Control of root nodule formation ensures sufficient shoot water availability in Lotus japonicus.

Leguminous plants provide carbon to symbiotic rhizobia in root nodules to fuel the energy-consuming process of nitrogen fixation. The carbon investment pattern from the acquired sources is crucial for shaping the growth regime of the host plants. The autoregulation of nodulation (AON) signaling pathway tightly regulates the number of nodules that form. AON disruption leads to excessive nodule formation and stunted shoot growth. However, the physiological role of AON in adjusting the carbon investment pattern is unknown. Here, we show that AON plays an important role in sustaining shoot water availability, which is essential for promoting carbon investment in shoot growth in Lotus japonicus. We found that AON-defective mutants exhibit substantial accumulation of non-structural carbohydrates, such as sucrose. Consistent with this metabolic signature, resilience against water-deficit stress was enhanced in the shoots of the AON-defective mutants. Furthermore, the water uptake ability was attenuated in the AON-defective mutants, likely due to the increased ratio of nodulation zone, which is covered with hydrophobic surfaces, on the roots. These results increase our physiological understanding of legume-rhizobia symbiosis by revealing a trade-off between root nodule formation and shoot water availability.

IMA peptides regulate root nodulation and nitrogen homeostasis by providing iron according to internal nitrogen status.

Legumes control root nodule symbiosis (RNS) in response to environmental nitrogen availability. Despite the recent understanding of the molecular basis of external nitrate-mediated control of RNS, it remains mostly elusive how plants regulate physiological processes depending on internal nitrogen status. In addition, iron (Fe) acts as an essential element that enables symbiotic nitrogen fixation; however, the mechanism of Fe accumulation in nodules is poorly understood. Here, we focus on the transcriptome in response to internal nitrogen status during RNS in Lotus japonicus and identify that IRON MAN (IMA) peptide genes are expressed during symbiotic nitrogen fixation. We show that LjIMA1 and LjIMA2 expressed in the shoot and root play systemic and local roles in concentrating internal Fe to the nodule. Furthermore, IMA peptides have conserved roles in regulating nitrogen homeostasis by adjusting nitrogen-Fe balance in L. japonicus and Arabidopsis thaliana. These findings indicate that IMA-mediated Fe provision plays an essential role in regulating nitrogen-related physiological processes.

The Roles of Functional Amino Acids in Plant Growth and Development.

Plants incorporate acquired carbon and nitrogen into amino acid metabolism, whereby the building blocks of proteins and the precursors of various metabolites are produced. This fundamental demand requires tight amino acid metabolism to sustain physiological homeostasis. There is increasing evidence that amino acid metabolism undergoes plastic alteration to orchestrate specific growth and developmental events. Consequently, there has been a gradual exploration of the interface at which amino acid metabolism and plant morphogenesis are mutually affected. This research progress offers an opportunity to explore amino acid metabolism, with the goal to understand how it can be modulated to serve special cellular needs and regulate specific growth and developmental pathways. Continuous improvements in the sensitivity and coverage of metabolomics technology, along with the development of chemoinformatics, have allowed the investigation of these research questions. In this review, we summarize the roles of threonine, serine, arginine and γ-aminobutyric acid as representative examples of amino acids relevant to specific developmental processes in plants ('functional amino acids'). Our objective is to expand perspectives regarding amino acid metabolism beyond the conventional view that it is merely life-supporting machinery.

Tissue-targeted inorganic pyrophosphate hydrolysis in a fugu5 mutant reveals that excess inorganic pyrophosphate triggers developmental defects in a cell-autonomous manner.

