The dicer-like1 Homolog fuzzy tassel Is Required for the Regulation of Meristem Determinacy in the Inflorescence and Vegetative Growth in Maize

Plant architecture is determined by meristems that initiate leaves during vegetative development and flowers during reproductive development. Maize (Zea mays) inflorescences are patterned by a series of branching events, culminating in floral meristems that produce sexual organs. The maize fuzzy tassel (fzt) mutant has striking inflorescence defects with indeterminate meristems, fasciation, and alterations in sex determination. fzt plants have dramatically reduced plant height and shorter, narrower leaves with leaf polarity and phase change defects. We positionally cloned fzt and discovered that it contains a mutation in a dicer-like1 homolog, a key enzyme required for microRNA (miRNA) biogenesis. miRNAs are small noncoding RNAs that reduce target mRNA levels and are key regulators of plant development and physiology. Small RNA sequencing analysis showed that most miRNAs are moderately reduced in fzt plants and a few miRNAs are dramatically reduced. Some aspects of the fzt phenotype can be explained by reduced levels of known miRNAs, including miRNAs that influence meristem determinacy, phase change, and leaf polarity. miRNAs responsible for other aspects of the fzt phenotype are unknown and likely to be those miRNAs most severely reduced in fzt mutants. The fzt mutation provides a tool to link specific miRNAs and targets to discrete phenotypes and developmental roles.     

Evidence That Chlorinated Auxin Is Restricted to the Fabaceae But Not to the Fabeae

Auxin is a pivotal plant hormone, usually occurring in the form of indole-3-acetic acid (IAA). However, in maturing pea (Pisum sativum) seeds, the level of the chlorinated auxin, 4-chloroindole-3-acetic acid (4-Cl-IAA), greatly exceeds that of IAA. A key issue is how plants produce halogenated compounds such as 4-Cl-IAA. To better understand this topic, we investigated the distribution of the chlorinated auxin. We show for the first time, to our knowledge, that 4-Cl-IAA is found in the seeds of Medicago truncatula, Melilotus indicus, and three species of Trifolium. Furthermore, we found no evidence that Pinus spp. synthesize 4-Cl-IAA in seeds, contrary to a previous report. The evidence indicates a single evolutionary origin of 4-Cl-IAA synthesis in the Fabaceae, which may provide an ideal model system to further investigate the action and activity of halogenating enzymes in plants.The chlorinated form of auxin, 4-chloroindole-3-acetic acid (4-Cl-IAA), is a highly active hormone that is thought to play a key role in early pericarp growth (Reinecke et al., 1995, 1999; Ozga et al., 2009). Exogenous 4-Cl-IAA, for example, has been shown to promote the pericarp elongation of deseeded pea (Pisum sativum) pods (Reinecke et al., 1999). Johnstone et al. (2005) reported that 4-Cl-IAA and bioactive GA (GA₃ or GA₁) act synergistically on pericarp growth when applied simultaneously, and a growth regulatory role has been proposed for 4-Cl-IAA through induction of GA biosynthesis and inhibition of ethylene action. In other species, e.g. tomato (Solanum lycopersicum), the nonchlorinated form of auxin, indole-3-acetic acid (IAA), also stimulates fruit growth via GAs (Serrani et al., 2008; Tang et al., 2015). The chlorinated auxin is mainly found in reproductive structures (Katayama et al., 1988), in which its levels often exceed those of the more widespread IAA (Tivendale et al., 2012). The chlorinated form is thought to be restricted to members of the leguminous tribe Fabeae (Reinecke 1999), which includes the genera Vicia, Pisum, Lathyrus, Lens, and Vavilovia (Schaefer et al., 2012). However, there is a curious exception: 4-Cl-IAA has been reported also from Scots pine (Pinus sylvestris; Ernstsen and Sandberg, 1986).We previously published evidence that most 4-Cl-IAA in maturing pea seeds is synthesized from 4-Cl-tryptophan (4-Cl-Trp) via 4-Cl-indole-3-pyruvic acid (Tivendale et al., 2012, 2014). 4-Cl-Trp has been identified in extracts from pea and broad bean (Vicia faba) seeds (Kettner et al., 1992; Manabe et al., 1999), but whether the precursors of Trp can be chlorinated is unknown.Virtually nothing is known about the enzymes that catalyze halogenation reactions in plants. In bacteria, fungi, and marine algae, there are six types of enzymes responsible for the addition of halogen atoms to organic molecules. These include heme haloperoxidases, vanadium-dependent haloperoxidases, mononuclear nonheme iron halogenases, flavin-dependent halogenases, S-adenosyl-l-Met-dependent chlorinases and fluorinases, and methyl halide transferases (Butler and Sandy, 2009; Wagner et al., 2009). However, in the genomes of angiosperms, the only type of halogenating enzyme that has been annotated are haloperoxidases, but very little is known about these enzymes. To further understand the activity and action of halogenating enzymes in plants, a comparative system is required.In this study, we investigated the distribution of 4-Cl-IAA and 4-Cl-Trp in the Fabaceae by monitoring these compounds in the seeds of representative species spanning the phylogeny of this family. Most of these species have not been previously tested for the presence of the chlorinated compounds. In addition, we reexamined the reported occurrence of 4-Cl-IAA outside the Fabaceae, namely in Scots pine; several other Pinus species were investigated here as well. We also examined the endogenous levels of 4-Cl-IAA in both vegetative tissues and seeds of broad bean to address the question of whether 4-Cl-IAA is largely restricted to seeds (Pless et al., 1984; Katayama et al., 1988).     

