Correction: Suppression of class I compensated cell enlargement by xs2 mutation is mediated by salicylic acid signaling.

[This corrects the article DOI: 10.1371/journal.pgen.1008873.].

Characterization of the Arabidopsis mutant oligocellula6-D reveals the importance of leaf initiation in determining final leaf size.

The leaf is a determinate organ with a final size under genetic control. Numerous factors that regulate final leaf size have been identified in Arabidopsis thaliana; although most of these factors play their roles during the growth of leaf primordia, much less is known about leaf initiation and its effects on final leaf size. In this study, we characterized oligocellula6-D (oli6-D), a semidominant mutant of Arabidopsis thaliana with smaller leaves than the wild type due to its reduced leaf cell numbers. A time-course analysis showed that oli6-D had approximately 50% fewer leaf cells, even immediately after leaf initiation; this difference was maintained throughout leaf development. Next-generation sequencing showed that oli6-D had chromosomal duplication involving 2-kbp and 3-Mbp regions of chromosomes 2 and 4, respectively. Several duplicated genes examined had approximately twofold higher expression levels, and at least one gene acquired a new intron/exon structure due to a chromosome fusion event. oli6-D showed reduced auxin responses in leaf primordia, primary roots, and embryos as well as reduced apical dominance and partial auxin-resistant root growth. CRISPR/Cas9-mediated genome editing enabled the removal of a 3-Mbp duplicated segment, the largest targeted deletion in plants thus far. As a result, oli6-D restored the wild-type leaf phenotypes, demonstrating that oli6-D is a gain-of-function mutant. Our results suggest a new regulatory point of leaf size determination that functions at a very early stage of leaf development and is negatively regulated by one or more genes located in the duplicated chromosomal segments.

Stem integrity in Arabidopsis thaliana requires a load-bearing epidermis.

Because plant cells are glued to each other via their cell walls, failure to coordinate growth among adjacent cells can create cracks in tissues. Here, we find that the unbalanced growth of inner and outer tissues in the clavata3 de-etiolated3 (clv3 det3) mutant of Arabidopsis thaliana stretched epidermal cells, ultimately generating cracks in stems. Stem growth slowed before cracks appeared along clv3 det3 stems, whereas inner pith cells became drastically distorted and accelerated their growth, yielding to stress, after the appearance of cracks. This is consistent with a key role of the epidermis in restricting growth. Mechanical property measurements recorded using an atomic force microscope revealed that epidermal cell wall stiffness decreased in det3 and clv3 det3 epidermises. Thus, we hypothesized that stem integrity depends on the epidermal resistance to mechanical stress. To formally test this hypothesis, we used the DET3 gene as part of a tissue-specific strategy to complement cell expansion defects. Epidermis-driven DET3 expression restored growth and restored the frequency of stem cracking to 20% of the clv3 det3 mutant, demonstrating the DET3-dependent load-bearing role of the epidermis.

Suppression of class I compensated cell enlargement by xs2 mutation is mediated by salicylic acid signaling.

The regulation of leaf size has been studied for decades. Enhancement of post-mitotic cell expansion triggered by impaired cell proliferation in Arabidopsis is an important process for leaf size regulation, and is known as compensation. This suggests a key interaction between cell proliferation and cell expansion during leaf development. Several studies have highlighted the impact of this integration mechanism on leaf size determination; however, the molecular basis of compensation remains largely unknown. Previously, we identified extra-small sisters (xs) mutants which can suppress compensated cell enlargement (CCE) via a specific defect in cell expansion within the compensation-exhibiting mutant, angustifolia3 (an3). Here we revealed that one of the xs mutants, namely xs2, can suppress CCE not only in an3 but also in other compensation-exhibiting mutants erecta (er) and fugu2. Molecular cloning of XS2 identified a deleterious mutation in CATION CALCIUM EXCHANGER 4 (CCX4). Phytohormone measurement and expression analysis revealed that xs2 shows hyper activation of the salicylic acid (SA) response pathway, where activation of SA response can suppress CCE in compensation mutants. All together, these results highlight the regulatory connection which coordinates compensation and SA response.

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.

Evidence for a Role of ANAC082 as a Ribosomal Stress Response Mediator Leading to Growth Defects and Developmental Alterations in Arabidopsis.

