Coordination of meristem and boundary functions by transcription factors in the SHOOT MERISTEMLESS regulatory network.

The Arabidopsis homeodomain transcription factor SHOOT MERISTEMLESS (STM) is crucial for shoot apical meristem (SAM) function, yet the components and structure of the STM gene regulatory network (GRN) are largely unknown. Here, we show that transcriptional regulators are overrepresented among STM-regulated genes and, using these as GRN components in Bayesian network analysis, we infer STM GRN associations and reveal regulatory relationships between STM and factors involved in multiple aspects of SAM function. These include hormone regulation, TCP-mediated control of cell differentiation, AIL/PLT-mediated regulation of pluripotency and phyllotaxis, and specification of meristem-organ boundary zones via CUC1. We demonstrate a direct positive transcriptional feedback loop between STM and CUC1, despite their distinct expression patterns in the meristem and organ boundary, respectively. Our further finding that STM activates expression of the CUC1-targeting microRNA miR164c combined with mathematical modelling provides a potential solution for this apparent contradiction, demonstrating that these proposed regulatory interactions coupled with STM mobility could be sufficient to provide a mechanism for CUC1 localisation at the meristem-organ boundary. Our findings highlight the central role for the STM GRN in coordinating SAM functions.

Comparative Transcriptomics Analysis of Brassica napus L. during Seed Maturation Reveals Dynamic Changes in Gene Expression between Embryos and Seed Coats and Distinct Expression Profiles of Acyl-CoA-Binding Proteins for Lipid Accumulation.

Production of vegetable oils is a vital agricultural resource and oilseed rape (Brassica napus) is the third most important oil crop globally. Although the regulation of lipid biosynthesis in oilseeds is still not fully defined, the acyl-CoA-binding proteins (ACBPs) have been reported to be involved in such metabolism, including oil accumulation, in several plant species. In this study, progressive changes in gene expression in embryos and seed coats at different stages of seed development were comprehensively investigated by transcriptomic analyses in B. napus, revealing dynamic changes in the expression of genes involved in lipid biosynthesis. We show that genes encoding BnACBP proteins show distinct changes in expression at different developmental stages of seed development and show markedly different expression between embryos and seed coats. Both isoforms of the ankyrin-repeat BnACBP2 increased during the oil accumulation period of embryo development. By contrast, the expression of the three most abundant isoforms of the small molecular mass BnACBP6 in embryos showed progressive reduction, despite having the highest overall expression level. In seed coats, BnACBP3, BnACBP4 and BnACBP5 expression remained constant during development, whereas the two major isoforms of BnACBP6 increased, contrasting with the data from embryos. We conclude that genes related to fatty acid and triacylglycerol biosynthesis showing dynamic expression changes may regulate the lipid distribution in embryos and seed coats of B. napus and that BnACBP2 and BnACBP6 are potentially important for oil accumulation.

DEFECTIVE KERNEL 1 promotes and maintains plant epidermal differentiation.

During plant epidermal development, many cell types are generated from protodermal cells, a process requiring complex co-ordination of cell division, growth, endoreduplication and the acquisition of differentiated cellular morphologies. Here we show that the Arabidopsis phytocalpain DEFECTIVE KERNEL 1 (DEK1) promotes the differentiated epidermal state. Plants with reduced DEK1 activity produce cotyledon epidermis with protodermal characteristics, despite showing normal growth and endoreduplication. Furthermore, in non-embryonic tissues (true leaves, sepals), DEK1 is required for epidermis differentiation maintenance. We show that the HD-ZIP IV family of epidermis-specific differentiation-promoting transcription factors are key, albeit indirect, targets of DEK1 activity. We propose a model in which DEK1 influences HD-ZIP IV gene expression, and thus epidermis differentiation, by promoting cell adhesion and communication in the epidermis.

The Arabidopsis homeobox gene SHOOT MERISTEMLESS has cellular and meristem-organisational roles with differential requirements for cytokinin and CYCD3 activity.

