Early flowering phenotype of the Arabidopsis altered meristem program1 mutant is dependent on the FLOWERING LOCUS T-mediated pathway.

Controlling the flowering time is crucial for propagating plant species and crop production. ALTERED MERISTEM PROGRAM1 (AMP1) in Arabidopsis thaliana encodes a putative carboxypeptidase, and an AMP1 mutant (amp1) was found to cause highly pleiotropic phenotypes including a short plastochron, an enlarged shoot apical meristem, and reduced apical dominance. Although amp1 also shows an early flowering phenotype, its mechanism has not been investigated in detail. The most important floral integrator or florigen gene, FLOWERING LOCUS T (FT), has a close relative, TWIN SISTER OF FT (TSF). In this report, we generated a new allele of tsf using a genome-editing technique and produced ft tsf double and amp1 ft tsf triple mutants. The flowering time of amp1 ft tsf was equally as late as ft tsf under long-day conditions. In addition, the expression level of FT in amp1 was 2.4-fold higher than that in wild-type, even five days after germination under long-day conditions. These results suggest that the elevated expression level of FT is responsible for the early flowering phenotype of amp1. Furthermore, expression of FLOWERING LOCUS C (FLC), a negative regulator of FT expression, is severely repressed in amp1, raising the possibility that low expression levels of FLC contributes to upregulation of FT expression and the early flowering phenotype of amp1.

Reduction in organ-organ friction is critical for corolla elongation in morning glory.

In complex structures such as flowers, organ-organ interactions are critical for morphogenesis. The corolla plays a central role in attracting pollinators: thus, its proper development is important in nature, agriculture, and horticulture. Although the intraorgan mechanism of corolla development has been studied, the importance of organ-organ interactions during development remains unknown. Here, using corolla mutants of morning glory described approximately 200 years ago, we show that glandular secretory trichomes (GSTs) regulate floral organ interactions needed for corolla morphogenesis. Defects in GST development in perianth organs result in folding of the corolla tube, and release of mechanical stress by sepal removal restores corolla elongation. Computational modeling shows that the folding occurs because of buckling caused by mechanical stress from friction at the distal side of the corolla. Our results suggest a novel function of GSTs in regulating the physical interaction of floral organs for macroscopic morphogenesis of the corolla.

Highly pleiotropic functions of CYP78As and AMP1 are regulated in non-cell-autonomous/organ-specific manners.

The cytochrome P450 CYP78A5/KLUH in Arabidopsis thaliana is predicted to be involved in the synthesis of a mobile signal molecule that has a pleiotropic function that is distinct from classical phytohormones. CYP78A5 has five close relatives in Arabidopsis. We first investigated their functions, focusing on the plastochron, leaf size, and leaf senescence. Our analyses revealed that CYP78A5 and CYP78A7 are involved in the plastochron and leaf size, and CYP78A6 and CYP78A9 are involved in leaf senescence. Complementation analyses using heterologous promoters and expression analyses suggested that CYP78A isoforms have a common biochemical function and are functionally differentiated via organ-specific expression. The altered meristem program1 (amp1) carboxypeptidase mutant shows a phenotype very similar to that of the cyp78a5 mutant. Complementation analyses using boundary and organizing center-specific promoters suggested that both CYP78A5 and AMP1 act in a non-cell-autonomous manner. Analyses of multiple cyp78a mutants and crosses between cyp78a and amp1 mutants revealed that AMP1/LIKE AMP1 (LAMP1) and CYP78A isoforms regulate plastochron length and leaf senescence in the same genetic pathway, whereas leaf size is independently regulated. Furthermore, we detected feedback regulation between CYP78A6/CYP78A9 and AMP1 at the gene expression level. These observations raise the possibility that AMP1 and CYP78A isoforms are involved in the synthesis of the same mobile signal molecule, and suggest that AMP1 and CYP78A signaling pathways have a very close, albeit complex, functional relationship.