STM/BP-Like KNOXI Is Uncoupled from ARP in the Regulation of Compound Leaf Development in Medicago truncatula

Class I KNOTTED-like homeobox (KNOXI) genes are critical for the maintenance of the shoot apical meristem. The expression domain of KNOXI is regulated by ASYMMETRIC LEAVES1/ROUGHSHEATH2/PHANTASTICA (ARP) genes, which are associated with leaf morphology. In the inverted repeat-lacking clade (IRLC) of Fabaceae, the orthologs of LEAFY (LFY) function in place of KNOXI to regulate compound leaf development. Here, we characterized loss-of-function mutants of ARP (PHAN) and SHOOTMERISTEMLESS (STM)- and BREVIPEDICELLUS (BP)-like KNOXI in the model IRLC legume species Medicago truncatula. The function of ARP genes is species specific. The repression of STM/BP-like KNOXI genes in leaves is not mediated by PHAN, and no suppression of PHAN by STM/BP-like KNOXI genes was observed either, indicating that STM/BP-like KNOXI genes are uncoupled from PHAN in M. truncatula. Furthermore, comparative analyses of phenotypic output in response to ectopic expression of KNOXI and the M. truncatula LFY ortholog, SINGLE LEAFLET1 (SGL1), reveal that KNOXI and SGL1 regulate parallel pathways in leaf development. We propose that SGL1 probably functions in a stage-specific manner in the regulation of the indeterminate state of developing leaves in M. truncatula.     

The Small GTPase ROP10 of Medicago truncatula Is Required for Both Tip Growth of Root Hairs and Nod Factor-Induced Root Hair Deformation

Rhizobia preferentially enter legume root hairs via infection threads, after which root hairs undergo tip swelling, branching, and curling. However, the mechanisms underlying such root hair deformation are poorly understood. Here, we showed that a type II small GTPase, ROP10, of Medicago truncatula is localized at the plasma membrane (PM) of root hair tips to regulate root hair tip growth. Overexpression of ROP10 and a constitutively active mutant (ROP10CA) generated depolarized growth of root hairs, whereas a dominant negative mutant (ROP10DN) inhibited root hair elongation. Inoculated with Sinorhizobium meliloti, the depolarized swollen and ballooning root hairs exhibited extensive root hair deformation and aberrant infection symptoms. Upon treatment with rhizobia-secreted nodulation factors (NFs), ROP10 was transiently upregulated in root hairs, and ROP10 fused to green fluorescent protein was ectopically localized at the PM of NF-induced outgrowths and curls around rhizobia. ROP10 interacted with the kinase domain of the NF receptor NFP in a GTP-dependent manner. Moreover, NF-induced expression of the early nodulin gene ENOD11 was enhanced by the overexpression of ROP10 and ROP10CA. These data suggest that NFs spatiotemporally regulate ROP10 localization and activity at the PM of root hair tips and that interactions between ROP10 and NF receptors are required for root hair deformation and continuous curling during rhizobial infection.     