The Arabidopsis class-1 KNOX gene SHOOT MERISTEMLESS (STM) encodes a homeodomain transcription factor essential for shoot apical meristem (SAM) formation and sustained activity. STM activates cytokinin (CK) biosynthesis in the SAM, but the extent to which STM function is mediated through CK is unclear. Here we show that STM inhibits cellular differentiation and endoreduplication, acting through CK and the CK-inducible CYCD3 cell cycle regulators, establishing a mechanistic link to cell cycle control which provides sustained mitotic activity to maintain a pool of undifferentiated cells in the SAM. Equivalent functions are revealed for the related KNOX genes KNAT1/BP and KNAT2 through ectopic expression. STM is also required for proper meristem organisation and can induce de novo meristem formation when expressed ectopically, even when CK levels are reduced or CK signaling is impaired. This function in meristem establishment and organisation can be replaced by KNAT1/BP, but not KNAT2, despite its activation of CK responses, suggesting that promotion of CK responses alone is insufficient for SAM organisation. We propose that STM has dual cellular and meristem-organisational functions that are differentially represented in the class-1 KNOX gene family and have differing requirements for CK and CYCD3.

Evaluating repellence properties of catnip essential oil against the mosquito species Aedes aegypti using a Y-tube olfactometer.

The mosquito species Aedes aegypti (L.) is known to act as a vector in the transmission of various diseases, including dengue fever and yellow fever. The use of insect repellents is one of precautionary measures used to mitigate the risk of these diseases in humans by reducing mosquito biting. Nepetalactone, a potent natural insect repellent primarily found in catnip (Nepeta cataria) essential oil, has emerged as a promising candidate for mosquito repellence. Here, we evaluated the potential of catnip essential oil (> 95% nepetalactone) for use as a mosquito repellent. Using a Y-tube olfactometer and human hands as an attractant, we analysed the effectiveness of catnip oil at repelling the mosquito species Aedes aegypti. We tested a range of dilutions of catnip essential oil and found that concentrations as low as 2% were effective at repelling > 70% of mosquitoes for between one and four hours after repellent application. These findings suggest that nepetalactone could potentially be used as a natural, effective alternative to synthetic mosquito repellents, thereby offering protection against vector-borne diseases.

The D-type cyclin CYCD4;1 modulates lateral root density in Arabidopsis by affecting the basal meristem region.

Root cell division occurs primarily in the apical meristem, from which cells are displaced into the basal meristem, where division decreases and cell length increases before the final differentiation zone. The organization of the root in concentric files implies coordinated division and differentiation of cell types, including the xylem pole pericycle cells, which uniquely can resume division to initiate lateral roots (LR). Here, we show that D-type cyclin CYCD4;1 is expressed in meristematic pericycle protoxylem poles and is required for normal LR density. Cycd4;1 mutants also show a displacement of the apical/basal meristem boundary in the pericycle and longer pericycle basal meristem cells, whereas other cell layers and overall meristem size and root growth are unaffected. Auxin is proposed to separately prepattern and stimulate LR initiation. Stimulation is unimpaired in cycd4;1, suggesting CYCD4;1 requirement for normal spacing but not initiation. Both pericycle cell length and LR density phenotypes of cycd4;1 are rescued by low concentrations of applied auxin, suggesting that the basal meristem has a role in determining LR density. We further show CYCD4;1 is rate-limiting for sucrose-dependent LR formation, since CYCD4;1 expression is sucrose-dependent and wild-type roots fully phenocopy cycd4;1 in sucrose absence. We conclude that CYCD4;1 links meristem pericycle cell behavior to LR density consistent with a basal meristem prepatterning model and that D-type cyclins can confer division potential of defined cell types through cell-specific expression patterns.

Optimising expression and extraction of recombinant proteins in plants.

Recombinant proteins are of paramount importance for research, industrial and medical use. Numerous expression chassis are available for recombinant protein production, and while bacterial and mammalian cell cultures are the most widely used, recent developments have positioned transgenic plant chassis as viable and often preferential options. Plant chassis are easily maintained at low cost, are hugely scalable, and capable of producing large quantities of protein bearing complex post-translational modification. Several protein targets, including antibodies and vaccines against human disease, have been successfully produced in plants, highlighting the significant potential of plant chassis. The aim of this review is to act as a guide to producing recombinant protein in plants, discussing recent progress in the field and summarising the factors that must be considered when utilising plants as recombinant protein expression systems, with a focus on optimising recombinant protein expression at the genetic level, and the subsequent extraction and purification of target proteins, which can lead to substantial improvements in protein stability, yield and purity.

