A Raf-like kinase is required for smoke-induced seed dormancy in Arabidopsis thaliana.

Plants sense and integrate diverse stimuli to determine the timing for germination. A smoke compound, 3,4,5-trimethylfuran-2(5H)-one (trimethylbutenolide, TMB), has been identified to inhibit the seed germination of higher plants. To understand the mode of action, we examined various physiological and molecular aspects of the TMB-dependent inhibition of seed germination in Arabidopsis thaliana The results indicated that the effect of TMB is due to the enhanced physiological dormancy, which is modulated by other dormancy regulatory cues such as after-ripening, stratification, and ABA/GA signaling. In addition, gene expression profiling showed that TMB caused genome-wide transcriptional changes, altering the expression of a series of dormancy-related genes. Based on the TMB-responsive physiological contexts in Arabidopsis, we performed mutant screening to isolate genetic components that underpin the TMB-induced seed dormancy. As a result, the TMB-RESISTANT1 (TES1) gene in Arabidopsis, encoding a B2 group Raf-like kinase, was identified. Phenotypic analysis of the tes1 mutant implicated that TES1 has a critical role in the TMB-responsive gene expression and the inhibition of seed germination. Taken together, we propose that plants have been equipped with a TMB sensory pathway through which the TMB induces the seed dormancy in a TES1-dependent way.

A myosin XI adaptor, TAPE, is essential for pollen tube elongation in rice.

Pollen tube (PT) elongation is important for double fertilization in angiosperms and affects the seed-setting rate and, therefore, crop productivity. Compared to Arabidopsis (Arabidopsis thaliana L.), information on PT elongation in rice (Oryza sativa L.) is limited by the difficulty in obtaining homozygous mutants. In a screen of T-DNA insertional mutants, we identified a mutant in the Tethering protein of actomyosin transport in pollen tube elongation (TAPE) gene with an unusual segregation ratio by genotyping analysis. A CRISPR/Cas9 knockout mutant of TAPE that produced a short PT was sterile, and TAPE was expressed specifically in pollen grains. TAPE is a homolog of a myosin XI adaptor in Arabidopsis with three tetratricopeptide repeat and Phox and Bem1 protein domains. TAPE showed latrunculin B-sensitive, actin-dependent localization to the endoplasmic reticulum. Yeast two-hybrid screening and transcriptome analysis revealed that TAPE interacted with pollen-specific LIM protein 2b and elongation factor 1-alpha. Loss of TAPE affected transcription of 1,259 genes, especially genes related to cell organization, which were downregulated. In summary, TAPE encodes a myosin XI adaptor essential for rice PT elongation.

Rice pollen-specific OsRALF17 and OsRALF19 are essential for pollen tube growth.

Pollen tube growth is essential for successful double fertilization, which is critical for grain yield in crop plants. Rapid alkalinization factors (RALFs) function as ligands for signal transduction during fertilization. However, functional studies on RALF in monocot plants are lacking. Herein, we functionally characterized two pollen-specific RALFs in rice (Oryza sativa) using multiple clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9-induced loss-of-function mutants, peptide treatment, expression analyses, and tag reporter lines. Among the 41 RALF members in rice, OsRALF17 was specifically expressed at the highest level in pollen and pollen tubes. Exogenously applied OsRALF17 or OsRALF19 peptide inhibited pollen tube germination and elongation at high concentrations but enhanced tube elongation at low concentrations, indicating growth regulation. Double mutants of OsRALF17 and OsRALF19 (ralf17/19) exhibited almost full male sterility with defects in pollen hydration, germination, and tube elongation, which was partially recovered by exogenous treatment with OsRALF17 peptide. This study revealed that two partially functionally redundant OsRALF17 and OsRALF19 bind to Oryza sativa male-gene transfer defective 2 (OsMTD2) and transmit reactive oxygen species signals for pollen tube germination and integrity maintenance in rice. Transcriptomic analysis confirmed their common downstream genes, in osmtd2 and ralf17/19. This study provides new insights into the role of RALF, expanding our knowledge of the biological role of RALF in regulating rice fertilization.

Analysis of sticky generative cell mutants reveals that suppression of callose deposition in the generative cell is necessary for generative cell internalization and differentiation in Arabidopsis.

