The iRoCS Toolbox – 3D analysis of the plant root apical meristem at cellular resolution

To achieve a detailed understanding of processes in biological systems, cellular features must be quantified in the three‐dimensional (3D) context of cells and organs. We described use of the intrinsic root coordinate system (iRoCS) as a reference model for the root apical meristem of plants. iRoCS enables direct and quantitative comparison between the root tips of plant populations at single‐cell resolution. The iRoCS Toolbox automatically fits standardized coordinates to raw 3D image data. It detects nuclei or segments cells, automatically fits the coordinate system, and groups the nuclei/cells into the root's tissue layers. The division status of each nucleus may also be determined. The only manual step required is to mark the quiescent centre. All intermediate outputs may be refined if necessary. The ability to learn the visual appearance of nuclei by example allows the iRoCS Toolbox to be easily adapted to various phenotypes. The iRoCS Toolbox is provided as an open‐source software package, licensed under the GNU General Public License, to make it accessible to a broad community. To demonstrate the power of the technique, we measured subtle changes in cell division patterns caused by modified auxin flux within the Arabidopsis thaliana root apical meristem.     

条件启动子pCTR4在隐球菌基因表达调控中的应用

目的通过条件启动子pCTR4的质粒构建以及其在新生隐球菌中的同源置换,研究其在隐球菌基因表达调控中的应用。方法应用套叠PCR,构建含报告基因NEO的铜离子抑制性启动子质粒pNEO/CTR4和启动子同源重组框,并利用基因枪将其转化入新生隐球菌感受态细胞,常规及实时定量PCR检测条件启动子对目的基因的转录调控效应。结果成功构建了质粒pNEO/CTR和隐球菌条件启动子重建菌株,条件启动子pCTR4对目的基因具有预期的转录诱导和抑制效果。结论新建铜离子抑制性启动子质粒pNEO/CTR4可以应用于对隐球菌目的基因表达水平的调控;隐球菌泛素编码基因UBI 1并非致死性关键基因。我们的研究为今后新生隐球菌泛素系统的分子致病机制研究奠定了基础。     

ERECTA Family Genes Regulate Auxin Transport in the Shoot Apical Meristem and Forming Leaf Primordia

