The Arabidopsis Callose Synthase Gene GSL8 Is Required for Cytokinesis and Cell Patterning

Cytokinesis is the division of the cytoplasm and its separation into two daughter cells. Cell plate growth and cytokinesis appear to require callose, but direct functional evidence is still lacking. To determine the role of callose and its synthesis during cytokinesis, we identified and characterized mutants in many members of the GLUCAN SYNTHASE-LIKE (GSL; or CALLOSE SYNTHASE) gene family in Arabidopsis (Arabidopsis thaliana). Most gsl mutants (gsl1–gsl7, gsl9, gsl11, and gsl12) exhibited roughly normal seedling growth and development. However, mutations in GSL8, which were previously reported to be gametophytic lethal, were found to produce seedlings with pleiotropic defects during embryogenesis and early vegetative growth. We found cell wall stubs, two nuclei in one cell, and other defects in cell division in homozygous gsl8 insertional alleles. In addition, gsl8 mutants and inducible RNA interference lines of GSL8 showed reduced callose deposition at cell plates and/or new cell walls. Together, these data show that the GSL8 gene encodes a putative callose synthase required for cytokinesis and seedling maturation. In addition, gsl8 mutants disrupt cellular and tissue-level patterning, as shown by the presence of clusters of stomata in direct contact and by islands of excessive cell proliferation in the developing epidermis. Thus, GSL8 is required for patterning as well as cytokinesis during Arabidopsis development.Cytokinesis divides the cytoplasm of a plant cell by the deposition of plasma membrane and a cell wall during late mitosis. This process requires the phragmoplast, a dynamic, plant-specific cytoskeletal and membranous array, which delivers vesicles containing lipids, proteins, and cell wall components to the division plane to construct the cell plate. Cell plate formation involves several stages: initiation through vesicle fusion, the formation of a tubular-vesicular network, a transition to a solely tubular phase, and then further fusion to form a fenestrated sheet (Samuels et al., 1995). The outward growth of the cell plate leads to its fusion with the parental cell wall (Jürgens, 2005a, 2005b; Backues et al., 2007).Key regulators of cytokinesis include KNOLLE, KEULE, KORRIGAN, and HINKEL, which when defective induce pleiotropic phenotypes and seedling lethality (Lukowitz et al., 1996; Nicol et al., 1998; Zuo et al., 2000; Assaad et al., 2001; Strompen et al., 2002). KNOLLE, a syntaxin homolog, is required for the fusion of exocytic vesicles via a SNARE/SNAP33 complex (Lukowitz et al., 1996; Heese et al., 2001). KEULE, a homolog of yeast Sec1p, regulates syntaxin function by interacting with KNOLLE (Waizenegger et al., 2000; Assaad et al., 2001). KORRIGAN is an endo-1,4-β-glucanase required for cell wall biogenesis during cytokinesis (Zuo et al., 2000). And HINKEL is a kinesin-related protein required for the reorganization of phragmoplast microtubules during cytokinesis (Strompen et al., 2002).Additional regulators include Formin5, TWO-IN-ONE (TIO), and Arabidopsis (Arabidopsis thaliana) dynamin-like proteins (ADLs; Kang et al., 2001, 2003; Hong et al., 2003; Collings et al., 2005; Ingouff et al., 2005; Oh et al., 2005). Formin5 localizes to the cell plate and is an actin-organizing protein involved in cytokinesis and cell polarity. TIO, a Ser/Thr protein kinase, functions in cytokinesis in plant meristems and in gametogenesis (Oh et al., 2005). Members of the Arabidopsis DRP family associate with the developing cell plate, whereas DRP1a (ADL1A) locally constricts tubular membranes, interacts with callose synthase, and may facilitate callose deposition into the lumen.Callose, a β-1,3-glucan polymer with β-1,6-branches (Stone and Clarke, 1992), is synthesized in both sporophytic and gametophytic tissues and appears to play various roles. Callose accumulates at the cell plate during cytokinesis, in plasmodesmata, where it regulates cell-to-cell communication, and in dormant phloem, where it seals sieve plates after mechanical injury, pathogen attack, and metal toxicity (Stone and Clarke, 1992; Samuels et al., 1995; Lucas and Lee, 2004).Twelve GLUCAN SYNYHASE-LIKE (GSL) genes (also known as CALLOSE SYNTHASE [CalS]) have been identified in the Arabidopsis genome based on sequence homology (Richmond and Somerville, 2000; Hong et al., 2001; Enns et al., 2005). A GSL that functions in callose deposition after injury and pathogen treatment is GSL5 (Jacobs et al., 2003). Five other members of the Arabidopsis GSL family are required for microgametogenesis. GSL1 and GSL5 act redundantly to produce a callosic wall that prevents microspore degeneration, and both are needed for fertilization (Enns et al., 2005). GSL2 is required for the callosic wall around pollen mother cells, for the patterning of the pollen exine (Dong et al., 2005), and for callose deposition in the wall and plugs of pollen tubes (Nishikawa et al., 2005). GSL8 and GSL10 are independently required for the asymmetric division of microspores and for the entry of microspores into mitosis (Töller et al., 2008; Huang et al., 2009).Callose is a major component of the cell plate, especially during later plate development (Kakimoto and Shibaoka, 1992; Samuels et al., 1995; Hong et al., 2001). Callose appears to structurally reinforce the developing cell plate after the breakdown of the phragmoplast microtubule array and during plate consolidation (Samuels and Staehelin, 1996; Rensing et al., 2002). It is likely that callose is synthesized at the cell plate rather than in the endoplasmic reticulum and in the Golgi (Kakimoto and Shibaoka, 1988). GSL6 (CalS1) appears to be involved in callose synthesis at the cell plate, since a 35S∷GFP-GSL6 fusion in transgenic BY-2 tobacco (Nicotiana tabacum) cells increases callose accumulation, and GFP fluorescence was found specifically at the cell plate (Hong et al., 2001). However, functional and genetic data on the role of any GSL in Arabidopsis sporophytic cytokinesis are still lacking.Here, we report that GSL8 (CalS10) is required for normal cytokinesis. In addition, gsl8 mutants exhibit excessive cell proliferation and abnormal cell patterning, phenotypes not previously reported for cytokinesis-defective mutants.     