In flowering plants, double fertilization between male and female gametophytes, which are separated by distance, largely depends on the unique pattern of the male gametophyte (pollen): two non-motile sperm cells suspended within a tube-producing vegetative cell. A morphological screen to elucidate the genetic control governing the strategic patterning of pollen has led to the isolation of a sticky generative cell (sgc) mutant that dehisces abnormal pollen with the generative cell immobilized at the pollen wall. Analyses revealed that the sgc mutation is specifically detrimental to pollen development, causing ectopic callose deposition that impedes the timely internalization and differentiation of the generative cell. We found that the SGC gene encodes the highly conserved domain of unknown function 707 (DUF707) gene that is broadly expressed but is germline specific during pollen development. Additionally, transgenic plants co-expressing fluorescently fused SGC protein and known organelle markers showed that SGC localizes in the endoplasmic reticulum, Golgi apparatus and vacuoles in pollen. A yeast two-hybrid screen with an SGC bait identified a thaumatin-like protein that we named GCTLP1, some homologs of which bind and/or digest ß-1,3-glucans, the main constituent of callose. GCTLP1 is expressed in a germline-specific manner and colocalizes with SGC during pollen development, indicating that GCTLP1 is a putative SGC interactor. Collectively, our results show that SGC suppresses callose deposition in the nascent generative cell, thereby allowing the generative cell to fully internalize into the vegetative cell and correctly differentiate as the germline progenitor, with the potential involvement of the GCTLP1 protein, during pollen development in Arabidopsis.

OsMTD2-mediated reactive oxygen species (ROS) balance is essential for intact pollen-tube elongation in rice.

The highly specialized haploid male gametophyte-pollen consist of two sperm cells and a large vegetative cell. Successful fertilization requires proper growth timing and rupture of the pollen tube until it delivers sperm cells, which occur immediately after a pollen grain hydrates. Although a tight regulation on polar cell-wall expansion of the pollen tube is fundamentally important, the underlying molecular mechanism remains largely unknown, especially in crop plants. Here, we characterized the function of male-gene transfer defective 2 (OsMTD2) gene in rice (Oryza sativa), which belongs to the plant-specific receptor-like kinase, the CrRLK1L family. We demonstrated that OsMTD2 is an essential male factor participating in pollen-tube elongation based on genetic evidence and physiological observations. Because of unavailability of homozygous mutant via conventional methods, we used CRISPR-Cas9 system to obtain homozygous knockout mutant of OsMTD2. We were able to identify phenotypic changes including male sterility due to early pollen-tube rupture in the mutant. We observed that the production of reactive oxygen species (ROS) was dramatically reduced in mutants of OsMTD2 pollen grain and tubes with defective pectin distribution. Transcriptome analysis of osmtd2-2 versus wild-type anthers revealed that genes involved in defense responses, metabolic alteration, transcriptional and protein modification were highly upregulated in the osmtd2-2 mutant. Through yeast-two-hybrid screening, we found that OsMTD2 kinase interacts with E3 ligase SPL11. Taken together, we propose that OsMTD2 has crucial functions in promoting pollen-tube elongation through cell-wall modification, possibly by modulating ROS homeostasis during pollen-tube growth.

GORI, encoding the WD40 domain protein, is required for pollen tube germination and elongation in rice.

Successful delivery of sperm cells to the embryo sac in higher plants is mediated by pollen tube growth. The molecular mechanisms underlying pollen germination and tube growth in crop plants remain rather unclear, although these mechanisms are crucial to plant reproduction and seed formation. By screening pollen-specific gene mutants in rice (Oryza sativa), we identified a T-DNA insertional mutant of Germinating modulator of rice pollen (GORI) that showed a one-to-one segregation ratio for wild type (WT) to heterozygous. GORI encodes a seven-WD40-motif protein that is homologous to JINGUBANG/REN4 in Arabidopsis. GORI is specifically expressed in rice pollen, and its protein is localized in the nucleus, cytosol and plasma membrane. Furthermore, a homozygous mutant, gori-2, created through CRISPR-Cas9 clearly exhibited male sterility with disruption of pollen tube germination and elongation. The germinated pollen tube of gori-2 exhibited decreased actin filaments and altered pectin distribution. Transcriptome analysis revealed that 852 pollen-specific genes were downregulated in gori-2 compared with the WT, and Gene Ontology enrichment analysis indicated that these genes are strongly associated with cell wall modification and clathrin coat assembly. Based on the molecular features of GORI, phenotypical observation of the gori mutant and its interaction with endocytic proteins and Rac GTPase, we propose that GORI plays key roles in forming endo-/exocytosis complexes that could mediate pollen tube growth in rice.