Leaves are produced postembryonically at the flanks of the shoot apical meristem. Their initiation is induced by a positive feedback loop between auxin and its transporter PIN-FORMED1 (PIN1). The expression and polarity of PIN1 in the shoot apical meristem is thought to be regulated primarily by auxin concentration and flow. The formation of an auxin maximum in the L1 layer of the meristem is the first sign of leaf initiation and is promptly followed by auxin flow into the inner tissues, formation of the midvein, and appearance of the primordium bulge. The ERECTA family genes (ERfs) encode leucine-rich repeat receptor-like kinases, and in Arabidopsis (Arabidopsis thaliana), this gene family consists of ERECTA (ER), ERECTA-LIKE1 (ERL1), and ERL2. Here, we show that ERfs regulate auxin transport during leaf initiation. The shoot apical meristem of the er erl1 erl2 triple mutant produces leaf primordia at a significantly reduced rate and with altered phyllotaxy. This phenotype is likely due to deficiencies in auxin transport in the shoot apex, as judged by altered expression of PIN1, the auxin reporter DR5rev::GFP, and the auxin-inducible genes MONOPTEROS, INDOLE-3-ACETIC ACID INDUCIBLE1 (IAA1), and IAA19. In er erl1 erl2, auxin presumably accumulates in the L1 layer of the meristem, unable to flow into the vasculature of a hypocotyl. Our data demonstrate that ERfs are essential for PIN1 expression in the forming midvein of future leaf primordia and in the vasculature of emerging leaves.Leaves are formed during postembryonic development by the shoot apical meristem (SAM), a dome-shaped organ with a stem cell reservoir at the top and with leaf initiation taking place slightly below in the peripheral zone. The initiation of leaf primordia depends on the establishment of auxin maxima at the site of initiation (Braybrook and Kuhlemeier, 2010). Auxin is polarly transported through the epidermal layer of the meristem to the incipient primordium initiation site (Heisler et al., 2005) and then moves inward, where it promotes the formation of a vascular strand (Scarpella et al., 2006; Bayer et al., 2009). The developing vascular tissue acts as an auxin sink, depleting auxin in the epidermal layer (Scarpella et al., 2006). PIN1, an auxin efflux protein, is a central player in the formation of auxin maxima and is involved in the transport of auxin in both the epidermis and the forming vascular strand during leaf initiation (Benková et al., 2003; Reinhardt et al., 2003). PIN1 is the earliest marker for midvein formation (Scarpella et al., 2006), which starts to form before a leaf primordium bulges out of the meristem. The mechanisms determining PIN1 expression and polar localization in the SAM are central to understanding leaf initiation. In the L1 layer of the SAM, PIN1 is polarly localized in the plasma membrane toward cells with higher auxin concentration (Jönsson et al., 2006; Smith et al., 2006). Formation of the vein is explained by the canalization hypothesis, in which high auxin flux reinforces PIN1 expression (Kramer, 2008). Of all plasma membrane-localized PIN family transporters, only PIN1 has been detected in the vegetative SAM and linked with the initiation of rosette leaves (Guenot et al., 2012). At the same time, rosette leaves are positioned nonrandomly in pin1 mutants, suggesting that additional PIN1-independent mechanisms also have a role in regulating leaf initiation (Guenot et al., 2012).Here, we investigate the role of ERECTA family receptor-like kinases during leaf initiation in Arabidopsis (Arabidopsis thaliana). Previously, ERECTA family genes (ERfs) have been shown to be involved in the regulation of epidermis development and of plant growth along the apical-basal/proximodistal axis in aboveground organs (Torii et al., 1996; Shpak et al., 2004, 2005). Triple erecta (er), erecta-like1 (erl1), and erl2 mutants (er erl1 erl2) form a rosette with small, round leaves that lack petiole elongation. During the reproductive stage, the main inflorescence stem exhibits striking elongation defects and reduced apical dominance. ER has been implicated in vascular development, with the er mutation causing radial expansion of xylem (Ragni et al., 2011) and premature differentiation of vascular bundles (Douglas and Riggs, 2005). Recently, the dwarfism of described mutants was attributed to the function of ERf genes in the phloem, where they perceive signals from the endodermis (Uchida et al., 2012a). In the epidermis, all three genes inhibit the initial decision of protodermal cells to become meristemoid mother cells (Shpak et al., 2005). In addition, ERL1 and to a lesser extent ERL2 are important for maintaining cell proliferative activity in stomata lineage cells and for preventing terminal differentiation of meristemoids into guard mother cells. The activity of ERf receptors in the epidermis is regulated by a different set of peptides than in the phloem. EPIDERMAL PATTERNING FACTOR1 (EPF1) and EPF2 are expressed in stomatal precursor cells. They inhibit the development of new stomata in the vicinity of a forming stoma (Hara et al., 2007, 2009; Hunt and Gray, 2009). EPF-LIKE9 (EPFL9)/stomagen is expressed in the mesophyll, and, in contrast, it promotes the development of stomata (Kondo et al., 2010; Sugano et al., 2010). EPFL4 and EPFL6/CHALLAH are expressed in the endodermis, and their perception by phloem-localized ERfs is critical for stem elongation (Uchida et al., 2012a).While ERfs are very strongly expressed in the vegetative SAM and in forming leaf primordia, only recently has it become clear that these genes are involved in the regulation of meristem size and leaf initiation (Uchida et al., 2012b, 2013). It was suggested that ERfs regulate stem cell homeostasis in the SAM via buffering its cytokinin responsiveness by an unknown mechanism (Uchida et al., 2013). Here, we further investigate the involvement of ERfs in the control of leaf initiation and phyllotaxy. Our data suggest that ERfs are essential for PIN1 expression in the vasculature of forming leaf primordia. Based on analysis of the DR5rev::GFP reporter, auxin may accumulate in the L1 layer of the SAM in the mutant but is not able to move into the vasculature, consistent with drastically reduced PIN1pro:PIN1-GFP expression there. These data suggest that the convergence of PIN1 expression in the inner tissues of the SAM during leaf initiation is a complex process involving intercellular communications enabled by ERfs. The importance of ERfs for efficient auxin transport is further supported by reduced phototropic response in the er erl1 erl2 mutant.     

The origin of interspersed repeats in the human genome

Over a third of the human genome consists of interspersed repetitive sequences which are primarily degenerate copies of transposable elements. In the past year, the identities of many of these transposable elements were revealed. The emerging concept is that only three mechanisms of amplification are responsible for the vast majority of interspersed repeats and that with each autonomous element a number of dependent non-autonomous sequences have co-amplified.     