拟南芥胼胝质合酶基因GSL8参与细胞壁形成和根端静止中心建立与维持

用T-DNA插入和RNA干扰技术敲除拟南芥(Arabidopsis thaliana)胼胝质合酶基因GSL8, 在光学和透射电子显微镜下观察野生型和突变体种子的细胞壁和胚根结构, 比较未敲除和敲除该基因幼苗细胞壁及根端分生组织结构。结果表明, 敲除该基因可导致细胞壁发育不良, 壁上出现大小不等的缺口, 缺口处没有质膜将相邻细胞分隔。用T-DNA插入法抑制该基因表达, 发现在种子发育阶段胚根不能形成正常的静止中心。用小RNA干扰技术抑制该基因表达, 结果显示根端失去静止中心。综合以上结果, 认为该基因不仅参与细胞壁发育, 也参与根端静止中心的建立与维持。     

Arabidopsis Decapping 5 Is Required for mRNA Decapping,P-Body Formation,and Translational Repression during Postembryonic Development

Eukaryotic processing bodies (P-bodies) are implicated in mRNA storage and mRNA decapping. We previously found that a decapping complex comprising Decapping 1 (DCP1), DCP2, and Varicose in Arabidopsis thaliana is essential for postembryonic development, but the underlying mechanism is poorly understood. Here, we characterized Arabidopsis DCP5, a homolog of human RNA-associated protein 55, as an additional P-body constituent. DCP5 associates with DCP1 and DCP2 and is required for mRNA decapping in vivo. In spite of its association with DCP2, DCP5 has no effect on DCP2 decapping activity in vitro, suggesting that the effect on decapping in vivo is indirect. In knockdown mutant dcp5-1, not only is mRNA decapping compromised, but the size of P-bodies is also significantly decreased. These results indicate that DCP5 is required for P-body formation, which likely facilitates efficient decapping. During wild-type seed germination, mRNAs encoding seed storage proteins (SSPs) are translationally repressed and degraded. By contrast, in dcp5-1, SSP mRNAs are translated, leading to accumulation of their products in germinated seedlings. In vitro experiments using wheat germ extracts confirmed that DCP5 is a translational repressor. Our results showed that DCP5 is required for translational repression and P-body formation and plays an indirect role in mRNA decapping.     

