David Bird

Plant ABC proteins – a unified nomenclature and updated inventory

The ABC superfamily comprises both membrane-bound transporters and soluble proteins involved in a broad range of processes, many of which are of considerable agricultural, biotechnological and medical potential. Completion of the Arabidopsis and rice genome sequences has revealed a particularly large and diverse complement of plant ABC proteins in comparison with other organisms. Forward and reverse genetics, together with heterologous expression, have uncovered many novel roles for plant ABC proteins, but this progress has been accompanied by a confusing proliferation of names for plant ABC genes and their products. A consolidated nomenclature will provide much-needed clarity and a framework for future research.

Arabidopsis LTPG Is a Glycosylphosphatidylinositol-Anchored Lipid Transfer Protein Required for Export of Lipids to the Plant Surface

Plant epidermal cells dedicate more than half of their lipid metabolism to the synthesis of cuticular lipids, which seal and protect the plant shoot. The cuticle is made up of a cutin polymer and waxes, diverse hydrophobic compounds including very-long-chain fatty acids and their derivatives. How such hydrophobic compounds are exported to the cuticle, especially through the hydrophilic plant cell wall, is not known. By performing a reverse genetic screen, we have identified LTPG, a glycosylphosphatidylinositol-anchored lipid transfer protein that is highly expressed in the epidermis during cuticle biosynthesis in Arabidopsis thaliana inflorescence stems. Mutant plant lines with decreased LTPG expression had reduced wax load on the stem surface, showing that LTPG is involved either directly or indirectly in cuticular lipid deposition. In vitro 2-p-toluidinonaphthalene-6-sulfonate assays showed that recombinant LTPG has the capacity to bind to this lipid probe. LTPG was primarily localized to the plasma membrane on all faces of stem epidermal cells in the growing regions of inflorescence stems where wax is actively secreted. These data suggest that LTPG may function as a component of the cuticular lipid export machinery.

Identification of the Wax Ester Synthase/Acyl-Coenzyme A:Diacylglycerol Acyltransferase WSD1 Required for Stem Wax Ester Biosynthesis in Arabidopsis

Wax esters are neutral lipids composed of aliphatic alcohols and acids, with both moieties usually long-chain (C16 and C18) or very-long-chain (C20 and longer) carbon structures. They have diverse biological functions in bacteria, insects, mammals, and terrestrial plants and are also important substrates for a variety of industrial applications. In plants, wax esters are mostly found in the cuticles coating the primary shoot surfaces, but they also accumulate to high concentrations in the seed oils of a few plant species, including jojoba (Simmondsia chinensis), a desert shrub that is the major commercial source of these compounds. Here, we report the identification and characterization of WSD1, a member of the bifunctional wax ester synthase/diacylglycerol acyltransferase gene family, which plays a key role in wax ester synthesis in Arabidopsis (Arabidopsis thaliana) stems, as first evidenced by severely reduced wax ester levels of in the stem wax of wsd1 mutants. In vitro assays using protein extracts from Escherichia coli expressing WSD1 showed that this enzyme has a high level of wax synthase activity and approximately 10-fold lower level of diacylglycerol acyltransferase activity. Expression of the WSD1 gene in Saccharomyces cerevisiae resulted in the accumulation of wax esters, but not triacylglycerol, indicating that WSD1 predominantly functions as a wax synthase. Analyses of WSD1 expression revealed that this gene is transcribed in flowers, top parts of stems, and leaves. Fully functional yellow fluorescent protein-tagged WSD1 protein was localized to the endoplasmic reticulum, demonstrating that biosynthesis of wax esters, the final products of the alcohol-forming pathway, occurs in this subcellular compartment.

Arabidopsis ABCG Transporters, Which Are Required for Export of Diverse Cuticular Lipids, Dimerize in Different Combinations

Arabidopsis ATP binding cassette (ABC) transporters, ABCG11 and ACBG12, are required for export of different lipids from the epidermis to the cuticle. This study shows that ABCG11 and ABCG12 can form an obligate heterodimer and that ABCG11 can homodimerize. Live-cell imaging in epidermal cells demonstrates that localization of these ABCGs to the plasma membrane is dependent upon dimerization. ATP binding cassette (ABC) transporters play diverse roles, including lipid transport, in all kingdoms. ABCG subfamily transporters that are encoded as half-transporters require dimerization to form a functional ABC transporter. Different dimer combinations that may transport diverse substrates have been predicted from mutant phenotypes. In Arabidopsis thaliana, mutant analyses have shown that ABCG11/WBC11 and ABCG12/CER5 are required for lipid export from the epidermis to the protective cuticle. The objective of this study was to determine whether ABCG11 and ABCG12 interact with themselves or each other using bimolecular fluorescence complementation (BiFC) and protein traffic assays in vivo. With BiFC, ABCG11/ABCG12 heterodimers and ABCG11 homodimers were detected, while ABCG12 homodimers were not. Fluorescently tagged ABCG11 or ABCG12 was localized in the stem epidermal cells of abcg11 abcg12 double mutants. ABCG11 could traffic to the plasma membrane in the absence of ABCG12, suggesting that ABCG11 is capable of forming flexible dimer partnerships. By contrast, ABCG12 was retained in the endoplasmic reticulum in the absence of ABCG11, indicating that ABCG12 is only capable of forming a dimer with ABCG11 in epidermal cells. Emerging themes in ABCG transporter biology are that some ABCG proteins are promiscuous, having multiple partnerships, while other ABCG transporters form obligate heterodimers for specialized functions.

Arabidopsis cyclin-dependent kinase inhibitors are nuclear-localized and show different localization patterns within the nucleoplasm.