Genetic Manipulation of Biosynthetic Pathways in Mint.

In recent years, the study of aromatic plants has seen an increase, with great interest from industrial, academic, and pharmaceutical industries. Among plants attracting increased attention are the Mentha spp. (mint), members of the Lamiaceae family. Mint essential oils comprise a diverse class of molecules known as terpenoids/isoprenoids, organic chemicals that are among the most diverse class of naturally plant derived compounds. The terpenoid profile of several Mentha spp. is dominated by menthol, a cyclic monoterpene with some remarkable biological properties that make it useful in the pharmaceutical, medical, cosmetic, and cleaning product industries. As the global market for Mentha essential oils increases, the desire to improve oil composition and yield follows. The monoterpenoid biosynthesis pathway is well characterised so metabolic engineering attempts have been made to facilitate this improvement. This review focuses on the Mentha spp. and attempts at altering the carbon flux through the biosynthetic pathways to increase the yield and enhance the composition of the essential oil. This includes manipulation of endogenous and heterologous biosynthetic enzymes through overexpression and RNAi suppression. Genes involved in the MEP pathway, the menthol and carvone biosynthetic pathways and transcription factors known to affect secondary metabolism will be discussed along with non-metabolic engineering approaches including environmental factors and the use of plant growth regulators.

Transgenic manipulation of triacylglycerol biosynthetic enzymes in B. napus alters lipid-associated gene expression and lipid metabolism.

Oilseed rape (Brassica napus) is an important crop that is cultivated for the oil (mainly triacylglycerol; TAG) it produces in its seeds. TAG synthesis is controlled mainly by key enzymes in the Kennedy pathway, such as glycerol 3-phosphate acyltransferase (GPAT), lysophosphatidate acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT) but can also be produced from phosphoglycerides such as phosphatidylcholine (PC) by the activity of the enzyme phospholipid: diacylglycerol acyltransferase (PDAT). To evaluate the potential for these enzymes to alter oil yields or composition, we analysed transgenic B. napus lines which overexpressed GPAT, LPAT or PDAT using heterologous transgenes from Arabidopsis and Nasturtium and examined lipid profiles and changes in gene expression in these lines compared to WT. Distinct changes in PC and TAG abundance and spatial distribution in embryonic tissues were observed in some of the transgenic lines, together with altered expression of genes involved generally in acyl-lipid metabolism. Overall our results show that up-regulation of these key enzymes differentially affects lipid composition and distribution as well as lipid-associated gene expression, providing important information which could be used to improve crop properties by metabolic engineering.

Control of division and differentiation of plant stem cells and their derivatives.

The core mechanism of the plant cell cycle is conserved with all other eukaryotes but several aspects are unique to plant cells. Key characteristics of plant development include indeterminate growth and repetitive organogenesis derived from stem cell pools and they may explain the existence of the high number of cell cycle regulators in plants. In this review, we give an overview of the plant cell cycle and its regulatory components. Furthermore, we discuss the cell cycle aspects of plant stem cell maintenance and how the cell cycle relates to cellular differentiation during development. We exemplify this transition by focusing on organ initiation in the shoot.

Dissecting regulatory pathways of G1/S control in Arabidopsis: common and distinct targets of CYCD3;1, E2Fa and E2Fc.

Activation of E2F transcription factors at the G1-to-S phase boundary, with the resultant expression of genes needed for DNA synthesis and S-phase, is due to phosphorylation of the retinoblastoma-related (RBR) protein by cyclin D-dependent kinase (CYCD-CDK), particularly CYCD3-CDKA. Arabidopsis has three canonical E2F genes, of which E2Fa and E2Fb are proposed to encode transcriptional activators and E2Fc a repressor. Previous studies have identified genes regulated in response to high-level constitutive expression of E2Fa and of CYCD3;1, but such plants display significant phenotypic abnormalities. We have sought to identify targets that show responses to lower level induced changes in abundance of these cell cycle regulators. Expression of E2Fa, E2Fc or CYCD3;1 was induced using dexamethasone and the effects analysed using microarrays in a time course allowing short and longer term effects to be observed. Overlap between CYCD3;1 and E2Fa modulated genes substantiates their action in a common pathway with a key role in controlling the G1/S transition, with additional targets for CYCD3;1 in chromatin modification and for E2Fa in cell wall biogenesis and development. E2Fc induction led primarily to gene downregulation, but did not antagonise E2Fa action and hence E2Fc appears to function outside the CYCD3-RBR pathway, does not have a direct effect on cell cycle genes, and promoter analysis suggests a distinct binding site preference.