Zyxin-mediated Actin Assembly Is Required for Efficient Wound Closure

Cytoskeletal regulation of cell adhesion is vital to the organization of multicellular structures. The focal adhesion protein zyxin emerged as a key regulator of actin assembly because zyxin recruits Enabled/vasodilator-stimulated phospho-proteins (Ena/VASP) to promote actin assembly. Zyxin also localizes to the sites of cell-cell adhesion and is thought to promote actin assembly with Ena/VASP. Using shRNA targeted to zyxin, we analyzed the roles of zyxin at adhesive contacts. In zyxin-deficient cells, the actin assembly at both focal adhesion and cell-cell adhesion was limited, but their migration rate was unchanged. Cell spreading on E-cadherin-coated surfaces and the formation of cell clusters were slower for zyxin-deficient cells than wild type cells. By ablating a single cell within a cell monolayer, we quantified the rate of wound closure driven by a contractile circumferential actin ring. Zyxin-deficient cells failed to recruit VASP to cell-cell junctions at the wound edge and had a slower wound closure rate than wild type cells. Our results suggest that, by recruiting VASP, zyxin regulates actin assembly at the sites of force-bearing cell-cell adhesion.     

CTP Synthase Is Required for Optic Lobe Homeostasis in Drosophila

CTP synthase(CTPsyn) is a metabolic enzyme responsible for the de novo synthesis of the nucleotide CTP. Several recent studies have shown that CTPsyn forms filamentous subcellular structures known as cytoophidia in bacteria, yeast, fruit flies and humans. However, it remains elusive whether and how CTPsyn and cytoophidia play a role during development. Here, we show that cytoophidia are abundant in the neuroepithelial stem cells in Drosophila optic lobes. Optic lobes are underdeveloped in CTPsyn mutants as well as in CTPsyn RNAi. Moreover, overexpressing CTPsyn impairs the development of optic lobes, specifically by blocking the transition from neuroepithelium to neuroblast. Taken together, our results indicate that CTPsyn is critical for optic lobe homeostasis in Drosophila.     

DEX1, a Novel Plant Protein, Is Required for Exine Pattern Formation during Pollen Development in Arabidopsis

To identify factors that are required for proper pollen wall formation, we have characterized the T-DNA-tagged, dex1 mutation of Arabidopsis, which results in defective pollen wall pattern formation. This study reports the isolation and molecular characterization of DEX1 and morphological and ultrastructural analyses of dex1 plants. DEX1 encodes a novel plant protein that is predicted to be membrane associated and contains several potential calcium-binding domains. Pollen wall development in dex1 plants parallels that of wild-type plants until the early tetrad stage. In dex1 plants, primexine deposition is delayed and significantly reduced. The normal rippling of the plasma membrane and production of spacers observed in wild-type plants is also absent in the mutant. Sporopollenin is produced and randomly deposited on the plasma membrane in dex1 plants. However, it does not appear to be anchored to the microspore and forms large aggregates on the developing microspore and the locule walls. Based on the structure of DEX1 and the phenotype of dex1 plants, several potential roles for the protein are proposed.     

The Arabidopsis Endosomal Sorting Complex Required for Transport III Regulates Internal Vesicle Formation of the Prevacuolar Compartment and Is Required for Plant Development