Excess PPi triggers developmental defects in a cell-autonomous manner. The level of inorganic pyrophosphate (PPi) must be tightly regulated in all kingdoms for the proper execution of cellular functions. In plants, the vacuolar proton pyrophosphatase (H+-PPase) has a pivotal role in PPi homeostasis. We previously demonstrated that the excess cytosolic PPi in the H+-PPase loss-of-function fugu5 mutant inhibits gluconeogenesis from seed storage lipids, arrests cell division in cotyledonary palisade tissue, and triggers a compensated cell enlargement (CCE). Moreover, PPi alters pavement cell (PC) shape, stomatal patterning, and functioning, supporting specific yet broad inhibitory effects of PPi on leaf morphogenesis. Whereas these developmental defects were totally rescued by the expression of the yeast soluble pyrophosphatase IPP1, sucrose supply alone canceled CCE in the palisade tissue but not the epidermal developmental defects. Hence, we postulated that the latter are likely triggered by excess PPi rather than a sucrose deficit. To formally test this hypothesis, we adopted a spatiotemporal approach by constructing and analyzing fugu5-1 PDF1 pro ::IPP1, fugu5-1 CLV1 pro ::IPP1, and fugu5-1 ICL pro ::IPP1, whereby PPi was removed specifically from the epidermis, palisade tissue cells, or during the 4 days following seed imbibition, respectively. It is important to note that whereas PC defects in fugu5-1 PDF1 pro ::IPP1 were completely recovered, those in fugu5-1 CLV1 pro ::IPP1 were not. In addition, phenotypic analyses of fugu5-1 ICL pro ::IPP1 lines demonstrated that the immediate removal of PPi after seed imbibition markedly improved overall plant growth, abolished CCE, but only partially restored the epidermal developmental defects. Next, the impact of spatial and temporal removal of PPi was investigated by capillary electrophoresis time-of-flight mass spectrometry (CE-TOF MS). Our analysis revealed that the metabolic profiles are differentially affected among all the above transgenic lines, and consistent with an axial role of central metabolism of gluconeogenesis in CCE. Taken together, this study provides a conceptual framework to unveil metabolic fluctuations within leaf tissues with high spatio-temporal resolution. Finally, our findings suggest that excess PPi exerts its inhibitory effect in planta in the early stages of seedling establishment in a tissue- and cell-autonomous manner.

Leaf-size control beyond transcription factors: Compensatory mechanisms.

Plant leaves display abundant morphological richness yet grow to characteristic sizes and shapes. Beginning with a small number of undifferentiated founder cells, leaves evolve via a complex interplay of regulatory factors that ultimately influence cell proliferation and subsequent post-mitotic cell enlargement. During their development, a sequence of key events that shape leaves is both robustly executed spatiotemporally following a genomic molecular network and flexibly tuned by a variety of environmental stimuli. Decades of work on Arabidopsis thaliana have revisited the compensatory phenomena that might reflect a general and primary size-regulatory mechanism in leaves. This review focuses on key molecular and cellular events behind the organ-wide scale regulation of compensatory mechanisms. Lastly, emerging novel mechanisms of metabolic and hormonal regulation are discussed, based on recent advances in the field that have provided insights into, among other phenomena, leaf-size regulation.

Metabolic Control of Gametophore Shoot Formation through Arginine in the Moss Physcomitrium patens.

Shoot formation is accompanied by active cell proliferation and expansion, requiring that metabolic state adapts to developmental control. Despite the importance of such metabolic reprogramming, it remains unclear how development and metabolism are integrated. Here, we show that disruption of ANGUSTIFOLIA3 orthologs (PpAN3s) compromises gametophore shoot formation in the moss Physcomitrium patens due to defective cell proliferation and expansion. Trans-omics analysis reveals that the downstream activity of PpAN3 is linked to arginine metabolism. Elevating arginine level by chemical treatment leads to stunted gametophores and causes Ppan3 mutant-like transcriptional changes in the wild-type plant. Furthermore, ectopic expression of AtAN3 from Arabidopsis thaliana ameliorates the defective arginine metabolism and promotes gametophore formation in Ppan3 mutants. Together, these findings indicate that arginine metabolism is a key pathway associated with gametophore formation and provide evolutionary insights into the establishment of the shoot system in land plants through the integration of developmental and metabolic processes.

an3-Mediated Compensation Is Dependent on a Cell-Autonomous Mechanism in Leaf Epidermal Tissue.