A homolog of Arabidopsis SDP1 lipase in Nannochloropsis is involved in degradation of de novo-synthesized triacylglycerols in the endoplasmic reticulum.

Organisms of the microalgal genus Nannochloropsis produce high levels of triacylglycerols (TAGs), an efficient raw material for biofuels. A complete understanding of the TAG-breakdown pathway is critical for improving the productivity of TAGs to meet future needs. Among a number of lipases annotated as TAG lipase in the genomes of every organism, Arabidopsis SUGAR-DEPENDENT 1 (AtSDP1) lipases are characterized as a type of crucial TAG lipase in plants, similar to ScTgl3-5 in Saccharomyces cerevisiae. Homologs of the AtSDP1 TAG lipases are universally found in the genomes of plants, fungi, and algae. Here we identified two homologs of AtSDP1 TAG lipases in the oleaginous microalga species Nannochloropsis oceanica, NoTGL1 and NoTGL2. We generated single- and double-knockout strains for these lipases by homologous recombination. Whereas overall TAG content in the NoTGL2 single-knockout mutant was identical to that of wild type, the NoTGL1 knockout showed a two-fold increase in TAG content per cell in early log phase under nutrient-sufficient conditions without affecting growth. Homologs of AtSDP1 in S. cerevisiae are localized to the surface of lipid droplets, and AtSDP1 is transported from peroxisomes to the surface of lipid droplets. In contrast, NoTGL1 localized to the endoplasmic reticulum in both Nannochloropsis and yeast. We suggest that homologs of AtSDP1 lipases in Nannochloropsis modulate de novo TAG biosynthesis in the endoplasmic reticulum, unlike the roles of these lipases in other organisms. These results provide important insights into the mechanisms of TAG metabolism catalyzed by homologs of AtSDP1 lipase, which are highly conserved across species.

Betaine Lipid Is Crucial for Adapting to Low Temperature and Phosphate Deficiency in Nannochloropsis.

Diacylglyceryl-N,N,N-trimethylhomo-Ser (DGTS) is a nonphosphorous, polar glycerolipid that is regarded as analogous to the phosphatidylcholine in bacteria, fungi, algae, and basal land plants. In some species of algae, including the stramenopile microalga Nannochloropsis oceanica, DGTS contains an abundance of eicosapentaenoic acid (EPA), which is relatively scarce in phosphatidylcholine, implying that DGTS has a unique physiological role. In this study, we addressed the role of DGTS in N. oceanica We identified two DGTS biosynthetic enzymes that have distinct domain configurations compared to previously identified DGTS synthases. Mutants lacking DGTS showed growth retardation under phosphate starvation, demonstrating a pivotal role for DGTS in the adaptation to this condition. Under normal conditions, DGTS deficiency led to an increase in the relative amount of monogalactosyldiacylglycerol, a major plastid membrane lipid with high EPA content, whereas excessive production of DGTS induced by gene overexpression led to a decrease in monogalactosyldiacylglycerol. Meanwhile, lipid analysis of partial phospholipid-deficient mutants revealed a role for phosphatidylcholine and phosphatidylethanolamine in EPA biosynthesis. These results suggest that DGTS and monogalactosyldiacylglycerol may constitute the two major pools of EPA in extraplastidic and plastidic membranes, partially competing to acquire EPA or its precursors derived from phospholipids. The mutant lacking DGTS also displayed impaired growth and a lower proportion of EPA in extraplastidic compartments at low temperatures. Our results indicate that DGTS is involved in the adaptation to low temperatures through a mechanism that is distinct from the DGTS-dependent adaptation to phosphate starvation in N. oceanica.

Differently localized lysophosphatidic acid acyltransferases crucial for triacylglycerol biosynthesis in the oleaginous alga Nannochloropsis.