We have established an efficient transient expression system with several vacuolar reporters to study the roles of endosomal sorting complex required for transport (ESCRT)-III subunits in regulating the formation of intraluminal vesicles of prevacuolar compartments (PVCs)/multivesicular bodies (MVBs) in plant cells. By measuring the distributions of reporters on/within the membrane of PVC/MVB or tonoplast, we have identified dominant negative mutants of ESCRT-III subunits that affect membrane protein degradation from both secretory and endocytic pathways. In addition, induced expression of these mutants resulted in reduction in luminal vesicles of PVC/MVB, along with increased detection of membrane-attaching vesicles inside the PVC/MVB. Transgenic Arabidopsis (Arabidopsis thaliana) plants with induced expression of ESCRT-III dominant negative mutants also displayed severe cotyledon developmental defects with reduced cell size, loss of the central vacuole, and abnormal chloroplast development in mesophyll cells, pointing out an essential role of the ESCRT-III complex in postembryonic development in plants. Finally, membrane dissociation of ESCRT-III components is important for their biological functions and is regulated by direct interaction among Vacuolar Protein Sorting-Associated Protein20-1 (VPS20.1), Sucrose Nonfermenting7-1, VPS2.1, and the adenosine triphosphatase VPS4/SUPPRESSOR OF K⁺ TRANSPORT GROWTH DEFECT1.Endomembrane trafficking in plant cells is complicated such that secretory, endocytic, and recycling pathways are usually integrated with each other at the post-Golgi compartments, among which, the trans-Golgi network (TGN) and prevacuolar compartment (PVC)/multivesicular body (MVB) are best studied (Tse et al., 2004; Lam et al., 2007a, 2007b; Müller et al., 2007; Foresti and Denecke, 2008; Hwang, 2008; Otegui and Spitzer, 2008; Robinson et al., 2008; Richter et al., 2009; Ding et al., 2012; Gao et al., 2014). Following the endocytic trafficking of a lipophilic dye, FM4-64, the TGN and PVC/MVB are sequentially labeled and thus are defined as the early and late endosome, respectively, in plant cells (Lam et al., 2007a; Chow et al., 2008). While the TGN is a tubular vesicular-like structure that may include several different microdomains and fit its biological function as a sorting station (Chow et al., 2008; Kang et al., 2011), the PVC/MVB is 200 to 500 nm in size with multiple luminal vesicles of approximately 40 nm (Tse et al., 2004). Membrane cargoes destined for degradation are sequestered into these tiny luminal vesicles and delivered to the lumen of the lytic vacuole (LV) via direct fusion between the PVC/MVB and the LV (Spitzer et al., 2009; Viotti et al., 2010; Cai et al., 2012). Therefore, the PVC/MVB functions between the TGN and LV as an intermediate organelle and decides the fate of membrane cargoes in the LV.In yeast (Saccharomyces cerevisiae), carboxypeptidase S (CPS) is synthesized as a type II integral membrane protein and sorted from the Golgi to the lumen of the vacuole (Spormann et al., 1992). Genetic analyses on the trafficking of CPS have led to the identification of approximately 17 class E genes (Piper et al., 1995; Babst et al., 1997, 2002a, 2002b; Odorizzi et al., 1998; Katzmann et al., 2001) that constitute the core endosomal sorting complex required for transport (ESCRT) machinery. The evolutionarily conserved ESCRT complex consists of several functionally different subcomplexes, ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III and the ESCRT-III-associated/Vacuolar Protein Sorting4 (VPS4) complex. Together, they form a complex protein-protein interaction network that coordinates sorting of cargoes and inward budding of the membrane on the MVB (Hurley and Hanson, 2010; Henne et al., 2011). Cargo proteins carrying ubiquitin signals are thought to be passed from one ESCRT subcomplex to the next, starting with their recognition by ESCRT-0 (Bilodeau et al., 2002, 2003; Hislop and von Zastrow, 2011; Le Bras et al., 2011; Shields and Piper, 2011; Urbé, 2011). ESCRT-0 recruits the ESCRT-I complex, a heterotetramer of VPS23, VPS28, VPS37, and MVB12, from the cytosol to the endosomal membrane (Katzmann et al., 2001, 2003). The C terminus of VPS28 interacts with the N terminus of VPS36, a member of the ESCRT-II complex (Kostelansky et al., 2006; Teo et al., 2006). Then, cargoes passed from ESCRT-I and ESCRT-II are concentrated in certain membrane domains of the endosome by ESCRT-III, which includes four coiled-coil proteins and is sufficient to induce the membrane invagination (Babst et al., 2002b; Saksena et al., 2009; Wollert et al., 2009). Finally, the ESCRT components are disassociated from the membrane by the adenosine triphosphatase (ATPase) associated with diverse cellular activities (AAA) VPS4/SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1 (SKD1) before releasing the internal vesicles (Babst et al., 1997, 1998).Putative homologs of ESCRT-I–ESCRT-III and ESCRT-III-associated components have been identified in plants, except for ESCRT-0, which is only present in Opisthokonta (Winter and Hauser, 2006; Leung et al., 2008; Schellmann and Pimpl, 2009). To date, only a few plant ESCRT components have been studied in detail. The Arabidopsis (Arabidopsis thaliana) AAA ATPase SKD1 localized to the PVC/MVB and showed ATPase activity that was regulated by Lysosomal Trafficking Regulator-Interacting Protein5, a plant homolog of Vps Twenty Associated1 Protein (Haas et al., 2007). Expression of the dominant negative form of SKD1 caused an increase in the size of the MVB and a reduction in the number of internal vesicles (Haas et al., 2007). This protein also contributes to the maintenance of the central vacuole and might be associated with cell cycle regulation, as leaf trichomes expressing its dominant negative mutant form lost the central vacuole and frequently contained multiple nuclei (Shahriari et al., 2010). Double null mutants of CHARGED MULTIVESICULAR BODY PROTEIN, chmp1achmp1b, displayed severe growth defects and were seedling lethal. This may be due to the mislocalization of plasma membrane (PM) proteins, including those involved in auxin transport such as PINFORMED1, PINFORMED2, and AUXIN-RESISTANT1, from the vacuolar degradation pathway to the tonoplast of the LV (Spitzer et al., 2009).Plant ESCRT components usually contain several homologs, with the possibility of functional redundancy. Single mutants of individual ESCRT components may not result in an obvious phenotype, whereas knockout of all homologs of an ESCRT component by generating double or triple mutants may be lethal to the plant. As a first step to carry out systematic analysis on each ESCRT complex in plant cells, here, we established an efficient analysis system to monitor the localization changes of four vacuolar reporters that accumulate either in the lumen (LRR84A-GFP, EMP12-GFP, and aleurain-GFP) or on the tonoplast (GFP-VIT1) of the LV and identified several ESCRT-III dominant negative mutants. We reported that ESCRT-III subunits were involved in the release of PVC/MVB’s internal vesicles from the limiting membrane and were required for membrane protein degradation from secretory and endocytic pathways. In addition, transgenic Arabidopsis plants with induced expression of ESCRT-III dominant negative mutants showed severe cotyledon developmental defects. We also showed that membrane dissociation of ESCRT-III subunits was regulated by direct interaction with SKD1.     