Conspicuity Equipment and Its Contribution to the Welfare of Horse and Rider Combinations Using the Road System in the United Kingdom.

Limited research exists regarding proximity of vehicles to "vulnerable road users" and effects of "conspicuity equipment". Previous studies stated over 60% of horse/rider combinations experience near-miss traffic collisions in any one year, whereas use of fluorescent/reflective (FR) equipment did not decrease occurrences. Similarly, research into vehicle proximity to bicycles reported no significance in cyclists wearing FR. This study undertook a similar field experiment using a horse/rider combination wearing different conspicuity equipment to test proximity distance. Two horses with similar height, age, base coat color, and temperament were ridden using four conspicuity measures 60 times each (n = 240) along a straight 0.8 km lane. Measures were chosen by random crossover with two selected for each separate trip. These were an FR "tabard", a novel black/white pattern (BW), flashing lights on a helmet (L), and control of mid-blue (C). A novel proximity meter was used on the saddle pommel and distance/approach either front or rear were logged into a voice recorder. Analysis reported a significant difference between all measures and proximities observed (P = .000). Mann-Whitney tests concluded significant differences in proximity in C versus L and BW (P = .000; P = .000), respectively. FR versus C showed no significant difference in proximity (P = .005). L versus BW reported no significant difference in proximity (W = 3640.0, P = .958). Results may suggest wearing conspicuity equipment of L or BW outperforms both C and FR. Further study is recommended to test these findings, and it is pertinent to still consider suggesting the use of FR equipment due to previous positive findings with other road users.

STM sustains stem cell function in the Arabidopsis shoot apical meristem and controls KNOX gene expression independently of the transcriptional repressor AS1.

The Arabidopsis KNOX gene SHOOT MERISTEMLESS (STM) is required for both the development and the sustained function of the shoot apical meristem (SAM) and can induce de novo meristem formation when expressed ectopically. STM acts through induction of cytokinin (CK) synthesis to inhibit cellular differentiation and additionally functions to organize undifferentiated cells into a self-sustaining meristem. STM has been shown to positively regulate the related KNOX genes KNAT1/BP and KNAT2, and it has been proposed that this is mediated through repression of the ARP-type transcriptional repressor ASYMMETRIC LEAVES1 (AS1). Here we investigate the role of STM in SAM organization, stem cell maintenance and the regulation of KNOX gene expression. We show that culture of stm mutant explants in high CK conditions does not restore proper sustained shoot growth, supporting the idea of STM having CK-independent roles in meristem function. Furthermore, we show that STM is required for continued stem cell function in the SAM by sustaining expression of the stem cell-promoting factor WUS and preventing cells of the meristem organizing center from adopting lateral organ-specific fates. We also demonstrate that transcriptional activation of class-1 KNOX genes by STM is independent of AS1, since AS1 transcript levels are not reduced in response to STM and STM is able to transactivate expression of both KNAT1/BP and KNAT2 in the as1 mutant background.

A model for Arabidopsis class-1 KNOX gene function.

The Arabidopsis class-1 KNOX genes STM, BP/KNAT1, KNAT2 and KNAT6 encode homeodomain transcriptional regulators important for shoot apical meristem (SAM) and carpel development. During vegetative growth, STM is required to establish and maintain the stem cell pool of the SAM, a function replaceable by ectopic expression of BP/KNAT1 but not of other class-1 KNOX genes. We recently demonstrated additional STM roles in the development of the central floral whorl and subsequent formation of carpels, a function replaceable by ectopic KNAT2 expression. However, STM is normally required for BP/KNAT1 and KNAT2 expression, explaining why it is essential for both SAM and carpel development. We propose therefore that STM provides the critical KNOX function in these processes and that the SAM- and carpel-promoting activities of STM are redundantly duplicated in BP/KNAT1 and KNAT2 respectively. Here we show that ectopic KNAT2 expression can restore carpel development to stm mutants, but fails to restore proper development of the central floral whorl, which requires a function analogous to the SAM-promoting activities of STM and BP/KNAT1. Similarly, we show that ectopic KNAT2 expression does not restore normal meristem organisation to the SAM. We propose a model for discrete and overlapping class-1 KNOX gene function in Arabidopsis.