Leaves are formed by coordinated growth of tissue layers driven by cell proliferation and expansion. Compensation, in which a defect in cell proliferation induces compensated cell enlargement (CCE), plays an important role in cell-size determination during leaf development. We previously reported that CCE triggered by the an3 mutation is observed in epidermal and subepidermal layers in Arabidopsis thaliana (Arabidopsis) leaves. Interestingly, CCE is induced in a non-cell autonomous manner between subepidermal cells. However, whether CCE in the subepidermis affects cell size in the adjacent epidermis is still unclear. We induced layer-specific expression of AN3 in an3 leaves and found that CCE in the subepidermis had little impact on cell-size determination in the epidermis, and vice versa, suggesting that CCE is induced in a tissue-autonomous manner. Examination of the epidermis in an3 leaves having AN3-positive and -negative sectors generated by Cre/loxP revealed that, in contrast to the subepidermis, CCE occurred exclusively in AN3-negative epidermal cells, indicating a cell autonomous action of an3-mediated compensation in the epidermis. These results clarified that the epidermal and subepidermal tissue layers have different cell autonomies in CCE. In addition, quantification of cell-expansion kinetics in epidermal and subepidermal tissues of the an3 showed that the tissues exhibited a similar temporal profile to reach a peak cell-expansion rate as compared to wild type. This might be one feature representing that the two tissue layers retain their growth coordination even in the presence of CCE.

Quantitative Imaging Reveals Distinct Contributions of SnRK2 and ABI3 in Plasmodesmatal Permeability in Physcomitrella patens.

Cell-to-cell communication is tightly regulated in response to environmental stimuli in plants. We previously used a photoconvertible fluorescent protein Dendra2 as a model reporter to study this process. This experiment revealed that macromolecular trafficking between protonemal cells in Physcomitrella patens is suppressed in response to abscisic acid (ABA). However, it remains unknown which ABA signaling components contribute to this suppression and how. Here, we show that ABA signaling components SUCROSE NON-FERMENTING 1-RELATED PROTEIN KINASE 2 (PpSnRK2) and ABA INSENSITIVE 3 (PpABI3) play roles as an essential and promotive factor, respectively, in regulating ABA-induced suppression of Dendra2 diffusion between cells (ASD). Our quantitative imaging analysis revealed that disruption of PpSnRK2 resulted in defective ASD onset itself, whereas disruption of PpABI3 caused an 81-min delay in the initiation of ASD. Live-cell imaging of callose deposition using aniline blue staining showed that, despite this onset delay, callose deposition on cross walls remained constant in the PpABI3 disruptant, suggesting that PpABI3 facilitates ASD in a callose-independent manner. Given that ABA is an important phytohormone to cope with abiotic stresses, we further explored cellular physiological responses. We found that the acquisition of salt stress tolerance is promoted by PpABI3 in a quantitative manner similar to ASD. Our results suggest that PpABI3-mediated ABA signaling may effectively coordinate cell-to-cell communication during the acquisition of salt stress tolerance. This study will accelerate the quantitative study for ABA signaling mechanism and function in response to various abiotic stresses.

Author Correction: Pyrophosphate inhibits gluconeogenesis by restricting UDP-glucose formation in vivo.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Pyrophosphate inhibits gluconeogenesis by restricting UDP-glucose formation in vivo.