Ribosome-related mutants in Arabidopsis thaliana share several notable characteristics regarding growth and development, which implies the existence of a common pathway that responds to disorders in ribosome biogenesis. As a first step to explore this pathway genetically, we screened a mutagenized population of root initiation defective2 (rid2), a temperature-sensitive mutant that is impaired in pre-rRNA processing, and isolated suppressor of root initiation defective two1 (sriw1), a suppressor mutant in which the defects of cell proliferation observed in rid2 at the restrictive temperature was markedly rescued. sriw1 was identified as a missense mutation of the NAC transcription factor gene ANAC082 The sriw1 mutation greatly alleviated the developmental abnormalities of rid2 and four other tested ribosome-related mutants, including rid3 However, the impaired pre-rRNA processing in rid2 and rid3 was not relieved by sriw1 Expression of ANAC082 was localized to regions where phenotypic effects of ribosome-related mutations are readily evident and was elevated in rid2 and rid3 compared with the wild type. These findings suggest that ANAC082 acts downstream of perturbation of biogenesis of the ribosome and may mediate a set of stress responses leading to developmental alterations and cell proliferation defects.

tRNA Wobble Modification Affects Leaf Cell Development in Arabidopsis thaliana.

The tRNA modification at the wobble position of Lys, Glu and Gln (wobbleU* modification) is responsible for the fine-tuning of protein translation efficiency and translation rate. This modification influences organism function in accordance with growth and environmental changes. However, the effects of wobbleU* modification at the cellular, tissue, or individual level have not yet been elucidated. In this study, we show that sulfur modification of wobbleU* of the tRNAs affects leaf development in Arabidopsis thaliana. The sulfur modification was impaired in the two wobbleU*-modification mutants: the URM1-like protein-defective mutant and the Elongator complex-defective mutants. Analyses of the mutant phenotypes revealed that the deficiency in the wobbleU* modification increased the airspaces in the leaves and the leaf size without affecting the number and the area of palisade mesophyll cells. On the other hand, both mutants exhibited increased number of leaf epidermal pavement cells but with reduced cell size. The deficiency in the wobbleU* modification also delayed the initiation of the endoreduplication processes of mesophyll cells. The phenotype of ASYMMETRIC LEAVES2-defective mutant was enhanced in the Elongator-defective mutants, while it was unchanged in the URM1-like protein-defective mutant. Collectively, the findings of this study suggest that the tRNA wobbleU* modification plays an important role in leaf morphogenesis by balancing the development between epidermal and mesophyll tissues.

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.

Double-stranded RNA-binding protein DRB3 negatively regulates anthocyanin biosynthesis by modulating PAP1 expression in Arabidopsis thaliana.

The model plant Arabidopsis thaliana has five double-stranded RNA-binding proteins (DRB1-DRB5), two of which, DRB1 and DRB4, are well characterized. In contrast, the functions of DRB2, DRB3 and DRB5 have yet to be elucidated. In this study, we tried to uncover their functions using drb mutants and DRB-over-expressed lines. In over-expressed lines of all five DRB genes, the over-expression of DRB2 or DRB3 (DRB2ox or DRB3ox) conferred a downward-curled leaf phenotype, but the expression profiles of ten small RNAs were similar to that of the wild-type (WT) plant. Phenotypes were examined in response to abiotic stresses. Both DRB2ox and DRB3ox plants exhibited salt-tolerance. When these plants were exposed to cold stress, drb2 and drb3 over-accumulated anthocyanin but DRB2ox and DRB3ox did not. Therefore, the over-expression of DRB2 or DRB3 had pleiotropic effects on host plants. Microarray and deep-sequencing analyses indicated that several genes encoding key enzymes for anthocyanin biosynthesis, including chalcone synthase (CHS), dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS), were down-regulated in DRB3ox plants. When DRB3ox was crossed with the pap1-D line, which is an activation-tagged transgenic line that over-expresses the key transcription factor PAP1 (Production of anthocyanin pigmentation1) for anthocyanin biosynthesis, over-expression of DRB3 suppressed the expression of PAP1, CHS, DFR and ANS genes. DRB3 negatively regulates anthocyanin biosynthesis by modulating the level of PAP1 transcript. Since two different small RNAs regulate PAP1 gene expression, a possible function of DRB3 for small RNA biogenesis is discussed.