Anomalous Cases of Conjugation and Spontaneous Autogamy in Stylonychia mytilus: A Note on the Asexuality of Early Conjugants

Four types of anomalous conjugation were documented in Stylonychia mytilus. Type I pairs were formed between mates of different sizes. These pairs exhibited an abnormal site of fusion in at least one of the mates, and the mates might face each other ventrally throughout conjugation instead of the normal side-by-side position. Type I pairs underwent sexual nuclear development and proceeded with the first cortical reorganization as in normal conjugants. Type II involved pairing at the anterior ends of mates with ventral surfaces facing the same direction. These pairs also underwent sexual nuclear development. Hence, aberrant orientation of the mates, and also ectopic sites of cytoplasmic fusion, if extensive, would permit sexual development. Type III pairs were united ventral-to-ventral with their anterior-left sides at the adoral zone of membranelles, and remained as such throughout conjugation. In these pairs, nuclear and cortical events were typical of the asexual development of physiological reorganization. In Type IV pairs, one mate of the pair possessed a fission furrow and developed two sets of ciliature typical of binary fission, while the other mate might undergo physiological reorganization or binary fission. Type III and Type IV pairs thus reveal the asexual state of early conjugants, which can pursue either one of the two modes of asexual cortical reorganization; these cases reinforce the notion of overlap of asexual and sexual cycles during conjugation of hypotrichs. Spontaneous autogamy was documented for the first time for this genus. The autogamonts proceeded with nuclear development and with the first cortical reorganization. Some probably underwent second and third reorganizations, as in conjugants, but accompanied by abnormalities, particularly in the stages beyond fertilization. Post-autogamous clones were nonviable except for one dubious case.     

Competitive antagonism between IAA and indole alkaloid hypaphorine must contribute to regulate ontogenesis

Since 1995 the role of fungal hypaphorine in plants has been widely investigated and its IAA-antagonist activity recognized. Evidence of competitive antagonism includes organ development, gene expression or molecule–molecule interaction levels. Based on present knowledge, three sites of hypaphorine/IAA competition and subsequent signalling pathways have been hypothesized: the extracellular signalling pathway, the intracellular signalling pathway, and the transmembrane signalling pathway. Hypaphorine with other active indole alkaloids should be regarded as a new class of IAA antagonist finely regulating specific steps of plant growth or development.     

Multicellular compartmentation of catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate

In situ RNA hybridization and immunocytochemistry were used to establish the cellular distribution of monoterpenoid indole alkaloid biosynthesis in Madagascar periwinkle (Catharanthus roseus). Tryptophan decarboxylase (TDC) and strictosidine synthase (STR1), which are involved in the biosynthesis of the central intermediate strictosidine, and desacetoxyvindoline 4-hydroxylase (D4H) and deacetylvindoline 4-O-acetyltransferase (DAT), which are involved in the terminal steps of vindoline biosynthesis, were localized. tdc and str1 mRNAs were present in the epidermis of stems, leaves, and flower buds, whereas they appeared in most protoderm and cortical cells around the apical meristem of root tips. In marked contrast, d4h and dat mRNAs were associated with the laticifer and idioblast cells of leaves, stems, and flower buds. Immunocytochemical localization for TDC, D4H, and DAT proteins confirmed the differential localization of early and late stages of vindoline biosynthesis. Therefore, we concluded that the elaboration of the major leaf alkaloids involves the participation of at least two cell types and requires the intercellular translocation of a pathway intermediate. A basipetal gradient of expression in maturing leaves also was shown for all four genes by in situ RNA hybridization studies and by complementary studies with dissected leaves, suggesting that expression of the vindoline pathway occurs transiently during early leaf development. These results partially explain why attempts to produce vindoline by cell culture technology have failed.     

犁头霉11α-羟基化制备16β-甲基-11α,17α,21-三羟基孕甾-1,4-二烯-3,20-二酮

选育到一株对16β-甲基,17α,21-二羟基孕甾-1,4-二烯-3,20-二酮(Ⅱa)11α-羟基化活性强的犁头霉A28菌株,并发现底物21-乙酰化(Ⅱb)可明显提高11α-羟基化的能力.在适宜的转化条件下,Ⅱb投料浓度0.5%,产物16β-甲基-11α,11α,21-三羟基孕甾-1,4-二烯-3,20-二酮(Ⅲ)收率为73%,结构经波谱分析确认.