CELLULOSE SYNTHASE INTERACTIVE1 Is Required for Fast Recycling of Cellulose Synthase Complexes to the Plasma Membrane in Arabidopsis

Plants are constantly subjected to various biotic and abiotic stresses and have evolved complex strategies to cope with these stresses. For example, plant cells endocytose plasma membrane material under stress and subsequently recycle it back when the stress conditions are relieved. Cellulose biosynthesis is a tightly regulated process that is performed by plasma membrane-localized cellulose synthase (CESA) complexes (CSCs). However, the regulatory mechanism of cellulose biosynthesis under abiotic stress has not been well explored. In this study, we show that small CESA compartments (SmaCCs) or microtubule-associated cellulose synthase compartments (MASCs) are critical for fast recovery of CSCs to the plasma membrane after stress is relieved in Arabidopsis thaliana. This SmaCC/MASC-mediated fast recovery of CSCs is dependent on CELLULOSE SYNTHASE INTERACTIVE1 (CSI1), a protein previously known to represent the link between CSCs and cortical microtubules. Independently, AP2M, a core component in clathrin-mediated endocytosis, plays a role in the formation of SmaCCs/MASCs. Together, our study establishes a model in which CSI1-dependent SmaCCs/MASCs are formed through a process that involves endocytosis, which represents an important mechanism for plants to quickly regulate cellulose synthesis under abiotic stress.     

myo-Inositol-1-phosphate Synthase Is Required for Polar Auxin Transport and Organ Development

myo-Inositol-1-phosphate synthase is a conserved enzyme that catalyzes the first committed and rate-limiting step in inositol biosynthesis. Despite its wide occurrence in all eukaryotes, the role of myo-inositol-1-phosphate synthase and de novo inositol biosynthesis in cell signaling and organism development has been unclear. In this study, we isolated loss-of-function mutants in the Arabidopsis MIPS1 gene from different ecotypes. It was found that all null mips1 mutants are defective in embryogenesis, cotyledon venation patterning, root growth, and root cap development. The mutant roots are also agravitropic and have reduced basipetal auxin transport. mips1 mutants have significantly reduced levels of major phosphatidylinositols and exhibit much slower rates of endocytosis. Treatment with brefeldin A induces slower PIN2 protein aggregation in mips1, indicating altered PIN2 trafficking. Our results demonstrate that MIPS1 is critical for maintaining phosphatidylinositol levels and affects pattern formation in plants likely through regulation of auxin distribution.     