The Arabidopsis genome contains seven cyclin-dependent kinase (CDK) inhibitors (ICK for inhibitor/interactor with cyclin-dependent kinase) which share a small conserved C-terminal domain responsible for the CDK-inhibition activity by these proteins. Different ICK/KRPs have been shown to have unique expression patterns within tissues, organs and during the cell cycle. Previous studies have shown that overexpressing one of the ICK/KRPs inhibits CDK activity, cell division, and profoundly affects plant growth and development. In this study, we investigated the subcellular localization of the seven Arabidopsis ICK proteins and domains responsible for this localization. Using transgenic expression in Arabidopsis plants and transient expression in tobacco leaf cells, all ICK/KRPs fused to green fluorescent protein (GFP) were localized to the nucleus, suggesting that the nucleus is the cellular compartment for the plant CDK inhibitors to function. While ICK2/KRP2, ICK4/KRP6, and ICK5/KRP7 were localized to the nucleoplasm in a homogeneous manner, ICK1/KRP1, ICK3/KRP5, ICK6/KRP3, and ICK7/KRP4 showed a punctate pattern of localization. A small motif conserved amongst the latter group of ICK/KRPs is required to confer this subcellular pattern as deletion of this motif from ICK7/KRP4 resulted in a shift from a punctate to a homogeneous pattern of localization. While a single nuclear localization signal (NLS) is responsible for the nuclear localization of ICK2/KRP2, multiple mechanisms for nuclear localization are suggested to exist for the other six ICK/KRPs since deletion mutants lacking predicted NLS motifs and the conserved C-terminal domain are still localized in the nucleus.

Functions, regulation and cellular localization of plant cyclin-dependent kinase inhibitors

The cell cycle is regulated by the cyclin‐dependent kinase (CDK), and CDK inhibitors can bind to CDKs and inhibit their activities. This review examines plant CDK inhibitors, with particular emphasis on their molecular and cellular functions, regulation and cellular localization. In plants, a family of ICK/KRP CDK inhibitors represented by ICK1 is known and another type of CDK inhibitor represented by the SIMESE (SIM) has recently been reported. Considerable understanding has been gained with the ICK/KRP CDK inhibitors. These plant CDK inhibitors share only limited sequence similarity in the C‐terminal region with the KIP/CIP family of mammalian CDK inhibitors. The ICK/KRP CDK inhibitors thus provide good tools to understand the basic machinery as well as the unique aspects of the plant cell cycle. The ICK/KRP CDK inhibitors interact with D‐type cyclins or A‐type CDKs or both. Several functional regions and motifs have been identified in ICK1 for CDK inhibition, nuclear localization and protein instability. Clear evidence shows that ICK/KRP proteins are important for the cell cycle and endoreduplication. Preliminary evidence suggests that they may also be involved in cell differentiation and cell death. Results so far show that plant CDK inhibitors are exclusively localized in the nucleus. The molecular sequences regulating the localization and functional significance will be discussed.

Arabidopsis ketoacyl-CoA synthase 16 forms C36/C38 acyl precursors for leaf trichome and pavement surface wax

The aliphatic waxes sealing plant surfaces against environmental stress are generated by fatty acid elongase complexes, each containing a β-ketoacyl-CoA synthase (KCS) enzyme that catalyses a crucial condensation forming a new C─C bond to extend the carbon backbone. The relatively high abundance of C35 and C37 alkanes derived from C36 and C38 acyl-CoAs in Arabidopsis leaf trichomes (relative to other epidermis cells) suggests differences in the elongation machineries of different epidermis cell types, possibly involving KCS16, a condensing enzyme expressed preferentially in trichomes. Here, KCS16 was found expressed primarily in Arabidopsis rosette leaves, flowers and siliques, and the corresponding protein was localized to the endoplasmic reticulum. The cuticular waxes on young leaves and isolated leaf trichomes of ksc16 loss-of-function mutants were depleted of C35 and C37 alkanes and alkenes, whereas expression of Arabidopsis KCS16 in yeast and ectopic overexpression in Arabidopsis resulted in accumulation of C36 and C38 fatty acid products. Taken together, our results show that KCS16 is the sole enzyme catalysing the elongation of C34 to C38 acyl-CoAs in Arabidopsis leaf trichomes and that it contributes to the formation of extra-long compounds in adjacent pavement cells.

Defining the Diverse Cell Populations Contributing to Lignification in Arabidopsis thaliana Stems

Xylem vessel lignification in young Arabidopsis stems requires neighboring xylary parenchyma, but, as stems mature, fibers primarily produce their own lignin monomers. Many land plants evolved tall and sturdy growth habits due to specialized cells with thick lignified cell walls: tracheary elements that function in water transport and fibers that function in structural support. The objective of this study was to define how and when diverse cell populations contribute lignin precursors, monolignols, to secondary cell walls during lignification of the Arabidopsis (Arabidopsis thaliana) inflorescence stem. Previous work demonstrated that, when lignin biosynthesis is suppressed in fiber and tracheary element cells with thickened walls, fibers become lignin-depleted while vascular bundles still lignify, suggesting that nonlignifying neighboring xylem cells are contributing to lignification. In this work, we dissect the contributions of different cell types, specifically xylary parenchyma and fiber cells, to lignification of the stem using cell-type-specific promoters to either knock down an essential monolignol biosynthetic gene or to introduce novel monolignol conjugates. Analysis of either reductions in lignin in knockdown lines, or the addition of novel monolignol conjugates, directly identifies the xylary parenchyma and fiber cell populations that contribute to the stem lignification and the developmental timing at which each contribution is most important.