MYB81, a microspore-specific GAMYB transcription factor, promotes pollen mitosis I and cell lineage formation in Arabidopsis.

Sexual reproduction in flowering plants relies on the production of haploid gametophytes that consist of germline and supporting cells. During male gametophyte development, the asymmetric mitotic division of an undetermined unicellular microspore segregates these two cell lineages. To explore genetic regulation underlying this process, we screened for pollen cell patterning mutants and isolated the heterozygous myb81-1 mutant that sheds ~50% abnormal pollen. Typically, myb81-1 microspores fail to undergo pollen mitosis I (PMI) and arrest at polarized stage with a single central vacuole. Although most myb81-1 microspores degenerate without division, a small fraction divides at later stages and fails to acquire correct cell fates. The myb81-1 allele is transmitted normally through the female, but rarely through pollen. We show that myb81-1 phenotypes result from impaired function of the GAMYB transcription factor MYB81. The MYB81 promoter shows microspore-specific activity and a MYB81-RFP fusion protein is only expressed in a narrow window prior to PMI. Ectopic expression of MYB81 driven by various promoters can severely impair vegetative or reproductive development, reflecting the strict microspore-specific control of MYB81. Our data demonstrate that MYB81 has a key role in the developmental progression of microspores, enabling formation of the two male cell lineages that are essential for sexual reproduction in Arabidopsis.

Physiological Importance of Pectin Modifying Genes During Rice Pollen Development.

Although cell wall dynamics, particularly modification of homogalacturonan (HGA, a major component of pectin) during pollen tube growth, have been extensively studied in dicot plants, little is known about how modification of the pollen tube cell wall regulates growth in monocot plants. In this study, we assessed the role of HGA modification during elongation of the rice pollen tube by adding a pectin methylesterase (PME) enzyme or a PME-inhibiting catechin extract (Polyphenon 60) to in vitro germination medium. Both treatments led to a severe decrease in the pollen germination rate and elongation. Furthermore, using monoclonal antibodies toward methyl-esterified and de-esterified HGA epitopes, it was found that exogenous treatment of PME and Polyphenon 60 resulted in the disruption of the distribution patterns of low- and high-methylesterified pectins upon pollen germination and during pollen tube elongation. Eleven PMEs and 13 PME inhibitors (PMEIs) were identified by publicly available transcriptome datasets and their specific expression was validated by qRT-PCR. Enzyme activity assays and subcellular localization using a heterologous expression system in tobacco leaves demonstrated that some of the pollen-specific PMEs and PMEIs possessed distinct enzymatic activities and targeted either the cell wall or other compartments. Taken together, our findings are the first line of evidence showing the essentiality of HGA methyl-esterification status during the germination and elongation of pollen tubes in rice, which is primarily governed by the fine-tuning of PME and PMEI activities.

GROWTH-REGULATING FACTOR and GRF-INTERACTING FACTOR Specify Meristematic Cells of Gynoecia and Anthers.