Impaired Auxin Biosynthesis in the defective endosperm18 Mutant Is Due to Mutational Loss of Expression in the ZmYuc1 Gene Encoding Endosperm-Specific YUCCA1 Protein in Maize

The phytohormone auxin (indole-3-acetic acid [IAA]) plays a fundamental role in vegetative and reproductive plant development. Here, we characterized a seed-specific viable maize (Zea mays) mutant, defective endosperm18 (de18) that is impaired in IAA biosynthesis. de18 endosperm showed large reductions of free IAA levels and is known to have approximately 40% less dry mass, compared with De18. Cellular analyses showed lower total cell number, smaller cell volume, and reduced level of endoreduplication in the mutant endosperm. Gene expression analyses of seed-specific tryptophan-dependent IAA pathway genes, maize Yucca1 (ZmYuc1), and two tryptophan-aminotransferase co-orthologs were performed to understand the molecular basis of the IAA deficiency in the mutant. Temporally, all three genes showed high expression coincident with high IAA levels; however, only ZmYuc1 correlated with the reduced IAA levels in the mutant throughout endosperm development. Furthermore, sequence analyses of ZmYuc1 complementary DNA and genomic clones revealed many changes specific to the mutant, including a 2-bp insertion that generated a premature stop codon and a truncated YUC1 protein of 212 amino acids, compared with the 400 amino acids in the De18. The putative, approximately 1.5-kb, Yuc1 promoter region also showed many rearrangements, including a 151-bp deletion in the mutant. Our concurrent high-density mapping and annotation studies of chromosome 10, contig 395, showed that the De18 locus was tightly linked to the gene ZmYuc1. Collectively, the data suggest that the molecular changes in the ZmYuc1 gene encoding the YUC1 protein are the causal basis of impairment in a critical step in IAA biosynthesis, essential for normal endosperm development in maize.The phytohormone auxin, as a signaling molecule, controls and coordinates numerous aspects of plant growth and development. Indole-3-acetic acid (IAA) is the most predominant in planta auxin and regulates diverse processes, including cell division, cell elongation, formation and maintenance of meristems, vascular tissue differentiation, phototropism, flowering, and endosperm and embryo growth in developing seeds (Davies, 2010). Despite its critical roles, basic components of IAA biosynthesis are poorly understood, compared with transport and signaling aspects. However, the use of appropriate genetic screens in Arabidopsis (Arabidopsis thaliana) and the use of sensitive analytical tools in the identification of metabolic intermediates have led to significant advancements toward a better understanding of biosynthesis. Currently, there are four proposed Trp-dependent pathways of de novo IAA biosynthesis in plants (Woodward and Bartel, 2005; Pollmann et al., 2009; Normanly, 2010); of these, indole-3-pyruvic acid (IPA) was recently suggested to predominate in Arabidopsis (Mashiguchi et al., 2011; Won et al., 2011; Stepanova et al., 2011) and in pea (Pisum sativum) seeds (Tivendale et al., 2012).The first step of the IPA pathway involves the conversion of Trp to IPA by Trp aminotransferases, first demonstrated in Arabidopsis by Stepanova et al. (2008) and Tao et al. (2008). The mutants of Arabidopsis Trp-aminotransferase (taa1) are defective in shade avoidance syndrome due to reduced levels of IAA. In maize (Zea mays), orthologs of the TAA1 gene include an endosperm-specific gene, ZmTar1 (for TA-Related1; Chourey et al., 2010) and Vanishing tassel2 (Vt2), which encode grass-specific Trp aminotransferases (Phillips et al., 2011). The vt2 mutant is marked by severe developmental abnormality, attributed to approximately 60% reduced IAA levels in the mutant seedlings. These results are significant in showing the functionality of the TAR enzyme and the IPA pathway in IAA biosynthesis in maize. Recently, it was suggested that the IPA pathway also involves the YUCCA (YUC) genes, which encode flavin monooxygenases that are now believed to catalyze the conversion of IPA to IAA (Phillips et al., 2011; Mashiguchi et al., 2011; Stepanova et al., 2011; Won et al., 2011; Kriechbaumer et al., 2012). This