Stable establishment of cotyledon identity during embryogenesis in Arabidopsis by ANGUSTIFOLIA3 and HANABA TARANU.

In seed plants, the shoot apical and root apical meristems form at the apical and basal poles of the embryonic axis, and leaves form at the flanks of the shoot apical meristem. ANGUSTIFOLIA3/GRF INTERACTING FACTOR1 (AN3/GIF1) encodes a putative transcriptional co-activator involved in various aspects of shoot development, including the maintenance of shoot apical meristems, cell proliferation and expansion in leaf primordia, and adaxial/abaxial patterning of leaves. Here, we report a novel function of AN3 involved in developmental fate establishment. We characterised an an3-like mutant that was found to be an allele of hanaba taranu (han), named han-30, and examined its genetic interactions with an3. an3 han double mutants exhibited severe defects in cotyledon development such that ectopic roots were formed at the apical region of the embryo, as confirmed by pWOX5::GFP expression. Additionally, gif2 enhanced the ectopic root phenotype of an3 han. Although the auxin accumulation pattern of the embryo was correct in an3 han-30, based on DR5rev::GFP expression at the globular stage, expression of the PLETHORA1 (PLT1), a master regulator of root development, expanded from the basal embryonic region to the apical region during the same developmental stage. Furthermore, the plt1 mutation suppressed ectopic root formation in an3 han. These data suggest that establishing cotyledon identity requires both AN3 and HAN to repress ectopic root formation by repressing PLT1 expression.

Nitrogen dioxide regulates organ growth by controlling cell proliferation and enlargement in Arabidopsis.

• To gain more insight into the physiological function of nitrogen dioxide (NO2), we investigated the effects of exogenous NO2 on growth in Arabidopsis thaliana. • Plants were grown in air without NO2 for 1 wk after sowing and then grown for 1-4 wk in air with (designated treated plants) or without (control plants) NO2. Plants were irrigated semiweekly with a nutrient solution containing 19.7 mM nitrate and 10.3 mM ammonium. • Five-week-old plants treated with 50 ppb NO2 showed a ≤ 2.8-fold increase in biomass relative to controls. Treated plants also showed early flowering. The magnitude of the effects of NO2 on leaf expansion, cell proliferation and enlargement was greater in developing than in maturing leaves. Leaf areas were 1.3-8.4 times larger on treated plants than corresponding leaves on control plants. The NO2-induced increase in leaf size was largely attributable to cell proliferation in developing leaves, but was attributable to both cell proliferation and enlargement in maturing leaves. The expression of different sets of genes for cell proliferation and/or enlargement was induced by NO2, but depended on the leaf developmental stage. • Collectively, these results indicated that NO2 regulates organ growth by controlling cell proliferation and enlargement.

The ATM-dependent DNA damage response acts as an upstream trigger for compensation in the fas1 mutation during Arabidopsis leaf development.

During leaf development, a decrease in cell number often triggers an increase in cell size. This phenomenon, called compensation, suggests that some system coordinates cell proliferation and cell expansion, but how this is mediated at the molecular level is still unclear. The fugu2 mutants in Arabidopsis (Arabidopsis thaliana) exhibit typical compensation phenotypes. Here, we report that the FUGU2 gene encodes FASCIATA1 (FAS1), the p150 subunit of Chromatin Assembly Factor1. To uncover how the fas1 mutation induces compensation, we performed microarray analyses and found that many genes involved in the DNA damage response are up-regulated in fas1. Our genetic analysis further showed that activation of the DNA damage response and the accompanying decrease of cell number in fas1 depend on ATAXIA TELANGIECTASIA MUTATED (ATM) but not on ATM AND RAD3 RELATED. Kinematic analysis suggested that the delay in the cell cycle leads to a decrease in cell number in fas1 and that loss of ATM partially restores this phenotype. Consistently, both cell size phenotypes and high ploidy phenotypes of fas1 are also suppressed by atm, supporting that the ATM-dependent DNA damage response leads to these phenotypes. Altogether, these data suggest that the ATM-dependent DNA damage response acts as an upstream trigger in fas1 to delay the cell cycle and promote entry into the endocycle, resulting in compensated cell expansion.