The production of renewable bioenergy will be necessary to meet rising global fossil fuel demands. Members of the marine microalgae genus Nannochloropsis produce large quantities of oils (triacylglycerols; TAGs), and this genus is regarded as one of the most promising for biodiesel production. Recent genome sequencing and transcriptomic studies on Nannochloropsis have provided a foundation for understanding its oleaginous trait, but the mechanism underlying oil accumulation remains to be clarified. Here we report Nannochloropsis knock-out strains of four extraplastidic lysophosphatidic acid acyltransferases (LPAT1-LPAT4) that catalyze a major de novo biosynthetic step of TAGs and membrane lipids. We found that the four LPATs are differently involved in lipid metabolic flow in Nannochloropsis. Double knock-outs among the LPATs revealed the pivotal LPATs for TAG biosynthesis, and localization analysis indicated that the stramenopile-specific LPATs (LPAT3 and LPAT4) associated with TAG synthesis reside at the perimeter of lipid droplets. No homologous region has been found with other lipid droplet-associated proteins, however. Lipid droplets are an organelle found in nearly all organisms, and recently they were shown to play important roles in cellular metabolism and signaling. Our results provide direct evidence for the importance of the perimeter of lipid droplet in TAG synthesis in addition to its known role in maintaining TAG stability, and these findings suggest that the oleaginous trait of Nannochloropsis is enabled by the acquisition of LPATs at the perimeter of lipid droplets.

Primitive Extracellular Lipid Components on the Surface of the Charophytic Alga Klebsormidium flaccidum and Their Possible Biosynthetic Pathways as Deduced from the Genome Sequence.

Klebsormidium flaccidum is a charophytic alga living in terrestrial and semiaquatic environments. K. flaccidum grows in various habitats, such as low-temperature areas and under desiccated conditions, because of its ability to tolerate harsh environments. Wax and cuticle polymers that contribute to the cuticle layer of plants are important for the survival of land plants, as they protect against those harsh environmental conditions and were probably critical for the transition from aquatic microorganism to land plants. Bryophytes, non-vascular land plants, have similar, but simpler, extracellular waxes and polyester backbones than those of vascular plants. The presence of waxes in terrestrial algae, especially in charophytes, which are the closest algae to land plants, could provide clues in elucidating the mechanism of land colonization by plants. Here, we compared genes involved in the lipid biosynthetic pathways of Arabidopsis thaliana to the K. flaccidum and the Chlamydomonas reinhardtii genomes, and identified wax-related genes in both algae. A simple and easy extraction method was developed for the recovery of the surface lipids from K. flaccidum and C. reinhardtii. Although these algae have wax components, their surface lipids were largely different from those of land plants. We also investigated aliphatic substances in the cell wall fraction of K. flaccidum and C. reinhardtii. Many of the fatty acids were determined to be lipophilic monomers in K. flaccidum, and a Fourier transform infrared spectroscopic analysis revealed that their possible binding mode was distinct from that of A. thaliana. Thus, we propose that K. flaccidum has a cuticle-like hydrophobic layer composed of lipids and glycoproteins, with a different composition from the cutin polymer typically found in land plant cuticles.

Tangled evolutionary processes with commonality and diversity in plastidial glycolipid synthesis in photosynthetic organisms.

In photosynthetic organisms, the photosynthetic membrane constitutes a scaffold for light-harvesting complexes and photosynthetic reaction centers. Three kinds of glycolipids, namely monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol, constitute approximately 80-90% of photosynthetic membrane lipids and are well conserved from tiny cyanobacteria to the leaves of huge trees. These glycolipids perform a wide variety of functions beyond biological membrane formation. In particular, the capability of adaptation to harsh environments through regulation of membrane glycolipid composition is essential for healthy growth and development of photosynthetic organisms. The genome analysis and functional genetics of the model seed plant Arabidopsis thaliana have yielded many new findings concerning the biosynthesis, regulation, and functions of glycolipids. Nevertheless, it remains to be clarified how the complex biosynthetic pathways and well-organized functions of glycolipids evolved in early and primitive photosynthetic organisms, such as cyanobacteria, to yield modern photosynthetic organisms like land plants. Recently, genome data for many photosynthetic organisms have been made available as the fruit of the rapid development of sequencing technology. We also have reported the draft genome sequence of the charophyte alga Klebsormidium flaccidum, which is an intermediate organism between green algae and land plants. Here, we performed a comprehensive phylogenic analysis of glycolipid biosynthesis genes in oxygenic photosynthetic organisms including K. flaccidum. Based on the results together with membrane lipid analysis of this alga, we discuss the evolution of glycolipid synthesis in photosynthetic organisms. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.