is based in part on evidence that the Arabidopsis YUC2 protein, expressed in Escherichia coli, converted IPA to IAA in vitro (Mashiguchi et al., 2011). In Arabidopsis, three Yuc genes, Yuc-1, -4, and -10, are expressed in an overlapping fashion in developing seeds and are considered essential in embryogenesis (Cheng et al., 2007); however, single or double mutant yuc1 yuc4 do not show detectable defects in embryogenesis or seed phenotype.Orthologs of the AtYuc genes are now described in several plant groups, including maize (Gallavotti et al., 2008; LeClere et al., 2010). The first Yuc-like gene in maize was isolated through positional cloning of the sparse inflorescence1 (spi1) locus; spi1 mutants showed auxin-deficient-related characteristics in the male inflorescence (Gallavotti et al., 2008). The second gene, ZmYuc1, is highly endosperm specific and its temporal expression pattern coincided with IAA biosynthesis at various stages of seed development (LeClere et al., 2010). In pea, two highly similar PsYuc-like genes, PsYuc1 and PsYuc2, showed seed- and root-specific expression, respectively (Tivendale et al., 2010). Metabolic studies in pea, however, showed that only the roots but not seeds can metabolize Trp to IAA through the proposed TAM pathway (Quittenden et al., 2009; Tivendale et al., 2010).In contrast with many studies on auxin-related mutants that affect vegetative parts of the plant, very limited data are available on auxin mutants affecting seed development, even though seeds accumulate higher levels of IAA than any other tissue of the plant. In maize, endosperm synthesizes nearly 100- to 500-fold higher levels of IAA relative to vegetative tissues (Jensen and Bandurski, 1994; LeClere et al., 2008; Phillips et al., 2011). The significance of the large abundance of IAA in developing endosperm remains to be understood, except that it may be used during the very early stages of seed germination because >90% of the total IAA is in biologically inactive conjugated storage form (Jensen and Bandurski, 1994; LeClere et al., 2008). Such a role in germination is consistent with the fact that there are very few viable seed mutants reported in maize that are linked to IAA deficiency, although single-locus recessive mutants (defective kernels [dek]) with various abnormalities in either embryo or endosperm development and with low IAA levels (measured by ELISA) were reported by Lur and Setter (1993). It is significant in this regard that a viable defective endosperm-B18 (hereafter, de18) was identified as associated with IAA deficiency (Torti et al., 1986). Although not quantified by mass spectrometry, de18 endosperms contained total IAA levels (including conjugates) in the range of 6% to 0.3% of the wild type B37 (hereafter, De18) values, during 12 to 40 d after pollination (DAP). At the early stages, the mutant seed phenotype is <50% of the wild type in seed weight, and throughout seed development, mutant seeds are reduced in kernel size and accumulate less dry matter. Furthermore, application of the synthetic auxin, naphthalene acetic acid, to developing seeds largely rescued the de18 mutant phenotype, indicating impairment in IAA biosynthesis or metabolism as the cause of the phenotypic changes (Torti et al., 1986). Recent cellular-level studies also indicated the IAA deficiency of the de18 endosperm; high levels of immunosignal for IAA were detected in the basal endosperm transfer layer (BETL), aleurone, embryo surrounding region domains, and maternal chalazal tissue in De18 but not in the mutant (Forestan et al., 2010). Overall, the maize de18 and the pea tar2 (Tivendale et al., 2012) mutants are thus far the only seed-specific viable mutants linked to auxin deficiency. The objective of this study is to further extend our knowledge on IAA deficit in the de18 kernels, to specifically analyze temporal expression of two major IAA biosynthetic genes and to elucidate the possible molecular basis of the mutant. Our collective data, based on the cloning and sequencing of ZmYuc1 and on mapping studies, indicate that ZmYuc1 and De18 are tightly associated and that the aberrant YUC1 protein in de18 is the causal basis of IAA deficiency and the small seed phenotype in that mutant.     