We investigated the biological roles of the Arabidopsis (Arabidopsis thaliana) GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF) transcriptional complex in the development of gynoecia and anthers. There are nine GRFs and three GIFs in Arabidopsis, and seven GRFs are posttranscriptionally silenced by microRNA396 (miR396). We found that overexpression of MIR396 in the gif1 gif2 double mutant background (gif1 gif2 35S:MIR396) resulted in neither ovary nor pollen. Histological and molecular marker-based analyses revealed that the mutant gynoecial primordia failed to develop carpel margin meristems and mature flowers lacked the ovary, consisting only of the stigma, style, and replum-like tissues. The mutant anther primordia were not able to form the pluripotent archesporial cells that produce pollen mother cells and microsporangia. Multiple combinations of GRF mutations also displayed the same phenotypes, indicating that the GRF-GIF duo is required for the formation of those meristematic and pluripotent cells. Most GRF proteins are localized and abundant in those cells. We also found that the weak gynoecial defects of pinoid-3 (pid-3) mutants were remarkably exacerbated by gif1 gif2 double mutations and 35S:MIR396, so that none of the gynoecia produced by gif1 gif2 pid-3 and 35S:MIR396 pid-3 developed ovaries at all. Moreover, gif1 gif2 double mutations and 35S:MIR396 also acted synergistically with 1-N-naphthylphthalamic acid in forming aberrant gynoecia. The results altogether suggest that the GRF-GIF duo regulates the meristematic and pluripotent competence of carpel margin meristems and the archesporial cell lineage and that this regulation is implemented in association with auxin action, ultimately conferring reproductive competence on Arabidopsis.

PACLOBUTRAZOL-RESISTANCE Gene Family Regulates Floral Organ Growth with Unequal Genetic Redundancy in Arabidopsis thaliana.

A PACLOBUTRAZOL-RESISTANCE (PRE) gene family, consisting of six genes in Arabidopsis thaliana, encodes a group of helix-loop-helix proteins that act in the growth-promoting transcriptional network. To delineate the specific role of each of the PRE genes in organ growth, we took a reverse genetic approach by constructing high order pre loss-of-function mutants of Arabidopsis thaliana. In addition to dwarf vegetative growth, some double or high order pre mutants exhibited defective floral development, resulting in reduced fertility. While pre2pre5 is normally fertile, both pre2pre6 and pre5pre6 showed reduced fertility. Further, the reduced fertility was exacerbated in the pre2pre5pre6 mutant, indicative of the redundant and critical roles of these PREs. Self-pollination assay and scanning electron microscopy analysis showed that the sterility of pre2pre5pre6 was mainly ascribed to the reduced cell elongation of anther filament, limiting access of pollens to stigma. We found that the expression of a subset of flower-development related genes including ARGOS, IAA19, ACS8, and MYB24 was downregulated in the pre2pre5pre6 flowers. Given these results, we propose that PREs, with unequal functional redundancy, take part in the coordinated growth of floral organs, contributing to successful autogamous reproduction in Arabidopsis thaliana.

An Arabidopsis divergent pumilio protein, APUM24, is essential for embryogenesis and required for faithful pre-rRNA processing.

Pumilio RNA-binding proteins are largely involved in mRNA degradation and translation repression. However, a few evolutionarily divergent Pumilios are also responsible for proper pre-rRNA processing in human and yeast. Here, we describe an essential Arabidopsis nucleolar Pumilio, APUM24, that is expressed in tissues undergoing rapid proliferation and cell division. A T-DNA insertion for APUM24 did not affect the male and female gametogenesis, but instead resulted in a negative female gametophytic effect on zygotic cell division immediately after fertilization. Additionally, the mutant embryos displayed defects in cell patterning from pro-embryo through globular stages. The mutant embryos were marked by altered auxin maxima, which were substantiated by the mislocalization of PIN1 and PIN7 transporters in the defective embryos. Homozygous apum24 callus accumulates rRNA processing intermediates, including uridylated and adenylated 5.8S and 25S rRNA precursors. An RNA-protein interaction assay showed that the histidine-tagged recombinant APUM24 binds RNAin vitro with no apparent specificity. Overall, our results demonstrated that APUM24 is required for rRNA processing and early embryogenesis in Arabidopsis.

Exo-ethylene application mitigates waterlogging stress in soybean (Glycine max L.).

