mutants, respectively, as compared to the wild-type Col-0 plants. Additionally, our results show that the loss of the GNAT enzymes results in increased accumulation of Rubisco and Rubisco activase (RCA) at the thylakoids. Nevertheless, the reallocation of Rubisco and RCA did not have consequent effects on carbon assimilation under the studied conditions. Taken together, our results show that chloroplast GNATs affect diverse aspects of plant metabolism and pave way for future research into the role of protein acetylation.

Shade avoidance syndrome (SAS) is a strategy of major adaptive significance and typically includes elongation of the stem and petiole, leaf hyponasty, reduced branching and phototropic orientation of the plant shoot toward canopy gaps. Both cryptochrome 1 and phytochrome B (phyB) are the major photoreceptors that sense the reduction in the blue light fluence rate and the low red:far-red ratio, respectively, and both light signals are associated with plant density and the resource reallocation when SAS responses are triggered. The B-box (BBX)-containing zinc finger transcription factor BBX24 has been implicated in the SAS as a regulator of DELLA activity, but this interaction does not explain all the observed BBX24-dependent regulation in shade light. Here, through a combination of transcriptional meta-analysis and large-scale identification of BBX24-interacting transcription factors, we found that JAZ3, a jasmonic acid signaling component, is a direct target of BBX24. Furthermore, we demonstrated that joint loss of BBX24 and JAZ3 function causes insensitivity to DELLA accumulation, and the defective shade-induced elongation in this mutant is rescued by loss of DELLA or phyB function. Therefore, we propose that JAZ3 is part of the regulatory network that controls the plant growth in response to shade, through a mechanism in which BBX24 and JAZ3 jointly regulate DELLA activity. Our results provide new insights into the participation of BBX24 and JA signaling in the hypocotyl shade avoidance response in Arabidopsis.

Class II water-soluble chlorophyll proteins (WSCPs) from Brassicaceae are non-photosynthetic proteins that bind with chlorophyll (Chl) and its derivatives. The physiological function of WSCPs is still unclear, but it is assumed to be involved in stress responses, which is likely related to their Chl-binding and protease inhibition (PI) activities. Yet, the dual function and simultaneous functionality of WSCPs must still be better understood. Here, the biochemical functions of Brassica napus drought-induced 22-kDa protein (BnD22), a major WSCP expressed in B. napus leaves, were investigated using recombinant hexahistidine-tagged protein. We showed that BnD22 inhibited cysteine proteases, such as papain, but not serine proteases. BnD22 was able to bind with Chla or Chlb to form tetrameric complexes. Unexpectedly, BnD22-Chl tetramer displays higher inhibition toward cysteine proteases, indicating (i) simultaneous Chl-binding and PI activities and (ii) Chl-dependent activation of PI activity of BnD22. Moreover, the photostability of BnD22-Chl tetramer was reduced upon binding with the protease. Using three-dimensional structural modeling and molecular docking, we revealed that Chl binding favors interaction between BnD22 and proteases. Despite its Chl-binding ability, the BnD22 was not detected in chloroplasts but rather in the endoplasmic reticulum and vacuole. In addition, the C-terminal extension peptide of BnD22, which cleaved off post-translationally in vivo, was not implicated in subcellular localization. Instead, it drastically promoted the expression, solubility and stability of the recombinant protein.

Programmed cell death (PCD) in lateral root caps (LRCs) is crucial for maintaining root cap functionality. Endoplasmic reticulum (ER) bodies play important roles in plant immunity and PCD. However, the distribution of ER bodies and their communication with vacuoles in the LRC remain elusive. In this study, we investigated the ultrastructure of LRC cells of wild-type and transgenic Arabidopsis lines using an auto-acquisition transmission electron microscope (TEM) system and high-pressure freezing. Gigapixel-scale high-resolution TEM imaging of the transverse and longitudinal sections of roots followed by three-dimensional imaging identified sausage-shaped structures budding from the ER. These were subsequently identified as ER bodies using GFPh transgenic lines expressing green fluorescent protein (GFP) fused with an ER retention signal (HDEL). Immunogold labeling using an anti-GFP antibody detected GFP signals in the ER bodies and vacuoles. The fusion of ER bodies with vacuoles in LRC cells was identified using correlative light and electron microscopy. Imaging of the root tips of a GFPh transgenic line with a PYK10 promoter revealed the localization of PYK10, a member of the β-glucosidase family with an ER retention signal, in the ER bodies in the inner layer along with a fusion of ER bodies with vacuoles in the middle layer and collapse of vacuoles in the outer layer of the LRC. These findings suggest that ER bodies in LRC directly transport β-glucosidases to the vacuoles, and that a subsequent vacuolar collapse triggered by an unknown mechanism releases protective substances to the growing root tip to protect it from the invaders.