Long-Term Evolution of Nucleotide-Binding Site-Leucine-Rich Repeat Genes: Understanding Gained from and beyond the Legume Family

Proper utilization of plant disease resistance genes requires a good understanding of their short- and long-term evolution. Here we present a comprehensive study of the long-term evolutionary history of nucleotide-binding site (NBS)-leucine-rich repeat (LRR) genes within and beyond the legume family. The small group of NBS-LRR genes with an amino-terminal RESISTANCE TO POWDERY MILDEW8 (RPW8)-like domain (referred to as RNL) was first revealed as a basal clade sister to both coiled-coil-NBS-LRR (CNL) and Toll/Interleukin1 receptor-NBS-LRR (TNL) clades. Using Arabidopsis (Arabidopsis thaliana) as an outgroup, this study explicitly recovered 31 ancestral NBS lineages (two RNL, 21 CNL, and eight TNL) that had existed in the rosid common ancestor and 119 ancestral lineages (nine RNL, 55 CNL, and 55 TNL) that had diverged in the legume common ancestor. It was shown that, during their evolution in the past 54 million years, approximately 94% (112 of 119) of the ancestral legume NBS lineages experienced deletions or significant expansions, while seven original lineages were maintained in a conservative manner. The NBS gene duplication pattern was further examined. The local tandem duplications dominated NBS gene gains in the total number of genes (more than 75%), which was not surprising. However, it was interesting from our study that ectopic duplications had created many novel NBS gene loci in individual legume genomes, which occurred at a significant frequency of 8% to 20% in different legume lineages. Finally, by surveying the legume microRNAs that can potentially regulate NBS genes, we found that the microRNA-NBS gene interaction also exhibited a gain-and-loss pattern during the legume evolution.To combat the constant challenges by pathogens, plants have evolved a sophisticated two-layered defense system, in which proteins encoded by disease RESISTANCE (R) genes serve to sense pathogen invasion signals and to elicit defense responses (Jones and Dangl, 2006; McDowell and Simon, 2006; Bent and Mackey, 2007). Over 140 R genes have been characterized from different flowering plants, which confer resistance to a large array of pathogens, including bacteria, fungi, oomycetes, viruses, and nematodes (Liu et al., 2007; Yang et al., 2013). Among these, about 80% belong to the NBS-LRR class, which encodes a central nucleotide-binding site (NBS) domain and a C-terminal leucine-rich repeat (LRR) domain. Based on whether their N termini are homologous to the Toll/Interleukin1 receptor (TIR), the angiosperm NBS-LRR genes are further divided into the TIR-NBS-LRR (TNL) subclass and the non-TIR-NBS-LRR (nTNL) subclass (Meyers et al., 1999; Bai et al., 2002; Cannon et al., 2002). The latter has been also called CC-NBS-LRR (CNL) subclass, since a coiled-coil (CC) structure is often detected at the N terminus (Meyers et al., 2003). Interestingly, a small group of nTNL genes have an N-terminal RPW8-like domain with a transmembrane region before the CC structure (Xiao et al., 2001). This group of RPW8-NBS-LRR (RNL) genes has been usually viewed as a specific lineage of CNLs (Bonardi et al., 2011; Collier et al., 2011); however, its real phylogenetic relationship with CNLs and TNLs requires further investigation.NBS-LRR genes have been surveyed in many sequenced genomes of flowering plants, including four monocots: rice (Oryza sativa), maize (Zea mays), sorghum (Sorghum bicolor), and Brachypodium distachyon; one basal eudicot: Nelumbo nucifera; two asterid species: potato (Solanum tuberosum) and tomato (Solanum lycopersicum); and 14 rosids: Vitis vinifera, Populus trichocarpa, Ricinus communis, Medicago truncatula, soybean (Glycine max), Lotus japonicus, Cucumis sativus, Cucumis melo, Citrullus lanatus, Gossypium raimondii, Carica papaya, Arabidopsis (Arabidopsis thaliana), Arabidopsis lyrata, and Brassica rapa (Bai et al., 2002; Meyers et al., 