Arabidopsis PTD Is Required for Type I Crossover Formation and Affects Recombination Frequency in Two Different Chromosomal Regions

In eukaryotes, crossovers together with sister chromatid cohesion maintain physical association between homologous chromosomes, ensuring accurate chromosome segregation during meiosis I and resulting in exchange of genetic information between homologues. The Arabidopsis PTD （Parting Dancers） gene affects the level of meiotic crossover formation, but its functional relationships with other core meiotic genes, such as AtSP011-1, AtRAD51, and AtMSH4, are unclear; whether PTD has other functions in meiosis is also unknown. To further analyze PTD function and to test for epistatic relationships, we compared the meiotic chromosome behaviors ofAtspoll-1 ptd and Atrad51 ptd double mutants with the relevant single mutants. The results suggest that PTD functions downstream of AtSP011-1 and AtRAD51 in the meiotic recombination pathway. Furthermore, we found that meiotic defects in rck pM and Atmsh4 ptd double mutants showed similar meiotic phenotypes to those of the relevant single mutants, providing genetic evidences for roles of PTD and RCK in the type I crossovers pathway. Moreover, we employed a pollen tetrad-based fluorescence method and found that the meiotic crossover frequencies in two genetic intervals were significantly reduced from 6.63% and 22.26% in wild-type to 1.14% and 6.36%, respectively, in the ptd~2 mutant. These results revealed new aspects of PTD function in meiotic crossover formation.     

拟南芥编码亮氨酸拉链蛋白的AS2基因在叶发育中具有控制极性建立的作用

在拟南芥 (Arabidopsisthaliana (L .)Heynh .)叶发育研究中 ,as2是一个经典突变体。as2典型的表型是叶片开裂或形成一种小叶状结构。遗传学和分子生物学实验证明 ,AS2基因具有抑制KNOX基因在叶中表达的功能。在本文中 ,我们着重研究了新得到的在Landsbergerecta (Ler)遗传背景下的as2突变体。除了前人报道过的as2表型外 ,新as2突变体的部分叶柄长在叶片的下方 ,形成一种荷叶状结构 ,更严重的甚至长成花丝状叶结构。这两种结构都反映了不正常的叶腹背轴极性分化。在我们所收集到的as2等位突变体中 ,只有在Ler背景下这两种结构才以高频率出现。我们通过图位克隆方法分离了AS2基因。该基因编码一个含有亮氨酸拉链结构的蛋白。在拟南芥中 ,AS2同源基因共 4 3个 ,除AS2外 ,其他基因的功能都不清楚。AS2在叶和花中表达 ,在茎中无表达 ,这种表达模式和as2突变体的表型是吻合的。     

Tam41 Is a CDP-Diacylglycerol Synthase Required for Cardiolipin Biosynthesis in Mitochondria

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拟南芥编码亮氨酸拉链蛋白的AS2基因在叶发育中具有控制极性建立的作用

在拟南芥(Arabidopsis thaliana (L.) Heynh.)叶发育研究中,as2是一个经典突变体.as2典型的表型是叶片开裂或形成一种小叶状结构.遗传学和分子生物学实验证明,AS2基因具有抑制KNOX基因在叶中表达的功能.在本文中,我们着重研究了新得到的在Landsberg erecta (Ler)遗传背景下的as2突变体.除了前人报道过的as2表型外,新as2突变体的部分叶柄长在叶片的下方,形成一种荷叶状结构,更严重的甚至长成花丝状叶结构.这两种结构都反映了不正常的叶腹背轴极性分化.在我们所收集到的as2等位突变体中,只有在Ler背景下这两种结构才以高频率出现.我们通过图位克隆方法分离了AS2基因.该基因编码一个含有亮氨酸拉链结构的蛋白.在拟南芥中,AS2同源基因共43个,除AS2外,其他基因的功能都不清楚.AS2在叶和花中表达,在茎中无表达,这种表达模式和as2突变体的表型是吻合的.     