BACKGROUND: Waterlogging (WL) is a key factor hindering soybean crop productivity worldwide. Plants utilize various hormones to avoid various stress conditions, including WL stress; however, the physiological mechanisms are still not fully understood. RESULTS: To identify physiological mechanisms during WL stress, different phytohormones, such as ethephon (ETP; donor source of ethylene), abscisic acid, gibberellins, indole-3-acetic acid, kinetin, jasmonic acid, and salicylic acid were exogenously applied to soybean plants. Through this experiment, we confirmed the beneficial effects of ETP treatment. Thus, we selected ETP as a candidate hormone to mitigate WL. Further mechanistic investigation of the role of ETP in waterlogging tolerance was carried out. Results showed that ETP application mitigated WL stress, significantly improved the photosynthesis pigment, and increased the contents of endogenous GAs compared to those in untreated plants. The amino acid contents during WL stress were significantly activated by EPT treatments. The amino acid contents were significantly higher in the 100 µM ETP-treated soybean plants than in the control. ETP application induced adventitious root initiation, increased root surface area, and significantly increased the expressions of glutathione transferases and relative glutathione activity compared to those of non-ETP-treated plants. ETP-treated soybeans produced a higher up-regulation of protein content and glutathione S-transferase (GSTs) than did soybeans under the WL only treatment. CONCLUSIONS: In conclusion, the current results suggest that ETP application enabled various biochemical and transcriptional modulations. In particular, ETP application could stimulate the higher expression of GST3 and GST8. Thus, increased GST3 and GST8 induced 1) increased GSH activity, 2) decreased reactive oxygen species (ROS), 3) mitigation of cell damage in photosynthetic apparatus, and 4) improved phenotype consecutively.

A missense allele of KARRIKIN-INSENSITIVE2 impairs ligand-binding and downstream signaling in Arabidopsis thaliana.

A smoke-derived compound, karrikin (KAR), and an endogenous but as yet unidentified KARRIKIN INSENSITIVE2 (KAI2) ligand (KL) have been identified as chemical cues in higher plants that impact on multiple aspects of growth and development. Genetic screening of light-signaling mutants in Arabidopsis thaliana has identified a mutant designated as ply2 (pleiotropic long hypocotyl2) that has pleiotropic light-response defects. In this study, we used positional cloning to identify the molecular lesion of ply2 as a missense mutation of KAI2/HYPOSENSITIVE TO LIGHT, which causes a single amino acid substitution, Ala219Val. Physiological analysis and genetic epistasis analysis with the KL-signaling components MORE AXILLARY GROWTH2 (MAX2) and SUPPRESSOR OF MAX2 1 suggested that the pleiotropic phenotypes of the ply2 mutant can be ascribed to a defect in KL-signaling. Molecular and biochemical analyses revealed that the mutant KAI2ply2 protein is impaired in its ligand-binding activity. In support of this conclusion, X-ray crystallography studies suggested that the KAI2ply2 mutation not only results in a narrowed entrance gate for the ligand but also alters the structural flexibility of the helical lid domains. We discuss the structural implications of the Ala219 residue with regard to ligand-specific binding and signaling of KAI2, together with potential functions of KL-signaling in the context of the light-regulatory network in Arabidopsis thaliana.

Analysis of gemini pollen 3 mutant suggests a broad function of AUGMIN in microtubule organization during sexual reproduction in Arabidopsis.

In flowering plants, male gametes arise via meiosis of diploid pollen mother cells followed by two rounds of mitotic division. Haploid microspores undergo polar nuclear migration and asymmetric division at pollen mitosis I to segregate the male germline, followed by division of the germ cell to generate a pair of sperm cells. We previously reported two gemini pollen (gem) mutants that produced twin-celled pollen arising from polarity and cytokinesis defects at pollen mitosis I in Arabidopsis. Here, we report an independent mutant, gem3, with a similar division phenotype and severe genetic transmission defects through pollen. Cytological analyses revealed that gem3 disrupts cell division during male meiosis, at pollen mitosis I and during female gametophyte development. We show that gem3 is a hypomorphic allele (aug6-1) of AUGMIN subunit 6, encoding a conserved component in the augmin complex, which mediates microtubule (MT)-dependent MT nucleation in acentrosomal cells. We show that MT arrays are disturbed in gem3/aug6-1 during male meiosis and pollen mitosis I using fluorescent MT-markers. Our results demonstrate a broad role for the augmin complex in MT organization during sexual reproduction, and highlight gem3/aug6-1 mutants as a valuable tool for the investigation of augmin-dependent MT nucleation and dynamics in plant cells.

LATERAL ORGAN BOUNDARIES DOMAIN (LBD)10 interacts with SIDECAR POLLEN/LBD27 to control pollen development in Arabidopsis.