Keep an eye on PPi: the vacuolar-type H+-pyrophosphatase regulates postgerminative development in Arabidopsis.

Postgerminative growth of seed plants requires specialized metabolism, such as gluconeogenesis, to support heterotrophic growth of seedlings until the functional photosynthetic apparatus is established. Here, we show that the Arabidopsis thaliana fugu5 mutant, which we show to be defective in AVP1 (vacuolar H(+)-pyrophosphatase), failed to support heterotrophic growth after germination. We found that exogenous supplementation of Suc or the specific removal of the cytosolic pyrophosphate (PPi) by the heterologous expression of the cytosolic inorganic pyrophosphatase1 (IPP1) gene from budding yeast (Saccharomyces cerevisiae) rescued fugu5 phenotypes. Furthermore, compared with the wild-type and AVP1(Pro):IPP1 transgenic lines, hypocotyl elongation in the fugu5 mutant was severely compromised in the dark but recovered upon exogenous supply of Suc to the growth media. Measurements revealed that the peroxisomal ß-oxidation activity, dry seed contents of storage lipids, and their mobilization were unaffected in fugu5. By contrast, fugu5 mutants contained ~2.5-fold higher PPi and ~50% less Suc than the wild type. Together, these results provide clear evidence that gluconeogenesis is inhibited due to the elevated levels of cytosolic PPi. This study demonstrates that the hydrolysis of cytosolic PPi, rather than vacuolar acidification, is the major function of AVP1/FUGU5 in planta. Plant cells optimize their metabolic function by eliminating PPi in the cytosol for efficient postembryonic heterotrophic growth.

Enhanced cell expansion in a KRP2 overexpressor is mediated by increased V-ATPase activity.

Decreased cell numbers during leaf development often trigger increased cell size, a phenomenon called compensation. In compensation-exhibiting mutants, the unusually high cell expansion activity occurs through two different mechanisms during the post-mitotic stage of leaf development, except in the KIP-RELATED PROTEIN 2-overexpressing line (KRP2 o/e), whose cell sizes are 2-fold greater during proliferative growth. However, the molecular basis of compensated cell expansion (CCE) has not been characterized. The det3-1 mutant has a mutation in the C-subunit of the vacuolar-type H(+)-ATPase (V-ATPase) complex that causes a 50% decrease in its activity and cell size. To determine the contribution of V-ATPase activity to CCE, the cellular phenotypes of double mutants between det3-1 and compensation-exhibiting fugu5-1, an3-4, fas1-5 and KRP2 o/e were analyzed in detail. Interestingly, while decreased V-ATPase activity caused by det3-1 did not suppress CCE in fugu5-1, fas1-5 and an3-4, CCE in KRP2 o/e was totally suppressed. Furthermore, measurements revealed that the activity and quantity of the A-subunit of the V-ATPase complex were significantly increased in the shoots of KRP2 o/e plants. Importantly, the unusually increased size of actively dividing KRP2 o/e cells was restored to normal in the det3-1 background. Taken together, our data strongly suggest that CCE in KRP2 o/e, but not in other compensation-exhibiting mutants, occurs exclusively through the increase of V-ATPase activity.

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.

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.

How do 'housekeeping' genes control organogenesis?--Unexpected new findings on the role of housekeeping genes in cell and organ differentiation.

In recent years, an increasing number of mutations in what would appear to be 'housekeeping genes' have been identified as having unexpectedly specific defects in multicellular organogenesis. This is also the case for organogenesis in seed plants. Although it is not surprising that loss-of-function mutations in 'housekeeping' genes result in lethality or growth retardation, it is surprising when (1) the mutant phenotype results from the loss of function of a 'housekeeping' gene and (2) the mutant phenotype is specific. In this review, by defining housekeeping genes as those encoding proteins that work in basic metabolic and cellular functions, we discuss unexpected links between housekeeping genes and specific developmental processes. In a surprising number of cases housekeeping genes coding for enzymes or proteins with functions in basic cellular processes such as transcription, post-transcriptional modification, and translation affect plant development.

Differential contributions of ribosomal protein genes to Arabidopsis thaliana leaf development.