Pyrophosphate (PPi) is produced by anabolic reactions and serves as an energy donor in the cytosol of plant cells; however, its accumulation to toxic levels disrupts several common biosynthetic pathways and is lethal. Before acquiring photosynthetic capacity, young seedlings must endure a short but critical heterotrophic period, during which they are nourished solely by sugar produced from seed reserves by the anabolic process of gluconeogenesis. Previously, we reported that excess PPi in H+-PPase-knockout fugu5 mutants of Arabidopsis thaliana severely compromised gluconeogenesis. However, the precise metabolic target of PPi inhibition in vivo remained elusive. Here, CE-TOF MS analyses of major metabolites characteristic of gluconeogenesis from seed lipids showed that the Glc6P;Fru6P level significantly increased and that Glc1P level was consistently somewhat higher in fugu5 compared to wild type. In contrast, the UDP-Glc level decreased significantly in the mutants. Importantly, specific removal of PPi in fugu5, and thus in AVP1pro:IPP1 transgenic lines, restored the Glc1P and the Glc6P;Fru6P levels, increased the UDP-Glc level ~2.0-fold, and subsequently increased sucrose synthesis. Given the reversible nature of the Glc1P/UDP-Glc reaction, our results indicate that UGP-Glc pyrophosphorylase is the major target when excess PPi exerts inhibitory effects in vivo. To validate our findings, we analyzed metabolite responses using a mathematical theory called structural sensitivity analysis (SSA), in which the responses of concentrations in reaction systems to perturbations in enzyme activity are determined from the structure of the network alone. A comparison of our experimental data with the results of pure structural theory predicted the existence of unknown reactions as the necessary condition for the above metabolic profiles, and confirmed the above results. Our data support the notion that H+-PPase plays a pivotal role in cytosolic PPi homeostasis in plant cells. We propose that the combination of metabolomics and SSA is powerful when seeking to identify and predict metabolic targets in living cells.

The cytochrome P450 CYP77A4 is involved in auxin-mediated patterning of the Arabidopsis thaliana embryo.

Metabolism often plays an important role in developmental control, in addition to supporting basal physiological requirements. However, our understanding of this interaction remains limited. Here, we performed quantitative phenome analysis of Arabidopsis thaliana cytochrome P450 mutants to identify a novel interaction between development and metabolism. We found that cyp77a4 mutants exhibit specific defects in cotyledon development, including asymmetric positioning and cup-shaped morphology, which could be rescued by introducing the CYP77A4 gene. Microscopy revealed that the abnormal patterning was detected at least from the 8-cell stage of the cyp77a4 embryos. We next analysed auxin distribution in mutant embryos, as the phenotypes resembled those of auxin-related mutants. We found that the auxin response pattern was severely perturbed in the cyp77a4 embryos owing to an aberrant distribution of the auxin efflux carrier PIN1. CYP77A4 intracellularly localised to the endoplasmic reticulum, which is consistent with the notion that this enzyme acts as an epoxidase of unsaturated fatty acids in the microsomal fraction. We propose that the CYP77A4-dependent metabolic pathway is an essential element for the establishment of polarity in plant embryos.

Spatially Different Tissue-Scale Diffusivity Shapes ANGUSTIFOLIA3 Gradient in Growing Leaves.

The spatial gradient of signaling molecules is pivotal for establishing developmental patterns of multicellular organisms. It has long been proposed that these gradients could arise from the pure diffusion process of signaling molecules between cells, but whether this simplest mechanism establishes the formation of the tissue-scale gradient remains unclear. Plasmodesmata are unique channel structures in plants that connect neighboring cells for molecular transport. In this study, we measured cellular- and tissue-scale kinetics of molecular transport through plasmodesmata in Arabidopsis thaliana developing leaf primordia by fluorescence recovery assays. These trans-scale measurements revealed biophysical properties of diffusive molecular transport through plasmodesmata and revealed that the tissue-scale diffusivity, but not the cellular-scale diffusivity, is spatially different along the leaf proximal-to-distal axis. We found that the gradient in cell size along the developmental axis underlies this spatially different tissue-scale diffusivity. We then asked how this diffusion-based framework functions in establishing a signaling gradient of endogenous molecules. ANGUSTIFOLIA3 (AN3) is a transcriptional co-activator, and as we have shown here, it forms a long-range signaling gradient along the leaf proximal-to-distal axis to determine a cell-proliferation domain. By genetically engineering AN3 mobility, we assessed each contribution of cell-to-cell movement and tissue growth to the distribution of the AN3 gradient. We constructed a diffusion-based theoretical model using these quantitative data to analyze the AN3 gradient formation and demonstrated that it could be achieved solely by the diffusive molecular transport in a growing tissue. Our results indicate that the spatially different tissue-scale diffusivity is a core mechanism for AN3 gradient formation. This provides evidence that the pure diffusion process establishes the formation of the long-range signaling gradient in leaf development.