A dual-color marker system for in vivo visualization of cell cycle progression in Arabidopsis.

Visualization of the spatiotemporal pattern of cell division is crucial to understand how multicellular organisms develop and how they modify their growth in response to varying environmental conditions. The mitotic cell cycle consists of four phases: S (DNA replication), M (mitosis and cytokinesis), and the intervening G1 and G2 phases; however, only G2/M-specific markers are currently available in plants, making it difficult to measure cell cycle duration and to analyze changes in cell cycle progression in living tissues. Here, we developed another cell cycle marker that labels S-phase cells by manipulating Arabidopsis CDT1a, which functions in DNA replication origin licensing. Truncations of the CDT1a coding sequence revealed that its carboxy-terminal region is responsible for proteasome-mediated degradation at late G2 or in early mitosis. We therefore expressed this region as a red fluorescent protein fusion protein under the S-specific promoter of a histone 3.1-type gene, HISTONE THREE RELATED2 (HTR2), to generate an S/G2 marker. Combining this marker with the G2/M-specific CYCB1-GFP marker enabled us to visualize both S to G2 and G2 to M cell cycle stages, and thus yielded an essential tool for time-lapse imaging of cell cycle progression. The resultant dual-color marker system, Cell Cycle Tracking in Plant Cells (Cytrap), also allowed us to identify root cells in the last mitotic cell cycle before they entered the endocycle. Our results demonstrate that Cytrap is a powerful tool for in vivo monitoring of the plant cell cycle, and thus for deepening our understanding of cell cycle regulation in particular cell types during organ development.

Klebsormidium flaccidum genome reveals primary factors for plant terrestrial adaptation.

The colonization of land by plants was a key event in the evolution of life. Here we report the draft genome sequence of the filamentous terrestrial alga Klebsormidium flaccidum (Division Charophyta, Order Klebsormidiales) to elucidate the early transition step from aquatic algae to land plants. Comparison of the genome sequence with that of other algae and land plants demonstrate that K. flaccidum acquired many genes specific to land plants. We demonstrate that K. flaccidum indeed produces several plant hormones and homologues of some of the signalling intermediates required for hormone actions in higher plants. The K. flaccidum genome also encodes a primitive system to protect against the harmful effects of high-intensity light. The presence of these plant-related systems in K. flaccidum suggests that, during evolution, this alga acquired the fundamental machinery required for adaptation to terrestrial environments.

Restriction of cell proliferation in internal tissues via the synthesis of very-long-chain fatty acids in the epidermis.

Very-long-chain fatty acids (VLCFAs) are major components of cuticular wax and are also contained in seed storage triacylglycerols and sphingolipids. Arabidopsis mutants with severe defects in VLCFA synthesis produce fused leaves because of impaired cuticle formation. We recently reported that a small decrease in VLCFA content did not cause growth defects, but instead led to enhanced cell proliferation in internal tissues. We observed that this overproliferation was induced by elevated expression of cytokinin biosynthesis genes, which in turn increased the cytokinin level. Interestingly, VLCFAs are specifically synthesized in the epidermis for cuticular wax secretion, whereas cytokinin biosynthesis mainly occurs in the vasculature. Our results indicate the requirement of VLCFA synthesis in the epidermis for sending non-autonomous signals, thereby suppressing cytokinin biosynthesis in the vasculature. We propose that the interaction between the surface (epidermis) and axis (vasculature) of the plant body fine-tunes cell division activity and restricts organ size in determinate growth.