The KNOX gene SHOOT MERISTEMLESS is required for the development of reproductive meristematic tissues in Arabidopsis.

In Arabidopsis, the central stem cells of the shoot apical meristem (SAM) are indefinitely maintained, whereas those in floral meristems differentiate into female gametophyte-bearing organs termed carpels. The class 1 KNOX genes encode homeodomain transcription factors that function variously in the establishment and maintenance of the SAM, and have also been implicated in carpel development. Here we show that the KNOX gene SHOOT MERISTEMLESS (STM) induces carpel formation and promotes homeotic conversion of ovules to carpels when ectopically expressed in flowers, as previously reported for the related gene KNAT2. In contrast to KNAT2, loss of which confers no phenotype, we show using inducible RNA interference and mutational analysis that progressive loss of STM causes floral phenotypes ranging from reduced formation of placental tissues and inhibited carpel fusion to complete loss of carpel development. These effects result neither from failure to establish the central stem cell niche nor from reduced floral homeotic gene expression, but rather indicate a specific requirement for STM in carpel initiation, as further supported by the loss of leafy carpelloid features in stm leafy double mutants. Activation of carpel development by STM is independent of LEAFY and WUSCHEL, but requires the function of AGAMOUS. The essential role for STM in carpel development, together with its previous reported role in the SAM, shows that, despite the existence of several partially redundant paralogous genes, STM provides the critical KNOX function in the development of both vegetative and reproductive meristematic tissues.

Arabidopsis CYCD3 D-type cyclins link cell proliferation and endocycles and are rate-limiting for cytokinin responses.

Current understanding of the integration of cell division and expansion in the development of plant lateral organs such as leaves is limited. Cell number is established during a mitotic phase, and subsequent growth into a mature organ relies primarily on cell expansion accompanied by endocycles. Here we show that the three Arabidopsis cyclin D3 (CYCD3) genes are expressed in overlapping but distinct patterns in developing lateral organs and the shoot meristem. Triple loss-of-function mutants show that CYCD3 function is essential neither for the mitotic cell cycle nor for morphogenesis. Rather, analysis of mutant and reciprocal overexpression phenotypes shows that CYCD3 function contributes to the control of cell number in developing leaves by regulating the duration of the mitotic phase and timing of the transition to endocycles. Petals, which normally do not endoreduplicate, respond to loss of CYCD3 function with larger cells that initiate endocycles. The phytohormone cytokinin regulates cell division in the shoot meristem and developing leaves and induces CYCD3 expression. Loss of CYCD3 impairs shoot meristem function and leads to reduced cytokinin responses, including the inability to initiate shoots on callus, without affecting endogenous cytokinin levels. We conclude that CYCD3 activity is important for determining cell number in developing lateral organs and the relative contribution of the alternative processes of cell production and cell expansion to overall organ growth, as well as mediating cytokinin effects in apical growth and development.

KNOX gene function in plant stem cell niches.

Homeobox genes encode transcriptional regulators that control development in multicellular eukaryotes. In plants, post-embryonic shoot growth relies on the activity of indeterminate cell populations termed shoot meristems, within which members of the class-1 KNOX sub-family of homeobox genes are expressed. KNOX genes are differentially required for meristem development and function to inhibit cell expansion and differentiation associated with organogenesis. Mechanisms must therefore be employed to prevent KNOX gene expression in developing lateral organs such as leaves. This review focuses on the expression patterns, meristematic functions and regulation of KNOX genes, and how the activities of these genes are integrated within the framework of pathways that control plant development.

The developmental context of cell-cycle control in plants.

Plant growth is characterised both by continued growth and organogenesis throughout development, as well as by environmental influences on the rate and pattern of these processes. This necessitates a close relationship between cell cycle control, differentiation and development that can be readily observed and studied. The sequencing of the Arabidopsis genome has revealed the full complexity of cell cycle regulators in plants, creating a challenge to understand how these genes control plant growth and differentiation, and how they are integrated with intrinsic and external signals. Here, we review the control of the cell cycle and examine how it is integrated with proliferative activity within meristems, and during the differentiation processes leading to leaf and lateral root formation.