Probing the stochastic property of endoreduplication in cell size determination of Arabidopsis thaliana leaf epidermal tissue.

Cell size distribution is highly reproducible, whereas the size of individual cells often varies greatly within a tissue. This is obvious in a population of Arabidopsis thaliana leaf epidermal cells, which ranged from 1,000 to 10,000 µm2 in size. Endoreduplication is a specialized cell cycle in which nuclear genome size (ploidy) is doubled in the absence of cell division. Although epidermal cells require endoreduplication to enhance cellular expansion, the issue of whether this mechanism is sufficient for explaining cell size distribution remains unclear due to a lack of quantitative understanding linking the occurrence of endoreduplication with cell size diversity. Here, we addressed this question by quantitatively summarizing ploidy profile and cell size distribution using a simple theoretical framework. We first found that endoreduplication dynamics is a Poisson process through cellular maturation. This finding allowed us to construct a mathematical model to predict the time evolution of a ploidy profile with a single rate constant for endoreduplication occurrence in a given time. We reproduced experimentally measured ploidy profile in both wild-type leaf tissue and endoreduplication-related mutants with this analytical solution, further demonstrating the probabilistic property of endoreduplication. We next extended the mathematical model by incorporating the element that cell size is determined according to ploidy level to examine cell size distribution. This analysis revealed that cell size is exponentially enlarged 1.5 times every endoreduplication round. Because this theoretical simulation successfully recapitulated experimentally observed cell size distributions, we concluded that Poissonian endoreduplication dynamics and exponential size-boosting are the sources of the broad cell size distribution in epidermal tissue. More generally, this study contributes to a quantitative understanding whereby stochastic dynamics generate steady-state biological heterogeneity.

Compensation: a key to clarifying the organ-level regulation of lateral organ size in plants.

Leaves are ideal model systems to study the organ size regulation of multicellular plants. Leaf cell number and cell size are determinant factors of leaf size which is controlled through cell proliferation and post-mitotic cell expansion, respectively. To achieve a proper leaf size, cell proliferation and post-mitotic cell expansion should be co-ordinated during leaf morphogenesis. Compensation, which is enhanced post-mitotic cell expansion associated with a decrease in cell number during lateral organ development, is suggestive of such co-ordination. Genetic and kinematic studies revealed at least three classes of modes of compensation, indicating that compensation is a heterogeneous phenomenon. Recent studies have increased our understanding about the molecular basis of compensation by identifying the causal genes of each compensation-exhibiting mutant. Furthermore, analyses using chimeric leaves revealed that a type of compensated cell expansion requires cell-to-cell communication. Information from recent advances in molecular and genetic studies on compensation has been integrated here and its role in organ size regulation is discussed.

Mobility of signaling molecules: the key to deciphering plant organogenesis.

Signaling molecules move between cells to form a characteristic distribution pattern within a developing organ; thereafter, they spatiotemporally regulate organ development. A key question in this process is how the signaling molecules robustly form the precise distribution on a tissue scale in a reproducible manner. Despite of an increasing number of quantitative studies regarding the mobility of signaling molecules, the detail mechanism of organogenesis via intercellular signaling is still unclear. We here review the potential advantages of plant development to address this question, focusing on the cytoplasmic continuity of plant cells through the plasmodesmata. The plant system would provide a unique opportunity to define the simple transportation mode of diffusion process, and, hence, the mechanism of organogenesis via intercellular signaling. Based on the advances in the understanding of intercellular signaling at the molecular level and in the quantitative imaging techniques, we discuss our current challenges in measuring the mobility of signaling molecules for deciphering plant organogenesis.