Stearoyl-CoA Desaturase 1 Activity Is Required for Autophagosome Formation

Autophagy is one of the major degradation pathways for cytoplasmic components. The autophagic isolation membrane is a unique membrane whose content of unsaturated fatty acids is very high. However, the molecular mechanisms underlying formation of this membrane, including the roles of unsaturated fatty acids, remain to be elucidated. From a chemical library consisting of structurally diverse compounds, we screened for novel inhibitors of starvation-induced autophagy by measuring LC3 puncta formation in mouse embryonic fibroblasts stably expressing GFP-LC3. One of the inhibitors we identified, 2,5-pyridinedicarboxamide, N2,N5-bis[5-[(dimethylamino)carbonyl]-4-methyl-2-thiazolyl], has a molecular structure similar to that of a known stearoyl-CoA desaturase (SCD) 1 inhibitor. To determine whether SCD1 inhibition influences autophagy, we examined the effects of the SCD1 inhibitor 28c. This compound strongly inhibited starvation-induced autophagy, as determined by LC3 puncta formation, immunoblot analyses of LC3, electron microscopic observations, and p62/SQSTM1 accumulation. Overexpression of SCD1 or supplementation with oleic acid, which is a catalytic product of SCD1 abolished the inhibition of autophagy by 28c. Furthermore, 28c suppressed starvation-induced autophagy without affecting mammalian target of rapamycin activity, and also inhibited rapamycin-induced autophagy. In addition to inhibiting formation of LC3 puncta, 28c also inhibited formation of ULK1, WIPI1, Atg16L, and p62/SQSTM1 puncta. These results suggest that SCD1 activity is required for the earliest step of autophagosome formation.     

Arabidopsis RETINOBLASTOMA-RELATED Is Required for Stem Cell Maintenance, Cell Differentiation, and Lateral Organ Production

Several genes involved in the regulation of postembryonic organ initiation and growth have been identified. However, it remains largely unclear how developmental cues connect to the cell cycle. RETINOBLASTOMA RELATED (RBR) is a plant homolog of the tumor suppressor Retinoblastoma (pRb), which is a key regulator of the cell cycle. Using inducible RNA interference (RNAi) against Arabidopsis thaliana RBR (RBRi), we reduced RBR expression levels at different stages of plant development. Conditional reduction or loss of RBR function disrupted cell division patterns, promoted context-dependent cell proliferation, and negatively influenced establishment of cell differentiation. Several lineages of toti- and pluripotent cells, including shoot apical meristem stem cells, meristemoid mother cells, and procambial cells, failed to produce appropriately differentiated cells. Meristem activity was altered, leading to a disruption of the CLAVATA-WUSCHEL feedback loop and inhibition of lateral organ formation. Release of RBR from RNAi downregulation restored meristem activity. Gene profiling analyses soon after RBRi induction revealed that a change in RBR homeostasis is perceived as a stress, even before genes regulated by RBR-E2F become deregulated. The results establish RBR as a key cell cycle regulator required for coordination of cell division, differentiation, and cell homeostasis.     

Gibberellin Is Required for Flowering in Arabidopsis thaliana under Short Days

Mutants of Arabidopsis thaliana deficient in gibberellin synthesis (ga1-3 and ga1-6), and a gibberellin-insensitive mutant (gai) were compared to the wild-type (WT) Landsberg erecta line for flowering time and leaf number when grown in either short days (SD) or continuous light (CL). The ga1-3 mutant, which is severely defective in ent-kaurene synthesis because it lacks most of the GA1 gene, never flowered in SD unless treated with exogenous gibberellin. After a prolonged period of vegetative growth, this mutant eventually underwent senescence without having produced flower buds. The gai mutant and the “leaky” ga1-6 mutant did flower in SD, but took somewhat longer than WT. All the mutants flowered readily in CL, although the ga1-3 mutant showed some delay. Unlike WT and ga1-3, the gai mutant failed to respond to gibberellin treatment by accelerating flowering in SD. A cold treatment promoted flowering in the WT and gai, but failed to induce flowering in ga1-3. From these results, it appears that gibberellin normally plays a role in initiating flowering of Arabidopsis.