2003; Monosi et al., 2004; Zhou et al., 2004; Yang et al., 2006, 2008b; Ameline-Torregrosa et al., 2008; Mun et al., 2009; Porter et al., 2009; Chen et al., 2010; Li et al., 2010a, 2010b; Guo et al., 2011; Zhang et al., 2011; Jupe et al., 2012; Lozano et al., 2012; Luo et al., 2012; Tan and Wu, 2012; Andolfo et al., 2013; Jia et al., 2013; Lin et al., 2013; Wan et al., 2013; Wei et al., 2013; Wu et al., 2014). Variable numbers (from dozens to hundreds) of NBS-LRR genes were reported from these genomes, making one wonder: how did these genes evolve so variably during flowering plant speciation?Comparative genomic studies conducted in the available genome sequences of closely related species or subspecies revealed that a significant proportion of NBS-LRR genes are not shared. For example, 70 NBS-LRR genes between Arabidopsis and A. lyrata show the presence/absence of polymorphisms (Chen et al., 2010; Guo et al., 2011). Moreover, synteny analysis revealed that, among 363 NBS-LRR gene loci in indica (cv 93-11) and japonica (cv Nipponbare) rice, 124 loci exist in only one genome (Luo et al., 2012). Unequal crossover, homologous repair, and nonhomologous repair are the three ways that NBS-LRR gene deletions are caused in rice genomes (Luo et al., 2012).In many surveyed genomes, the majority of NBS-LRR genes are found in a clustered organization (physically close to each other), with the rest exhibited as singletons. Many clusters are homogenous, with only duplicated members occupying the same phylogenetic lineage, whereas heterogenous clusters comprise members from distantly related clades (Meyers et al., 2003). Leister (2004) defined three types of NBS gene duplications: local tandem, ectopic, and segmental duplications. Although a general agreement on the widespread occurrence of local tandem duplications can be reached by various genome survey studies, the relative importance of ectopic and segmental duplications has been seldom investigated since the earliest surveys of the Arabidopsis genome (Richly et al., 2002; Baumgarten et al., 2003; Meyers et al., 2003; McDowell and Simon, 2006).With more genomic data available in certain angiosperm families, NBS-LRR genes should be further investigated among phylogenetically distant species to fill the gaps in the understanding of their long-term evolutionary patterns. The legume family contains many economically important crop species, such as clover (Trifolium spp.), soybean, peanut (Arachis hypogaea), and common bean (Phaseolus vulgaris). Although these legumes are constantly threatened by various pathogens, only a few functional legume R genes have been characterized, and all of them belong to the NBS-LRR class (Ashfield et al., 2004; Hayes et al., 2004; Gao et al., 2005; Seo et al., 2006; Yang et al., 2008a; Meyer et al., 2009). Therefore, it would be interesting to investigate the NBS-LRR gene repertoire among different legume species. Here, we carried out a comprehensive analysis of NBS-LRR genes in four divergent legume genomes, M. truncatula, pigeon pea (Cajanus cajan), common bean, and soybean, which shared a common ancestor approximately 54 million years ago (MYA; Fig. 1; Lavin et al., 2005). Approximately 1,000 nTNL and 667 TNL subclass NBS-encoding genes were identified in our study. Their genomic distribution, organization modes, phylogenetic relationships, and syntenic patterns were examined to obtain insight into the long-term evolutionary patterns of NBS-LRR genes.Open in a separate windowFigure 1.The phylogenetic tree of four investigated legume species (M. truncatula, pigeon pea, common bean, and soybean). Two WGD events are indicated with triangles: one occurred approximately 59 MYA in the common ancestor of the four investigated legumes, and the other occurred approximately 13 MYA in the Glycine spp. lineage alone (Schmutz et al., 2010). The numbers at the branch nodes indicate divergence times (Lavin et al., 2005; Stefanovic et al., 2009). [See online article for color version of this figure.]     