Promotion of chloroplast proliferation upon enhanced post-mitotic cell expansion in leaves.

BACKGROUND: Leaves are determinate organs; hence, precise control of cell proliferation and post-mitotic cell expansion is essential for their growth. A defect in cell proliferation often triggers enhanced post-mitotic cell expansion in leaves. This phenomenon is referred to as 'compensation'. Several lines of evidence from studies on compensation have shown that cell proliferation and post-mitotic cell expansion are coordinately regulated during leaf development. Therefore, compensation has attracted much attention to the mechanisms for leaf growth. However, our understanding of compensation at the subcellular level remains limited because studies of compensation have focused mainly on cellular-level phenotypes. Proper leaf growth requires quantitative control of subcellular components in association with cellular-level changes. To gain insight into the subcellular aspect of compensation, we investigated the well-known relationship between cell area and chloroplast number per cell in compensation-exhibiting lines, and asked whether chloroplast proliferation is modulated in response to the induction of compensation. RESULTS: We first established a convenient and reliable method for observation of chloroplasts in situ. Using this method, we analyzed Arabidopsis thaliana mutants fugu5 and angustifolia3 (an3), and a transgenic line KIP-RELATED PROTEIN2 overexpressor (KRP2 OE), which are known to exhibit typical features of compensation. We here showed that chloroplast number per cell increased in the subepidermal palisade tissue of these lines. We analyzed tetraploidized wild type, fugu5, an3 and KRP2 OE, and found that cell area itself, but not nuclear ploidy, is a key parameter that determines the activity of chloroplast proliferation. In particular, in the case of an3, we uncovered that promotion of chloroplast proliferation depends on the enhanced post-mitotic cell expansion. The expression levels of chloroplast proliferation-related genes are similar to or lower than that in the wild type during this process. CONCLUSIONS: This study demonstrates that chloroplast proliferation is promoted in compensation-exhibiting lines. This promotion of chloroplast proliferation takes place in response to cell-area increase in post-mitotic phase in an3. The expression of chloroplast proliferation-related genes were not promoted in compensation-exhibiting lines including an3, arguing that an as-yet-unknown mechanism is responsible for modulation of chloroplast proliferation in these lines.

ANGUSTIFOLIA3 signaling coordinates proliferation between clonally distinct cells in leaves.

Coordinated proliferation between clonally distinct cells via inter-cell-layer signaling largely determines the size and shape of plant organs. Nonetheless, the signaling mechanism underlying this coordination in leaves remains elusive because of a lack of understanding of the signaling molecule (or molecules) involved. ANGUSTIFOLIA3 (AN3, also called GRF-INTERACTING FACTOR1) encodes a putative transcriptional coactivator with homology to human synovial sarcoma translocation protein. AN3 transcripts accumulate in mesophyll cells but are not detectable in leaf epidermal cells. However, we found here that in addition to mesophyll cells, epidermal cells of an3 leaves show defective proliferation. This spatial difference between the accumulation pattern of AN3 transcripts and an3 leaf phenotype is explained by AN3 protein movement across cell layers. AN3 moves into epidermal cells after being synthesized within mesophyll cells and helps control epidermal cell proliferation. Interference with AN3 movement results in abnormal leaf size and shape, indicating that AN3 signaling is indispensable for normal leaf development. AN3 movement does not require type II chaperonin activity, which is needed for movement of some mobile proteins. Taking these findings together, we present a novel model emphasizing the role of mesophyll cells as a signaling source coordinating proliferation between clonally independent leaf cells.

Key proliferative activity in the junction between the leaf blade and leaf petiole of Arabidopsis.