In Arabidopsis thaliana, mutations in genes encoding ribosomal proteins (r-proteins) perturb various developmental processes. Whether these perturbations are caused by overall ribosome insufficiency or partial dysfunction of the ribosome caused by deficiency of a particular ribosomal protein is not known. To distinguish these possibilities, a comparative study using several r-protein mutants was required. Here, we identified mutations in 11 r-protein genes from previously isolated denticulata and pointed-leaves mutants. Most of these mutations were associated with pointed leaves, with reduced growth due to a decrease in the number or size of palisade mesophyll and pavement cells. In addition, leaf abaxialization was usually observed when these r-protein mutations were combined with asymmetric leaves1 (as1) and as2 mutations. These results suggest that the establishment of leaf polarity is highly sensitive to ribosome functionality in general. However, several r-protein mutants showed a preference towards a specific developmental defect. For example, rpl4d mutations did not affect cell proliferation but caused strong abaxialization of leaves in the as1 and as2 backgrounds. On the other hand, rps28b enhanced leaf abaxialization of as2 to a weaker extent than expected on the basis of its negative effect on cell proliferation. In addition, hypomorphic rps6a alleles had the strongest effects on most of the phenotypes examined. These findings suggest that deficiencies in these three r-protein genes lead to production of dysfunctional ribosomes. Depending on their structural abnormalities, dysfunctional ribosomes may affect translation of specific transcripts involved in the regulation of some leaf developmental processes.

ANGUSTIFOLIA, a plant homolog of CtBP/BARS, functions outside the nucleus.

CtBP/BARS is a unique protein family in having quite diversified cellular functions, intercellular localizations, and developmental roles. ANGUSTIFOLIA (AN) is the sole homolog of CtBP/BARS from Arabidopsis thaliana, although it has plant AN-specific motifs and a long C-terminus. Previous studies suggested that AN would function in the nucleus as a transcriptional co-repressor, as CtBPs function in animals; however, precise verification has been lacking. In this paper, we isolated a homologous gene (MAN) of AN from liverwort, Marchantia polymorpha. Transformation of the Arabidopsis an-1 mutant with 35S-driven MAN completely complemented the an-1 phenotype, although it lacks the putative nuclear localization signal (NLS) that exists in AN proteins isolated from other plant species. We constructed several plasmids for expressing modified ANs with amino acid substitutions in known motifs. The results clearly indicated that modified AN with mutations in the putative NLS-like domain could complement the an-1 phenotype. Therefore, we re-examined localization of AN using several techniques. Our results demonstrated that AN localizes on punctuate structures around the Golgi, partially overlapping with a trans-Golgi network resident, which highlighted an unexpected link between leaf development and membrane trafficking. We should reconsider the roles and evolutionary traits of AN based on these findings.

Berberine enhances defects in the establishment of leaf polarity in asymmetric leaves1 and asymmetric leaves2 of Arabidopsis thaliana.

Leaves develop as flat lateral organs from the indeterminate shoot apical meristem. The establishment of polarity along three-dimensional axes, proximal-distal, medial-lateral, and adaxial-abaxial axes, is crucial for the growth of normal leaves. The mutations of ASYMMETRIC LEAVES1 (AS1) and AS2 of Arabidopsis thaliana cause defects in repression of the indeterminate state and the establishment of axis formation in leaves. Although many mutations have been identified that enhance the adaxial-abaxial polarity defects of as1 and as2 mutants, the roles of the causative genes in leaf development are still unknown. In this study, we found that wild-type plants treated with berberine produced pointed leaves, which are often observed in the single mutants that enhance phenotypes of as1 and as2 mutants. The berberine-treated as1 and as2 mutants formed abaxialized filamentous leaves. Berberine, an isoquinoline alkaloid compound naturally produced in various plant sources, has a growth inhibitory effect on plants that do not produce berberine. We further showed that transcript levels of meristem-specific class 1 KNOX homeobox genes and abaxial determinant genes were increased in berberine-treated as1 and as2. Berberine treated plants carrying double mutations of AS2 and the large subunit ribosomal protein gene RPL5B showed more severe defects in polarity than did the as2 single mutant plants. We suggest that berberine inhibits (a) factor(s) that might be required for leaf adaxial cell differentiation through a pathway independent of AS1 and AS2. Multiple pathways might play important roles in the formation of flat symmetric leaves.