Role of Aminoalcoholphosphotransferases 1 and 2 in Phospholipid Homeostasis in Arabidopsis

Aminoalcoholphosphotransferase (AAPT) catalyzes the synthesis of phosphatidylcholine (PC) and phosphotidylethanolamine (PE), which are the most prevalent membrane phospholipids in all eukaryotic cells. Here, we show that suppression of AAPTs results in extensive membrane phospholipid remodeling in Arabidopsis thaliana. Double knockout (KO) mutants that are hemizygous for either aapt1 or aapt2 display impaired pollen and seed development, leading to embryotic lethality of the double KO plants, whereas aapt1 or aapt2 single KO plants show no overt phenotypic alterations. The growth rate and seed yield of AAPT RNA interference (RNAi) plants are greatly reduced. Lipid profiling shows decreased total galactolipid and phospholipid content in aapt1-containing mutants, including aapt1, aapt1/aapt1 aapt2/AAPT2, aapt1/AAPT1 aapt2/aapt2, and AAPT RNAi plants. The level of PC in leaves was unchanged, whereas that of PE was reduced in all AAPT-deficient plants, except aapt2 KO. However, the acyl species of PC was altered, with increased levels of C34 species and decreased C36 species. Conversely, the levels of PE and phosphatidylinositol were decreased in C34 species. In seeds, all AAPT-deficient plants, including aapt2 KO, displayed a decrease in PE. The data show that AAPT1 and AAPT2 are essential to plant vegetative growth and reproduction and have overlapping functions but that AAPT1 contributes more than AAPT2 to PC production in vegetative tissues. The opposite changes in molecular species between PC and PE and unchanged PC level indicate the existence of additional pathways that maintain homeostatic levels of PC, which are crucial for the survival and proper development of plants.     