During male gametophyte development in Arabidopsis thaliana, the microspores undergo an asymmetric division to produce a vegetative cell and a generative cell, which undergoes a second division to give rise to two sperm cells. SIDECAR POLLEN/LATERAL ORGAN BOUNDARIES DOMAIN (LBD) 27 plays a key role in the asymmetric division of microspores. Here we provide molecular genetic evidence that a combinatorial role of LBD10 with LBD27 is crucial for male gametophyte development in Arabidopsis. Expression analysis, genetic transmission and pollen viability assays, and pollen development analysis demonstrated that LBD10 plays a role in the male gametophyte function primarily at germ cell mitosis. In the mature pollen of lbd10 and lbd10 expressing a dominant negative version of LBD10, LBD10:SRDX, aberrant microspores such as bicellular and smaller tricellular pollen appeared at a ratio of 10-15% with a correspondingly decreased ratio of normal tricellular pollen, whereas in lbd27 mutants, 70% of the pollen was aborted. All pollen in the lbd10 lbd27 double mutants was aborted and severely shrivelled compared with that of the single mutants, indicating that LBD10 and LBD27 are essential for pollen development. Gene expression and subcellular localization analyses of LBD10:GFP and LBD27:RFP during pollen development indicated that posttranscriptional and/or posttranslational controls are involved in differential accumulation and subcellular localization of LBD10 and LBD27 during pollen development, which may contribute in part to combinatorial and distinct roles of LBD10 with LBD27 in microspore development. In addition, we showed that LBD10 and LBD27 interact to form a heterodimer for nuclear localization.

THO2, a core member of the THO/TREX complex, is required for microRNA production in Arabidopsis.

The THO/TREX complex mediates transport of nascent mRNAs from the nucleus towards the cytoplasm in animals, and has a role in small interfering RNA-dependent processes in plants. Here we describe five mutant alleles of Arabidopsis thaliana THO2, which encodes a core subunit of the plant THO/TREX complex. tho2 mutants present strong developmental defects resembling those in plants compromised in microRNA (miRNA) activity. In agreement, not only were the levels of siRNAs reduced in tho2 mutants, but also those of mature miRNAs. As a consequence, a feedback mechanism is triggered, increasing the amount of miRNA precursors, and finally causing accumulation of miRNA-targeted mRNAs. Yeast two-hybrid experiments and confocal microscopy showed that THO2 does not appear to interact with any of the known miRNA biogenesis components, but rather with the splicing machinery, implying an indirect role of THO2 in small RNA biogenesis. Using an RNA immunoprecipitation approach, we found that THO2 interacts with miRNA precursors, and that tho2 mutants fail to recruit such precursors into the miRNA-processing complex, explaining the reduction in miRNA production in this mutant background. We also detected alterations in the splicing pattern of genes encoding serine/arginine-rich proteins in tho2 mutants, supporting a previously unappreciated role of the THO/TREX complex in alternative splicing.

Genome-wide identification and analysis of rice genes preferentially expressed in pollen at an early developmental stage.

Microspore production using endogenous developmental programs has not been well studied. The main limitation is the difficulty in identifying genes preferentially expressed in pollen grains at early stages. To overcome this limitation, we collected transcriptome data from anthers and microspore/pollen and performed meta-expression analysis. Subsequently, we identified 410 genes showing preferential expression patterns in early developing pollen samples of both japonica and indica cultivars. The expression patterns of these genes are distinguishable from genes showing pollen mother cell or tapetum-preferred expression patterns. Gene Ontology enrichment and MapMan analyses indicated that microspores in rice are closely linked with protein degradation, nucleotide metabolism, and DNA biosynthesis and regulation, while the pollen mother cell or tapetum are strongly associated with cell wall metabolism, lipid metabolism, secondary metabolism, and RNA biosynthesis and regulation. We also generated transgenic lines under the control of the promoters of eight microspore-preferred genes and confirmed the preferred expression patterns in plants using the GUS reporting system. Furthermore, cis-regulatory element analysis revealed that pollen specific elements such as POLLEN1LELAT52, and 5659BOXLELAT5659 were commonly identified in the promoter regions of eight rice genes with more frequency than estimation. Our study will provide new sights on early pollen development in rice, a model crop plant.

The Arabidopsis thaliana NGATHA transcription factors negatively regulate cell proliferation of lateral organs.