Leaves are the most important, fundamental units of organogenesis in plants. Although the basic form of a leaf is clearly divided into the leaf blade and leaf petiole, no study has yet revealed how these are differentiated from a leaf primordium. We analyzed the spatiotemporal pattern of mitotic activity in leaf primordia of Arabidopsis (Arabidopsis thaliana) in detail using molecular markers in combination with clonal analysis. We found that the proliferative zone is established after a short interval following the occurrence of a rod-shaped early leaf primordium; it is separated spatially from the shoot apical meristem and seen at the junction region between the leaf blade and leaf petiole and produces both leaf-blade and leaf-petiole cells. This proliferative region in leaf primordia is marked by activity of the ANGUSTIFOLIA3 (AN3) promoter as a whole and seems to be differentiated into several spatial compartments: activities of the CYCLIN D4;2 promoter and SPATULA enhancer mark parts of it specifically. Detailed analyses of the an3 and blade-on-petiole mutations further support the idea that organogenesis of the leaf blade and leaf petiole is critically dependent on the correct spatial regulation of the proliferative region of leaf primordia. Thus, the proliferative zone of leaf primordia is spatially differentiated and supplies both the leaf-blade and leaf-petiole cells.

Non-cell-autonomously coordinated organ size regulation in leaf development.

The way in which the number and size of cells in an organ are determined poses a central challenge in our understanding of organ size control. Compensation is an unresolved phenomenon, whereby a decrease in cell proliferation below some threshold level triggers enhanced postmitotic cell expansion in leaf primordia. It suggests an interaction between these cellular processes during organogenesis and provides clues relevant to an understanding of organ size regulation. Although much attention has been given to compensation, it remains unclear how the cellular processes are coordinated. Here, we used a loss-of-function mutation in the transcriptional coactivator gene ANGUSTIFOLIA3 (AN3), which causes typical compensation in Arabidopsis thaliana. We established Cre/lox systems to generate leaves chimeric for AN3 expression and investigated whether compensation occurs in a cell-autonomous or non-cell-autonomous manner. We found that an3-dependent compensation is a non-cell-autonomous process, and that an3 cells seem to generate and transmit an intercellular signal that enhances postmitotic cell expansion. The range of signalling was restricted to within one-half of a leaf partitioned by the midrib. Additionally, we also demonstrated that overexpression of the cyclin-dependent kinase inhibitor gene KIP-RELATED PROTEIN2 resulted in cell-autonomous compensation. Together, our results revealed two previously unknown pathways that coordinate cell proliferation and postmitotic cell expansion for organ size control in plants.

Transcriptional control of two ribosome-inactivating protein genes expressed in spinach (Spinacia oleracea) embryos.

SoRIP1 and SoRIP2 are ribosome-inactivating protein (RIP: EC 3.2.2.22) genes identified in spinach (Spinacia oleracea). They are differentially expressed in a development-dependent manner during spinach somatic embryogenesis. Here, we isolated genomic clones of SoRIP1 and SoRIP2. These two RIP genes have different genomic organization. Phylogenetic analysis of predicted amino acid sequences of RIPs in Caryophyllales plants revealed that they are divided into two major subfamilies, corresponding to SoRIP1 and SoRIP2. To gain further insight into the transcriptional control of SoRIP1 and SoRIP2, we obtained their 5'-flanking sequences by inverse PCR. Comparison of two 5'-flanking sequences revealed the characteristic cis elements in each region that confer differential transcriptional control. In the 5'-flanking region of SoRIP1, we found several motifs with functions related to embryonic development. The 5'-flanking region of SoRIP2 contains some defense-responsive motifs. Expression of SoRIP1 was detected in various tissues. In particular, SoRIP1 was highly expressed in the early immature fruits, and immunohistochemistry showed that SoRIP1 accumulated in the peripheral region of the immature embryo, with weaker expression in internal cells. During fruit development, the expression of SoRIP2 was low. However, the accumulation of SoRIP2 was conspicuous in the epidermis of the immature embryo. The expression of SoRIP2, but not SoRIP1, in leaves was induced by salicylic acid treatment. This differential transcriptional regulation of SoRIP1 and SoRIP2 suggests that the corresponding proteins may have different functions, one being related to embryonic development and the other to embryo defense.