Stomatal Blue Light Response Is Present in Early Vascular Plants

Light is a major environmental factor required for stomatal opening. Blue light (BL) induces stomatal opening in higher plants as a signal under the photosynthetic active radiation. The stomatal BL response is not present in the fern species of Polypodiopsida. The acquisition of a stomatal BL response might provide competitive advantages in both the uptake of CO₂ and prevention of water loss with the ability to rapidly open and close stomata. We surveyed the stomatal opening in response to strong red light (RL) and weak BL under the RL with gas exchange technique in a diverse selection of plant species from euphyllophytes, including spermatophytes and monilophytes, to lycophytes. We showed the presence of RL-induced stomatal opening in most of these species and found that the BL responses operated in all euphyllophytes except Polypodiopsida. We also confirmed that the stomatal opening in lycophytes, the early vascular plants, is driven by plasma membrane proton-translocating adenosine triphosphatase and K⁺ accumulation in guard cells, which is the same mechanism operating in stomata of angiosperms. These results suggest that the early vascular plants respond to both RL and BL and actively regulate stomatal aperture. We also found three plant species that absolutely require BL for both stomatal opening and photosynthetic CO₂ fixation, including a gymnosperm, C. revoluta, and the ferns Equisetum hyemale and Psilotum nudum.Stomata regulate gas exchange between plants and the atmosphere (Zeiger, 1983; Assmann, 1993; Roelfsema and Hedrich, 2005; Shimazaki et al., 2007; Kim et al., 2010). Acquisition of stomata was a key step in the evolution of terrestrial plants by allowing uptake of CO₂ from the atmosphere and accelerating the provision of nutrients via the transpiration stream within the plant (Hetherington and Woodward, 2003; McAdam and Brodribb, 2013). Stomatal aperture is regulated by changes in the turgor of guard cells, which are induced by environmental factors and endogenous phytohormones. Light is a major factor in the promotion of stomatal opening, and the opening is mediated via two distinct light-regulated pathways that are known as photosynthesis- and blue light (BL)-dependent responses under photosynthetic active radiation (PAR; Vavasseur and Raghavendra, 2005; Shimazaki et al., 2007; Lawson et al., 2014).The photosynthesis-dependent stomatal opening is induced by a continuous high intensity of light, and the action spectrum for the stomatal opening resembles that of photosynthetic pigments in leaves (Willmer and Fricker, 1996). Both mesophyll and guard cells possess photosynthetically active chloroplasts, and their photosynthesis has been suggested to contribute to stomatal opening in leaves. The decrease in the concentration of intercellular CO₂ (Ci) caused by photosynthetic CO₂ fixation or some unidentified mediators and metabolites from mesophyll cells is supposed to elicit stomatal opening, although the exact nature of the events is unclear (Wong et al., 1979; Vavasseur and Raghavendra, 2005; Roelfsema et al., 2006; Mott et al., 2008; Lawson et al., 2014).BL-dependent stomatal opening requires a strong intensity of PAR as a background: weak BL solely scarcely elicits stomatal opening, but the same intensity of BL induces the fast and large stomatal opening in the presence of strong red light (RL; Ogawa et al., 1978; Shimazaki et al., 2007). Since such stomatal opening requires BL under the RL or PAR, we call the opening reaction a BL-dependent response of stomata. BL-dependent stomatal response takes place and proceeds in natural environments because the sunlight contains both BL and RL and facilitates photosynthetic CO₂ fixation (Assmann, 1988; Takemiya et al., 2013a). In this stomatal response, BL and PAR (BL, RL, and other wavelengths of light) seem to act as a signal and an energy source, respectively.The BL-dependent stomatal opening is initiated by the absorption of BL by phototropin1 and phototropin2 (Kinoshita et al., 2001), the plant-specific BL receptors, in guard cells followed by activation of the plasma membrane proton-translocating adenosine triphosphatase (H+-ATPase; Kinoshita and Shimazaki, 1999). Two newly identified proteins, protein phosphatase1 and blue light signaling1 (BLUS1), mediate the signaling between phototropins and H+-ATPase (Takemiya et al., 2006, 2013a, 2013b). The activated H+-ATPase evokes a plasma membrane hyperpolarization, which drives K⁺ uptake through the voltage-gated, inward-rectifying K⁺ channels (Assmann, 1993; Shimazaki et al., 2007; Kim et al., 2010; Kollist et al., 2014). The accumulation of K⁺ causes water uptake and increases turgor pressure of guard cells, and finally results in stomatal opening. The BL-dependent opening is enhanced by RL, and BL at low intensity is effective in the presence of RL (Ogawa et al., 1978; Iino et al., 1985; Shimazaki et al., 2007; Suetsugu et al., 2014). These stomatal responses by RL and BL are commonly observed in a number of seed plants so far examined.Fine control of stomatal aperture to various environmental factors has been characterized in many angiosperms. Although morphological and mechanical diversity of stomata is widely documented, little is known about the functional diversity (Willmer and Fricker, 1996; Hetherington and Woodward, 2003). Our previous study indicated that BL-dependent stomatal response is absent in the major fern species of Polypodiopsida, including Adiantum capillus-veneris, Pteris cretica, Asplenium scolopendrium, and Nephrolepis auriculata, but the stomata of these species open by PAR including RL (Doi et al., 2006). When the epidermal peels isolated from A. capillus-veneris are treated with photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1dimethylurea (Doi and Shimazaki, 2008), the response is completely inhibited, but the responses in the seed plants of Vicia faba and Commelina communis are relatively insensitive to 3-(3,4-dichlorophenyl)-1,1dimethylurea (Schwartz and Zeiger, 1984). These findings suggest that there is functional diversity in light-dependent stomatal response in different lineages of land plants. In accord with this notion, the different sensitivities of stomatal response to abscisic acid and CO₂ have been reported among the plant species of angiosperm, gymnosperm, ferns, and lycophytes (Mansfield and Willmer, 1969; Brodribb and McAdam, 2011), although the exact responsiveness to abscisic acid and CO₂ is still debated (Chater et al., 2011, 2013; Ruszala et al., 2011; McAdam and Brodribb, 2013).To address the origin and distribution of stomatal light responses, we investigated the presence of a stomatal response using a gas exchange method and various lineages of vascular plants, including euphyllophytes and lycophytes. Unexpectedly, all plant lineages except Polypodiopsida in monilophytes exhibited a stomatal response to BL in the presence of RL, suggesting that the response was present in the early evolutionary stage of vascular plants. We also report the stomatal opening in response to RL in these plant species.