The cell proliferation process of aerial lateral organs, such as leaves and flowers, is coordinated by complex genetic networks that, in general, converge on the cell cycle. The Arabidopsis thaliana NGATHA (AtNGA) family comprises four members that belong to the B3-type transcription factor superfamily, and has been suggested to be involved in growth and development of aerial lateral organs, although its role in the cell proliferation and expansion processes remains to be resolved in more detail. In order to clarify the role of AtNGAs in lateral organ growth, we took a systematic approach using both the loss- and gain-of-functional mutants of all four members. Our results showed that overexpressors of AtNGA1 to AtNGA4 developed small, narrow lateral organs, whereas the nga1 nga2 nga3 nga4 quadruple mutant produced large, wide lateral organs. We found that cell numbers of the lateral organs were significantly affected: a decrease in overexpressors and, inversely, an increase in the quadruple mutant. Kinematic analyses on leaf growth revealed that, compared with the wild type, the overexpressors displayed a lower activity of cell proliferation and yet the mutant a higher activity. Changes in expression of cell cycle-regulating genes were well in accordance with the cell proliferation activities, establishing that the AtNGA transcription factors act as bona fide negative regulators of the cell proliferation of aerial lateral organs.

Genetic identification of ACC-RESISTANT2 reveals involvement of LYSINE HISTIDINE TRANSPORTER1 in the uptake of 1-aminocyclopropane-1-carboxylic acid in Arabidopsis thaliana.

1-Aminocyclopropane-1-carboxylic acid (ACC) is a biosynthetic precursor of ethylene, a gaseous plant hormone which controls a myriad of aspects of development and stress adaptation in higher plants. Here, we identified a mutant in Arabidopsis thaliana, designated as ACC-resistant2 (are2), displaying a dose-dependent resistance to exogenously applied ACC. Physiological analyses revealed that mutation of are2 impaired various aspects of exogenous ACC-induced ethylene responses, while not affecting sensitivity to other plant hormones during seedling development. Interestingly, the are2 mutant was normally sensitive to gaseous ethylene, compared with the wild type. Double mutant analysis showed that the ethylene-overproducing mutations, eto1 or eto3, and the constitutive ethylene signaling mutation, ctr1 were epistatic to the are2 mutation. These results suggest that the are2 mutant is not defective in ethylene biosynthesis or ethylene signaling per se. Map-based cloning of ARE2 demonstrated that LYSINE HISTIDINE TRANSPORTER1 (LHT1), encoding an amino acid transporter, is the gene responsible. An uptake experiment with radiolabeled ACC indicated that mutations of LHT1 reduced, albeit not completely, uptake of ACC. Further, we performed an amino acid competition assay and found that two amino acids, alanine and glycine, known as substrates of LHT1, could suppress the ACC-induced triple response in a LHT1-dependent way. Taken together, these results provide the first molecular genetic evidence supporting that a class of amino acid transporters including LHT1 takes part in transport of ACC, thereby influencing exogenous ACC-induced ethylene responses in A. thaliana.

Endoplasmic reticulum- and Golgi-localized phospholipase A2 plays critical roles in Arabidopsis pollen development and germination.

The phospholipase A(2) (PLA(2)) superfamily of lipolytic enzymes is involved in a number of essential biological processes, such as inflammation, development, host defense, and signal transduction. Despite the proven involvement of plant PLA(2)s in many biological functions, including senescence, wounding, elicitor and stress responses, and pathogen defense, relatively little is known about plant PLA(2)s, and their genes essentially remain uncharacterized. We characterized three of four Arabidopsis thaliana PLA(2) paralogs (PLA(2)-ß, -γ, and -δ) and found that they (1) are expressed during pollen development, (2) localize to the endoplasmic reticulum and/or Golgi, and (3) play critical roles in pollen development and germination and tube growth. The suppression of PLA(2) using the RNA interference approach resulted in pollen lethality. The inhibition of pollen germination by pharmacological PLA(2) inhibitors was rescued by a lipid signal molecule, lysophosphatidyl ethanolamine. Based on these results, we propose that plant reproduction, in particular, male gametophyte development, requires the activities of the lipid-modifying PLA(2)s that are conserved in other organisms.