Synthesis of very-long-chain fatty acids in the epidermis controls plant organ growth by restricting cell proliferation.

Plant organ growth is controlled by inter-cell-layer communication, which thus determines the overall size of the organism. The epidermal layer interfaces with the environment and participates in both driving and restricting growth via inter-cell-layer communication. However, it remains unknown whether the epidermis can send signals to internal tissue to limit cell proliferation in determinate growth. Very-long-chain fatty acids (VLCFAs) are synthesized in the epidermis and used in the formation of cuticular wax. Here we found that VLCFA synthesis in the epidermis is essential for proper development of Arabidopsis thaliana. Wild-type plants treated with a VLCFA synthesis inhibitor and pasticcino mutants with defects in VLCFA synthesis exhibited overproliferation of cells in the vasculature or in the rib zone of shoot apices. The decrease of VLCFA content increased the expression of IPT3, a key determinant of cytokinin biosynthesis in the vasculature, and, indeed, elevated cytokinin levels. These phenotypes were suppressed in ipt3;5;7 triple mutants, and also by vasculature-specific expression of cytokinin oxidase, which degrades active forms of cytokinin. Our results imply that VLCFA synthesis in the epidermis is required to suppress cytokinin biosynthesis in the vasculature, thus fine-tuning cell division activity in internal tissue, and therefore that shoot growth is controlled by the interaction between the surface (epidermis) and the axis (vasculature) of the plant body.

Very-long-chain fatty acids have an essential role in plastid division by controlling Z-ring formation in Arabidopsis thaliana.

Recent studies have showed the essential mechanisms for plastid division that have bacterial orthologues, such as FtsZ and Min system proteins; however, causal factors regulating plastid division in plant cells are poorly understood. Here, we show that plastid division is inhibited in Arabidopsis by reduced amounts of very-long-chain fatty acids (VLCFAs), which have an acyl chain length of more than 20 carbons and are used for cuticular wax formation. The number of amyloplasts and chloroplasts decreased in the mutant defective in VLCFA synthesis and in wild-type plants treated with an inhibitor of VLCFA synthesis. Although similar inhibition of plastid division was observed in transgenic plants that over-expressed PDV2 , one of the outer membrane proteins at the plastid division site, dot-like aggregates of FtsZ protein and disordered placement of multiple Z-rings were found in wild-type chloroplasts treated the inhibitor of VLCFA synthesis. Expression analysis showed that ARC3 , one of the Min system genes, was down-regulated under low VLCFA conditions. Our results indicate that VLCFAs or VLCFA-containing lipids have an essential role in plastid division by controlling Z-ring formation, showing a novel function of plant VLCFAs.

Quantitative and cell type-specific transcriptional regulation of A-type cyclin-dependent kinase in Arabidopsis thaliana.

A-type cyclin-dependent kinase (CDKA) is an ortholog of yeast Cdc2/Cdc28p, and is assumed to have an essential function in plant growth and organogenesis. Previous studies revealed that its kinase activity is controlled by post-translational modifications, such as binding to cyclins and phosphorylations, but its transcriptional regulation is poorly understood. Here, we generated a promoter dissection series of Arabidopsis (Arabidopsis thaliana) CDKA;1, and used beta-glucuronidase (GUS) gene-fused reporter constructs for expression analyses in planta. The results revealed two types of transcriptional control in shoots: general quantitative regulation and cell type-specific regulation. We identified a promoter region that promotes CDKA;1 expression in the leaf epidermis, but not in the L1 layer of the shoot apical meristem. This region also directed abaxial side-biased expression, which may be linked to the adaxial/abaxial side specification. Another reporter construct showed that CDKA;1 expression in the inner layers of leaves is controlled by a distinct regulatory region in the promoter. These results suggest that the transcriptional regulation of CDKA;1 may play a key role in proper development of leaves by coordinating cell division and differentiation of different cell types.